Dichloroacetate as a novel pharmaceutical treatment for cancer-related fatigue in melanoma.

Cancer-related fatigue (CRF) is one of the most common complications in patients with multiple cancer types and severely affects patients' quality of life. However, there have only been single symptom-relieving adjuvant therapies but no effective pharmaceutical treatment for the CRF syndrome. Dichloroacetate (DCA), a small molecule inhibitor of pyruvate dehydrogenase kinase, has been tested as a potential therapy to slow tumor growth, based largely on its effects in vitro to halt cell division. We found that although DCA did not affect rates of tumor growth or the efficacy of standard cancer treatment (immunotherapy and chemotherapy) in two murine cancer models, DCA preserved physical function in mice with late-stage tumors by reducing circulating lactate concentrations. In vivo liquid chromatography-mass spectrometry/mass spectrometry studies suggest that DCA treatment may preserve membrane potential, postpone proteolysis, and relieve oxidative stress in muscles of tumor-bearing mice. In all, this study provides evidence for DCA as a novel pharmaceutical treatment to maintain physical function and motivation in murine models of CRF.NEW & NOTEWORTHY We identify a new metabolic target for cancer-related fatigue, dichloroacetate (DCA). They demonstrate that in mice, DCA preserves physical function and protects against the detrimental effects of cancer treatment by reducing cancer-induced increases in circulating lactate. As DCA is already FDA approved for another indication, these results could be rapidly translated to clinical trials for this condition for which no pharmaceutical therapies exist beyond symptom management.

A Sub-6 nm MnFe2O4-dichloroacetic acid nanocomposite modulates tumor metabolism and catabolism for reversing tumor immunosuppressive microenvironment and boosting immunotherapy.

Adenosine and lactate accumulated in tumor microenvironment are two major causes of immunosuppression, their concurrent downregulation holds promise in effective cancer immunotherapy, but remains challenging. Here, a sub-6 nm MnFe2O4 conjugated with dichloroacetic acid (DCA) nanoparticle is developed to modulate tumor glucose metabolism and ATP catabolism for reversing the tumor immunosuppressive microenvironment. The ultrasmall MnFe2O4-DCA nanoparticle can efficiently enter mitochondria and supply oxygen, improving the bioactivity of DCA to regulate glucose metabolism and reduce lactate production ca. 100 times higher than free DCA itself. Moreover, this design significantly downregulates CD39 and CD73 expression than DCA or MnFe2O4 alone, which consequently decreases the extracellular ATP catabolism. The concurrent regulation of glucose metabolism and ATP catabolism leads to increased immunostimulatory ATP level and decreased immunosuppressive adenosine and lactate levels in tumor microenvironment, eventually amplified dendritic cells maturation, enhanced cytotoxic T lymphocyte response, and improved cancer immunotherapy efficacy.

[Anti-cancer properties of dichloroacetate]. / Przeciwnowotworowe wlasciwosci dichlorooctanu.

One of the hallmarks of cancer cells is aerobic glycolysis (the Warburg effect). The effect of dichloroacetate (DCA) is to switch glucose metabolism (cellular respiration) to a more efficient process involving oxygen, reduce the production of lactic acid, activate the respiratory chain, change the potential of the mitochondrial membrane, and release pro-apoptotic mediators (cytochrome c and AIF) into the cytosol. As a result, the control over the mutated cells is improved, their sensitivity to various drugs or radiotherapy and their sensitivity to apoptosis increase. In the study the review of data on the mechanism of action of DCA on neoplastic cells was performed to indicate the side effects associated with the possible introduction of this compound to cancer therapy.

Concentration-dependent effects of dichloroacetate in type 2 diabetic hearts assessed by hyperpolarized [1-13 C]-pyruvate magnetic resonance imaging.

Personalized medicine or individualized therapy promises a paradigm shift in healthcare. This is particularly true in complex and multifactorial diseases such as diabetes and the multitude of related pathophysiological complications. Diabetic cardiomyopathy represents an emerging condition that could be effectively treated if better diagnostic and, in particular, better therapeutic monitoring tools were available. In this study, we investigate the ability to differentiate low and high doses of metabolically targeted therapy in an obese type 2 diabetic rat model. Low-dose dichloroacetate (DCA) treatment was associated with increased lactate production, while no or little change was seen in bicarbonate production. High-dose DCA treatment was associated with a significant metabolic switch towards increased bicarbonate production. These findings support further studies using hyperpolarized [1-13 C]-pyruvate magnetic resonance imaging to differentiate treatment effects and thus allow for personalized titration of therapeutics.

Mitigation of doxorubicin-induced cardiotoxicity by dichloroacetate: potential roles of restoration of PGC-1α/SIRT3 signaling and suppression of oxidative stress and apoptosis.

OBJECTIVE: Doxorubicin (DOX) is an effective chemotherapeutic agent used in the treatment of various neoplasms. Nevertheless, its therapeutic efficacy is hampered by life-threatening heart failure. Therefore, the current study was undertaken to investigate whether dichloroacetate (DCA), a metabolic and mitochondrial modulator, when administered at a therapeutically feasible dose could potentially reverse acute DOX cardiotoxicity. Furthermore, the possible underlying mechanisms of cardioprotection were also assessed. MATERIALS AND METHODS: Different techniques were performed to assess cardiac injury like echocardiography, histopathology, transmission electron microscope, biomarkers of cardiac injury, and oxidative stress markers. Further, the expression levels of mRNA and protein were quantified by PCR and immunohistochemistry, respectively. RESULTS: Echocardiography showed that mice that received DOX/DCA combination were protected against heart failure. Additionally, histopathology and transmission electron microscopy revealed structural damage alleviation by DOX/DCA combination, which was confirmed biochemically via significant suppression of elevated CK-MB and AST levels. Mechanistically, DOX dysregulated the expression of PGC-1α and SIRT-3 genes which are key to normal mitochondrial functioning. Of note, co-treatment with DCA effectively restored PGC-1α/SIRT-3 signaling and normalized the mitochondrial DNA index. Moreover, events downstream of DOX-triggered mitochondrial dysfunction such as oxidative stress and p53-dependent apoptosis were all abrogated by combination with DCA. CONCLUSIONS: The present study is the first to provide in vivo evidence that DCA is effective in protecting against acute DOX cardiotoxicity. Additionally, the study highlights the potential of administering metabolic modulators to safeguard against DOX cardiotoxicity.

Evaluation of combination protocols of the chemotherapeutic agent FX-9 with azacitidine, dichloroacetic acid, doxorubicin or carboplatin on prostate carcinoma cell lines.

The isoquinolinamine FX-9 is a novel potential chemotherapeutic agent showing antiproliferative effects against hematologic and prostate cancer cell lines such as B- and T-acute lymphoblastic leukemia and prostate cancer (PC) of different species. Interestingly, FX-9 shows no hemolytic activity and low toxicity in benign adherent cells. The detailed FX-9 molecular mode of action is currently not fully understood. But application on neoplastic cells induces pro-apoptotic and antimitotic effects. Canine prostate cancer (cPC) represents a unique spontaneous occurring animal model for human androgen-independent PC. Human androgen-independent PC as well as cPC are currently not satisfactorily treatable with chemotherapeutic protocols. Accordingly, the evaluation of novel agent combinations bears significant potential for identifying novel treatment strategies. In this study, we combined FX-9 with the currently approved therapeutic agents doxorubicin, carboplatin, the demethylating substance azacitidine as well as further potentially antitumorigenic agents such as dichloroacetic acid (DCA) in order to evaluate the respective synergistic potential. The combinations with 1-5 µM FX-9 were evaluated regarding the effect after 72 hours on cell viability, cell count and apoptotic/necrotic cells in two human prostate cancer cell lines (LNCaP, PC-3) and a canine prostate cancer cell line (Adcarc1258) representing androgen-dependent and -independent PC/cPC forms. FX-9 in combination with azacitidine decreases cell viability and increases cell death with positive Bliss values. Furthermore, this decreases the cell count with neutral Bliss values on PC-3. Carboplatin in combination with FX-9 reduces cell viability with a neutral Bliss value and increases cell death on LNCaP with calculated positive Bliss values. DCA or doxorubicin in combination with FX-9 do not show synergistic or additive effects on the cell viability. Based on these results, azacitidine or carboplatin in combination with FX-9 offers synergistic/additive efficacy against prostate adenocarcinoma cell lines in vitro. The beneficial effects of both combinations are worth further investigation.

Sodium dichloroacetate attenuates the growth of B16-F10 melanoma in vitro and in vivo: an opportunity for drug repurposing.

Sodium dichloroacetate (DCA) is a metabolic regulator used to treat diabetes. Since DCA inhibits pyruvate dehydrogenase kinase, decreasing lactic acid formation, it can reverse the Warburg effect in cancer cells, promoting apoptosis. Therefore, this study aimed to investigate the potential of DCA as a drug repurposing candidate for the treatment of melanoma. For the in-vitro assay, murine B16-F10 melanoma cells were treated with 0.5, 1, 5, 10, 20 or 50 mM DCA for 3 days, analyzed with the crystal violet method. The in-vivo effect of DCA was evaluated in B16-F10 tumor-bearing C57BL/6 mice treated with different doses of DCA (0, 25, 75 or 150 mg/kg) by gavage for 10 days, followed by measurement of tumor volume. Upon necropsy, representative slices of lung, liver, kidney, spleen and intestine were collected, processed and submitted for histopathological examination. The DCA concentrations of 10, 20 and 50 mM reduced B16-F10 cell viability after 48 and 72 h of treatment, whereas 20 and 50 mM were effective after 24 h of treatment. A significant reduction in tumor growth was observed in B16-F10 melanoma bearing mice at all doses, with no change in body weight or histology. DCA attenuates the growth of B16-F10 melanoma in vitro and in vivo, without systemic toxic effects. Therefore, DCA is a candidate for drug repurposing against melanomas.

Targeting Cancer Cell Metabolism with Metformin, Dichloroacetate and Memantine in Glioblastoma (GBM).

AIM: To investigate the effects of metformin, dichloroacetate (DCA), and memantine on T98G and U87-MG human glioblastoma (GBM) cells to target tumor cell metabolism in a multi-directional manner. MATERIAL AND METHODS: IC50 levels for metformin, DCA, metformin+DCA and memantine were determined by MTT assay in T98G and U87-MG cells in vitro. Casp3, Bcl-2, Bax, c-Myc and GSK-3B protein expressions were investigated post treatments. Fifteen GBM+ tumor tissues were assessed for Casp-3, Bcl-2, Bad, Bax for apoptotic protein expression patterns. RESULTS: Cancer cell metabolism targeting drugs metformin, DCA, metformin+DCA and memantine induced cytotoxicity in a dose-dependent manner in T98G and U87-MG cells. IC50 for memantine is found as 0.5 mM (p < 0.01) which is nearly 10 times lower concentration than that of metformin. Fifteen GBM+ tumor tissues had differential apoptotic protein expressions. CONCLUSION: Memantine exerted anti-cancer mechanism of action in T98G and U87-MG cells, however, such a mechanism requires deeper investigation for GBM treatment.

Antitumor Therapy under Hypoxic Microenvironment by the Combination of 2-Methoxyestradiol and Sodium Dichloroacetate on Human Non-Small-Cell Lung Cancer.

A hypoxic microenvironment is a hallmark in different types of tumors; this phenomenon participates in a metabolic alteration that confers resistance to treatments. Because of this, it was proposed that a combination of 2-methoxyestradiol (2-ME) and sodium dichloroacetate (DCA) could reduce this alteration, preventing proliferation through the reactivation of aerobic metabolism in lung adenocarcinoma cell line (A549). A549 cells were cultured in a hypoxic chamber at 1% O2 for 72 hours to determine the effect of this combination on growth, migration, and expression of hypoxia-inducible factors (HIFs) by immunofluorescence. The effect in the metabolism was evaluated by the determination of glucose/glutamine consumption and the lactate/glutamate production. The treatment of 2-ME (10 µM) in combination with DCA (40 mM) under hypoxic conditions showed an inhibitory effect on growth and migration. Notably, this reduction could be attributed to 2-ME, while DCA had a predominant effect on metabolic activity. Moreover, this combination decreases the signaling of HIF-3α and partially HIF-1α but not HIF-2α. The results of this study highlight the antitumor activity of the combination of 2-ME 10 µl/DCA 40 mM, even in hypoxic conditions.

Synergisms of genome and metabolism stabilizing antitumor therapy (GMSAT) in human breast and colon cancer cell lines: a novel approach to screen for synergism.

BACKGROUND: Despite an improvement of prognosis in breast and colon cancer, the outcome of the metastatic disease is still severe. Microevolution of cancer cells often leads to drug resistance and tumor-recurrence. To target the driving forces of the tumor microevolution, we focused on synergistic drug combinations of selected compounds. The aim is to prevent the tumor from evolving in order to stabilize disease remission. To identify synergisms in a high number of compounds, we propose here a three-step concept that is cost efficient, independent of high-throughput machines and reliable in its predictions. METHODS: We created dose response curves using MTT- and SRB-assays with 14 different compounds in MCF-7, HT-29 and MDA-MB-231 cells. In order to efficiently screen for synergies, we developed a screening tool in which 14 drugs were combined (91 combinations) in MCF-7 and HT-29 using EC25 or less. The most promising combinations were verified by the method of Chou and Talalay. RESULTS: All 14 compounds exhibit antitumor effects on each of the three cell lines. The screening tool resulted in 19 potential synergisms detected in HT-29 (20.9%) and 27 in MCF-7 (29.7%). Seven of the top combinations were further verified over the whole dose response curve, and for five combinations a significant synergy could be confirmed. The combination Nutlin-3 (inhibition of MDM2) and PX-478 (inhibition of HIF-1α) could be confirmed for all three cell lines. The same accounts for the combination of Dichloroacetate (PDH activation) and NHI-2 (LDH-A inhibition). Our screening method proved to be an efficient tool that is reliable in its projections. CONCLUSIONS: The presented three-step concept proved to be cost- and time-efficient with respect to the resulting data. The newly found combinations show promising results in MCF-7, HT-29 and MDA-MB231 cancer cells.

Dichloroacetate restores colorectal cancer chemosensitivity through the p53/miR-149-3p/PDK2-mediated glucose metabolic pathway.

The development of chemoresistance remains a major challenge that accounts for colorectal cancer (CRC) lethality. Dichloroacetate (DCA) was originally used as a metabolic regulator in the treatment of metabolic diseases; here, DCA was assayed to identify the mechanisms underlying the chemoresistance of CRC. We found that DCA markedly enhanced chemosensitivity of CRC cells to fluorouracil (5-FU), and reduced the colony formation due to high levels of apoptosis. Using the microarray assay, we noted that miR-149-3p was involved in the chemoresistance of CRC, which was modulated by wild-type p53 after DCA treatment. In addition, PDK2 was identified as a direct target of miR-149-3p. Mechanistic analyses showed that overexpression of miR-149-3p enhanced 5-FU-induced apoptosis and reduced glucose metabolism, similar to the effects of PDK2 knockdown. In addition, overexpression of PDK2 partially reversed the inhibitory effect of miR-149-3p on glucose metabolism. Finally, both DCA treatment and miR-149-3p overexpression in 5-FU-resistant CRC cells were found to markedly sensitize the chemotherapeutic effect of 5-FU in vivo, and this effect was also validated in a small retrospective cohort of CRC patients. Taken together, we determined that the p53/miR-149-3p/PDK2 signaling pathway can potentially be targeted with DCA treatment to overcome chemoresistant CRC.

News and views in myalgic encephalomyelitis/chronic fatigue syndrome (ME/CFS): The role of co-morbidity and novel treatments.

Though affecting many thousands of patients, myalgic encephalomyelitis/chronic fatigue syndrome (ME/CFS) should be considered an orphan disease, since the cause remains elusive and no treatment is available that can provide complete cure. There is reasonable insight into the pathogenesis of signs and symptoms, and treatments specifically directed to immunological, inflammatory and metabolic processes offer relief to an increasing number of patients. Particular attention is given to the importance of co-morbidity requiring appropriate therapy. Promising results are obtained by treatment with Metformin, or possibly Momordica charantia extract, which will correct insulin resistance, with Meldonium improving the transportation of glucose into the mitochondria, with sodium dichloroacetate activating pyruvate dehydrogenase, and with nutraceutical support reducing oxidative and inflammatory impairment.

Targeting aerobic glycolysis by dichloroacetate improves Newcastle disease virus-mediated viro-immunotherapy in hepatocellular carcinoma.

BACKGROUND: Oncolytic viro-immunotherapy holds promise for cancer treatment. While immune activation can be robustly triggered by oncolytic viruses, negative feedback is often upregulated in the tumour microenvironment (TME). Lactate accumulation, signal transducer and activator of transcription 3 (STAT3) activation, indoleamine 2,3-dioxygenase 1 (IDO1) expression, and myeloid-derived suppressor cell (MDSC) infiltration coordinate to shape the immunosuppressive TME. METHODS: Representative hepatocellular carcinoma (HCC) cell lines and HCC-bearing mice were treated with oncolytic Newcastle disease virus (NDV), alone or in combination with dichloroacetate (DCA, a pyruvate dehydrogenase kinase (PDK) inhibitor). RESULTS: We found that infection with oncolytic NDV led to significant induction of the aforementioned suppressive factors. Interestingly, DCA significantly reduced lactate release, STAT3 activation, IDO1 upregulation, and MDSC infiltration in NDV-treated HCC. Consequently, DCA significantly enhanced the antitumour immune responses, leading to improved antitumour efficacy and prolonged survival in mouse models of ascitic and subcutaneous HCC. Furthermore, DCA increased NDV replication in a PDK-1-dependent manner in HCC. CONCLUSIONS: Targeting aerobic glycolysis by DCA improves NDV-mediated viro-immunotherapy in HCC by mitigating immune negative feedback and promoting viral replication. These findings provide a rationale for targeting reprogrammed metabolism together with oncolytic virus-mediated viro-immunotherapy for HCC treatment.

Acidosis-induced metabolic reprogramming in tumor cells enhances the anti-proliferative activity of the PDK inhibitor dichloroacetate.

Altered metabolic pathways in cancer such as exacerbated glycolytic flux and increased glutamine metabolism are promising targets for anti-cancer therapies. While commonly observed in glycolytic tumors, extracellular acidosis has never been considered as a potential modulator of anti-metabolic drug activity such as dichloroacetate (DCA). Using cancer cells from various origins selected for their ability to proliferate under acidic conditions, we found that DCA exerts greater inhibitory effects on the growth of these acid-adapted cells than in parental cells. Moreover, daily DCA administration to mice led to a significant decrease in tumor growth from acid-adapted cells but not from parental cells. 13C-tracer studies revealed that DCA induced a double metabolic shift, diminishing glycolysis and increasing intracellular glutamine in acid-adapted cells. As a consequence, DCA reduced the pentose phosphate pathway activity more extensively and increased apoptosis in acid-adapted cells. Finally, the combination of DCA with a glutaminase inhibitor significantly enhanced the cytotoxic effects of DCA. Overall, the interplay between acidosis and DCA exposure leads to metabolic reprogramming that considerably alters cellular fitness.

Dichloroacetate (DCA) and Cancer: An Overview towards Clinical Applications.

An extensive body of literature describes anticancer property of dichloroacetate (DCA), but its effective clinical administration in cancer therapy is still limited to clinical trials. The occurrence of side effects such as neurotoxicity as well as the suspicion of DCA carcinogenicity still restricts the clinical use of DCA. However, in the last years, the number of reports supporting DCA employment against cancer increased also because of the great interest in targeting metabolism of tumour cells. Dissecting DCA mechanism of action helped to understand the bases of its selective efficacy against cancer cells. A successful coadministration of DCA with conventional chemotherapy, radiotherapy, other drugs, or natural compounds has been tested in several cancer models. New drug delivery systems and multiaction compounds containing DCA and other drugs seem to ameliorate bioavailability and appear more efficient thanks to a synergistic action of multiple agents. The spread of reports supporting the efficiency of DCA in cancer therapy has prompted additional studies that let to find other potential molecular targets of DCA. Interestingly, DCA could significantly affect cancer stem cell fraction and contribute to cancer eradication. Collectively, these findings provide a strong rationale towards novel clinical translational studies of DCA in cancer therapy.

Comparison of Antitumor Effects of Combined and Separate Treatment with NO Synthase Inhibitor T1023 and PDK1 Inhibitor Dichloroacetate.

Combined chronic treatment of Ehrlich solid carcinoma (EC) with an NOS inhibitor 1-isobutanoyl-2-isopropylisothiourea hydrobromide (T1023) and a PDK1 inhibitor dichloroacetate was accompanied by statistically significant synergetic antitumor effects manifested in a significant and stable suppression of neoplasm growth (by 55-65%). Separate treatment with T1023 and dichloroacetate induced moderate short-term inhibition of tumor growth (by 30-35%) followed by weakening of tumor sensitivity to these substances. These results attest to synergetic antitumor effects NOS inhibitor T1023 and PDK1 inhibitor dichloroacetate producing antiangiogenic and hypoxia-targeted cytotoxic effects, during their combined administration, which allows overcoming the adaptive potential of the tumors.

Pyruvate dehydrogenase deficiency. / Pyruvatdehydrogenase-mangel.

Pyruvate dehydrogenase deficiency is a rare mitochondrial disease leading to energy deficiency in the cells. This particularly affects the central nervous system, resulting in a broad range of neurological symptoms.

Myalgic Encephalomyelitis/Chronic Fatigue Syndrome Induced by Repeated Forced Swimming in Mice.

Myalgic encephalomyelitis/chronic fatigue syndrome (ME/CFS) is characterized by disabling fatigue of at least 6 months, in addition to symptoms such as muscle pain and muscle weakness. There is no treatment provides long-term benefits to most patients. Recently, clinical research suggested the involvement of pyruvate dehydrogenase (PDH) in ME/CFS. PDH is a crucial enzyme in the mitochondria matrix that links glycolysis to the tricarboxylic acid cycle and oxidative phosphorylation. However, it is little known whether PDH could be a therapeutic target. The purpose of this study was to establish ME/CFS in mice and to investigate the involvement of PDH in ME/CFS. To induce the chronic fatigue in mice, a repeated forced swimming test was conducted. To evaluate fatigue, we measured immobility time in forced swimming test and starting time of grooming. An open field test was conducted on day 8. After 25 d of the forced swimming test, the mitochondrial fraction in gastrocnemius muscle was isolated and PDH activity was measured. Moreover, we evaluated the effect of PDH activation by administering sodium dichloroacetate (DCA). In ME/CFS mice group, the immobility time and starting time of grooming increased time-dependently. In addition, the moved distance was decreased in ME/CFS mice. PDH activity was decreased in the mitochondrial fraction of the gastrocnemius muscle of the forced swimming group. DCA treatment may be beneficial in preventing fatigue-like behavior in ME/CFS. These findings indicate that ME/CFS model was established in mice and that a decrease in mitochondrial PDH activity is involved with the symptom of ME/CFS.

Dichloroacetate-induced peripheral neuropathy.

Dichloroacetate (DCA) has been the focus of research by both environmental toxicologists and biomedical scientists for over 50 years. As a product of water chlorination and a metabolite of certain industrial chemicals, DCA is ubiquitous in our biosphere at low µg/kg body weight daily exposure levels without obvious adverse effects in humans. As an investigational drug for numerous congenital and acquired diseases, DCA is administered orally or parenterally, usually at doses of 10-50mg/kg per day. As a therapeutic, its principal mechanism of action is to inhibit pyruvate dehydrogenase kinase (PDK). In turn, PDK inhibits the key mitochondrial energy homeostat, pyruvate dehydrogenase complex (PDC), by reversible phosphorylation. By blocking PDK, DCA activates PDC and, consequently, the mitochondrial respiratory chain and ATP synthesis. A reversible sensory/motor peripheral neuropathy is the clinically limiting adverse effect of chronic DCA exposure and experimental data implicate the Schwann cell as a toxicological target. It has been postulated that stimulation of PDC and respiratory chain activity by DCA in normally glycolytic Schwann cells causes uncompensated oxidative stress from increased reactive oxygen species production. Additionally, the metabolism of DCA interferes with the catabolism of the amino acids phenylalanine and tyrosine and with heme synthesis, resulting in accumulation of reactive molecules capable of forming adducts with DNA and proteins and also resulting in oxidative stress. Preliminary evidence in rodent models of peripheral neuropathy suggest that DCA-induced neurotoxicity may be mitigated by naturally occurring antioxidants and by a specific class of muscarinic receptor antagonists. These findings generate a number of testable hypotheses regarding the etiology and treatment of DCA peripheral neuropathy.

Dichloroacetate Affects Mitochondrial Function and Stemness-Associated Properties in Pancreatic Cancer Cell Lines.

Targeting metabolism represents a possible successful approach to treat cancer. Dichloroacetate (DCA) is a drug known to divert metabolism from anaerobic glycolysis to mitochondrial oxidative phosphorylation by stimulation of PDH. In this study, we investigated the response of two pancreatic cancer cell lines to DCA, in two-dimensional and three-dimension cell cultures, as well as in a mouse model. PANC-1 and BXPC-3 treated with DCA showed a marked decrease in cell proliferation and migration which did not correlate with enhanced apoptosis indicating a cytostatic rather than a cytotoxic effect. Despite PDH activation, DCA treatment resulted in reduced mitochondrial oxygen consumption without affecting glycolysis. Moreover, DCA caused enhancement of ROS production, mtDNA, and of the mitophagy-marker LC3B-II in both cell lines but reduced mitochondrial fusion markers only in BXPC-3. Notably, DCA downregulated the expression of the cancer stem cells markers CD24/CD44/EPCAM only in PANC-1 but inhibited spheroid formation/viability in both cell lines. In a xenograft pancreatic cancer mouse-model DCA treatment resulted in retarding cancer progression. Collectively, our results clearly indicate that the efficacy of DCA in inhibiting cancer growth mechanistically depends on the cell phenotype and on multiple off-target pathways. In this context, the novelty that DCA might affect the cancer stem cell compartment is therapeutically relevant.