Reversal Effect Of Tyroservatide (YSV) On Multi-drug Resistance And Its Mechanism In Multi-drug Resistant Tumor Cells

Objective:To investigate the potential reversal effect of YSV on multi-drug resistance of tumor cells, and explore its mechanism of action in vitro.Methods:1. The cross-resistance assays of drug resistant human hepatocellular carcinoma BEL-7402/5-FU cells and human breast cancer MCF-7/ADR cells to 5-fluorouracil, doxorubicin and vincristine were determined by MTS method.2. The drug-resistant human hepatocellular carcinoma BEL-7402/5-FU cells and human breast cancer MCF-7/ADR cells were used to explore the intrinsic toxicity of YSV by MTS assay. In order to investigate the reversal effect of YSV on multi-drug resistant cells in vitro, YSV was administered to cells at weakly cytotoxic concentration.3. The effects of YSV on intracellular accumulation of doxorubicin and Rhodamine-123 in BEL-7402/5-FU cells and MCF-7/ADR cells were determined by flow cytometry.4. By Real-time PCR and Western blot methods, the effects of YSV on mRNA and protein expression of MDR1 in BEL-7402/5-FU cells and MCF-7/ADR cells were observed. The effect of YSV on protein expression of PKCa which regulate MDR1 gene expression was explored by Western blot method.5. By Real-time PCR and Western blot methods, the effects of YSV on mRNA and protein expression of MRP1, LRP, GST-Ï€in BEL-7402/5-FU cells and MCF-7/ADR cells were observed.6. By flow cytometry and DNA Ladder methods, the relationship between drug resistance and decrease of apoptosis in drug-resistant human hepatocellular carcinoma BEL-7402/5-FU cells was evaluated. The apoptosis-inducing function of YSV combined with chemotherapeutics in BEL-7402/5-FU cells were observed.7. Western Blot method was used to explore the effects of YSV on protein expression of apoptosis inhibitor Bcl-2, Bcl-xL and anti-oncogene P 53 in drug resistant human hepatocellular carcinoma BEL-7402/5-FU cells.Results:1. The resistance index (RI) to 5-fluorouracil and doxorubicin were 287.39 and 5.89 respectively in drug-resistant human hepatocellular carcinoma BEL-7402/5-FU cells. The resistance index to doxorubicin and vincristine were 18.74 and 8.08 respectively in drug-resistant human breast cancer MCF-7/ADR cells.2. YSV (0.1-1.6mg/ml) inhibited the growth of BEL-7402/5-FU cells and MCF-7/ADR cells in a dose-dependent manner. YSV at below 0.8 mg/ml was weakly cytotoxic in BEL-7402/5-FU cells. YSV at below 1.6 mg/ml was weakly cytotoxic in MCF-7/ADR cells.3. YSV (0.4,0.8mg/ml) exerted a partial reversal effect against 5-fluorouracil and doxorubicin in BEL-7402/5-FU cells. YSV (0.8,1.6mg/ml) had a partial reversal effect against doxorubicin and vincristine in MCF-7/ADR cells.4. The intracellular accumulation of doxorubicin and rhodamine-123 were increased remarkably after BEL-7402/5-FU cells and MCF-7/ADR cells were incubated in the presence of YSV.5. YSV could inhibit the mRNA and protein expression of MDR1 and decrease the protein level of PKCa which was involved in regulating MDR1 gene expression. Moreover, the protein level of MRP 1, the mRNA and protein expression of LRP and GST-Ï€were significantly decreased in BEL-7402/5-FU cells and MCF-7/ADR cells after treatment with YSV.6. The apoptosis-inducing effect of 5-fluorouracil was weaker in drug-resistant human hepatocellular carcinoma BEL-7402/5-FU than in parental BEL-7402 cells from the results of flow cytometry. The DNA Ladders were detected and apoptotic cells amount were increased in BEL-7402/5-FU cells after treatment with YSV (0.4,0.8mg/ml) combined with 5-fluorouracil.7. YSV could decrease the protein level of anti-apoptotic factors Bcl-2 and Bcl-xL and increase the protein expression of anti-oncogene P53 in drug-resistant human hepatocellular carcinoma BEL-7402/5-FU cells.Conclusion: Administration of YSV reversed the multi-drug resistance of BEL-7402/5-FU cells and MCF-7/ADR cells significantly and enhanced drug sensitivity partially. The mechanism of its reversal effect could be as follows:YSV could decrease expression of MDR1, LRP,MRP1, GST-Ï€and PKCa genes, inhibite the transmembrane pump activity of P-gp, decrease the protein expression of apoptosis inhibitor Bcl-2 and Bcl-xL, increase the protein level of P53, enhance apoptosis-inducing effect of chemotherapeutic drugs.