Salah Mohamed El Sayed1, 2 , Hussam Baghdadi1 , Nagwa Sayed Ahmed2 , Hamdi H. Almaramhy3 , Ahmed Al-Amir Mahmoud2 , Samer El-Sawy2 , Mongi Ayat1 , Momen Elshazley4,5, Wafaa Abdel-Aziz6 , Haitham Mahmoud Abdel-Latif6 , Walaa Ibrahim6 .
1 Department of Clinical Biochemistry and Molecular Medicine, Taibah College of Medicine, Taibah University, Al-Madinah Al-Munawwarah, Saudi Arabia.
2 Department of Medical Biochemistry, Sohag Faculty of Medicine, Sohag University, Egypt.
3 Department of surgery, Taibah College of Medicine, Taibah University, AlMadinah Al-Munawwarah, Saudi Arabia.
4 Department of Medicine, Taibah College of Medicine, Taibah University, AlMadinah Al-Munawwarah, Saudi Arabia.
5 Department of occupational diseases and toxigenomics, Sohag Faculty of Medicine, Sohag University, Egypt.
6 Department of Medical Pharmacology, Sohag Faculty of Medicine, Sohag University, Egypt.
Assist. Professor/ Salah Mohamed El Sayed
*Department of Clinical Biochemistry and Molecular Medicine, Taibah College of Medicine, Taibah University, Al-Madinah Al-Munawwarah, Saudi Arabia.
* Department of Medical Biochemistry, Sohag Faculty of Medicine, Sohag University, Egypt
Tel.: 00966503551588, 00966542927804.
*Department of Medical Biochemistry, Sohag Faculty of Medicine, Sohag University, Egypt.
Email: firstname.lastname@example.org, email@example.com
Received: 18 July 2018
Accepted: 20 November 2018
Available online: 22 November 2018
Dichloroacetate (DCA) is a promising safe anticancer drug that cured a patient with chemoresistant non-Hodgkin’s lymphoma and treated lactic acidosis effectively. The well-known mechanism of DCA action is through stimulating Krebs cycle (stimulating pyruvate dehydrogenase via inhibiting pyruvate dehydrogenase kinase). This prevents lactate formation (Warburg effect) depriving cancer cells of lactatebased benefits e.g. angiogenesis, chemoresistance and radioresistance. Here, we introduce novel evidence-based hypotheses to explain DCA-induced anticancer effects. On pharmacological and biochemical bases, we hypothesize that DCA is a structural antagonist of acetate competing with it for target enzymes and biological reactions. We hypothesize that DCA exerts its anticancer effects via depriving cancer of acetate benefits. We hypothesize also that acetate is an antidote of DCA capable of treating DCA toxicity. Many reports support our hypotheses. Acetate is vital for cancer cells (tumors depend on acetate) and DCA is structurally similar to acetate. DCA exerts opposite effects to acetate. Acetate caused a decrease in serum potassium, phosphorus and glucose, and an increase in serum lactate, citrate, free fatty acids and ketone bodies (serum acetoacetate and beta-hydroxybutyrate levels). Acetate decreased the proportion of active (dephosphorylated) pyruvate dehydrogenase in perfused rat heart. DCA produced quite opposite effects. Intravenous infusion of acetate produced metabolic alkalemia while DCA caused minimal effects on acidbase status. Acetate is important for cancer cells metabolism and survival as elevated acetate can drive resistance to targeted cancer treatments. Acetate is required for epidermal growth factor receptor vIII mutation in lethal brain tumors. Experimentally, DCA inhibited acetate oxidation in hearts of normal rats and reversed inhibitory effects of acetate on the oxidation of glucose. During presence of DCA with no glucose in heart perfusions with [1-14C]acetate, DCA decreased the specific radioactivity of acetyl CoA and its product citrate. This proves our hypotheses that DCA is an antimetabolite that antagonizes acetate for vital reactions in cancer cells. Acetate may be used as an antidote to combat DCA toxicity.
Keywords: Dichloroacetate, anticancer mechanisms, acetate
Running title: Dichloroacetate: an antimetabolite that antagonizes acetate
Monochloroacetate (MCA), dichloroacetate (DCA), and trichloroacetate (TCA) are chlorinated acetates that are formed during water disinfection processes . DCA results in daily life from water chlorination  and is also a promising drug treatment for treating mitochondrial diseases, pulmonary arterial hypertension and cancer . In clinical oncology, DCA emerged as a promising safe antineoplastic agent causing cancer cells apoptosis via targeting the enzyme pyruvate dehydrogenase kinase (PDK) that regulates initiation of Krebs cycle using pyruvate . Depriving cancer cells of lactate is vital as a cancer therapeutic strategy due to the numerous lactate-based benefits to cancer cells and tumors e.g. angiogenesis, chemoresistance and radioresistance. . Interestingly, DCA was reported as a successful treatment to a young child having metabolic hyperlactacidemia . A major hope in cancer treatment is based upon introduction of DCA in clinical oncology based on the promising success of DCA in treating many human patients having different types of tumors [7-9]. The promising report by Strum et al. in curing a patient having chemoresistant non-Hogkin lymphoma using DCA  urged us to search for potential novel anticancer mechanisms beyond DCA . Herein, we introduce novel evidence-based hypotheses and mechanisms of DCA anticancer effects based on earlier findings through antagonizing acetate.
Table 1. Hypotheses points:
- DCA competes with acetate for target enzymes and biological reactions based
on structural similarity (structural antagonism) (figure 1).
- DCA deprives cancer cells of acetate benefits i.e. DCA is an antimetabolite.
- Acetate is an antidote to DCA that can treat DCA toxicity.
On evidence-based pharmacological and biochemical bases, we introduce here our novel evidence-based hypotheses regarding new anticancer mechanisms of action of DCA. We hypothesize that DCA is an antimetabolite (based on structural similarity to acetate). We hypothesize that DCA antagonizes acetate for target enzymes and biological reactions based on structural similarity (structural antagonism). DCA is structurally similar to acetate. Substitution of two hydrogens in acetate by two chloride ions produces DCA. We hypothesize that DCA is a structural analog to acetate that may antagonize its biochemical effects and compete with it for its target enzymes and biochemical reactions Evidences supporting that are summarized in Table 2 where DCA produces quite opposite effects to acetate in many situations .
|Effects of intravenous infusion on pH||Metabolic alkalemia||Minimal effects on acid-base state and oxygen consumption|
|Effects on serum potassium and phosphorus||Marked decrease||Marked increase|
|Effects on serum lactate and citrate levels||Increase||Decrease|
|Effects on serum free fatty acids||Increase||Decrease|
|Effects on serum acetoacetate and beta-hydroxybutyrate levels||Increased markedly||Less increase|
|Effects on serum glucose level||Decreases significantly||Decreases|
|Effects on the proportion of active (dephosphorylated) pyruvate dehydrogenase in perfused rat heart||Decreases||Increases|
Evaluation of the hypothesis/idea
We searched the literature extensively to formulate our suggested new concepts regarding the novel anticancer mechanisms of action of DCA. Acetate caused a decrease in serum potassium, phosphorus and glucose, and an increase in serum lactate, citrate, free fatty acids and ketone bodies (serum acetoacetate and beta-hydroxybutyrate levels). Acetate decreased the proportion of active (dephosphorylated) pyruvate dehydrogenase in perfused rat heart. DCA produced quite opposite effects . Regarding the effects of intravenous acetate infusion on pH, acetate produced metabolic alkalemia while DCA infusion caused minimal effects on acid-base state and oxygen consumption . All these evidences prove that DCA is a competitive antagonist of acetate.
There are many reports that support our idea that DCA works through antagonizing acetic acid (Table 2). DCA inhibited extraction of plasma free fatty acids (as acetic acid) in heart tissues from normally fed rats and also from alloxan-induced diabetic rats. In heart perfusions with glucose, insulin and acetate (stimulators of glycolysis and subsequently Krebs cycle), DCA decreased the cell citrate concentration (product of Krebs cycle). DCA was also reported not to be metabolized to dichloroacetyl-CoA, dichloroacetylcarnitine, citrate or CO(2) , which supports the hypothesis that DCA acts as an antagonist that inhibits the enzyme activity without being metabolized. This supports also that DCA is an enzyme inhibitor that does not undergo further metabolism. In the absence of glucose and insulin (stimulators of glycolysis and subsequently Krebs cycle), DCA inhibited glycolysis as a source of acetyl CoA causing no increase in acetyl CoA levels . Taking into account that acetate is vital for many tumor types, its possible antagonism by DCA can be a novel mechanism for DCA-induced cancer cell cytotoxicity.
The vital question that arises now: is acetate itself important for cancer cells metabolism and survival?
Acetate promotes tumor growth
Acetate is a bioenergetic substrate for human glioblastoma and brain metastases. Elevated acetate levels can drive resistance to targeted cancer treatments. Treatment with sequentially increasing concentrations of acetic acid was reported to persistently enhance RNA, protein, and DNA synthesis. That was followed by extensive epidermal hyperplasia . Based on that, acetate promotes synthesis of DNA and macromolecules essential for cancer cell division. Either glucose or acetate is required for the activation of epidermal growth factor receptor vIII (a common growth factor receptor mutation in the fatal brain cancer glioblastoma multiforme). Moreover, elevated energetic nutrient levels (acetate or glucose) can induce resistance to targeted cancer treatments . Also, acetate is an epigenetic regulator of posttranslational protein modification, and is a carbon source for cancer cell biomass accumulation. Acetate plays a major role in protein acetylation and acetate oxidation plays an important role in carcinogenesis, cancer progression and treatment .
Acetate fuels tumor growth where acetate is a fuel for promoting the growth of tumors. This was confirmed by positron emission tomography (PET) imaging studies using [11C]acetate where many tumor types are dependent on acetate as a source of carbon to get acetyl-CoA. Excessive acetate uptake was reported in prostate, lung, liver, and brain cancers , , , . Tumors exhibit acetate dependence. Acetate constitutes a vital supply of acetyl-CoA for feeding these tumors . Interestingly, [11C]acetate PET imaging is preferred than [18F]fluorodeoxyglucose (FDG) PET imaging as [11C]acetate PET imaging is more accurate and sensitive. Acetate consumption by human tumors is evident by nuclear magnetic resonance (NMR). Acetyl coA synthase seems to be of paramount importance to consume acetate as a supply of acetyl-CoA. Primary and metastatic mouse orthotopic brain tumors have the capacity to oxidize acetate and glucose. Acetate consumption in such tumors correlated with the expression of acetyl-CoA synthetase enzyme .
Consequences of the hypothesis and discussion
DCA exerts potent promising anticancer effects. DCA exerted anti-proliferative and pro-apoptotic effects against many cancer cells. DCA was reported to reverse the glycolytic phenotype and inhibit metastasis of breast cancer cells both in vitro and in vivo . DCA treatment was reported to reduce cancer cell-induced production of lactate and to sensitize malignant cells to 5-fluorouracil in gastric cancer cells . Moreover, DCA reduced cancer cells (hypoxic HeLa cells) proliferation, viability and colony counts . Unlike many chemotherapeutics, hypoxia (a common criterion of cancer biology) enhances the antitumor activity of DCA .
Interestingly, DCA killed cancer stem cells effectively e.g. embryonal carcinoma stem cells (shares in teratocarcinoma development that exhibited strong dependence on glycolysis with decreased mitochondrial biogenesis) . Till now, DCA-induced anticancer effects are looked at as through stimulating Krebs cycle entry via inhibiting PDK causing stimulation of pyruvate dehydrogenase complex where acetyl CoA starts Krebs cycle through combination with oxaloacetate to form citrate. This necessitates that glycolysis reaction becomes aerobic and not anaerobic (ending in formation of lactate, Warburg effect). Tumor glycolysis-induced pyruvate is converted into lactate (Warburg effect) that confers many lactate-based benefits to cancer cells e.g. angiogenesis, chemoresistance and radioresistance . Based on that, DCA clearly reverses cancer glycolytic phenotype and exerts potent anticancer effects.
Future perspectives based on our evidence-based hypothesis include many points. Our hypotheses will guide scientific research in this direction to open a new research frontier towards many investigations based on our hypothesized points that DCA antagonizes acetate. The following practical research points and applications may benefit medical practice:
1. An experimental design to investigate acetate-induced inhibition of DCA-induced cell death.
2. An experimental biochemical design to investigate the enzyme kinetics of DCA-induced antagonistic effects of acetate and the effects of DCA on acetate-based enzyme systems e.g. acetyl CoA synthase enzyme .
3. Future possibility of using acetic acid-based treatments as antidotes and antagonists of DCA to combat DCA accidental toxicity and poisoning.
4. Acetate dependence of tumors can be utilized for developing sensitive diagnostic tools and radiotracers.
That started to be recently utilized e.g. 11C-acetate and 18F-fluorodeoxyglucose positron emission tomography/computed tomography dual imaging for the prediction of response and prognosis after transarterial chemoembolization.
We conclude that DCA is an antimetabolite that antagonizes acetate for vital reactions in cancer cells.
Conflict of interest
The authors declare that there is no conflict of interest.
The authors declare that there is no financial support from any source to this work. Authors themselves support the whole work.
We are very grateful for the College of Medicine, Taibah University, Saudi Arabia for kindly providing the library and scientific facilities to achieve this work. We are grateful to Mr. Adel Joraibei Al-Obaidi, Sultan Al-Hussaini, Mohamed Abdel-Samad, Wael Barakat and Mr.Raed Ali Al-Raddadi from the administrative department, College of Medicine, Taibah University for their technical help and support to the work.
REFERENCES1 M.M. Schmidt, A. Rohwedder, R. Dringen Effects of chlorinated acetates on the glutathione metabolism and on glycolysis of cultured astrocytes Neurotox Res, 19 (4) (2011), pp. 628-637
2 P.W. Stacpoole, Y.J. Greene Dichloroacetate Diabetes Care, 15 (6) (1992), pp. 785-791
3 H.W. Maisenbacher 3rd, A.L. Shroads 3rd, G. Zhong, A.D. Daigle, et al. Pharmacokinetics of oral dichloroacetate in dogs J Biochem Mol Toxicol, 27 (12) (2013), pp. 522-525
4 J. Tong, G. Xie, J. He, J. Li, F. Pan, et al. Synergistic antitumor effect of dichloroacetate in combination with 5-fluorouracil in colorectal cancer J Biomed Biotechnol, 2011 (2011), p. 740564
5 S.M. El Sayed, W.G. Mohamed, M.A. Seddik, et al. Safety and outcome of treatment of metastatic melanoma using 3-bromopyruvate: a concise literature review and case study Chin J Cancer., 33 (7) (2014), pp. 356-364
6 O.N. Elpeleg, W. Ruitenbeek, C. Jakobs, et al. Congenital lacticacidemia caused by lipoamide dehydrogenase deficiency with favorable outcome J Pediatr, 126 (1) (1995), pp. 72-74
7 E.D. Michelakis, G. Sutendra, P. Dromparis, et al. Metabolic modulation of glioblastoma with dichloroacetate Sci Transl Med, 2 (31) (2010), p. 31ra34
8 G. Sutendra, E.D. Michelakis Pyruvate dehydrogenase kinase as a novel therapeutic target in oncology Front Oncol, 7 (3) (2013), p. 38
9 A. Khan, D. Marier, E. Marsden, et al. A novel form of dichloroacetate therapy for patients with advanced cancer: a report of 3 cases Altern Ther Health Med, 20 (Suppl 2) (2014), pp. 21-28
10 S.B. Strum, O. Adalsteinsson, R.R. Black, et al. Case report: Sodium dichloroacetate (DCA) inhibition of the “Warburg Effect” in a human cancer patient: complete response in non-Hodgkin’s lymphoma after disease progression with rituximab-CHOP J Bioenerg Biomembr, 45 (3) (2013), pp. 307-315
11 D.M. Jaworski, A.M. Namboodiri, J.R. Moffett Acetate as a metabolic and epigenetic modifier of cancer therapy J Cell Biochem, 117 (3) (2016), pp. 574-588
12 R.A. Ward, R.L. Wathen, G.B. Harding, et al. Comparative metabolic effects of acetate and dichloroacetate infusion in the anesthetized dog Metabolism., 34 (7) (1985), pp. 680-687
13 A. McAllister, S.P. Allison, P.J. Randle Effects of dichloroacetate on the metabolism of glucose, pyruvate, acetate, 3-hydroxybutyrate and palmitate in rat diaphragm and heart muscle in vitro and on extraction of glucose, lactate, pyruvate and free fatty acids by dog heart in vivo Biochem J, 134 (4) (1973), pp. 1067-1081
14T.J. Slaga, G.T. Bowden, R.K. Boutwell Acetic acid, a potent stimulator of mouse epidermal macromolecular synthesis and hyperplasia but with weak tumor-promoting ability J Natl Cancer Inst, 55 (4) (1975), pp. 983-987
15K. Masui, K. Tanaka, S. Ikegami, et al.Glucose-dependent acetylation of Rictor promotes targeted cancer therapy resistance Proc Natl Acad Sci USA, 112 (30) (2015), pp. 9406-9411
16 C.L. Ho, S.C. Yu, D.W. Yeung 11C-acetate PET imaging in hepatocellular carcinoma and other liver masses J Nucl Med, 44 (2) (2003), pp. 213-221
17 H. Nomori, H. Shibata, K. Uno, et al. 11C-Acetate can be used in place of 18F-fluorodeoxyglucose for positron emission tomography imaging of non-small cell lung cancer with higher sensitivity for well-differentiated adenocarcinoma J Thorac Oncol, 3 (12) (2008), pp. 1427-1432
18 N. Oyama, H. Akino, H. Kanamaru, et al. 11C-acetate PET imaging of prostate cancer J Nucl Med, 43 (2) (2002), pp. 181-186
19 T. Tsuchida, H. Takeuchi, H. Okazawa, et al. Grading of brain glioma with 1–11C-acetate PET: comparison with 18F-FDG PET Nucl Med Biol, 35 (2) (2008), pp. 171-176
20 S.A. Comerford, Z. Huang, X. Du, et al. Acetate dependence of tumors Cell, 159 (7) (2014), pp. 1591-1602
21 T. Mashimo, K. Pichumani, V. Vemireddy, et al. Acetate is a bioenergetic substrate for human glioblastoma and brain metastases Cell, 159 (7) (2014), pp. 1603-1614.
22 R.C. Sun, M. Fadia, J.E. Dahlstrom, et al. Reversal of the glycolytic phenotype by dichloroacetate inhibits metastatic breast cancer cell growth in vitro and in vivoBreast Cancer Res Treat, 120 (1) (2010), pp. 253-260
23 H. Hur, Y. Xuan, Y.B. Kim, et al. Expression of pyruvate dehydrogenase kinase-1 in gastric cancer as a potential therapeutic target Int J Oncol, 42 (1) (2013 Jan), pp. 44-54
24 K.M. Anderson, J. Jajeh, P. Guinan, M. Rubenstein In vitro effects of dichloroacetate and CO2 on hypoxic HeLa cells Anticancer Res, 29 (11) (2009), pp. 4579-4588
25 O.N. Pyaskovskaya, D.L. Kolesnik, A.G. Fedorchuk, et al. 2-Deoxy-D-glucose enhances dichloroacetate antitumor action against Lewis lung carcinoma Exp Oncol, 38 (3) (2016), pp. 176-180
26 I. Vega-Naredo, R. Loureiro, K.A. Mesquita, et al. Mitochondrial metabolism directs stemness and differentiation in P19 embryonal carcinoma stem cells Cell Death Differ, 21 (10) (2014), pp. 1560-1574.
27S. Park, T.S. Kim, S.H. Kang, H.B. Kim, J.W. Park, S.K. Kim 11C-acetate and 18F-fluorodeoxyglucose positron emission tomography/computed tomography dual imaging for the prediction of response and prognosis after transarterial chemoembolization Medicine (Baltimore)., 97 (37) (2018 Sep), Article e12311