Effects of hyperthermia on biological behavior of human laryngeal cancer Hep-2 cisplatin-resistant cell line / 肿瘤研究与临床

ABSTRACT

Objective:To investigate the effects of hyperthermia on the biological behavior of human laryngeal cancer Hep-2 cisplatin-resistant (Hep-2/CDDP) cell line and its possible mechanism.Methods:Hep-2/CDDP cell line was induced by high impact combined with increasing concentration method. Cell count method was used to detect the cell proliferation ability of Hep-2 parental cell group (Hep-2 cells without cisplatin-resistance and the cells were cultured with RPMI 1640 cultured medium without cisplatin), Hep-2/CDDP cell group and Hep-2/CDDP+cisplatin group (using RPMI 1640 cultured medium including 4 mg/L cisplatin). Hep-2/CDDP cell group and Hep-2 parental cell group were treated with cultured medium including 0, 0.004, 0.04, 0.4, 4, 40 mg/L cisplatin, respectively. The sensitivity of Hep-2/CDDP cells to cisplatin, vincristine and 5-fluorouracil was determined by using methyl thiazolyl tetrazolium (MTT) method. The half inhibitory concentration ( IC50) and resistance index (RI) were also calculated. Hep-2/CDDP cell group was divided into 4 subgroups the cells in the control group were cultured for 24 h at 37 ℃; the cells in hyperthermia group were treated at 43 ℃ for 2 h and then re-cultured at 37 ℃ for 22 h; the cells in cisplatin group were cultured at 37 ℃ for 24 h in cultured medium containing 4 mg/L cisplatin. The cells in hyperthermia combined with cisplatin group were cultured in cultured medium containing 4 mg/L cisplatin, treated at 43 ℃ for 2 h and then re-cultured at 37 ℃ for 22 h. The effects of hyperthermia combined with cisplatin on the proliferation and early apoptosis of Hep-2/CDDP cells were detected by using MTT and flow cytometry. The interaction of hyperthermia combined with cisplatin on the proliferation and early apoptosis of HEP-2/CDDP cells was observed by using factorial analysis. Western blotting was used to detect the effect of hyperthermia combined with cisplatin on the expressions of wild-type p53 and PI3K in Hep-2/CDDP cells. Hep-2/CDDP cells were divided into 4 groups the control group (Hep-2/CDDP cells were cultured for 24 h at 37 ℃); chemotherapy group was treated with 12 mg/L vincristine or 9 mg/L 5-fluorouracil; in the hyperthermia group, Hep-2/CDDP cells were treated at 43℃ for 2 h and then re-cultured at 37 ℃ for 22 h; in hyperthermia combined with chemotherapy group, the cells were cultured in a medium containing 12 mg/L vincristine or 9 mg/L 5-fluorouracil, treated at 43 ℃ for 2 h and then re-cultured at 37 ℃ for 22 h. MTT method was used to detect the effect of hyperthermia combined with vincristine and 5-fluorouracil on the proliferation of Hep-2/CDDP cells. Results:Hep-2/CDDP cell line was successfully established. There were no significant differences in the number of cells in Hep-2/CDDP cell group, Hep-2 parental cell line group and Hep-2/CDDP + cisplatin cell group at different time points (all P > 0.05), and the doubling time was 43.8, 40.6 and 43.5 h, respectively. The IC50 of Hep-2 parental cell line group and Hep-2/CDDP cell group to cisplatin was 4.771 mg/L and 42.749 mg/L, respectively, and the RI was 8.960. Hyperthermia combined with cisplatin could inhibit the proliferation of Hep-2/CDDP cells ( F = 327.91, P < 0.05) and promote the early apoptosis of Hep-2/CDDP cells ( F = 724.63, P < 0.05). Factorial analysis showed that hyperthermia combined with cisplatin had an interaction effect on the proliferation and early apoptosis of Hep-2/CDDP cells ( F = 185.68, 472.51, all P < 0.05). Western blotting showed that the relative expression levels of wild-type p53 protein and PI3K protein in the control group, hyperthermia group, cisplatin group and hyperthermia combined with cisplatin group were significantly different ( F = 547.76, 404.44, all P < 0.01). Hyperthermia combined with vincristine or 5-fluorouracil could inhibit the proliferation of Hep-2/CDDP cells ( F = 33.06, 34.61, all P < 0.05). Factorial analysis showed that hyperthermia combined with vincristine and 5-fluorouracil had no interaction effect on the proliferation of Hep-2/CDDP cells ( F = 0.64,0.60, all P > 0.05). Conclusions:Hyperthermia may reverse the resistance of Hep-2/CDDP cell line to cisplatin by upregulating wild-type p53 expression and inhibiting the PI3K pathway. Hep-2/CDDP cell line has cross-resistance to vincristine and 5-fluorouracil. Hyperthermia can increase the sensitivity of Hep-2/CDDP cell line to vincristine and 5-fluorouracil.