Shikonin Inhibits The Growth Of Squamous Cell Carcinoma Of The Skin And Its Effect On PI3K/AKT Signaling Pathway

Objective:In this study,we investigated the effects of shikonin on proliferation,cell cycle and apoptosis of CSCC cells in vitro,and studied the mechanism of PI3K/AKT signaling pathway in order to clarify the inhibition of growth and related mechanisms of shikonin against CSCC.Methods: A431 cells were cultured in vitro and randomly divided into control group,low-dose shikonin group,medium-dose shikonin group and high-dose shikonin group.The proliferation activity of the above groups was detected by CCK-8 method.The cytometer detects cell cycle and apoptosis.At the same time,the expression of proliferating cell nuclear antigen(PCNA)and Cyclin B1 mRNA in each group was detected by real-time PCR.The expression of(serine/threonine kinas,AKT)and p-AKT was detected by western blot.Results: The vitality of each A431 cells group was detected on 24 h,48h,72 h by CCK-8 kit.The results of CCK-8 assay showed that the vitality of shikonin processed A431 cells was lower than it of control group and the vitality declined with time prolonging(P<0.05).There was significantly different vitality among the groups.2.Flow cytometry assayed the cell cycle of each group.The percentage of A431 cells in S phase and G2/M phase increased(P<0.05)and the ratio of G0/G1 phase decreased(P<0.05)in shikonin processed A431 cells.The percentage of medium-dose group in S phase and G2/M phase increased and the ratio of G0/G1 phase decreased more than it in low-dose group(P<0.05).The percentage of high-dose A431 cells group in S phase and G2/M phase increased and the G0/G1 phase decreased more than it in medium-dose(P<0.05).The results of flow cytometry showed that shikonin could arrest the cell cycle of A431 cells in S phase and G2/M phase(P<0.05).Dose dependent.3.Apoptosis of A431 cells was detected by flow cytometry.The apoptosis increased in shikonin processed group.The apoptosis in medium-dose shikonin group and high-dose shikonin group increased more than it in low-dose shikonin group(P<0.05).The apoptosis in high-dose shikonin group increased more than it in medium-dose shikonin group(P<0.05).The results of flow cytometry showed that shikonin could induce apoptosis of A431 cells(P<0.05).Dose dependent.4.Real-time PCR detected the expression of PCNA and Cyclin B1 mRNA in A431 cells.The expression of PCNA and Cyclin B1 mRNA was inhibited in shikonin processed A431 cells(P<0.05).The expression of PCNA and Cyclin B1 mRNA in medium-dose shikonin group and high-dose shikonin group decreased more than it in low-dose shikonin group(P<0.05).The expression of PCNA and Cyclin B1 mRNA in high-dose shikonin group decreased more than it in medium-dose shikonin group(P<0.05).5.Expression of AKT and p-AKT was detected by western blot.The expression of p-AKT in medium-dose shikonin group and high-dose shikonin group was decreased more than it in low-dose shikonin group(P<0.05).The expression of f p-AKT in high-dose shikonin group decreased more than it in medium-dose shikonin group(P<0.05).There is no significantly different expression of AKT among all groups(P>0.05).The results of western blot showed that shikonin could down-regulate the expression of p-AKT protein in cells(P<0.05).Conclusions: Shikonin can inhibit the growth of CSCC cells,and its mechanism may be related to inhibition of PI3K/AKT signaling pathway activation,and inhibition of cell proliferation and expression of cell cycle related genes PCNA and CyclinB 1.