This in vivo study was designed to investigate the efficacy of ENMD-2076, a small-molecule kinase inhibitor with activity against the Aurora kinases A and B, and several other tyrosine kinases linked to cancer, including vascular endothelial growth factor receptor 2, cKit, and fibroblast growth factor receptor 1, against murine xenograft models of human colorectal cancer (CRC).
HT-29 CRC cell line xenografts were treated with either vehicle or ENMD-2076 (100 or 200 mg/kg) orally daily for 28 days. Tumor growth inhibition, dynamic contrast-enhanced magnetic resonance imaging, and 18FDG-positron emission tomography were conducted to assess the anti-proliferative, antiangiogenic, and antimetabolic responses, respectively. Effects on proliferation were also analyzed by immunohistochemical methods. Additionally, three patient-derived xenografts from primary and metastatic sites were treated with ENMD-2076 (100 mg/kg) and assessed for tumor growth inhibition.
In the HT-29 xenograft model, ENMD-2076 induced initial tumor growth inhibition followed by regression. Treatment was associated with significant tumor blanching, indicating a loss of vascularity and substantial reductions in tumor vascular permeability and perfusion as measured by dynamic contrast-enhanced magnetic resonance imaging. Positron emission tomography scanning showed significant decreases in 18FDG uptake at days 3 and 21 of treatment, which was associated with a marked reduction in proliferation as assessed by Ki-67. All three of the patient-derived xenografts tested were sensitive to treatment with ENMD 2076 as measured by tumor growth inhibition.
ENMD-2076 showed robust antitumor activity against cell line and patient-derived xenograft models of CRC that is detectable by functional imaging, supporting clinical investigation of this agent in CRC.
The Aurora kinases are a family of conserved serine-threonine kinases with key roles in mitotic cell division. As with other promising anti-cancer targets, patient selection strategies to identify a responsive subtype will likely be required for successful clinical development of Aurora kinase inhibitors. The purpose of this study was to evaluate the antitumor activity of the Aurora and angiogenic kinase inhibitor ENMD-2076 against preclinical models of breast cancer with identification of candidate predictive biomarkers.
Twenty-nine breast cancer cell lines were exposed to ENMD-2076 and the effects on proliferation, apoptosis, and cell cycle distribution were evaluated. In vitro activity was confirmed in in MDA-MB-468 and MDA-MB-231 triple-negative breast cancer xenografts. Systematic gene expression analysis was used to identify up- and down-regulated pathways in the sensitive and resistant cell lines, including within the triple-negative breast cancer subset.
ENMD-2076 demonstrated anti-proliferative activity against breast cancer cell lines, with more robust activity against cell lines lacking estrogen receptor expression and those without increased HER2 expression. Within the triple-negative breast cancer subset, cell lines with a p53 mutation and increased p53 expression were more sensitive to the cytotoxic and pro-apoptotic effects of ENMD-2076 exposure than cell lines with decreased p53 expression.
ENMD-2076 exhibited robust anticancer activity against models of triple-negative breast cancer and the candidate predictive biomarkers identified in this study may be useful in selecting patients for Aurora kinase inhibitors in the future.
triple-negative breast cancer; aurora kinase; ENMD-2076; angiogenesis; vascular endothelial growth factor
Signal transducers and activators of transcription (STAT) 1 plays a pivotal role in cell-cycle and cell-fate determination, and vascular endothelial growth factor (VEGF) also contributes tumor growth. Recently interferon (IFN) α has been reported to be effective for prevention of hepatocellular carcinomas (HCCs) recurrence, but the detailed mechanisms remain elusive. In vitro, cobalt chloride–treated VEGF induction and hypoxia responsive element (HRE) promoter activity were inhibited by IFNs and this abrogation was cancelled by introduction of small interfering RNA for STAT1. Immunoprecipitation /Chromatin Immunoprecipitation analyses showed STAT1 bound to hypoxia-inducible factor (HIF)-1α and dissociated HIF-complex from HRE promoter lesion. In a xenograft model using Balb/c nude mice, tumor growth was suppressed by IFNα through inhibition of VEGF expression and it was oppositely enhanced when STAT1-deleted cells were injected. This augmentation was due to upregulation of VEGF and hyaluronan synthase 2. In human samples, 29 HCCs were resected, divided into two groups based on STAT1 activation in tumor and the clinical features were investigated. Patients with suppressed STAT1 activity had a shorter recurrence-free survival. Histological and RT-PCR analyses showed portal vein microinvasion and increased VEGF levels in tumors from suppressed STAT1 group. These human samples also showed a reverse correlation between VEGF and STAT1-regulated genes expression. These results in vitro and in vivo suggested that IFNα are potential candidates for prevention of vessel invasion acting through inhibition of VEGF expression and need to be properly used when STAT1 expression is suppressed.
STAT1; VEGF; HCC; HIF-1; IFNα
Hepatitis C virus (HCV) infection is one of the major causes of chronic hepatitis, liver cirrhosis, which subsequently leads to hepatocellular carcinoma (HCC). The overexpression of the angiogenic factors has been demonstrated in HCC. In this study, we investigated the potential of HCV gene expression in inducing angiogenesis. Our results show that HCV infection leads to the stabilization of hypoxia-inducible factor 1α (HIF-1α). We further show that this stabilization was mediated via oxidative stress induced by HCV gene expression. The activation of NF-κB, STAT-3, PI3-K/AkT, and p42/44 mitogen-activated protein kinase was necessary for HIF-1α stabilization. HIF-1α induction in turn led to the stimulation of vascular endothelial growth factor. By using the chick chorioallantoic membrane assay, we show that HCV-infected cells released angiogenic cytokines, leading to neovascularization in vivo. These results indicate the potential of HCV gene expression in angiogenesis.
Hypoxia-inducible factor-1 (HIF-1) is a ubiquitously expressed oxygen-regulated transcription factor composed of α and β subunits. HIF-1 activates the transcription of various genes including those involved in the formation and metastatic growth of hepatocellular carcinoma (HCC).
To investigate the levels of hepatic and circulating HIF-1α expression in a range of patients with liver disease in order to determine how it can be used in the diagnosis of HCC and in establishing prognosis.
Patients and Methods
Total RNA was extracted from a self-controlled HCC and paracancerous specimen. HIF-1α mRNA was amplified by nested RT-PCR and confirmed by sequencing. Tissue HIF-1α was analyzed by immunohistochemistry. The levels of HIF-1α, vascular endothelial growth factor (VEGF), and angiopoietin-2 (Ang-2) expression in the sera of 220 patients with liver disease were quantitatively detected by ELISA.
The positive staining of liver HIF-1α was brown and granule-like and was mainly present in the cytoplasm, with lower levels in the nucleus of hepatocytes. Its incidence was 80% in HCC cells and 100% in paracancerous tissues, with no significant difference in HIF-1α expression in relation to tumor number, degree of differentiation, or hepatitis B surface antigen (HBsAg) positivity, but with some correlation between HIF-1α and tumor size. HIF-1α expression was detected in the sera of HCC patients at a significantly higher level than in cases of benign liver disease, with pathological characteristics associated with the levels of circulating VEGF and Ang-2 expression, the size of the tumor, and the level of extrahepatic metastasis, but not with patients’ gender, age, or alpha-fetoprotein (AFP) levels.
Hepatic HIF-1α expression is associated with the development and prognosis of HCC, and circulating HIF-1α level is a useful marker for HCC diagnosis and prognosis.
Hypoxia-Inducible Factor 1, alpha Subunit; Carcinoma, Hepatocellular; Gene Expression; Vascular Endothelial Growth Factor; Angiopoietin-2
We investigated the molecular mechanisms underlying the effect of sorafenib and SC-59, a novel sorafenib derivative, on hepatocellular carcinoma (HCC). Sorafenib activated autophagy in a dose- and time-dependent manner in the HCC cell lines PLC5, Sk-Hep1, HepG2 and Hep3B. Sorafenib downregulated phospho-STAT3 (P-STAT3) and subsequently reduced the expression of myeloid cell leukemia-1 (Mcl-1). Inhibition of Mcl-1 by sorafenib resulted in disruption of the Beclin 1-Mcl-1 complex; however, sorafenib did not affect the amount of Beclin 1, suggesting that sorafenib treatment released Beclin 1 from binding with Mcl-1. Silencing of SHP-1 by small interference RNA (siRNA) reduced the effect of sorafenib on P-STAT3 and autophagy. Ectopic expression of Mcl-1 abolished the effect of sorafenib on autophagy. Knockdown of Beclin 1 by siRNA protected the cells from sorafenib-induced autophagy. Moreover, SC-59, a sorafenib derivative, had a more potent effect on cancer cell viability than sorafenib. SC-59 downregulated P-STAT3 and induced autophagy in all tested HCC cell lines. Furthermore, our in vivo
data showed that both sorafenib and SC-59 inhibited tumor growth, downregulated P-STAT3, enhanced the activity of SHP-1 and induced autophagy in PLC5 tumors, suggesting that sorafenib and SC-59 activate autophagy in HCC. In conclusion, sorafenib and SC-59 induce autophagy in HCC through a SHP-1-STAT3-Mcl-1-Beclin 1 pathway.
SC-59; sorafenib; STAT3; HCC
Hepatocellular carcinoma (HCC) is the third cause of cancer-related death worldwide. When these tumors are in advanced stages, few therapeutic options are available. Therefore, it is essential to search for new treatments to fight this disease. In this study, we investigated the effects of cannabinoids – a novel family of potential anticancer agents – on the growth of HCC. We found that Δ9-tetrahydrocannabinol (Δ9-THC, the main active component of Cannabis sativa) and JWH-015 (a cannabinoid receptor 2 (CB2) cannabinoid receptor-selective agonist) reduced the viability of the human HCC cell lines HepG2 (human hepatocellular liver carcinoma cell line) and HuH-7 (hepatocellular carcinoma cells), an effect that relied on the stimulation of CB2 receptor. We also found that Δ9-THC- and JWH-015-induced autophagy relies on tribbles homolog 3 (TRB3) upregulation, and subsequent inhibition of the serine–threonine kinase Akt/mammalian target of rapamycin C1 axis and adenosine monophosphate-activated kinase (AMPK) stimulation. Pharmacological and genetic inhibition of AMPK upstream kinases supported that calmodulin-activated kinase kinase β was responsible for cannabinoid-induced AMPK activation and autophagy. In vivo studies revealed that Δ9-THC and JWH-015 reduced the growth of HCC subcutaneous xenografts, an effect that was not evident when autophagy was genetically of pharmacologically inhibited in those tumors. Moreover, cannabinoids were also able to inhibit tumor growth and ascites in an orthotopic model of HCC xenograft. Our findings may contribute to the design of new therapeutic strategies for the management of HCC.
cannabinoid; CB2 receptor; AMPK; autophagy; hepatocellular carcinoma
Hepatocellular carcinoma (HCC) displays a characteristic hypervascularity and depends on angiogenesis for tumor growth, which thus provides a potential target for therapeutic approaches to HCC. In this study, through the use of combined micro-positron emission tomography (PET)/computed tomography (CT), we investigate if such a combined targeting of vascular endothelial growth factor (VEGF) activity and expression might retard HCC growth in an orthotopic intrahepatic xenograft model.
Xenograft models were created by intraportal vein injection of HepG2 cell suspensions in severe combined immunodeficient mice. The mice were then treated with (1) rapamycin (RAPA), a mammalian target of rapamycin pathway inhibitor; (2) bevazicumab (BEV), a VEGF monoclonal antibody; and (3) a RAPA/BEV combination.
Assessment of HCC progression using CT with Omnipaque and PET with 2-deoxy-2-(F-18)-fluoro-d-glucose showed that mice treated with RAPA/BEV had the lowest standardized uptake values (SUVs). At week 2, mice treated with RAPA/BEV, RAPA, and BEV all showed a marked decrease in the SUVmax readings with the greatest drop being observed in the RAPA/BEV group (1.33 + 0.26, 1.81 + 0.2, 2.05 + 0.4 vs. vehicle control 2.11 + 0.53).
Our results, supported by micro-PET/CT, suggest that RAPA/BEV represents a potential novel antiangiogenic therapy for the treatment of HCC.
Hepatocellular carcinoma; Orthotopic xenograft model; Tumor vasculature; Rapamycin; Bevacizumab; 18F-FDG-PET; Micro-CT; Micro-PET
Patients with advanced stages of hepatocellular carcinoma (HCC) face a poor prognosis. Although encouraging clinical results have been obtained with multikinase inhibitor sorafenib, the development of improved therapeutic strategies for HCC remains an urgent goal. Aurora kinases are key regulators of the cell cycle, and their uncontrolled expression promotes aneuploidy and tumor development. In tissue microarray analyses, we detected aurora-A kinase expression in all of the examined 93 human HCC samples, whereas aurora-B kinase expression levels significantly correlated with the proliferation index of HCCs. In addition, two human HCC cell lines (Huh-7 and HepG2) were tested positive for aurora-A and -B and revealed Ser10 phosphorylation of histone H3, indicating an increased aurora-B kinase activity. The antiproliferative features of a novel aurora kinase inhibitor, PHA-739358, currently under investigation in phase 2 clinical trials for other solid tumors, were examined in vitro and in vivo. At concentrations exceeding 50nM, PHA-739358 completely suppressed tumor cell proliferation in cell culture experiments and strongly decreased histone H3 phosphorylation. Cell cycle inhibition and endoreduplication were observed at 50 nM, whereas higher concentrations led to a complete G2/M-phase arrest. In vivo, administration of PHA-739358 resulted in significant tumor growth inhibition at a well-tolerated dose. In combination with sorafenib, additive effects were observed. Remarkably, when tumors restarted to grow under sorafenib monotherapy, subsequent treatment with PHA-739358 induced tumor shrinkage by up to 81%. Thus, targeting aurora kinases with PHA-739358 is a promising therapeutic strategy administered alone or in combination with sorafenib for patients with advanced stages of HCC.
Background & Aims
Epidemiological studies have shown that obesity is a risk factor for hepatocellular carcinoma (HCC). Lower adiponectin levels are associated with poor prognosis in obese HCC patients hence it is plausible that adiponectin acts as a negative regulator of HCC. Here, we investigated the effects of adiponectin on HCC development and elucidated the underlying molecular mechanisms.
We utilized various in vitro assays using Huh7 and HepG2 HCC cells to examine the signal transduction pathways involved in protective function of adiponectin in HCC. These studies were followed by in vivo approaches using HCC xenografts and tumor analysis. Results from in vitro and in vivo findings were corroborated using human HCC tissue micro-array (TMA) and analysis of clinicopathological characteristics.
Adiponectin treatment resulted in increased apoptosis of HCC cells via activation of caspase-3. Adiponectin increased phosphorylation of c-Jun-N-terminal kinase (JNK) and inhibition of JNK-phosphorylation inhibited adiponectin-induced apoptosis and caspase-3 activation. Adiponectin increased phosphorylation of AMP-activated protein kinase (AMPK) and tumor suppressor TSC2 and inhibited mammalian target of rapamycin (mTOR) phosphorylation. Inhibition of AMPK phosphorylation not only inhibited adiponectin-induced JNK phosphorylation but also effectively blocked biological effects of adiponectin. In vivo study showed that adiponectin treatment substantially reduced liver tumorigenesis in nude mice. Importantly, analysis of adiponectin expression levels in TMA of human HCC patients revealed an inverse correlation of adiponectin expression with tumor size.
These novel findings show protective role of adiponectin in liver tumorigenesis and could help explain poor prognosis of obese HCC patients who typically have low adiponectin levels.
Hepatocellular carcinoma; Adiponectin; TSC2; mTOR
Hepatocellular carcinoma (HCC) is an aggressive and often fatal neoplasm. HepG2 cells are a cell line derived from HCC. This investigation shows that vasoactive intestinal peptide (VIP) inhibits HepG2 cell proliferation in vitro. In addition, VIP decreases the expression of signal transducers and activators of transcription-3 (STAT-3) and phosphorylated STAT-3 (pSTAT-3). Transfection of HepG2 cells with STAT-3 siRNA also dose-dependently inhibits proliferation. These findings suggest that VIP-mediated inhibition of HepG2 proliferation may be mediated by STAT-3. Further studies demonstrate that VIP increases HepG2 cAMP levels and 8-cl-cAMP inhibits HepG2 proliferation as well as pSTAT-3 and STAT-3 levels, suggesting that cAMP is also involved in the inhibition of HepG2 proliferation. VIP also attenuates the proliferative effects of hepatocyte growth factor (HGF) and interleukin-6 (IL-6) on HepG2 cells. These preliminary studies suggest that the antiproliferative actions of VIP may offer a new and promising means of suppressing HCC.
hepatocellular carcinoma; hepatocyte proliferation; VIP; IL-6; HGF
The members of inhibitor of apoptosis proteins (IAPs) family are key negative regulators of apoptosis. Overexpression of IAPs are found in hepatocellular carcinoma (HCC), and can contribute to chemotherapy resistance and recurrence of HCC. Small-molecule Second mitochondria-derived activator of caspases (Smac) mimetics have recently emerged as novel anticancer drugs through targeting IAPs. The specific aims of this study were to 1) examine the anticancer activity of Smac mimetics as a single agent and in combination with chemotherapy in HCC cells, and 2) investigate the mechanism of anticancer action of Smac mimetics.
Four HCC cell lines, including SMMC-7721, BEL-7402, HepG2 and Hep3B, and 12 primary HCC cells were used in this study. Smac mimetic SM-164 was used to treat HCC cells. Cell viability, cell death induction and clonal formation assays were used to evaluate the anticancer activity. Western blotting analysis and a pancaspase inhibitor were used to investigate the mechanisms.
Although SM-164 induced complete cIAP-1 degradation, it displayed weak inhibitory effects on the viability of HCC cells. Nevertheless, SM-164 considerably potentiated Apo2 ligand or TNF-related apoptosis-inducing ligand (APO2L/TRAIL)- and Doxorubicin-mediated anticancer activity in HCC cells. Mechanistic studies demonstrated that SM-164 in combination with chemotherapeutic agents resulted in enhanced activation of caspases-9, -3 and cleavage of poly ADP-ribose polymerase (PARP), and also led to decreased AKT activation.
Smac mimetics can enhance chemotherapeutic-mediated anticancer activity by enhancing apoptosis signaling and suppressing survival signaling in HCC cells. This study suggests Smac mimetics are potential therapeutic agents for HCC.
IFNs are a group of cytokines that possess potent antiviral and antitumor activities, while β-catenin pathway is a proliferative pathway involved in carcinogenesis. Interaction between these two pathways has not been well elaborated in hepatocellular carcinoma (HCC).
HCC cell lines, HepG2 and Huh7, were used in this study. β-catenin protein levels and corresponding signaling activities were observed by flow cytometry and luciferase assay, respectively. Cell proliferation was quantified by counting viable cells under microscope, and apoptosis by TUNEL assay. DKK1 and GSK3β levels were determined by flow cytometry. Secreted DKK1 was tested by ELISA. FLUD, S3I and aDKK1 were used to inhibit STAT1, STAT3 and DKK1 activities, respectively.
Our findings show that all three types of IFNs, IFNα, IFNγ and IFNλ, are capable of inhibiting β-catenin signaling activity in HepG2 and Huh7 cells, where IFNγ was the strongest (p<0.05). They expressed suppression of cellular proliferation and induced apoptosis. IFNγ expressed greater induction ability when compared to IFNα and IFNλ (p<0.05). All tested IFNs could induce DKK1 activation but not GSK3β in HepG2 and Huh7 cells. IFNs induced STAT1 and STAT3 activation but by using specific inhibitors, we found that only STAT3 is vital for IFN-induced DKK1 activation and apoptosis. In addition, DKK1 inhibitor blocked IFN-induced apoptosis. The pattern of STAT3 activation by different IFNs is found consistent with the levels of apoptosis with the corresponding IFNs (p<0.05).
In hepatocellular carcinoma, all three types of IFNs are found to induce apoptosis by inhibiting β-catenin signaling pathway via a STAT3- and DKK1-dependent pathway. This finding points to a cross-talk between different IFN types and β-catenin signaling pathways which might be carrying a biological effect not only on HCC, but also on processes where the two pathways bridge.
AIM: To investigate the anti-angiogenic and anti-tumor activities of recombinant vascular basement membrane-derived multifunctional peptide (rVBMDMP) in hepatocellular carcinoma (HCC).
METHODS: HepG2, Bel-7402, Hep-3B, HUVE-12 and L-02 cell lines were cultured in vitro and the inhibitory effect of rVBMDMP on proliferation of cells was detected by MTT assay. The in vivo antitumor efficacy of rVBMDMP on HCC was assessed by HepG2 xenografts in nude mice. Distribution of rVBMDMP, mechanism by which the growth of HepG2 xenografts is inhibited, and microvessel area were observed by proliferating cell nuclear antigen (PCNA) and CD31 immunohistochemistry.
RESULTS: MTT assay showed that rVBMDMP markedly inhibited the proliferation of human HCC (HepG2, Bel-7402, Hep-3B) cells and human umbilical vein endothelial (HUVE-12) cells in a dose-dependent manner, with little effect on the growth of L-02 cells. When the IC50 was 4.68, 7.65, 8.96, 11.65 and 64.82 μmol/L, respectively, the potency of rVBMDMP to HepG2 cells was similar to 5-fluorouracil (5-FU) with an IC50 of 4.59 μmol/L. The selective index of cytotoxicity to HepG2 cells of rVBMDMP was 13.8 (64.82/4.68), which was higher than that of 5-FU [SI was 1.9 (8.94/4.59)]. The VEGF-targeted recombinant humanized monoclonal antibody bevacizumab (100 mg/L) did not affect the proliferation of HepG2, Bel-7402, Hep-3B and L-02 cells, but the growth inhibitory rate of bevacizumab (100 mg/L) to HUVE-12 cells was 87.6% ± 8.2%. Alternis diebus intraperitoneal injection of rVBMDMP suppressed the growth of HepG2 xenografts in a dose-dependent manner. rVBMDMP (1, 3, 10 mg/kg) decreased the tumor weight by 12.6%, 55.9% and 79.7%, respectively, compared with the vehicle control. Immunohistochemical staining of rVBMDMP showed that the positive area rates (2.2% ± 0.73%, 4.5% ± 1.3% and 11.5% ± 3.8%) in rVBMDMP treated group (1, 3, 10 mg/kg) were significantly higher than that (0.13% ± 0.04%) in the control group (P < 0.01). The positive area rates (19.0% ± 5.7%, 12.2% ± 3.5% and 5.2% ± 1.6% ) of PCNA in rVBMDMP treated group (1, 3, 10 mg/kg) were significantly lower than that (29.5% ± 9.4%) in the control group (P < 0.05). rVBMDMP at doses of 1, 3 and 10 mg/kg significantly reduced the tumor microvessel area levels (0.26% ± 0.07%, 0.12% ± 0.03% and 0.05% ± 0.01% vs 0.45% ± 0.15%) in HepG2 xenografts (P < 0.01), as assessed by CD31 staining.
CONCLUSION: rVBMDMP has effective and unique anti-tumor properties, and is a promising candidate for the development of anti-tumor drugs.
Hepatocellular carcinoma; Recombinant vascular basement membrane-derived multifunctional peptide; Proliferating cell nuclear antigen; CD31; Therapeutic action
Hepatocellular carcinomas (HCCs) are tumors with a highly developed vascular architecture. HCC cells require access to blood vessels for growth and metastasis; therefore, the inhibition of angiogenesis represents a potential therapeutic target for HCC that may reduce the mortality and morbidity from HCC. Various attempts to develop an anti-angiogenic therapy have been made in past decades; however, modest results have been achieved in clinical trials and the challenge of HCC treatment remains. Single-chain antibodies (scFv) are characterized by low molecular weight, low immunogenicity, high penetration and a short half-life, and are easy to produce on a large scale by genetic engineering. Accordingly, an scFv against a specific angiogenic regulator, such as angiopoietin (Ang), may be a promising anti-angiogenic therapy for HCC. Our previous study indicated that an imbalanced expression of angiopoietin-2 (Ang-2) vs. angiopoietin-1 (Ang-1) in HCCs contributes to initiation of neovascularization and promotes the angiogenesis and progression of HCCs. Therefore, we suggest that specific Ang-2-targeting interventions may be valuable in the treatment of HCC via remodeling the neovascular network and changing the tumor microenvironment. In this study, a prokaryotic expression vector of Ang-2 was constructed and purified human Ang-2 protein was isolated. An scFv against human Ang-2 (scFv-Ang2) was identified and purified via phage display technology, and the effects of scFv-Ang2 in vitro and in vivo on HCC in nude mice were evaluated. The results show that scFv-Ang2 inhibits vascular endothelial growth factor (VEGF) and Ang-2 induces the proliferation, migration and tubule formation of human umbilical vein endothelial cells (HUVECs) in vitro. In the in vivo assay, statistical indices, including tumor weight and volume, metastases to lungs, CD31 expression and the microvessel density (MVD) count in the scFv-Ang2-treated group of mice were significantly lower than those in the control group (P<0.05). In conclusion, the successfully generated scFv-Ang2 showed significant inhibitory effects on the angiogenesis and tumor growth of human HCC in vitro and in vivo.
angiopoietin-2; phage display peptide library; single-chain antibody; neovascularization; liver tumor
Traditional systemic chemotherapy does not provide survival benefits in patients with hepatocellular carcinoma (HCC). Molecular targeted therapy shows promise for HCC treatment, however, the duration of effectiveness for targeted therapies is finite and combination therapies offer the potential for improved effectiveness.
Sorafenib, a multikinase inhibitor, and YC-1, a soluble guanylyl cyclase (sGC) activator, were tested in HCC by proliferation assay, cell cycle analysis and western blot in vitro and orthotopic and ectopic HCC models in vivo.
In vitro, combination of sorafenib and YC-1 synergistically inhibited proliferation and colony formation of HepG2, BEL-7402 and HCCLM3 cells. The combination also induced S cell cycle arrest and apoptosis, as observed by activated PARP and caspase 8. Sorafenib and YC-1 respectively suppressed the expression of phosphorylated STAT3 (p-STAT3) (Y705) in a dose- and time-dependent manner. Combination of sorafenib and YC-1 significantly inhibited the expression of p-STAT3 (Y705) (S727), p-ERK1/2, cyclin D1 and survivin and SHP-1 activity compared with sorafenib or YC-1 used alone in all tested HCC cell lines. In vivo, sorafenib-YC-1 combination significantly suppressed the growth of HepG2 tumor xenografts with decreased cell proliferation and increased apoptosis observed by PCNA and PARP. Similar results were also confirmed in a HCCLM3 orthotopic model. There was a reduction in CD31-positive blood vessels and reduced VEGF expression, which suggested a combinational effect of sorafenib and YC-1 on angiogenesis. The reduced expression of p-STAT3, cyclin D1 and survivin was also observed with the combination of sorafenib and YC-1.
Our data show that sorafenib-YC-1 combination is a novel potent therapeutic agent that can target the STAT3 signaling pathway to inhibit HCC tumor growth.
YC-1; Sorafenib; Hepatocellular carcinoma; STAT3
Molecular targeted therapy has shown promise as a treatment for advanced hepatocellular carcinoma (HCC). Sorafenib, a multikinase inhibitor, recently received FDA approval for the treatment of advanced HCC. However, although sorafenib is well tolerated, concern for its safety has been expressed. Celecoxib (Celebrex®) is a selective cyclooxygenase-2 (COX-2) inhibitor which exhibits antitumor effects in human HCC cells. The present study examined the interaction between celecoxib and sorafenib in two human liver tumor cell lines HepG2 and Huh7. Our data showed that each inhibitor alone reduced cell growth and the combination of celecoxib with sorafenib synergistically inhibited cell growth and increased apoptosis. To better understand the molecular mechanisms underlying the synergistic antitumor activity of the combination, we investigated the expression profile of the combination-treated liver cancer cell lines using microarray analysis. Combination treatment significantly altered expression levels of 1,986 and 2,483 transcripts in HepG2 and Huh7 cells, respectively. Genes functionally involved in cell death, signal transduction and regulation of transcription were predominantly up-regulated, while genes implicated in metabolism, cell-cycle control and DNA replication and repair were mainly down-regulated upon treatment. However, combination-treated HCC cell lines displayed specificity in the expression and activity of crucial factors involved in hepatocarcinogenesis. The altered expression of some of these genes was confirmed by semi-quantitative and quantitative RT-PCR and by Western blotting. Many novel genes emerged from our transcriptomic analyses, and further functional analyses may determine whether these genes can serve as potential molecular targets for more effective anti-HCC strategies.
Hepatocellular carcinoma (HCC) is a hypervascular highly angiogenic tumor usually associated with liver cirrhosis. Vascular endothelial growth factor plays a critical role in vascular development in HCC. In contrast to the treatment of early-stage HCC, the treatment options for advanced HCC are limited and prognosis is often poor, which contributes to this tumor type being the third leading cause of cancer-related deaths worldwide. Metronomic chemotherapy, which was originally designed to inhibit angiogenesis, involves low-dose chemotherapeutic agents administered in a frequent regular schedule with no prolonged breaks and minimizes severe toxicities. We reviewed the potential effects and impact of metronomic chemotherapy in preclinical studies with HCC models and in patients with advanced HCC, especially when combined with a molecular targeted agent. Metronomic chemotherapy involves multiple mechanisms that include antiangiogenesis and antivasculogenesis, immune stimulation by reducing regulatory T cells and inducing dendritic cell maturation, and possibly some direct tumor cell targeting effects, including the cancer stem cell subpopulation. The total number of preclinical studies with HCC models shows impressive results using metronomic chemotherapy-based protocols, especially in conjunction with molecular targeted agents. Four clinical trials and two case reports evaluating metronomic chemotherapy for HCC indicate it to be a safe and potentially useful treatment for HCC. Several preclinical and clinical HCC studies suggest that metronomic chemotherapy may become an alternative type of chemotherapy for advanced unresectable HCC and postsurgical adjuvant treatment of HCC.
The prognosis of patients suffering from primary hepatocellular carcinoma (HCC) is unfavourable because the tumour usually develops in cirrhosis-affected liver and is typically not diagnosed until an advanced stage of the disease. The 5-year survival rate for HCC patients in Europe does not exceed 9%. On the basis of a clinical case, the present article discusses the strategy of treatment of HCC patients. Patients with advanced HCC, stage C according to the Barcelona Clinic Liver Cancer (BCLC) staging system, typically receive systemic chemotherapy with sorafenib. The standard management in the treatment of intermediate-stage HCC, i.e. BCLC's stage B, is chemoembolization (TACE). However, repeated TACE sessions activate factors involved in the process of angiogenesis such as hypoxia-inducible factor-1 (HIF-1) and vascular endothelial growth factor (VEGF), which can render the procedure ineffective. Therefore, there are scientific foundations for combining TACE with antiangiogenic agents such as sorafenib. Results of studies conducted to date indicate that the combination of sorafenib with TACE in patients with BCLC's stage B brings tangible therapeutic effects while being safe. The value of this therapeutic strategy is confirmed by the case described below, in which TACE + sorafenib have induced a partial regression of HCC.
hepatocellular carcinoma; HCC; sorafenib; TACE
Hepatocellular carcinoma (HCC) is an aggressive cancer, and is the third leading cause of cancer death worldwide. Standard therapy is ineffective partly because HCC is intrinsically resistant to conventional chemotherapy. Its poor prognosis and limited treatment options make it critical to develop novel and selective chemotherapeutic agents. Since the Wnt/β-catenin pathway is essential in HCC carcinogenesis, we studied the inhibition of Wnt-1-mediated signaling as a potential molecular target in HCC.
We demonstrated that Wnt-1 is highly expressed in human hepatoma cell lines and a subgroup of human HCC tissues compared to paired adjacent non-tumor tissues. An anti-Wnt-1 antibody dose-dependently decreased viability and proliferation of Huh7 and Hep40 cells over-expressing Wnt-1 and harboring wild type β-catenin, but did not affect normal hepatocytes with undetectable Wnt-1 expression. Apoptosis was also observed in Huh7 and Hep40 cells after treatment with anti-Wnt-1 antibody. In these two cell lines, the anti-Wnt-1 antibody decreased β-catenin/Tcf4 transcriptional activities, which were associated with down-regulation of the endogenous β-catenin/Tcf4 target genes c-Myc, cyclin D1, and survivin. Intratumoral injection of anti-Wnt-1 antibody suppressed in vivo tumor growth in a Huh7 xenograft model, which was also associated with apoptosis and reduced c-Myc, cyclin D1, and survivin expressions.
Our results suggest that Wnt-1 is a survival factor for HCC cells, and that the blockade of Wnt-1-mediated signaling may offer a potential pathway-specific therapeutic strategy for the treatment of a subgroup of HCC that over-expresses Wnt-1.
The MEK-ERK growth signaling pathway is important in human hepatocellular carcinoma (HCC). To evaluate the targeting of this pathway in HCC, we characterized a novel, orally-active MEK inhibitor, PD184161, using human HCC cells (HepG2, Hep3B, PLC, and SKHep) and in vivo human tumor xenografts. PD184161 inhibited MEK activity (IC50 = 10–100 nM) in a time- and concentration-dependent manner more effectively than PD098059 or U0126. PD184161 inhibited cell proliferation and induced apoptosis at concentrations of > 1.0 µM in a time- and concentration-dependent manner. in vivo, tumor xenograft P-ERK levels were significantly reduced 3 to 12 hours after an oral dose of PD184161 (P < .05). Contrarily, tumor xenograft P-ERK levels following long-term (24 days) daily dosing of PD184161 were refractory to this signaling effect. PD184161 significantly suppressed tumor engraftment and initial growth (P < .0001); however, established tumors were not significantly affected. In conclusion, PD184161 has antitumor effects in HCC in vitro and in vivo that appear to correlate with suppression of MEK activity. These studies demonstrate that PD184161 is unable to suppress MEK activity in HCC xenografts in the long term. Thus, we speculate that the degree of success of MEK-targeted treatment in HCC and other cancers may, in part, depend on the discovery of mechanisms governing MEK inhibitor signaling resistance.
Hepatocellular carcinoma; liver cancer; MEK; ERK; PD184161
Hepatitis B virus X (HBx) protein has been known to play an important role in development of hepatocellular carcinoma (HCC). The aim of this study is to find out whether HBx protein expression affects antiproliferative effect of an epidermal growth factor receptor-tyrosine kinase (EGFR-TK) inhibitor and a MEK inhibitor in HepG2 and Huh-7 cell lines. We established HepG2 and Huh-7 cells transfected stably with HBx gene. HBx protein expression increased pERK and pAkt expression as well as β-catenin activity in both cells. Gefitinib (EGFR-TK inhibitor) inhibited pERK and pAkt expression and β-catenin activity in both cells. Selumetinib (MEK inhibitor) reduced pERK level and β-catenin activity but pAkt expression was rather elevated by selumetinib in these cells. Reduction of pERK levels was much stronger with selumetinib than gefitinib in both cells. The antiproliferative efficacy of selumetinib was more potent than that of gefitinib. However, the antiproliferative effect of gefitinib, as well as selumetinib, was not different between cell lines with or without HBx expression. Signal pathway activation by HBx might not be strong enough to attenuate the antiproliferative effect of EGFR-TK inhibitor. Future experiments are needed to understand the role of HBx protein expression in HCC treatment using molecular targeting agent.
Hepatocellular Carcinoma; HBx protein; Gefitinib; Selumetinib; EGFR; MEK
Hepatocellular carcinoma (HCC) has a dismal 5-year-survival rate of 10%, so
novel strategies are warranted. IL-24 mediates anti-tumor activity reducing
STAT3 expression, which suggests that interferon (IFN) alpha may augment
tumor cell lysis and reduce angiogenesis. We investigated the antitumor
activity of treatment with IFN-α, with the oncolytic adenovirus
SG600-IL-24, or the combination of both in HCC in vitro and in
RT-PCR, ELISA assay and Western-blot confirmed that the exogenous IL-24 gene
was highly expressed in HCC cells infected with SG600-IL-24. Treatment with
combined IFN-α and SG600-IL-24 suppressed growth and promoted apoptosis
of the HepG2, MHCC97L, and HCCLM3 cell lines compared with the normal cell
line L02. The combined therapy increased STAT1 and SOCS1 and apoptosis, but
decreased the expression of the metastatic and angiogenic proteins MMP-2,
XIAP, OPN, and VEGF, which are regulated by STAT3 in HCC cells in
vitro. To assess the effects in vivo, the HCC cell line
HCCLM3 was transplanted subcutaneously into the right flanks of nude mice.
Mice in the IFN-α group, the SG600-IL-24 group, or the combined therapy
group had significantly suppressed growth of the HCC xenografted tumors
compared to the PBS control group of mice. Among the mice treated with the
combination of IFN-α and SG600-IL-24, three of those eight mice had
long-term survival and no evidence of a tumor. These mice also had decreased
expression of the metastatic and angiogenic proteins MMP-2, XIAP, OPN, and
The present study demonstrated for the first time the potential antitumor
activity of IFN-α combined with the oncolytic adenovirus SG600-IL-24 in
HCC both in vitro and in vivo, and suggests its further
development as a potential candidate for HCC cancer gene therapy.
Gene therapy; Hepatocellular carcinoma; Interferon (IFN); Mda-7/IL-24; Nude mice; Oncolytic adenovirus
Sirtuins and hypoxia-inducible transcription factors (HIF) have well-established roles in regulating cellular responses to metabolic and oxidative stress. Recent reports have linked these two protein families by demonstrating that sirtuins can regulate the activity of HIF-1 and HIF-2. Here we investigated the role of SIRT1, a NAD+-dependent deacetylase, in the regulation of HIF-1 activity in hypoxic conditions. Our results show that in hepatocellular carcinoma (HCC) cell lines, hypoxia did not alter SIRT1 mRNA or protein expression, whereas it predictably led to the accumulation of HIF-1α and the up-regulation of its target genes. In hypoxic models in vitro and in in vivo models of systemic hypoxia and xenograft tumor growth, knockdown of SIRT1 protein with shRNA or inhibition of its activity with small molecule inhibitors impaired the accumulation of HIF-1α protein and the transcriptional increase of its target genes. In addition, endogenous SIRT1 and HIF-1α proteins co-immunoprecipitated and loss of SIRT1 activity led to a hyperacetylation of HIF-1α. Taken together, our data suggest that HIF-1α and SIRT1 proteins interact in HCC cells and that HIF-1α is a target of SIRT1 deacetylase activity. Moreover, SIRT1 is necessary for HIF-1α protein accumulation and activation of HIF-1 target genes under hypoxic conditions.
Cannabinoids exert antiproliferative effects in a wide range of tumoral cells, including hepatocellular carcinoma (HCC) cells. In this study, we examined whether the PPARγ-activated pathway contributed to the antitumor effect of two cannabinoids, Δ9-tetrahydrocannabinol (THC) and JWH-015, against HepG2 and HUH-7 HCC cells. Both cannabinoids increased the activity and intracellular level of PPARγ mRNA and protein, which was abolished by the PPARγ inhibitor GW9662. Moreover, genetic ablation with small interfering RNA (siRNA), as well as pharmacological inhibition of PPARγ decreased the cannabinoid-induced cell death and apoptosis. Likewise, GW9662 totally blocked the antitumoral action of cannabinoids in xenograft-induced HCC tumors in mice. In addition, PPARγ knockdown with siRNA caused accumulation of the autophagy markers LC3-II and p62, suggesting that PPARγ is necessary for the autophagy flux promoted by cannabinoids. Interestingly, downregulation of the endoplasmic reticulum stress-related protein tribbles homolog 3 (TRIB3) markedly reduced PPARγ expression and induced p62 accumulation, which was counteracted by overexpression of PPARγ in TRIB3-knocked down cells. Taken together, we demonstrate for the first time that the antiproliferative action of the cannabinoids THC and JWH-015 on HCC, in vitro and in vivo, are modulated by upregulation of PPARγ-dependent pathways.
cannabinoids; THC; JWH-015; PPARγ; TRIB3; HepG2