Expression of TNFAIP8 and ATP7A in cisplatin-adapted HeLa cell line

Abstract

Cisplatin is one of the most-commonly used platinum-based treatments for chemotherapy in various types of cancer. However, the high incidence of chemo-resistance has become the main limitation. TNFAIP8 (an anti-apoptotic protein) and ATP7A (a copper-efflux transporter protein) have been associated with the development of cisplatin resistance. Both proteins are highly expressed in cancer cells that are resistant to platinum drugs such as cisplatin. The objective of this study is to generate a cisplatin-adapted Hela cell line and determine the expression level of TNFAIP8 and ATP7A. The IC50 of cisplatin on parental HeLa cells was 1,75 μM as determined by MTT assay. Cisplatin-adapted cells were induced by continuously exposing the HeLa cell line to 1 and 2 μM cisplatin until stable cell growth was observed. The growth curve analysis showed that in the normal complete media, the cisplatin- adapted Hela cell line grows a bit slower as compared to the growth of the parental cell line. Cisplatin-adapted HeLa cell line showed higher IC50 compared to the parental Hela cell line by 2.4-fold as measured by trypan blue dye exclusion assay. Quantitative RT-PCR was used to measure the level of TNFAIP8 and ATP7A expression against GAPDH as the control. TNFAIP8 and ATP7A expression increased when the parental cells were treated with an incremental amount of cisplatin. The basal expression of TNFAIP8 and ATP7A is higher in cisplatin-adapted cells compare to those in the parental cell line. The expression level of TNFAIP8 and ATP7A was higher in the cisplatin- adapted cells as compared to the parental cells that were treated briefly with 1 μM and 2 μM of cisplatin. The results of this preliminary study corroborated the previous reports in the literature about the role of TNFAIP8 and ATP7A in the cancer resistance to cisplatin and suggest that the two proteins might be drug targets. Generation of cisplatin- resistance HeLa cell lines with higher IC50 can be used to screen for new cytotoxic drugs that might reverse resistance into sensitive cell lines, as well as investigating the mechanism and pathways involved in resistance and its reversal.