The Combined Effect of Dichloroacetate and 3- Bromopyruvate in Colorectal Cancer Cell Line, HT- 29; The Mitochondrial Pathway Apoptosis

Hojatolla Nikravesh Ahvaz Jundishapur University of Medical Sciences Mohammad Javad Khodayar Ahvaz Jundishapur University of Medical Sciences Babak Behmanesh Ahvaz Jundishapur University of Medical Sciences Masoud Mahdavinia (  mahdavimasoud@yahoo.com ) Ahvaz Jundishapur University of Medical Sciences Ali Teimoori Hamadan University of Medical Sciences Soheila Alboghobeish Ahvaz Jundishapur University of Medical Sciences Leila Zeidooni Ahvaz Jundishapur University of Medical Sciences


Abstract
Background 5-Fluorouracil (5-FU) is regarded as the rst line treatment for colorectal cancer; however, its effectiveness is limited by drug resistance. The ultimate goal of cancer therapy is induction of cancer cell death to achieve an effective outcome with minimal side effects. The present work aimed to assess the anticancer activities of mitocans which can be considered as an effective anticancer drug due to high speci city in targeting cancer cells.
Results treatment with mitocans (3Br-P + DCA) inhibited the growth of HT29. Moreover, 3Br-P + DCA signi cantly induced apoptosis and necrosis, activation of caspase 3 activity, depolarize the mitochondrial membrane potential, and ROS production. At a molecular level, 3Br-P + DCA treatment remarkably down-regulated the expression of Bcl-2, while up-regulated the expression of Bax. Conclusion mitocans, in particular the combined drug, 3Br-P + DCA, could be regarded and more evaluated as a safe and effective compound for CRC treatment. Targeting hexokinase and pyruvate dehydrogenase kinase enzymes may be an option to overcome 5-FU -mediated chemo-resistant in colorectal cancer.

Background
Cancer, a pathology characterized by unchecked division of cells, is one of the fatal diseases with an increasing number of cases all over the world (1). Malignant neoplasms with the source of the intestine, rectum and anal canal, classi ed as colorectal cancer (CRC), are the third and second leading causes of death in males and females in the world, respectively (2). Approximately, up to 1.8 million new cases of CRC were reported to occur yearly in the world, with an estimated 881000 cases dying of this malignancy (3). Despite substantial progresses in CRC screening measures and treatment, the prognosis of patients with this cancer remains poor, demanding novel therapeutic strategies to manage the disease (4). In this respect, 5-Fluorouracil (5-FU) is a well-known anti-cancer drug commonly used to treat CRC; the agent interferes with DNA and RNA to disrupt the metabolism of the uracil, which lead to the breakdown of DNA and apoptosis of the cancer cells. Nonetheless, it is reported that the resistance to 5-FU during the course of CRC treatment plays a major role in treatment failure (5)(6)(7). Therefore, the expansion and development of new and more effective drug combinations for CRC treatment is guaranteed. Studies show that some anti-cancer agents, known as mitocans, which exert their anti-tumor activities by modulating mitochondria, can be considered as an effective anti-cancer drug due to high speci city in targeting cancer cells (8). Dichloroacetate (DCA) and 3-bromopyruvate (3Br-P) are two such anti-tumor agents, which belong to class 1 (hexokinase inhibitors) and class 7 (Krebs cycle inhibitors) of mitocans, respectively (8). DCA activates pyruvate dehydrogenase by inhibiting the pyruvate dehydrogenase kinase enzyme, which ultimately boosts the production of Acetyl-CoA, as a precursor of the Krebs cycle. The enhancement of the Krebs' cycle activity in turn increases the activity of the electron transfer chain; a function that increases the production of reactive oxygen species with mitochondrial origin, which lead to selective killing of cancer cells (9). The another agent, 3Br-P, acts as a potent inhibitor of hexokinase; an enzyme that is overexpressed in cancer cells and is essential for the metabolism of cancer cells. The enzyme binds to voltage-dependent anion channel (VDAC, or porin) in the outer mitochondrial membrane, which results in resistance to chemotherapy by inhibiting glycolysis (10). Recent studies reported that multi-target therapy acts more effective than single-target one (11). Numerous studies have shown a synergistic action of DCA or 3Br-P with other compounds (12) (13). However, the present work assessed the anticancer effects of 3Br-P in combined with DCA in a colorectal cancer cell line, HT-29.

Cell culture and treatment
The cell lines, HT29 and HEK-293, were cultured in DMEM media with 10% FBS at 37°C, under a humidi ed atmosphere with 5% CO2.

Cell counting
The trypan blue exclusion dye assay was applied to determine the number of living cells in a cell suspension. Shortly, 20 μl of trypan blue dye 0.4% was added and mixed with equal parts of cell suspension in a micro tube. Subsequently, one side of a hemacytometer counter was lled with the cell suspension by placing the tip of the pipette at the notch, followed by counting the cells on the stage of a light microscope. The Calculation of cell percentage per milliliter was done via the following formula: cells/ml = average count per square × dilution factor × 10 4 . Moreover, the experiments were performed at least for three times (14).

Cytotoxicity assays
Brie y, the HT-29 and HEK-293 cells were grown in 96-well plate (5000cells/well) in 100µl medium and left overnight to adhere. Then, cells were treated with different concentrations of 3Br-P, DCA and 5-FU alone and combined with together, and incubate in media containing 10% FBS at 37°C and 5 -6.5% CO2 for 48 hours. After treatment, 10 μl of the MTT (5 mg/ml in PBS) was added to each well and incubated for 3.5 hours in 37 °C. Subsequently, the medium was removed and the formazan crystals were solubilized in 150µl of DMSO. Plates were covered with foil and shacked on an orbital shaker for 15 minutes, and in the nal step complete solubilization of the purple formazan crystals and reduced MTT were measured spectrophotometrically by microplate enzyme-linked immunosorbent assay (ELISA) reader at 570 nm according to the lters available for the ELISA reader. Viability inhibition was calculated as follows: cell viability (%) = Ab (test) /Ab (control) ×100 (15).

Measurement of apoptosis and necrosis
This was measured according to ApopNexin™ FITC/PI Kit instructions. In summary, 1.5×10 5 cells were seeded and treated with different concentrations of 3Br-P, DCA and 5-FU alone and combined together in HT-29 cells for 48 hours. After drug exposure, the cells were harvested, and then were washed with icecold PBS and stained with ApopNexin TM FITC kit for 15 minutes at room temperature in the dark. Stained samples were analyzed by Galaxy ow cytometer (ser. No: 0105362) (16).

Caspase 3 Assay
Caspase 3 activity was determined via using the speci c caspase-3 substrate, prepared by Caspase-3 Assay Kit, Colorimetric Qiagen (Hilden, Germany). In short, after treating the cells with 3Br-P, DCA and 5-FU alone and/or combined together, the caspase-3 activity was measured by ELISA at 504 nm, according to the protocol of the kit (17).

Mitochondrial membrane potential (ΔΨm)
MitoTracker Green is a uorescent color that selectively attaches covalently to mitochondrial proteins in the mitochondrial matrix by reacting with free thiol groups of cysteine roots. Brie y, in each well of 24-well plates, about 10 5 cells were seeded and incubated overnight, and subsequently treated with 3Br-P, DCA and 5-FU alone and/or combined together for 48 hours. Then, the cells were harvested and incubated with 10 µl of MitoTracker Green for 30 minutes at 37°C in a 5% CO2 incubator. After incubation, the samples were placed on ice and immediately were assessed by ow cytometry (18).

Determination of ROS production
To perform this, 24-well plates were seeded with 10 5 cells in a complete culture medium and allowed to adhere overnight. In all wells (90% con uence) treated with 3Br-P, DCA and 5-FU alone and/or combined together, cells were washed twice with PBS, followed by staining with 10 µM DCFH-DA for 45 min. Then, after 2, 4, 8, and 12 hours, the formation of uorescent-oxidized DCF was measured by uorimetry (535 nm excitation and 635 nm emission) (19). The fold change of gene expression was calculated using 2 −ΔΔCt after normalizing to the expression level of GAPDH.

Statistical analysis
All experiments were performed at least through 3 trials. All quantitative data are expressed as mean ± SD. One-way analysis of variance (ANOVA) followed by Tukey's multiple comparisons were executed for comparison of different parameters between the groups using a GraphPad Prism 5 software (GraphPad Prism, San Diego, California, The U.S.A.).

In vitro ROS activity
The ROS activity in the treated group was shown to be signi cantly higher than the untreated ones, at the times of 2, 4 and 8 hours after treatment. Furthermore, among the mentioned times in the treated group, the ROS amount was found to be higher during 4 hours after treatment. Moreover, the combinations of 5-FU+3Br-P and 3Br-P+DCA in the treated HT-29 cells produced more ROS amount than the other compounds ( Figure 2).
In vitro assessment of mitochondrial membrane potential (ΔΨm) Based on our results, the compound 3Br-P signi cantly promoted ΔΨm reduction in HT-29 cells in comparison with the negative control and other treated groups. These results show that the combined agents, including 5-FU+3Br-P, 5-FU+ DCA, and 3Br-P+DCA signi cantly increased ΔΨm decrease in HT-29 cells in compared to the negative control (Figure 3, 4, and 5).

Induction of apoptosis and necrosis by ow cytometry
Annexin-V FITC/PI staining con rmed that 5-FU, 3Br-P, DCA, 5-FU+DCA, 5-FU+3Br-P, and 3Br-P+DCA could induce apoptosis in HT-29 cells. All the treated HT-29 cells had a signi cant higher necrotic pattern as compared to untreated cells (p≤ 0.05). It was found that the treatment with 5-FU signi cantly increases the percentage of apoptosis from 0.465% in the control cells to 32.35% in the treating cells. Moreover, the 5-FU+DCA, 5-FU+3Br-P, and 3Br-P+DCA groups had a signi cantly increased apoptosis in compared to the control, 3Br-P and DCA groups (Figure 8, 9 and 10).

Discussion
Globally, CRC is the second and third leading causes of death in women and men, respectively (20). The aim of cancer therapy is to inhibit proliferation (cytostatic effects) and to induce cell death (cytotoxic effects) of cancer cells (21), to achieve an effective outcome with minimal side effects (23). In most cancers, the rst therapeutic strategy used to treat is surgical removal. Obviously, surgery for some types of cancers is relatively simple and for other types is impractical (22). Therefore, chemotherapy and radiotherapy are used to control or eradicate metastatic cells. Traditional chemotherapy is the use of drugs to target the cell cycle, DNA, RNA, and proteins in cancer cells. 5-FU is the rst traditional chemotherapy and the rst choice for CRC therapy, whose effectiveness is limited by drug resistance (23). Mitochondria have emerged as an attractive target for anticancer drugs, so that the compounds such as mitocans that target mitochondria with anti-cancer activity has become a focus of recent research due to their great clinical potential (24) (25). In our study, we have described the cytotoxic effect of the mitochondria-targeted drugs, 3Br-P + DCA, in HT-29 cell line and assessed the mechanism of its action.
Recent studies have shown that therapies having multiple targets result in greater bene ts than singletargeted therapies (26). There are several studies suggesting a synergistic action of 3Br-P or DCA with other compounds. For instance, the selective inhibitor of glycolytic enzymes, 3Br-P, when combined with sodium citrate (SCT), resulted in reduced ATP level, induced mitochondrial-mediated apoptosis, and suppressed tumor growth in gastric cancer cells (27). In the same way, when cisplatin or oxaliplatin combined with 3Br-P, amazingly boosted the antiproliferative effects of the platinum drugs in HCT116 cells (28). Moreover, antitumor effects of tamoxifen, the rst-line adjuvant endocrine therapy for estrogen receptor-positive breast cancer, were markedly improved when used with 3Br-P, as a sensitizer to target glycolysis (29). On the other hand, in colorectal cancer cells parallel results were obtained using traditional chemotherapy drugs with DCA. Ayyanathan et al. demonstrated that the combination of DCA and sulindac augments the selective killing of A549 and SCC25 cancer cells via ROS production, mitochondrial dysfunction, and apoptosis (30). Similarly, Florio et al., reported that the combination of three metabolic drugs, DCA, GW6471, and metformin resulted in a remarkable enhanced ROS production, modulated cell cycle progression, and promoted apoptosis in PGL cells (31). These ndings are in line with the results reported in our study. In the present study, we found that DCA and 3Br-P together or in combined with 5-FU signi cantly reduced cell viability and proliferation of HT-29 cell lines (Fig. 8). Apoptosis plays an important role in treatment of cancer, as it is a popular target of many treatment strategies (32). Accordingly, we showed that the combined drug therapy, DCA + 3Br-P, promotes apoptosis more effectively than the single therapies in HT-29 cell lines (Fig. 3); a mechanism that may be induced by modulating pathways such as ROS-dependent apoptosis in HT-29 cell lines. In the mitochondrial (caspase-dependent intrinsic) pathway that induces apoptosis, caspases, in particular caspase-3, are crucial mediators for the apoptotic machinery in many cell types (33). This pathway is controlled by B-cell lymphoma-2 (BCL2) family proteins, composed of members that either promote or inhibit apoptosis. A major checkpoint in cellular fate is a balance formed between pro-apoptotic (Bax) and anti-apoptotic (Bcl-2) members (34). Increased Bax/Bcl-2 ratio up-regulates caspase-3, which leads to apoptosis of tumor cells (35). In the current study, we evaluated caspase 3 release, Bax/Bcl-2 ratio expression and mitochondrial membrane potential (ΔΨm). The results demonstrated that the three mentioned parameters increased by the combined drug therapy (DCA + 3Br-P) in comparison to single drug therapy. As a result, more studies are needed to investigate the mechanisms underlying the anticancer effect of combined 3Br-P + DCA and a combination of other mitocans treatment on CRC cells (Fig. 11).

Conclusion
We have indicated that a combination of 3Br-P + DCA exerts an antitumor effect in vitro by activation of mitochondria-dependent apoptosis. The rst goal of target therapy is to ght cancer cells with more precision and fewer side effects potentially. Targeted cancer therapies were designed to target a speci c molecule that is critical for the growth and expansion of cancer cell, hence, blocking these molecules can kill cancer cells without any harm to normal cells. According to the mitocans features, we can consider them as apoptosis inducers. However, the results of the combination of 3Br-P + DCA in this study have no signi cant difference with the 5-FU alone; however, we should never ignore the primary goal of this study to show that combination therapy has lower side effects and resistance to treatment.    Mitochondrial membrane potential changes in colorectal cancer cell line (HT-29) measured via a uorescence microscope (20X) 48h after exposure to 3Br-P, DCA, and 5-FU alone and/or combined together.   Effect of 3Br-P, DCA, and 5-FU alone and/or combined together on necrosis in HT-29 cell line after 48 h. Data presented as Mean ± SEM. a: (p 0.05) designates signi cant difference with control, b: (p 0.05) designates signi cant difference with 5-FU, 3Br-P, and DCA.