Effects Of Sinomenine On The Proliferation And Apoptosis Of Lung Cancer NCI-H460 Cells And Research On Its Mechanism

PrefaceLung cancer is the leading cause of cancer mortality in the United States and worldwide in both men and women. Although multimodality therapies and some molecular targeted therapies have been applied, the clinical responses to chemotherapy in patients with lung cancer are still unsatisfactory. The 5-year overall survival in many countries generally is less than 15%. More effective chemo preventive and therapeutic approaches are apparently needed for those patients to improve 5-year survival rate.Sinomenine (7,8-didehydro-4-hydroxyl-3,7-dimethoxy-17-meth ylmorphinan-6-one; SIN Figure 1) is a principal alkaloid isolated from the stem and root of Chinese medical plant Sinomenium acutum Rehd. et Wils (Family Menispermaceae), which has been successfully used for centuries in the treatment of various autoimmune diseases in Chinese folk medicine. Generally, SIN hydrochloride is the main chemical form for pharmaceutical purposes. Previous reports have demonstrated that SIN had a wide range of pharmacological actions, including anti-inflammatory, antirheumatic, analgesic, antiarrhythmic, anti-angiogenesis, anti-lipid-peroxidation and immunosuppressive effects.Furthermore, the antitumor effect of sinomenine has been reported in a few papers.Apoptosis, or programmed cell death is defined as an active physiological process of cell self-destruction with specific morphological and biochemical changes. Some agents suppress the proliferation of malignant cells by inducing apoptosis. The mechanism of apoptosis mainly involves the mitochondrial, endoplasmic-reticulum-specific and cell death receptor signaling pathways. The key element in the mitochondrial pathway is the efflux of cytochrome C from mitochondria to the cytosol, where it subsequently forms a complex (apoptosome) with Apaf-1 and caspase-9, leading to activation of caspase-3. The cell death receptor pathway is characterized by the binding of cell death ligands and receptors, with subsequent activation of caspase-8 and caspase-3. The endoplasmic reticulum pathway is characterized by the activation of caspase-12.In the present study we evaluated the effects of sinomenine on the growth of human lung cancer cell NCI-H460 and investigated its mechanism involved in the apoptosis induced by sinomenine. It will supply experimental basis for the futher invesitigation of anti-tumor mechanism of Sinomenine and will provide the theoretical basis for clinical treatment of lung cancer.Materials and Methods1 The research on the effects of sinomenine on the proliferation and apoptosis of lung cancer NC(?)H460 cellsThe effect of SIN on NCI-H460 cell proliferation was determined by MTT assay. Annexin V-FITC and PI double staining was used to analyze apoptosis. Morphological changes of cells was observed by TUNNEL assay. Changes of Cell Ultrastructure stucture was observed by transmission electron microscopy.2 The research on the mechanism of lung cancer cell line NCI-H460 apoptosis induced by SinomenineThe mitochondrial membrane potential (△ψm) stained with Rhodamine123 were examined using flow cytometry. Caspase-3,-8 and-9 were detected by chromogenic substrate assay. The expression of apoptosis-related proteins,such as Cytochrome C,Bcl-2,Bax were evaluated by Western blot.3 The research on the MEK/ERK和PI3K/Akt signal passway involved in the apoptosis induced by SinomenineThe expression of ERK1/2,p-ERK1/2,Akt,p-Akt were evaluated by Western blot. The apoptosis induced by signal passway blockers combined with Sinomenine were detected by PI staining used flow cytometry.Results1 Effects of sinomenine on the proliferation and apoptosis of lung cancer NCI-H460 cellsSIN inhibited cell proliferation in NCI-H460 cell lines in a concentration-dependent and time-dependent manner. Maximal proliferation inhibition was observed at 72 h with 200μg/ml SIN, which inhibited proliferation in 85.89% of NCI-H460 cells. To further confirm the induction of apoptosis by SIN, we stained cells with Annexin V /PI, TUNEL assay and TEM were performed at the same time also. The proportion of Annexin V positive cells in SIN-treated cells were increased in a dose-dependent manner(*p<0.05 vs. the control group), which supports the finding that SIN-induced NCIH-460 cell death by apoptosis. As demonstrated by the results, treatment with SIN (120μg/ml and 200μg/ml) for 48 h significantly induced the apoptotic cell death with condensed nuclei and increase of TUNEL positive cells, suggesting that the DNA fragmentation was occurring in these cells. There were no corresponding changes in the control group. TEM analysis exhibited different morphological alterations inNCI-H460 cells after treatment with SIN (200μg/ml) for 48 h. In the control group, NCI-H460 cell remained regularity morphology with large nucleus, rich in mitochondria, membrane phase structural integrity. Cell volume shrinking, intracytoplasmic vacuoles increasing, Chromatin condensation and nuclear fragmentation mitochondrial swelling were observed in treated NCI-H460 cells.2 The mechanism of lung cancer cell line NC(?)H460 apoptosis induced by SinomenineThe activities of caspase-3, caspase-8 and caspase-9 were measured using caspase-3/-8/-9 activity Assay kit. Activation of caspase-3 and caspase-9 were significantly increased after SIN-treated 24 h, while no alteration of caspase-8 activity was observed in SIN treated cells. In order to further confirm the mitochondrial involvement in SIN-induced apoptotic cell death, we examined disruption in the mitochondrial membrane potential (△ψm) and release of cytochrome C from mitochondria into cytoplasm. As compared with controls, the proportion of depolarized cells in SIN-treated cells were increased in a dose-dependent manner, cells shifted towards left in case of SIN (120μg/ml and 200μg/ml) treatment for 48 h.The result indicated that SIN treatment induce significant disruption of△ψm. Western blot analysis revealed an increase in cytosolic cytochrome C after SIN treatment in NCI-H460 cells. Relative density of cytochrome C in mitochondrial fraction was decreased for SIN treated fraction as compared to control. These results suggest that cytochrome C release is involved in SIN-induced apoptosis. The Western blot analysis Western blot analysis showed that SIN treatment leads to decrease in Bcl-2 levels; increase in Bax levels as compared to control cells. The ratios of Bax/Bcl-2 increased as the concentration of SIN increased. These results indicated that SIN up-regulation Bax protein expression and down-regulation Bcl-2 protein expression.3 MEK/ERK和PI3K/Akt signal passway involved in the apoptosis induced by SinomenineWith gradual extension of time, the expression of ERK1/2,p-ERK1/2,Akt,p-Akt were increased after treated with Sinominen. The results indicated that Sinomenine could induce MEK/ERK and PI3K-AKT pathway activation. PD98059+SIN group, LY294002+SIN group compared with the pure Sinomenine group, the apoptosis significantly increased(*p<0.05 vs. the control group). The MEK/ERK and PI3K/AKT signal passway blockers performed anti-apoptotic effect by inhibited the activition of MEK/ERK and PI3K/AKT signal passway and enhanced the induction of apoptosis effect by Sinomenine.Conclusion1 Sinomenine could inhibted the proliferation of NCI-H460 cell lines in a concentration-dependent and time-dependent manner. Sinomenine inhibit lung cancer cell line NCI-H460 proliferation through the induction of apoptosis.2 Sinomenine induced lung cancer cell line NCI-H460 apoptosis by mitochondrial pathway. Sinomenine possibly regulated the expression of Bcl-2 family protein and changed the mitochondrial membrane permeability. Cytochrome C released from mitochondria into the cytoplasm and activated the mitochondrial pathway and then induced apoptosis.3 Sinomenine induced apoptosis of NCI-H460 cells followed by the activation of MEK/ERK and PI3K/AKT signal passway. Application of MEK/ERK and the PI3K/AKT pathway blockers could antagonize its anti-apoptotic effects and improve the SIN-induced apoptosis in NCI-H460 cells.