Warburg effect in chemosensitivity: targeting lactate dehydrogenase-A re-sensitizes taxol-resistant cancer cells to taxol

Abstract

Background: Taxol is one of the most effective chemotherapeutic agents for the treatment of patients with breast cancer. Despite impressive clinical responses initially, the majority of patients eventually develop resistance to Taxol. Lactate dehydrogenase-A (LDH-A) is one of the predominant isoforms of LDH expressed in breast tissue, which controls the conversion of pyruvate to lactate and plays an important role in glucose metabolism. In this study we investigated the role of LDH-A in mediating Taxol resistance in human breast cancer cells.

Results: Taxol-resistant subclones, derived from the cancer cell line MDA-MB-435, sustained continuous growth in high concentrations of Taxol while the Taxol-sensitive cells could not. The increased expression and activity of LDH-A were detected in Taxol-resistant cells when compared with their parental cells. The downregulation of LDH-A by siRNA significantly increased the sensitivity of Taxol-resistant cells to Taxol. A higher sensitivity to the specific LDH inhibitor, oxamate, was found in the Taxol-resistant cells. Furthermore, treating cells with the combination of Taxol and oxamate showed a synergistical inhibitory effect on Taxol-resistant breast cancer cells by promoting apoptosis in these cells.

Conclusion: LDH-A plays an important role in Taxol resistance and inhibition of LDH-A re-sensitizes Taxol-resistant cells to Taxol. This supports that Warburg effect is a property of Taxol resistant cancer cells and may play an important role in the development of Taxol resistance. To our knowledge, this is the first report showing that the increased expression of LDH-A plays an important role in Taxol resistance of human breast cancer cells. This study provides valuable information for the future development and use of targeted therapies, such as oxamate, for the treatment of patients with Taxol-resistant breast cancer.

Figure 1

Characterization of Taxol-resistant cells. A, MDA-435, 435TR1 and 435TRP cells were treated with 20 nM Taxol for 24 hrs and their morphology was observed under fluorescence microscope. The phase image of these cells was shown at the top and the nucleus stained by DAPI was shown at the bottom (200 ×). B, MDA-435, 435TR1 and 435TRP cells were treated with 20 nM Taxol for 48 hrs and apoptosis was examined by flow cytometry using Annexin V/PI staining and by Cell Death Detection ELISA PLUS Kit. Fold induction value was calculated following the formula: mU of the sample (cells treated with Taxol)/mU of the corresponding negative control (cells without Taxol treatment). C, Taxol-resistant cells and their parental cells were treated without or with 20 nM Taxol for 48 hrs, then poly (ADP-ribose) polymerase (PARP) and its cleaved protein (c-PARP) were analyzed by Western blotting with specific antibodies, respectively. β-actin was used as a loading control. D, Cell viability analysis was performed to evaluate cytotoxicity of Taxol to MDA-435 and Taxol-resistant 435TR1 and 435TRP cells under treatment with indicated concentrations of Taxol for 48 hrs.

Figure 2

Increased LDH-A expression and activity in Taxol-resistant cells. A, Western blot was performed with an anti-LDH-A antibody of total cell extract from MDA-435, 435TR1 and 435TRP cells. The β-actin protein was used as a loading control. B, LDH activity in MDA-435, 435TR1 and 435TRP were examined. C, MDA-435 cells were treated with increasing concentrations of Taxol for 24 hrs. The cell lysates were prepared and Western blotting was carried out with antibodies against to LDH-A and β-actin. D, LDHA protein stability assay was performed in MDA-435 cells under the treatments of Taxol at 4 nM and CHX at 50 ug/ml followed by Western blotting assay to exam the protein expression level of LDHA at 0 and 8 hrs (top). The relative intensity of LDHA band was normalized to its β-actin loading (bottom). E, LDHA mRNA level was detected by real-time PCR under 2 nM Taxol in MDA-435 cells. The LDHA primers used for PCR are: forward, 5'-tgg agt gga atg aat gtt gc-3'; reverse: 5'-ata gcc cag gat gtg tag cc-3'. Columns, mean of three independent experiments; bars, SE. ***, P < 0.001.

Figure 3

Knockdown of LDH-A increases the sensitivity of Taxol-resistant 435TR1 cells to Taxol. A, MDA-435 and 435TR1 cells were transfected with scramble siRNA (Ctr) or LDH-A siRNA. 48 hrs after siRNA transfection, cell lysates were prepared and Western blotting was performed with antibodies against LDH-A. The β-actin protein was used as a loading control. B, LDH activity was examined from lysates of MDA-435 and 435TR1 48 hrs after siRNA transfection. C and D, 24 hrs after siRNA transfection, MDA-435 and 435TR1 cells were seeded into 96-well plates at the density of 8 × 10³ cells per well, and treated with Taxol (5 nM and 10 nM for MDA-435, 50 nM and 100 nM for 435TR1) for 48 hrs. Then the cell viability was detected using a MTS reagent. Data are presented as the percentage of viability inhibition measured in cells treated without Taxol. Columns, mean of three independent experiments; bars, SE. *, P < 0.05, **, P < 0.01, ***, P < 0.001.

Figure 4

Knockdown of LDH-A increases the sensitivity of breast cancer cell lines BT474 and MDA-231 to Taxol. A, BT474 cells were transfected with scramble siRNA (Ctr) or si-LDHA. 48 hrs after siRNA transfection, cell lysates were prepared and immunoblot analyses were carried out with antibodies against LDHA and β-actin. B, 48 hrs after siRNA transfection, cell lysates were prepared and LDHA activity was examined. Data are shown in percentage of LDH activity relative to Ctr-transfected cells. C, 48 hrs after siRNA transfection, cells were seeded into 96-well plates at the density of 8 × 10³ cells per well. 12 hrs after incubation, the cells were then treated with various concentrations of Taxol for 48 hrs. Then the cell viability was detected using a MTS reagent. Data are presented as the percentage of viability inhibition measured in cells treated without Taxol. D, LDHA protein expression in MDA-MB-231 (Ctr) and stable LDHA knowdown MDA-MB-231 cells (sh-LDHA) were detected to evaluate the efficiency of LDH-A knockdown by using an LDHA antibody. β-actin was used as a loading control. E, LDH activity was examined in MDA-MB-231 cells with and without stably knockdown of LDH-A. Data are shown in percentage of LDH activity relative to Ctr cells. F, MDA-MB-231 cells with or without stably knockdown of LDH-A were seeded into 96-well plates at the density of 8 × 10³ cells per well. 12 hrs after incubation, the cells were treated with various concentrations of Taxol for 48 hrs. Then the cell viability was detected using a MTS reagent. Data are presented as the percentage of viability inhibition measured in cells treated without Taxol. Columns, mean of three independent experiments; bars, SE.*, P < 0.05, **, P < 0.01, ***, P < 0.001.

Figure 5

Taxol-resistant cells are more sensitive to glycolysis inhibitor oxamate. A, MDA-435 and 435TR1 cells were treated with various concentrations of oxamate for 48 hrs, then LDH activity was detected. Data are shown as the percentage of LDH activity inhibition detected in cells treated without oxamate. B, MDA-435 and 435TR1 were treated with various concentrations of oxamate for 48 hrs. Then the cell viability was detected using a MTS reagent. Data are presented as the percentage of viability inhibition measured in cells treated without oxamate. Columns, mean of three independent experiments; bars, SE. *, P < 0.05. **, P < 0.01.

Figure 6

Combination of Taxol with oxamate shows synergistic inhibitory effects of Taxol-resistant and BT474 cells. A and B, 8 × 10³ per well of 435TRP and 435TR1 cells were plated in 96-well plates and then treated with Taxol (Tax), Oxamate (Oxa) alone or Taxol plus Oxamate (Tax+ Oxa) with the indicated concentrations for 48 hrs. Cell viability was examined by MTS assay. Data are presented as the percentage of viability inhibition measured in cells treated without Tax and Oxa. C, BT474 cells were treated with Tax, Oxa alone or Tax plus Oxa with the indicated concentrations for 48 hrs.. Cell viability was measured by direct cell counting. Data are presented as the percentage of viability inhibition counted in cells treated without Tax and Oxa. D, 435TR1 cells were treated with 100 nM Taxol or/and 40 mM oxamate for 48 hrs, cell lysates were prepared and Western blotting were carried out with antibodies against total PARP (Top) or its cleaved protein c-PARP (Middle). β-actin was used as a loading control (Bottom). Columns, mean of three independent experiments; bars, SE. ***, P < 0.001.