Establishment of cisplatin-resistant melanoma cell line with stable expression of T-cadherin and its biological characteristics

Abstract

To establish cisplatin (CDDP)-resistant melanoma B16F10 (CDDP-R B16F10) cell line with stable expression of T-cadherin, and to study its biological characteristics. Methods: CDDP-R B16F10 cell line was exposure to high and gradually increased dose of CDDP. 3-(4.5-Dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) was used to test the proliferation of CDDP-R B16F10 cell line, and the sensitivity of CDDP-R B16F10 cell line to CDDP and paclitaxel was examined. The pEGFP-N1-T-cadherin, a plasmid vector encoding human T-cadherin, was generated by inserting T-cadherin cDNA into a pEGFP-N1 vector. The pEGFP-N1-T-cadherin was transfected into CDDP-R B16F10 cell line. The expression of T-cadherin mRNA and protein were measured by reverse transcription polymerase chain reaction (RT-PCR), Western blotting and immunohistochemistry SP method, respectively. The effect of T-cadherin combined with CDDP on proliferation of CDDP-R B16F10 cell line was determined by MTT assay. The sensitivity of CDDP-R B16F10 cell line with stably transfected T-cadherin to paclitaxel was examined by MTT assay. Results: The CDDP-R B16F10 cell line was established successfully. There was no difference in proliferation between the CDDP-R B16F10 cell line and B16F10 cell line (P>0.05). The IC50 of CDDP-R B16F10 cell line and B16F10 cell line to CDDP were 268.706 and 19.748 mg/L, respectively, and the resistance index was 13.61. The IC50 of CDDP-R B16F10 cell line and B16F10 cells to paclitaxel were 11.415 and 7.799 mg/L, respectively, and the resistance index was 1.46. The expression vector pEGFP-N1-T-cadherin was constructed successfully. RT-PCR, Western blotting and immunohistochemistry SP method showed that T-cadherin could be transcribed and expressed. MTT assay showed that T-cadherin combined with CDDP could inhibit the proliferation of CDDP-R B16F10 cell line (P<0.05). Factorial analysis showed that there was interaction between T-cadherin and CDDP in inhibiting the proliferation of CDDP-R B16F10 cell line (P<0.05). T-cadherin combined with paclitaxel could inhibit the proliferation of CDDP-R B16F10 cell line (P<0.05). Factorial analysis showed that there was no interaction between T-cadherin and paclitaxel in inhibiting the proliferation of CDDP-R B16F10 cell line (P>0.05). Conclusion: The CDDP-R B16F10 cell line with stable expression of T-cadherin is established successfully. T-cadherin can reverse the CDDP resistance to CDDP-R B16F10 cell line, and promote its sensitivity to paclitaxel.