Elsevier

Lung Cancer

Volume 33, Issues 2–3, August 2001, Pages 221-228
Lung Cancer

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

https://doi.org/10.1016/S0169-5002(01)00205-7Get rights and content

Abstract

After exposure of H460 cells to an increasing concentrations of cis-diammine-dichloroplatinum(II) (cisplatin, CDDP) for 6 months, cisplatin resistant cells were isolated (H460/CIS). The biologic behaviors of H460 and H460/CIS cells were tested using animal experiments. Only the resistant cells developed lung metastases despite cisplatin treatment. The characteristics of H460/CIS cells are as follows, MTT analyses revealed that H460/CIS cells were markedly resistant to cisplatin compared with their parental cells. Also, H460/CIS cells exhibited cross-resistance to DNA damaging agents such as doxorubicin (DXR) and etoposide. Cisplatin treatment dramatically increased p53 expression in parental cells but not in H460/CIS cells which expressed basal levels of p53. Without cisplatin treatment, Bcl-2 and Bax were expressed in H460/CIS cells, but not in parental cell. Our data suggested that p53, Bax and Bcl-2 were up-regulated in H460/CIS cells. These changes could explain some of the mechanisms of cisplatin resistance. Thus, H460/CIS could be useful to investigate the mechanisms of drug resistance to cisplatin including apoptotic gene expressions conferring drug resistance, thereby making progress in the treatment of cisplatin-resistant tumor cells.

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.

Section snippets

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).

References (34)

  • H. Eichholtz-Wirth

    Reversal of radiation-induced cisplatin resistance in murine fibrosarcoma cells by selective modulation of the cyclic GMP-dependent transduction pathway

    Br. J. Cancer

    (1995)
  • Ferreira CG, Tolis C, Giaccone G. p53 and chemosensitivity. Ann...
  • D.P. Gately et al.

    Cellular accumulation of the anticancer agent cisplatin

    Br. J. Cancer

    (1993)
  • M.M. Heim et al.

    Differential modulation of chemosensitivity to alkylating agents and platinum compounds by DNA repair modulators in human lung cancer cell lines

    J. Cancer Res. Clin. Oncol.

    (2000)
  • B.T. Hill et al.

    Characterization of a cisplatin-resistant human ovarian carcinoma cell line expressing cross-resistance to 5-fluorouracil but collateral sensitivity to methotrexate

    Cancer Res.

    (1992)
  • A. Hirao et al.

    DNA damage-induced activation of p53 by the checkpoint kinase Chk2

    Science

    (2000)
  • W.S. Hong et al.

    Establishment and characterization of cisplatin-resistant sublines of human lung cancer cell lines

    Int. J. Cancer

    (1988)
  • Cited by (0)

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