Crebanine N-oxide Induces Autophagy And Apoptosis In Human Gastric Cancer Cells Via AKT/mTOR Pathway

BackgroundGastric cancer is one of the major public health issues and the leading cause of cancer-related death worldwide.As patients with early gastric cancer have no symptoms or unexpected symptoms,most cases are not diagnosed until the advanced stages of the disease.Moreover,the current therapeutic effect and prognosis are still unsatisfactory,despite multimodal approaches involving surgery,chemotherapy,and radiotherapy.Thus,the development of novel anticancer agents or adjuvants for patients with advanced gastric cancer is urgently needed.Recently,therapeutic targeting of autophagy in gastric cancer has emerged as a 'hot topic' in cancer biology.Phytochemicals have been regarded as an invaluable source of potential therapeutic agents,especially targeting many cellular signaling pathways such as apoptosis.Stephania hainanensis is traditionally used for the treatment of toothache,neuralgia and stomach pain.And it contains a variety of natural aporphine alkaloids including crebanine and crebanine N-oxide.Although several studies have demonstrated the cytotoxic effects of crebanine in several cancer cell lines,little is known about the effect and mechanism of crebanine N-oxide in cancer cells.Hence,in the present study,the cytotoxic activity of crebanine N-oxide in human gastric cancer SGC-7901 cells was investigated.ObjectiveAntitumor drug crebanine N-oxide(also oxocrebanine,OXO)isolated from Stephania hainanensis is a natural aporphine alkaloid.In this study,we investigated whether crebanine N-oxide could effectively inhibit the growth of gastric cancer cell line SGC-7901 invitro and xenograft nude mice in vivo.We also explored the molecular mechanism of crebanine N-oxide inhibiting the growth of SGC-7901 cells in terms of apoptosis and autophagy.These experiments maybe provide evidence for developing and utilizing crebanine N-oxide as a candidate drug for the treatment of gastric cancer.MethodCrebanine N-oxide was separated from Stephania hainanensis by column chromatography.The structure of crebanine N-oxide was identified by nuclear magnetic resonance method and the purity was assessed by high performance liquid chromatography(HPLC).In order to verify whether Crebanine N-oxide has an inhibitory effect on SGC-7901 cells in vitro,we performed 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide(MTT)experiments,BrdU cell proliferation assay,clone formation experiments,and morphological observation experiments.The nude mice xenograft model was employed to investigate the inhibitory effect of crebanine N-oxide on gastric cancer cell SGC-7901 in vivo.When tumor masses were palpable(about 50mm3),the nude mice were treated with crebanine N-oxide(10 mg/Kg)intraperitoneally daily for 14 days and observed another 21 days.Subsequently,the tumor tissue was completely peeled off and fixed in 4%paraformaldehyde.The expression of Ki67 in tumor specimens was detected by immunohistochemical staining after routine paraffin embedding and sectioning.In order to verify whether the relative inhibitory effect of crebanine N-oxide on SGC-7901 cell growth is involved in the induction of apoptosis,we first used the Annexin V-FITC/PI double staining kit to detect the apoptosis level of SGC-7901 cells,and then used Western blot to detect the shear levels of two effector molecules of apoptosis(Caspase-3 and PARP)and changes in apoptosis-related proteins(Mcl-1 and Bcl-2,two anti-apoptotic proteins;Bax and Bak,two pro-apoptotic proteins).In order to verify the relationship between crebanine N-oxide and autophagy,Western blot was used to detect the regulates of crebanine N-oxide on the conversion of LC3-? to LC3-? when presence or absence of autophagy inhibitor 3-MA,and the expression levels of autophagy-related proteins Beclin-1 and the level phosphorylations of proteins associated with Akt/mTOR pathways.Moreover,the formation of LC3 fluorescent spots was detected by immunofluorescence experiments,and immunohistochemical staining was used to detect LC3B expression in tumor paraffin specimens.All charts in this article are made using software GraphPad Prism.ResultsThe results of MTT experiments,BrdU cell proliferation assay,clone formation experiments,and morphological observation experiments indicate that Crebanine N-oxide can effectively inhibit the growth of human gastric cancer cell SGC-7901 in vitro.The tumor-bearing nude mice were treated with Crebanine N-oxide(10 mg/Kg)intraperitoneally daily for 14 days and observed another 21 days indicate that Crebanine N-oxide can effectively inhibit the growth of human gastric cancer cell SGC-7901 in vivo.Mechanically,the results of Annexin-V-FITC/PI double staining and Western blot detecting the cleavage levels of two effector molecules of apoptosis(Caspase-3 and PARP)and changes in apoptosis-related proteins(Mcl-1 and Bcl-2;Bax and Bak)indicate that Crebanine N-oxide relative proliferation inhibition in human gastric cancer cell SGC-7901 due to apoptosis.Meanwhile,the conversion of LC3-? to LC3-? when presence or absence of autophagy inhibitor 3-MA,and the expression levels of autophagy-related proteins Beclin-1 demonstrate that the effect of proliferation inhibition of Crebanine N-oxide was performed through obvious induction of protective autophagy.Further research found that autophagy in SGC-7901 cells induced by Crebanine N-oxide mainly achieved by inhibiting the Akt/mTOR signaling pathway.These findings suggest crebanine N-oxide as a potential agent for treating gastric cancer,the mechanism by which Crebanine N-oxide inhibits the growth of human gastric cancer cell line SGC-7901,and show that the combined treatment of Crebanine N-oxide and autophagy inhibitors can significantly improve the effect of Crebanine N-oxide against gastric cancer.Conclusion1.Crebanine N-oxide can inhibit the growth of human gastric cancer cell line SGC-7901 in vitro and in vivo,and its mechanism may be related to its induction of gastric cancer cell apoptosis.2.Crebanine N-oxide can induce autophagy in the human gastric cancer cell line SGC-7901 through the Akt/mTOR signaling pathway.This autophagy may contribute to the survival and proliferation of gastric cancer cells.