Effects Of Cucurbitacin Ⅱa On Proliferation Of Human Lung Cancer Cell Lines NCI-H460 And A549 And Its Mechanism

Background:Lung cancer is one of the most common malignant tumor which its morbidity and mortality are the first place in the global malignant tumor.Accroding to the clinicopathological organization,it can be divided into small cell lung cancer and non-small cell lung cancer and non-small cell lung cancer which accounted for 80%.Recently the treatment method of lung cancer mainly includes surgical treatment,radiotherapy,chemotherapy and targeted therapy.The five-year survival rate of lung cancer is still lower than 15%.As the lung cancer early symptom is not obvious,those who have been diagnosed are mostly mid-to-late has lost chance of the surgery.Chemotherapy is dominant of clinical therapy for lung cancer which is highly cost and greatly toxic effect.It is urgent to study new anti-tumor drugs that make cancer patients reduce the cost of treatment and improve the survival rate.In recent years,natural products have been significantly improved in anti-tumor research with the improvement of drug extraction and separation technology.Our country is richful in natural products which provides a variety of raw materials for research.CucurbitacinⅡa（CuⅡa）which is extracted from cucurbitaceae is a tetracyclic triterpene compound.The pharmacological activity of CuⅡa has the properties of anti-bacterial and anti-inflammatory.In recent years,the effect of CuⅡa on proliferation of non-small cell lung cancer A549 has been reported,but its molecular mechanism has not been elucidation.In this paper,the inhibitory effect of CuⅡa on tumor cell proliferation was confirmed and its molecular mechanism was further explored by using two kinds of non-small cell lung cancer NCI-H460 and A549 which can provide a theoretical and experimental basis for the application of CuⅡa as an antitumor drug in the clinical treatment of tumor.Objective:To study the effects of CuⅡa on proliferation of human lung cancer cell lines NCI-H460 and A549 and its mechanism.Methods:1.Treated the non-small cell lung cancer NCI-H460 and A549 with0,1.6,8,40,2000,10000 and 50000nM concentrations of CuⅡa for 48 hours,the survival was detected by the CCK-8 and the IC₅₀0 values was obtained.2.Treated with 200nM concentratios of CuⅡa for 24h,the rate of colony formation was obtained.3.Treated with 0,0.4,0.8,1.6μM concentration of CuⅡa for 12 hours,the proportion of the early apoptosis was stained by Annexin V-PE/7-AAD and detected by flow cytometry.The expression of the apoptosis related proteins caspase3,cleaved-PARP was tested by the Westerm-Blot.4.Treated with 0,0.4,0.8,1.6μM concentration CuⅡa for 48 hours,the proportion of cell cycle was stained by PI and detected by flow cytometry.5.Treated with 0,0.4,0.8,1.6 and 3.2μM concentration of CuⅡa for 12 hours,the expression and the phosphorylation level of Aurora A、STAT3 and cofilin was detected by Western-Blot.The S2270 and Ruxolitinib was positive control for P-Aurora A and P-STAT3 respectively.Results:1.The proliferation of the non-small lung cancer cell lines NCI-H460 and A549 was inhibited by CuⅡa which showed cytotoxic activity with IC₅₀0 values of 108.3 and224.9 nM against NCI-H460 and A549 respectively.2.The clone formation rate of lung cancer cell lines NCI-H460 and A549 treated with200nM concentratios of CuⅡa for 24 hours was 6.7%±2.4%and 10.1%±2.5%,respectively.3.The result of the detection of cell apoptosis which was treated with 0,0.4,0.8 and1.6μM concentration of CuⅡa for 12 hours showed that the proportion of early apoptosis of NCI-H460 increased from 4.3%to 30.3%,the proportion of apoptosis of A549 rate increased from 2.4%to 2.4%.The result of detetion by Western-Blot showed that the expression of caspase3 and cleaved-PARP was significantly increased with a dose-independence.4.The result of the detection of cell cycle which was treated by with 0,0.4,0.8 and 1.6μM concentration of CuⅡa for 48 hours showed that the proportion of G2/M phase apoptosis of NCI-H460 increased from 22.2%to 45.3%,the proportion of G2/M phase of A549 rate increased from 26.5%to 51.2%.5.The results of detetion by Western-Blot showed that and the phosphorylation level of the protein of Aurora A、STAT3 and cofilin were decreased by CuⅡa.Conclusion:1.The proliferation of lung cancer cell lines NCI-H460 and A549 was inhibited significantly by CuⅡa with a dose-dependence.2.One of the mechanism of apoptosis induced by CuⅡa is to increase the expression of caspase3 and activated caspase3 signaling pathway and to block the JAK/STAT signaling pathway by inhibiting the phosphorylation of STAT3.At the same time,it is associated with the reduction of phosphorylation of cofilin which destoryed of the cytoskeleton microstructure.3.The lung cancer cell arrested at G2/M phase by inhibited the phosphorylation of the protein kinase Aurora A by CuⅡa.