RBTI Inhibits Proliferation Of Hep G2 Cells By Up-regulating The Expression Of PTEN

Protease inhibitors are small molecular compounds or peptides and they have concentrated inhibition on corresponding proteases.They are widespread in plants,animals,and microorganisms.Besides sustaining organism internal environment stability,protease inhibitors have anti-HIV,anti-insect,anti-fungal activities and inhibite tumor cell proliferation.Buckwheat trypsin inhibitor(BTI)isolated from buckwheat seeds,belonging to the potato I-type protease inhibitors family.It is composed of 69 amino acids and the molecular weight is 7.9 kDa.The primary investigation indicated that recombinant buckwheat trypsin inhibitor(rBTI)could inhibit the proliferation of some tumor cells,such as EC9706,K562,Hep G2,Hela,and so on,and had no effect on normal hepatocyte HL7702.However,the specific molecular mechanism of rBTI inhibiting tumor cell proliferation and molecular target are not clear.In this paper,we studied the possible molecular mechanism of rBTI inhibiting Hep G2 cells proliferation.The research mainly revolved around three aspects to carry out:1.The effects of rBTI on the proliferation and cell cycle of Hep G2 cells were monitored by MTT assays and flow cytometry.The subcellular localization and the expression of PTEN and p-PTEN were determined by immunofluorescence and western blotting.And the change of factors involved in cell cycle was detected by qRT-PCR and western blotting.The results showed that rBTI could significantly inhibit the proliferation of Hep G2 cells and the cell cycle was arrested at G0/G1 phase in a dose-and time-dependent manner.Meanwhile,rBTI significantly increased the expression of PTEN in the cytoplasm and p-PTEN in the nucleus.Further study found that the overexpression of p-PTEN was co-localized with nucleolus in Hep G2 cells.The assessment of cycle-related proteins indicated that the transcription and expression of p53 and p21 were increased after treatment with rBTI.Therefore,all results show that rBTI inhibits Hep G2 cells proliferation by up-regulating the expression of PTEN to arrest cell cycle in G0/G1 phase.2.qRT-PCR was used to detect the effect of rBTI on the expression of PTEN.The change of reactive oxygen species(ROS)was measured by flow cytometry and fluorescent microscope.Western blot was used to detect the expressions of PTEN and p-PTEN.The results showed that the expression of PTEN and the level of ROS were all up-regulated when Hep G2 cells were treated with rBTI.Western blot results also showed that the expressions of PTEN and p-PTEN were elevated.While Hep G2 cell was treated with NAC,the level of ROS was decreased.Meanwhile,the expressions of PTEN and p-PTEN were down-regulated.As a result,we can conclude that the up-regulated the expression of PTEN is mediated by ROS.3.MTT assays was used to detect the effects of rBTI and autophagy inhibitor 3-MA on proliferation of Hep G2 cells.The distribution of EGFP-LC3 and the expression of autophagy-related protein,such as LC3,p62,were measured by plasmid transfection and western blot.The expression of PI3K/AKT/mTOR signaling pathway related protein was assessed by western blot.The results showed that the proliferation inhibition of Hep G2 treated with rBTI was caused by induction of autophagy.After pEGFP-LC3 transfected Hep G2 cells were treated with rBTI,the expression of EGFP-LC3 was up-regulated,as well as the aggregation state of LC3 having obvious change and formation of EGFP-LC3 punctate structures.Western blot results showed that LC3?/?ratio raised and the expression of autophagy substrate p62 was decreased,meanwhile,we found the expression of PI3K/AKT/mTOR signaling pathway related proteins,such as,p-AKT,p-mTOR were decreased when cells treated with rBTI.We also found that cells treated with rBTI combined phen had down-regulated expression of EGFP-LC3 as compared to cells treated with rBTI alone.Western blot result also showed LC3 II/I ratio was decreased and the expression of p62 was elevated.Meanwhile,the expression of p-AKT and p-mTOR were up-regulated when cells treated with rBTI combined phen.Therefore,we infer that rBTI can induce autophagy in Hep G2 cells by raising the expression of PTEN and inhibiting the pathway of PI3K/AKT/mTOR.Above all,rBTI inhibits the proliferation of Hep G2 cells by arresting cell cycle in G0/G1;rBTI stimulates the generation of cellular ROS;increased ROS elevates the expression of tumor suppressor protein PTEN;and then up-regulated PTEN affects the PI3K/AKT/mTOR signaling pathway;at last,autophagy is induced.These studies provide a theoretical foundation for this kind of protein inhibiting tumor cell proliferation at the molecular level,the molecular mechanism of them promoting cell autophagy and revealing the possible pattern of molecular interaction.