Study On The Inhibitory Effect Of Evodiamine On Stat3Signaling Pathway In Hepg2Cells

The activation of STAT3has been linked with the survival, proliferation,angiogenesis and immunosuppression of cancer cells. Many previous studies foundthat STAT3is constitutively activated and overexpressed in various tumor types suchas breast carcinoma, prostate cancer, melanoma, multiple myeloma, and leukemia.Agents that can suppress STAT3activation have potential to be cancer therapeutics.Evodiamine, an alkaloidal component extracted from the fruit of evodia rutaecarpa(Juss.) Benth, has anti-inflammatory effect and immunoregulatory effect. In addition,many previous studies have strongly proved that evodiamine exhibited anti-tumoractivities.In this study, Western blot, MTT assay, elctrophoretic mobility shift assay(EMSA), small interference RNA technology, real-time quantitative polymerase chainreaction (RT-qPCR) and flow cytometric detection technology were used toinvestigate the effects of evodiamine on JAK2/STAT3signaling pathway in HepG2cells and the anti-cancer effects of evodiamine in vitro. We studied the anti-tumoreffects of evodiamine on hepatocellular carcinoma in vivo by establishing the nudemice xenograft model with HepG2cells.We found that evodiamine suppressed both constitutive and IL-6-inducedactivation of STAT3effectively. The phosphorylation of JAK2, Src and ERK1/2which were upstream components of STAT3pathway were suppressed by evodiamine.Interestingly, treatment of cells with sodium pervanadate, a tyrosine phosphataseinhibitor, abrogated the inhibition of evodiamine on IL-6-induced STAT3activationindicating the involvement of a protein tyrosine phosphatase. Indeed, furtherexpirements showed that evodiamine induced the expression of SHP-1, which mayplay a key role in dephosphorylation of STAT3. Moreover, deletion of the SHP-1gene by siRNA abolished the ability of evodiamine to inhibit IL-6-induced STAT3activation. Furthermore, evodiamine upregulated mRNA levels of SOCS3, which is ainhibitory protein of STAT3signaling pathway. Evodiamine also suppressed STAT3DNA binding activity and down-regulated the expression of STAT3-mediated genes,such as Bcl-2, Mcl-1, cyclin D1, HIF1-α, MMP-9, VEGF and survivin, leading to thesuppression of proliferation, induction of apoptosis and accumulation of cells in G2/Mphase of cell cycle. In vivo, evodiamine significantly supressed tumor growth in asubcutaneous xenograft model with HepG2cells. In a word, evodiamine blocked STAT3signaling pathway by inhibiting upstreamkinases of STAT3, inducing a protein phosphatase SHP-1and a inhibitory protein ofSTAT3(SOCS-3). Eventually, evodiamine downregulated protein expressionregulated by STAT3and induced hepatocellular carcinoma cell apoptosis in vitro andexhibited anti-tumor effect in vivo. Our results indicated that evodiamine is a novelnatual inhibitor of STAT3signaling pathway and has potential to be hepatocellularcarcinoma cancer therapeutics.