Antitumor Effect of Dichloroacetate in Combination with 5-Fluorouracil in Colorectal Cancer
Jingtao Tong, Ganfeng Xie, Jinxia He, Jianjun Li, Feng Pan, and Houjie Liang
Department of Oncology, Southwest Hospital, Third Military Medical University,
29 Gaotanyan Street,
Correspondence should be addressed to Houjie Liang, firstname.lastname@example.org
Received 27 May 2010; Revised 29 December 2010; Accepted 13 January 2011
Academic Editor: Miguel A. Andrade
Copyright © 2011 Jingtao Tong et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
Dichloroacetate (DCA), an inhibitor of pyruvate dehydrogenase kinase (PDK), has been recently demonstrated as a promising nontoxic antineoplastic agent that promotes apoptosis of cancer cells. In the present study, we aimed to investigate the antitumor eﬀect of DCA combined with 5-Fluorouracil (5-FU) on colorectal cancer (CRC) cells. Four human CRC cell lines were treated with DCA or 5-FU, or a combination of DCA and 5-FU. The cell viability was determined by 3-(4,5-dimethylthiazol-2-yl)2,5-diphenyltetrazolium bromide assay. The interaction between DCA and 5-FU was evaluated by the median eﬀect principle. Immunocytochemistry with bromodeoxyuridine (BrdU) was carried out to determine the proliferation of CRC cells. Cell cycle and apoptosis were measured by ﬂow cytometry, and the expression of apoptosis-related molecules was assessed by western blot. Our results demonstrated that DCA inhibited the viability of CRC cells and had synergistic antiproliferation in combination with 5-FU. Moreover, compared with 5-FU alone, the apoptosis of CRC cells treated with DCA and 5-FU was enhanced and demonstrated with the changes of Bcl-2, Bax, and caspase-3 proteins. Our results suggest that DCA has a synergistic antitumor eﬀect with 5-FU on CRC cell lines in vitro.
Colorectal cancer is one of the most common malignancies worldwide . Other than surgery, treatment for CRC patients relies primarily on chemotherapy, especially the patients with advanced CRC. Among the chemotherapeutic agents for CRC, 5-Fluorouracil (5-FU), which is a classical chemotherapy agent, has been the ﬁrst line regimen for treating CRC over several decades [2, 3]. However, 5-FU alone is poorly selective to tumor as well as highly toxic to bone marrow, gastrointestinal tract, and skin when used at the therapeutic dose .
Metabolic abnormity is one of critical hallmarks of cancer . As early as 1920s, Otto Warburg observed that cancer cells generally use glycolysis rather than oxidative phosphorylation for energy . Thus, the metabolic switch to anaerobic respiration through glycolysis from pyruvate, rather than pyruvate conversion to acetyl-CoA by action of pyruvate dehydrogenase (PDH) in aerobic glucose metabolism, becomes a preferential phenotype of cancer progress. PDHcan be inactivated by pyruvate dehydrogenase kinase (PDK) in many glycolytic phenotypes including cancer, while inhibition of PDK switches metabolism to aerobic oxidation which is proved to be disadvantageous to tumor growth .
Dichloroacetate (DCA) is a prototypical inhibitor of mitochondrial PDK. By blocking the enzyme, DCA decreases lactate production by shifting the metabolism of pyruvate from glycolysis towards oxidation in the mitochondria. This property has led to trials of DCA for the treatment of lactic acid accumulation disorders . Recently, studies have demonstrated that DCA suppresses tumor growth via inhibition of PDK [9–11]. Michelakis and his colleagues found that DCA restored mitochondrial function, thus restoring apoptosis, killing cancer cells in vitro, and shrinking the tumors in the rats .
Combination chemotherapy has been used widely. 5-FU is usually combined with other antineoplastic agents and radiation to enhance its antitumor eﬀect. The clinical insuﬃciency appears to be caused from resistance of 5-FU andseveresideeﬀects. The strong and selective induction of apoptosis suggests that the PDK inhibitor DCA may potentiate the inhibitory eﬀect of anticancer drugs, thus exceeding the eﬃcacy of current treatment. Herein, we aimed to examine the combined antitumor eﬀects of DCA with 5-FU on the CRC cells, in hoping of assessing a relatively eﬀective and safe regimen potentiated for CRC treatment.
2. Material and Methods
2.1. Cells and Regents. The human colon cancer cell lines LS174T, LoVo, SW620, and HT29 were purchased from American Type Culture Collection (Manassas, VA, USA). Cell culture reagents were purchased from Gibco-Invitrogen (Carlsbad, CA, USA). Cell lines were maintained in Dulbecco’s Modiﬁed Eagle’s medium or Leibovitz L-15 medium containing 10% fetal bovine serum (FBS), 100 U/mL penicillin, and 100 μg/mL streptomycin in a 37◦C, 5% CO humidiﬁed incubator. 5-FU and DCA was purchased from Sigma-Aldrich Co. ltd. (St. Louis, MO, USA), dissolved in deionized water to make 1 mol/L working solution, ﬁltersterilized, and subsequently diluted in growth medium for treatment.
2.2. Cell Viability Assay. Cell viability was determined using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay (Sigma-Aldrich). Cells were seeded into 96-well plates (5×104 cells per well) and incubated under standard growth conditions overnight from 60% to 70% conﬂuence. Cells were then treated with DCA alone (ﬁnal concentration 0–90 mM) or with DCA combined with 5-FU (5–200 μM). After a 48-hour treatment, cells were incubated at 37◦C for another 4 hours with MTT (20 μLper well) and the absorbance at 490 nm was measured in a BioRad Model 550 plate reader (Hercules, CA, USA).
2.3. Analysis of Drug Interaction. The interaction between DCA and 5-FU was analyzed by using the median eﬀect principle described by Chou and Talalay [13, 14]. The program enables calculation of combination indices (CIs) which, when smaller than 1, equal to 1, or greater than 1, indicate synergism, additivity, or antagonism, respectively, between two drugs. CIs were calculated by
where (Dx)1 and (Dx)2 are the concentrations of DCA alone or 5-FU alone, giving x% growth inhibition, and (D)1 and (D)2 are the drug concentrations in combination inhibiting cell growth also x%. (Dx)1 and (Dx)2 were calculated by the median-eﬀect equation:
where Dm is the median-eﬀect dose, fa is the fraction aﬀected, and mrepresents the slope of the median-eﬀect plot.
2.4. Cell Proliferation Assays. Immunocytochemistry was carried out with bromodeoxyuridine (BrdU) (BDBioscience, San Jose, CA, USA) in vitro. Cells were propagated on coverslips in 12-well plates under standard growth conditions. After 24 hours, various concentrations of DCA, 5-FU, or a combination of two drugs were added. Cells were serumstarved for 12 hours in growth media containing 0.5% FBS to reset the cell cycle to G0 phase, and then cells were pulsed for 2 hours with 10 μmol/L BrdU in growth media. Subsequently, the cells were ﬁxed, washed, and stained according to the manufacturer’s instruction.
Cell cycle analysis was determined indirectly using propidium iodine (PI, BD Bioscience) staining by ﬂow cytometry (FACScan, Becton Dickinson, San Jose, CA, USA). Cells were seeded into 6-well plates and cultured with or without 10 mM DCA or 20 μM 5-FU. After incubating for 48 hours, cells were harvested with 0.25% Trypsin-EDTA (Invitrogen, Carlsbad, CA, USA). Then, cells were ﬁxed with 70% alcohol for 24 hours at 4◦C and washed twice with phosphate buﬀer saline (PBS). RNAase (100 μL; 1 mg/mL) (BD Bioscience) was added, and cells were incubated in a 37◦ Cwaterbathfor 30 minutes. After staining with 200 μLPI(50μg/mL), cells were held at 4◦C for 30 minutes. Finally, cells were analyzed by ﬂow cytometry.
2.5. Apoptosis Assay. Apoptosis was detected by ﬂow cytometry with annexin-V-FITC (BD Bioscience) and PI. Cells were seeded into 6-well plates. After a 48-hour incubation with or without drugs, cells were washed and resuspended in 0.5 mL PBS buﬀer. After staining with annexin-V-FITC and PI, cells were analyzed by ﬂow cytometry in three independent experiments.
2.6. Western Blot. Cells were harvested and total proteins were extracted with RIPA buﬀer containing protease inhibitors. Total protein (50 μg) was subjected to 10% or 12% SDS/PAGE, and the resolved proteins were transferred electrophoretically to PVDF membranes (Millipore, Bedford, MA, USA). Membranes were blocked for 2 hours with 5% nonfat milk in TBS buﬀer containing 0.05% Tween20 (TBST) at 4◦C. Membranes were then incubated with antibodies to Bax, Bcl-2, Caspase-3, and GADPH overnight at 4◦C. After washing in TBST, the membranes were incubated with their respective secondary antibodies for 1 hour. Membranes were then incubated with SuperSignal West Femto Maximum Sensitivity Substrate (Pierce, Rockford, IL, USA) for 1 minute and imaged using a Gel Doc XR system (Bio-Rad). All of the antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA).
2.7. Statistics. All data are expressed as means ± standard deviation (SD). Statistical analysis was performed using the SPSS 13.0 software (SPSS, Chicago, IL, USA). Diﬀerences between two groups were determined by paired-samples ttest or independent-samples t-test (two-tailed) as indicated. Diﬀerences among groups were analyzed by one-way analysis of variance (ANOVA). P<.05 was considered statistically signiﬁcant.
3.1. Viability of CRC Cells Treated with DCA Alone or in Combination with 5-FU. To de t e r mi ne t he e ﬀect of DCA on CRC cells, cells were exposed to DCA (0–90 mM) for 48 hours. The result showed that inhibitory eﬀect was dose dependent. As shown in Figure 1, the inhibition of viability of cancer cell lines treated with 50 mM DCA was as follows: SW620 (46.73% ± 5.21%), LoVo (30.94% ± 3.57%), LS174t (54.59% ± 3.93%), and HT29 (55.31% ± 3.35%). We treated cells with 5-FU (20–100 μM) and found that the viability of SW620, LoVo, LS174t was inhibited signiﬁcantly except HT29, with its viability inhibition not obvious under 80 μM 5-FU. When treated with these two drugs simultaneously, the viability of aforementioned CRC cells was signiﬁcantly decreased compared with DCA or 5FU alone (IC50 values are shown in Table 1).
3.2. Synergistic Eﬀects of DCA Combined with 5-FU in CRC Cell Lines. The viability of CRC cells were decreased in the presence of 5-FU and DCA. DCA potentiated the inhibitory eﬀects of 5-FU on CRC cells, and the inﬂuence of DCA on eﬀects of 5-FU was dose dependent (Figure 1). Interaction between 5-FU and DCA was analyzed with median eﬀects method. The combination of DCA and 5-FU produced synergistic or additive eﬀects depending on the range of cell kill level (Fa). All of four CRC cell lines indicated synergistic eﬀect with 5-FU and DCA. The synergism was statistically signiﬁcant in LS174t at inhibition levels of 50% and 80%, achieving CI values from 0.46–0.61, and in HT-29 cell line at inhibition levels of 50 and 80%, with CI values in the range from 0.42 to 0.52. In SW620 and LoVo cells, CI values at 50% and 80% inhibition levels were 0.64–0.75 and 0.75–0.94, respectively, (Figure 2).
3.3. DCA Increased the Eﬃciency of Antiproliferative Eﬀect of 5-FU. To conﬁrm that decreased cell viability was due to reduced proliferation, immunocytochemistry, and ﬂow cytometry were employed. Cells were treated with 10 mM DCA combined with 20 μM 5-FU. As expected, cells treated with DCA demonstrated reduced proliferation compared with untreated cells. The number of BrdU positive cells on four CRC cell lines after treatment with 5-FU and DCA were 11.4 ± 2.12, 13.55 ± 3.10, 12.84 ± 1.34, and 18.83 ± 1.45, respectively, which were lower than that treated with 5-FU or DCA alone (P<.01, see Table 2). In addition, treatment with DCA potentiated the cell cycle arrest in G1 phase. When treated with DCA and 5-FU, cells blocked in G1 /S phase were more than that of incorporated with DCA or 5-FU alone (Figure 3).
Table 1: IC50 values (concentrations required to reduce the viability of cells by 50% as compared with the control cells) were computed using linear or nonlinear regression (three parametric Hill function) (R2 > 0.9). They are presented as mean ± SD from at least three independent experiments. IC50 values of the studied drugs for inhibition of growth of various cell lines (cells were incubated with drugs for 48 hours).
|5-FU (μM)||99.50 ± 2.31||127.23 ± 1.28||177.45 ± 2.16||798.38 ± 1.25|
|DCA (mM)||37.65 ±1.03||29.15 ± 0.93||56.97 ± 0.84||47.02 ± 0.52|
Table 2: SW620, LoVo, LS174t, HT29 cells, and 293T noncancerous controls were treated with 10 mM DCA and 20 μM5-FUalone or in combination for 48 hours, and then pulsed with BrdU. Cells were then harvested and stained, and the numbers of BrdU+ cells were calculated as the mean number of the positive cells in eight diﬀerent ﬁelds of view in one image (magniﬁcation, 400X). This calculation was repeated three times. ∗P<.05, compared with control. BrdU SD).
|untreated||39.70 ± 2.13||40.54 ± 1.28||38.53 ± 0.94||42.14 ± 3.18|
|5-FU||25.2 ± 2.40||27.33 ± 1.47||27.94 ± 0.83||40.53 ± 2.71|
|DCA||30.4 ± 1.25||28.25 ± 2.17||31.72 ± 0.73||29.94 ± 2.16|
|5-FU+DCA||11.4 ± 2.12||13.55 ± 3.10||12.84 ± 1.34||18.83 ± 1.45|
3.4. Increased Apoptosis Induced by DCA Combined with 5-FU in CRC Cells. To further investigate the decreased viability of CRC cells treated with combination regimen, apoptosis was determined by ﬂow cytometry. As shown in Figure 4,DCA alone increased the proportion of apoptosis CRC cells. When treated with 10 mM DCA, the apoptosis rates of four CRC cell lines were 15.72 ± 1.63%, 11.32 ± 0.74%, 9.77 ± 0.53%, and 14.52 ± 1.00%, respectively, while the apoptosis rates were 9.14 ± 119%, 8.82 ± 0.41%, 10.31 ± 0.71%, and 7.27 ± 0.96% with 5-FU. When combination of DCA and 5-FU was applied, the apoptosis rate was much higher than 5-FU or DCA alone (P<.05), which indicated apoptotic eﬀect was increased via combination DCA and 5-FU (Figure 4). 3.5. Changes on Apoptosis-Associated Molecules Stimulated by DCA and 5-FU. To conﬁrm that increased apoptosis induced by combination therapy was due to modiﬁed expressions of apoptosis-associated molecules, western blot assay was applied. In Figure 5, the results indicated that 5-FU or DCA decreased the expression of Bcl-2 in four CRC cell lines compared to PBS controls, and the combination of DCA and 5-FU decreased Bcl-2 expression signiﬁcantly as compared with DCA or 5-FU alone. Conversely, the expressions of Bax and caspase-3 were signiﬁcantly increased in the four CRC cell lines treated with combination of DCA and 5-FU compared to their single usage. The most obvious increasing of Bax expression was detected in LS174t cells, while in LoVo it appeared that caspase-3 expression increased most (Figure 5).
Figure 1: Viability of four CRC cell lines in response to treatment with dichloroacetate (DCA) and 5-ﬂuorouracil (5-FU). The viability of SW620, LoVo, LS174t, and HT29 was decreased signiﬁcantly with diﬀerent concentration of 5-FU and DCA alone or in combination. Each experiment was performed in triplicate; ∗P<.05
In the present study, we demonstrate that DCA not only reduced cell viability and proliferation, but also have the synergistic antitumor eﬃciency with chemotherapeutic agent 5-FU in vitro in CRC cells. Meanwhile, DCA has no signiﬁcant eﬀects on noncancerous cells. Furthermore, we showed that DCA-induced apoptosis contributes to its synergistic antitumor eﬀect. Compared with DCA or 5FU alone, combination usage of these two drugs promotes apoptosis of CRC cells.
5-FU is a chemotherapy drug used to treat several types of cancer, including colorectal, breast, esophageal, and stomach . However, 5-FU–related toxicity is a serious and common issue for many cancer patients, with myelosuppression and gastrointestinal toxicity being the most commonly observed side eﬀects . The clinical activity of 5-FU is modest at standard doses, and in general, dosing is limited by the safety proﬁle. As a result, we are usually left with the dilemma of making decisions about the therapeutic dose of 5-FU. Various strategies have been developed to enhance the clinical activity of 5-FU, such as biochemical modulation , alterations in scheduling of administration , and the use of combination therapy [19–22].
|Figure 2: Mean values of the combination index at the aﬀected fractions of 50% (IC50 ) and 80% (IC80 ) when 5-ﬂuoruracil (5-FU) was combined with dichloroacetate (DCA) in HT-29, LoVo, LS174t, and SW620 cells. Mean IC50 and IC80 ± SD (n = 3) are shown. A CI value signiﬁcantly less than 1 indicates synergism, a CI not signiﬁcantly diﬀerent from 1 indicates addition, and a CI signiﬁcantly higher than 1 indicates antagonism; ∗P<.05.|
|Figure 3: Changes in cell cycle progression in SW620, LoVo, LS174t, and HT29 cells after 48-hour treatment with 5-ﬂuoruracil (5-FU) and dichloroacetate (DCA) applied alone or in combination. Each bar represents the mean ± SD (n = 3). The data obtained from FACS were analyzed using SPSS13.0; ∗P<.05.|
DCA is an odorless, colorless, inexpensive, relatively nontoxic, small molecule. It has been in clinical use since 1969 for treatment of lactic acidosis via boosting the ability of mitochondria to generate energy and reducing lactic acid accumulation . DCA has been considered as a potential cancer therapy regimen since a Canadian group found that it caused regression in several cancers, including lung, breast, and brain tumors [24, 25]. When given to cancer cells, the cells switched from glycolysis to mitochondrial energy production. What’s more, functional mitochondria help cells recognize functional abnormalities and trigger cell death due to its inhibition of tumor growth and induction of apoptosis in certain cancer. In the present study, we found the same viability inhibitory eﬀect in CRCs, which was dose-dependent and various with diﬀerent degrees of tumor diﬀerentiation. The results also indicated that low-dose DCA exerts a synergistic eﬀect with chemotherapeutic agent 5FU in halting growth of CRC cells, which was quantitatively analyzed according to Chou-Talalay method.
The results of cell proliferation also demonstrated that DCA enhanced the antiproliferational eﬀects of 5-FU. The number of BrdU positive cells was decreased when treated with 5-FU or DCA, while the BrdU-stained cells were decreased signiﬁcantly when treated with combination of 5FU and DCA compared with their single usage. Meanwhile, the growth inhibition of CRC cell was accompanied by cell cycle arrest. Combination of DCA and 5-FU induced cell cycle arrest in G1 /S phase in CRC cell lines, whereas 5-FU induced arrest in G1 phase was not obvious. The induction of cell cycle arrest may result from inhibitory ability to synthesize or repair DNA, which may lead to cell apoptosis.
Figure 4: Induction of apoptosis in SW620, LoVo, LS174t, and HT29 cells after 48-hour treatment with 5-ﬂuoruracil (5-FU) and dichloroacetate (DCA) alone or in combination. Each bar represents the mean ± SD (n = 3); ∗P<.05.
DCA appeared to exert biochemical eﬀects consistent with reversing the Warburg eﬀect and killing cancer cells. We found that DCA-induced CRC cells apoptosis, which is orchestrated with the previous DCA studies. Importantly, the combination of DCA with 5-FU increased the number of apoptotic cells compared with 5-FU alone, demonstrating that DCA inhibited cell growth via apoptosis.
Many factors mediating apoptosis converge to activate the critical eﬀector the caspase-3, which is considered as the key protease of caspase family in mammalian cell apoptosis . It always exists as a 23 kD inactive precursor in cytoplasm, which is activated during apoptosis and takes part in apoptosis induced by multiple factors. Caspasedependent apoptosis pathway mainly includes mitochondria pathway, death receptor pathway, and endoplasmic reticulum pathway . And the mitochondria pathway is controlled and regulated by the Bcl-2 family of proteins [28, 29], which are divided into two parts, the antiapoptotic members (Bcl-2) and proapoptotic members (Bax) . Recent study indicates that the Bcl-2 inhibits apoptosis via inhibiting Bax removing to mitochondrial outer membrane . We investigated the expression of caspase-3, Bcl-2, and Bax in protein level and the result of western blot showed that the expressions of caspase-3 and Bax were increased, while the expression of Bcl-2 was decreased in the combination of DCA with 5-FU treatment, comparing with the single treatments. These results suggested that the apoptosis induced by the combination of DCA and 5-FU might be related with caspase-dependent mitochondria pathway. Previous investigations suggested that the induction of apoptosis by DCA resulted from returning the dysfunction of the mitochondria and NFAT-Kv1.5 pathway [9, 12], which focused on the same point as present study that was mitochondria-mediated apoptosis.
Figure 5: Eﬀects of 5-ﬂuorouracil (5-FU) and dichloroacetate (DCA) on apoptosis-associated molecules expression. Bcl-2 expression was signiﬁcantly decreased by DCA in SW620, LoVo, and LS174t and HT29 cells. Bax and caspase-3 expression levels were higher after exposure to 5-FU and DCA compared with control.
This work was supported by the National Natural Science Foundation of China (NSFC, no. 30873015). J. Tong and G. Xie contributed equally to this work.
 A.Jemal,R.Siegel,E.Ward,Y.Hao,J.Xu,andM.J.Thun, “Cancer statistics, 2009,” CA Cancer Journal for Clinicians,vol. 59, no. 4, pp. 225–249, 2009.
J.A.MeyerhardtandR.J.Mayer,“Drugtherapy:systemic therapy for colorectal cancer,” The New England Journal of Medicine, vol. 352, no. 5, pp. 476–487, 2005.
 N. C. Tebbutt, E. Cattell, R. Midgley, D. Cunningham, and D. Kerr, “Systemic treatment of colorectal cancer,” European Journal of Cancer, vol. 38, no. 7, pp. 1000–1015, 2002.
M.Gusella,A.C.Frigo,C.Bolzonellaetal.,“Predictorsof survival and toxicity in patients on adjuvant therapy with 5ﬂuorouracil for colorectal cancer,” British Journal of Cancer, vol. 100, no. 10, pp. 1549–1557, 2009.
 D. Hanahan and R. A. Weinberg, “The hallmarks of cancer,” Cell, vol. 100, no. 1, pp. 57–70, 2000.
 Z. Chen, W. Lu, C. Garcia-Prieto, and P. Huang, “The Warburg eﬀect and its cancer therapeutic implications,” Journal of Bioenergetics and Biomembranes, vol. 39, no. 3, pp. 267–274, 2007.
 Y. Chen, R. Cairns, I. Papandreou, A. Koong, and N. C. Denko, “Oxygen consumption can regulate the growth of tumors, a new perspective on the Warburg eﬀect,” PLoS ONE,vol.4,no. 9, Article ID e7033, 2009.
A.AynsleyGreen,A.M.Weindling,G.Soltesz,andP. A. Jenkins, “Transient lactic acidosis and hyperalaninaemia associated with neonatal hyperinsulinaemic hypoglycaemia: the eﬀects of dichloroacetate (DCA),” European Journal of Pediatrics, vol. 141, no. 2, pp. 114–117, 1983.
 J.Y.Y.Wong,G.S.Huggins,M.Debidda,N.C.Munshi,and I. De Vivo, “Dichloroacetate induces apoptosis in endometrial cancer cells,” Gynecologic Oncology, vol. 109, no. 3, pp. 394– 402, 2008.
 W. Cao, S. Yacoub, K. T. Shiverick et al., “Dichloroacetate (DCA) sensitizes both wild-type and over expressing bcl-2 prostate cancer cells in vitro to radiation,” Prostate, vol. 68, no. 11, pp. 1223–1231, 2008.
 E. D. Michelakis, L. Webster, and J. R. Mackey, “Dichloroacetate (DCA) as a potential metabolic-targeting therapy for cancer,” British Journal of Cancer, vol. 99, no. 7, pp. 989–994, 2008.
 S. Bonnet, S. L. Archer, J. Allalunis-Turner et al., “A mitochondria-K+ channel axis is suppressed in cancer and its normalization promotes apoptosis and inhibits cancer growth,” Cancer Cell, vol. 11, no. 1, pp. 37–51, 2007.
 T. C. Chou and P. Talalay, “Analysis of combined drug eﬀects: a new look at a very old problem,” Trends in Pharmacological Sciences, vol. 4, pp. 450–454, 1983.
 T. C. Chou and P. Talalay, “Quantitative analysis of doseeﬀect relationships: the combined eﬀects of multiple drugs or enzyme inhibitors,” Advances in Enzyme Regulation,vol.22, pp. 27–55, 1984.
 D. B. Longley, D. P. Harkin, and P. G. Johnston, “5Fluorouracil: mechanisms of action and clinical strategies,” Nature Reviews Cancer, vol. 3, no. 5, pp. 330–338, 2003.
M.W.Saif,A.Choma,S.J.Salamone,andE.Chu,“Pharmacokinetically guided dose adjustment of 5-ﬂuorouracil: a rational approach to improving therapeutic outcomes,” Journal of the National Cancer Institute, vol. 101, no. 22, pp. 1543–1552, 2009.
 C.G.Leichman,K.Chansky,J.S.Macdonaldetal.,“Biochemical modulation of 5-ﬂuorouacil through dihydropyrimidine dehydrogenase inhibition: a Southwest Oncology Group phase IItrialofeniluraciland5-ﬂuorouracilinadvancedresistant colorectal cancer,” Investigational New Drugs,vol.20,no.4,pp. 419–424, 2002.
 F. A. Levi, R. Zidani, J. M. Vannetzel et al., “Chronomodulated versus ﬁxed-infusion-rate delivery of ambulatory chemotherapy with oxaliplatin, ﬂuorouracil, and folinic acid (leucovorin) in patients with colorectal cancer metastases: a randomized multi-institutional trial,” Journal of the National Cancer Institute, vol. 86, no. 21, pp. 1608–1617, 1994.
G.Melen-Mucha,E.Balcerczak,S.Mucha,M.Panczyk,S. Lipa, and M. Mirowski, “Expression of p65 gene in experimental colon cancer under the inﬂuence of 5-ﬂuorouracil given alone and in combination with hormonal modulation,” Neoplasma, vol. 51, no. 4, pp. 319–324, 2004.
 F. Richards II, L. D. Case, and D. R. White, “Combination chemotherapy (5-ﬂuorouracil, methyl-CCNU, mitomycin C) versus 5-ﬂuorouracil alone for advanced previously untreated colorectal carcinoma. A phase III study of the piedmont oncology association,” Journal of Clinical Oncology,vol.4,no. 4, pp. 565–570, 1986.
A.Aquino,S.P.Prete,J.W.Greineretal.,“Eﬀect of the combined treatment with 5-ﬂuorouracil, γ-interferon or folinic acid on carcinoembryonic antigen expression in colon cancer cells,” Clinical Cancer Research, vol. 4, no. 10, pp. 2473– 2481, 1998.
 S. Obi, H. Yoshida, R. Toune et al., “Combination therapy of intraarterial 5-ﬂuorouracil and systemic interferon-alpha for advanced hepatocellular carcinoma with portal venous invasion,” Cancer, vol. 106, no. 9, pp. 1990–1997, 2006.
 P. W. Stacpoole, “Review of the pharmacologic and therapeutic eﬀects of diisopropylammonium dichloroacetate (DIPA),” The Journal of Clinical Pharmacology, vol. 9, no. 5, pp. 282– 291, 1969.
 S. Bonnet, S. L. Archer, J. Allalunis-Turner et al., “A mitochondria-K+ channel axis is suppressed in cancer and its normalization promotes apoptosis and inhibits cancer growth,” Cancer Cell, vol. 11, no. 1, pp. 37–51, 2007.
 E.D.Michelakis,G.Sutendra,P.Dromparisetal.,“Metabolic modulation of glioblastoma with dichloroacetate,” Science Translational Medicine, vol. 2, no. 31, pp. 31–ra34, 2010.
 T. Fernandes-Alnemri, G. Litwack, and E. S. Alnemri, “CPP32, a novel human apoptotic protein with homology to Caenorhabditis elegans cell death protein Ced-3 and mammalian interleukin-1β-converting enzyme,” The Journal of Biological Chemistry, vol. 269, no. 49, pp. 30761–30764, 1994.
 H. Mehmet, “Caspases ﬁnd a new place to hide,” Nature,vol. 403, no. 6765, pp. 29–30, 2000.
 E. Yang and S. J. Korsmeyer, “Molecular thanatopsis: a discourse on the BCL2 family and cell death,” Blood, vol. 88, no. 2, pp. 386–401, 1996.
 D. R. Green and J. C. Reed, “Mitochondria and apoptosis,” Science, vol. 281, no. 5381, pp. 1309–1312, 1998.
 J. C. Reed, “Double identity for proteins of the Bcl-2 family,” Nature, vol. 387, no. 6635, pp. 773–776, 1997.
 B. Antonsson, S. Montessuit, B. Sanchez, and J. C. Martinou, “Bax is present as a high molecular weight oligomer/complex in the mitochondrial membrane of apoptotic cells,” The Journal of Biological Chemistry, vol. 276, no. 15, pp. 11615– 11623, 2001.