gant-61 and Liver-Neoplasms

Lenvatinib has been approved as a first-line treatment for advanced hepatocellular carcinoma (HCC) in recent years. However, Lenvatinib resistance hinders its therapeutic effect, and the underlying mechanism of action of Lenvatinib needs to be better understood. Increasing studies have suggested that cancer stem cells (CSCs) are an important driving force. Hedgehog signalling is important for the maintenance of hepatocellular carcinoma stemness. In the present study, we investigated the therapeutic role of the Hedgehog signalling inhibitor in reversing Lenvatinib resistance in CD133-positive HCC cells. First, we examined the inhibitory impact of Lenvatinib against CD133 expression in HCC cell lines through Western blot. The CCK8 assay showed that GANT61, a Hedgehog signalling inhibitor, has a suppression advantage over other CSCs-related signalling inhibitors regarding cell viability. Moreover, Lenvatinib and GANT61 combined had better inhibitory effects on cell viability and malignant properties, both in vivo and in vitro. In addition, GANT61 reversed the upregulation of CD133 and Hedgehog signalling caused by Lenvatinib in SK-Hep-1 and MHCC97H. Thus, our results suggested that GANT61 reversed Lenvatinib resistance by suppressing Hedgehog signalling in HCC cells, especially in CD133-positive cells and combining Lenvatinib with Hedgehog signalling inhibitors could improve its therapeutic efficacy in HCC patients with high CD133 expression levels.

Topics: AC133 Antigen; Animals; Antineoplastic Combined Chemotherapy Protocols; Carcinoma, Hepatocellular; Cell Line, Tumor; Cell Proliferation; Drug Resistance, Neoplasm; Hedgehog Proteins; Humans; Liver Neoplasms; Mice; Phenylurea Compounds; Pyridines; Pyrimidines; Quinolines; Signal Transduction; Up-Regulation; Xenograft Model Antitumor Assays

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.

Topics: ATP Binding Cassette Transporter, Subfamily B, Member 2; Carcinogenesis; Carcinoma, Hepatocellular; Cell Line, Tumor; DNA-Binding Proteins; Drug Resistance, Neoplasm; Female; Gene Expression Regulation, Neoplastic; Hedgehog Proteins; Humans; Liver Neoplasms; Male; Nuclear Proteins; Promoter Regions, Genetic; Pyridines; Pyrimidines; Signal Transduction; Zinc Finger Protein GLI1; Zinc Finger Protein Gli2

The Hedgehog (HH)-GLI pathway plays an important role in cell dedifferentiation and is therefore pivotally involved in the malignant transformation of cancer cells. GANT61, a selective inhibitor of GLI1 and GLI2, was reported as a promising treatment for cancer in various tissues; however, the biological impact of GANT61 in hepatocellular carcinoma (HCC), especially in undifferentiated HCC cells, remains unclear. In this study, we investigated the antitumor effect of GANT61 using two undifferentiated hepatoma cell lines: HLE and HLF. Quantitative PCR and RT-PCR analyses revealed that these cells express GLI transcripts, showing mesenchymal phenotypes characterized by the loss of epithelial and hepatic markers and specific expression of epithelial-mesenchymal transition (EMT)-related genes. GANT61 significantly reduced the proliferation and cell viability after drug treatment using 5-FU and Mitomycin C. We showed that GLI transcript levels were down-regulated by the MEK inhibitor U0126 and the Raf inhibitor sorafenib, suggesting that non-canonical signaling including the Ras-Raf-MEK-ERK pathway is involved. Sphere formation and migration were significantly decreased by GANT61 treatment, and it is suggested that the underlying molecular mechanisms are the down-regulation of stemness-related genes (Oct4, Bmi1, CD44, and ALDH) and the EMT-related gene Snail1. The data presented here showed that direct inhibition of GLI might be beneficial for the treatment of dedifferentiated HCC.

Topics: Carcinoma, Hepatocellular; Cell Line, Tumor; Cell Proliferation; Cell Survival; Epithelial-Mesenchymal Transition; Gene Expression Regulation, Neoplastic; Hep G2 Cells; Humans; Liver Neoplasms; Nuclear Proteins; Pyridines; Pyrimidines; Signal Transduction; Up-Regulation; Zinc Finger Protein GLI1; Zinc Finger Protein Gli2

Cholangiocarcinoma represents the second most common primary liver tumor after hepatocellular carcinoma. Mahanine, a carbazole alkaloid derived from Murraya koenigii (Linn.) Spreng, has been used as folk medicine in Thailand, where the liver fluke-associated cholangiocarcinoma is common. The expression of microphthalmia-associated transcription factor (MITF) is maintained at immunohistochemically undetectable levels in hepatocytes and cholangiocytes. To explore the regulation of MITF expression in the liver, we immunohistochemically analyzed the MITF expression using hepatocellular carcinoma and cholangiocarcinoma specimens of the human liver cancer tissue array. MITF immunoreactivity was detected in subsets of hepatocellular carcinoma (6 out of 38 specimens; 16%) and cholangiocarcinoma (2/7 specimens; 29%). Moreover, immunoreactivity for glioma-associated oncogene 1 (GLI1), a transcription factor of the Hedgehog signaling pathway, was detected in 55% of hepatocellular carcinoma (21/38 specimens) and 86% of cholangiocarcinoma (6/7 specimens). Importantly, MITF was detectable only in the GLI1-positive hepatocellular carcinoma and cholangiocarcinoma, and MITF immunoreactivity is associated with poor prognosis in patients with hepatocellular carcinoma. Subsequently, the effect of mahanine was analyzed in HepG2 human hepatocellular carcinoma and HuCCT1 and KKU-100 human cholangiocarcinoma cells. Mahanine (25 µM) showed the potent cytotoxicity in these hepatic cancer cell lines, which was associated with increased expression levels of MITF, as judged by Western blot analysis. MITF is over-expressed in subsets of hepatocellular carcinoma and cholangiocarcinoma, and detectable MITF immunoreactivity is associated with poor prognosis in patients with hepatocellular carcinoma. MITF expression levels may be determined in hepatic cancer cells by the balance between the Hedgehog signaling and the cellular stress.

Topics: Carbazoles; Carcinoma, Hepatocellular; Cell Line, Tumor; Cholangiocarcinoma; Female; Gene Expression Profiling; Gene Expression Regulation, Neoplastic; Humans; Liver Neoplasms; Male; Microphthalmia-Associated Transcription Factor; Middle Aged; Pyridines; Pyrimidines; Zinc Finger Protein GLI1

Hedgehog (Hh) signaling plays an important role in embryonic development and in the regulation of a variety of cellular functions. Aberrant activation of Hh signaling has been implicated in several human cancers including hepatocellular carcinoma (HCC). In this study we examined the pathobiological functions and molecular mechanisms of the Hh signaling pathway in HCC cells. Treatment of cultured human HCC cells (Huh7, Hep3B, and HepG2) with the Hh signaling ligand (recombinant Shh) or agonist, SAG and purmorphamine, prevented the induction of autophagy. In contrast, GANT61 (a small molecule inhibitor of Gli1 and Gli2) induced autophagy, as determined by immunoblotting for microtubule-associated protein light chain 3 (LC3) and p62, GFP-LC3 puncta, monodansylcadaverine (MDC) staining, and transmission electron microscopy. Hh inhibition-induced autophagy was associated with up-regulation of Bnip3, as determined by immunoblotting and real-time polymerase chain reaction (PCR) assay. Knockdown of Bnip3 by RNAi impaired GANT61-induced autophagy. Additionally, Hh inhibition-induced autophagy was associated with Bnip3-mediated displacement of Bcl-2 from Beclin-1, as determined by immunoblotting and immunoprecipitation assays. Furthermore, inhibition of Hh signaling increased HCC cell apoptosis and decreased cell viability, as determined by caspase and WST-1 assays. Pharmacological or genetic inhibition of autophagy by 3-methyladenine (3-MA) or Beclin-1 small interfering RNA (siRNA) partially suppressed GANT61-induced cell apoptosis and cytotoxicity. In a tumor xenograft model using SCID mice inoculated with Huh7 cells, administration of GANT61 inhibited tumor formation and decreased tumor volume; this effect was partially blocked by the autophagy inhibitor, 3-MA.. These findings provide novel evidence that Hh inhibition induces autophagy through up-regulation of Bnip3 and that this mechanism contributes to apoptosis. Therefore, the status of autophagy is a key factor that determines the therapeutic response to Hh-targeted therapies.

Topics: Animals; Apoptosis; Apoptosis Regulatory Proteins; Autophagy; Beclin-1; Carcinoma, Hepatocellular; Cell Line, Tumor; Cells, Cultured; Cyclohexylamines; Hedgehog Proteins; Humans; Liver Neoplasms; Male; Membrane Proteins; Mice; Mice, SCID; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-bcl-2; Pyridines; Pyrimidines; Signal Transduction; Thiophenes; Xenograft Model Antitumor Assays