Down regulation of lactate dehydrogenase initiates apoptosis in HeLa and MCF-7 cancer cells through increased voltage-dependent anion channel protein and inhibition of BCL2

Malignant cells commonly use aerobic glycolysis for ATP production; this is known as the Warburg effect, where pyruvate is converted to lactate, by enzyme lactate dehydrogenase A (LDH-A). In this study, we have investigated the effect of inhibition of LDH-A on cells viability and identifying the mechanism of cell death in HeLa and MCF-7 cancer cells. Human cervical cancer HeLa cell line and breast cancer MCF-7 cell line were used to investigate the effect of inhibition of LDH-A by sodium oxamate on cell survival and proliferation using western blot, spectrophotometry, and immunofluorescent study. There was significant reduction in LDH-A (P < 0.001) and cell viability (P < 0.001) in a dose-dependent mode in both HeLa and MCF-7 SO-treated cancer cells. The voltage-dependent anion channel (VDAC) protein was significantly increased (P < 0.001) in association with decreased LDH-A. The proapoptotic proteins; cytochrome C (P < 0.001), BAX (P < 0.001), cleaved caspase-3 (P < 0.001), cleaved caspase-8 (P < 0.001), and cleaved caspase-9 (P < 0.001) were significantly increased in association with decreased LDH-A. While, the anti-apoptotic protein Bcl2 was significantly decreased (P < 0.001) in association with decreased LDH-A. We conclude that Inhibition of LDH-A can decrease cells viability through activation of intrinsic apoptotic pathway via increased VDAC protein and inhibition of Bcl2 as well as activation of the extrinsic apoptotic pathway through activation of caspase-8.


INTRODUCTION
Malignant cells commonly use aerobic glycolysis for ATP production; this is known as the Warburg effect [1]. Pyruvate is converted to lactate by enzyme lactate dehydrogenase A (LDH-A) where nicotinamide adenine dinucleotide (NAD+) is generated from NADH in this process [2]. The production of NAD + is required by glyceraldehyde 3-phosphate dehydrogenase to maintain glycolysis and ATP production [2]. LDH, which owns two subunits LDH-A and LDH-B, is an enzyme commonly existing in human cells [3]. LDH-A catalyzes the conversion of pyruvate to lactate with the liberation of NAD + , which possesses a vital role in glycolysis. It has long been noted that LDH-A expression is upregulated in human neoplastic tissues [3].
Recently, many studies have shown that LDH-A plays a vital role in maintaining tumor growth and progression [4]. Furthermore, relevant studies have demonstrated that inhibition of LDH-A induces oxidative stress and suppresses tumor growth in a variety of cancer cell lines [5,6].
Since aerobic glycolysis is the favored way of ATP production in cancer cells, it becomes a very attractive target for cancer therapies [7].
Sodium oxamate (SO) is a competitive inhibitor of LDH-A, hence, we use SO to block aerobic glycolysis and reduces the main energy source in cancer cells [8]. However, the detailed mechanism remains largely unclear. In this study we will investigate changes in apoptotic and oxidative stress pathways in association with the use of sodium oxamate in cervical (HeLa) and breast (MCF-

Research Paper
Oncotarget 924 www.oncotarget.com 7) cancer cell lines aiming in identifying mechanism of cancer cell death following SO treatment.

Effect of sodium oxamate on cell proliferation
The effect of SO on cell proliferation was determined by MTT assay (Figure 1). The proliferation of HeLa and MCF-7 cells was significantly inhibited by various concentration of SO (20-100 mmol/L). The inhibitory effect was observed after 24-hour of incubation. Figure 1 reveals treatment with SO can lead to significant inhibition of proliferation. The SO inhibitory effect was dose-related. The treatment of 60 mmol/L of SO revealed almost 40% cell inhibition on both HeLa and MCF-7 cells (Table 1). Hence, we have selected the 40 and 60 mmol/L concentrations of SO for further analysis. Treatment with SO has shown dose-related inhibition of cell growth of both HeLa cells and MCF-7 cells (Figures 1 and 2).

Changes in the activities of antioxidant enzymes SO reduces superoxide dismutase (SOD) activity
SO (40 and 60 mmol/L) treatment significantly reduced the activity of SOD in HeLa and MCF-7 cells, when compared with untreated control cells ( Figure 3). The two tested doses, 40 and 60 mmol/L of SO, significantly decreased SOD levels in both HeLa and MCF7 cancer cells, suggesting that SO increased reactive oxygen species (ROS) content in both HeLa and MCF7 cancer cells.

SO decreases reduced glutathione (GSH)
Reduced GSH is an important cellular antioxidant through donating thiol groups. There was a significant reduction of reduced GSH levels in SO treated HeLa and MCF7 cancer cells ( Figure 4). The two tested doses, 40 and 60 mmol/L of SO, significantly decreased GSH levels in both HeLa and MCF7 cancer cells, suggesting that SO increased ROS content in both HeLa and MCF7 cancer cells.

Changes in the LDH-A level
In this study, LDH-A was significantly decreased in SO-treated groups when compared to control cancer cells in both HeLa and MCF-7 ( Figure 5). We have noticed LDH-A reduction is even greater in 60 mmol/L compared to 40 mmol/L treated groups, suggesting a dose-response activity of SO on LDH-A. Accordingly, SO treatment significantly inhibited LDH-A activity in cancer cells, while ROS content was distinctly increased.

Correlation between the expression of LDH-A and VDAC
The effect of SO on the expression pattern of VDAC, LDH-A and pyruvate dehydrogenase (PDH) in HeLa and MCF-7 cells was analyzed by western blot. The expressions of VDAC was found to be significantly increased in 40 and 60 mmol SO-treated HeLa and MCF-7 cells when compared to control cells. Whereas, similar concentrations of SO had shown significant decrease in the expression of LDH-A and PDH when compared to control non-treated HeLa and MCF-7 cancer cells ( Figure 6).

DISCUSSION
Cancer cells produce a large amount of energy through glycolysis even in the presence of sufficient oxygen [1][2][3]. LDH-A, a key regulator of glycolysis, reversibly catalyzes the conversion of pyruvate to lactate. In this study, we show SO significantly reduces LDH-A in a dose-dependent mode.
We also show SO significantly inhibits the viability of human cervical (HeLa) and breast (MCF-7) cancer cells in a dose-dependent fashion.
Increased levels of LDH-A have been reported in several types of cancers [9][10][11], and their levels of expression have been correlated with clinical stages in esophageal, pancreatic, prostate carcinomas; suggesting that overexpression of LDH-A has proliferative role on cancer cells. These studies [9][10][11] further support the concept that therapies targeting LDH-A may provide useful strategies in controlling cancer progression. Our immunofluorescent staining results have also shown a decrease in the expression of growth signals markers; p-NFK-B, BMI-1, VEGF and ki-67 in SO treated HeLa cells and MCF-7 cells suggesting an association with the decreased LDH-A in these cells.
In addition, we have shown treatment with SO significantly augments ROS production leading to a significant reduction in SOD and GSH levels in SO-treated cancer cells suggesting that inhibition of LDH-A plays an important role in the induction of ROS. Zhao Z et al. have also shown that SO treatment significantly augments ROS production [12]. Reports have shown increased ROS can induce apoptosis [13][14][15].
We have also shown SO significantly increases proapoptotic proteins BAX and cytochrome c. In addition,  there is significant increase in the initiating active caspases; cleaved caspase 8 and 9 and terminal executer cleaved caspase 3 which is accompanied by significant reduction in antiapoptotic protein Bcl2. These findings support apoptotic role of SO. Chandan et al. have shown inhibition of LDH-A can lead to apoptosis, which supports our finding [16].
Interestingly, we also show SO-treated cancer cells have significant increase in VDAC protein. VDAC protein has also been shown to play an important role in apoptosis  Oncotarget 928 www.oncotarget.com [17]. Following the initiation of apoptotic signals there is increased permeability of VDAC, which allows the release of proapoptotic proteins such as cytochrome c from the mitochondria. Although cytochrome c plays a crucial role in oxidative phosphorylation within the mitochondria, in the cytosol it activates caspases, which play a major role in programed cell death [18]. While the mechanism of VDAC-enabled cytochrome c release has not yet been fully understood, there have been reports suggest that oligomerization between individual subunits can create  a large stretchy pores through which cytochrome c can pass [19]. Remarkably, the release of cytochrome c is also regulated by Bax, which interacts directly with VDAC to increase pore size and promote cytochrome c release, whereas anti-apoptotic Bcl2 produces the opposite effect [20]. In fact, it has been shown that antibodies which are capable of inhibiting VDAC also interfere with Baxmediated cytochrome c release [21].
Zalk et al. have suggested that VDAC overexpression can lead to increased cell death since higher VDAC expression favors VDAC oligomerization [19]. Moreover, it has been advocated that oligomeric VDAC mediates the release of cytochrome c [19], because the internal diameter of a single VDAC pore is 2.5-3.0 nm, which is insufficient to pass a folded protein [22]. The increased VDAC has been shown to assemble to form polymers that increases the diameter of VDAC and allows the release of cytochrome c [22]. Reports have shown upregulation of Bcl2 prevents VDAC oligomerization and then apoptosis [23,24].
Our study has shown for the first time a significant dose-related increase in VDAC protein in SO-treated HeLa and MCF7 cells as compared with control cancer cells; suggesting SO has a crucial effect on VDAC production Oncotarget 930 www.oncotarget.com either directly or through the increase in free radicals or a decrease in LDH-A.
The increase in VDAC can lead to increase in cell death through activation of the intrinsic pathway of apoptosis by enhancing the release of cytochrome c and inactivation of Bcl2, which may be the main mechanism of apoptosis in our study [19].
We have also shown a significant increase in cleaved caspase-8 suggesting activation of extrinsic pathway of apoptosis in SO-treated HeLa and MCF7 cancer cells. There was no previous reports on activation of extrinsic pathway of apoptosis in association with SO-treated cancer cells. It is possible that the activation of caspase-8 is related to increase in free radical production associated with SO treatment. This is supported by other studies, which also show activation of caspase-8 by oxidative stress [25,26].
In conclusion, the Inhibition of LDH-A can decrease cells viability through activation of intrinsic apoptotic pathway via increased VDAC protein and inhibition of Bcl2 as well as activation of the extrinsic apoptotic pathway through activation of caspase-8.

Chemicals
Sodium oxamate was supplied by Sigma-Aldrich (St. Louis, MO, USA). All other chemicals were procured from Merck (analytical grade).

Cell culture
The human cervical cancer cell line (HeLa) and breast cancer cell line (MCF-7) were obtained Oncotarget 932 www.oncotarget.com from ATCC, Rockville, MD, USA. Entire cell culture experiments were conducted using pre-sterile consumables and followed aseptic techniques already in place. The HeLa cell was grown as a monolayer in Dulbecco's Modified Eagle's Medium (DMEM) and the MCF-7 cell was grown as a monolayer in MEM with 10% FBS, 200 mM L-glutamine, 10,000 U/mL penicillin, and 10 mg/mL streptomycin at 37°C in 5% CO 2 . Cells were harvested by trypsinization once 80% confluency was achieved during subculture. Stocks were maintained in 25 cm 2 tissue culture flasks.

MTT (3-(4,5-dimethylthiazol-2-yl)-2,5diphenyltetrazolium bromide) assay
The MTT assay, which detects mitochondrial dehydrogenase activity in living cells, was used to assess cytotoxicity [27]. Briefly, cells (5 × 10 3 cells/well) plated in 96-well flat-bottom plates were exposed with an increasing range of concentration of SO. After incubation, 10 μl of MTT (5 mg/ml) in PBS were added to each well and allowed to develop color. DMSO was added to stop the reaction and the absorbance at 570 nm was read using Tecan infinite microplate reader.

Cell treatments
Both HeLa and MCF-7 cells were treated with SO in different concentrations and incubated at 37°C in 5% CO 2 incubator. After 24 h incubation, the cells were harvested by trypsinization for further experiments.

Biochemical estimations
The cells were harvested by trypsinization. The obtained cell pellet was suspended in PBS and that suspension was used for biochemical estimations. Superoxide dismutase (SOD) was assayed by the method of Kakkar et al. [27]. Nicotinamide adenine dinucleotide (NADH), phenazine methosulphate, and nitroblue tetrazolium formazon formation are all inhibited in this assay. The reaction was initiated by the addition of NADH. For a set period of time, a known amount of enzyme preparation was permitted to react with H 2 O 2 in the presence of glutathione (GSH). The GSH material that remained after the reaction was then calculated. The total GSH content was measured based on the development of a yellow color when 5,5′-dithiobis-2-nitrobenzoic acid was added to compound containing sulfhydryl groups [28].

Lactate dehydrogenase A activity
LDH-A Assay kit (Sigma, USA) was used to determine the intracellular LDHA activity. In this test, LDH-A reduces NAD to NADH, which interacts with a specific probe to produce a color (λ max = 450 nm), LDH-A level was measured using enzymatic colorimetric methods on Roche/Hitachi Cobas C systems (Integra 400 Plus, Germany). Results were expressed as percentage of LDH-A activity normalized to protein concentration.

Immunofluorescent staining
Both HeLa cells and MCF7 cells were grown on coverslips in 6-well flat-bottom plates as non-treated wells and SO 60 mmol treated wells. Following the attachment of the cells to the coverslips and become confluent, treatment with SO 60 mmol will be started for 24 hours followed by washing the coverslips with PBS for 3 times then appropriate primary antibodies (anti-Bax antibody antibody (mouse monoclonal, 1:50, DAKO, Agilent, Santa Clara, CA, USA), will be added to the treated and untreated wells and kept for 24 hours. For double labeling a second primary antibody will be added for another 24 hour followed by washing. Then coverslips will be washed with PBS for 3 times and then Alexa Fluor 488 labeled donkey anti-rabbit antibody will be added for one hour followed by washing with PBS for 3 times. For double labeling Alexa Fluor 488 labeled donkey anti-rabbit antibody will be added for one hour followed by washing with PBS for 3 times and then Rhodamine labeled donkey anti-mouse antibody will be added followed by washing with PBS for 3 times. Afterward, coverslips will be mounted on slides using DAPI IF mounting media.

Statistical analysis
The statistical analyses were performed using GraphPad Prism Software version 5. The comparisons between the various groups were achieved by one-way analysis of variance (ANOVA), followed by Newman-Keuls multiple range tests. Data were presented in mean ± standard error (S.E). P values < 0.05 were considered significant.

Author contributions
All authors made a significant contribution in the scientific quality of the paper, fulfilling the ICMJE criteria. All authors approved the submitted version of the manuscript and agreed to be accountable for all aspects of