Multidrug Resistance and its Reversal in Cancer Chemotherapy

• Main Authors: Chih-Hsin Yang, 楊志新
• Other Authors: Yao-Chang Chen
• Format: Others
• Language: en_US
• Published: 2000
• Online Access: http://ndltd.ncl.edu.tw/handle/52528306924352660135

博士 === 國立臺灣大學 === 臨床醫學研究所 === 88 === Cancer cells exposed to certain chemotherapeutic agents can acquire cross-resistance to other structurally or functionally related or unrelated drugs, a phenomenon known as multidrug resistance. Multiple factors are known to confer resistance to chemotherapeutic agents. This thesis explores the emergence of multidrug resistance in cancer cells and the possibility of reversal of this resistance by relatively non-toxic drugs. In the first part of my thesis studies, the role of multidrug-resistance associated protein 1 (MRP1) in multidrug resistant cancer cells was investigated. We tested MRP1 expression in several pairs of drug-sensitive and -resistant cancer cells by quantitative RT-PCR. MRP1 mRNA was expressed in all cells tested. Overexpression of MRP1 was found in MCF7/VP, MCF7/ADR and HL60/AR multidrug resistant cancer cells. Southern blot analysis demonstrated that MRP1 gene was amplified in MCF7/VP cells. My study supports that MRP1 overexpression confers resistance to multidrug resistance in cancer cells. In the second part of the present study, we explored the mechanism of resistance in a novel chemotherapeutic agent, arsenic trioxide. The possibility of using arsenic trioxide in the treatment of solid tumor was tested. Cytotoxicity assay of arsenic trioxide in 17 cancer cell lines demonstrated that bladder cancer and gastrointestinal cancer cells were most sensitive to arsenic trioxide in addition to acute promyelocytic leukemic cells. Arsenic trioxide may induce apoptosis in NTU-B1 bladder cancer cells. On the other hand, non-small cell lung cancer cells and hepatocellular carcinoma cells were intrinsically most resistant to arsenic. The mechanism of arsenic resistance was investigated. through the correlation between glutathione, glutathione-s-transferase and MRP1 expression and arsenic toxicity. MRP1 was overexpressed in two cell lines (H460 and BFTC909) that were resistant to arsenic. But it is not overexpressed in all other resistant cell lines. There was no correlation of GST-pi expression and arsenic toxicity. One the other hand, GSH content of the cancer cells correlated well to intrinsic arsenic toxicity. Multidrug resistant MCF7/VP, MCF7/ADR and NTU-B1/P14 cells were cross-resistant to arsenic. MRP1 was overexpressed in MCF7/VP and MCF7/ADR cells but not in NTU-B1/P14 cells, while glutathione content was overexpressed in NTU-B1/P14 cells. Therefore, both MRP1 and glutathione overexpression may confer acquired resistance to cancer cells. Ten micromolar of BSO may completely reverse arsenic resistance in both NTU-B1/P14 and MCF7/VP cells. In conclusion, glutathione content may be the main determinant of arsenic toxicity. GSH-MRP1 system is the major route of arsenic detoxification. It is warranted to conduct arsenic trioxide clinical trials in solid tumors. Measurement of GSH content in cancer and normal tissues should be considered in the clinical trials. In the third part of the study, we investigated a novel multidrug resistance phenotype, MX/TPT resistance. MCF7/MX cells are mitoxantrone-resistant and MCF7/TPT cells are topotecan-resistant human breast carcinoma cells. They contain several common resistance phenotypes. Both cells were resistant to mitoxantrone, topotecan and CPT-11 and were only slightly cross-resistant to camptothecin, doxorubicin and VP16. There was no P-glycoprotein or MRP1 overexpression in MCF7/MX or MCF7/TPT cells. Prior studies on topoisomerase I toxin resistance suggested that down-regulation and/or mutation of topoisomerase I was the main mechanisms of resistance. Our study, however, showed that DNA topoisomerase I activity and amount were not changed in MCF7/MX cells compared to MCF7/WT cells. Flowcytometric analysis demonstrated that topotecan accumulation was decreased in MCF7/MX as well as in MCF7/TPT cells. On the other hand, camptothecin accumulation was only slightly increased in MCF7/MX cells compared to MCF7/WT cells. Topotecan efflux was markedly enhanced in MCF7/TPT cells. Topotecan efflux was inhibited by pre-incubation in sodium azide in glucose free media to deplete cellular ATP. These results suggest that a novel ATP-binding cassette protein may confer MX/TPT resistance in these cells. We also found that novobiocin may partially reverse topotecan and mitoxantrone resistance in MCF7/TPT cells. The mechanism of novobiocin may be through inhibition of topotecan efflux of MCF7/TPT cells. Breast cancer resistance protein (BCRP) is a novel half ABC protein that was linked to mitoxantrone resistance. We found that BCRP was over-expressed in MCF7/MX cells but only vaguely expressed in MCF7/TPT300 cells. Further investigations are needed to decipher the role of BCRP in MX/TPT resistance. High dose tamoxifen has been shown in the laboratory to reverse p-glycoprotein dependent and independent multidrug resistance. In the fourth part of the studies, we designed a phase II study to test the feasibility of using high dose tamoxifen plus cisplatin and VP16 in the treatment of inoperable non-small cell lung cancer patients. Forty patients were accrued to the study. The response rate was 37.5%. One-year survival was 44% and median survival was 47 weeks. Progression-free survival was 21 weeks. Our study suggests that reversing agents with chemotherapy may provide a new avenue to improve the effect of chemotherapy in the future. In conclusion, we have demonstrated that MRP1 is involved in multidrug resistance in cancer cells. Arsenic trioxide is cytotoxic to bladder and gastrointestinal cancer cell lines. The main determinant of arsenic sensitivity is cellular glutathione content. GSH-MRP system is the main arsenic detoxification system in cancer cells. BSO may reverse arsenic resistance and can be tested in clinical trials. We also discover a novel topotecan/mitoxantrone resistance in MCF7/MX and MCF7/TPT cells. It is a non-p-glycoprotein, non-MRP1, energy-dependent topotecan efflux mechanism. The possibility of breast cancer resistance protein (BCRP) overexpression in these cells is explored. We also demonstrate that novobiocin may reverse MX/TPT resistance in these cells. The feasibility of using tamoxifen as a reversing agent for chemotherapy in clinical trials was also demonstrated in the study.