Multidrug resistance is one of the reasons for low survival of advanced hepatocellular carcinoma (HCC). Our previous studies indicate that the hedgehog signalling is involved in hepatic carcinogenesis, metastasis and chemo-resistance. The present study aims to uncover molecular mechanisms underlying hepatoma chemo-resistance. TAP1 and GLI1/2 gene expression was assessed in both poorly differentiated hepatoma cells and HCC specimens. Potential GLI-binding site in the TAP1 promoter sequence was validated by molecular assays. Approximately 75% HCC specimens exhibited an elevated expression of hedgehog GLI1 transcription factor compared with adjacent liver tissue. Both GLI1/2 and TAP1 protein levels were significantly elevated in poorly differentiated hepatoma cells. Both Huh-7-trans and Huh-7-DN displayed more karyotypic abnormalities and differential gene expression profiles than their native Huh-7 cells. Sensitivity to Sorafenib, doxorubicin and cisplatin was remarkably improved after either GLI1 or TAP1 gene was inhibited by an RNAi approach or by a specific GLI1/2 inhibitor, GANT61. Further experiments confirmed that hedgehog transcription factor GLI1/2 binds to the TAP1 promoter, indicating that TAP1 is one of GLI1/2 target genes. In conclusion, TAP1 is under direct transcriptional control of the hedgehog signalling. Targeting hedgehog signalling confers a novel insight into alleviating drug resistance in the treatment of refractory HCC. K E Y W O R D Schemo-resistance, GANT61, hedgehog signalling, hepatocellular carcinoma, TAP1 | 4299 ZHOU et al.
The discovery of new therapeutic drugs with the ability of preventing inflammation and joint destruction with less adverse effects is urgently needed for rheumatoid arthritis (RA). Carnosic acid (CA), a major phenolic compound isolated from the leaves of Rosemary (Rosmarinus officinalis L.), has been reported to have antioxidative and antimicrobial properties. However, its effects on RA have not been elucidated. Here, we investigated the effects of CA on osteoclasts and fibroblast-like synoviocytes in vitro and on collagen-induced arthritis (CIA) in Wistar rats in vivo. Our in vitro and in vivo studies showed that CA suppressed the expression of pro-inflammatory cytokines including TNFɑ, IL-1β, IL-6, IL-8, IL-17 and MMP-3, and downregulated the production of RANKL. More importantly, we observed that CA inhibited osteoclastogenesis and bone resorption in vitro and exerted therapeutic protection against joint destruction in vivo. Further biochemical analysis demonstrated that CA suppressed RANKL-induced activations of NF-κB and MAPKs (JNK and p38) leading to the downregulation of NFATc1. Taken together, our findings provide the convincing evidence that rosemary derived CA is a promising natural compound for the treatment of RA.
BACKGROUND: It has been demonstrated in our previous study that poorly-differentiated hepatoma cells with low α-feto-protein (AFP) expression and a CD133-/EpCAM- surface profile exhibited aberrant hedgehog signaling (Hh) activity, epithelial-mesenchymal transition and chemoresistance. However, expression of canonical Hh molecules (Sonic ligand and SMO) was not increased in poorly-differentiated hepatoma cells in contrast to well-differentiated hepatoma cells, indicating that Hh transcription factors Gli-1/2 may be activated through a non-classic pathway. The present study aims to investigate whether the TGF-β/Smad3 pathway is responsible for Gli-2 activation through a non-canonical pathway. METHODS: Expression of Sonic Hh (Shh) and Smad3 was assessed in both hepatoma cells and HCC specimens. Gli-2 and Smad3 expression was suppressed by lentiviral vector-mediated RNA interference in poorly-differentiated hepatoma Huh-7-DN and HLE cells. Chemosensitivity was evaluated in hepatoma cells with a Gli-2 or Smad3 inhibitor. RESULTS: Significantly increased Gli-1 and Gli-2 protein levels were observed in 78% (11 out of 14) and 50% (7 out of 14) HCC specimens. In 7 HCC patients with a normal serum AFP value (<20 μg/L), Gli-1/2 protein levels were obviously higher than their pericancerous tissue. Moreover, increased Gli-2 and ABCC1 protein levels were accompanied with increased TGF-β1 and p-Smad3 protein levels in 5 out 7 HCC specimens; whereas Shh protein levels were similar to paired pericancerous tissue. Consistent with the findings from HCC specimens, significantly higher Gli-2 and p-Smad3 nuclear protein levels were associated with increased ABCC1 protein levels in poorly-differentiated hepatoma cells (Huh-7-trans, Huh-7-DN, HLE and HLF); whereas Shh and SMO protein levels were not significantly different from well-differentiated hepatoma Huh-7 and Hep3B cells. Suppressing Gli-2 expression by specific shRNA caused marked decreased expression of Gli-2 and ABCC1 in Huh-7-DN, Huh-7-trans and HLE cells. In addition, expression of Smad3, Gli-2 and ABCC1 was significantly down-regulated in HLE cells after infected with a lentiviral vector harboring shSmad3, indicating that Gli-2 is a downstream target of Smad3. Sorafenib IC50 was significantly reduced from 35.76 to 17.45 μM or 14.04 μM in HLE cells when treated with a GLI inhibitor (GANT-61) or a Smad3 inhibitor (SIS3) at 5 μM. A potential GLI-binding site in the ABCC1 promoter was identified. ABCC1-Gli-luciferase reporter activity was increased up to 39-fold in HLE cells when transfected with constructs containing the potential GLI-binding site, compared to the construct that lacked a GLI-binding site. CONCLUSION: TGF-β/Smad3 activated Gli-2 and its direct transcriptional target ABCC1 in poorly-differentiated and chemoresistant hepatoma cells. Suppressing TGF-β/Smad3/Gli-2 signaling activity confers a potential molecular target in partially alleviating Sorafenib resistance for the treatment of refractory hepatoma. Citation Format: Jia Ding, Hui-yan Li, Li Zhang, Xiao-tian Zhou, Jian Wu. Non-canonical activation of Gli-2 by TGF-β/Smad3 signaling pathway in modulation of chemoresistance of hepatocellular carcinoma [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 6336.
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