Hedgehog signalling mediates drug resistance through targeting TAP1 in hepatocellular carcinoma.

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.

Hedgehog signaling pathway affects the sensitivity of hepatoma cells to drug therapy through the ABCC1 transporter.

The poor response to drug therapy often seen in hepatocellular carcinoma requires insight into the molecular interplay responsible for intrinsic or acquired drug resistance. We previously demonstrated that the CD133-/EpCAM- subpopulation of the Huh-7 hepatoma cell line features aberrant activation of the hedgehog signaling (Hh) pathway and chemoresistance. The prevailing hypothesis of the present study is that hedgehog signaling may govern expression of ATP-binding cassette (ABC) transporters, which are responsible for drug resistance in the CD133-/EpCAM- subpopulation. Our aim is to reveal the molecular interplay in the mediation of drug resistance with a newly established Huh-7 subpopulation featuring high Hh signaling activity and drug resistance. In this study, chemoresistance was determined in a newly established Huh-7-DN subpopulation featuring the CD133-/EpCAM- surface marker profile, aberrant expression of Hh pathway, and epithelial-mesenchymal transition (EMT). Expression of ABC transporter proteins (ABCB1, ABCC1, and ABCG2) and Hh transcription factor Gli-1/2 was evaluated with and without Hh signaling antagonists LDE225 or itraconazole. We found that hedgehog signaling activity as determined by transfection with a Gli-Lux reporter cassette and gene expression levels tended to increase from Huh-7 CD133+/EpCAM+ to CD133-/EpCAM-, and the highest levels were found in Huh-7-DN cells. The Huh-7-DN subpopulation exhibited characteristics of EMT as evidenced by increased expression of vimentin and loss of E-cadherin. Sorafenib significantly inhibited the viability of all subpopulations except the Huh-7-DN subpopulation. Compared with other sorafenib-sensitive subpopulations, the Huh-7-DN subpopulation showed enhanced expression of Hh transcription factor Gli-2 and ABCC1 transporter protein. Silencing Gli-2 by lentivirus harboring shRNA against Gli-2 or LDE225 significantly suppressed expression of Gli-2 and ABCC1 genes in Huh-7-DN subpopulation. In conclusion, aberrant hedgehog signaling activation is linked to poor differentiation, epithelial-mesenchymal transition, and chemoresistance in the Huh-7-DN subpopulation. Hedgehog signaling transcription factor Gli-2 appears to be the primary regulator for drug sensitivity of hepatoma through the ABCC1 transporter.

Synthesis and biological evaluation of 1-phenyl-1,2,3,4-dihydroisoquinoline compounds as tubulin polymerization inhibitors.

A series of 1-phenyl-3,4-dihydroisoquinoline derivatives and several 1-phenyl-1,2,3,4-tetrahydroisoquinoline, 1-phenyl-isoquinoline analogues were synthesized, and their cytotoxicity and tubulin polymerization inhibitory activity were evaluated. The 1-phenyl-3,4-dihydroisoquinoline compounds were found to be potential tubulin polymerization inhibitors. Compound 5n, bearing a 3'-OH and 4'-OCH(3) substituted 1-phenyl B-ring, was shown to confer optimal bioactivity. The single-crystal structure of 5n was further determined by X-ray diffraction, and the binding mode of 5n to tubulin was obtained by molecular docking, which can explain the structure-activity relationships. The studies presented here provide a new structural type for the development of novel antitumor agents.

Imidazolone-amide bridges and their effects on tubulin polymerization in cis-locked vinylogous combretastatin-A4 analogues: synthesis and biological evaluation.

A series of novel combretastatin-A4 analogues in which the cis-olefinic bridge is replaced by an imidazolone-amide were synthesized, and their cytotoxicity and tubulin-polymerization inhibitory activities were evaluated. These compounds appear to be potential tubulin-polymerization inhibitors. Compounds 10, 9b and 9c, bearing 3'-NH2-4'-OCH3, 4'-CH3 and 3'-CH3-substituted 1-phenyl B-ring, confer optimal bioactivity. The binding modes of these compounds to tubulin were obtained by molecular docking, which can explain the compounds' structure-activity relationship. The studies presented here provide a new structural type for the development of novel antitumor agents.

Biomarkers of altered coagulation and fibrinolysis as measures of disease activity in active inflammatory bowel disease: a gender-stratified, cohort analysis.

BACKGROUND: Growing evidence recognizes inflammatory bowel disease (IBD) as a chronic inflammatory condition characterized by a hypercoagulable state and prothrombotic conditions. The aims of our study were to evaluate the abnormalities in coagulation and fibrinolysis status in patients with IBD, and to analyze parameters of altered coagulation and fibrinolysis status which can correlated with and predict inflammatory parameters of disease activity. METHODS: A cohort of 271 consecutive IBD patients was compared with healthy controls for coagulation and fibrinolysis status. Associations between altered coagulation and fibrinolysis status stratified by gender and inflammatory parameters were analyzed. RESULTS: The mean levels of platelet, platelet distribution width, prothrombin time, fibrinogen, activated partial thromboplastin time were significantly higher in IBD patients than in healthy controls (all P<0.05). Mean platelet volume was lower in male patients with IBD than in healthy controls (P<0.01). Furthermore, multiple linear regression indicated that fibrinogen was an independent predictor of ESR (beta=1.316, P=<0.001) and CRP (beta=1.233, P=0.015) in male patients with active ulcerative colitis. Platelet (beta=0.436, P=0.037) and prothrombin time (beta=0.810, P=<0.001) were predictors of Crohn's Disease Activity Index in female patients with Crohn's disease. CONCLUSIONS: To our knowledge, this study provides characteristics on altered coagulation and fibrinolysis status in active IBD patients using the largest number of cases assembled in one study to date. Our data suggest that in IBD patients, abnormalities in coagulation and fibrinolysis status were associated with disease activity. Fibrinogen, platelet and prothrombin time were predictors of inflammation.

[Investigation of the effect of nuclear factor κB on inflammatory cell recruitment phenotype of oral cancer associated macrophage].

PURPOSE: To explore the ability of nuclear factor κB (NFκB) in sustaining inflammatory cell recruitment phenotype of oral cancer associated macrophages, by using NFκB inhibitor(-Bay11-7082). METHODS: By primary culture, murine macrophages were harvested. Cal27 conditioned medium (CM) and Bay11-7082 were applied for stimulation of the macrophages. RT-PCR and ELISA were used for detecting the inflammatory cell recruitment related chemotactic factors. GraphPadPrism5 was used for statistical analysis. RESULTS: Bay11-7082 prevented the contour change into a spindle shape via Cal 27 CM. It also attenuated MCP-1, GM-CSF, MCP-5 and CCL-5 mRNA increase after Cal 27 CM stimulation (P<0.05). At protein level, impeding NFκB activation could significantly prevent MCP-1 and GM-CSF secretion from oral cancer associated macrophage (P<0.001). CONCLUSIONS: NFκB signaling may play a key role in sustaining the inflammatory cell recruitment phenotype of oral cancer associated macrophages.