The Effect And Mechanism Research On The Inhibition Of Human Glioblastoma Cell Proliferation,Invasion And Migration By Sinomenine

Glioblastoma is one of the most aggressive and malignant brain tumors in the central nervous system,and it shows substantial migration and invasion,resulting in frequent metastases into the surrounding tissues.Because of its inherent apoptosis-resistant phenotype of the malignancy and highly invasive nature,patients with glioblastoma often have a poor prognosis.Conventional cancer therapies have failed to exert positive effects on human glioblastoma.Therefore,the identification of potentially novel therapeutic agents that effectively inhibit human glioblastoma cell proliferation and metastasis is urgently needed.Autophagy is a cellular process that induces the recycling of cytoplasmic macromolecules and structures via the formation of double or multi-membrane-bound vacuoles called autophagosomes that degrade and engulf large portions of cells.Dysregulation of autophagy is related to the suppression of tumorigenesis and malignant transformation;however,its role in glioblastoma is still unclear.The currently available studies on autophagy in different cell types under various cellular conditions show conflicting evidence regarding its role in cellular death.Although the biological functions of autophagy may be context-dependent,there are many reports showing that some natural compounds or therapeutic drugs can induce caspase-independent autophagic cell death by activating autophagy signaling pathways.The level of intracellular reactive oxygen species(ROS)is increased under stress conditions,and ROS could act as signaling molecules inducing caspase-independent autophagy-mediated cancer cell death.Moreover,recent reports have indicated that the protein kinase B(Akt)-mammalian target of rapamycin(mTOR)pathway inhibition has been confirmed to activate autophagy and suppress cancer cell growth.Phosphorylation of c-Jun NH2-terminal kinase(JNK)is another crucial factor in autophagy-mediated cell death in different cancer cells.In addition to the two signaling pathways mentioned above,the lysosome has a key function in the process of autophagic flux.The major signaling molecule regulating lysosomal biogenesis is transcription factor EB(TFEB).Meanwhile,autophagy has been reported to exert both anti-and pro-metastatic effects,which may be context-dependent in cancer cells.Autophagy activation is associated with the degradation of Snail,a protein that regulates epithelial-mesenchymal transition(EMT)in breast cancer models Additionally,autophagy has been reported to be an adaptive response under endoplasmic reticulum(ER)stress conditions.Moreover,ER stress responses have recently been shown to inhibit tumor cell migration and invasion.Matrix metalloproteinases(MMPs)play a central role in the invasion process,and suppression of MMP-2/-9 expression represents a potential strategy for preventing tumor cell invasion.Nuclear factor kappa B(NF?B)is one of the major components of the intracellular signaling pathways responsible for MMP-2/-9 activation.The inflammatory microenvironment promotes EMT progression to facilitate metastasis Based on the aforementioned observations,we hypothesized that sinomenine suppresses human glioblastoma cell proliferation and metastasis by regulating these factors.The traditional Chinese medicinal plant Sinomenium acutum Rehd.et Wils.(Fam.Menispermaceae)has been employed to effectively remedy rheumatoid arthritis for centuries.Sinomenine is the major effective alkaloid derived from the plant and is widely applied in the clinical treatment of rheumatoid diseases due to its anti-immune and anti-inflammatory effects.Sinomenine hydrochloride(SH)is a hydrochloride form of sinomenine.The anti-tumor activity of sinomenine has received increasing attention,and it exerts its potent anticancer effects by enhancing apoptosis and autophagy,blocking metastasis,normalizing angiogenesis,overcoming drug resistance and coordinating with chemotherapeutic drugs.However.the underlying mechanisms of the anti-cancer effects of sinomenine remain unclear.Many studies have found that sinomenine shows positive activity in diseases of the central nervous system,such as neurodegenerative disorders,ischemia/reperfusion brain damage and experimental autoimmune encephalomyelitis,and our previous studies have shown that the prescription of CQM,whose main active ingredient is SH,has a significant analgesic effect on neurogenic pain and cancerous pain.Therefore,the aims of this study were to determine whether SH could be used to inhibit human glioblastoma cell proliferation,to investigate whether SH exerts inhibitory effects on human glioblastoma cell metastasis and to explore its potential mechanisms of actions.The results showed that SH potently inhibited U87 and SF767 cell viability and did not cause caspase-dependent cell death,as demonstrated by the absence of significant early apoptosis and caspase-3 cleavage.Instead,SH activated an autophagy-mediated cell death pathway,as indicated by the accumulated microtubule-associated protein light chain 3B(LC3B)-II,triggered autophagic flux and enhanced cell viability after pretreatment with autophagy inhibitors.SH-mediated autophagy in the two cell lines was implicated in ROS generation,Akt-mTOR pathway suppression and JNK pathway activation.The ROS antioxidant N-acetylcysteine(NAC),the Akt-specific activator insulin-like growth factor-1(IGF-1)and the JNK-specific inhibitor SP600125 attenuated SH-induced autophagy.Moreover,ROS activated autophagy via the Akt-mTOR and JNK pathways.Additionally,SH treatment may promote lysosome biogenesis through activating TFEB.The in vivo study found that SH effectively suppressed glioblastoma growth without exhibiting significant toxicity.Meanwhile,SH promoted G0/G1 phase arrest,inhibited the migration and invasion of the two cell lines,suppressed the activation ofNFKB and the expression of MMP-2/-9,triggered ER stress and inhibited the exogenous EMT induced by the inflammatory microenvironment and the endogenous EMT.Additionally,NF?B p65 overexpression blocked the SH-mediated inhibitory effects on MMP-2/-9 expression and cell invasion.SH-induced autophagy was reduced in CCAAT/enhancer binding protein(C/EBP)homologous protein(CHOP)or autophagy-related 5(ATG5)-silenced human glioblastoma cells and cells treated with 4-phenylbutyric acid(4-PBA)or 3-methyladenine(3-MA),as shown by the decreased levels of the LC3B-II and autophagic vacuoles(AVs)stained with monodansylcadaverine(MDC),respectively.Moreover,knockdown of CHOP or ATG5 and treatment with 4-PBA or 3-MA abolished the SH-mediated inhibition of mesenchymal markers(vimentin,Snail and Slug)expression and cell invasion,respectively.Importantly,SH also regulated the above related pathways in nude mice.In conclusion,this is the first time,to our knowledge,that the in vitro and in vivo anti-proliferative effects of SH on human glioblastoma have been observed,and a novel mechanism of action of SH on cancer cells in autophagy induction has been revealed.The major findings include the following:(1)SH treatment caused autophagy,but not apoptosis,in U87 and SF767 cells;(2)SH induced autophagy through the ROS-,Akt-mTOR-and JNK-dependent pathway;(3)SH may facilitate lysosome biogenesis by activating TFEB;and(4)SH inhibited the growth of human glioblastoma xenografts,without showing significant toxicity,when administered systemically.Meanwhile,this study is also the first to report that SH inhibits human glioblastoma cell proliferation by inducing cell cycle arrest and attenuates the metastasis of U87 and SF-767 cells through the following mechanisms:(1)suppressing the expression of MMP-2/-9 via NF?B inactivation;(2)reversing the endogenous EMT through ER stress-mediated autophagy;and(3)reversing the exogenous EMT stimulated by the tumor inflammatory microenvironment.Thus,these findings provide a theoretical basis for the further development of clinical applications of sinomenine in treating human glioblastoma.