Breast Cancer Metastasis Suppressor 1 Regulates Hepatocellular Carcinoma Cell Apoptosis via Suppressing Osteopontin Expression

Breast cancer metastasis suppressor 1 (BRMS1) was originally identified as an active metastasis suppressor in human breast cancer. Loss of BRMS1 expression correlates with tumor progression, and BRMS1 suppresses several steps required for tumor metastasis. However, the role of BRMS1 in hepatocellular carcinoma (HCC) remains elusive. In this study, we found that the expression level of BRMS1 was significantly down-regulated in HCC tissues. Expression of BRMS1 in SK-Hep1 cells did not affect cell growth under normal culture conditions, but sensitized cells to apoptosis induced by serum deprivation or anoikis. Consistently, knockdown of endogenous BRMS1 expression in Hep3B cells suppressed cell apoptosis. We identified that BRMS1 suppresses osteopontin (OPN) expression in HCC cells and that there is a negative correlation between BRMS1 and OPN mRNA expression in HCC tissues. Moreover, knockdown of endogenous OPN expression reversed the anti-apoptosis effect achieved by knockdown of BRMS1. Taken together, our results show that BRMS1 sensitizes HCC cells to apoptosis through suppressing OPN expression, suggesting a potential role of BRMS1 in regulating HCC apoptosis and metastasis.     

Novel cationic antimicrobial peptide GW-H1 induced caspase-dependent apoptosis of hepatocellular carcinoma cell lines

Due to its malignancy, the development of effective therapeutic strategies for hepatocellular carcinoma (HCC) is of urgent needs. Natural antimicrobial peptides (AMPs), also known as host defense peptides (HDPs), not only act as direct antimicrobial agents, but also represent important regulators of the innate immune system. It has been reported that cationic AMPs may exhibit cancer-selective toxicity. We have designed a series of novel AMPs with potent antimicrobial activity against a broad spectrum of bacterial pathogens. In the current study, we evaluate the antitumor potency of these AMPs toward HCC cell lines J5, Huh7, and Hep3B. Selected AMPs inhibit the viability of HCC cells in a dose-dependent fashion, while the normal 3T3 cells were significantly less susceptible to these AMPs. GW-H1 treatment (20μM) of J5 cells for 24-72h resulted in the induction of apoptosis, as revealed by flow cytometry (increased sub-G1 populations), and western blot analysis for the appearance of activated caspase-3, -7 and -9 cleavages. Two-dimensional gel electrophoresis was applied to further analyze the AMP-responsive protein profiles of HCC, down-regulation of Hsp27, phophoglycerate kinase 1 and triosephosphate isomerase indicated that GW-H1 may induce apoptosis, and further inhibit progression and metastasis of J5 HCC cells. FITC-labeled GW-H1 was found to attach to cell membrane initially, then translocated into the cytoplasm, and eventually membranous organelles or nucleus. GW-H1 induced a marked growth suppression of J5 xenografts in nude mice in a dose dependent manner. These findings provided support for future application of GW-H1 as potential therapeutic agent for HCC.     

Enhanced expression of matrix metalloproteinase-9 by hepatitis B virus infection in liver cells

The hepatitis B virus (HBV) is a major cause of human liver disease, including hepatocellular carcinoma (HCC). The prognosis for HCC is largely dependent on the clinicopathological characteristics regarding invasion and metastasis. Enhanced matrix metalloproteinase-9 (MMP-9) expression has been implicated as playing an important role in metastasis and invasion of HCC. However, the relationship between HBV infection and MMP-9 expression in HCC is currently poorly understood. We report here on a study of the levels of MMP-9 and MMP-2 expression in human fetal liver tissue, rat liver tissue, and Chang, HepG2, and Hep3B cells by gelatin zymography. Among these sources, Hep3B cells, which contain the integrated hepatitis B viral genome, continuously secrete the hepatitis B viral surface antigen, and express HBV genomic RNA, expressed high levels of proMMP-9, and a small amount of active MMP-9 was detected in Hep3B cells as assayed by zymography. We investigated the issue of whether HBV infection affects MMP-9 expression, which is known to play an important role in HCC invasion and metastasis. As a first step, human fetal hepatocyte (HFH) and HepG2 (HCC origin, HBV not detected) cells were subjected to infection with HBV, and the resulting infected cells successfully established are hereafter referred to as HFH-T2 and HepG2-HBV. The expression of MMP-9 was upregulated by the infected HBV in HFH-T2 and HepG2-HBV cells, as assayed by zymography, Northern blot, and Western blot analysis, and small amounts of active MMP-9 were detected in HFH-T2 and HepG2-HBV cells as assayed by zymography. The activation of the immature proMMP-9 to the mature MMP-9 could be induced by plasmin treatment. The activation of proMMP-9 was increased to a greater extent with plasmin treatment than without plasmin in HFH-T2 and HepG2-HBV cells but the addition of recombinant TIMP-1 inhibited the activation of proMMP-9. Finally, the addition of plasmin to the invasion assay using Matrigel resulted in an increase in invasiveness of HFH-T2 and HepG2-HBV cells, as well as MMP-9 activation, but the treatment with TIMP-1 inhibited the invasiveness of HFH-T2 and HepG2-HBV cells as well as MMP-9 activation. We conclude from these findings that HBV infection of hepatocytes and HepG2 cells affected the upregulation of MMP-9 expression and MMP-9 activation and, thus, increased the invasion potential by plasmin. To our knowledge, this is a first report showing that an HBV infection is linked to the upregulation of MMP-9 in HCC.     

Disulfiram combined with copper inhibits metastasis and epithelial–mesenchymal transition in hepatocellular carcinoma through the NF‐κB and TGF‐β pathways

Late‐stage hepatocellular carcinoma (HCC) usually has a low survival rate because of the high risk of metastases and the lack of an effective cure. Disulfiram (DSF) has copper (Cu)‐dependent anticancer properties in vitro and in vivo. The present work aims to explore the anti‐metastasis effects and molecular mechanisms of DSF/Cu on HCC cells both in vitro and in vivo. The results showed that DSF inhibited the proliferation, migration and invasion of HCC cells. Cu improved the anti‐metastatic activity of DSF, while Cu alone had no effect. Furthermore, DSF/Cu inhibited both NF‐κB and TGF‐β signalling, including the nuclear translocation of NF‐κB subunits and the expression of Smad4, leading to down‐regulation of Snail and Slug, which contributed to phenotype epithelial–mesenchymal transition (EMT). Finally, DSF/Cu inhibited the lung metastasis of Hep3B cells not only in a subcutaneous tumour model but also in an orthotopic liver metastasis assay. These results indicated that DSF/Cu suppressed the metastasis and EMT of hepatic carcinoma through NF‐κB and TGF‐β signalling. Our study indicates the potential of DSF/Cu for therapeutic use.     

LncRNA SNHG3 induces EMT and sorafenib resistance by modulating the miR-128/CD151 pathway in hepatocellular carcinoma

Dysregulation of long noncoding RNAs (lncRNAs) plays important roles in carcinogenesis and tumor progression, including hepatocellular carcinoma (HCC). Small nucleolar RNA host gene 3 (SNHG3) has been considered as an lncRNA to be associated with a poor prognosis in patients with HCC. Here, we reported that SNHG3 expression was significantly higher in the highly metastatic HCC (HCCLM3) cells compared with the lowly metastatic HCC cells (Hep3B and PLC/PRF/5). Furthermore, forced expression of SNHG3 promoted cell invasion, epithelial-mesenchymal transition (EMT), and sorafenib resistance in HCC. Moreover, SNHG3 overexpression induced HCC cells EMT via miR-128/CD151 cascade activation. Clinically, our data revealed that increased SNHG3 expression is correlated with poor HCC survival outcomes and sorafenib response. These data suggest that SNHG3 may be a novel therapeutic target and a biomarker for predicting response to sorafenib treatment of HCC.     

A PTEN inhibitor displays preclinical activity against hepatocarcinoma cells

Phosphatase and tensin homolog (PTEN) gene is considered a tumor suppressor gene. However, PTEN mutations rarely occur in hepatocellular carcinoma (HCC), whereas heterozygosity of PTEN, resulting in reduced PTEN expression, has been observed in 32–44% of HCC patients. In the present study, we investigated the effects of the small molecule PTEN inhibitor VO-OHpic in HCC cells. VO-OHpic inhibited cell viability, cell proliferation and colony formation, and induced senescence-associated β-galactosidase activity in Hep3B (low PTEN expression) and to a lesser extent in PLC/PRF/5 (high PTEN expression) cells, but not in PTEN-negative SNU475 cells. VO-OHpic synergistically inhibited cell viability when combined with PI3K/mTOR and RAF/MEK/ERK pathway inhibitors, but only in Hep3B cells, and significantly inhibited tumor growth in nude mice bearing xenografts of Hep3B cells. Therefore, we demonstrated for the first time that VO-OHpic inhibited cell growth and induced senescence in HCC cells with low PTEN expression, and that the combination of VO-OHpic with PI3K/mTOR and RAF/MEK/ERK inhibitors resulted in a more effective tumor cell kill. Our findings, hence, provide proof-of-principle evidence that pharmacological inhibition of PTEN may represent a promising approach for HCC therapy in a subclass of patients with a low PTEN expression.     

Hypoxia‐induced miR‐3677‐3p promotes the proliferation,migration and invasion of hepatocellular carcinoma cells by suppressing SIRT5

Hepatocellular carcinoma (HCC), with life‐threatening malignant behaviours, often develops distant metastases and is the fourth most common primary cancer in the world, having taken millions of lives in Asian countries such as China. The novel miR‐3677‐3p is involved in a high‐expression‐related poor prognosis in HCC tissues and cell lines, indicating oncogenesis functions in vitro and in vivo. Initially, we confirmed the inhibition of proliferation, migration and invasion in miR‐3677‐3p knock‐down MHCC‐97H and SMMC‐7721 cell lines, which are well known for their high degree of invasiveness. Then, we reversed the functional experiments in the low‐miR‐3677‐3p‐expression Hep3B cell line via overexpressing miR‐3677‐3p. In nude mice xenograft and lung metastasis assays, we found suppressor behaviours, smaller nodules and low density of organ spread, after injection of cells transfected with shRNA‐miR‐3677‐3p. A combination of databases (Starbase, TargetScan and MiRgator) illustrated miR‐3677‐3p targets, and it was shown to suppress the expression of SIRT5 in a dual‐luciferase reporter system. To clarify the conclusions of previous ambiguous research, we up‐regulated SIRT5 in Hep3B cells, and rescue tests were established for confirmation that miR‐3677‐3p suppresses SIRT5 to enhance the migration and invasion of HCC. Interestingly, we discovered hypoxia‐induced miR‐3677‐3p up‐regulation benefited HCC malignancy and invasiveness. In conclusion, the overexpression of miR‐3677‐3p mediated SIRT5 inhibition, which could increase proliferation, migration and invasion of HCC in hypoxic microenvironments.     

Hepatic stellate cell promoted hepatoma cell invasion via the HGF/c-Met signaling pathway regulated by p53

The biological behaviors of hepatocellular carcinoma (HCC) are complex mainly due to heterogeneity of progressive genetic and epigenetic mutations as well as tumor environment. Hepatocyte growth factor (HGF)/c-Met signaling pathway is regarded to be a prototypical example for stromal-epithelial interactions during developmental morphogenesis, wound healing, organ regeneration and cancer progression. And p53 plays as an important regulator of Met-dependent cell motility and invasion. Present study showed that 2 HCC cell lines, Hep3B and HepG2, displayed different invasive capacity when treated with HGF which was secreted by hepatic stellate cells (HSCs). We found that HGF promoted Hep3B cells invasion and migration as well as epithelial-mesenchymal transition (EMT) occurrence because Hep3B was p53 deficient, which leaded to the c-Met over-expression. Then we found that HGF/c-Met promoted Hep3B cells invasion and migration by upregulating Snail expression. In conclusion, HGF/c-Met signaling is enhanced by loss of p53 expression, resulting in increased ability of invasion and migration by upregulating the expression of Snail.     

MicroRNA‐301b‐3p contributes to tumour growth of human hepatocellular carcinoma by repressing vestigial like family member 4

MicroRNAs (miRNAs) are key regulators in the tumour growth and metastasis of human hepatocellular carcinoma (HCC). Increasing evidence suggests that miR‐301b‐3p functions as a driver in various types of human cancer. However, the expression pattern of miR‐301b‐3p and its functional role as well as underlying molecular mechanism in HCC remain poorly known. Our study found that miR‐301b‐3p expression was significantly up‐regulated in HCC tissues compared to adjacent non‐tumour tissues. Clinical association analysis revealed that the high level of miR‐301b‐3p closely correlated with large tumour size and advanced tumour‐node‐metastasis stages. Importantly, the high miR‐301b‐3p level predicted a prominent poorer overall survival of HCC patients. Knockdown of miR‐301b‐3p suppressed cell proliferation, led to cell cycle arrest at G2/M phase and induced apoptosis of Huh7 and Hep3B cells. Furthermore, miR‐301b‐3p knockdown suppressed tumour growth of HCC in mice. Mechanistically, miR‐301b‐3p directly bond to 3′UTR of vestigial like family member 4 (VGLL4) and negatively regulated its expression. The expression of VGLL4 mRNA was down‐regulated and inversely correlated with miR‐301b‐3p level in HCC tissues. Notably, VGLL4 knockdown markedly repressed cell proliferation, resulted in G2/M phase arrest and promoted apoptosis of HCC cells. Accordingly, VGLL4 silencing rescued miR‐301b‐3p knockdown attenuated HCC cell proliferation, cell cycle progression and apoptosis resistance. Collectively, our results suggest that miR‐301b‐3p is highly expressed in HCC. miR‐301b‐3p facilitates cell proliferation, promotes cell cycle progression and inhibits apoptosis of HCC cells by repressing VGLL4.     

Relationship between TRAF6 and deterioration of HCC: an immunohistochemical and in vitro study

Objective

To explore the relationship between tumor necrosis factor receptor-associated factor 6 (TRAF6) and the clinicopathological features in HCC as well as its biological function.

Methods

Totally, 412 liver tissues were collected, including 171 hepatocellular carcinoma (HCC) and their corresponding non-tumor tissues, 37 cirrhosis and 33 normal liver tissues. The expression of TRAF6 was assessed by immunohistochemistry. Then, analysis of the correlations between TRAF6 expression and clinicopathological parameters in HCC was conducted. Furtherer, in vitro experiments on HepG2 and Hep3B cells were performed to validate the biological function of TRAF6 on HCC cells. TRAF6 siRNA was transfected into HepG2 and Hep3B cell lines and TRAF6 expression was evaluated with RT-qPCR and western blot. The assays of cell viability, proliferation, apoptosis and caspase-3/7 activity were carried out to investigate the effects of TRAF6 on HCC cells with RNA interference. Cell viability was assessed with Cell Titer-Blue kit. Cell proliferation was tested with MTS kit. Cell apoptosis was checked through morphologic detection with fluorescence microscope, as well as caspase-3/7 activity was measured with fluorogenic substrate detection.

Results

The positive expression rate of TRAF6 protein was 49.7 % in HCC, significantly higher than that of normal liver (12.1 %), cirrhosis (21.6 %) and adjacent non-cancerous tissues (36.3 %, all P < 0.05). Upregulated TRAF6 was detected in groups with metastasis (Z = ?2.058, P = 0.04) and with low micro-vessel density (MVD) expression (Z = ?2.813, P = 0.005). Spearman correlation analysis further showed that the expression of TRAF6 was positively correlated with distant metastasis (r = 0.158, P = 0.039) and negatively associated with MVD (r = ?0.249, P = 0.004). Besides, knock-down of TRAF6 mRNA in HCC cell lines HepG2 and Hep3B both resulted in cell viability and proliferation inhibition, also cell apoptosis induction and caspase-3/7 activity activation.

Conclusions

TRAF6 may contribute to metastasis and deterioration of the HCC via influencing cell growth and apoptosis. Thus, TRAF6 might become a predictive and therapeutic biomarker for HCC.

     

Potentiating the Efficacy of Molecular Targeted Therapy for Hepatocellular Carcinoma by Inhibiting the Insulin-Like Growth Factor Pathway

Insulin-like growth factor (IGF) signaling pathway is an important regulatory mechanism of tumorigenesis and drug resistance in many cancers. The present study explored the potential synergistic effects between IGF receptor (IGFR) inhibition and other molecular targeted agents (MTA) in HCC cells. HCC cell lines (Hep3B, PLC5, and SK-Hep1) and HUVECs were tested. The MTA tested included sorafenib, sunitinib, and the IGFR kinase inhibitor NVP-AEW541. The potential synergistic antitumor effects were tested by median dose effect analysis and apoptosis assay in vitro and by xenograft models in vivo. The activity and functional significance of pertinent signaling pathways and expression of apoptosis-related proteins were measured by RNA interference and Western blotting. We found that IGF can activate IGFR and downstream AKT signaling activities in all the HCC cells tested, but the growth-stimulating effect of IGF was most prominent in Hep3B cells. NVP-AEW541 can abrogate IGF-induced activation of IGFR and AKT signaling in HCC cells. IGF can increase the resistance of HCC cells to sunitinib. The apoptosis-inducing effects of sunitinib, but not sorafenib, were enhanced when IGFR signaling activity was inhibited by NVP-AEW541 or IGFR knockdown. Chk2 kinase activation was found contributory to the synergistic anti-tumor effects between sunitinib and IGFR inhibition. Our data indicate that the apoptosis-potentiating effects of IGFR inhibition for HCC may be drug-specific. Combination therapy of IGFR inhibitors with other MTA may improve the therapeutic efficacy in HCC.     

Blockade of ARHGAP11A reverses malignant progress via inactivating Rac1B in hepatocellular carcinoma

Background

The molecular signaling events involving in high malignancy and poor prognosis of hepatocellular carcinoma (HCC) are extremely complicated. Blockade of currently known targets has not yet led to successful clinical outcome. More understanding about the regulatory mechanisms in HCC is necessary for developing new effective therapeutic strategies for HCC patients.

Methods

The expression of Rho GTPase-activating protein 11A (ARHGAP11A) was examined in human normal liver and HCC tissues. The correlations between ARHGAP11A expression and clinicopathological stage or prognosis in HCC patients were analyzed. ARHGAP11A was downregulated to determine its role in the proliferation, invasion, migration, epithelial-to-mesenchymal transition (EMT) development, and regulatory signaling of HCC cells in vitro and in vivo.

Results

ARHGAP11A exhibited high expression in HCC, and was significantly correlated with clinicopathological stage and prognosis in HCC patients. Moreover, ARHGAP11A facilitated Hep3B and MHCC97-H cell proliferation, invasion, migration and EMT development in vitro. ARHGAP11A knockdown significantly inhibited the in vivo growth and metastasis of HCC cells. Furthermore, ARHGAP11A directly interacted with Rac1B independent of Rho GTPase- activating activity. Rac1B blockade effectively interrupted ARHGAP11A-elicited HCC malignant phenotype. Meanwhile, upregulation of Rac1B reversed ARHGAP11A knockdown mediated mesenchymal-to-epithelial transition (MET) development in HCC cells.

Conclusion

ARHGAP11A facilitates malignant progression in HCC patients via ARHGAP11A-Rac1B interaction. The ARHGAP11A/Rac1B signaling could be a potential therapeutic target in the clinical treatment of HCC.

     

Motile hepatocellular carcinoma cells preferentially secret sugar metabolism regulatory proteins via exosomes

Exosomes are deliverers of critically functional proteins, capable of transforming target cells in numerous cancers, including hepatocellular carcinoma (HCC). We hypothesize that the motility of HCC cells can be featured by comparative proteome of exosomes. Hence, we performed the super‐SILAC‐based MS analysis on the exosomes secreted by three human HCC cell lines, including the non‐motile Hep3B cell, and the motile 97H and LM3 cells. More than 1400 exosomal proteins were confidently quantified in each MS analysis with highly biological reproducibility. We justified that 469 and 443 exosomal proteins represented differentially expressed proteins (DEPs) in the 97H/Hep3B and LM3/Hep3B comparisons, respectively. These DEPs focused on sugar metabolism‐centric canonical pathways per ingenuity pathway analysis, which was consistent with the gene ontology analysis on biological process enrichment. These pathways included glycolysis I, gluconeogenesis I and pentose phosphate pathways; and the DEPs enriched in these pathways could form a tightly connected network. By analyzing the relative abundance of proteins and translating mRNAs, we found significantly positive correlation between exosomes and cells. The involved exosomal proteins were again focusing on sugar metabolism. In conclusion, motile HCC cells tend to preferentially export more sugar metabolism‐associated proteins via exosomes that differentiate them from non‐motile HCC cells.     

Reexpression of ARHI inhibits tumor growth and angiogenesis and impairs the mTOR/VEGF pathway in hepatocellular carcinoma

The Ras-related tumor suppressor gene aplasia Ras homolog member I (ARHI) is frequently downregulated in many types of cancer, including hepatocellular carcinoma (HCC). In this study, we sought to explore the therapeutic implications of ARHI reconstitution in the treatment of HCC. We generated stable cell lines overexpressing ARHI in Hep3B and SK-Hep1 cells, both of which lack endogenous ARHI. The effects of ARHI reexpression on tumor growth and angiogenesis were assessed. Given the key role of mammalian target of rapamycin (mTOR) signaling in HCC progression, we also tested whether ARHI overexpression affected the mTOR pathway. Forced expression of ARHI resulted in a significant inhibition of the proliferation of both Hep3B and SK-Hep1 cells compared to control cells (P<0.01). Cell cycle analysis revealed a G0-G1 arrest induced by ARHI reexpression. Moreover, ARHI reexpression significantly retarded Hep3B xenograft growth in vivo, and caused a marked reduction in tumor angiogenesis assessed by CD31-stained microvessel count. Western blot analysis of the xenografts showed that ARHI overexpression substantially reduced the phosphorylation of two mTOR substrates, S6K1 and 4E-BP1, indicative of an inactivation of the mTOR pathway. Accompanying with the mTOR inactivation, the angiogenic factors, hypoxia-inducible factor 1 alpha and vascular endothelial growth factor, were significantly downregulated. These data highlighted an important role for ARHI in controlling HCC growth and angiogenesis, therefore offering a possible therapeutic strategy against this malignancy.     

Epigenetic activation of α4, β2 and β6 integrins involved in cell migration in trichostatin A-treated Hep3B cells

Summary The epigenetic modulation by histone deacetylase (HDAC) inhibitors including trichostatin A (TSA) has been known to block cell proliferation, induce apoptosis and inhibit cell migration in human cancer cells that represents the potential therapeutic agents for cancers and fibrosis. However, more than 55% of Hep3B cells remained alive after our initial study of 100 nM TSA treatment. To further study the epigenetic modulation and the biological function of newly activated genes by HDAC inhibitor involved in HCC progression and metastasis, we profiled 23 integrin genes including 15α and 8β in TSA-treated Hep3B cells. Six integrins including three down-regulated α6, α10, β8 and three significant up-regulated α4, β2, β6 integrins were revealed after semi-quantitative RT-PCR. To confirm the epigenetic modulation and explore their biological functions, we selected the three significantly up-regulated integrins for confirmation of protein up-regulation, hyperacetylated-histones by ChIP assays, and functional inhibition by specific neutralizing antibodies of integrins. Our results indicated that epigenetic modulation in TSA-treated Hep3B cells up-regulated new integrins including α4, β2 and β6 and reduced migration activities by specific neutralizing antibodies to 61.3%, 42.4% and 34.5%, respectively. Our novel findings provided a better understanding of the epigenetic modulation of integrins and suggested that targeting the epigenetic up-regulated integrins to abrogate the migration activity might be a promising strategy to prevent HCC progression.     

Parthenolide sensitizes hepatocellular carcinoma cells to TRAIL by inducing the expression of death receptors through inhibition of STAT3 activation

This article shows that HepG2, Hep3B, and SK-Hep1 cells, three lines of human hepatocellular carcinoma (HCC) cells, are resistant to apoptosis induced by tumor necrosis factor-related apoptosis-inducing ligand (TRAIL). Parthenolide, a sesquiterpene lactone found in European feverfew, has been shown to exert both anti-inflammatory and anti-cancer activities. This article demonstrates that co-treatment with parthenolide and TRAIL-induced apoptosis with synergistic interactions in the three lines of HCC cells. In order to explain these effects we ascertained that parthenolide increased either at protein or mRNA level the total content of death receptors TRAIL-R1 and -R2 as well as their surface expression. These effects were found in the three cell lines in the case of TRAIL-R2, while for TRAIL-R1 they were observed in HepG2 and SK-Hep1 cells, but not in Hep3B cells. We suggest that the effects of parthenolide on death receptors depend on the decrease in the level of phosphorylated and active forms of STAT proteins, an event which could be a consequence of the inhibitory effect exerted by parthenolide on the activation of JAK proteins. In agreement with this hypothesis treatment with STAT3 siRNA increased in HCC cells the effect of parthenolide on the expression of death receptors. Sensitization by parthenolide to TRAIL stimulated in the three cell lines the extrinsic mechanism of apoptosis with the activation of both caspases 8 and 3, whereas mitochondria were not involved in the process. Our results suggest that co-treatment with parthenolide and TRAIL could represent a new important therapeutic strategy for hepatic tumors.     

An EpCAM/CD3 bispecific antibody efficiently eliminates hepatocellular carcinoma cells with limited galectin-1 expression

There have been several studies suggesting that cancer stem cells (CSCs) contribute to the high rates of recurrence and resistance to therapies observed in hepatocellular carcinoma (HCC). Epithelial cell adhesion molecule (EpCAM) has been demonstrated to be a biomarker of CSCs and a potential therapeutic target in HCC. Here, we prepared two anti-EpCAM monoclonal antibodies (1H8 and 2F2) and an anti-EpCAM bispecific T cell engager (BiTE) 1H8/CD3, which was derived from 1H8, and used them to treat HCC in vitro and in vivo. The results demonstrated that all of the developed anti-EpCAM antibodies specifically bound to EpCAM. Neither anti-EpCAM monoclonal antibody had obvious anti-HCC activities in vitro or in vivo. However, anti-EpCAM BiTE 1H8/CD3 induced strong peripheral blood mononuclear cell-dependent cellular cytotoxicity in Huh-7 and Hep3B cells but not EpCAM-negative SK-Hep-1 cells. Notably, 1H8/CD3 completely inhibited the growth of Huh-7 and Hep3B xenografts in vivo. Treatment of the Huh-7 HCC xenografts with 1H8/CD3 significantly suppressed tumor proliferation and reduced the expression of most CSC biomarkers. Intriguingly, galectin-1 (Gal-1) overexpression inhibited 1H8/CD3-induced lymphocytotoxicity in HCCs while knockdown of Gal-1 increased the lymphocytotoxicity. Collectively, these results indicate that anti-EpCAM BiTE 1H8/CD3 is a promising therapeutic agent for HCC treatment. Gal-1 may contribute to the resistance of HCC cells to 1H8/CD3-induced lysis.     

GPC3-targeted CAR-T cells secreting B7H3-targeted BiTE exhibit potent cytotoxicity activity against hepatocellular carcinoma cell in the in vitro assay

Hepatocellular carcinoma (HCC), the most common primary liver cancer has a high mortality in China, and it is usually diagnosed at a late stage, thereby leaving patients with few effective treatment options. Chimeric antigen receptor-T (CAR-T) cell therapy, a novel immunotherapy that has shown promising results in leukemia, lymphoma and multiple myeloma, is also expected to work well in solid tumors, including HCC. However, the ideal therapeutic efficacy has not yet been achieved, in part due to tumor antigen escape caused by antigen heterogeneity. To overcome such challenge, we screened a panel of biomarkers in HCC cell lines and found that GPC3 and B7H3 were highly expressed on HCC with expression heterogeneity. Then we developed a novel bispecific T cell engagers CAR-T (CAR.T-BiTEs) that drives the expression of a CAR specific for GPC3 and BiTEs against CD3 and B7H3, herein referred to as “GPC3-BiTE CAR.” We found that BiTEs promoted the increased activation of untransduced T cells and IFN-γ release. Moreover, BiTEs secreted by GPC3-BiTE CAR-HEK293T cells promoted increased cytotoxicity activity of untransduced T cells against GPC3+/B7H3+ (GPC3 positive/B7H3 positive) and GPC3-/B7H3+(GPC3 negative/B7H3 positive) HCC cell lines. In vitro function assays showed that GPC3-BiTE CAR-T cells exhibited greater cytotoxicity activity against GPC3+/B7H3+ HCC cell lines than GPC3 CAR-T cells (GPC3-targeted CAR-T cells) and B7H3 CAR-T cells (B7H3-targeted CAR-T cells). Furthermore, GPC3-BiTE CAR-T cells exhibited superior cytotoxicity against GPC3 negative HCC cell lines compared with GPC3 CAR T cells. In conclusion, our study showed that GPC3-BiTE CAR T cells exhibited superior antitumor activity than single-target CAR-T cells and can overcome tumor escape induced by antigen heterogeneity, suggesting that this could be a promising therapeutic strategy for HCC.