Intrinsic resistance to chemotherapeutic agents in murine osteosarcoma cells

Background: There are two general categories of drug resistance: acquired a nd intrinsic. The mechanisms involved in acquired drug resistance have been extensively studied, and several mechanisms have been described. However, the mechanisms responsible for intrinsic drug resistance have not been eluc idated, to our knowledge. The purpose of the present study was to investiga te the cytological and biochemical differences between acquired and intrins ic drug resistance in osteosarcoma cells. Methods: We previously isolated a clonal cell line (MOS/ADR1) to study acqu ired resistance in osteosarcoma by exposure of parental murine osteosarcoma cells (MOS) to doxorubicin. In the present study, we cloned a new intrinsi cally resistant cell line (MOS/IR1) by single-cell culture of MOS cells and we investigated the differences in cell phenotype and the mechanisms of re sistance in both of these resistant clones. Results: The MOS/ADR1 and MOS/IR1 cells were sevenfold and fivefold more re sistant to doxorubicin than the parental murine osteosarcoma cells. Morphol ogically, the MOS/ADR1 cell line was composed of polygonal cells, whereas t he MOS/IR1 cell line consisted of plump spindle cells with long cytoplasmic processes. The MOS/IR1 cells showed a much lower level of alkaline phospha tase activity than did the MOS/ADR1 and MOS cells. There were no substantia l differences in the cellular DNA content or the doubling time among these three lines. Overexpression of the P-glycoprotein involved in the function of an energy- dependent drug-efflux pump was detected in the MOS/ADR1 cells but not in th e MOS/IR1 cells. After the cells were incubated with doxorubicin for one ho ur, the two resistant lines had less accumulation of the drug than did the parent line (p < 0.05). The addition of a P-glycoprotein antagonist, verapa mil, or the depletion of cellular adenosine triphosphate resulted in a mark ed increase in the accumulation of doxorubicin in the MOS/ADR1 cells (p < 0 .05) but not in the MOS/IR1 cells. The MOS/ADR1 cells were found to exhibit cross-resistance only to substrates for P-glycoprotein (such as doxorubici n, vincristine, and etoposide), whereas the MOS/IR1 cells were resistant to all of the drugs studied (including cisplatin and methotrexate). The degree of drug resistance in the MOS/IR1 cells was found to be associat ed,vith the molecular weight of the drugs (p < 0.05). Permeabilization of t he plasma membrane by saponin increased both the accumulation of doxorubici n (p < 0.05) and the cytotoxic activity of this drug in all lines, but the effects were most pronounced in the MOS/IR1 cells. Conclusions: Taken together, this data suggests that reduced drug accumulat ion in the MOS/IR1 cells may be due to the effect of decreased permeability of the plasma membrane on the transport of drugs from the extracellular en vironment into the cytosol of the cell and that this may be the mechanism r esponsible for intrinsic resistance to multiple drugs in the MOS/IR1 cell l ine. Clinical Relevance: Current drug treatment for human osteosarcoma may inclu de multiple chemotherapeutic agents, such as doxorubicin, cisplatin, and me thotrexate. These drugs exhibit different cytotoxic actions and, thus, the mechanisms of resistance to individual drugs vary. Clinical resistance to m ultidrug chemotherapy may be observed in tumors that recur after repetitive chemotherapy and in previously untreated tumors. In the former group, a tu rner cell may express multidrug resistance by combining several different m echanisms due to its exposure to various drugs. In the latter group, howeve r, this is not likely. Decreased intracellular drug accumulation due to red uced permeability of the plasma membrane, found in the MOS/IR1 cells, is on e possible mechanism and may explain the intrinsic resistance to multidrug chemotherapy for the treatment of osteosarcoma. Further study regarding the resistance mechanism in the MOS/IR1 cells may help to overcome the intrins ic drug resistance in osteosarcoma.