In vitro establishment of cis-diammine-dichloroplatinum(II) resistant lung cancer cell line and modulation of apoptotic gene expression as a mechanism of resistant phenotype
Introduction
cis-diammine-dichloroplatinum(II) (cisplatin, CDDP) is a DNA damaging agent, one of the most active anti-cancer agents, used in a wide variety of malignancies [26]. Despite initial tumor response to chemotherapeutic agents, resistance develops sooner or later in most instances. To elucidate the mechanisms of drug resistance, several groups established drug resistant cell lines and characterized their biologic behaviours [13], [17], [18], [21], [30]. Many studies have suggested that the mechanisms of drug resistance are closely associated with the altered transmembrane drug transport [19], an increase in drug detoxification [9], [16] and an increase in DNA repair [10], [13], [31]. Other lines of evidence have suggested that drug resistance was associated with alternations of the cGMP-dependent transduction pathway and could be overcome by enhanced cGMP formation [7]. In addition to these mechanisms, any cellular changes that may affect cell-survival or apoptosis should be considered as possible contributing factors for drug resistance. In detail, cross-resistance to a variety of chemotherapeutic agents with different intracellular targets has led to the hypothesis that a defective apoptotic pathway may be responsible for the development of resistant phenotype.
Responses to cisplatin, a DNA damaging agent, are mainly mediated through p53 dependent apoptotic pathways and partially mediated via p53 independent apoptosis [4], [10], [24], [25], [28]. Most studies have reported that lung cancer cells having wt p53 are more sensitive to cisplatin than lung cancer cells having either mt p53 or null p53 [25]. H460 cells are relatively sensitive to cisplatin and overexpression of p53 induced by cisplatin may trigger apoptotic pathway via transactivation of Bax gene [8]. To investigate the anti-apoptotic mechanisms resulting in development of cisplatin resistance, we have developed cisplatin resistant cells (H460/CIS) using H460 cells bearing wild-type p53 gene via stepwise exposure to increasing concentrations of cisplatin. These cells were injected into nude mice via lateral tail vein injection. Cell cultures were established from lung metastases. The availability of H460/CIS cells is crucial to better understand the mechanisms of cisplatin resistance. So far, there have been no reports on the mechanisms of cisplatin resistance in H460 cells expressing wtp53. In this study, we characterized a biological behavior of a resistant lung cancer cell line (H460/CIS) to cisplatin and demonstrated the expression patterns of apoptosis related genes in H460/CIS cells treated with cisplatin.
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Cell lines and cell culture
Cisplatin was obtained from Bristol-Meyers Squib (Syracuse, NY). All other chemicals and reagents were obtained from Sigma. Cell culture reagents were obtained from GIBCO-BRL. H460 cells having wtp53 were obtained from American Type Culture Collection (ATCC) and maintained in RPMI 1640 supplemented with 10% fetal bovine serum (FBS), 100 U/ml penicillin, and 100 μg/ml streptomycin at 37°C, in a humid atmosphere of 5% CO2/95% air. Exposing them to gradually increasing concentrations of cisplatin
Establishment and characterization of cisplatin resistant cells (H460/CIS)
By continuous exposure to stepwise increasing concentrations of cisplatin, cisplatin resistant cell line (H460/CIS) was established from H460 cells expressing wtp53. It took 3 months to develop the cisplatin resistant cells in vitro. In MTT assay, H460/CIS cells were 20 times more resistant to cisplatin than H460 cells. H460/CIS cells were also cross-resistant to doxorubicin and etoposide, by 10.2- and 6.7-folds, respectively (Table 1). Since the sensitivity to cisplatin could be influenced by
Discussion
Cisplatin is one of major drugs in the treatment of lung cancer, but the acquisition of drug resistance by tumor cells is a key problem preventing the successful outcome of cancer chemotherapy. Changes in biologic behaviors may be due to genetic or epigenetic alterations. Many studies have been focused on elucidating the mechanisms of cisplatin resistance using cisplatin resistant cells exposed to cisplatin in vitro. Despite recent progresses, the mechanisms controlling drug resistance are
Acknowledgements
This paper was supported by NON DIRECTED RESEARCH FUND, Korea Research Foundation, 1998, (KRF, 1998-019-F00063).
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