Whether cytidine deaminase of Mycoplasma hyorhinis promotes drug resistance by decomposing gefitinib

Abstract

Abstract Objective: To investigate whether cytidine deaminase (CDD) of Mycoplasma hyorhinis induces gefitinib resistance in epidermal growth factor receptor (EGFR)-mutated lung cancer cells by decomposing gefitinib. Method: Download the gene sequence of CDD from NCBI (Gene ID: 61335421) and redesign the CDD sequence. The prokaryotic expression vector and eukaryotic expression vector of CDD were constructed respectively. The H1650 cell line with stable expression of CDD(H1650-CDD) was obtained by lentiviral infection and verified by western blotting. The toxicity of gefitinib to different cells was detected by CCK8 assay. The prokaryotic expression vector was transformed into escherichia coli, through protein induction and protein purification, CDD protein was obtained. High performance liquid chromatography (HPLC) was used to detect whether the CDD can decompose gefitinib. Results: The prokaryotic expression vector and the eukaryotic expression vector were successfully constructed by molecular cloning, and the H1650-CDD cell line was obtained by lentiviral infection. The cytotoxicity of gefitinib on H1650-CDD cells and H1650wt cells was detected by CCK8 assay. The cell viability of H1650-CDD cells show significant differences with H1650wt in 30 (t=4.223, P= 0.0134), 40 (t=15.05, P=0.0001), 50 (t=2.919, P= 0.0433), 60 (t=12.28, P= 0.0003), 70 (t=22.97, P<0.0001), 80 (t=6.648, P= 0.0027) μmol/L gefitinib; HPLC suggests that there was no difference among the control group, the CDD group and the gefitinib group. Conclusion: H1650-CDD cells were resistant to gefitinib, but CDD protein can’t decompose gefitinib. Since the common mechanism of gefitinib resistance includes changes in EGFR downstream signals, it is speculated that the mechanism of CDD promoting drug resistance in H1650-CDD cells may be related to cell signaling pathway, which requires further study.