Study On Apoptosis Mechanism Of Human Melanoma A375 Cells Induced By Shikonin

Objective: Melanoma is the deadliest form of skin cancer with a low but rising incidence,high malignancy and poor prognosis.At present,surgical clearance followed by adjuvant chemotherapy remains the best option among all therapies.Unfortunately,most patients develop primary or acquired resistance to chemotherapy,which will result in unsatisfactory curative effect.However,some natural drugs have the advantages of less side effects,mild efficacy and small adverse reactions in tumor treatment,and some have been developed into anti-tumor drugs for clinical application.Shikonin,an active component extracted from the roots of Lithospermum erythrorhizon,exerts excellent anti-tumor effects in different cancer cells.However,the exact mechanism underlying the anti-cancer effect of Shikonin in human melanoma A375 cells has not been thoroughly investigated.The study preliminarily discussed the apoptosis mechanism of human melanoma A375 cells induced by Shikonin,and aimed to provide theoretical and experimental basis for Shikonin as a potential drug in the treatment of melanoma.Methods: A375 cells were treated with different concentrations of Shikonin,the effect of Shikonin on A375 cell proliferation was determined by CCK8 assay and inverted microscope observation.Flow cytometry was used to detect the cell cycle,apoptosis,and ROS level of A375 cells,and the effect of Shikonin on apoptosis of A375 cells were detected by Hoechst 32258 staining.WB was used to analyze the levels of protein related with cell cycle,apoptosis,autophagy,ERS,and p38 pathway.In addition,Flow cytometry,WB,and CCK8 assay were used to detect the effect of inhibitors(NAC,3-MA,and SB203580)on apoptosis,autophagy,and cell viability induced by Shikonin in A375 cells.Results: 1.CCK8 assay showed that A375 cell viability decreased significantly with the increasing Shikonin concentrations.It was observed that the Shikonin obviously increased the number of dead A375 cells in a concentration dependent manner under an inverted microscope in a concentration dependent manner.2.The results of Flow cytometry showed that Shikonin significantly increased the proportion of G2/M-phase cells in a concentration dependent manner.Meanwhile,WB results showed that Shikonin reduced the expression of Cyclin B1 and increased the expression of p21 in A375 cells in a time-dependent manner.3.The results of Hoechst 32258 staining showed that more enhanced blue fluorescence could be observed in A375 cells with the increasing Shikonin concentrations under a fluorescence microscope.The results of Flow cytometry showed that Shikonin significantly induced apoptosis and ROS generation in A375 cells in a concentration dependent manner.WB results showed that Cleaved Caspase-3 was detected in A375 cells treated with Shikonin,and Shikonin increased the expression of p-e If2α and CHOP in A375 cells in a time-dependent manner,and both of the expression of these proteins and Shikonin-induced apoptosis were reversed by NAC.4.WB results showed that Shikonin increased the expression of LC3B-II,Beclin-1 and p-p38 in A375 cells in a time-dependent manner,which was reversed by NAC pretreatment.In addition,3-MA pretreatment significantly enhanced the apoptosis of A375 cells induced by Shikonin,and further inhibited the proliferation of A375 cells,which was consistent with the result of SB203580 pretreatment.Conclusions: The results above suggest that Shikonin effectively inhibits A375 cell growth by inducing G2/M phase arrest,and induces apoptosis and protective autophagy via activation of ER stress and p38 pathways,which are modulated by ROS generation.In addition,inhibition of autophagy significantly enhances apoptosis induced by Shikonin.Therefore,Shikonin can serve as a potential anti-melanoma drug,and reasonable inhibition of autophagy can effectively increase the sensitivity of A375 cells to Shikonin,which may be a promising adjuvant chemotherapy.