The Effect Of Nicotine On Increase Proliferation And Variation Of PI3K/Akt Pathway In NCI-H1975 And NCI-H460 Cell

Background: Lung cancer is the most common malignant tumors witch with high mortality and low five-year survival rate. World Health Organization(WHO) divided lung cancer into non-small cell lung cancer(NSCLC) and small cell lung cancer(SCLC) according to the histological type. The causes of lung cancer is very complicated. Smoking is directly related to lung cancer especially NSCLC. Many studies have shown that nicotine, the main alkaloid in cigarettes, has no direct carcinogenic effect but has tumor promotion and cells proliferation effect. Phosphoinositide 3-kinase(PI3K) is important kinase of inositol and phosphatidylinositol(PI). PI3 K activation causes a conformational change and phosphorylated of Akt protein causing its activation. Phosphorylated Akt can activate or inhibit downstream target proteins like Bad, Caspase 9, NF-kappa B, Forkhead, m TOR to promote cell survival.PI3K/Akt signaling pathway plays an important role in cell proliferation and survival, many kind of tumors existing PI3K/Akt signaling pathway excessive activation. The basic idea of this study is to observe the different work of nicotine on different pathological NSCLCs and PI3K/Akt pathway to search for possible mechanisms of nicotine reaches a difference role in different pathological origin NSCLCs, looking for different treatment ideas for different pathological types NSCLC.Objective: To observe the different work of nicotine on different pathological NSCLCs and PI3K/Akt pathway to search for possible mechanisms of nicotine reaches a difference role in different pathological origin NSCLCs, looking for different treatment ideas for different pathological types NSCLC.Methods: Use the cell counting kit-8(CCK-8) and flow cytometry test nicotine influences on NCI-H1975 cell and NCI-H460 cell proliferation and apoptosis inhibition. Use Westernblot and RT-PCR technique to detect changes of PI3K/Akt pathway key genes m RNA and protein level of NCI-H1975/NCI-H460 cell after nicotine treated, suggesting possible mechanism of different changes of different pathological types NSCLC cells after nicotine treated.Results: 1、By CCK-8 detection, nicotine promoted NCI-H1975/NCI-H460 cells proliferation and the control group with a statistically significant difference(P<0.05), both have time-dependent(NCI-H1975, y=102.5e0.056 x, R2=0.982; NCI-H460, y=102.5e0.056 x, R2=0.976) and dose-dependent(NCI-H1975, y=11.72ln(X)+100.4, R2=0.994; NCI-H460, y=10.90ln(x)+99.68, R2=0.996), but the NCI-H1975 cell more responsive to nicotine. 2、By flow cytometry detection, nicotine can significantly reduce NCI-H1975/ NCI-H460 cells apoptosis caused by cisplatin(P<0.05), and both have time-dependent(NCI-H1975, y=66.48x0.84, R2=0.979; NCI-H460, y=63.98x0.84, R2=0.985), but the NCI-H1975 more sensitive to nicotine. 3、By western blot and RT-PCR detection, NCI-H1975 cells PI3 K gene m RNA level increased, Bad/Caspase 9/Fox O3a/m TOR genes m RNA levels decreased, PI3K/p-Akt/p-Bad/p-Caspase 9/p-Fox O3a/m TOR protein levels increased, Bad/Fox O3a/m TOR protein levels decreased, with the increase of concentration of nicotine. NCI-H1975 cells PI3 K gene m RNA level increased, Bad/Fox O3a/m TOR m RNA levels decreased, PI3 K protein level increased, Bad/Fox O3a/m TOR protein levels decreased, with the increase of work time of nicotine. NCI-H460 cells PI3 K gene m RNA level increased, Bad/Fox O3a/m TOR genes m RNA levels decreased, PI3K/p-Akt/p-Bad/m TOR protein levels increased, Bad/Fox O3a/m TOR protein levels decreased, with the increase of concentration of nicotine. NCI-H460 cells PI3 K gene m RNA level increased, Bad/Fox O3a/m TOR m RNA levels decreased, PI3 K protein level increased, Bad/Fox O3a/m TOR protein levels decreased, with the increase of work time of nicotine. 4、Nicotine have different work on Caspase 9 and Fox O3 a of NCI-H1975/NCI-H460 cells.Conclusions: 1、Nicotine promotes NCI-H1975/NCI-H460 cells proliferation and inhibit apoptosis, and the effect was dose-dependent and time-dependent.2、NCI-H1975/NCI-H460 cells have different sensitivity to nicotine. 3、Nicotine may be adjusted NCI-H1975/NCI-H460 cells proliferation and apoptosis by regulating the PI3K/Akt pathway downstream of the multiple genes m RNA levels, protein levels and phosphorylation levels. 4、NCI-H1975/NCI-H460 cells differet sensitivity to nicotine may due to Caspase 9 and Fox O3 a two proteins.