Inhibited Hepatocellular Carcinoma Growth Research Articles

Galectin-9 suppresses the growth of hepatocellular carcinoma via apoptosis in vitro and in vivo.

Galectin-9, a soluble β-galactoside-binding animal lectin, evokes apoptosis in various human cancer cell lines. The galectin-9 antitumor effect against hepatocellular carcinoma (HCC) is, however, unknown. We investigated whether galectin-9 suppresses HCC growth in vitro and in vivo. We assessed the antitumor effect of galectin-9 on HCC cells by conducting WST-8 assay in vitro and xenograft model analysis in vivo. Galectin-9-induced apoptosis was evaluated by FACS and ELISA in vitro and by TUNEL stain in vivo. Cell cycle alteration was profiled by FACS. Caspases were profiled by colorimetry. MicroRNAs related to the galectin-9 antitumor effects were determined using microarrays, and their antitumor effect was confirmed in a transfection study in vitro. The expression levels of the target proteins of the miRNAs extracted above were analyzed by western blot analysis. To summarize the results, galectin-9 inhibited the growth of the HCC cell lines HLE and Li-7 in vitro and Li-7 in vivo inducing apoptosis. Cell cycle turnover was not arrested in HLE and Li-7 cells in vitro. miR-1246 was similarly extracted both in vitro and in vivo, which sensitized Li-7 cells to apoptosis when transfected into the cells. DYRK1A, a target protein of miR-1246 was downregulated in Li-7 cells. Caspase-9 was upregulated in Li-7 cells in vitro and in vivo. In conclusion, galectin-9 inhibited the growth of HCC cells by apoptosis, but not cell cycle arrest, in vitro and in vivo. miR-1246 mediated signals of galectin-9, possibly through miR-1246-DYRK1A-caspase-9 axis. Galectin-9 might be a candidate agent for HCC chemotherapy.

ψ-Bufarenogin, a novel anti-tumor compound, suppresses liver cancer growth by inhibiting receptor tyrosine kinase-mediated signaling.

Resistance of hepatocellular carcinoma (HCC) to existing chemotherapeutic agents largely contributes to the poor prognosis of patients, and discovery of novel anti-HCC drug is in an urgent need. Herein we report ψ-Bufarenogin, a novel active compound that we isolated from the extract of toad skin, exhibited potent therapeutic effect in xenografted human hepatoma without notable side effects. In vitro, ψ-Bufarenogin suppressed HCC cells proliferation through impeding cell cycle progression, and it facilitated cell apoptosis by downregulating Mcl-1 expression. Moreover, ψ-Bufarenogin decreased the number of hepatoma stem cells through Sox2 depression and exhibited synergistic effect with conventional chemotherapeutics. Mechanistic study revealed that ψ-Bufarenogin impaired the activation of MEK/ERK pathway, which is essential in the proliferation of hepatoma cells. ψ-Bufarenogin notably suppressed PI3-K/Akt cascade, which was required in ψ-Bufarenogin-mediated reduction of Mcl-1 and Sox2. ψ-Bufarenogin inhibited the auto-phosphorylation and activation of epithelial growth factor receptor (EGFR) and hepatocyte growth factor receptor (c-Met), thereafter suppressed their primary downstream cascades Raf/MEK/ERK and PI3-K/Akt signaling. Taken together, ψ-Bufarenogin suppressed HCC growth via inhibiting, at least partially, receptor tyrosine kinases-regulated signaling, suggesting that ψ-Bufarenogin could be a novel lead compound for anti-HCC drug.

MiR-26a Promoted by Interferon-Alpha Inhibits Hepatocellular Carcinoma Proliferation and Migration by Blocking EZH2

Interferon (IFN)-α has been used to treat hepatocellular carcinoma (HCC). Here, we report that the IFN-α-induced microRNA-26a (miR-26a) can inhibit HCC proliferation and invasion by suppressing enhancer of zeste homologue 2 (EZH2) expression in tumor cells. First, the miR-26a transcription level was quantified by real-time quantitative PCR in the HCC specimens from IFN-α-treated HCC patients. Next, we transfected HepG2 cells with miR-26a mimics and miR control, and then we investigated the influence of miR-26a mimic transfection on HepG2 cell proliferation and invasion. It was shown that there was increased miR-26a accompanied with downregulated EZH2 expression in the HCC specimens, and EZH2 mRNA levels were inversely correlated with miR-26a expression. There was a dose-response correlation between the IFN-α dosage and EZH2 expression. In addition, the miR-26a mimic transfection decreased the EZH2 expression level significantly in the transfected HepG2 cells and inhibited HepG2 cell proliferation and invasion effectively. Our results indicate that miR-26a exerts growth inhibition in HCC and that its inhibitory effect is mediated briefly by blocking EZH2 expression.

MicroRNA-486-5p, which is downregulated in hepatocellular carcinoma, suppresses tumor growth by targeting PIK3R1.

Deregulated microRNAs and their roles in carcinogenesis and cancer progression have attracted much attention. In previous studies conducted in our laboratory, the Illumina Solexa massively parallel signature sequencing of miRNomes in nontumor and hepatocellular carcinoma (HCC) tissues revealed that miR-486-5p was significantly downregulated in HCC, but its role in HCC development remains unknown. In this study, miR-486-5p levels in HCC tissues and matched control tissues, and in seven HCC cell lines (QGY-7701, QGY-7703, QGY-7404, SMMC-7721, Huh7, HepG2, and PCL/PRF/5) and human normal liver cells (HL-7702), were tested by real-time quantitative RT-PCR. We found that the level of miR-486-5p was significantly decreased in HCC tissue and in all seven HCC cell lines. Overexpression of miR-486-5p markedly suppressed HCC cell proliferation, migration and invasion in vitro, and inhibited HCC growth in vivo. Mechanistically, miR-486-5p was confirmed to directly target PIK3R1 expression, thereby suppressing phosphatidylinositol 3-kinase-AKT pathway activation, by dual luciferase reporter assay and real-time quantitative RT-PCR and western blot analysis. In addition, PIK3R1 knockdown mimicked the effects of miR-486-5p overexpression by inhibiting HCC growth, migration, and invasion. Furthermore, correlation analysis, Kaplan-Meier estimates and Cox proportional hazard models showed an inverse correlation between miR-486-5p and PIK3R1, as well as a shorter time to recurrence after HCC resection, in patients with lower miR-486-5p expression. Hence, we conclude that miR-486-5p, which is frequently downregulated in HCC, inhibits HCC progression by targeting PIK3R1 and phosphatidylinositol 3-kinase-AKT activation.

Abstract 685: Antagonism of sorafenib and regorafenib-mediated HCC growth inhibition by platelet factors

Abstract Background. Circulating blood platelet numbers are frequently altered in hepatocellular carcinoma (HCC) patients. Thrombocytopenia is associated with smaller tumors and thrombocytosis with larger tumors (Oncology 2013;85:153-9). Recently, platelet extracts have been shown to enhance HCC cell growth and have anti-apoptotic effects. Methods. Human HCC cell lines were used to evaluate the actions of time-expired mixed human platelet lysates on the inhibition of HCC growth by MTT assay, as well as on migration and Matrigel invasion mediated by Sorafenib or Regorafenib in vitro and Western blots were performed for cell signaling and apoptosis. Results. Sorafenib or Regorafenib-mediated inhibition of cell growth, migration and invasion was antagonized by platelet extracts. The multi-kinase inhibitor-mediated induction of apoptosis and decrease in phospho-ERK levels were both blocked by the platelet extracts, which also increased anti-apoptotic phospho-STAT, Bax and Bcl-XL levels. Conclusions. In addition to their effects on coagulation and wound healing, platelet factors can antagonize growth inhibition and apoptosis induction by multi-kinase inhibitors Sorafenib or Regorafenib. This could be due to several growth factors or inflammatory cytokines that are produced by platelets. Targeting platelets might be a novel approach to increasing multi-kinase inhibitor actions. Citation Format: Brian I. Carr, Rosalba D'Alessandro, Maria Grazie Refolo, Catia Lippolis, Caterina Messa, Aldo Cavallini. Antagonism of sorafenib and regorafenib-mediated HCC growth inhibition by platelet factors. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 685. doi:10.1158/1538-7445.AM2014-685

Abstract 3547: FoxD3-regulated microRNA-137 suppresses tumour growth and metastasis in human hepatocellular carcinoma by targeting AKT2

Abstract Background and Aims: microRNAs, which are frequently deregulated in human cancer, have been implicated in the progression of hepatocarcinogenesis. Our study aims to investigate the role of miR-137 in cell proliferation and metastasis of hepatocellular carcinoma and the underlying molecular mechanisms. Methods: Real-time PCR and western blot were used to examine the expressions of miR-137, Akt2 and FoxD3 in HCC tissues and cells. The in vitro and in vivo effects of miR-137 were determined. Luciferase reporter assay was conducted to confirm the association of miR-137 and AKT2. CHIP assay was performed to test the regulation of miR-137 by FoxD3. Results: We show that microRNA (miR)-137 is significantly down-regulated in hepatocellular carcinoma (HCC), and its decreased expression is associated with vein invasion, incomplete Involucrum, and tumour metastasis. Multivariate analysis suggests that miR-137 is an independent indicator for poor survival. To determine its role in HCC development, we showed that overexpression of miR-137 suppresses cell proliferation, invasion and metastasis in vitro. Conversely, miR-137 inhibition promoted HCC cell growth. We also identified AKT2 as a key target of miR-137 in this context. Statistical data revealed a reverse correlation of AKT2 and miR-137 expression in HCC patients. Silencing of AKT2 phenotypically copied miR-137-induced phenotypes, whereas re-expression of AKT2 reversed the suppressive effects of miR-137. Further investigation showed that miR-137 exerted its anti-tumour activity via inhibiting the AKT2/mTOR pathway. Moreover, we demonstrated that FoxD3 binds to the promoter of miR-137 and activates its transcription. In vivo studies confirmed that FoxD3-regulated miR-137 inhibition of HCC growth and metastasis via directly targeting AKT2. Conclusions: Our findings indicated that miR-137 is a valuable biomarker for HCC prognosis and demonstrated that the FoxD3/miR-137/AKT2 regulatory network plays an important role in HCC progression. Citation Format: Li-Li Liu, Shi-Xun Lu, Min Li, Jia Fu, Chris Zhiyi Zhang, Jing-Ping Yun. FoxD3-regulated microRNA-137 suppresses tumour growth and metastasis in human hepatocellular carcinoma by targeting AKT2. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3547. doi:10.1158/1538-7445.AM2014-3547

Abstract 5215: miR-200b/c/429 sub-family negatively regulating Rho/ROCK signaling pathway and suppresses HCC metastasis

Abstract MicroRNAs (miRNAs) are a class of small (∼22 nt) noncoding regulatory RNA molecules that can regulate gene expression by either cleaving or repressing the translation of their mRNA targets. Deregulation of miRNA expression is common in human cancers, including hepatocellular carcinoma (HCC). Recent studies have indicated that the miR-200 family (miR-200a, miR-200b, miR-200c, miR-141 and miR-429) negatively regulates epithelial-mesenchymal transition (EMT) and cell motility by targeting E-cadherin repressors, ZEB1 and ZEB2. However, the roles of the miR-200 family in HCC remain elusive. In this study, we found that miR-200 family was frequently down-regulated in human HCC, which was contributed by the frequently CpG hypermethylation of miR-200 promoters. Functionally, although all miR-200 members suppressed ZEB1/2 expression in HCC, only overexpression of miR-200b/c/429 sub-family impeded HCC cell migration. These findings implied that, in addition to ZEB1/2, other yet unidentified miR-200b/c/429 specific targets may play an important role in HCC metastasis. In silico analyses revealed that miR-200b/c/429 sub-family may have a profound role in modulating cell cytoskeletal reorganization. Along with this notion, we further identified RhoA and ROCK2 as down-stream targets of miR-200b/c/429 sub-family. By using immunofluorescence staining, scanning electron microscope and live cell imaging, we demonstrated that overexpression of miR-200b/c/429 subfamily but not miR-200a/141 sub-family suppressed Rho/ROCK signaling mediated stress fiber and focal adhesion formations, which in turn led to a dramatic cell morphology change and loss of cell migration ability. Furthermore, by employed an orthotopic liver implantation model, we showed that overexpression of miR-200b was sufficient to suppress HCC growth and metastasis in nude mice. Our results together suggest that miR-200b/c/429 sub-family functions as a metastatic suppressor in human HCC by negatively regulating Rho/ROCK signaling pathway. Citation Format: Chun-Ming Wong, Lai Wei, Sandy Leung-Kuen Au, Sandy Leung-Kuen Au, Dorothy Ngo-Yin Fan, Joyce Man-Fong Lee, Carmen Chak-Lui Wong, Irene Oi-Lin Ng. miR-200b/c/429 sub-family negatively regulating Rho/ROCK signaling pathway and suppresses HCC metastasis. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 5215. doi:10.1158/1538-7445.AM2014-5215

Abstract 2453: Transketolase (TKT) is a critical component of the metabolic machinery of hepatocellular carcinoma (HCC)

Abstract Hepatocellular carcinoma, HCC, is one of the commonest malignant cancers in human liver. The prognosis of HCC patients is extremely poor mainly because of late symptom presentation and absence of effective therapy. Liver is the metabolic engine that maintains the homeostasis of various nutrients; nevertheless, how the metabolic reprogramming of HCC cells benefits tumor progression remains largely unknown. Understanding the metabolic machinery of HCC is important for the development of new therapeutic approaches for HCC. Increased glucose intermediates through glycolysis and the pentose phosphate pathway (PPP) is a metabolic signature of cancer. The PPP works collaboratively with glycolysis to support rapid tumor growth by producing NADPH, the major antioxidant in the cells, and ribose-5-phosphate, the backbone of RNA. One of the key enzymes that connect PPP with glycolysis is the transketolase family (TK). The TK family comprises 3 members namely transketolase (TKT), TKT-like 1 (TKTL1) and TKTL2. Many studies have indicated that strong TKTL1 protein expression was correlated with invasive features and poor clinical outcome in other cancers. However, our transcriptome sequencing data from 16 pairs of human HCC and non-tumorous liver (NT) tissues revealed that (1) the TKT is the predominant TK member in the liver and HCC; (2) TKT, but not other TK family members, is overexpressed in HCC. In an expanded HCC cohort consisting of 60 human HCC cases, we confirmed that TKT is significantly overexpressed and is closely associated with aggressive HCC clinicopathological features including tumor microsatellite formation (P = 0.04), venous invasion (P = 0.01), and large tumor size (P = 0.04). Stable knockdown of TKT by various short hairpin RNA (shRNA) sequences in multiple HCC cell lines (MHCC97L, SMMC-7721, and BEL-7402) consistently suppressed cell growth, elevated intracellular ROS level (CM-H2DCFDA staining), and decreased glucose uptake (2-NBDG staining). In line with the in vitro observations, stable knockdown of TKT drastically impeded HCC growth in orthotropic and subcutaneous tumor models. More interestingly, pharmacological inhibition and genetic knockdown of TKT by oxythiamine (OT) and shRNA, respectively, sensitized HCC cells to the treatment of 2-deoxyglucose (2-DG), a glycolysis inhibitor. Inhibition of PPP and glycolysis by co-treatment of OT and 2-DG completely suppressed HCC growth. Taken together, our present data suggested that TKT overexpression strongly favors HCC cell growth in vitro and in vivo. Our data also showed that TKT is a critical component of the metabolic machinery of HCC and therefore an attractive therapeutic target. Citation Format: Mingjing Xu, Carmen Chak-Lui Wong, Irene Oi-Lin Ng. Transketolase (TKT) is a critical component of the metabolic machinery of hepatocellular carcinoma (HCC). [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2453. doi:10.1158/1538-7445.AM2014-2453

Synergistic suppression effect on tumor growth of hepatocellular carcinoma by combining oncolytic adenovirus carrying XAF1 with cisplatin.

The potent anticancer efficacy of oncolytic viruses has been verified in Clinic in recent years. Cisplatin (DDP) is one of most common chemotherapeutic drugs, but is accompanied by side effects and drug resistance. Our previous studies have shown the strategy of cancer -targeting gene-viro-therapy (CTGVT) mediated by the oncolytic virus ZD55 containing the XAF1 cDNA (ZD55-XAF1), which exhibited potent antitumor effects in various tumor cells and no apparent toxicities on normal cells. In the study, the CTGVT strategy is broadened by combining DDP with ZD55-XAF1 for growth inhibition of hepatocellular carcinoma (HCC) cells. The transgenic expression was evaluated by both in vitro and in vivo experiments, and the enhanced inhibitory effect of ZD55-XAF1 combined with cisplatin was assessed in HCC cells. The cytotoxicity on normal liver cells was evaluated by MTT assay and apoptotic cell staining. Activation of caspase-9 and PARP for apoptosis was further detected by Western blot analysis. The in vivo antitumor efficacy of combination treatment with cisplatin and ZD55-XAF1 was estimated in an HCC xenograft mouse model. We found that the combination of ZD55-XAF1 and cisplatin showed enhanced inhibitory effects on the proliferation of HCC cells in vitro and tumor growth in mice. Furthermore, the combined treatment of ZD55-XAF1 and DDP decreases the chemotherapy dose needed to achieve the same inhibitory effect without overlapping toxicities on normal liver cells and induces tumor cell apoptosis via the activation of caspase-9/PARP pathway. Thus, these data suggest that the chemo-gene-viro-therapeutic strategy by combining ZD55-XAF1 and DDP reveals a novel therapeutic strategy for hepatocellular carcinoma.

Clinical relevance and therapeutic potential of angiopoietin-like protein 4 in hepatocellular carcinoma.

BackgroundDevelopment of novel adjuvant therapy to eradicate tumor angiogenesis and metastasis is a pressing need for patients with advanced hepatocellular carcinoma (HCC). We aimed to investigate the clinical relevance and therapeutic potential of angiopoietin-like 4 (ANGPTL4) in HCC.MethodsANGPTL4 mRNA levels in tumor and non-tumor liver tissues of HCC patients were analyzed to investigate its clinical relevance. The mechanisms of deregulation of ANGPTL4 in HCC were studied by copy number variation (CNV) and CpG methylation analyses. The orthotopic liver tumor nude mice model was applied using a human metastatic cell line. ANGPTL4-overexpressing adenovirus (Ad-ANGPTL4) was injected via portal vein to investigate its anti-tumorigenic and anti-metastatic potentials.ResultsHCC tissues expressed significantly lower levels of ANGPTL4 mRNA than non-tumor tissues. The copy number of ANGPTL4 gene in tumor tissues was significantly lower than in non-tumor tissues of HCC patients. Higher frequency of methylation of CpG sites of ANGPTL4 promoter was detected in tumor tissues compared to non-tumor tissues. Downregulation of ANGPTL4 mRNA in HCC was significantly associated with advanced tumor stage, presence of venous infiltration, poor differentiation, higher AFP level, appearance of tumor recurrence, and poor postoperative overall and disease-free survivals of HCC patients. Treatment with Ad-ANGPTL4 significantly inhibited the in vivo tumor growth, invasiveness and metastasis by promoting tumoral apoptosis, inhibiting tumoral angiogenesis and motility, and suppressing tumor-favorable microenvironment. Moreover, administration of recombinant ANGPTL4 protein suppressed the motility of HCC cells and altered the secretion profile of cytokines from macrophages.ConclusionANGPTL4 is a diagnostic and prognostic biomarker for HCC patients and a potential therapeutic agent to suppress HCC growth, angiogenesis and metastasis.

FoxD3-regulated microRNA-137 suppresses tumour growth and metastasis in human hepatocellular carcinoma by targeting AKT2.

microRNAs, frequently deregulated in human cancer, have been implicated in the progression of hepatocarcinogenesis. Here, we show that microRNA (miR)-137 is significantly down-regulated in hepatocellular carcinoma (HCC). Its decreased expression is associated with vein invasion, incomplete Involucrum, and distant metastasis. Multivariate analysis suggests that miR-137 is an independent indicator for poor survival. We next show that over-expression of miR-137 suppresses cell proliferation, migration and invasion in vitro. Conversely, miR-137 inhibition promotes HCC cell growth. We also identify AKT2 as a key target of miR-137 in this context. Statistical data reveal a reverse correlation of AKT2 and miR-137 expression in HCC patients. Silencing of AKT2 phenotypically copied miR-137-induced phenotypes, whereas re-expression of AKT2 reversed the suppressive effects of miR-137. Further investigations showed that miR-137 exerted its anti-tumour activity via inhibiting the AKT2/mTOR pathway. Moreover, we demonstrate that FoxD3 directly binds to the promoter of miR-137 and activates its transcription. In vivo studies confirm that FoxD3-regulated miR-137 inhibited HCC growth and metastasis via targeting AKT2. Together, our findings indicate that miR-137 is a valuable biomarker for HCC prognosis and the FoxD3/miR-137/AKT2 regulatory network plays an important role in HCC progression.

MiR-185 Inhibits Hepatocellular Carcinoma Growth by Targeting the DNMT1/PTEN/Akt Pathway

miRNAs have recently been implicated in hepatocarcinogenesis, although the actions and mechanisms of individual miRNAs remain incompletely understood. We examined the biological functions and molecular mechanisms of miR-185 in hepatocellular carcinoma (HCC). The expression of miR-185 is decreased in human HCC tissues compared with the nonneoplastic liver parenchyma. Quantitative RT-PCR showed a reduction of miR-185 in human HCC cells compared with primary hepatocytes. miR-185 overexpression in human HCC cells inhibited cell proliferation and invasion invitro and prevented tumor growth in SCID mice. miR-185 overexpression inhibited DNMT1 3' untranslated region luciferase reporter activity in HCC cells; this effect was abolished when the miR-185 binding site was mutated. miR-185 mimic or overexpression decreased the level of DNMT1 protein in HCC cells. These findings establish DNMT1 as a bona fide target of miR-185 in HCC cells. The role of DNMT1 in miR-185-induced inhibition of HCC growth was further supported by the fact that DNMT1 overexpression prevented miR-185-induced inhibition of HCC cell proliferation/invasion. miR-185 mimic or overexpression reduced PTEN promoter DNA methylation and enhanced PTEN expression, leading to the inhibition of Akt phosphorylation; these effects were partially reversed by DNMT1 overexpression. These results provide novel evidence that miR-185 inhibits HCC cell growth by targeting DNMT1, leading to PTEN induction and Akt inhibition. Thus, reactivation or induction of miR-185 may represent a novel therapeutic strategy for HCC treatment.

P0105 Inhibition of hepatocellular carcinoma growth by adenovirus-mediated expression of human telomerase reverse transcriptase COOH-27 terminal polypeptide in mice

Background A 27-kDa C-terminal fragment of human telomerase reverse transcriptase, hTERTC27, has previously been reported to inhibit the growth and tumorigenicity of HeLa human cervical cancer cells and U87-MG human glioblastoma multiforme cells. However, the antitumour effects of hTERTC27 in hepatoma and its underlying mechanisms are unclear. Methods In the current study, the therapeutic effect of hTERTC27, mediated by recombinant adenovirus, in hepatocellular carcinoma was explored in vitro and in vivo to investigate the possible mechanisms. Findings Recombinant adenovirus carrying hTERTC27 (rAdv-hTERTC27) effectively inhibited the growth and induced apoptosis of Hepa 1–6 cell lines. Dendritic cells transduced with rAdv-hTERTC27 were highly effective at inducing antigen-specific T cell proliferation and increasing the activated cytotoxicity of T cells against Hepa 1–6 cells. Hepatocellular carcinoma was inhibited significantly when a single dose of 5 × 10 7 pfu rAdv-hTERTC27 was administered intravenously. Interpretation Our results suggest that rAdv-hTERTC27 may serve as a reagent for intravenous administration when combined with telomerase-based gene therapy and immunotherapy for cancer.

Differential effects of sorafenib on liver versus tumor fibrosis mediated by stromal-derived factor 1 alpha/C-X-C receptor type 4 axis and myeloid differentiation antigen-positive myeloid cell infiltration in mice.

Sorafenib--a broad kinase inhibitor--is a standard therapy for advanced hepatocellular carcinoma (HCC) and has been shown to exert antifibrotic effects in liver cirrhosis, a precursor of HCC. However, the effects of sorafenib on tumor desmoplasia--and its consequences on treatment resistance--remain unknown. We demonstrate that sorafenib has differential effects on tumor fibrosis versus liver fibrosis in orthotopic models of HCC in mice. Sorafenib intensifies tumor hypoxia, which increases stromal-derived factor 1 alpha (SDF-1α) expression in cancer and stromal cells and, subsequently, myeloid differentiation antigen-positive (Gr-1(+)) myeloid cell infiltration. The SDF-1α/C-X-C receptor type 4 (CXCR4) pathway directly promotes hepatic stellate cell (HSC) differentiation and activation through the mitogen-activated protein kinase pathway. This is consistent with the association between SDF-1α expression with fibrotic septa in cirrhotic liver tissues as well as with desmoplastic regions of human HCC samples. We demonstrate that after treatment with sorafenib, SDF-1α increased the survival of HSCs and their alpha-smooth muscle actin and collagen I expression, thus increasing tumor fibrosis. Finally, we show that Gr-1(+) myeloid cells mediate HSC differentiation and activation in a paracrine manner. CXCR4 inhibition, using AMD3100 in combination with sorafenib treatment, prevents the increase in tumor fibrosis--despite persistently elevated hypoxia--in part by reducing Gr-1(+) myeloid cell infiltration and inhibits HCC growth. Similarly, antibody blockade of Gr-1 reduces tumor fibrosis and inhibits HCC growth when combined with sorafenib treatment. Blocking SDF-1α/CXCR4 or Gr-1(+) myeloid cell infiltration may reduce hypoxia-mediated HCC desmoplasia and increase the efficacy of sorafenib treatment.

FOXO/TXNIP pathway is involved in the suppression of hepatocellular carcinoma growth by glutamate antagonist MK-801

BackgroundAccumulating evidence has suggested the importance of glutamate signaling in cancer growth, yet the signaling pathway has not been fully elucidated. N-methyl-D-aspartic acid (NMDA) receptor activates intracellular signaling pathways such as the extracellular-signal-regulated kinase (ERK) and forkhead box, class O (FOXO). Suppression of lung carcinoma growth by NMDA receptor antagonists via the ERK pathway has been reported. However, series of evidences suggested the importance of FOXO pathways for the regulation of normal and cancer cell growth. In the liver, FOXO1 play important roles for the cell proliferation such as hepatic stellate cells as well as liver metabolism. Our aim was to investigate the involvement of the FOXO pathway and the target genes in the growth inhibitory effects of NMDA receptor antagonist MK-801 in human hepatocellular carcinoma.MethodsExpression of NMDAR1 in cancer cell lines from different tissues was examined by Western blot. NMDA receptor subunits in HepG2, HuH-7, and HLF were examined by reverse transcriptase polymerase chain reaction (RT-PCR), and growth inhibition by MK-801 and NBQX was determined using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. The effects of MK-801 on the cell cycle were examined by flow cytometry and Western blot analysis. Expression of thioredoxin-interacting protein (TXNIP) and p27 was determined by real-time PCR and Western blotting. Activation of the FOXO pathway and TXNIP induction were examined by Western blotting, fluorescence microscopy, Chromatin immunoprecipitation (ChIP) assay, and reporter gene assay. The effects of TXNIP on growth inhibition were examined using the gene silencing technique.ResultsNMDA receptor subunits were expressed in all cell lines examined, and MK-801, but not NBQX, inhibited cell growth of hepatocellular carcinomas. Cell cycle analysis showed that MK-801 induced G1 cell cycle arrest by down-regulating cyclin D1 and up-regulating p27. MK-801 dephosphorylated Thr24 in FOXO1 and induced its nuclear translocation, thus increasing transcription of TXNIP, a tumor suppressor gene. Knock-down of TXNIP ameliorated the growth inhibitory effects of MK-801.ConclusionsOur results indicate that functional NMDA receptors are expressed in hepatocellular carcinomas and that the FOXO pathway is involved in the growth inhibitory effects of MK-801. This mechanism could be common in hepatocellular carcinomas examined, but other mechanisms such as ERK pathway could exist in other cancer cells as reported in lung carcinoma cells. Altered expression levels of FOXO target genes including cyclin D1 and p27 may contribute to the inhibition of G1/S cell cycle transition. Induction of the tumor suppressor gene TXNIP plays an important role in the growth inhibition by MK-801. Our report provides new evidence that FOXO-TXNIP pathway play a role in the inhibition of the hepatocellular carcinoma growth by MK-801.

Fn14•Trail Effectively Inhibits Hepatocellular Carcinoma Growth

BackgroundNew strategies for the treatment of hepatocellular carcinoma (HCC) are needed, given that currently available chemotherapeutics are inefficient. Since tumor growth reflects the net balance between pro-proliferative and death signaling, agents shifting the equilibrium toward the latter are of considerable interest. The TWEAK:Fn14 signaling axis promotes tumor cell proliferation and tumor angiogenesis, while TRAIL:TRAIL-receptor (TRAIL-R) interactions selectively induce apoptosis in malignant cells. Fn14•TRAIL, a fusion protein bridging these two pathways, has the potential to inhibit tumor growth, by interfering with TWEAK:Fn14 signaling, while at the same time enforcing TRAIL:TRAIL-R-mediated apoptosis. Consequently, Fn14•TRAIL's capacity to inhibit HCC growth was tested.ResultsFn14•TRAIL induced robust apoptosis of multiple HCC cell lines, while sparing non-malignant hepatocyte cell lines. Differential susceptibility to this agent did not correlate with expression levels of TRAIL, TRAIL-R, TWEAK and Fn14 by these lines. Fn14•TRAIL was more potent than soluble TRAIL, soluble Fn14, or a combination of the two. The requirement of both of Fn14•TRAIL's molecular domains for function was established using blocking antibodies directed against each of them. Subcutaneous injection of Fn14•TRAIL abrogated HCC growth in a xenograft model, and was well tolerated by the mice.ConclusionsIn this study, Fn14•TRAIL, a multifunctional fusion protein originally designed to treat autoimmunity, was shown to inhibit the growth of HCC, both in vitro and in vivo. The demonstration of this fusion protein’s potent anti-tumor activity suggests that simultaneous targeting of two signaling axes by a single fusion can serve as a basis for highly effective anti-cancer therapies.

LBH589 Inhibits proliferation and metastasis of hepatocellular carcinoma via inhibition of gankyrin/stat3/akt pathway

BackgroundGankyrin has shown to be overexpressed in human liver cancers and plays a complex role in hepatocarcinogenesis. Panobinostat (LBH589), a new hydroxamic acid-derived histone deacetylase inhibitor has shown promising anticancer effects recently. Here, we investigated the potential of LBH589 as a form of treatment for hepatocellular carcinoma (HCC).MethodsGankyrin plasmid was transfected into HCC cells, and the cells were selected for more than 4 weeks by incubation with G418 for overexpression clones. The therapeutic effects of LBH589 were evaluated in vitro and in vivo. Cell proliferation, apoptosis, cell cycle, invasive potential, and epithelial-mesenchy-mal transition (EMT) were examined.ResultsLBH589 significantly inhibited HCC growth and metastasis in vitro and in vivo. Western blotting analysis indicated that LBH589 could decrease the expression of gankyrin and subsequently reduced serine-phosphorylated Akt and tyrosine-phosphorylated STAT3 expression although the total Akt and STAT3 were unaffected. LBH589 inhibited metastasis in vitro via down-regulation of N-cadherin, vimentin, TWIST1, VEGF and up-regulation of E-cadherin. LBH589 also induced apoptosis and G1 phase arrest in HCC cell lines. Ectopic expression of gankyrin attenuated the effects of LBH589, which indicates that gankyrin might play an important role in LBH589 mediated anticancer effects. Lastly, in vivo study indicated that LBH589 inhibited tumor growth and metastasis, without discernable adverse effects comparing to control group, with abrogating gankyrin/STAT3/Akt pathway.ConclusionsOur results suggested that LBH589 could inhibit HCC growth and metastasis through down-regulating gankyrin/STAT3/Akt pathway. LBH589 may present itself as a novel therapeutic strategy for HCC.

Mitochondria-targeted antioxidant and glycolysis inhibition

Hepatocellular carcinoma (HCC) is the third leading cause of cancer-related deaths worldwide. Mito-carboxy proxyl (Mito-CP), a lipophilic cationic nitroxide, accumulates in the mitochondria because of the large negative transmembrane potential. Studies have shown that these agents act by disrupting the energy-producing mechanism, inducing mitochondrial-mediated apoptosis, and also enhancing the action of other chemotherapeutic agents in cancer cells. We hypothesized that the combination of Mito-CP and glycolysis inhibitor, 2-deoxyglucose (2-DG), would synergistically inhibit HCC in vitro. HepG2 cells and primary hepatocytes were treated with various combinations of Mito-CP and 2-DG. Cell cytotoxicity was measured using the methylthiazolyldiphenyl-tetrazolium bromide assay and ATP bioluminescence assay. In addition, caspase 3/7 enzymatic activity was examined after treatment. Mito-CP and 2-DG induced synergistic cytotoxicity in HepG2 cells in a dose-dependent and time-dependent manner, whereas primary cells remained viable and unaffected after treatment. The intracellular ATP levels of HepG2 cells were suppressed within 6 h of combination treatment, whereas primary cells maintained higher levels of ATP. Dose-dependent increases in caspase 3/7 activity occurred in HepG2 cells in a time-dependent manner, showing the initiation of cell death through the apoptotic pathway. These findings indicate that a combination of Mito-CP and 2-DG effectively inhibits HCC growth in vitro. The increase in caspase 3/7 activity supports the occurrence of 2-DG-induced and Mito-CP-induced apoptotic death in HCC. The inability of the compounds to induce cytotoxicity or suppress the production of ATP in primary hepatocytes provides a selective and synergistic approach for the treatment of HCC.

Inhibition of hepatocellular carcinoma growth using immunoliposomes for co-delivery of adriamycin and ribonucleotide reductase M2 siRNA

The chemotherapy combined with gene therapy has received great attention. We developed targeted LPD (liposome-polycation-DNA complex) conjugated with anti-EGFR (epidermal growth factor receptor) Fab' co-delivering adriamycin (ADR) and ribonucleotide reductase M2 (RRM2) siRNA (ADR-RRM2-TLPD), to achieve combined therapeutic effects in human hepatocellular carcinoma (HCC) overexpressing EGFR. The antitumor activity and mechanisms of ADR-RRM2-TLPD were investigated. The results showed that RRM2 expression was higher in HCC than in non-HCC tissue, and RRM2 siRNA inhibited HCC cell proliferation, suggesting that RRM2 is a candidate target for HCC therapy. ADR-RRM2-TLPD delivered ADR and RRM2 siRNA to EGFR overexpressing HCC cells specifically and efficiently both in vitro and in vivo, resulting in enhanced therapeutic effects (cytotoxicity, apoptosis and senescence-inducing activity) compared with single-drug loaded or non-targeted controls, including ADR-NC-TLPD (targeted LPD co-delivering ADR and negative control siRNA), RRM2-TLPD (targeted LPD delivering RRM2 siRNA) and ADR-RRM2-NTLPD (non-targeted LPD co-delivering ADR and RRM2 siRNA). Mechanism studies showed that p21 is involved in the combined therapeutic effect of ADR-RRM2-TLPD. The average weight of the orthotopic HCC in mice treated with ADR-RRM2-TLPD was significantly lighter than that of mice treated with other controls. Thus, ADR-RRM2-TLPD represents a potential strategy for combined therapy of HCC overexpressing EGFR.

Interferon-β gene-modified human bone marrow mesenchymal stem cells attenuate hepatocellular carcinoma through inhibiting AKT/FOXO3a pathway

Objective:This study aims to investigate the using of bone marrow mesenchymal stem cells (BMSCs) genetically engineered to produce interferon-β (IFN-β) as a gene delivery system to treat hepatocellular carcinoma (HCC) in vitro and in vivo.Methods:To measure the effects on tumour cell growth in vitro, IFN-β-producing BMSCs (BMSC/IFN-β) were co-cultured with the HCC cell line HepG2 and Huh7. Enzyme-linked immunosorbent assay (ELISA) was used to detect the IFN-β secretion in the BMSC culture condition medium (CM). The effect of BMSC/IFN-β on HCC cells proliferation was examined both in vitro and in vivo by using MTT, colony formation assay, BrdU staining, cell cycle analysis, and xenografted NOD/SCID mouse tumour model. To examine the impact of BMSC/IFN-β on the AKT/FOXO3a signalling, RT–PCR and western blotting were performed.Results:The BMSC/IFN-β cells can stably secrete high levels of IFN-β. Both MTT and colony forming assay showed that HCC cells had a lower growth rate when cultured in BMSC/IFN-β-CM as compared with that in BMSC/vector-CM or DMEM culture group. Co-culture with BMSC/IFN-β-CM dramatically decreased the percentages of cells with incorporated BrdUrd. In BMSC/IFN-β-CM-treated HCC cells, the proportion of G1-phase cells increased but it decreased in the S phase of the cell. The BMSC/IFN-β inhibited HCC growth in NOD/SCID mice and proved the survival period of these mice. Compared with the control group, p21 and p27 expression of hepatoma cells increased, whereas cyclin D1 and phosphorylation of Rb expression decreased when co-cultured with BMSC/IFN-β-CM. It was associated with suppression of Akt activity and enhanced transcriptional activity of FOXO3a.Conclusion:The IFN-β gene-modified BMSCs can effectively inhibit the proliferation of HCC cells in vitro and in vivo through inhibiting AKT/FOXO3a pathway. These results indicate that BMSC/IFN-β are a powerful anticancer cytotherapeutic tool for HCC.