Mechanisms of resistance to chemosensitizers in a multidrug resistant human multiple myeloma cell line

Abstract

Tumor cells in cancer patients acquire drug resistance as a result of chemotherapy. One type of acquired drug resistance is multidrug resistance (MDR) caused by the overexpression of P-glycoprotein, a transmembrane efflux protein. Inhibitors of P-glycoprotein or chemosensitizers such as verapamil are used to reverse MDR in cancer patients. Clinical studies have shown that some patients with P-glycoprotein positive cancer cells respond to the chemosensitizing effect of verapamil. However, this response is short lived and tumor cells become resistant to chemosensitizers. In order to study the mechanism of resistance to chemosensitizers, a human myeloma cell line, 8226/MDR₁₀V, was selected from a P-glycoprotein positive cell line, 8226/Dox₄₀, in the continuous presence of doxorubicin and verapamil. MDR₁₀V cells are consistently more resistant to MDR drugs than the parent cells, Dox₄₀. Chemosensitizers were less effective in reversing resistance in the MDR₁₀V compared to D0X₄₀ cells. Despite higher resistance to cytotoxic agents, MDR₁₀V expresses less P-glycoprotein in the plasma membrane compared to Dox₄₀. However, total cellular P-glycoprotein was the same in both cell lines suggesting a relocation of P-glycoprotein from plasma membrane into cytoplasm. Confocal immunofluorescence microscopy showed 2.5X more P-glycoprotein in the cytoplasm of MDR₁₀V cells as compared to D0X₄₀ cells. The relocation of P-glycoprotein was associated with a redistribution of doxorubicin. In D0X40 cells, doxorubicin was concentrated in the nucleus, whereas in MDR₁₀V cells, 90% of doxorubicin was found in the cytoplasm. We hypothesized that P-glycoprotein trafficking from the endoplasmic reticulum to the plasma membrane may be interrupted resulting in a higher concentration in the cytoplasm. To test this hypothesis, endoglycosidase H sensitivity of newly sensitized P-glycoprotein was examined. Medial Golgi processing of P-glycoprotein was identical between the two cell lines and the N-glycosylation of P-glycoprotein was complete by 3 hours. No mutations were found in MDR1 cDNA from MDR₁₀V cells compared to Dox₄₀ cells. These results suggest that increased resistance to cytotoxic drugs and chemosensitizers is associated with an altered intracellular location of P-glycoprotein which in turn causes a redistribution of doxorubicin.