Exploring The Anticancer Mechanisms Of Scutellaria Barbata Extract Targeting STAT3Signaling Pathway

Scutellaria barbata is a clinically widely used detoxification herb, which is also widely used for the adjuvant therapy of various cancers. Modern studies also proved Scutellaria barbata extract can effectively inhibit a variety of cancer cell growth and survival, and cause apoptosis, while influences of Scutellaria barbata on tumor-associated signaling pathways have yet been reported. STAT3signaling pathway is one of the cancer-promoting signaling pathways that have been well understood, which is invoved in almost all the key steps in cancer development and progression. Numerous studies have demonstrated that inhibition of STAT3signaling pathway can significantly inhibit cancer cell survival, proliferation and metastasis. Therefore, exploring the inhibitory effect of Scutellaria barbata extract on STAT3signaling pathway will be helpful to understand the molecular mechanism of its anti-cancer activity.Firstly, we performed alcohol extraction and solvent extraction of the whole barbata plant, by which we got different extraction components. Western Blotting tests showed that the ethyl acetate phase of Scutellaria barbata extract (BE) showed a specific inhibition on the tyrosine phosphorylation of STAT3. What’s more, BE can selectively inhibit the proliferation of STAT3-dependent DU145human prostate cancer cells, while exhibited little inhibitory effect on cancer cells and normal-derived cells that have low levels of constitutive STAT3activation, which primarily proved that BE could exert its anti-cancer activity through inhibiting the STAT3signaling pathway. By analyzing the chemical composition of BE by HPLC tests, we found two known flavonoid STAT3inhibitor, the Luteolin and Apigenin, but their superimposed effect on signal and growth inhibition was not better than BE, which indicates that the efficient inhibition of STAT3signal by Scutellaria barbata is not a simple superposition from single chemical compositions.Based on the above results, the in vitro signal inhibitory activity and anti-cancer effects of BE were verified at the cellular level. In STAT3-dependent DU145cells, BE significantly inhibited the constitutive activation of STAT3in time-and dose-dependent manners, and apparent inhibition occurred at15min when treatment with50μg/mL concentration; In LNCaP cells that have low levels of constitutive STAT3activation, pretreatment with10μg/mL BE for2h could significantly hold back the IL-6activated STAT3phosphorylation, and also in a dose dependent manner; BE also strongly inhibited IL-6-stimulated STAT3nuclear translocation. BE also down-regulated STAT3mediated cell cycle related proteins (CDK2and cyclin B) and anti-apoptotic proteins (Bc1-2, Bcl-XL and Survivin), leading to G2/M-phase arrest and Caspase-dependent apoptosis. In the classic STAT3signaling pathway, STAT3activation is often dependent on the activation of its upstream JAK kinase. Further studies have shown that BE can effectively inhibit both the constitutive and IL-6-activated phosphorylation of JAK2. What’s more, apparent p-JAK2inhibition by BE appeared earlier than the p-STAT3inhibition, and the concentrations causing significant p-JAK2is also lower than that of p-STAT3inhibition, indicating that the BE induced STAT3signal blockage may be dependent on JAK2inhibition.To further verify the STAT3signal suppression and anti-cancer effects of BE, we constructed a BABC/c-4T1mouse breast cancer model, and used both the prevention (low-dose gavage before tumor cell inoculation) and treatment (high-dose gavage after tumor formation) administration to evaluate the in vivo anti-tumor activity of BE. Both prophylactic and therapeutic BE administration could significantly inhibit the growth of4T1tumors, with inhibition rate reached45.1%and31.6%respectively, and prophylaxis can play better inhibitory effect. At the molecular level, the serum CA153, a specific breast cancer marker, was significantly reduced in BE-treated mice; Immunohistochemistry and analysis of tumor tissue proteins by Western Blotting showed a significant reduction of KI-67and Survivin, and an increase of Caspase-3activation by BE. Immunostained frozen sections and immunohistochemistry jointly proved that BE also significantly inhibited the expression and nuclear translocation of p-STAT3in tumor tissues. This more potently proved the STAT3targeted anti-cancer effect of Scutellaria barbata.In summary, by in vitro and in vivo tests, we proved that BE could effectively suppress the JAK2/STAT3signal activation, thereby inhibiting STAT3-dependent tumor growth and promote apoptosis. These findings laid a foundation for further exploring the anticancer mechanism of Scutellaria barbata extracts, and provided a new theoretical basis for modernization of Chinese herbal medicine, and subsequent active ingredients analysis is continuing on research.