Necroptosis Induced By Shikonin Is Associated With The Upgrade Of RIP1 And RIP3 In Lung Cancer

Objective: Drug resistance of lung cancer to traditional chemotherapeutic drugs mainly inducing apoptosis has become increasingly prominent.Previous studies have found that shikonin can play an anti-tumor role through programmed necrosis,so we sought whether shikonin can induce the death of lung adenocarcinoma A549 cells and clarify its mechanism.Methods: Shikonin and human lung adenocarcinoma A549 cell lines were used in this study;MTT assay was used to detect cell viability;Annexin V/PI double staining was used to detect cell death mode;Western blotting was used to detect the changes of programmed necrosis-related protein levels;DCFH-DA probe was used to detect the changes of ROS levels in cells.One-way ANOVA analysis was performed using SPSS 19.0.The count data is described by the composition ratio(%),the measurement data conforms to the normal distribution,and the mean ± standard deviation(x ± s)is used to describe the non-normal distribution of the measurement data by the median + quartile.P < 0.05 was considered to be statistically different.Results: With the increase of shikonin concentration,the mortality rate of lung adenocarcinoma A549 cells also increased.Annexin V/PI double staining showed that A549 cells treated with shikonin presented necrosis-predominant cytology.Western-blotting results showed that the expression of RIP1 and RIP3 in A549 cells pretreated with shikonin increased significantly(p < 0.001),while the application of shikonin was specific.After the inhibitors Nec-1 and Gsk,the lethal effect of shikonin on A549 was alleviated both in morphology and protein expression.The ROS expression was increased by shikonin,and NAC,Nec-1 and Gsk could effectively inhibit the effect of shikonin on reactive oxygen species(p < 0.001).Conclusions: 1.Shikonin has a killing effect on A549 cells and is related to shikonin concentration.2.Shikonin-induced A549 cell death is mainly necrosis.3.The expression of RIP1 and RIP3 was increased by shikonin,and ROS was also overexpressed in A549 cells.4.It provides a new theoretical basis for the treatment of lung adenocarcinoma.