Where to buy Sodium Dichloroacetate DCA (NaDCA)

Sodium Dichloroacetate NaDCA available on line will cost $2-$3 per day.

You have obviously arrived here because yourself or someone in your life has cancer. Please take a minute and go to the home page of this site and read some of the published medical journal articles on DCA. Get an understanding of the Warburg effect the scientific principle behind this discovery, and you will then not question whether DCA works! This is not a hoax, this is not just one major University making the claim, there are papers published on this site from many major Universities.

The American Cancer Society warns you that these sellers are out to fleece desperate people of their money”.If you do your research you will be disappointed to find that these organizations you should be able to trust are actually working hand in hand with the Pharma companies to suppress any non pharma cancer treatment.

As we have said on this site NaDCA is a very simple molecule resembling vinegar and is documented in peer reviewed medical journal studies as being as safe to take for healthy people as it is for sick people.

If you are still questioning whether DCA works you need to only answer for yourself 2 questions.

1) Is the Warburg effect really the law of cancer? (meaning that all normal cells achieve respiration from Oxygen and all cancer cells receive respiration from glucose) PROOF….. The Warburg effect is the science behind the PET scan machine (patient is injected with radio active glucose and cancer cells light up on the screen) What Wargurg discovered was that when a cell became a cancer cell (reverted to glucose respiration) the mitochondria shut down. The mitochondria is the control center of a cell, every day as our cells divide the mitochondria looks at the cell and if it’s dna is not correct, (a bad cell) it kills itself through a process known as apoptosis. Every day our bodies expel millions of bad cells through apoptosis.  Therefore a cancer cell is simply a cell that achieves energy from glucose and continues to divide and multiply without our bodies being able to kill it through the normal process of apoptosis.

2) As the University of Alberta research claims does DCA turn the mitochondria of the cancer cell back on, allowing it to recognize itself as a bad cell and trigger apoptosis? The answer is YES! Read the published articles on the home page and believe in your own research, not what someone you think should know is telling you. At the very least use this information to start questioning your treatment or the treatment of a loved one suffering from cancer.

NaDCA advocate Martin C. Winer has come up with a protocol combining NaDCA with a supplement called Avemar, the protocol can be found here, this may be a  a good option for anyone in later stage cancer or has had orthodox treatment and a weakened immune system.http://www.martincwiner.com/dca-and-avemar-a-theoretical-protocol-for-cancer/

This protocol has been  tested by the Medicor Cancer Clinic in Toronto with great results.

There appear to be 3 main sites on line selling NaDCA. One thing you need to know is that these sites can not buy the NaDCA in North America or Europe. From what we have been able to find, the only supply sources at present are in China. This is due to the FDA , Health Canada and the European health agencies restrictions placed on Sodium dichloroacetate in 2007. The claimed intent of restricting NaDCA , (which is about as harmful as taking to much baking soda) being to protect us from ourselves.

For years we have seen many media reports of safety issues involving Chinese products. However, with what we have discovered about the ethics of North American drug companies and their ability to influence what the media reports to us, we began to question how bad quality issues could be. If you look into the Chinese pharmaceutical industry you will find they operate under very strict guidelines, with as much or more oversight as facilities in North America.

The truth is if there was as much medical journal published evidence that cocaine cured cancer, as there is regarding DCA, you would be asking everyone you knew if they knew a dealer! If the quality is your concern one of the online companies has every batch randomly tested in Canada. www.certifieddca.com

The NaDCA clinical trails are supplied world wide by a company called TCI America http://www.tciamerica.com/catalog/D1719.html and have been for decades, they do not sell to the general public. It also would appear from TCI’s sales site that the product they sell also comes from Asia. Medicor Cancer Center in Toronto claims to be purchasing their NaDCA from the USA which we would guess comes from TCI.

If you are going to take NaDCA, 100 grams is about a 90 day supply for most people treating cancer. As a supplement to prevent a cancer recurrence or just to keep you healthy 100 grams is about a 200 day supply at a half gram per day.

Keep in mind that there has not been a large demand for DCA and world wide supply is limited, one pharmaceutical company we spoke with indicated their excess available supply at 900 kg per year, that would only treat  9,000 people for 3 months. We also found that the difference in price varies in china, we found chemical companies that manufacture technical grade product are also now offering pharmaceutical grade, however pharmaceutical grade from an actual pharmaceutical company is more expensive which can be the price difference between the 3 major suppliers.

Since the amount taken is based on body weight it is not necessary to spend the extra for the product in capsules as you would only be breaking them apart to get the proper weight.

The other thing to consider is that all sites selling NaDCA were closed down by the FDA in 2007 and this could happen again, our point is if you are thinking of using NaDCA it may become hard to get as more people find out about it.

There have been some issues reported on The DCA Site regarding problems with DCA coming in from Mexico, if the price seems too cheap there could be good reason for it.

The www.dcasite.com is your best source for getting dosage information from others that have experience with NaDCA. Do keep in mind that social media people from the Pharma industry commonly take part in the chats and online discussions just to confuse people.

The top sites supplying NaDCA to the public are below,  However It has recently come to our attention that  www.shouldyoubuydca.com which has been a site used by Pharma DCA as a “we don’t sell DCA but buy this one site”  has claimed recently that they are an anonymous crusader for DCA and tested all the other suppliers product and only Pharma DCA and Sigma Aldrich passed, which of course Sigma Aldrich does not sell to the public. The site recommends people do not buy the other suppliers DCA now claiming their products have high Bromine counts however they provide no testing results. (their claim according to their site February 15,2015 as they may change it) What tells me no testing ever took place is that they report the Pure DCA and Certified dca as having 170mg per kg and 148mg per kg of bromine.  Had they actually tested, firstly the results would be reported as PPM (parts per million) or ug not mg per kg. They report Pharma dca and Sigma Aldrich as <2. less than 2 what? it should be less than 2 ug/kg which is also what the other 2 suppliers bromine levels are had they reported correctly  .170ug and .148 ug both <2 if reported in the same unit of measure as their product and Sigma Aldrich (information directly from Sigma Aldrich’s web site).

We have over the last three years received various comments and complaints about suppliers. We do not approve them without proper evidence that there is an issue with the supplier that could harm people. We set up this site as a place where people could find relative published medical Journal studies and form their own conclusions. We have long recommended these 3 sites as a source for DCA as they have been around the longest. What people may not realize is that these sites get regular orders from government buildings where the product is tested not for your protection but in an effort to catch a supplier selling something other than +99% pure dca. Believe me when I say if one of these suppliers was offering unsafe product you would have read about it everywhere, it would be a huge media circus.

We did have product tested from these 3 suppliers about 2 months ago and all passed, which I reported on this site, however I decided to take it down due to liability if there was an unexpected problem with a batch they put out. The sad part is that DCA does work and yet it is hard to get people to read through the medical journal articles and reach their own conclusions without second guessing themselves. There is a huge amount of propaganda put out by the Pharma companies disguised as Medical Websites and literally hundreds of pharma employees participating in social media sites using fear to keep people away from DCA. The last thing DCA needed is some idiot supplier trying to spread more false fear about other suppliers!

We still recommend the 3 suppliers below  strictly based on the fact that they have been around for more than 3 years and we have tested the products ourselves in the past.




We will continue to purchase from www.certifieddca.com simply because we have always had great service and each batch is tested in Canada for purity and any solvents or heavy metals. They also send you a test report for the batch once you have ordered. It is a little more expensive, about 20-40 cents per day. However they have always been very help full with questions. I don’t want to be seen as promoting them it is just the only supplier we have experience with, and as I said above, as far as quality goes It is my opinion that all 3 are safe to buy from.

Having the product tested yourself can cost up to $750 depending on where you go. If it is out of your budget by all means buy from whichever company you can afford.

If you do start taking NaDCA please let us know about your progress as it may help others as they try to make a decision that is best for them.

DCA Papers and Clinical Trials

DCA papers and clinical trials

For almost a decade there has been a growing interest in Dichloroacetate potential to successfully get rid of cancer while causing minimal harm to healthy organs. DCA is a relatively cheap substance which cannot be patented by the pharmaceutical industry, thus it could not generate profit for private drug companies. Right now, because of this reason, Dichloroacetate isn’t receiving enough funding and attention which it deserves.

Despite that, there are plenty of ongoing and completed studies which examine the facts of DCA appliance for therapy. This site will present a handful of completed scientific investigations and will constantly update you with the most recent publications related to the subject.

MEDLINE is the largest medical database in the world, and contains information on published DCA research. This database can be searched free of charge for those interested in reading DCA research, or the summaries of the DCA publications.

Further research to determine how well DCA works against various cancers within the human body is ongoing. 

DCA research

Further research


DCA History

DCA history

Since 1973 Sodium Dichloroacetate (DCA) was used to treat various mitochondrial disorders. It inhibits the activity of pyruvate dehydrogenase kinase, and reduces the accumulation of lactate in body tissues. Its usage for treating lactic acidosis has been successful and is still continued to this day, it is used in several research and medical centers in the United States and Canada.

The majority of the people who have used DCA are children with congenital mitochondrial disorders. The use of the drug could resume the normal function of the cellular enzymes and prevent further neurological damage, mental disability, microcephaly, blindness and movement disorders. Dichloroacetate safety has been confirmed long before the idea, that it could be helpful for someone who has cancer.
In 1920s German biochemist Otto Warburg found abnormalities in metabolism in cancer cells. Normal cells obtain energy by glucose oxidation, which requires the presence of oxygen. Cancer cells depend on glycolysis to obtain energy, and it can occur without the presence of oxygen, but is dependent on the availability of sugar. Cancer cells favor glycolysis even in the presence of adequate oxygen for oxidative phosphorylation, leading to a voracious appetite for glucose. This phenomenon prompted Warburg to propose that mitochondrial malfunction was the primary cause of cancer. Sodium Dichloroacetate (DCA) works by inhibiting the
“Warburg Effect”.

DCA forces cancer cell to abandon its preferred metabolic process and also induces apoptosis, or cellular suicide. The reason cancer is so fast growing is that the mitochondria have been deactivated, so the cells evade apoptosis and are able to grow in the absence of oxygen. DCA reverses this. In effect, DCA directly causes cancer cell apoptosis and works synergistically with other cancer therapies.

• In 2007 dr. Evangelos Michelakis of the University of Alberta in Canada published a research paper that renewed interest in DCA. It showed potential of DCA to shrink cancer tumors. In the study DCA was administered to rats with transplanted tumor cells (brain, breast and lung). DCA killed cancer cells without affecting healthy cells. The rats’ tumors decreased by up to 70 percent in three weeks of DCA treatment:
A Mitochondria-K Channel Axis Is Suppressed in Cancer and Its Normalization Promotes Apoptosis and Inhibits Cancer Growth

Other researchers have followed and confirmed anti-cancer effects of DCA. Yet most of the studies have been done on cell cultures in the lab, and not on the cancer patients themselves. But results are very consistent, suggesting DCA is effective against a wide variety of cancer types.

• In 2013, Phase 1 clinical trial of dichloroacetate (DCA) was completed in Canada. It showed that DCA is feasible and well tolerated in patients with recurrent malignant gliomas and other tumors metastatic to the brain using the dose range established for metabolic diseases:
Phase 1 trial of dichloroacetate (DCA) in adults with recurrent malignant brain tumors

• In another study, five glioblastoma multiforme patients were treated with oral DCA for up to 15 months. The research showed clinically promising results in four of the five patients:
Metabolic modulation of glioblastoma with dichloroacetate

• Medicor Cancer Center in Canada is a cancer clinic currently offering DCA therapy for it’s patients. It has published several case studies about the safety and effectiveness of DCA. Its real-world Observational DCA patient data is available to the public.

• Till this day, there are several ongoing clinical studies and a lot of pre-clinical research going on. Recently it has been noted that DCA can work by itself, however, it provides the maximum results in combination therapy with other drugs for a prolonged time period.

Methods and Supplements for Preventing DCA Side Effects

Methods and supplements for preventing DCA side effects

When you begin your Sodium dichloroacetate regimen, it is crucial that you take supplementation which provides protective benefits. This way you minimize the chance for developing reversible peripheral neuropathy asas well as other adverse reactions related to the nervous system.

Below you will find a list of supplements which are essential or recommended for a pleasant DCA usage experience with the lowest achievable side effect probability.

▪ Vitamin B1 – thiamine.(Necessary)
(take one and a half 100mg capsules / tablets twice a day. Take it before breakfast and before lunch.
An alternative way – take 100 mg three times a day. Total – 300 mg)

The B group vitamin thiamine appears to have a protective effect against peripheral neuropathy. This food supplement can be used not only for DCA induced neuropathy but also for other neuropathies which are caused by diabetes and chronic alcohol abuse. (Ref.)

We recommend using benfotiamine because it can be absorbed over five times better than the ordinary thiamine form.

In addition, the newest research claims that Vitamin B1 can have an antiproliferative effect on malignant cells. (Ref.)

▪ Alpha–Lipoic acid. (Necessary)
(take one 300 mg R+/S- capsule/tablet three times a day or take one 150 mg R+ capsule/tablet three times a day. Take it before breakfast, before lunch and before dinner. Total – 900 mg (R+/S-) or 450 mg (R+))

α-Lipoic acid is a strong antioxidant, it helps avoiding and controlling symptoms related to neuropathy. The supplement can lower anxiety, memory problems as well as help keeping away from peripheral neuropathy manifestations such as tingling, burning, painful sensations and numbness. (Ref.)

You can use smaller doses if you’re taking R-form α-Lipoic acid.
If you have Racemate α-Lipoic acid (which is a mix of R and L forms), you should take a twice larger dose to fulfill your daily goal.

Don’t take α-Lipoic acid if you’re receiving chemotherapy or radiotherapy.
α-Lipoic acid has a strong antioxidative effect that can interfere with the effectiveness of chemotherapy. For this reason, we recommend staying away from this supplement a couple of days before the chemo, during the treatment and 1 week after the chemotherapy. (Ref.)

α-Lipoic acid also can decrease the effectiveness of radiotherapy. This is why we recommend avoiding taking it for several days before, during and 2 week after these procedures.

▪ Acetyl L-Carnitine. (Recommended)
(take on 600mg capsule / tablet three times a day. Take it before breakfast, before lunch and before dinner. Total – 1800 mg)

The majority of scientific studies claims that Carnitine can be an effective aid to lower peripheral neuropathy. Acetyl L-Carnitine is also an attractive option because its longtime usage does not cause any side effects and carries no health risk. (Ref1.), (Ref2.), (Ref3.)

α-Lipoic acid and Acetyl L-Carnitine both appear to have a synergistic effect at preventing neuropathy.

On rare occasions, Sodium dichloroacetate administration can result in heartburn or nausea. If this is the case, try taking DCA after you eat a little bit of food and drink some fluids to avoid your stomach becoming irritated.

If that did not resolve the problem, you should try taking medications that lower gastric acid secretion – proton pump inhibitors.
Any type of PPI is acceptable provided the fact that they don’t have any major differences.

▪ Pantoprazole. 
(take one 40mg tablet per day, at the same time. Take it at least 30 minutes before your meal and DCA.)

For convenience purposes, we recommend using Pantoprazole because it doesn’t seem to have any poor interactions with other medications.

If you began experiencing moderate side effects or develop a stronger form of peripheral polyneuropathy – stop taking DCA until the adverse reactions become acceptable or disappear completely.

All Sodium dichloroacetate side effects are reversible.

When you stop taking DCA, the majority of the side effects disappear in several days. Peripheral neuropathy can take up to a week or, in rare occasions, a couple of weeks to resolve completely. (Ref.)

Additionally, if you have an opportunity – we recommend regularly performing blood tests and checking the blood serum for tumor marker levels.

UltrasoundComputer tomography scansMagnetic resonance imagingPositron emission tomography are imaging tests that can provide more information about the dynamics of your overall health, and most importantly, the size changes of your cancer.

DCA Safety and Side Effects

DCA safety and side effects

Sodium dichloroacetate is considered to be a fairly safe alternative cancer treatment. There have been no cases recorded for DCA to be a cause of death.

Before we begin, we should bear in mind that Sodium dichloroacetate has already demonstrated  success in dealing with ‘‘Lactic acidosis in children with congenital mitochondrial defects“  for some time. The first scientific studies and the usage of the drug began over 40 years ago. (Ref.)

In this time period, DCA has been constantly used as a medication for congenital mitochondrial diseases. The research done by Peter Stacpoole and his colleagues proved that when used for therapy, Sodium dichloroacetate can cause nonemild or moderate side effects. (Ref.)

The probability of adverse reactions is dependent on the dosing and the age of the patient. Larger DCA doses and  older patient age (above 40 years) are related to a higher side effect occurrence. (Ref.)

On exceptionally rare occasions, a small portion of the population can metabolize DCA more slowly than the average. For this reason, even the standard DCA doses can cause adverse reactions to appear faster and more prominent in this group of people. In this case, lowering the DCA dose should fix the issues.

If you stop taking DCA, almost all of the side effects disappear in less than a week. The reversible peripheral neuropathy can sometimes take up to 7 or 14 days (rarely) to resolve completely. (Ref.)

According to one of the most famous DCA clinics and their observational data, 44 % of the patients who have taken DCA did not experience side effects.

The most common side effects caused by Dichloroacetate:

▪ Peripheral neuropathy.
(experienced by up to 20% of people who use DCA).

This group of symptoms begins in the fingers, hands and feet. Depending on the intensity of the neuropathy, it can manifest as tingling, numbness, tremor, painful sensations and slightly increased difficulty of coordinated movement.
On less common occasions, neuropathy can emerge in other places and appear as the tingling of eyes, lips and tongue.

Typically, at least a couple of weeks or months are needed for peripheral neuropathy to develop.
This side effect is reversible – its intensity can decrease or it can disappear completely upon lowering the DCA dose or stopping DCA usage. (Ref.)

▪ Sleepiness, mental fogginess, confusion
(experienced by up to 20% of people who use DCA).

This group of symptoms is reversible – you can decrease their intensity or completely make them disappear by lowering the DCA dose or stopping DCA usage.

The rare side effects caused by Dichloroacetate:

▪ Heartburn, nausea, digestive disorders.

Administering Dichloroacetate through the mouth can sometimes cause GI irritability.

▪ Pain at the tumor site (temporary and then resolves).

A very rare adverse reaction. It indicates that due increased apoptosis a lot of cancer cells are dying and indicates that DCA therapy is effective. However, only a couple of Tumor lysis syndrome cases were documented in the most popular DCA administering clinics. This situation is more likely to happen to people who have leukemia, lymphoma or big volume tumors. (Ref.1Ref.2)

▪ Mild liver enzyme (AST, ALT, GGT) elevation, without symptoms.

A majority of medications can cause mild liver enzyme changes in the blood. DCA can cause minimal liver transaminase and transpeptidase elevations (about 50 – 60 U/l) for 1 % of the patients. These little alterations should not cause any worries.
A similar or bigger liver enzyme increase can be influenced by antibiotic, paracetamol (acetaminophen), some types of medicinal herbs and birth control pills. (Ref.)

▪ Increased anxiety, mood changes, hallucinations.

These effects are temporary and should disappear with the discontinued use of DCA. They are more likely to appear in patients that are using drugs which strongly influence the Central nervous system.

Dichloroacetate influence on different organ systems:

▪ DCA and the brain.
If you are currently using cannabinoids, benzodiazepines, opioids or other drugs which affect the Central nervous system, keep in mind that DCA can amplify the adverse reactions caused by these medications (eg. Delirium, memory problems).
This scenario is more likely to happen if the prescriptions have already caused side effects. If the patient is not experiencing any issues with the CNS affecting drugs – the risk for such interactions with DCA is low.

To minimize the probability of these drugs interacting, we recommend starting with low Sodium dichloroacetate doses and to gradually increase them. (Ref.)

▪ DCA and the heart.
Dichloroacetate seems to have a positive effect for the heart function without increasing the additional demand for oxygen. It also improves the efficiency of energy generation in the heart muscle. The drug is safe to use for people with heart failure and increased risk of cardiac ischemia. (Ref.)

▪ DCA and the liver.
In case of liver failure and severe jaundice don’t use high doses of DCA because Dichloroacetate is metabolised in the liver. In situations like these, DCA should be administered intravenously and not through the mouth. (Ref.)

▪ DCA and the kidneys.
Dichloroacetate is safe for patients who have kidney failure. The drug has no toxicity for the kidneys.

▪ DCA and diabetes.
Patients who have diabetes can achieve better blood glycemic control with the help of Sodium Dichloroacetate. DCA seems to lower the blood sugar in between meals. (Ref.)

This is the current accurate information on how DCA affects the major organs in the body. We can come to a conclusion that if Sodium Dichloroacetate is administered with care and adequate basic knowledge, its health risks are low and can be almost entirely prevented.

We hope this article answers the most important questions.

How DCA Works

For almost half a century, DCA has been a relatively basic substance used for treating people with congenital mitochondrial diseases. Nearly a decade ago, the interest in this drug spiked up because of new research and claims that it could be able to serve those who have cancer. Since then, there has been a lot of interest generated towards this medication.

In this long article, we will attempt to briefly cover what we know about dichloroacetate and how it works. Keep in mind – we‘ll try to explain the complex mechanisms as simply as possible. We encourage every person interested in DCA therapy to read on.

So… How does a small, inexpensive and a relatively nontoxic molecule like dichloroacetate work ?

How cancer cells act differently ?

To better understand the mechanism of DCA, we must be aware of the different processes that thrive in a cancerous cell. Cancer is considered to be a genetic disease in which genes that control how our cells grow and divide start behaving abnormally. Due to error in our DNA, the cells become chaotic, multiply uncontrollably and change their normal metabolic activity.

Every cell contains important organelles called mitochondria. These structures can be called “cellular power plants” because they produce the energy needed for live organisms to function properly. Besides, mitochondria are important in the cells life cycle – they play key roles in activating apoptosis.

Unfortunately, cancer cells have reduced mitochondrial function. This means that cancerous cells mostly produce energy by extremely high rates of glycolysis outside the mitochondria, rather than oxidative phosphorylation inside the mitochondria (Warburg effect) which also causes a massive increase in uptake of glucose and the exhaustion of the patient.

Because of the intracellular metabolic changes apoptosis (natural cell death) is stopped and it makes the malignant cells “immortal”.

Suppressed mitochondrion function leads to a lot of advantages for the tumor – it can survive and grow without oxygen in anaerobic conditions (eg. the cells in the middle of a cancerous mass), it promotes biosynthesis (cancer growth and division), it evades immune cells and disrupts the normal architecture of tissues (the cancer becomes more malignant and dangerous).

On the top of that, the Warburg effect produces an acidic environment. Such conditions damage the intercellular matrix, set the cancerous cells free into the bloodstream or lymph and promote metastasis (the cancer can spread and become deadly).

As we can see, the Warburg effect causes metabolic changes that make cancer a hardly manageable illness. Nevertheless, recently there have been ideas to begin perceiving and approaching cancer as a metabolic disease and this is where the molecule of DCA comes in handy.

How DCA affects cancer ?

So far we can understand how the cellular metabolism of tumors differs from that of our healthy, normal cells. Malignant cells switch off their mitochondria and start producing energy mainly through cytoplasmic glycolysis and these changes generate a lot of advantages for the tumor.

Dichloroacetate works by restoring the suppressed mitochondrial function and rendering the “bad cells” non cancerous. The normalized mitochondria then are able to resume the halted apoptosis process (the natural intracellular suicide system) and the harmful cells start dying on their own. What’s more important, the drug is selective. It doesn’t poison healthy tissues and cause significant effects on non carcinogenic cells like cytotoxic chemotherapy drugs.

The way DCA achieves these results is by reversing the Warburg effect. The substance inhibits an important enzyme which is essential for cancer proliferation – pyruvate dehydrogenase kinase (PDK). Once again the cell starts producing most of its energy in a normal way (through oxidative phosphorylation). The mechanism restores normal cellular metabolic activity.

Notably, Sodium Dichloroacetate has a lot of characteristics of an ideal antitumor therapy. We will discuss these characteristics further.

Why DCA is a good anticancer medication ?

To begin with, as a result of increased apoptosis, the substance effectively stops tumor growth (proliferation) and can even cause them to shrink in size or disappear.

To our surprise, DCA can also reduce the vascularity of tumors (by inhibiting angiogenesis). This prevents the nutrients from reaching and feeding the “bad cells”. Less blood vessel deposition on the cancerous masses also means that there are fewer pathways for cancer to spread – this lowers the probability of metastasis and disease progression.

Last but not least, since dichloroacetate is a small molecule, it crosses the blood-brain barrier and can help manage brain tumors. Currently, there are only few prescriptions that can reach the cerebral matter, making DCA a considerable option for therapy.

However, we are used to the reality that anticancer medications cause severe outcomes. Chemotherapy can have a very harsh effect on the body and provide unpleasant experiences. This is why patients are specifically prepared and receive medications prior to the administration of chemotherapy, to help minimize this

Despite that, DCA isn’t considered to be a cytotoxic chemotherapy drug and it appears to cause minimal systemic toxicity. Dichloroacetate is a gentle non-chemo treatment option that can have none, little or mild side reactions.

Then again, all the side effects are reversible which makes it the most appealing characteristic of using this molecule.

To put in simply, DCA induces intracellular as well as macroscopic changes that can help you accomplish successful therapy against cancer and achieve good improvements. Many people start feeling better in weeks.

What positive improvements people can expect ?

Given the fact that now we understand enough things about this relatively new cancer treatment,  we can turn our attention from a scientific perspective to a more practical point of view. What are the possible experiences when using Sodium Dichloroacetate ?

Bare in mind that the information we present is based on real and open observational data gathered from the clinical practise of top DCA therapy centers in the world. We must remember the main point which is true to every cancer case there is – the earlier the disease is caught and diagnosed, the sooner we take action, the better results we will achieve. The DCA treatment will not always provide positive outcomes and help everyone.

The lowest positive response is disease stabilization. This means that the tumor stops spreading and growing. There are no further signs of cancer progression.

As a result of taking DCA, a much better positive response can be improved symptoms. Patients regain their appetite, feel more energized, reduce their fatigue, regain weight and feel less pain. These things tend to last for a sustained period of time.

More importantly, people suffering from cancer obtain an improvement in blood tests and a reduction of tumor markers.

The best results of using DCA are measurable tumour size reduction or complete cancer remission. DCA users have their tumours screened by imaging techniques such as CT scans,  Magnetic resonance imaging, Ultrasonography and report significant cancer size reduction. Some of them even report complete cancer recovery.

Half of the people who take DCA experience mild side effects that most of the time are neurological and can be improved by a couple of dietary supplements (eg. Vitamin B1Alpha-Lipoic Acid)  or by taking a break from the treatment.

When all the things are considered, we must emphasize that sodium dichloroacetate can be taken alone or in combination with other anticancer medications. Naturally, a lot of people are eager to know – is DCA acceptable with other forms of cancer therapy ?

In short – yes. It is possible and even encouraged (with a couple of exceptions).

DCA and Cancer: Non-Hodgkin’s lymphoma cured in 4 months (Case presentation)

Today we would like to present you our first article based on Dichloroacetate usage benefits as described in case series. This writing will focus on a middle aged man who managed to cure his IV stage Non-Hodgin lymphoma with the use of DCA.

But first – let’s briefly remember some important aspects of this cancer and Sodium dichloroacetate.

Non-Hodgkin lymphoma is a type of cancer that begins in the body‘s immune system cells – T or B lymphocytes. The main histological difference between this malignancy and an other similar illness, the Hodgkin disease, is that the latter cancer has Reed-Sternberg cells present in biopsies. Hodgkin lymphoma is a lot rarer between the two diseases. Besides that, it is also one of the most successfully treated cancers today – 5-year survival rate is about 90 %. (Ref.)

Often one of the first considerably important symptoms of Non-Hodgkin lymphoma is enlarged non-painful lymph nodes. The other signs of the disease include fever, loss of weight, fatigue, cough, shortness of breath and night sweats. This cancer is first suspected when the associated symptoms appear (especially the painless swelling in the lymph nodes) or by accidentally discovering abnormal blood test results. Then the person has to get his blood checked for further analysis.

Above all, computed tomography scans and biopsies play a major role in determining the final diagnosis and the best possible choice of treatment for such patient.

Non-Hodgkin Lymphoma Hodgkin lymphoma
 Symptoms Non-painful lymph node swelling, night sweats, weight loss, fever, fatigue, cough, shortness of breath
 Diagnosis Confirmed via a biopsy of an abnormal lymph node, bone marrow or suspected tumor tissue
 Occurrence More common (6th most widespread cancer in the world) Less common (about ten times fewer cases than Non-Hodgkin lymphoma
 Age groups 45 years and older 15 – 24 years old or 60 years and older
 Treatment Mostly chemotherapy
(R-CHOP chemo regimen)
I or II stage – radiotherapy, ± chemotherapy
III or IV stage – always chemotherapy
(ABVD chemo regimen)
 Prognosis Depends on the type,
recovery is less frequent than in Hodgkin lymphoma
One of the most treatable cancers

Did you know that Non-Hodgkin lymphoma is a quite common cancer ?

Non-Hodgkin lymphoma accounts for 4 % of all new cancer cases. About 20 men and women of 100 000 develop this disease annually.
In 2014 there were about 660 000 people in America who were living with this diagnosis. (Ref.)

The numbers of new Non-Hodgkin lymphoma cases are increasing every year. However, this could be because the diagnostic capabilities of such diseases are getting better. (Ref.)

Non-Hodgkin Lymphoma symphoms and risk factors I DCA and Cancer


A lot of cancers, including Non-Hodgkin lymphoma, are treated with chemotherapy which can help achieve full remission (cured cancer).

Unfortunately, treatment with such medications can result in uncomfortable adverse reactions and long-term health problems. Sometimes the malignancy can relapse (come back) and you have to take chemo drugs all over again.

Not surprisingly, this can be a couple of reasons why people seek alternative cancer therapies by themselves or with the help of other specialists.
One of such alternative treatment choices is Sodium dichloroacetate. Before the idea that this drug could be used to help someone with oncological illnesses, the substance has been used for several decades as a medication for children suffering from congenital mitochondrial diseases.

DCA pharmacokinetics, pharmacodynamics and side effects were studied and discovered long before the accidental findings that this medication could be useful for treating cancer. (Ref.1), (Ref.2)

But how does DCA work ? To put it simply, the most important thing that you should know about the mechanism is that the drug inhibits an important enzyme for cancer – pyruvate dehydrogenase kinase. This leads to various changes in tumor cells.

Firstly, the Warburg effect is diminished. This resumes normal cellular respiration from aerobic glycolysis and lowers elevated intracellular acidity. Secondly, dichloroacetate promotes selective cancer cell apoptosis (cell death) which means that it stops tumor growth and shrinks their volume. Last but not least, DCA can even lower the risk of metastasis. (Ref.)

These sound like great accomplishments considering the fact that the risk of side effects is significantly lower when you take DCA with a couple of other food supplements.

If, however, the adverse reactions do show up – they‘re mostly mild and do not cause much discomfort. These issues are entirely reversible and go away in a couple of days when you stop taking dichloroacetate. (Ref.)

So, this appears to be a promising alternative cancer treatment. And it‘s already helping people who are dealing with oncological diseases. (Ref.1), (Ref.2)

We believe that it could help even more people. That is why we would like to present a case in which a currently 52 year old man cured his cancer.

He had IV stage Non-Hodgkin follicular lymphoma, which was completely resolved as a result of self-medication with DCA (Sodium dichloroacetate).

Non-Hodgkin Lymhpoma I Before and after DCA treatment

The person was at the age of 46 when he started feeling strange. In the last 5 months he lost a lot of weight (50 pounds), had constant fever and drenching night sweats. On top of that, he had enlarged cervical lymph nodes that extended all the way from the top of his neck to the collarbone.

Finally, the man decided to wait no more and got his health checked. The results of a computed tomography scan concluded that there were pathologic lymph nodes in his head, neck, chest, abdomen and pelvis. This was poor news.

Once the male had biopsies taken from his bone marrow and lymph nodes, the diagnosis became clear – he had IV stage Non-Hodgkin follicular lymphoma.

Shortly afterwards the doctors gave him six cycles of R-CHOP chemotherapy (rituximab,cyclophosphamidedoxorubicin,vincristine and prednisolone).

The chemo treatment took several intense months. When he once again had a CT scan performed, the doctors confirmed that he was clear from cancer. He had achieved his first remission (his tumors disappeared).

The man resumed to his former way of life, continued his ordinary daily activities and regularly performed health checks at the physician‘s office. For a year he was completely healthy.

However, after some time the fever, coughing and night sweats came back. Not only that, but he had also lost 11 pounds in two weeks.

Non-Hodgkin Lymphoma I DCA usage protocol for cancerThe male went straight to the doctors and after a couple of diagnostic procedures he received bad news.

He had enlarged lymph nodes on the right side of his head and neck. The cancer was back.

When the man received offers to repeat the treatment, he refused, claiming that chemotherapy and its side effects, especially nausea and vomiting, made him extremely upset and that he would rather avoid the experience.

As a result of his situation, he started searching for alternative cancer treatments that could improve his condition.

Shortly, he learned about DCA, bought this substance online and began treating himself.

His every day ‘‘DCA protocol“ consisted of:
• 1 000 mg Sodium dichloroacetate,
• Vitamin B1 500 mg,
• Alpha-Lipoic acid 1200 mg,
• Green tea leaf extract (Jarrow) 500 mg,
• 10 oz of  Mountain Dew (he would mix it with DCA and drink it afterwards).

The man took this regimen every day. These were the results:

✓ After two weeks his fever, night sweats, fatigue and weight loss started to improve.
✓ After a month of taking DCA his enlarged lymph nodes started to shrink. Two months later no lymph nodes were palpable.
✓ After 71 days of the regimen his symptoms disappeared completely. He recovered his sense of well-being, was full of energy, regained normal appetite and was once again able to have good quality sleep.

Eventually, something astonishing happened. There were no remaining signs of cancer left in his body. This was confirmed by a PET scan at 2008 December (4 months after he had begun his DCA therapy).

All of the previous tumor was gone. The man was cured from Non-Hodgkin lymphoma cancer.

Last time they contacted this person who beat cancer was at the end of 2012. Then he claimed that he still takes 1 000 mg of DCA three times a week together with Thiamine and Alpha-Lipoic acid for prophylaxis.

The 52 year old man feels great. He enjoys his life and works full-time. The last PET scan did not detect any cancerous tissues and cells in the body. Aside from his slightly elevated triglyceride and cholesterol levels, all of his blood tests are normal (the fat and cholesterol are probably higher due to other reasons).

Non-Hodgkin Lymphoma I Before and After DCA treatment PET scan

We prepared this case for presentation based on voluntary medical document distribution to the researchers.

Multi-modality imaging to assess metabolic response to dichloroacetate treatment in tumor models


Reverting glycolytic metabolism is an attractive strategy for cancer therapy as upregulated glycolysis is a hallmark in various cancers. Dichloroacetate (DCA), long used to treat lactic acidosis in various pathologies, has emerged as a promising anti-cancer drug. By inhibiting the pyruvate dehydrogenase kinase, DCA reactivates the mitochondrial function and decreases the glycolytic flux in tumor cells resulting in cell cycle arrest and apoptosis. We recently documented that DCA was able to induce a metabolic switch preferentially in glycolytic cancer cells, leading to a more oxidative phenotype and decreasing proliferation, while oxidative cells remained less sensitive to DCA treatment. To evaluate the relevance of this observation in vivo, the aim of the present study was to characterize the effect of DCA in glycolytic MDA-MB-231 tumors and in oxidative SiHa tumors using advanced pharmacodynamic metabolic biomarkers. Oxygen consumption, studied by 17O magnetic resonance spectroscopy, glucose uptake, evaluated by 18F-FDG PET and pyruvate transformation into lactate, measured using hyperpolarized 13C-magnetic resonance spectroscopy, were monitored before and 24 hours after DCA treatment in tumor bearing mice. In both tumor models, no clear metabolic shift was observed. Surprisingly, all these imaging parameters concur to the conclusion that both glycolytic tumors and oxidative tumors presented a similar response to DCA. These results highlight a major discordance in metabolic cancer cell bioenergetics between in vitro and in vivo setups, indicating critical role of the local microenvironment in tumor metabolic behaviors.

Keywords: tumor metabolism, DCA, 17O MRS, hyperpolarized 13C-MRI, 18F-FDG PET


Warburg metabolism (enhanced glycolysis in the presence of oxygen) is a common feature of several malignant tumors and is associated with cancer aggressiveness, invasiveness and poor prognosis []. Because of this high glycolytic rate in various cancers, targeting glucose metabolism is presented as an attractive anticancer approach endowed with a high specificity and limited undesirable side effects []. Indeed, conventional treatments rely on the rapid proliferation process present in cancer cells but also in healthy cells. Treatments targeting glycolytic metabolism should instead specifically alter metabolic adaptations that support the Warburg malignant phenotype, adaptations that are not shared by normal cells. To support drug development and assessment in clinical trials, there is a critical need for dedicated criteria evaluating tumor response to these emerging therapies. Moreover, for new cytostatic agents targeting tumor metabolism, the use of conventional anatomical imaging techniques is not optimal for treatment response assessment [] and only functional and molecular imaging techniques may offer the possibility of an early assessment of the tumor response [].

Recently, we have investigated the effects of dichloroacetate (DCA) in tumor cell lines presenting different metabolic profiles []. DCA is a promising molecule that promotes glucose oxidation over glycolysis by inhibiting the mitochondrial pyruvate dehydrogenase kinase (PDK) and has successfully reached clinical trials []. We found that 5 mM DCA was more effective in glycolytic-phenotype cancer cells, where reduction in cell proliferation was mediated by a reactivation of mitochondrial function and a decrease in glycolytic and pentose phosphate pathway fluxes. Our data suggested that DCA may benefit to patients with highly glycolytic tumors. Therefore, the objective of the present study was to assess the effect of DCA in these prototypical tumor models in vivo, namely the glycolytic MDA-MB-231 human breast cancer model and the oxidative SiHa human cervical cancer model. For this purpose, we used a multi-modality molecular imaging approach using several pharmacodynamic metabolic biomarkers. Oxygen consumption, studied by 17O magnetic resonance spectroscopy (17O MRS), glucose uptake, evaluated by 18F-fluorodeoxyglucose positron emission tomography (18F-FDG PET) and pyruvate transformation into lactate, measured during hyperpolarized 13C-magnetic resonance imaging (hyperpolarized 13C-MRI), were monitored before and after DCA treatment in tumor bearing mice. Surprisingly, in vivo models did not recapitulate the previously observed in vitro behavior.


To assess the impact of DCA treatment on the metabolism of the models in vivo, oxygen consumption (Figure ​(Figure1),1), glucose uptake (Figure ​(Figure2)2) and lactate flux (Figure ​(Figure3)3) were measured in MDA-MB-231 and SiHa tumors before and 24 hours after DCA treatment.


Figure 1

Effect of dichloroacetate on tumor oxygen consumption in vivo

Tumor H217O signal from representative MDA-MB-231 tumors A. and SiHa tumors B. acquired before, during and after a 2 min inhalation period of the 17O2gas. H217O signal is expressed as relative to the mean baseline signal before 17O2delivery. 17O2 metabolismis not modified by DCA treatment. C. Comparison of the rate of H217O signal after 17O2 delivery in tumors pre and post-treatment. Data are expressed as means ± SEM. Paired tests were two-sided.


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Figure 2

Effect of dichloroacetate on tumor glucose uptake in vivo

Representative 18F-FDG PET images showing MDA-MB-231 A-B. and SiHa C-D.tumor-bearing mouse imaged before and 24 hours after DCA treatment. Tumors are indicated by thin arrows. 18F-FDG uptake is expressed in %ID/g. Images were normalized. DCA does not alter 18F-FDG uptake in MDA-MB-231 and SiHa tumors. E. Comparison of 18F-FDG uptake before and after treatment. Data are expressed as means ± SEM. Paired tests were two-sided.


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Figure 3

Effect of dichloroacetate on tumor lactate production in vivo

Tumor lactate and pyruvate peak intensities after i.v. injection of hyperpolarized 1-13C pyruvate from representative MDA-MB-231 tumors A-B. and SiHa tumors C-D. Lactate production, measured by the Lac/Pyr ratio, in MDA-MB-231 and SiHa tumors before and after treatment E. Data are expressed as means ± SEM. Paired tests were two-sided.

In Figure ​Figure1,1, tumor H217O spectra are presented for representative MDA-MB-231 (Figure ​(Figure1A)1A) and SiHa tumors (Figure ​(Figure1B)1B) during 17O MRS experiments. The results were highly reproducible under the same conditions tested. The evolution of H217O signal, demonstrating the 17O2 metabolism in tumors, was similar before and after DCA treatment in MDA-MB-231 tumors (Figure ​(Figure1A)1A) and in SiHa tumors (Figure ​(Figure1B).1B). In both tumors models, we found that DCA treatment did not majorly impact oxygen consumption, as assessed by the rate of increase in H217O signal (Figure ​(Figure1C).1C). MDA-MB-231 tumors exhibited a slope of 1.02 10-3 ± 0.16 10-3 s-1and 0.91 10-3 ± 0.09 10-3 s-1 before and after treatment respectively (n=5, P=0.5366). For SiHa tumors, the slopes were 0.85 10-3 ± 0.14 10-3 s-1 under baseline condition and 0.79 10-3 ± 0.11 10-3 s-1 post-treatment (n =5, P=0.2892).

18F-FDG uptake (%ID/g) measured in both tumor models under baseline and post-treatment conditions are presented in Figure ​Figure2.2. In both tumor models, we found that DCA treatment did not lead to a significant change in the uptake of 18F-FDG (Figure 2A-2D), assessing a limited impact of DCA on glucose uptake (Figure ​(Figure2E).2E). %ID/g measured on PET images (mean ± SEM) were 1.88 ± 0.12 under baseline condition and 1.78 ± 0.11 after treatment for MDA-MB-231 tumors (n=7, P=0.2120) and 1.79 ± 0.21 under baseline condition and 1.89 ± 0.19 after treatment for SiHa tumors (n=7, P=0.0813).

The influence of DCA treatment on pyruvate transformation into lactate was measured after hyperpolarized 1-13C pyruvate injection during hyperpolarized 13C-MRS studies (Figure ​(Figure3).3). Representative pyruvate and lactate peak intensities over time of MDA-MB-231 tumors and SiHa tumors captured before and 24 hours after DCA treatment are shown in Figure 3A-3D. Lactate production was reduced after DCA treatment only in SiHa tumors (Figure ​(Figure3E).3E). Lactate/pyruvate ratio (Lac/Pyr) shifted from 0.55 ± 0.05 to 0.48 ± 0.04 in MDA-MB-231 tumors (n=7, P=0.3105) and from 0.82 ± 0.05 to 0.63 ± 0.07 in SiHa tumors (n=7, P=0.0348).

We also evaluated the magnitude of response to the treatment by measuring the variation within the above biomarkers between baseline and post-treatment conditions (Figure ​(Figure4).4). No differences in oxygen consumption and lactate flux measurements were observed between MDA-MB-231 and SiHa tumors during the treatment (P>0.05) (Figure 4A, 4C). Only a small but significant difference in behavior was observed for 18F-FDG uptake measurements (P=0.0240) (Figure ​(Figure4B).4B). MDA-MB-231 tumors decreased their 18F-FDG uptake after treatment (n=7, -5.4 ± 3.5 %) compared to SiHa tumors (n=7, +6.7 ± 3.1 %).


Figure 4

DCA does not significantly influence the metabolism of glycolytic tumors compared to oxidative tumors, as assessed by 17O2 metabolism A., 18F-FDG uptake B. and pyruvate transformation into lactate C. measurements in vivo

The magnitude of response to dichloroacetate (variation) is identical in both models, only a small difference in behavior is observed for 18F-FDG uptake. Data are expressed as means ± SEM. Unpaired tests were two-sided.


In this study, the impact of DCA on tumors presenting different metabolic profiles was evaluated using molecular imaging in vivo. Recent findings identified that DCA preferentially impairs glycolytic cells compared to oxidative cells. The purpose of the present study was to establish the relevance of these findings in vivo using the same prototypical tumor models as in our in vitro study [], namely the MDA-MB-231 human breast cancer model reported as glycolytic [] and the SiHa human cervical cancer model documented as oxidative []. The dose and administration scheme were selected based on previous reports attesting the efficacy of DCA in tumors [].

In vitro, we previously identified clear effects of DCA treatment on oxygen consumption, glucose consumption and lactate uptake in glycolytic MDA-MB-231 human breast cancer cells. On the other hand, the metabolic activity of oxidative SiHa human cervical cancer cells was not altered by DCA treatment. Using a multi-modality imaging project, we were not able to recapitulate these findings in vivo. Pre-treatment, MDA-MB-231 and SiHa tumors exhibit the same metabolic profile. As MDA-MB-231 and SiHa tumors were previously described as hypoxic under baseline condition [], we highlighted here that both tumor models exhibit a glycolytic phenotype under anaerobic condition. Post-treatment, glycolytic MDA-MB-231 tumors do not appear more impacted than oxidative SiHa tumors (Figure 1-4). Also, some marginal metabolic changes were identified such as a significant decreased lactate production in SiHa tumors (Figure ​(Figure3E)3E) or a decreased 18F-FDG uptake in MDA-MB-231 tumors (Figure ​(Figure4B).4B). Together, those findings did not highlight a clear metabolic shift in MDA-MB-231 tumors or in SiHa tumors treated with DCA during 24 hours (Supplementary Figure S1). This inability to observe any treatment response in vivo could not be attributed to any differences in growth rate between the tumor models under study (Supplementary Figure S2). Also, the measurement repeatability was formerly established using the same tumor models []. Our study demonstrated that the tumor metabolic response to DCA was dramatically different between in vitro and in vivo conditions.

Because of its good tolerability and safety, DCA has been universally exploited to lower lactate levels in acquired or congenital forms of lactic acidosis []. In 2007, Bonnet and colleagues investigated the effects of DCA in cancer and discovered that DCA was promoting apoptosis in vitro and decreasing tumor growth in vivo []. Since then, this orally available and cheap molecule has been further investigated in vitro, in vivo and successfully reached clinical trials. The first data available from the clinical trials indicate that DCA appears to be efficient in adults in solid and brain tumors []. However, no firm conclusions stand out in advanced non-small cell lung cancer []. In another recent study of Feuerecker and co-workers, promotion of tumor growth was even observed after DCA treatment in neuroblastoma tumors []. These studies indicate that response to DCA treatment may drastically vary among tumor types.

The redirection of glucose metabolism from glycolysis to oxidation leading to the inhibition of proliferation and the induction of caspase-mediated apoptosis was initially proposed as the generic mechanism of action of DCA. In a recent phase I study in patients with advanced solid tumors, decreased 18F-FDG uptake was observed after DCA therapy, supporting the use of 18F-FDG uptake as a potential biomarker of response to DCA []. Also, hyperpolarized 13C-pyruvate MRI has already been used in several preclinical studies to monitor DCA effect in solid tumors [], but also in cardiac [] and brain studies []. In the present study, 18F-FDG uptake was unchanged after DCA treatment (Figure ​(Figure2E).2E). This suggests that DCA treatment does not impair glucose uptake and phosphorylation but could potentially impact downstream transformation of glucose. However, no effects on bicarbonate production were detected that could demonstrate changes in energy metabolism from glycolysis to oxidative phosphorylation (Supplementary Figure S3). Recent findings suggested that DCA may also act by other mechanisms. While a possible disruption of the balance between fatty acid β-oxidation and glucose oxidation has already been suggested as an additional mechanism involved in the overall effects of DCA in vivo (as reviewed by []), PDK inhibitors may potentially induce other compensatory mechanisms that could limit the impact of such drugs on global tumor metabolism. The anti-cancer effects of DCA appear to rely on multiple mechanisms depending on the drug concentration, drug administration scheme [] and cell type []. Also, a change in PDK isoform expression between in vitro and in vivo model could also greatly influence the effect of DCA on tumor metabolism in vivo. Indeed, oncogene regulation and tumor microenvironment, like extracellular acidosis, can affect PDK isoform expression [], possibly leading to the expression of a PDK isoform less sensitive to DCA effects. Our findings are consistent with a recent study highlighting that tumor microenvironment could be as important as the (epi) genetic profile to shape the tumor phenotype []. Further investigations using relevant isogenic cell clones with ability to form tumors in vivo should be considered to determine the effects of DCA on energy metabolism in vivo.

In conclusion, our multi-modality imaging study identified major discordances between in vitro and in vivo metabolic responses to DCA treatment, in cancer models presenting distinct metabolic profiles. Results suggest preferring implanted tumors and spontaneous cancer models to study DCA treatment within the milieu of the tumor microenvironment. Overall, further investigations are required to elucidate the impact of different tumor microenvironments on metabolic effects of DCA and its impact for clinical use.


Cell culture

MDA-MB-231 (human breast cancer) and SiHa (human cervix squamous cell carcinoma) cell lines (American Type Culture Collection [ATCC]), were routinely cultured in Dulbecco’s modified Eagle’s medium containing 4.5g/l glucose supplemented with 10% fetal bovine serum and 1% penicillin-streptomycin.

Animal housing

Animal studies were undertaken in accordance with Belgian and the Université catholique de Louvain ethical committee regulations (agreements number UCL/2010/MD/001 and UCL/2014/MD/026). Hyperpolarized 13C-MRI experiments were carried out in compliance with the Guide for the Care and Use of Laboratory Animal Resources (National Research Council, 1996) and approved by the National Cancer Institute (NCI) Animal Care and Use Committee.

Tumor implantation and animal experiments

A total of 107 MDA-MB-231 cells or 107 SiHa cells, amplified in vitro, were collected by trypsinization, washed three times with Hanks balanced salt solution and resuspended in 200 μL of a 1:1 mixture of Matrigel (BD Biosciences) and Hanks balanced salt solution. For 17O MRS and PET scan experiments, the tumor cells were inoculated subcutaneously into the hind thigh of nude NMRI female mice (Janvier Le Genest-Saint-Isle, France). For hyperpolarized 13C-MRI experiments, the tumor cells were inoculated subcutaneously into the hind thigh of athymic nude female mice (Frederick Cancer Research Center, Animal Production, Frederick, MD, USA). The experiments were performed when tumors reached 7 mm (at this tumor size, necrosis was less than 5% as characterized by Hematoxylin Eosin staining).

To assess the effects of DCA on tumor metabolism using biomarkers, all animals have undergone imaging before and after treatment, with one day between each measurement. Dichloroacetate sodium (Sigma-Aldrich) was administered intraperitoneally (200 mg/kg) after baseline measurements to the mouse. Another dose was given 24 hours after the first dose injection. Post-treatment measurements were initiated 1 hour after treatment administration. This administration scheme is consistent with previous studies attesting the effects of DCA in tumors using hyperpolarized 13C-MRI []. The imaging protocol is summarized in Figure ​Figure55.


Figure 5

Experimental protocol

Mice were anesthetized by isoflurane inhalation (Forene, Abbot, England) mixed with air in a continuous flow (2 L/min). Animals were warmed (approximately 35°C) throughout the anesthesia period.

17O MRS experiments

For oxygen consumption experiments, 17O MRS was performed on an 11.7 T (Bruker, Biospec) controlled by Paravision 6.0 (Bruker, Ettlingen, Germany). Experiments were carried out using a 1H/17O Bruker surface coil system positioned over the tumor mass. Anatomical images were firstly acquired using a T2-weighted axial turbo RARE sequence (TR = 2500ms; TE = 30ms; rare factor = 8; NA = 2; FOV = 25x25mm2; resolution: 98 x 98 μm2; 1 mm slice thickness). 17O MRS measurements were carried out using a nonlocalized, single-pulse sequence (TR = 16.5 ms; NA = 600; repetition: 120; Tacq = 20 min; Acq BW = 5000 Hz; FA = 20°). For 17O MRS sequence, the 90° reference pulse was optimized previously on natural abundance H217O samples.

To measure tumor oxygen consumption during the 17O2 delivery, a total of 120 17O-spectra were collected in about 20 min, before, during and after a 2 min inhalation period of the 17O2 mixture. The integrals of the H217O peaks over time were measured using a home-made program written in Matlab (The MathWorks Inc., Natick, MA, USA). H217O signal was then expressed as relative to the mean baseline signal before 17O2 delivery. The mean signal of the final steady state (sfinal) during the post-inhalation period was calculated between 1100 and 1200 s. We considered that the steady state was reached when the signal stood between sfinal ± 5 % of signal variation. The slope during the linear incorporation phase was measured between 600 sec and the time point when steady state was reached.

PET/CT imaging

Whole-body PET imaging was performed on a dedicated small-animal PET scanner (Mosaic, Philips Medical Systems, Cleveland, USA) with a spatial resolution of 2.5 mm (FWHM). The PET scans were followed by whole-body acquisitions using a helical CT scanner (NanoSPECT/CT Small Animal Imager, Bioscan Inc., DC, USA). For each breathing condition, anesthetized mice were injected 120 μl intraperitoneally with 11.1-14.8 MBq of 18F-FDG (Betaplus Pharma, Brussels, Belgium). A 10 min transmission scan was first obtained in a single mode using a 370 MBq 137Cs source for attenuation correction. A 10 min static PET acquisition was then performed after a 60 min resting period. After the correction with attenuation factors obtained from the transmission scan, images were reconstructed using a fully 3D iterative algorithm (3D-RAMLA) in a 128 x 128 x 120 matrix, with a voxel size of 1 mm3. After PET acquisition, anesthetized animals were transferred on the same bed from the PET scanner to the CT scanner (x-ray tube voltage: 55 kVp; number of projections: 180; exposure time 1000 ms) for anatomical reference. The CT projections were reconstructed with a voxel size of 0.221 x 0.221 x 0.221 mm3. Regions of Interest (ROIs) were delineated on PET images using PMOD software (PMOD™, version 3.403, PMOD technologies Ltd, Zurich, Switzerland). 2D ROIs were established on consecutive transversal slices using a 50% isocontour tool (ROI including the pixel values larger than 50% of the maximum pixel) that semi-automatically defined a 3D Volume of Interest (VOI) around the tissue of interest. To avoid overestimation of the uptake within the VOI, PET/CT fused images where used to discriminate hot pixels coming from the neighboring tissues like urinary bladder. Using the mean uptake within this VOI, the global tracer uptake was assessed in tumors and expressed as percentage of injected dose per gram of tissue (%ID/g).

Hyperpolarized 13C-MRI studies

1-13C pyruvic acid (30 μL), containing 15 mM OXO63 and 2.5 mM gadolinium chelate ProHance (Bracco Diagnostics, Milano, Italy), was hyperpolarized at 3.35T and 1.4K using the Hypersense DNP polarizer (Oxford Instruments, Abingdon, UK) according to the manufacturer’s instructions. After 60-90 min, the hyperpolarized sample was rapidly dissolved in 4.5 mL of a superheated alkaline buffer that consisted of 50 mM Tris(hydroxymethyl)aminomethane, 75 mM NaOH, and 100 mg/L ethylenediaminetetraacetic acid. The hyperpolarized 1-13C pyruvate solution (96 mM) was intravenously injected through a catheter placed in the tail vein of the mouse (12 μL/g body weight). Hyperpolarized 13C MRI studies were performed on a 3T scanner (MR Solutions, Guildford, UK) using a home-built 13C solenoid leg coil. After the rapid injection of hyperpolarized 1-13C pyruvate, spectra were acquired every second for 240 seconds using a single pulse sequence. Data were analyzed in a model free approach using the lactate/pyruvate ratio, calculated from the areas under the curves of the 1-13C lactate peak and the 1-13C pyruvate peak [].

Statistical analysis

Analysis was performed using the GraphPad Prism 7 software. Results are expressed as means value of parameter ± SEM. All statistical tests were two-sided. Paired t-test was used to compare mean changes between groups (baseline vs. post-treatment) for each tumor model, and unpaired t-test was used to compare mean changes between the two tumor models. Results with P < 0.05 (*), <0.01 (**), or <0.001 (***) were considered to be statistically significant.

Crosstalk among proteome, acetylome and succinylome in colon cancer HCT116 cell treated with sodium dichloroacetate

Crosstalk among proteome, acetylome and succinylome in colon cancer HCT116 cell treated with sodium dichloroacetate


Protein lysine acetylation and succinylation play important regulatory roles in cells, both of which or each other has a close relationship. Dichloroacetate (DCA), a well-known pyruvate dehydrogenase kinase (PDK) inhibitor, has the potential to be used as anti-cancer drugs for several tumors including colorectal cancer. However, little is known about the potential mechanism of DCA-based cancer therapy by protein posttranslational modifications (PTM) including global proteome, acetylome and succinylome. Here the combinations with stable isotope labeling (SILAC), antibody affinity enrichment and high resolution LC-MS/MS analysis were performed in human colon cancer HCT116 cells. The quantifiable proteome was annotated using bioinformatics. In total, 4,518 proteins, 1,436 acetylation sites, and 671 succinylation sites were quantified, respectively to DCA treatment. Among the quantified acetylated sites, 158 were with increased level (quantification ratio >1.5) and 145 with decreased level (quantification ratio <0.67). Meanwhile, 179 up-regulated and 114 down-regulated succinylated sites were identified. The bioinformatics analyses initially showed acetylation and succinylation were involved in a wide range of cellular functions upon DCA-based anti-cancer effects. Notably, protein-protein interaction network analyses demonstrated widespread interactions modulated by protein acetylation and succinylation. Taken together, this study may shed a light on understanding the mechanism of DCA-based cancer treatment.


Protein posttranslational modifications (PTMs), which can alter structural, conformational and physicochemical properties of proteins, are key cellular events involved in many biological processes1,2. Among all the amino acids, lysine is a frequent target to be modified, which can be subjected to a variety of PTMs3. With the advances in high-resolution mass spectrometry (MS) and antibody-based affinity enrichment of lysine (Lys) residues, a lot of novel PTMs have been identified, such as ubiquitylation, butyrylation, succinylation, and glutarylation.

The acetylation of Lys residues in proteins has a role in transferring acetyl moiety from acetyl-CoA to its amino groups. Early studies of Lys acetylation mainly focused on histones and other transcription factors in the nucleus4,5,6. However, they have been found recently to occur in almost every compartment of a cell, such as the cytoplasm and mitochondria, and play a major role in metabolic regulation, including glycolysis, tricarboxylic acid (TCA) cycle, fatty acid metabolism and so on7,8. Succinylation refers to the transfer of succinyl group from the succinyl donor succinyl-CoA to the ε-amino group of specific lysine residue of the target protein, which is also considered to occur frequently like acetylation in several cellular events9. Most of the identified succinylated proteins are enzymes involved in kinds of metabolic pathways, which are important for the regulation of central metabolism such as fatty acid metabolism, amino acid degradation. It was reported that acetylation and succinylation were closely linked to central carbon metabolism10. Brian et al.11 revealed that majority of succinylation sites in bacteria, yeast, and mouse liver were also acetylated at the same position11. Yuta Mizuno et al.12 found that lysine acetylation and succinylation target most enzymes in central carbon metabolic pathways that are directly linked to glutamate production, and the extent of modification changed in response to glutamate overproduction12.

Dichloroacetate (DCA), an inhibitor of pyruvate dehydrogenase kinase (PDK), can reverse the Warburg effect and induce apoptosis in tumor cell by increasing the flux of pyruvate into the mitochondria and promoting glucose oxidation13. The increasing evidence in preclinical in vitro and in vivo indicates that DCA may be a promising selective anti-cancer agent. However, the potential mechanism of DCA-based cancer treatment is still unclear.

Given the widespread regulatory role of PTMs and the close relationship between acetylation and succinylation, we are interested to explore the regulative mechanism of DCA treatment by PTM. Here, an integrated system of SILAC labeling, HPLC fractionation and affinity enrichment followed by high resolution LC-MS/MS analysis were performed for the quantitative comparison of the global proteome, acetylome and succinylome in HCT116 cells with or without DCA treatment. Intensive bioinformatics analyses were then used to annotate those quantifiable modified targets in response to DCA treatment. Our studies indicate that the approach described above is powerful for identification of modified proteins and peptides. Therefore, the results provided a novel insight into DCA treatment on colon cancer cells.


Analysis of lysine acetylation, and succinylation sites in HCT116 cells stimulated by DCA

To identify the profiles of the global proteome and lysine acetylome, succinylome, we carried out a 6-step workflow: (1) stable isotope labeling of HCT116 cells with or without DCA stimulation by SILAC; (2) protein extraction and trypsin digestion to yield peptides; (3) HPLC fractionation; (4) affinity enrichment of lysine acetylated and succinylated peptides; (5) LC-MS/MS analysis was used to identify the enriched peptides; (6) database search and bioinformatic analysis (Fig. 1).

Figure 1: Assaying DCA-based cancer treatment by PTM.

(A) The analytical strategy and method for DCA-responsive quantitative profiling of global proteome, acetylome and succinylome in HCT116 cell line33,34 (The Edraw Max V7.3 software is used to create the map in (A), https://www.edrawsoft.com/term-conditions.php). (B) Western blotting with anti-acetyl lysine antibody (up) and anti-succinyl lysine antibody (down).

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In this work, comparing cells with or without DCA treatment, 5,448 proteins identified and 4,518 proteins were quantified in aspects of global proteome. Among these, 244 proteins were increased and 269 proteins were decreased. In addition, we identified 1,484 lysine acetylation (Kac) sites in 860 proteins, among of which 1,436 Kac sites in 841 proteins were quantified. We also identified 680 lysine succinylation (Ksu) sites in 295 proteins, among of which 671 Ksu sites in 291 proteins were quantified. The identified acetylated and succinylated peptides showed different abundance depending on their lengths (Fig. 2B,D), match 860 acetylated and 295 succinylated proteins, respectively (Fig. 2A,C). Apparently, most of them were modified at sole site, consistent with the previous findings about post-translational modification14,15,16.

Figure 2: The distribution of acetylated proteins and succinylated proteins.

(A) Distribution of acetylated proteins based on their number of modified sites. (B) Distribution of acetylated peptides based on their length. (C) Distribution of succinylated proteins based on their number of modified sites. (D) Distribution of succinylated peptides based on their length.

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Figure 3 shows the MS/MS spectra of two representative acetylated and succinylated proteins, histone acetyltransferase p300 and 60 kDa heat shock protein, respectively. Histone acetyltransferase p300 is encoded by gene EP300, and functions as histone acetyltransferase, which can regulate transcription via chromatin remodeling by acetylating four core histones. 60 kDa heat shock protein encoded by gene HSPD1 is involved in mitochondrial protein import and macromolecular assembly. Meanwhile, it plays an important role in protein folding, refolding, and proper assembly of unfolded polypeptides generated under the stress conditions. The two proteins were obviously modified, implying their crucial function in response to DCA treatment.

Figure 3

Representative MS/MS spectra of Histone acetyltransferase p300 acetylation (A) and 60 kDa heat shock protein succinylation (B).

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DCA-responsive global proteome

DCA has been reported as anti-cancer drug. It is therefore intrigue to assay the DCA-responsive proteome in HCT116 cells. Here, 5,448 proteins were identified and 4,518 proteins were quantified in the cells. Among them, 244 proteins were DCA-increased and 269 proteins were DCA-decreased (Supplementary Information Table S1) when we set quantification ratio of >1.5 as up-regulated threshold and <0.67 as down-regulated threshold. To elucidate the possible roles of these proteins, we performed four types of enrichment-based clustering analyses: Gene Ontology (GO) functional classification, KEGG pathway analysis, protein domain enrichment-based analysis, and protein complex analysis. The quantified proteomic dataset were divided into four quantiles according to L/H ratio to generate four quantiles: Q1 (0~25%), Q2 (25~50%), Q3 (50~75%), and Q4 (75~100%). Enrichment analyses were performed separately based on the quantiles.

We analyzed the quantifiable proteome dataset for three enrichment GO categories: biological process, molecular function, and cellular compartment (Figure S1A~C). For the molecular function analysis (Figure S1A), we found that proteins with increased L/H ratios were enriched in carboxylic ester hydrolase activity, and structural constituent of cytoskeleton. Proteins with helicase activity, pyrophosphatase activity, and hydrolase activity were enriched in Q1 and Q2. The cellular compartment analysis was presented in Figure S1B, indicating that many proteins located in keratin filament, extracellular space, and cytosol were enriched in quantiles Q3 and Q4 with high L/H ratios, which may suggest that might indicate DCA treatment have obvious influence on cytoarchitecture located in these places. The decreased proteins were mainly focused on chromosome and spindle, condensed chromosome, suggesting DCA may participate in cell cycle and mitosis.

In the biological process category (Figure S1C), proteins in Q3 and Q4 quantiles were enriched in epidermis development, cell differentiation, and other developmental processes. In contrast, proteins in Q1 and Q2 quantiles were enriched in cell cycle process, cell division, mitosis and organelle fission. These findings were consistent with results of the cellular compartment analysis. Taken together, DCA-responsive protein modifications have impacts on various processes such as replication, transcription, cell proliferation and so on. Importantly, the influence of DCA on cell developmental process and differentiation suggest that the potential role of DCA may act as an anti-cancer agent.

The Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis of the quantified proteins was also performed (Figure S1D). DCA-decreased proteins were mainly focused on cell cycle, DNA replication, steroid biosynthesis, pyrimidine metabolism, and p53 signaling pathway. While DCA-increased proteins were mainly involved in galactose metabolism, amino sugar and nucleotide sugar metabolism, as well as glycerolipid metabolism. It has been proved that DCA is the inhibitor of pyruvate dehydrogenase kinase (PDK)17. Besides, DCA has the ability to activate AMPK signaling pathway, which has a close relationship with glycolysis or gluconeogenesis18. The findings indicate that DCA has potential regulatory roles of participating in other kinds of glycometabolism.

Finally, the protein domain analysis indicated that the protein function domains involved in MCM N-terminal, mini-chromosome maintenance, and DNA-dependent ATPase were enriched in Q1 and Q2 quantiles, while protein domains participated in EGF-like conserved site, MCM N-terminal domain were enriched in Q3 and Q4 quantiles (Figure S1E).

DCA-responsive acetylome

Acetylation is a dynamic and reversible PTMs, which can transfer acetyl moiety from acetyl-CoA or acetyl phosphate to lysine residues at ε-amino groups in proteins18,19. Lysine acetylation was found in histones20, and in non-histones21, which has diverse functions like regulation of gene transcription, cell cycle, apoptosis, metabolic flux and so on15,22. It has been reported that DCA may have a relationship with acetylation23, however the direct evidence needs to be further investigated.

Here, the acetylation level of proteins in response to DCA stimulation was investigated by the combination of SILAC labeling, lysine acetylation antibody enrichment and LC-MS/MS analysis. Notably, 1,484 lysine acetylation sites in 860 protein groups were assayed, among which 1,436 sites in 841 proteins were quantified (Table S2). When setting quantification ratio of >1.5 as up-regulated threshold and <0.67 as down-regulated threshold, 158 lysine acetylation sites in 116 proteins were quantified as up-regulated targets and 145 lysine acetylation sites in 112 proteins were quantified as down-regulated targets. To our knowledge, this is the first profiling of lysine acetylation dataset in HCT116 cells under DCA treatment.

The enrichment-based clusting analyses were then performed to compare the functions of corresponding DCA-responsive proteins. All these quantified acetylated proteins were divided into four quantiles (Q1~4) as described above, including Gene Ontology, KEGG pathway, protein domain and protein motif (Fig. 4A~EFigure S2A,C).

Figure 4: Clustering analysis of the quantified acetylome based on the functional enrichment.

(A) Molecular function, (B) cellular compartment, (C) biological process, (D) KEGG pathway, and (E) protein domain. (F) Protein-protein interaction network of acetylated proteins clustered in ribosome. (G) Protein-protein interaction network of acetylated proteins clustered in cell cycle process.

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In GO functional classification (Fig. 4A–C), the analysis of molecular function (Fig. 4A) showed that increased proteins involved in structural constituent of ribosome, structural molecule activity, ATPase activity were changed significantly, while the decreased proteins participated in nucleic acid binding, heterocyclic compound binding, and other bindings were severely affected. The results were consistent with the notion that DCA can lead to the inhibition of cell proliferation and cause cell death24. In cellular compartment (Fig. 4B), the increased proteins were mostly enriched in cytosolic ribosome, ribosome. In contrast, decreased proteins were mainly enriched in nucleolus, chromatin, and DNA bending complex. In the biological process category (Fig. 4C), nuclear-transcribed mRNA catabolic process, aromatic compound catabolic process, mRNA catabolic process were highly enriched, while the processes related to chromatin organization, regulation of skeletal muscle tissue development were significantly enriched. These results were consistent with the former analysis of molecular function, suggesting DCA may play a role in RNA and protein synthesis and therefore have influence on the growth of tumors.

To identify cellular pathways affected by DCA treatment, we then performed a pathway clustering analysis using KEGG (Fig. 4D). The results showed that ribosome, oxidative phosphorylation, carbon metabolism were the most prominent pathways enriched in quantiles with high L/H ratios, suggesting DCA functions as a regulatory factor of protein biosynthesis and energy metabolism. It is notable that these results were consistent with the notion DCA can alter the ‘Warburg Effect’ into normal oxidative phosphorylation and thus inhibit tumor growth. But in quantiles with low L/H ratios, protein expression in pathways of viral carcinogenesis, systemic lupus erythematosus, RNA transport were severely decreased in response to DCA stimulation.

As domain structure is one of the most important functional features of protein, we next analyzed the domain features of the proteins changed after DCA adding (Fig. 4E). We found that domains in proteins involved in Histone-fold, Histone core, Histone H2B were remarkably enriched upon DCA treatment in down-regulated quantiles, suggesting DCA have great influence on transcription. But Zinc finger, translation protein SH3-like domain, ribosomal protein L2 domain 2 were mainly enriched in up-regulated quantiles.

To determine if there were specific amino acids adjacent to acetylated lysines, we compared the sequences flanking acetylated sites by heat-map. A strong bias of amino acid sequence, namely, Tyrosine (Y), Phenylalanine (F), Histidine (H), Tryptophan (W), was found in our data. In addition, isoleucine (I) and arginine (R) were overrepresented in the two and four to five positions behind Kac sites (Figure S2A,C), suggesting aromatic groups were common to be modified by acetyltransferases. These results were consistent with former report using mycobacterium tuberculosis as test subjects15 and indicated lysine acetylating may be conserved and widespread.

Protein-protein interaction network of acetylome proteins was established by using Cytoscape software (Figure S4A and Fig. 4F~G). Two kinds of important pathways (ribosome and cell cycle process) were listed in Fig. 4F~G, indicating that proteins participated in cell cycle process have mostly been down-regulated except MCM3, which had been acetylated at multiple sites. The acetylation of this protein can inhibit the initiation of DNA replication and cell cycle progression. Meanwhile, MKI67, a nuclear protein that is associated with and may be necessary for cellular proliferation, was acetylated at multiple sites and their levels were down-regulated remarkably25. These findings can partly explain how DCA cause cell cycle arrest and inhibit cell proliferation.

The correlation of DCA-responsive global proteome and acetylome

Upon the dataset of DCA-responsive proteome and acetylome, we performed the overlapping analysis between them. According to the quantitative results, 664 proteins were quantified both in proteome and acetylome, including a number of 1484 Kac sites. The quantitative ratios of proteome and acetylome were compared (Table S4) and the scatter diagram was shown in Figure S3A. The pearson’s correlation coefficient and the Spearman’s rank correlation coefficient were 0.235 and 0.236, respectively. The results demonstrate the global proteome and acetylome have little correlation with each other.

DCA-responsive succinylome

The succinylome in HCT116 cells was identified by combining the SILAC, immuneaffinity enrichment by a high-specificity antibody (PTM Biolabs), and high-resolution mass spectrometry. We identified 680 lysine succinylation sites in 295 protein groups, among which 671 sites in 291 proteins were quantified. Among them, 179 lysine succinylation sites in 108 proteins were increased and 114 lysine succinylation sites in 71 proteins were decreased (Table S3). Then all the quantified succinylated proteins were divided into four quantiles (Q1~4) according to L/H ratios as described above. The clustering analyses included GO analysis, KEGG pathway analysis, protein domain analysis and motif analysis (Fig. 5A~E and Figure S2B,D).

Figure 5: Clustering analysis of the quantified succinylome based on the functional enrichment.

(A) Molecular function, (B) cellular compartment, (C) biological process, (D) KEGG pathway, and (E) protein domain. (F) Protein-protein interaction network of acetylated proteins clustered in ribosome. (G) Protein-protein interaction network of acetylated proteins clustered in oxidative phosphorylation.

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For the molecular function analysis (Fig. 5A), we first found that increased proteins were highly enriched in oxidoreductase activity, hydrogen ion transmembrane transporter activity, substrate-specific transmembrane transporter activity, but for decreased proteins, they were mainly enriched in unfolded protein binding, protein binding and calcium ion binding. It was reported that plasma membrane oxidoreductase activity had a relationship with mitochondrial function and oxidative stress26, and excessive reactive oxygen species (ROS) could ultimately lead to ROS-mediated genomic instability and cancer27 as well as induce apoptosis. We propose that the enhanced oxidoreductase activity induced by DCA may be associated with its anti-cancer effect.

In the cellular compartment category (Fig. 5B), proteins located in the mitochondrial parts such as matrix, membrane, inner membrane were highly enriched in Q3 and Q4 quantiles, indicating that DCA-induced succinylation may have important roles in mitochondrial. However, proteins in Q1 and Q2 quantiles were mainly focused on pigment granule, melanosome and cytosol. The biological process of succinylation was displayed in Fig. 5C, which indicates that the up-regulated proteins were mainly enriched in small molecule metabolic process, oxidation-reduction process, cellular respiration. The decreased proteins were mainly concentrated on protein folding, cellular localization, and cellular component organization.

The KEGG pathway analysis for the succinylate proteins showed a number of important pathways (Fig. 5D). For proteins in Q1 and Q2 quantiles, the ribosome, viral carcinogenesis, TCA cycle, and glycolysis/gluconeogenesis pathways were highly enriched. In contrast, pathways of oxidative phosphorylation, Huntington’s disease, Alzheimer’s disease, and metabolism were highly enriched in Q3 and Q4 quantiles. These results implied succinylation has a close relationship with diseases and some metabolic pathways like oxidative phosphorylation, indicating succinylation may play an important role in DCA’s effects of turning ‘Warburg effect’ into normal oxidative phosphorylation. In addition, the pathways were highly enriched in Q3 and Q4 quantiles of acetylated proteins, implying the close relationship between these two PTMs. This was associated with the previous report, suggesting that acetylation and succinylation are closely overlapped16.

Figure 5E shows that protein domains involved in Histone-fold, Histone core, and Histone H2B were highly enriched in proteins with decreased Ksu sites, while several function domains (i.e., Zinc finger, LIM-type, Translation protein SH3-like, and Ribosomal protein L2) were highly enriched in proteins with increased Ksu sites.

Protein-protein interaction network of succinylated proteins was established by using Cytoscape software, and the global network of Ksu proteins was displayed in Figure S4B. The network indicated that succinylated proteins actively participated in ribosome and oxidative phosphorylation (Fig. 5F~G). Almost all succinylated proteins involved in ribosome were decreased and proteins involved in oxidative phosphorylation were increased. These findings imply that DCA-responsive succinylation play a significant role in protein synthesis and cellular energy metabolism.

To test whether conserved lysine succinylation motifs exist among lysine- acetylated proteins, we carried out an analysis of global succinylated proteins (Figure S2B,D). Three preferred sequence patterns were found as V*Ksu, I*Ksu, and R******Ksu. To our knowledge, V*Ksu and I*Ksu have already been observed to be existed in human, rat and E. coli, but neither of them were reported to be among the most common ones in the previous reports11. In addition, EK and Ksu*****K, two of the most common sequence patterns, were overrepresented in this study, suggesting the preferred sites for succinylation have some similarities even in different species.

The correlation of DCA-responsive global proteome and succinylome.

We next assayed the features of DCA-responsive global proteome and succinylome. Totally, 295 quantified proteins were found in both of them, including 636 Ksu sites. The quantitative ratios of proteome and succinylome under DCA treatment were compared (Table S5), and the scatter diagram was shown in Figure S3B. To be accurate, the pearson’s correlation coefficient and the Spearman’s rank correlation coefficient were 0.255 and 0.292, respectively. The results demonstrate there is litte correlation between the global proteome and succinylome.

The correlation of DCA-responsive acetylome and succinylome.

It was reported that each PLM can crosstalk with at least one other PLM and the co-occurrences of different PLMs at the same site were abundant28. Here, the DCA-responsive acetylome and succinylome in HCT116 cells were assayed to investigate their relationships. 91 proteins were both acetylated and succinylated, but they were not modified at the same site exactly. Among them, there were 151 lysine sites were modified with both acetylation and succinylation (Fig. 6A,B). The correlation between the L/H ratios of acetylome and succinylome (Table S6), as well as the scatter diagram were shown in Fig. 6C. The Spearman’s correlation coefficient and Pearson’s was 0.347 and 0.478, respectively. This indicated that DCA-responsive patterns of acetylated and succinylated sites were positively related. Notably, some important proteins participated in glycolysis, the main pathway through which DCA regulates tumor cell metabolism, were acetylated and succinylated simultaneously, such as pyruvate kinase muscle (PKM), phosphoglycerate kinase 1 (PGK1), lactate dehydrogenase B (LDHB), and enolase 1 (ENO1). This implies that the two modifications are involved in DCA’s effect on HCT116 cells.

Figure 6: Correlation of global proteome, acetylome and succinylome.

(A) Overlap between acetylated proteins and succinylated proteins. (B) Overlap between acetylated sites and succinylated sites. (C) Correlation of acetylome and succinylome. (D) Protein-protein interaction network between acetylated proteins and succinylated proteins.

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For assaying the relationship of acetylome and succinylome, the protein-protein interaction network was established. The global overview of network among Kac and Ksu proteins was performed by using Cytoscape (Fig. 6D). The complicated network demonstrates that the acetylated and succinylated proteins have a closed crosstalk, indicating that some important proteins underwent both acetylation and succinylation, such as dihydrolipoamide dehydrogenase (DLD), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), and cytochrome C somatic (CYCS).


DCA has been reported to be a promising anti-cancer drug29, but its regulatory mechanisms in tumor cells are still unknown. In this study, a SILAC-based quantitative proteomic approach was used to investigate the anti-cancer effect of DCA on the proteome, acetylome and succinylome in HCT116 colon cancer cells.

The alternation of the whole proteome in HCT116 cells showed that proteins participated in cell cycle, cell division and proliferation apparently decreased, which linked gene expression levels with the DCA-induced anti-cancer effects. In addition, The KEGG pathway analysis of the quantified proteins showed that the increased proteins were highly enriched in galactose metabolism, amino sugar and nucleotide sugar metabolism, consistent with DCA’s role of regulating glucose metabolism.

The quantitative acetylome analysis revealed 1436 DCA-responsive Kac sites in HCT116 cells, which would be the most comprehensive Kac profiling in HCT116 cells. It indicated that the decreased proteins mainly located in nucleolus, chromatin, and DNA bending complex, participating in nucleic acid binding, which implys that DCA can inhibit DNA replication and regulate protein biosynthesis by lysine acetylation. In addition, we observed that some proteins encoded by important myconcogene was significantly down-regulated.

In addition, among 680 lysine succinylation sites in 295 proteins, the DCA-increased proteins were mostly localized in mitochondrial and enriched in oxidoreductase activity, hydrogen ion transmembrane transporter activity, and substrate-specific transmembrane transporter activity. It has been reported that mitochondrial membrane potential and ROS production are dependent on the flux of electrons down the electron transport chain (ETC). Decreased entry of pyruvate would eventually result in decreased flux of electrons in the ETC and therefore reduced ROS production, contributing to the close of the redox-sensitive mitochondrial transition pore (MTP) and mitochondrial hyperpolarization which are considered to be closely related with tumor genesis30. These findings suggest that DCA may result in lysine succinylation and then increase the delivery of pyruvate into the mitochondria, which may help mitochondria-based glucose oxidation and mitochondrial depolarizing, thus returning the membrane potential towards the levels of normal cells, and exert its anti-cancer effect.

Considering the growing evidence suggests that lysine acetylation and succinylation may have a close relationship, we focused on several pathways undergoing the two modifications. Generally, the overlap between acetylome and succinylome were mainly related to oxidative phosphorylation, metabolic pathways, citrate cycle (TCA cycle), and carbon metabolism. Here, we discussed the TCA cycle in detail for its close relationship with DCA’s anti-cancer effect. It is well known that DCA can target PDK, which can connect glycolysis with TCA cycle by promoting catalyzing pyruvate to acetyl-CoA. We checked the modification of PDH (regulated by PDK) and eight enzymes participated in TCA cycle, including citric synthase, aconitate hydratase, isocitrate dehydrogenase, ketoglurate dehydrogenase, succinyl-CoA synthase, succinate dehydrogenase, fumarate hydrogenase, malate dehydrogenase in our dataset. Some of them were acetylated or succinylated (Fig. 7). However, PDK itself was not acetylated or succinylated in our study. Isocitrate dehydrogenase, which catalyzes the conversion of isocitrate to α-keto-glutarate, was involved both in acetylation and succinylation. There were a total of eight Ksu sites and 5 Kac sites on this enzyme. According to the previous report, mutagenesis of the succinylated lysine residues of this enzyme has a crucial role of the activity of the enzyme31. It is reasonable to suppose that the modifications described above will induce important changes of TCA cycle, especially when this step is irreversible and rate-limiting. But we didn’t find another two enzymes in citric synthase and ketoglurate dehydrogenase were acetylated or succinylated. In total, among the nine enzymes, seven were succinylated and five were acetylated in HCT116 cells after DCA treatment, respectively. These results suggest that the enzyme activity in metabolism has been positively regulated by acetylome and succinylome, thus mediating the drug effect.

Figure 7: Acetylated and succinylated enzymes involved in TCA cycle.

The enzymes identified as lysine-acetylated are marked with an asterisk. The enzymes identified as lysine-succinylated are underlined.

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Recent studies have reported that histone acetylation could induce the change of global proteome by epigenetics while non-histone acetylation of transcription factors could regulate proteome level through transcriptiome32,33. Our classification was not clear enough to show the direct relationship among global proteome, acetylome and succinylome. Obviously, to further confirm the view, more explicit classification and bioinformatic analysis should be done.


Here we present a large-scale quantitative analysis of DCA-responsive global proteome, acetylome and succinylome in HCT116 cells by using high sensitivity mass spectrometry and bioinformatic analysis. In total, we identified DCA-responsive 860 Kac proteins and 295 Ksu proteins in cells. Our work provides a database that can be used to examine what effect DCA on cancer cells, drug resistance. Notably, the correlation of global proteome, acetylome and succinylome may expand our understanding of DCA’s anti-cancer effect. Although the underlying mechanism of acetylome and succinylome remains to be further explored, the current study shed a light on several biological processes like TCA cycle, glycolysis, and other functions.

Methods and Materials

HCT116 culture and SILAC labeling

Cells were grown to 80% confluence in high glucose Dulbecco’s modified Eagle’s medium (with glutamine and sodium pyruvate) (Pierce, MA, USA) containing 10% fetal bovine serum (Gibco, CA, USA) at 37 °C with 95% air and 5% CO2. Then cells were labeled with ‘light isotopic lysine’ (12C-Lysine) or ‘heavy isotopic lysine’ (13C-Lysine) using a SILAC Protein Quantitation Kit (Pierce, MA, USA) according to manufacturer’s instructions. After cells were expanded in SILAC media to our desired cell number (~5 × 108), the ‘light’ labeled cells were then treated with 20 mM DCA (Sigma, MO, USA) and cultured for another 24 hours in SILAC media before harvesting.

Protein extraction, Trypsin Digestion and HPLC fractionation

The harvested labeled cells were lysed with 2 × NETN buffer (200 mM NaCl, 100 mM Tris-Cl, 2 mM EDTA, 1.0% NP-40, pH 7.2) supplemented with 0.5% Triton X-100 on ice for 30 min. The unbroken cells or debris were removed after centrifuge at 20,000 g for 10 min at 4 °C. After concentration measurement, same amounts of proteins labeled with ‘heavy’ or ‘light’ were mixed and the crude proteins were precipitated. After washing twice with ice-cold acetone, the air-dried precipitate was dissolved in 100 mM NH4HCO3 (pH 8.0) and then digested with trypsin (Promega, WI, USA) at an enzyme-to substrate ratio (1:50) at 37 °C for 16 hours. After that, DTT was added to final concentration 5 mM and then incubated at 50 °C for 30 min, then IAA was added with final concentration 15 mM followed by incubation at room temperature in total darkness for 30 min. The alkylation reaction was terminated by 30 mM cysteine at room temperature for another 30 min. Again Trypsin was added with ratio of trypsin to protein at 1:100 for digestion at 37 °C for 4 hours to ensure the complete digestion. The sample was then fractionated into fractions by high pH reverse-phase HPLC by using Agilent 300Extend C18 column (5 μm particles, 4.6 mm ID, 250 mm length). In brief, peptides obtained were separated into 80 fractions with a gradient of 2% to 60% acetonitrile in 10 mM ammonium bicarbonate (pH 10) for over 80 min first, then they were combined into 18 fractions and dried.

Affinity Enrichment of Lysine Acetylated and Succinylated Peptides

Tryptic peptides dissolved in NETN buffer were incubated with pre-washed antibody beads (PTM Biolabs) (4 times with NETN buffer and 2 times with ddH2O) at 4 °C overnight with gentle shaking. And then, the bound peptides were eluted from the beads with 0.1% TFA, and the eluted fractions were combined and vacuum-dried. The peptides obtained were cleaned with C18 ZipTips (Millipore, MA, USA) according to the manufacturer’s instructions, followed by LC-MS/MS analysis.

LC-MS/MS Analysis

Three parallel analyses for each fraction were performed. Peptides were dissolved in 0.1% FA (Sigma Fluka, MO, USA), directly loaded onto a reversed-phase pre-column (Acclaim PepMap 100, Thermo, MA, USA). Peptides were separated by using a reversed-phase analytical column (Acclaim PepMap RSLC). The gradient was comprised of a span starting from 6% to 23% solvent B (0.1% FA in 98% ACN) for 24 min, 23% to 35% for 8 min, 80% in 4 min, and at last holding at 80% for another 4 min. All of the above procedures were at a constant flow rate of 280 nL/min on EASY-nLC 1000 UPLC system, and the resulting peptides were analyzed by Q ExactiveTM Plus hybrid quadrupole-Orbitrap mass spectrometer (Thermo Fisher, MA, USA).

The peptides were subjected to NSI source, followed by tandem mass spectrometry (MS/MS) in Q ExactiveTMPlus (Thermo, MA, USA) coupled online to the UPLC. We detected the intact peptides in the Orbitrap at a resolution of 70,000. Peptides were selected using NCE setting as 30; ion fragments were detected at a resolution of 17,500. A data-dependent procedure that alternated between one MS scan followed by 20 MS/MS scans was applied for the top 20 precursor ions above a threshold ion count of 1.5 E4 in the MS survey scan with 30.0 s dynamic exclusion. The electrospray voltage applied was 2.0 kV. Automatic gain control (AGC) was used to prevent overfilling of the ion trap; 5 E4 ions were accumulated for generation of MS/MS spectra. For MS scans, the m/z scan range was 350 to 1800.

Data processing

The data obtained was processed using MaxQuant with integrated Andromeda search engine (v. Tandem mass spectra were searched against SwissProt_human database concatenated with reverse decoy database. Mass error was set to 10 ppm for precursor ions and 0.02 Da for fragment ions. Carbamidomethylation on Cys was specified as fixed modification and oxidation on Met, acetylation or succinylation on Lys and protein N-terminal were specified as variable modifications. Minimum peptide length was set at 7. False discovery rate (FDR) thresholds for protein, peptides and modification sites were specified at 1%. All the other parameters in MaxQuant were the default values. The Kac and Ksu site localization probabilities were set as >0.75.

Bioinformatics Methods

Gene Ontology (GO) annotation proteome was derived from the UniProt-GOA database (http://www.ebi.ac.uk/GOA/). For proteins not annotated by UniProt-GOA database (http://www.ebi.ac.uk/GOA/), the InterProScan soft was used to annotated protein’s GO functional based on protein sequence alignment method. Kyoto Encyclopedia of Genes and Genomes (KEGG) database (http://www.genome.jp/kegg/) was used to annotate protein pathway. Online service tools KAAS was used to annotate protein’s KEGG description. KEGG mapper was used to map the annotation results. Functional Annotation Tool of DAVID Bioinformatics Resources 6.7 to identify enriched GO, KEGG pathway and protein domain against the background of Homo sapiens. A two-tailed Fisher’s exact test was employed to test the enrichment of the protein-containing IPI entries against all IPI proteins. Correction for multiple hypothesis testing was carried out using standard false discovery rate control methods. The GO with a corrected p-value < 0.05 is considered significant. The protein complex database CORUM was used for protein complex analysis. Corrected p-value < 0.05 was considered significant when performing the bioinformatics analysis.

Phase 1 trial of dichloroacetate (DCA) in adults with recurrent malignant brain tumors

Phase 1 trial of dichloroacetate (DCA) in adults with recurrent malignant brain tumors

  • M. Dunbar
  • S. Coats
  • L. Shroads
  • Langaee
  • Lew
  • R. Forder
  • J. Shuster
  • A. Wagner
  • W. Stacpoole


Background Recurrent malignant brain tumors (RMBTs) carry a poor prognosis. Dichloroacetate (DCA) activates mitochondrial oxidative metabolism and has shown activity against several human cancers. DesignWe conducted an open-label study of oral DCA in 15 adults with recurrent WHO grade III – IV gliomas or metastases from a primary cancer outside the central nervous system. The primary objective was detection of a dose limiting toxicity for RMBTs at 4 weeks of treatment, defined as any grade 4 or 5 toxicity, or grade 3 toxicity directly attributable to DCA, based on the National Cancer Institute’s Common Toxicity Criteria for Adverse Events, version 4.0. Secondary objectives involved safety, tolerability and hypothesis-generating data on disease status. Dosing was based on haplotype variation in glutathione transferase zeta 1/maleylacetoacetate isomerase (GSTZ1/MAAI), which participates in DCA and tyrosine catabolism. Results Eight patients completed at least 1 four week cycle. During this time, no dose-limiting toxicities occurred. No patient withdrew because of lack of tolerance to DCA, although 2 subjects experienced grade 0–1 distal parasthesias that led to elective withdrawal and/or dose-adjustment. All subjects completing at least 1 four-week cycle remained clinically stable during this time and remained on DCA for an average of 75.5 days (range 26–312). Conclusions Chronic, oral DCA is feasible and well-tolerated in patients with recurrent malignant gliomas and other tumors metastatic to the brain using the dose range established for metabolic diseases. The importance of genetic-based dosing is confirmed and should be incorporated into future trials of chronic DCA administration.