The Synergistic Effect Of PEITC Combine With Paxlitaxel On Breast Cancer Cells And Its Underlying Mechanisms

Breast cancer is the most common cancer and number 2 cancer killer in women. Paclitaxel (taxol PTX) inhibits microtubule depolymerization and is one of the most important chemotherapy agent for breast cancer and other solid tumors. The major limitation of PTX is the side effects and toxicity which restrict its clinical use. Phenethyl isothiocyanate (PEITC) is a novel Histone Deacetylase (HDAC) inhibitor with anti-cancer activity and has been found to also target tubulin. In addition, it has virtually no toxicity to normal tissues. With two compounds from different classes and different toxicity profiles, we wish to test whether combining the two drugs can enhance the anti-cancer effect while reducing toxicity.ObjectiveTo study the effect of combining paclitaxel with the novel histone deacetylase inhibitor (HDAC), phenethyl isothiocyanate (PEITC); To analyse the relationships among these datas and find out if there is any interaction between PEITC and PTX,To observe whether the combination yields a synergistic effect on breast cancer cells lines MCF7 and MDA-MB-231To explore the mechanisms of the synergistic effects, and supply the experimental basis for the clinical chemotherapy.MetholdsThe breast cancer cell lines MCF7 and MDA-MB231 cell lines were in exponential growth were exposed to PEITC and PTX at various concentrations. According to the experimental concentrations used before and the clinical achievable concentrations, we could find out the appropriate concentrations of PEITC and PTX. The control cultures were supplemented with the DMSO as the vehicle control of Paclitaxel. We combined PEITC with PTX and treated both cell lines in simultaneous for 48 hours. In the experiment, we assessed inhibition rate, the changes of cell cycle and apoptosis rate by MTT,flow cytometry and the TUNEL assay; At the same time we detected the level of cell cycle protein,cell apoptosis protein,HDAC6,tubutlin and acytelate-a-tubulin expression by Westerblotting,HDAC6 activity assay kit,confocal immunofluorescence microscopy. Statistical analysis Assays were set up in triplicate, and the results were expressed as the mean+/-SD. SPSS 10.0 was adopted. T-Test, ANOVAR, Chi-Square Test and LSD were used. P<0.05 was considered to be statistically significant. IC50, the concentration at which 50%of cell growth is inhibited, was calculated using Calcusyn software (Biosoft, Inc). Synergism was assessed by the dose-effect curves of single or combined drug treatment using Calcusyn software.Results1. Effect of PEITC and PTX on breast cancer cellsThe IC50 of PEITC for MCF7 cells at 48 hours is 5.6μM, the IC50 of PEITC for MDA-MB-231 cells at 48 hours is 15.6μM. It appears that 5μM and 10μM are the concentrations that can cause growth suppression in a linear fashion for MCF7, and MDA-MB-231 cells, respectively. These concentrations were therefore chosen for further combination studies. The IC50 of PTX for MCF7 and MDA-MB-231 cells at 48 hours is 111.3 nM and 410nM, respectively. The 10 nM and 100 nM concentrations of PTX were chosen for further combination studies for MCF7 and MDA-MB-231 cells, respectively. 2.Effect of PEI TC and PTX in combination on breast cancer cell growth When cells were treated with fixed concentration of PTX, PEITC IC50 for MCF7 and MDA-MB-231 cells decreased by more than 2.6-fold and 7.3-fold, respectively When cells were treated with fixed concentration of PEITC, PTX IC50 for MCF7 and MDA-MB-231 cells decreased by more than 37-fold and 50-fold, respectively. This makes it highly likely to significantly reduce side effects of PTX while maintaining clinical efficacy. This effect was further analyzed for synergism using computer modeling. For both MCF7 and MDA-MB-231 cells, there is clear synergistic effect when PEITC and PTX are combined.3. Effect of combination of PEI TC and PTX on cell cycle in breast cancer cells PTX and PEITC as single agent at low concentrations caused accumulation of cells in G2/M (PEITC:8.94%and 17.08%; PTX:6.43%and 14.35%, for MCF7 and MDA-MB-231, respectively). When PEITC and PTX were added concurrently in the cell culture for 48 hours, there was a significant increase (43.2%and 57.1%for MCF7 and MDA-MB-231, respectively) in the number of cells arrested in the G2/M phases (p=0.001) 4.. Effect of combination of PEI TC and PTX on apoptosis of breast cancer cells TUNEL assay, we examined the effect of PEITC and PTX on cell apoptosis. Compared with the single agent, combination of PEITC and PTX increased apoptosis by 3.4-and 2.8-fold, respectively, over PEITC and PTX single agent in MCF7 cells, and by more than two fold in MDA-MB-231 cells.5. PEI TC and PTX increased acetylation of alpha-tubulin in breast cancer cells Here we confirmed that PEITC can inhibit HDAC6 enzyme expression in both breast cancer cell lines. PEITC was found to inhibit the expression of both alpha-and beta-tubulins in a concentration-dependent manner in both breast cancer cell lines. We found that alpha-tubulin acetylation was increased with higher concentration of PEITC, even though the total level of tubulin protein is decreased. When the cells were treated with the combination of PEITC and PTX, the acetylation of alpha-tubulin was significantly increased in both MCF7 and MDA-MB-231 cells in comparison with that in single agent treated cells.PTX also led to the increase of acetylation of alpha-tubulin in both MCF7 and MDA-MB-231 cells. The cytoplasmic level of acetylated alpha-tubulin was clearly increased and can be directly visualized under confocal fluorescent microscope in both MCF7 and MDA-MB-231 cells after treatment with 5μM of PEITC for 48 hours.6. Effect of combination of PEITC and PTX on cyclin B1 and cdkl expression. When compared with single agent PEITC and PTX, the combination of both agents reduced the expression of cdkl more significantly than either agent alone. In the mean time, the cyclinB1 expression remained relatively stable, indicating a less significant effect from the treatment.7. Effect of combination of PEITC and PTX on Bax and Bcl-2 expression The levels of the Bax and Bcl-2 proteins expression in the breast cancer compared with single agent PEITC and PTX, the combination of both agents reduced Bcl-2 expression and increased Bax expression more than either agent alone.8. Effect of combination of PEI TC and PTX on PARP-1 and caspase expression The levels of the PARP-1 and caspase proteins in MCF7 and MDA-MB-231 cells were examined by Western blotting. When compared with single agent PEITC and PTX, the combination of both agents increased the PARP-1 cleavage (thus increase in degradation product) and degradation of caspase-3 and-9 proteins more than single agent in both cells.Conclusions1. The combination of the epigenetic agent PEITC with the chemotherapeutic agent PTX exhibits a synergistic effect on breast cancer cells growth inhibition;2. The combination of the epigenetic agent PEITC with the chemotherapeutic agent PTX exhibits a synergistic effect on induction of G2/M block and apoptosis in breast cancer cells;3. The epigenetic agent PEITC can inhibit the activity of HDAC6 in cytoplasm of Breast cancer cells;4. The epigenetic agent PEITC can increase a-tubulin acetylation expression in cytoplasm of Breast cancer cells;5. PETIC may increase paclitaxel cytotoxicity by inhibiting HDAC6 activity and enhance a-tubulin acetylation expression;6. This novel strategy deserves further study in vivo in animal models and may provide a new and enhanced treatment option for breast cancer patients.