Approach For Treatment Of Hepatocellular Carcinoma Research Articles

MiR-491 attenuates cancer stem cells-like properties of hepatocellular carcinoma by inhibition of GIT-1/NF-κB-mediated EMT.

Hepatocellular carcinoma (HCC) is the most common liver malignancy. Current standard practices for treatment of HCC are less than satisfactory because of CSCs-mediated recurrence. For this reason, targeting CSCs, or cancer cells with CSCs-like properties, is a new approach for HCC treatment. As we reported previously, microRNA-491 (miR-491) is lower expressed in poorly differentiated HCC tissues relative to well-differentiated HCC tissues. Here, we further evaluate the effects of miR-491 on the CSCs-like properties by using HCC cell lines and HCC tissue samples. Our data showed that miR-491 had a negative relationship with CSCs-like properties both in cell lines and tissue samples of HCC. Further, miR-491 levels of non-recurrence HCC tissues were higher than those of recurrence HCC tissues. In HCC cell lines, nuclear factor kappa B (NF-κB)/snail pathway was involved in the epithelial to mesenchymal transition and the maintenance of CSCs-like properties. Overexpression of miR-491 targeted G-protein-coupled receptor kinase-interacting protein 1 (GIT-1), which blocked the activation of NF-κB by the inhibition of extracellular signal-regulated kinases (ERKs). Such process attenuated the CSCs-like properties in HCC cells. Our results point to a previously undefined mechanism by which miR-491 decreases CSCs-like properties and help to identify potential targets for the therapy of HCCs.

Combined treatment with silibinin and either sorafenib or gefitinib enhances their growth-inhibiting effects in hepatocellular carcinoma cells.

Background/AimsSilibinin, the main component of silymarin, is used as a hepatoprotectant and exhibits anticancer effects against various cancer cells. This study evaluated the effects of a combination of silibinin with either gefitinib or sorafenib on hepatocellular carcinoma (HCC) cells.MethodsSeveral different human HCC cell lines were used to test the growth-inhibiting effects and cell toxicity of silibinin both alone and in combination with either gefitinib or sorafenib. The cell viability and growth inhibition were assessed using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay, trypan blue staining, and a colony-forming assay. Furthermore, changes in epidermal growth factor receptor (EGFR)-related signals were evaluated by Western blot analysis.ResultsGefitinib, sorafenib, and silibinin individually exhibited dose-dependent antiproliferative effects on HCC cells. Combined treatment with silibinin enhanced the gefitinib-induced growth-inhibiting effects in some HCC cell lines. The combination effect of gefitinib and silibinin was synergistic in the SNU761 cell line, but was only additive in the Huh-BAT cell line. The combination effect may be attributable to inhibition of EGFR-dependent Akt signaling. Enhanced growth-inhibiting effects were also observed in HCC cells treated with a combination of sorafenib and silibinin.ConclusionsCombined treatment with silibinin enhanced the growth-inhibiting effects of both gefitinib and sorafenib. Therefore, the combination of silibinin with either sorafenib or gefitinib could be a useful treatment approach for HCC in the future.

Targeting the microRNA-21/AP1 axis by 5-fluorouracil and pirarubicin in human hepatocellular carcinoma.

MicroRNAs function as oncomiRs and tumor suppressors in diverse cancers. However, the utility of specific microRNAs in predicting the clinical benefit of chemotherapy has not been well-established. Here, we investigated the correlation between microRNA-21 expression and hepatic arterial infusion chemotherapy with 5-fluorouracil and pirarubicin (HAIC) for hepatocellular carcinoma (HCC). We found that HCC patients with low microRNA-21 levels in tumors tended to have a longer time to recurrence and disease-free survival. We demonstrated that microRNA-21 suppression in combination with 5-fluorouracil and pirarubicin treatment inhibited tumor growth in subcutaneous xenograft mice models. Mechanistically, the AP-1 and microRNA-21-mediated axis was verified to be a therapeutic target of cytotoxic drugs and deregulation of this axis led to an enhanced cell growth in HCC. Taken together, our findings demonstrate that microRNA-21 is a chemotherapy responsive microRNA and can serve as a prognostic biomarker for HCC patients undergoing HAIC. Targeting microRNA-21 enhances the effect of chemotherapeutic drugs, thereby suggesting that microRNA-21 suppression in combination with HAIC may be a novel approach for HCC treatment.

Sodium Valproate Induces Cell Senescence in Human Hepatocarcinoma Cells

Hepatocarcinogenesis is associated with epigenetic changes, including histone deacetylases (HDACs). Epigenetic modulation by HDAC inhibition is a potentially valuable approach for hepatocellular carcinoma treatment. In present study, we evaluated the anticancer effects of sodium valproate (SVP), a known HDAC inhibitor, in human hepatocarcinoma cells. The results showed SVP inhibited the proliferation of Bel-7402 cells in a dose-dependent manner. Low dose SVP treatment caused a large and flat morphology change, positive SA-β-gal staining, and G0/G1 phase cell cycle arrest in human hepatocarcinoma cells. Low dose SVP treatment also increased acetylation of histone H3 and H4 on p21 promoter, accompanied by up-regulation of p21 and down-regulation of RB phosphorylation. These observations suggested that a low dose of SVP could induce cell senescence in hepatocarcinoma cells, which might correlate with hyperacetylation of histone H3 and H4, up-regulation of p21, and inhibition of RB phosphorylation. Since the effective concentration inducing cell senescence in hepatocarcinoma cells is clinically available, whether a clinical dose of SVP could induce cell senescence in clinical hepatocarcinoma is worthy of further study.

Clinical significance of therapy using branched‐chain amino acid granules in patients with liver cirrhosis and hepatocellular carcinoma

The liver is the major organ for the metabolism of protein, fat and carbohydrate. A nutritional approach is required in the treatment of cirrhosis, which is frequently complicated with protein-energy malnutrition. Several advanced treatment approaches for hepatocellular carcinoma (HCC) have been established in the past decade. HCC is often complicated by cirrhosis, so treatment of the underlying liver diseases is also necessary to improve the prognosis. Branched-chain amino acid (BCAA) granules were developed originally for the treatment of hypoalbuminemia associated with decompensated cirrhosis. However, subsequent studies found various other pharmacological actions of this agent. We review the clinical significance of therapy using BCAA granules in patients receiving different treatment approaches for cirrhosis and HCC based on the published work as well as our own data.

Liver transplantation and expanded Milan criteria: does it really work?

Orthotopic liver transplantation is an excellent treatment approach for hepatocellular carcinoma in well-selected candidates. Nowadays some institutions tend to Expand the Milan Criteria including tumor with more than 5 cm and also associate with multiple tumors none larger than 3 cm in order to benefit more patients with the orthotopic liver transplantation. The data collected were based on the online database PubMED. The key words applied on the search were "expanded Milan criteria" limited to the period from 2000 to 2009. We excluded 19 papers due to: irrelevance of the subject, lack of information and incompatibility of the language (English only). We compiled patient survival and tumor recurrence free rate from 1 to 5-years in patients with hepatocellular carcinoma submitted to orthotopic liver transplantation according to expanded the Milan criteria from different centers. Review compiled data from 23 articles. Fourteen different criteria were found and they are also described in detail, however the University of California - San Francisco was the most studied one among them. Expanded the Milan criteria is a useful attempt for widening the preexistent protocol for patients with hepatocellular carcinoma in waiting-list for orthotopic liver transplantation. However there is no significant difference in patient survival rate and tumor recurrence free rate from those patients that followed the Milan criteria.

Abstract 5620: Therapeutic targeting of glypican-3 using TGF-β2 as a novel treatment approach for HCC

Abstract Objective Glypican-3 (GPC3) is a valuable diagnostic marker and a potential therapeutic target in hepatocellular carcinoma (HCC). It is over-expressed in more than 60% of the HCC tumors. As a histological marker, GPC3 can be used to distinguish HCC cells from cirrhotic, non-tumorous liver. As a diagnostic marker, GPC3 be used to complement the diagnostic potential of AFP for screening of HCC patients. Previously we found that GPC3 knockdown can suppress HCC tumor growth both in vitro and in vivo. Gene expression analysis revealed that GPC3 suppression significantly up-regulated the expression of TGF-β2 at both RNA and protein levels. TGF-β2 itself possesses anti-proliferative effect in GPC3 positive HCC cell lines (HepG2 and Huh7). This study aims at evaluating the use of TGF-β2 as a novel treatment approach for HCC by targeting the GPC3/TGFβ signaling axis. Materials and methods To study the effects of TGF-β2 on HCC cells, cell lines with different level of GPC3 expression (HepG2, Huh7, Hep3B, Hep40, PLC/PRF/5, SNU-398, SNU-182, SNU-449, SNU-475) were treated with 1 or 5 ng/ml of rhTGF-β2 for 48 hours in their normal culture media supplemented with 10% FBS. Cell proliferation was determined by using MTT assay and colony formation assay. Cell cycle progression was measured by using flow cytometry. The presence of self-renewing cancer cells was determined by spheroid formation assay. Gene expression analysis was performed to identify the genes associated with TGF-β2 treatment in HCC cells. For in vivo study, orthotopic xenografts of HCC patient tumors in mice were established, and mice were injected with 100 ng of rhTGF-β2 every week via i.p. injection. Tumor growth was monitored by using non-invasive bioluminescence imaging. Results TGF-β2 effectively inhibited the growth of GPC3 positive cells as shown by the decreased cell proliferation and colony formation (HepG2, Huh7, Hep40, PLC.PRF/5, Hep3B and SNU-398). Additionally, GPC3 positive cells responded to TGF-β2 treatment with down-regulation of anti-apoptotic genes (Bcl-2, Bcl-xL and Mcl-1) and cell cycle arrest at G0/G1 phase. Cells with no endogenous GPC3 expression did not respond to TGF-β2 treatment. Moreover, TGF-β2 treatment inhibited self-renewal of GPC3 positive HCC cell lines as shown by inhibition of spheroid formation (P<0.01 compared to untreated cells). Gene expression analysis revealed that TGF-β2 treatment down-regulated the expression of GPC3 (72%, P<0.05), NDRG1 (56%, P<0.05) and CDC37 (56%, P<0.05), three genes that were reported to promote HCC tumor progression. In vivo, TGF-β2 inhibited HCC xenograft growth, after 3 weeks of treatment (P<0.05 compared to PBS control), as shown by the bioluminescence imaging. Conclusions In conclusion, molecular targeting of GPC3 by using rhTGF-β2 offers a novel treatment option for the clinical management of GPC3-positive HCC patients by targeting the GPC3/TGFβ signaling axis. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 5620. doi:1538-7445.AM2012-5620

Growth inhibition of human hepatocellular carcinoma cells by overexpression of G‐protein‐coupled receptor kinase 2

Hepatocellular carcinoma (HCC) is one of the deadliest forms of human liver cancer and does not respond well to conventional therapies. Novel effective treatments are urgently in need. G-protein-coupled kinase 2 (GRK2) is unique serine/threonine kinase that involves in many signaling pathways and regulates various essential cellular processes. Altered levels of GRK2 have been linked with several human diseases including cancer. In this study, we investigated a novel approach for HCC treatment by inducing overexpression of GRK2 in human HCC cells. We found that overexpression of GRK2 through recombinant adenovirus transduction inhibits the growth of human HCC cells. BrdU incorporation assay showed that the growth inhibition caused by elevated GRK2 level was due to reduced cell proliferation but not apoptosis. To examine the anti-proliferative function of increased GRK2 level, we performed cell cycle analysis using propidium iodide staining. We found that the proliferation suppression was associated with G2/M phase cell cycle arrest by the wild-type GRK2 but not its kinase-dead K220R mutant. Furthermore, increased levels of wild-type GRK2 induced upregulation of phosphor-Ser(15) p53 and cyclin B1 in a dose-dependent manner. Our data indicate that the anti-proliferative function of elevated GRK2 is associated with delayed cell cycle progression and is GRK2 kinase activity-dependent. Enforced expression of GRK2 in human HCC by molecular delivery may offer a potential therapeutic approach for the treatment of human liver cancer.

This Month in Gastroenterology

This Month in Gastroenterology

Serum high mobility group box chromosomal protein 1 is associated with clinicopathologic features in patients with hepatocellular carcinoma

Background/aims The role of high mobility group box chromosomal protein 1 in hepatocellular carcinoma is unknown. The aim of study was to evaluate contributions of high mobility group box chromosomal protein 1 in hepatocellular carcinoma, and analyse the correlation between high mobility group box chromosomal protein 1 and clinicopathologic outcomes. Patients/methods High mobility group box chromosomal protein 1 levels were analysed by Western blot analysis. Edmondson grade, TNM stage and the Cancer of the Liver Italian Program score were used as analysis variables. Results The serum high mobility group box chromosomal protein 1 levels in hepatocellular carcinoma (84.2 ± 50.4 ng/ml) was significantly higher than those in chronic hepatitis (39.8 ± 10.5 ng/ml), liver cirrhosis (40.2 ± 11.6 ng/ml) and healthy control (7.0 ± 5.9 ng/ml, p < 0.0001, respectively), and positive correlation were found between high mobility group box chromosomal protein 1 and α-fetoprotein ( r = 0.952, p < 0.0001), and between high mobility group box chromosomal protein 1 and the size of tumour ( r = 0.904, p < 0.0001). High mobility group box chromosomal protein 1 were significant differences among Edmondson grade I, II, III, IV; TNM stage I, II, III, IV and Cancer of the Liver Italian Program score 0–1 points, 2–4 points, >4 points ( p < 0.0001, respectively). Conclusions These results suggest that high mobility group box chromosomal protein 1 may be a useful marker for evaluating the tumour stage and predicting prognosis in hepatocellular carcinoma. Targeting high mobility group box chromosomal protein 1 production or release might have potential approaches for hepatocellular carcinoma treatment.

Epigenetic combination therapy as a tumor‐selective treatment approach for hepatocellular carcinoma

Innovative epigenetic therapeutics comprise histone deacetylase inhibitors (HDAC-I) and demethylating agents (DA). It was recently found that HDAC-I compounds exhibit profound therapeutic activities against hepatocellular carcinoma (HCC). A comprehensive preclinical investigation was performed on the potential of a combined HDAC-I/DA epigenetic regimen for the highly chemotherapy-resistant HCC entity. Human HCC-derived cell lines or primary human hepatocytes (PHH) were treated with HDAC-I compound suberoylanilide hydroxamic acid (SAHA) or DA compound 5-aza-2'-deoxycytidine (5-aza-dC) or both and examined for cellular damage, proliferation, histone acetylation pattern, and DNA methylation. In vivo activities were investigated in a xenograft hepatoma model. Monotherapeutic application of SAHA or 5-aza-dC was found to induce substantial antiproliferative effects in HCC-derived cells, strongly enhanced by combined SAHA and 5-aza-dC treatment. PHH from different human donors did not exhibit any relevant cellular damage even when applying high doses of the combination regimen, whereas HCC-derived cell lines showed a dose-dependent damage. In vivo testing demonstrated a statistical significant inhibition of hepatoma cell growth for the combined treatment regime. Because the combined HDAC-I/DA epigenetic approach was found to produce significant antitumor effects in HCC model systems and did not impair cellular integrity of untransformed hepatocytes, this combination therapy is now considered for further investigation in clinical trials.

Combinatorial epigenetic treatment approach for hepatocellular carcinoma

10066 Hepatocellular carcinoma (HCC) displays a striking resistance to chemotherapeutic drugs. Alternative approaches comprise employment of epigenetic compounds such as histone deacetylase inhibitors (HDAC-I) or demethylating agents (DA). Since we could recently demonstrate that HDAC-I exhibit inherent in vitro therapeutic activities against HCC cells, we now explored the potential of a HDAC-I / DA combined epigenetic treatment approach for the highly chemotherapy resistant tumor entity HCC. Methods: HCC cell lines (HepG2, Hep3B, HuH7) and primary human hepatocytes (PHH) were treated with a HDAC-I compound (Suberoylanilide hydroxamic acid - SAHA) together with or without a DA (5-aza-2dC) and examined for cellular damage (enzyme release), proliferation inhibition (SRB), apoptotic cells (FACS), histone acetylation pattern (Western blot) and methylation status (methylation specific PCR). The in vivo activity of our approach was investigated in a HuH7 xenograft mouse hepatoma model. Results: Both SAHA and 5-aza-2dC were found to induce substantial antiproliferative effects and apoptotic cell death in HCC cells; interestingly, combinatorial treatment (SAHA plus 5-aza-2dC) resulted in strongly enhanced anti-HCC effects. Interferences between DA and HDAC-I activities could be excluded by determining both (i) the methylation status of two model genes (p16, SOCS-1; known to be abberantly methylated in HCC) as well as (ii) patterns of histone acetylation. Most importantly, non-malignant primary human hepatocytes (5 different donors) did not exhibit any relevant cellular damage even when high doses of SAHA plus 5-aza-2dC were applied. Finally, in vivo testing of our combinatorial regimen (SAHA plus 5-aza-2dC) was found to be superior in suppression of tumor cell growth when compared to the application of single substances (SAHA or 5-aza-2dC) or placebo. Conclusion: Our combined epigenetic approach in chemotherapy resistant HCC not only has been found to be a highly active treatment option with profound anti-tumor effects both in vitro and in a small animal model in vivo, but did not impair primary human hepatocytes. Thus, this combination therapy now is considered for further investigation in clinical trials. No significant financial relationships to disclose.

Histone deacetylase inhibitors for treatment of hepatocellular carcinoma

Hepatocellular carcinoma (HCC) is one of the most common cancers in the world. Surgical resection has been considered the optimal treatment approach, but only a small proportion of patients are suitable candidates for surgery, and the relapse rate is high. Approaches to prevent recurrence, including chemoembolization before and adjuvant therapy after surgery, have proven to have a limited benefit; liver transplantation is successful in treating limited-stage HCC because only a minority of patients qualify for transplantation. Therefore, new therapeutic strategies are urgently needed. Because in addition to the classical genetic mechanisms of deletion or inactivating point mutations, epigenetic alterations, such as hyperacetylation of the chromatin-associated histones (responsible for gene silencing), are believed to be involved in the development and progression of HCC, novel compounds endowed with a histone deacetylase (HDAC) inhibitory activity are an attractive therapeutic approach. In particular, pre-clinical results obtained using HA-But, an HDAC inhibitor in which butyric acid residues are esterified to a hyaluronic acid backbone and characterized by a high affinity for the membrane receptor CD44, indicated that this class of compounds may represent a promising approach for hepatocellular carcinoma treatment.

Spontaneous tumor-specific humoral and cellular immune responses to NY-ESO-1 in hepatocellular carcinoma.

Hepatocellular carcinoma (HCC) is the fifth most common cancer around the world. Although several therapeutic approaches for treatment of HCC are available, survival rates for HCC patients are still very poor because of inefficient treatment options. For HCC, as well as other tumors, antigen-specific immunotherapy remains a viable approach that is dependent on the definition of tumor-associated antigens. NY-ESO-1, a member of the cancer testis antigen family, is one possible candidate for a tumor-specific antigen in HCC. The aim of this study was to show the relevance of NY-ESO-1 in hepatocellular carcinoma. Sera samples from 189 HCC patients were analyzed for NY-ESO-1-specific antibodies. Forty-nine HCC patients were screened for NY-ESO-1 mRNA expression in HCC tissue. Selected patients were followed for up to 3 years to correlate their immune response with their clinical course of events. NY-ESO-1-specific CD4+ and CD8+ T-cell responses from NY-ESO-1 seropositive patients were analyzed and a NY-ESO-1+ specific cytotoxic T-cell line was generated. Twelve of 49 analyzed tumor samples expressed NY-ESO-1 mRNA and 23 of 189 patients showed NY-ESO-1-specific antibody responses. These humoral immune responses were accompanied by NY-ESO-1-specific functional CD4+ and CD8+ T-cell responses. Finally, NY-ESO-1 humoral responses were dependent on the presence of NY-ESO-1-expressing tumors. This is the first report of a spontaneous immune response in HCC patients to a known tumor-specific antigen, NY-ESO-1 protein. Our data favor the possibility of immunotherapeutic strategies for the treatment of HCC.

Therapeutic strategies for hepatitis B virus-associated hepatocellular carcinoma.

Hepatocellular carcinoma (HCC) is a one of the most prevalent cancers worldwide, especially in the Asia Pacific region. At present, the five-year survival of individuals with HCC is low, mainly due to the late presentation of the disease, and limited therapeutic options. The major risk factors for HCC in the Asia Pacific region include hepatitis B and C viral infection. Removing or reducing these risk factors, such as by immunizing against the hepatitis B virus, may reduce the incidence of hepatitis B-associated HCC in the distant future. However, immunization does not decrease the risk of HCC in individuals who are currently infected with HBV worldwide highlighting the continued importance of examining strategies for the treatment of HCC. Current treatment strategies include surgical resection, liver transplantation, chemotherapy, transcatheter arterial chemoembolism and percutaneous injection. Except for surgical resection and liver transplantation, which represent the most viable treatment options, most of the other present treatments are mainly for palliation. Hence novel treatment modalities continue to be investigated and several of these are currently in clinical trials. One of the more promising novel approaches for HCC treatment is gene therapy. Potential promising gene therapeutic approaches for the treatment of HCC include augmentation of tumor suppressor genes, inhibition of abnormally over-expressed oncogenes as well as specifically inducing death of cancer cells either via "suicide" gene therapy, conditional replicative adenovirus strategy, immunomodulation or inhibiting tumor angiogenesis. Nonetheless, successful implementation of these gene therapeutic approaches is dependent on overcoming current practical and technical hurdles underscoring the need for a better understanding of the basic aspects of gene therapy.