Warburg effect in chemosensitivity: Targeting lactate dehydrogenase-A re-sensitizes Taxol-resistant cancer cells to Taxol

Breast cancer cells MDA-MB-435 (MDA-435), MDA-MB-231 (MDA-231), MCF7 and BT474 were purchased from American Type Culture Collection (ATCC). 435TR1 and 435TRP cells are Taxol-resistant single clone or pooled clones, which were developed from parental MDA-435 cells by treated with gradually increasing concentrations of Taxol in cell culture medium. MDA-231 cell line with stable knockdown of LDH-A was constructed through transfection of MSCV-based retroviral vector (MSCV/LTRmiR30-PIG). All of these cells were cultured in DMEM/F-12 (Mediatech Inc.) and supplemented with 10% FBS and Penicillin/Streptomycin.

The cells were seeded in 6-well plates at 3 × 10⁵ cells per well in duplicate. After 12 hr incubation, cells were treated with or without 20 nM Taxol for 24 hrs, with untreated cells serving as controls. The cells were washed twice with PBS and then fixed with methanol/acetone (1:1), subsequently stained with 4',6-diamidino-2-phenylindole (DAPI) in order to visualize the morphology of cell nucleus. The morphology of cells was observed with the fluorescence microscope.

The cancer cells were treated with 20 nM Taxol for 48 hrs. Two methods were used to detect apoptosis. 1) The early stage of apoptosis was detected by Annexin V/propidium iodide staining with the Apoptosis Detection Kit (BD PharMingen). Briefly, aliquots of 10⁵ Taxol-treated cells were incubated with Annexin V/propidium iodide for 15 min at room temperature. The cells were then analyzed by flow cytometry (BD LSR II). 2) The late stage of apoptosis was detected by Cell Death Detection ELISA PLUS kit (Roche) according to the manufacturer's instruction.

Cells were harvested and lysed in a buffer containing 50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 2 mM EDTA, 1% Triton, 1 mM PMSF and Protease Inhibitor Cocktail (Sigma) for 20 min on ice. Lysates were cleared by centrifugation at 14,000 rpm at 4°C for 10 min. Supernatants were collected and protein concentrations were determined by the Bradford assay (Bio-rad). The proteins were then separated with a SDS/polyacrylamide gel and transferred to a Nitrocellulose membrane (Bio-rad). After blocking in PBS with 5% non-fat dry milk for 1 hr, the membranes were incubated overnight at 4-8°C with the primary antibodies in PBS with 5% non-fat dry milk. The following antibodies were utilized: anti-LDHA rabbit antibody (1:1000, Cell Signaling); anti-PARP rabbit antibody (1:1000, Cell Signaling), anti-cleaved PARP Rabbit antibody (1:1000, Cell Signaling), anti-Bcl2 rabbit antibody (1:1000, Cell Signaling), anti-Bcl-XL rabbit monoclonal antibody (1:1000, Cell Signaling), anti-Cdc2 mouse monoclonal antibody (1:1000, Cell Signaling),, anti-p-Cdc2(Y15) rabbit monoclonal antibody (1:1000, Cell Signaling), and anti-β-actin monoclonal antibody (1:2000, Sigma). Membranes were extensively washed with PBS and incubated with horseradish peroxidase conjugated secondary anti-mouse antibody or anti-rabbit antibody (1:2,000, Bio-rad). After additional washes with PBS, antigen-antibody complexes were visualized with the enhanced chemiluminescence kit (Pierce).

The total LDH activity in cell lysates was examined according to the manufacturer's instructions of the LDH-cytotoxicity assay kit (BioVision). Briefly, 2 × 10⁵ cells were seeded in a 24-well plate one day before assaying and all samples were analyzed in triplicate. Then cells were collected, washed and extracted for protein to measure LDH activity. Results were normalized based upon total protein.

siRNA oligonucleotides for LDH-A was purchased from Sigma, with a scrambled siRNA (Sigma) used as a control. Transfection was performed using the Oligofectamine Transfection reagent (Invitrogen) according to the manufacturer's protocol. Forty-eight hours after transfection, whole-cell lysates were prepared for further analysis by Western blot, LDH activity and Taxol cytotoxicity assay.

A total of 5 × 10³ ~ 1 × 10⁴ cells/well were seeded in 96-well plates. Twenty-four hours later, the medium was replaced with fresh medium with or without Taxol and incubated for 24 or 48 hrs, respectively. Taxol in combination with various concentrations of oxamate were also used to treat the cells in order to investigate the effect of drug combinations. Cell viability was determined by two methods. 1) Using CellTiter 96 Aqueous One Solution Cell Proliferation Assay (Promega) according to the manufacturer's protocol; 2) by Typan Blue staining and direct cell counting using hematocytometer.

The unpaired Student's t-test was used for the data analysis. All data were shown as mean ± standard error (SE). A statistical difference of P < 0.05 was considered significant.

MDA-435 cells were treated with gradually increasing concentrations of Taxol in cell culture medium for selection of Taxol-resistant cells. After successive Taxol treatments for duration of 3 months, several resistant cell clones were developed from the MDA-435 cell line. Taxol-resistant clone 1 (435TR1) and Taxol-resistant pooled clones (435TRP) were used for all subsequent experiments in this study.

To compare the survival capacity of both Taxol-sensitive and Taxol-resistant cells, MDA-435, 435TR1 and 435TRP cells were treated with 20 nM Taxol for 24 hrs. Taxol-sensitive MDA-435 cells showed cell rounding and blebbing with empty spaces visualized within the cells. This suggested that a large portion of these cells were arrested in G2/M phase, with some of these cells undergoing apoptosis. However, no obvious morphological change was observed in Taxol-resistant 435TR1 and 435TRP cells (Fig. 1A). Early stage apoptosis was examined by flow cytometry analysis after staining with Annexin V/propidium iodide, and late stage apoptosis was detected by a Cell Death Detection ELISA PLUS kit, which examines the DNA fragmentation in the apoptotic cells. Both assays detected a smaller percentage of apoptotic cells in Taxol-resistant 435TR1 and 435TRP, compared to their parental MDA-435 cells after treatment with 20 nM Taxol for 48 hrs (Fig. 1B). The protein expression of the cleaved Poly (ADP-ribose) polymerase (c-PARP), an important marker of caspase-mediated apoptosis [19, 20], was also examined by Western blotting after the cells were treated with 20 nM Taxol for 48 hrs. We found much lower levels of cleaved PARP and correspondingly much higher levels of un-cleaved PARP in Taxol-resistant 435TR1 and 435TRP cells, compared to parental MDA-435 cells (Fig. 1C). Cell viability assay showed that 435TR1 and 435TRP cells could tolerate much higher concentrations of Taxol compared to MDA-435 cells, with their IC50 concentrations found to be more than 30-fold higher than those of MDA-435 cells (Fig. 1D).

To examine the role of LDH-A in mediating Taxol resistance in human breast cancer cells, the expression of LDH-A was examined in MDA-435, 435TR1 and 435TRP cells. We found that LDH-A levels were markedly increased in 435TR1 and 435TRP cells, compared to their parental MDA-435 cells (Fig. 2A). The activity of LDH was also increased about 2-fold in Taxol-resistant 435TR1 and 435TRP cells, compared to MDA-435 cells (Fig. 2B). These results indicated that Taxol resistance is correlated with the increased LDH-A expression and activity. Interestingly, treatment with Taxol resulted in the induction of LDH-A expression in a dose-dependent pattern in MDA-435 cells (Fig. 2C). We also identified that LDH activity could also be induced by Taxol in the Taxol-resistant cells (data not shown). To study the mechanism that may contribute to the increased expression and activity of LDH-A, MDA-435 cells were treated with CHX to block protein synthesis and the cells were further treated with or without Taxol for different times, the protein stability of LDH-A was measured by Western blot (Fig. 2D). The result showed that LDH-A protein is more stable in Taxol treated cells than that of untreated cells. We further compared the mRNA level of LDH-A in Taxol-treated and -untreated cells by qRT-PCR (Fig. 2E). The result showed that Taxol treatment increased the mRNA expression of LDH-A. These results suggest that both protein stability and mRNA induction by Taxol contribute to the up-regulation of LDH-A in these cells.

The increase of LDH-A expression and LDH activity detected in Taxol-resistant cells suggests that LDH-A may play a critical role in Taxol resistance. Therefore, the effect of LDH-A downregulation on the sensitivity of Taxol was investigated. After LDH-A was downregulated efficiently by specific siRNA to LDH-A (Fig. 3A), LDH activity was decreased about 40% in MDA-435 cells and about 55% in 435TR1 cells (Fig. 3B). Since the expression and activity of LDH-A was upregulated in Taxol-resistant cancer cells (Fig. 2), we hypothesized that the downregulation of LDH-A by siRNA might re-sensitize Taxol-resistant cells to Taxol. To this end, LDH-A was knocked down with siRNA in 435TR1 and parental MDA-435 cells respectively, and then the cells were treated with different concentrations of Taxol. The downregulation of LDH-A increased the sensitivity of these cells to Taxol, with Taxol-resistant 435TR1 cells showing about a 3-10 fold increase in cell growth inhibition under 50-100 nM Taxol treatment measured by both MTS assay (Fig. 3C) and direct cell counting (Additional file 1, Figure. S1). Interestingly, 435TR1 cells showed a much greater overall increased sensitivity to Taxol compared to their parental MDA-435 cells (Fig. 3C and 3D). Similar assays were performed in another breast cancer cell line BT474 (Fig. 4A-C), where the knockdown of LDH-A expression by siRNA increased the sensitivity to Taxol by at least 2-fold. To further confirm these results, MDA-231 cells with stable knockdown of LDH-A by short-hairpin RNA (shRNA) were used. Compared to those of control MDA-231 cells, LDH-A expression (Fig. 4D) and LDH activity (Fig. 4E) were dramatically decreased in LDH-A stably knockdown cells and these cells showed a much greater overall increased sensitivity to Taxol (Fig. 4F). These results demonstrated that LDH-A plays an important role in Taxol resistance. Since LDH is a critical enzyme in the glycolytic pathway, our results suggest that inhibition of glycolysis may re-sensitize Taxol-resistant cells to Taxol.

Oxamate is a pyruvate analog that directly inhibits the converting process of pyruvate to lactate by LDH, therefore, inhibits cell glycolysis [21]. We first examined the effect of oxamate on LDH activity and cell viability of MDA-435 and 435TR1 cells. Oxamate treatment led to a decrease of LDH activity (Fig. 5A) and an inhibition of cell viability (Fig. 5B) in a dose-dependant manner, in both MDA-435 and 435TR1 cells. Compared to MDA-435 cells, Taxol resistant 435TR1 cells showed a greater sensitivity to oxamate, consistent with the results of LDH-A knockdown by siRNA (Fig. 3). Since glycolysis and mitochondrial oxidative phosphorylation are linked processes [16], and we have previously shown that LDH-A is critical in regulating glycolysis and growth of breast cancer cells [18], we reasoned that the increased expression and activity of LDH-A in Taxol-resistant cells may lead to an increase of glycolysis and a decrease of mitochondrial oxidative phosphorylation. Thus, a specific inhibitor of the mitochondrial oxidative phosphorylation, oligomycin was utilized to treat these cells. As expected, Taxol-resistant 435TR1 cells were more resistant to oligomycin (Additional file 2, Figure. S2). These results further support the notion that increased Taxol sensitivity by oxamate is a consequence of the inhibition of cellular glycolysis.

Since downregulation of LDH-A by siRNA or oxamate significantly inhibited the viability of the Taxol-resistant cells, we further investigated the effects of combining Taxol with glycolysis inhibitor oxamate on Taxol-resistant breast cancer cells. In both Taxol-resistant 435TRP and 435TR1 cells (Fig. 6A and 6B; Additional file 3, Figure. S3), and in BT474 cells (Fig. 6C), Taxol combined with oxamate were much more effective in inhibiting cell viability compared with either agent given alone. Similar treatment combinations were performed in another breast cancer cell line, MCF7, with similar results obtained (Additional file 4, Figure. S4). Taken together, the combination of Taxol with oxamate has a greater capacity to inhibit Taxol-resistant cells compared to either agent given alone.

To further investigate the mechanism of oxamate-induced Taxol re-sensitization, we examined cellular apoptosis in these cells. PARP, a nuclear protein that can be easily cleaved by caspases, has been widely used as an apoptosis marker [19, 20]. The expression level of total PARP and cleaved PARP (c-PARP) were examined in 435TR1 cells after treatment with Taxol, oxamate, or their combination for 48 hrs, respectively. We found a significant increase of the levels of cleaved PARP after treatment with the combination of Taxol and oxamate compared to treatment with single agent (Fig. 6D). This indicates that cellular apoptosis is a mechanism involved in the increased cell growth inhibitory effect of the combination treatment of Taxol with oxamate.