Erastin Combined With Cisplatin Induce Ferroptosis In Cisplatin-resistant Nasopharyngeal Carcinoma Cells

Objective:As one of the basic treatment methods of nasopharyngeal carcinoma,chemotherapy has played a great role in clinical treatment.However,due to the different drug sensitivity of individuals,the occurrence of drug resistance need to be solved.Nowadays,relevant studies have confirmed that eratin,a small molecule that induces ferroptosis,can well enhance the therapeutic effect of traditional anticancer drugs such as azithromycin,cisplatin,cytarabine and temozolomide on some cancer cells,but the research of erastin on cisplatin-resistant nasopharyngeal carcinoma is still unclear.The purpose of this experiment is to study the chemosensitization effect of erastin on drug-resistant nasopharyngeal carcinoma cells,and to explore whether erastin combined with cisplatin（DDP）can induce the death of cisplatin resistant nasopharyngeal carcinoma cell lines,so as to provide a new idea for the selection of therapeutic drugs for nasopharyngeal carcinoma。Methods:The cisplatin resistant HNE-1 nasopharyngeal carcinoma cell line was cultured in 1640 medium contained cisplatin to maintain its resistance,and the sensitivity of common nasopharyngeal carcinoma cell lines such as HNE-1,Cen2z,HONE-1 and cisplatin-resistant cell line HNE-1/DDP to cisplatin was detected by CCK-8 method.The activity of each cell lines was observed to determine the cisplatin-resistant cell line HNE-1/DDP for subsequent experiments.CCK-8 method was used to detect the sensitivity of nasopharyngeal carcinoma common cell lines HNE-1,Cen2z,HONE-1 and HNE-1/DDP to erastin,determine whether the cisplatin-resistant cell lines are sensitive to erastin,and observe whether they can have anti-tumor effect on the cisplatin-resistant cell line.Erastin and DDP were combined to treat HNE-1/DDP,and then the changes of HNE-1/DDP cell activity were measured by CCK-8 method.In order to explore the specific role of erastin,the cell death of different treatments such as control group,DDP group,erastin group,DDP+erastin group,erastin+ferrostatin-1 group and erastin+DDP+ferrostatin-1 group were observed by flow cytometry of PI.In order to further verify whether erastin combined with DDP induced ferroptosis in cisplatin-resistant cell line,the changes of ROS levels in different groups of cells were detected by flow cytometry,Fe~2+and malondialdehyde（MDA）kit was used to detect the changes of Fe~2+and MDA level.Finally,in order to explore the mechanism of the anti-tumor effect of erastin combined with DDP on HNE-1/DDP,the m RNA and protein expression of ferroptosis related genes were detected by q RT-PCR and Western blot respectively,and the migration effect of erastin combined with DDP on HNE-1/DDP was verified by scratch test.Result:DDP and erastin significantly decreased the viability of common nasopharyngeal carcinoma cell lines in a dose-dependent manner,while the sensitivity of HNE-1/DDP to DDP was much lower than that of common nasopharyngeal carcinoma cell lines,and its IC50 was as high as 49.42±7.04μg/ml,which verified the formation of cisplatin-resistant cell line,and the sensitivity of cisplatin-resistant cell line to erastin was much lower than that of other ordinary cells.After combining erastin with DDP,the IC50 of erastin in HNE-1/DDP cells decreased from 45.89±6.89μmol/L to 17.56±4.07μmol/L（P=0.004）,suggesting that the combined treatment can indeed enhance the anti-tumor ability,which is also confirmed by the results of PI staining in flow cytometry.Compared with the death rate of HNE-1/DDP treated with erastin or DDP alone（more than 30%）,the death rate of HNE-1/DDP cells treated with erastin combined with cisplatin was 89.69%±9.48%,and ferroptosis inhibitor ferrostatin-1 can reverse the cell death induced by erastin combined with DDP,and reduce the mortality to 35.81%±8.2%,which further suggests that the anti-tumor ability of combined treatment is greatly enhanced compared with that of single treatment.The subsequent results show that when treated alone,the increase of ROS level is no more than 15%,while combined,the level of ROS is increased to 18.72%±3.05%.The combined treatment also increased the levels of intracellular Fe²⁺and MDA,and these changes could be reversed by ferroptosis inhibitor ferrostatin-1,which reduced the levels of intracellular ROS,Fe²⁺and MDA,which further confirmed the occurrence of ferroptosis.The results of q RT-PCR showed that the expression of ferroptosis related genes x CT and GPX4decreased and the the expression of antioxidant gene NRF2 and HO-1increased after the combined treatment,while the the results of Western Blot experiment showed that the protein expression level of x CT did not decrease significantly,suggesting that the combined treatment decreased the transcription of x CT,but it did not reduce the protein expression level by compensation in the end,and so do the NRF2,the whole protein level don’t change much as the m RNA level changed.The results of GPX4and HO-1 protein expression were consistent with that of q RT-PCR,and ferrostatin-1could also reverse the decrease of GPX4 expression and the increase of the HO-1.The results of scratch test showed that the combined treatment also reduced the migration ability of cisplatin-resistant cell line.Conclusion:The results of this study show that compared with cisplatin alone,erastin combined with cisplatin can significantly induce the death of cisplatin resistant cell line HNE-1/DDP and reduce its migration ability,and it is preliminarily verified that it is ferroptosis.NRF2-GPX4/HO-1 is involved in the process of ferroptosis,which means erastin is expected to be useful in the treatment of nasopharyngeal carcinoma.