The Inhibitory Effect Of The Combination Of Shikonin And Cisplatin In Non-small Cell Lung Cancer And Its Mechanism

BackgroundLung cancer is the major diseases that threaten human health, has the highest morbidity and mortality, while China has become the most serious country with lung cancer [1]. Although a variety of methods were used to treat lung cancer, such as surgical resection, chemotherapy, immunotherapy, biological therapy and molecular targeted, but the prognosis is poor,5-year survival rate is less than15%. Cisplatin as a chemotherapy drug is widely used to treat lung cancer, but its clinical application is limited due to its toxicity, poor anti-tumor selectivity, and prone to drug resistance after treatment.Shikonin mainly is from medicine comfrey, The current study is focused on its anti-tumor effect, the previous study found Shikonin may inhibit tumor cell growth by inducing tumor apoptosis, another study confirmed that shikonin can downregulation intergrinβl expression in A549cells thereby inhibiting cell adhesion, invasion and metastasis [2].PurposeIn this experiment, non-small cell lung cancer A549cells is for the study, observe the effect of Shikonin in combination with cisplatin on A549cell line, explore the inhibition mechanism of Shikonin in combination with cisplatin of on A549cells, provide experimental basis for shikonin’s clinical application.MethodMTT assay is used to screen optimum concentration of Shikonin which in combination of cisplatin, MTT assay is used to detect cell viability, flow cytometry to detect cell apoptosis and cell cycle, the mRNA of cyclin D1, PCNA, Rb and p16expression were detected by RT-PCR, immunofluorescence detected cyclin D1, PCNA, Rb and p16protein expression, Western Blot detected XIAP and Survivin protein expression. Result(1) The results of MTT assay show that The two-drug combination group showed more significant inhibitory effect, which Shikonin+5umol/L CDDP group showed the strongest inhibitory effect at72h, the inhibition rate was88.3%.(2) The flow cytometry results show that the group of Shikonin incombination of cisplatin can effectively induce apoptosis of A549cells’s apostosis, its apoptosis rate was significantly higher than the control group, CDDP group and L-Shikonin groups.(3) The results of cell cycle detected by flow cytometry showed that the percentage of cells in G0/G1phase of the combination group are significantly increased, the ratio of S phase were reduced, but had no significant effect on the G2/M phase (P<0.05).(4) The results RT-PCR showed that the mRNA expression levels of cyclin D1, PCNA, and Rb were significantly reduced, while p16mRNA expression was significantly increased, and two drugs combination group has the most obvious changes.(5) The result of immunofluorescence analysis showed that the protein of cyclinDl, PCNA, and Rb was significantly decreased, while the p16protein was significantly increased, and the combined group has the most obvious changes (P<0.05).(6) Western Blot test results showed the combined group can significantly reduced XIAP and Survivin protein expression (P<0.05).ConclusionShikonin can inhibit the proliferation of A549cells, Shikonin in combination with cisplatin could significantly inhibit the proliferation of A549cells, enhanced its effect of apoptosis induction, better than cisplatin alone group and Shikonin single-use group, and its mechanism may be:(1) by reducing cell cyclin D1, Rb,PCNA mRNA expression and protein expression, increased p16mRNA and protein expression, reduce inactivation of Rb phosphorylation, prevents cells into S phase, thereby inhibiting the proliferation;(2) by downregulated XIAP and Survivin protein expression to promote apoptosis.