Huaqiang Zhu

2-Methoxyestradiol synergizes with sorafenib to suppress hepatocellular carcinoma by simultaneously dysregulating hypoxia-inducible factor-1 and -2

Sorafenib is the approved systemic drug of choice for advanced hepatocellular carcinoma (HCC), but has demonstrated limited benefits because of drug resistance. 2-Methoxyestradiol (2ME2) has been shown to be a promising anticancer drug against various types of cancers and acts by dysregulating hypoxia-inducible factor (HIF)-1. Hypoxic cancer cells are extremely resistant to therapies since they elicit strong survival ability due to the cellular adaptive response to hypoxia, which is controlled by HIF-1 and HIF-2. The present study has demonstrated that sorafenib downregulated the expression of HIF-1α, making the hypoxic response switch from HIF-1α- to HIF-2α-dependent pathways, resulting in upregulation of HIF-2α, which contributes to the insensitivity of hypoxic HCC cells to sorafenib. HIF-2α played a dominant role in regulating VEGF, thus sorafenib in turn increased the expression of VEGF (a downstream molecule of both HIF-1 and HIF-2) and cyclin D1 (a downstream molecule of HIF-2), but reduced the expression of LDHA (a downstream molecule of HIF-1), in hypoxic HCC cells. 2ME2 significantly reduced the expression of both HIF-1α and HIF-2α, and their downstream molecules, VEGF, LDHA and cyclin D1, rendering hypoxic HCC cells to increased sensitivity to 2ME2. 2ME2 also inhibited the nuclear translocation of HIF-1α and HIF-2α proteins, but had no effect on their mRNA expression. 2M2 synergized with sorafenib to suppress the proliferation and induction of apoptosis of HCC cells in vitro and in vivo, and inhibited tumoral angiogenesis. These results indicate that 2ME2 given in combination with sorafenib acts synergistically for treating HCC.

Aberrant Expression of CCAT1 Regulated by c-Myc Predicts the Prognosis of Hepatocellular Carcinoma

BACKGROUND CCAT1 has been reported to be linked with pathogenesis of malignancies including colon cancer and gastric cancer. However, the regulatory effect of CCAT1 in hepatocellular carcinoma (HCC) remains unclear. The purpose of this research was to identify any role of CCAT1 in the progression of HCC. MATERIALS AND METHODS Real time-PCR was performed to test the relative expression of CCAT1 in HCC tissues. A computation screen of CCAT1 promoter was conducted to search for transcription-factor-binding sites. The association of c-Myc with CCAT1 promoter in vivo was tested by Pearson correlation analysis and chromatin immunoprecipitation assay. Additionally, Kaplan-Meier analysis and Cox proportional hazards analyses were performed. RESULTS c-Myc directly binds to the E-box element in the promoter region of CCAT, and when ectopically expressed increases promoter activity and expression of CCAT1. Moreover, Kaplan-Meier analysis demonstrated that the patients with low expression of CCAT1 demonstrated better overall and relapse-free survival compared with the high expression group. Cox proportional hazards analyses showed that CCAT1 expression was an independent prognostic factor for HCC patients. CONCLUSIONS The findings demonstrated CCAT1, acting as a potential biomarker in predicting the prognosis of HCC, is regulated by c-Myc.

Secretory clusterin contributes to oxaliplatin resistance by activating Akt pathway in hepatocellular carcinoma.

Secretory clusterin (sCLU) is expressed in numerous cancers and is associated with the resistance to chemotherapy. However, the role of sCLU in the resistance of hepatocellular carcinoma (HCC) to oxaliplatin (OXA), a recently used third‐generation platinum agent, remains unclear. The stable transfectants that are depleted of or overexpress sCLU and OXA‐resistant cells were generated using human HCC cells. Overexpression of sCLU abrogated OXA‐induced inhibition of cell growth and cell apoptosis, but depletion of sCLU synergized with OXA to inhibit cell growth and enhance cell apoptosis, by regulating proteins involved in mitochondrial apoptosis pathways, such as Bcl‐2, Bax, Bcl‐xL and caspase‐9, and affecting phosphorylation of Akt and GSK‐3β. Overexpression of sCLU in either OXA‐resistant cells or stable transfectants that overexpress sCLU significantly increased phosphorylated Akt. However, specific inhibition of Akt enhanced sensitivity of sCLU‐overexpressing cells to OXA, but had no effect on sCLU expression, suggesting that the regulatory effects between sCLU and pAkt may be in a one‐way manner in HCC cells. The expression levels of sCLU affected the therapeutic efficacy of OXA to treat HCC tumors established in immunodeficiency mice. The results have demonstrated that sCLU contributes to OXA resistance by activating Akt pathway, indicating that sCLU may be a novel molecular target for overcoming OXA resistance in HCC.

Low Expression of miR-448 Induces EMT and Promotes Invasion by Regulating ROCK2 in Hepatocellular Carcinoma.

Background/Aims: miR-448 has been reported to exhibit abnormal expression in hepatocellular carcinoma (HCC), however, the essential role of miR-448 in HCC progression is still unclear. Methods: real-time PCR was used to detect the expression of miRNAs and candidate genes in HCC samples (n=117). miR-448 mimics and inhibitor were tansfected in human HCC cells. The transwell assay was used to examine the cell invasive ability. The regulation mechanism was confirmed by luciferase reporter assay. The markers of EMT were detected by using Western blot. Results: miR-448 was decreased in HCC samples and associated with HCC development. Inhibition of miR-448 significantly promoted cell invasion, while the effect of miR-448 up-regulation was reverse. miR-448 could regulate ROCK2 in hepatocellular carcinoma. Knockdown of ROCK2 expression partially reversed the effect of miR-448 inhibitor. Abnormal expression of miR-448 could regulate the markers of epithelial-mesenchymal transition (EMT). Conclusions: miR-448 may contribute to the progression of HCC via regulating ROCK2 expression.

2ME2 inhibits the activated hypoxia-inducible pathways by cabozantinib and enhances its efficacy against medullary thyroid carcinoma

Cabozantinib is a multi-targeted tyrosine kinase inhibitor targeting vascular endothelial growth factor (VEGF) receptor (VEGFR)-2, MET (c-Met, also called hepatocyte growth factor (HGF) receptor), and other receptor tyrosine kinases. Cabozantinib has recently been approved for treating advanced medullary thyroid carcinoma (MTC), but its long-term benefit remains uncertain and dose-dependent adverse events are very common. The present study has demonstrated that 2-methoxyestradiol (2ME2), an inhibitor of hypoxia-inducible factors (HIFs) and a promising anticancer agent under investigation in clinical trials, strengthens anticancer activities of cabozantinib against MTC cells in vitro and in vivo. The activated hypoxia-inducible pathways, which are mainly regulated by HIF-1, contribute to the resistance of hypoxic MTC cells to cabozantinib. Cabozantinib upregulated HIF-1α expression at translational levels and increased the expression of the downstream factors including VEGF, lactate dehydrogenase A (LDHA), HGF, and MET. 2ME2 corrected the activated pathways by cabozantinib through downregulating HIF-1α expression and inhibiting its nuclear translocation in hypoxic MTC cells. Administration of 2ME2 enhanced the efficacy of cabozantinib in suppressing the growth of MTC cell line xenografts and patient-derived xenografts established in mice. Given that 2ME2 targets insensitive hypoxic cancer cells to cabozantinib and can inhibit the activated pathways by cabozantinib, the present results warrant further investigation of 2ME2, particularly in combination with cabozantinib, for the treatment of MTC.

MiR-1180 promoted the proliferation of hepatocellular carcinoma cells by repressing TNIP2 expression

MicroRNAs (miRNAs) are short, non-coding RNAs with post-transcriptional regulatory function, playing crucial roles in cancer development and progression of hepatocellular carcinoma (HCC). Previous studies have indicated that miR-1180 was implicated in diverse biological processes. However, the underlying mechanism of miR-1180 in HCC has not been intensively investigated. In this study, we aimed to investigate the role of miR-1180 and its target genes in HCC. We found that miR-1180 expression was significantly increased in HCC cells and clinical tissues compared with their corresponding controls. Overexpression of miR-1180 promoted cell proliferation in HCC cell line HepG2. TNFAIP3 interacting protein 2 (TNIP2), a potential target gene of miR-1180, and were validated by a luciferase assay. Further studies revealed that miR-1180 regulated cell proliferation of HCC by directly suppressing TNIP2 expression and the knockdown of TNIP2 expression reversed the effect of miR-1180-in on HCC cell proliferation. In summary, our data indicated that miR-1180 might act as a tumor promoter by targeting TNIP2 during development of HCC.

Hepatocyte nuclear factor 6 inhibits the growth and metastasis of cholangiocarcinoma cells by regulating miR-122

PurposeHepatocyte nuclear factor 6 (HNF6) is a liver-enriched transcription factor and highly expressed in mature bile duct epithelial cells. This study sought to investigate the role of HNF6, particularly the molecular mechanisms for how HNF6 is involved in the growth and metastasis of cholangiocarcinoma (CCA) cells.MethodsThe expression of HNF6, miR-122 and key molecules was examined by Western blot analysis and real-time RT-PCR. Stable transfectants, HCCC-HNFlow and RBE-HNFhigh, were generated from human CCA HCCC-9810 and RBE cells, respectively. The regulatory effect of HNF6 on miR-122 was evaluated by luciferase reporter assay. Cell proliferation, cycle distribution, migration and invasion were analyzed. The xenograft model was used to assess the effects of HNF6 overexpression on tumorigenesis, growth, metastasis and therapeutic potentials.ResultsHuman CCA tissues and cells expressed lower levels of HNF6, which positively correlated with miR-122. HNF6 regulated the expression of miR-122 by stimulating its promoter. HNF6 overexpression inhibited cell proliferation by inducing cell cycle arrest at G1 phase through regulating miR-122, cyclin G1 and insulin-like growth factor-1 receptor. HNF6 inhibited the migration and invasion of CCA cells by regulating matrix metalloproteinase-2 and metalloproteinase-9, reversion-inducing-cysteine-rich protein with kazal motifs, E-cadherin and N-cadherin. Co-transfection of anti-miR-122 abrogated the effects of HNF6. HNF6 overexpression inhibited the ability of cells to form tumors and to metastasize to the lungs of mice, and the growth of established tumors.ConclusionsThe results indicate that HNF6 may serve as a tumor suppressor by regulating miR-122, and its overexpression may represent a mechanism-based therapy for CCA.

PTEN and hTERT gene expression and the correlation with human hepatocellular carcinoma.

The aim of this study was to investigate the correlation between tumor suppressor gene phosphatase and tensin homolog (PTEN) expression levels and telomerase activity that mainly depends on telomerase reverse transcriptase (hTERT) in hepatocellular carcinoma (HCC) and paracancerous tissues. Immunohistochemistry and reverse transcriptase-polymerase chain reaction (RT-PCR) were used to detect the expression of PTEN and hTERT in 58 cases with HCC and the corresponding paracancerous tissues. The correlation between PTEN and hTERT was analyzed. The PTEN mRNA and protein expression was significantly lower in HCC, as compared with the paracancerous tissues (P<0.01), while those of hTERT demonstrated the opposite expression pattern (P<0.01). Additionally, both the PTEN and hTERT protein and mRNA levels demonstrated a significantly negative correlation with one another (P<0.05). In conclusion, PTEN and hTERT have different roles in the development of HCC. A significantly negative correlation between PTEN and hTERT gene expression indicates that hTERT activation and upregulation may be conferred by the loss of PTEN gene expression in HCC. The combined detection of PTEN and hTERT may provide critical clinical evidence for the diagnosis and biological behavior of HCC.

Correlation between the expression of hTERT gene and the clinicopathological characteristics of hepatocellular carcinoma

The aim of the present study was to detect the expression levels of the human telomerase reverse transcriptase (hTERT) gene in hepatocellular carcinoma (HCC), and investigate its significance in the incidence and development of the cancer. HCC tissues and corresponding para-carcinoma liver tissues were surgically collected from 78 patients with HCC who presented to Shandong Provincial Hospital (Jinan, Shandong, China). hTERT expression at the protein and mRNA levels were detected by immunohistochemistry (streptavidin peroxidase method) and reverse transcription polymerase chain reaction, respectively, in the HCC tissues and corresponding para-carcinoma liver tissues of 78 HCC patients and in 12 samples of normal liver tissue. The data were analyzed using SPSS 17.0 statistical software, and employing χ2 tests and t-tests. hTERT protein was mainly expressed in the HCC cell cytoplasm, but was occasionally observed in the cell nucleus. The positive rates of hTERT protein and mRNA expression in the HCC patients were 84.62% (66/78) and 78.21% (61/78), respectively, which was significantly higher compared with the rates of 10.26% (8/78) and 8.97% (7/78) in the paired para-carcinoma liver tissues (P<0.01). hTERT protein and mRNA were not expressed in the normal liver tissues (0/12). χ2 test and t-test analysis revealed that hTERT gene expression was correlated with tumor grade, the presence/absence of a portal vein tumor thrombus, hepatitis B surface antigen positivity and a high α-fetoprotein level (P<0.05) rather than patient age, gender or tumor size. Expression of the hTERT gene may play a pivotal role in the incidence and development of HCC. The hTERT gene potentially serves as an important molecular and biological index for diagnosing and predicating the biological behavior of HCC.

Interaction Between Polymorphisms of IFN-γ and MICA Correlated with Hepatocellular Carcinoma

Background We explored the relationship of interferon--γ (IFN-γ) and MHC class-I chain related gene A (MICA) genes polymorphisms with hepatocellular carcinoma (HCC) risk, and tried to determine whether the interaction existed between these two genes polymorphisms on the basis of HCC. Material/Methods Polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) method was used to detect the genotypes of the 3 single-nucleotide polymorphisms (SNPs) and to analyze the correlation of each SNP with HCC susceptibility in 120 HCC patients and 124 healthy people. The association strength between the 3 SNPs and HCC is represented with odds ratio (OR) and 95% confidence interval (95% CI). Hardy-Weinberg equilibrium (HWE) was tested by χ2 test in the control group. Results GG genotype of IFN-γ rs2069727 polymorphism had apparently different distributions in case and control groups (P<0.05), and might confer increased risk of HCC (OR=3.40, 95%CI=1.23–9.38). Analysis of MICA rs2596542 polymorphism also yielded the same result (OR=2.90, 95%CI=1.10–7.67), as did their risk alleles. Specifically, the interaction between rs2596542 and rs2069705 polymorphisms increased the HCC risk by 1.41 times and between rs2596542 and rs2069727 polymorphisms the increased risk of HCC by 5.56 times. Conclusions IFN-γ rs2069727 and MICA rs2596542 polymorphisms may be related to the incidence of HCC. Interaction exists between the polymorphisms of IFN-γ and MICA, which may increase risk of HCC.