Studies On The Radiosensitivity Of Cisplation-resistant Human Nasopharyngeal Carcinoma Cell Line

Background and ObjectiveNasopharyngeal carcinoma(NPC)is endemic in southern China and Southeast Asian. It is a tumor that is highly sensitive to both radiotherapy and chemotherapy. Radiotherapy is the treatment of choice in patients with early-stage NPC, whereas chemotherapy is important in the metastatic setting as well as in locally advanced disease when used in combination with radiotherapy. However, the outcome of nasopharyngeal carcinoma is far from satisfactory. Due to the multidrug resistance, many patients in advanced stages eventually died because of the failure of chemotherapy. Cisplatin(CDDP)is one of the widely used therapeutic agents and acts both as a cytotoxic agent and as a radiation sensitizer. The acquired resistance to cisplatin often leads to the failure of treatment. In the last few years, other agents have demonstrated significant single-agent activity, including paclitaxel, gemcitabine and so on. Paclitaxel is active as first-line therapy but not as secondline therapy. Gemcitabine could be promising in the setting of second-line chemotherapy, especially for recurrent or metastatic NPC patients who cisplatin-pretreated. In this study, we attempted to investigate the radiosensitivity of parental(HNE1)and cisplation-resistant(HNE1/CDDP)human nasopharyngeal carcinoma cells, and the difference of radiation sensitizing enhancement effect of cisplatin on these two cell lines, as well as to preliminarily determine the radiosensitizing effect of gemcitabine on HNE1/CDDP.MethodsHuman nasopharyngeal carcinoma cell line HNE1/CDDP and its parental cell line HNE1 were used as in vitro models. In the part one, colony formation assay was carried out to test the cytotoxicity of cisplatin. The IC₅₀ values were measured. HNE1 and HNE1/CDDP cells were treated in vitro with different doses of radiation alone or radiation following cisplatin(1.0ug/ml)exposure. Colony forming assay was used for survival fraction analysis. Survival curves were plotted by using radiomed software. In part two, colony formation assay and growth curves were carried out to test the cytotoxicity of gemcitabine. The IC₅₀ value also was measured. Immunochemistry was used to determine bax, p53 and bcl-2 expressions. Cell cycle distribution was analyzed by using flow cytometry. Colony forming assay was used for evaluating the radiation sensitizing effect of gemcitabine on cisplation- resistant human nasopharyngeal carcinoma cell line.ResultIn part one, after treated with cisplatin for one hour, the IC50 of HNE1 was 8.86±1.95ug/ml and that of HNE1/CDDP was 27.12±3.7ug/ml. The value of D₀ of HNE1 and HNE1/CDDP cells received radiation treatment alone was 0.8593Gy and 0.8757Gy, respectively. That of HNE1 cells received radiation alone or radiation plus cisplatin was 0.8593Gy and 0.7152Gy, respectively,with sensitization enhancement ratio(SER)of 1.2015. The value of D₀ of HNE1/CDDP received radiation alone or radiation plus cisplatin was 0.8757Gy and 0.9298Gy, respectively, indicating no sensitization effect of cisplation on HNE1/CDDP.In part two, after treated with gemcitabine for twenty-four hour, the IC50 of HNE1 was 1.3±0.1 ug/ml and that of HNE1/CDDP was 1.9±0.2 ug/ml. After treated for 24h, 1.5ug/ml gemcitabine has mild cytotoxic effect on HNE1/CDDP. Gemcitabine(1.5ug/ml)increased apoptosis and the expression of bax with no effect on p53 expression. However, expression of Bcl-2 was negative befor and after gemcitabine(1.Sug/ml)treated. Meanwhile, DNA flow cytometric analysis indicated that gemcitabine(1.5ug/ml)induced efficiently G1 phase arrest in this cell line. The value of D₀ of HNE1/CDDP received radiation alone or radiation plus gemcitabine was 0.8757Gy and 0.6350Gy respectively. The sensitization enhancement ratio(SER)of gemcitabine on HNE1/CDDP was 1.3791.Conclusions1 HNE1/CDDP was mild resistant to cisplatin and also mild resistance to gemcitabine.2 Cisplatin resistance does not predict radiation resistance in HNE1/CDDP cell line.3 Cisplatin has been shown a radiosensitizing effect in vitro on HNE1 cell line, but not in HNE1/CDDP cell line.4 Gemcitabine could significantly increase the radiation sensitivity of HNE1/CDDP cell line in vitro.5 Radiosensitization of gemcitabine(1.5ug/ml)in HNE1/CDDP cells is probably associated with apoptosis and G1 block. The apoptosis may be the consequence of regulation of p53, bcl-2 and bax.