| The lethality rate of Hepatocellar carcinoma (HCC) is ranked at the second place with low early diagnosis rate and local invasion and metastasis before it is confirmed. Tumor recurrence and metastasis are the main reasons cause death of patients with cancer. EMT is thought to be an important biological process that plays a critical role in tumor cell invasion or metastasis, and has been actively investigated in HCC. A number of studies have highlighted the role of inflammatory tumor microenvironment in promoting tumor metastasis. Interleukin-6 (IL-6)-mediated activation of Janus kinases/signal transducer and activator of transcription 3 (JAK/STAT3) signaling is frequently presented in human cancer EMT and metastasis. According to clinical and experimental studies, Traditional Chinese Medicine (TCM) has an obvious advantage on preventing tumor recurrence and metastasis. It also inhibits the formation of tumor EMT, However, the precise molecular events and underlying mechanisms that initiate this complex process are poorly understood and its elucidation could lead to further understanding of the role of TCM.Objective:To investigate the underlying mechanisms of modified of Biejia decoction in the process of EMT in HCC.Methods:1. To explore the effects of various concentrations BJJ on hepatocellular carcinoma cell (HCCLM3) proflifration and apotosis through MTT and Annexin V-FITC/PI.2. To investigate whether IL-6 could induce EMT in hepatocellular carcinoma cell, We established the model of IL-6 induced EMT in vitro in HCCLM3 and treated the HCCLM3 cell lined, which exhibite an epithelial phenotype. We addressed the expression of epithelial marker, E-cadherin, and the mesenchymal markers, N-cadherin and vimentin. Then, we used a JAK2/STAT3 specific inhibitor Cucurbitacin I (JSI-124) to suppress the activation of STAT3. To explore the underlying mechanisms on IL-6 induced EMT, we determined its effect on the activation of the JAK2/STAT3/TWIST signaling pathway. The expression of STAT3, phosphorylation of STAT3 (p-STAT3) and TWIST after IL-6 treatment were analyzed by Western Blot and qRT-PCR.3. To investigate the effects of various concentrations BJJ treatment on IL-6 induced EMT and the JAK2/STAT3/TWIST signaling pathway in HCCLM3 through the model of IL-6 induced EMT in vitro. The cells were treated with 100ng/ml IL-6 for 48h, western blot and qRT-PCR analysis showed that the expression of epithelial marker, E-cadherin, and mesenchymal markers, N-cadherin and vimentin following incubation with various BJJ concentrations and JSI-124. To explore the underlying mechanisms of how BJJ on IL-6 induced EMT, we determined its effect on the activation of the JAK2/STAT3/TWIST pathway. The expression of JAK2, phosphorylation of JAK2 (p-JAK2), STAT3, phosphorylation of STAT3 (p-STAT3) and TWIST after IL-6 treatment from the different concentration groups of BJJ treatment were analyzed by Western Blot and qRT-PCR.4. To investigate the effects of the various concentrations BJJ treatment and JSI-124 on EMT and the JAK2/STAT3/TWIST signal pathway in different stages(14d and 21d) in vivo. We established the model of subcutaneously implanted human HCCLM3 spontaneous lung metastasis in nude mice. Immunohistochemistry analysis showed that the location of epithelial marker, E-cadherin, and mesenchymal markers, N-cadherin and vimentin expression. Western blot and qRT-PCR analysis showed that the expression of epithelial marker, E-cadherin, and mesenchymal markers, N-cadherin and vimentin following intervation in different stages with various BJJ concentrations and JSI-124. To explore the underlying mechanisms of inhibitory activities of BJJ on HCC during the process of EMT, we determined its effect on the activation of the JAK2/STAT3/TWIST pathway. The expression of phosphorylation of JAK2, JAK2 (p-JAK2), STAT3, phosphorylation of STAT3 (p-STAT3) and TWIST from the different concentration groups of BJJ and JSI-124 treatment were analyzed by Western Blot and qRT-PCR.5. To investigate the effects of the different concentration BJJ treatment on the invasion of HCCLM3 and lung metastasis. The effects of the various concentrations BJJ and JSI-124 on IL-6 induced cell invasion in HCCLM3 were investigated by the Transwell assays in vitro. The effects of the different concentration BJJ and JSI-124 on lung metatstasis were investigated by the hematoxylin-eosin staining in lung.Results:1. The effects of the different concentration BJJ on hepatocellular carcinoma cell proflifration, the results showed that the different concentration BJJ treatment HCCLM3 after 24h has little effect on cell proliferation, the low and midlle dose BJJ compared with the Control group was not statistically significant(P>0.05). After treatment 48h with BJJ, the cell proliferation rate of the high and middle dose group were 87.86% and 84.60% respectively, and the cell proliferation rate of the high and middle dose group (BJJH and BJJM), compared with the Control group were statistically significant(P<0.05).2. The effects of the different concentration BJJ on hepatocellular carcinoma cell apotosis. The results showed that the low dose BJJL (BJJL) treatment HCCLM3 after 24h has little effect on cell apotosis, the difference compared with control was not statistically significant (P>0.05), but the middle, high doses BJJ (BJJL, BJJH) and JSI-124 (0.5μmol/l) apoptosis rate were 7.90%,12.22% and 15.36% respectively with significantly difference, compared with control(P<0.05). After treatment 48h, the low, middle, high doses BJJ (BJJL, BJJM, BJJH) and JSI-124(0.5μmol/l) apoptosis rate were 8.08%,11.95%,13.89% and 19.02% respectively, compared with the Control group, the difference was statistically significant(P<0.05).3. The cells treatment with 100ng/ml IL-6 for 24,48h, the results demonstrated that E-cadherin expression was significantly repressed, and N-cadherin and Vimentin expression were elevated with IL-6 treatment in HCCLM3 as compared to control group (absence of IL-6) (P<0.05). To test the underlying mechanism, we used a JAK2/STAT3 specific inhibitor Cucurbitacin I(JSI-124) to suppress the activation of STAT3. JSI-124 reduced STAT3 phosphorylation and TWIST expression induced by IL-6 in the process of EMT. IL-6 enhanced the invasion abilities of HCCLM3 and JAK2/STAT3 inhibitor (JSI-124) suppressed cell invasion abilities after treatment with IL-6.4. To investigate the effect of BJJ treatment on IL-6 induced EMT in HCCLM3, the cells were pretreated with 100ng/ml IL-6 for 48h, and then following incubation with various BJJ concentrations and JSI-124 for 48h. Western blot and qRT-PCR analysis showed that the expression of epithelial marker, E-cadherin was increasingly elevated in IL-6+BJJH and IL-6+JSI-124 groups, as compared to IL-6+, while mesenchymal markers, the expression of N-cadherin and vimentin were significantly downregulated in vitro(P<0.05). After treatment with various concentrations BJJ and JSI-124 in different stages(14d,21d), IHC, Western blot and qRT-PCR analysis showed that the expression of epithelial marker, E-cadherin was increasingly elevated in BJJH and JSI-124 groups, as compared to Control group, while mesenchymal markers, the expression of N-cadherin and vimentin were significantly downregulated in vivo(P<0.05).5. Transwell assays showed that IL-6+BJJM, IL-6+BJJH and IL-6+JSI-124 groups attenunated the invasion abilities of HCCLM3, compared with IL-6+(P<0.05). JAK2/STAT3 inhibitor(JSI-124) suppressed IL-6 induced cell invasion. The HE staining in lung results showed that BJJ suppressed lung metastasis in a time and dose-dependent manner.6. To investigate the underlying mechanism of BJJ treatment on IL-6 induced EMT in HCCLM3, the cells were pretreated with 100ng/ml IL-6 for 48h, and then following incubation with various BJJ concentrations and JSI-124 for 48h. Western blot and qRT-PCR analysis showed that the expression of JAK2, phosphorylation of JAK2(p-JAK2), STAT3, phosphorylation of STAT3(p-STAT3) and TWIST were significantly downregulated in IL-6+BJJH and IL-6+JSI-124 groups as compared to IL-6+(P<0.05). After treatment with various concentrations BJJ and JSI-124 in different stages (14d,21d), IHC, Western blot and qRT-PCR analysis showed that the expression of IL-6R, JAK2, STAT3, p-JAK2, p-STAT3 and TWIST were significantly downregulated in BJJH and JSI-124 groups as compared to Control group in vivo(P<0.05).Conclusions:1. The effects of the various concentrations BJJ on hepatocellular carcinoma cell proflifration and apotosis rate were in a dose-dependent manner in vitro.2. The model of IL-6 induced EMT in HCCLM3 was established in vitro and we demonstrated that IL-6 stimulated EMT in a time and dose-dependent manner accompanied by downregulation of E-cadherin and upregulation of N-cadherin and Vimentin in HCCLM3. JAK2/STAT3/TWIST may be activated in IL-6 induced EMT change. In addition, IL-6 mediated activation of JAK2/STAT3 implicated in EMT and the cell invasion ability was enhanced.3. The high dose of BJJ (BJJH) significantly hindered the process of EMT induced by IL-6 in HCCLM3, and reduced the invasion and lung metastasis abilities of HCCLM3.4. The high dose of BJJ(BJJH) significantly inhibited the activation of IL-6/JAK2/STAT3, and the EMT-related transcription factor TWIST was downregulated. The BJJ could block EMT via JAK2/STAT3/TWIST signaling pathway, which is an important mechanism underlying HCC development and metastasis.
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