Preclinical Evaluation of Liposomal C8 Ceramide as a Potent anti-Hepatocellular Carcinoma Agent

Hepatocellular carcinoma (HCC) remains a global health threat. The search for novel anti-HCC agents is urgent. In the current study, we synthesized a liposomal C8 ceramide, and analyzed its anti-tumor activity in pre-clinical HCC models. The liposomal C8 (ceramide) potently inhibited HCC cell (HepG2, SMMC-7721 and Huh-7 lines) survival and proliferation, more efficiently than free C8 ceramide. Yet, non-cancerous HL7702 human hepatocytes were resistant to the liposomal C8 treatment. Liposomal C8 activated caspase-dependent apoptosis in HCC cells, and HCC cytotoxicity by liposomal C8 was significantly attenuated with co-treatment of caspase inhibitors. At the molecular level, we showed that liposomal C8 activated ASK1 (apoptosis signal-regulating kinase 1)-JNK (Jun N-terminal protein kinase) signaling in HCC cells. On the other hand, JNK pharmacological inhibition or dominant negative mutation, as well as ASK1 shRNA-knockdown remarkably inhibited liposomal C8-induced apoptosis in HCC cells. Further studies showed that liposomal C8 inhibited AKT-mTOR (mammalian target of rapamycin) activation in HCC cells. Restoring AKT-mTOR activation by introducing a constitutively-active AKT alleviated HepG2 cytotoxicity by liposomal C8. In vivo, intravenous (i.v.) injection of liposomal C8 significantly inhibited HepG2 xenograft growth in severe combined immuno-deficient (SCID) mice, and mice survival was significantly improved. These preclinical results suggest that liposomal C8 could be further studied as a valuable anti-HCC agent.     

Knockdown of MACC1 expression suppressed hepatocellular carcinoma cell migration and invasion and inhibited expression of MMP2 and MMP9

Expression of MACC1 (metastasis-associated in colon cancer-1) protein is associated with metastasis of various human cancers. This study analyzed MACC1 protein expression in hepatocellular carcinoma (HCC) tissue specimens and then investigated the effects of MACC1 knockdown on HCC cell migration and invasion, and gene expression levels. Sixty pairs of HCC and adjacent normal liver tissues from HCC patients were analyzed for MACC1 expression immunohistochemically. The HCC cell lines Hep3B, Huh7, MHCC97H, SMMC-7721, Bel-7402, and HepG2 and the normal liver cell line LO2 were used to assess expressions of MACC1 mRNA and MACC1 protein using qRT-PCR and western blot, respectively. MACC1 short hairpin RNA (shRNA) was used to knockdown MACC1 protein expression in Huh7 cells. Changes in the tumor phenotype of these cells were analyzed with wound healing assay and invasion assays, and differences in gene expression were evaluated via western blot. Immunofluorescence was used to locate MACC1 protein in the above cell lines. MACC1 was highly expressed in HCC tissues and the nuclear expression of MACC1 protein was associated with poor tumor differentiation and intrahepatic metastasis or portal invasion. Moreover, MACC1 mRNA and MACC1 protein was also expressed in HCC cell lines. Immunostaining showed that MACC1 protein was localized in both nuclei and cytoplasm of HCC cell lines and the nuclear localization of MACC1 protein was associated with increased aggressiveness of HCC in cell lines. Knockdown of MACC1 expression using MACC1-shRNA reduced Huh7 cell migration and invasion abilities, which was associated with downregulation of MMP2, MMP9, and c-Met proteins in Huh7 cells. Localization of MACC1 protein to the nucleus may predict HCC progression. Knockdown of MACC1 expression using MACC1 shRNA warrants further evaluation as a novel therapeutic strategy for control of HCC.     

Isocorydine Targets the Drug-Resistant Cellular Side Population through PDCD4-Related Apoptosis in Hepatocellular Carcinoma

Isocorydine (ICD), an anticancer agent under current evaluation, decreased the percentage of side population (SP) cells significantly in hepatocellular carcinoma (HCC) cell lines. ICD treatment sensitized cancer cells to doxorubicin (DXR), a conventional clinical chemotherapeutic drug for HCC. We found that ICD decreased the percentage of SP cells in HCC cell lines by preferentially killing SP cells. In the early stage of treatment, ICD inhibited SP cell growth by arresting cells in G2/M; later, it induced apoptosis. Our xenograft model confirmed that ICD selectively reduced the size and weight of SP-induced tumor masses in vivo. Furthermore, it was found that programmed cell death 4 (PDCD4), a tumor suppressor gene, was relatively low when expressed in SP cells compared with non-SP cells, and its expression level was remarkably elevated when cells were treated with ICD. Taken together, these data suggest that ICD is a drug that may target the SP cells of HCC.     

Concurrent versus Sequential Sorafenib Therapy in Combination with Radiation for Hepatocellular Carcinoma

Sorafenib (SOR) is the only systemic agent known to improve survival for hepatocellular carcinoma (HCC). However, SOR prolongs survival by less than 3 months and does not alter symptomatic progression. To improve outcomes, several phase I-II trials are currently examining SOR with radiation (RT) for HCC utilizing heterogeneous concurrent and sequential treatment regimens. Our study provides preclinical data characterizing the effects of concurrent versus sequential RT-SOR on HCC cells both in vitro and in vivo. Concurrent and sequential RT-SOR regimens were tested for efficacy among 4 HCC cell lines in vitro by assessment of clonogenic survival, apoptosis, cell cycle distribution, and γ-H2AX foci formation. Results were confirmed in vivo by evaluating tumor growth delay and performing immunofluorescence staining in a hind-flank xenograft model. In vitro, concurrent RT-SOR produced radioprotection in 3 of 4 cell lines, whereas sequential RT-SOR produced decreased colony formation among all 4. Sequential RT-SOR increased apoptosis compared to RT alone, while concurrent RT-SOR did not. Sorafenib induced reassortment into less radiosensitive phases of the cell cycle through G₁-S delay and cell cycle slowing. More double-strand breaks (DSBs) persisted 24 h post-irradiation for RT alone versus concurrent RT-SOR. In vivo, sequential RT-SOR produced the greatest tumor growth delay, while concurrent RT-SOR was similar to RT alone. More persistent DSBs were observed in xenografts treated with sequential RT-SOR or RT alone versus concurrent RT-SOR. Sequential RT-SOR additionally produced a greater reduction in xenograft tumor vascularity and mitotic index than either concurrent RT-SOR or RT alone. In conclusion, sequential RT-SOR demonstrates greater efficacy against HCC than concurrent RT-SOR both in vitro and in vivo. These results may have implications for clinical decision-making and prospective trial design.     

Amplification of RAD54B promotes progression of hepatocellular carcinoma via activating the Wnt/β-catenin signaling

Liver cancer was reported to be the sixth most frequently diagnosed cancer, and hepatocellular carcinoma (HCC) accounts for 75%-85% of primary liver cancer. Nevertheless, the concrete molecular mechanisms of HCC progression remain obscure, which is essential to elucidate. The expression profile of RAD54B in HCC was measured using qPCR and western blotting. Moreover, the levels of RAD54B in paraffin-embedded samples were evaluated using immunohistochemistry (IHC). The effect of RAD54B on HCC progression was testified by in vitro experiments, and in vivo orthotopic xenograft tumor experiments. The mechanisms of RAD54B promoting HCC progression were investigated through molecular and function experiments. Herein, RAD54B are dramatically upregulated in HCC tissues and cell lines both on mRNA and protein levels, and RAD54B can servers as an independent prognostic parameter of 5-year overall survival and 5-year disease-free survival for patients with HCC. Moreover, up-regulation of RAD54B dramatically increases the capacity for in vitro cell viability and motility, and in vivo intrahepatic metastasis of HCC cells. Mechanistically, RAD54B promotes the HCC progression through modulating the wnt/β-catenin signaling. Notably, blocking the wnt/β-catenin signaling axis can counteract the activating effects of RAD54B on motility of HCC cells. Besides, further analysis illustrates that DNA amplification is one of the mechanisms leading to mRNA overexpression of RAD54B in HCC. Our findings indicate that RAD54B might be a promising potential prognostic marker and a candidate therapeutic target to therapy HCC.     

Tumor-derived secretory clusterin induces epithelial–mesenchymal transition and facilitates hepatocellular carcinoma metastasis

Hepatocellular carcinoma (HCC) is the third most common cause of cancer mortality. Metastasis is the major concern that causes death in HCC. The goal of this study was to identify tumor-derived proteins in serum during HCC metastasis using an orthotopic xenograft tumor model and explore the role of key protein in HCC metastasis. Serum samples collected from HCCLM3-R metastatic HCC tumor model at specific stages of metastasis (1 wk, 3 wks and 6 wks) were subjected to iTRAQ labeling followed by 2DLC-ESI-MS/MS analysis. Twenty tumor-derived proteins were identified through human specific peptides. Secretory clusterin (sCLU), which was significantly upregulated during cancer progression and metastasis, was chosen for further study. The expression of sCLU was significantly higher in metastatic HCC cell lines and samples from metastatic HCC patients. ShRNA-mediated down-regulation of sCLU resulted in a reduced migratory capacity in HCC cell lines, as well as a reduction in pulmonary metastasis in vivo. Overexpression of sCLU in HepG2 cell line showed increased cell migratory ability. Further study found that sCLU contributed to HCC migration and epithelial–mesenchymal transition (EMT) in vitro, and metastasis in vivo. In addition, sCLU also plays an important role in the regulation of TGF-β1-smad3 signaling. These findings suggest that sCLU may promote HCC metastasis via the induction of EMT process and may be a candidate target for HCC therapy.     

Switching of Pyruvate Kinase Isoform L to M2 Promotes Metabolic Reprogramming in Hepatocarcinogenesis

Hepatocellular carcinoma (HCC) is an aggressive tumor, with a high mortality rate due to late symptom presentation and frequent tumor recurrences and metastasis. It is also a rapidly growing tumor supported by different metabolic mechanisms; nevertheless, the biological and molecular mechanisms involved in the metabolic reprogramming in HCC are unclear. In this study, we found that pyruvate kinase M2 (PKM2) was frequently over-expressed in human HCCs and its over-expression was associated with aggressive clinicopathological features and poor prognosis of HCC patients. Furthermore, knockdown of PKM2 suppressed aerobic glycolysis and cell proliferation in HCC cell lines in vitro. Importantly, knockdown of PKM2 hampered HCC growth in both subcutaneous injection and orthotopic liver implantation models, and reduced lung metastasis in vivo. Of significance, PKM2 over-expression in human HCCs was associated with a down-regulation of a liver-specific microRNA, miR-122. We further showed that miR-122 interacted with the 3UTR of the PKM2 gene. Re-expression of miR-122 in HCC cell lines reduced PKM2 expression, decreased glucose uptake in vitro, and suppressed HCC tumor growth in vivo. Our clinical data and functional studies have revealed a novel biological mechanism involved in HCC metabolic reprogramming.     

Raf kinase inhibitor protein mediated signaling inhibits invasion and metastasis of hepatocellular carcinoma

BackgroundHepatocellular carcinoma (HCC) is the most common type of liver cancer with high mortality and poor prognosis. Mitogen-activated protein kinase (MAPK) and nuclear factor kappa B (NF-κB) signaling pathways have been implicated in promoting tumor cell proliferation and invasion of HCC cells.MethodsAs a potential inhibitor of tumor metastasis, the role of Raf kinase inhibitor protein (RKIP) in HCC development and the functional relevance with MAPK and NF-κB signaling pathways were investigated. The levels of RKIP expression were examined in human HCC tissues and correlated with tumor stages and metastatic status. Function of RKIP in cellular proliferation, migration, invasion and apoptosis was investigated in HCC cell lines by either overexpressing or knocking down RKIP expression. Mouse xenograft model was established to assess the effect of RKIP expression on tumor growth.ResultsOur results demonstrated decreased RKIP expression in HCC tissues and a strong correlation with tumor grade and distant metastasis. Manipulation of RKIP expression in HCCLM3 and HepG2 cells indicated that RKIP functioned to inhibit HCC cell motility and invasiveness, and contributed to tumor growth inhibition in vivo. Mechanistic studies showed that the function of RKIP was mediated through MAPK and NF-κB signaling pathways. However, cell type-dependent RKIP regulation on these two pathways was also suggested, indicating the complex nature of signaling network.ConclusionOur study provides a better understanding on the molecular mechanisms of HCC metastasis and sets the foundation for the development of targeted therapeutics for HCC.     

Generation of Subcutaneous and Intrahepatic Human Hepatocellular Carcinoma Xenografts in Immunodeficient Mice

In vivo experimental models of hepatocellular carcinoma (HCC) that recapitulate the human disease provide a valuable platform for research into disease pathophysiology and for the preclinical evaluation of novel therapies. We present a variety of methods to generate subcutaneous or orthotopic human HCC xenografts in immunodeficient mice that could be utilized in a variety of research applications. With a focus on the use of primary tumor tissue from patients undergoing surgical resection as a starting point, we describe the preparation of cell suspensions or tumor fragments for xenografting. We describe specific techniques to xenograft these tissues i) subcutaneously; or ii) intrahepatically, either by direct implantation of tumor cells or fragments into the liver, or indirectly by injection of cells into the mouse spleen. We also describe the use of partial resection of the native mouse liver at the time of xenografting as a strategy to induce a state of active liver regeneration in the recipient mouse that may facilitate the intrahepatic engraftment of primary human tumor cells. The expected results of these techniques are illustrated. The protocols described have been validated using primary human HCC samples and xenografts, which typically perform less robustly than the well-established human HCC cell lines that are widely used and frequently cited in the literature. In comparison with cell lines, we discuss factors which may contribute to the relatively low chance of primary HCC engraftment in xenotransplantation models and comment on technical issues that may influence the kinetics of xenograft growth. We also suggest methods that should be applied to ensure that xenografts obtained accurately resemble parent HCC tissues.     

Resveratrol as a Pan-HDAC Inhibitor Alters the Acetylation Status of Jistone Proteins in Human-Derived Hepatoblastoma Cells

The polyphenolic alcohol resveratrol has demonstrated promising activities for the prevention and treatment of cancer. Different modes of action have been described for resveratrol including the activation of sirtuins, which represent the class III histone deacetylases (HDACs). However, little is known about the activity of resveratrol on the classical HDACs of class I, II and IV, although these classes are involved in cancer development or progression and inhibitors of HDACs (HDACi) are currently under investigation as promising novel anticancer drugs. We could show by in silico docking studies that resveratrol has the chemical structure to inhibit the activity of different human HDAC enzymes. In vitro analyses of overall HDAC inhibition and a detailed HDAC profiling showed that resveratrol inhibited all eleven human HDACs of class I, II and IV in a dose-dependent manner. Transferring this molecular mechanism into cancer therapy strategies, resveratrol treatment was analyzed on solid tumor cell lines. Despite the fact that hepatocellular carcinoma (HCC) is known to be particularly resistant against conventional chemotherapeutics, treatment of HCC with established HDACi already has shown promising results. Testing of resveratrol on hepatoma cell lines HepG2, Hep3B and HuH7 revealed a dose-dependent antiproliferative effect on all cell lines. Interestingly, only for HepG2 cells a specific inhibition of HDACs and in turn a histone hyperacetylation caused by resveratrol was detected. Additional testing of human blood samples demonstrated a HDACi activity by resveratrol ex vivo. Concluding toxicity studies showed that primary human hepatocytes tolerated resveratrol, whereas in vivo chicken embryotoxicity assays demonstrated severe toxicity at high concentrations. Taken together, this novel pan-HDACi activity opens up a new perspective of resveratrol for cancer therapy alone or in combination with other chemotherapeutics. Moreover, resveratrol may serve as a lead structure for chemical optimization of bioavailability, pharmacology or HDAC inhibition.     

Interferon-α/β and anti-fibroblast growth factor receptor 1 monoclonal antibody suppress hepatic cancer cells in vitro and in vivo

Background

Hepatocellular carcinoma (HCC) is the most commonly occurring primary liver cancer and ranks as the fifth most frequently occurring cancer, overall, and the third leading cause of cancer deaths, worldwide. At present, effective therapeutic options available for HCC are limited; consequently, the prognosis for these patients is poor. Our aim in the present study was to identify a novel target for antibody therapy against HCC.

Methodology/Principal Findings

We used Western blot and flow cytometric and immunocytochemical analyses to investigate the regulation of FGFR1 expression by interferon-α/β in several human hepatic cancer cell lines. In addition, we tested the efficacy of combined treatment with anti-FGFR1 monoclonal antibody and interferon-α/β in a murine xenograft model of human HCC. We found that interferon-α/β induces expression of FGFR1 in human HCC cell lines, and that an anti-FGFR1 monoclonal antibody (mAb) targeting of the induced FGFR1 can effectively inhibit growth and survival of HCC cells in vitro and in vivo. Moreover, the combination of interferon-α, anti-FGFR1 mAb and peripheral blood mononuclear cells (PBMCs) exerted a significant antitumor effect in vitro.

Conclusions

Our results suggest that the combined use of an anti-FGFR1 antibody and interferon-α/β is a promising approach to the treatment of HCC.     

The Suppressive Role and Aberrent Promoter Methylation of BTG3 in the Progression of Hepatocellular Carcinoma

Background

BTG3 (B-cell translocation gene 3) has been identified as a tumor suppressor and hypermethylation contributes to its down-regulation in some tumors, but its role in hepatocellular carcinoma (HCC) remain unknown. This study aimed to detect the expression and methylation status of BTG3 in HCC cell lines or tissues, and determine its function in HCC progression.

Methodology

The expression of BTG3 was detected in HCC cell lines and HCC tissue by real-time RT-PCR, Western blot or immunohistochemistry. The promoter methylation status of BTG3 was measured by using methylation-specific PCR in HCC cell lines. A series of assays were performed to evaluate the effect of BTG3 on proliferation, invasion and cell cycle transition in vitro.

Results

BTG3 expression was lower in HCC cell lines than in hepatocyte cell line LO2 (P<0.05). BTG3 was also down-regulated in HCC tissues. Its expression was positively correlated with differentiation and distant metastasis (P<0.05). Patients with lower BTG3 expression had shorter overall survival time (P=0.029). DNA methylation directed repression of BTG3 mRNA expression in HCC cell lines. BTG3 suppressed proliferation, invasion and induces G1/S cycle arrest of HCC cells in vitro.

Conclusion

Down-regulation of BTG3 due to the promoter hypermethylation is closely associated with proliferation, invasion and cell cycle arrest of HCC cells. It may be a novel prognostic biomarker for HCC patients.     

CMTM5 is downregulated and suppresses tumour growth in hepatocellular carcinoma through regulating PI3K-AKT signalling

Background

Human chemokine like factor (CKLF)-like MAL and related proteins for vesicle trafficking transmembrane, domain-containing member 5 (CMTM5) has been shown to involved and may function as a tumour suppressor in tumorigenesis. The current study aimed to investigate the expression and function of CMTM5 in human hepatocellular carcinoma (HCC).

Methods

CMTM5 expression was examined by immunohistochemistry, and its clinical significance was analysed in 76 HCC specimens. The role and molecular mechanisms of CMTM5 in cell proliferation, apoptosis and invasion were examined in vitro and in vivo.

Results

CMTM5 expression was significantly downregulated in HCC tissues as well as cell lines. The expression of CMTM5 was absent in 77.6% of HCC tissues compared with 3.9% in normal liver tissues. Low CMTM5 expression was significantly correlated with poor overall survival in patients with HCC (P = 0.009). Restoring CMTM5 expression in Huh7 cells significantly inhibited cell growth, promoted cell apoptosis, and reduced cell metastatic and invasion ability compared with mock transfected cells in vitro. Overexpression of CMTM5 also suppressed xenograft tumour growth in vivo in a HCC xenograft model. Reduced cell growth and metastasis ability mediated by CMTM5 overexpression was associated with downregulation of PI3K/AKT and its downstream Bcl2, cyclinD1, cyclinE, MMP2 and MMP9 expressions, and an upregulation of p21, Bax, Bad, cleaved caspase3 expressions.

Conclusions

Our data suggest that CMTM5 might function as a tumour suppressor in human HCC, and represent a valuable potential therapeutic target for HCC.

     

SNHG16 as the miRNA let-7b-5p sponge facilitates the G2/M and epithelial-mesenchymal transition by regulating CDC25B and HMGA2 expression in hepatocellular carcinoma

Long noncoding RNAs (lncRNAs) play crucial roles in hepatocellular carcinoma (HCC). However, the underlying molecular mechanisms of small nucleolar RNA host gene 16 (SNHG16) for regulating the cell cycle and epithelial to mesenchymal transition (EMT) remain elusive. In this study, SNHG16 expression profiles of HCC tissues or cell lines were compared with those of normal tissues or hepatocyte cell line. The effect of SNHG16 knockdown in HCC cell lines was investigated by using in vitro loss-of-function experiments and in vivo nude mouse experiments. The potential molecular regulatory mechanism of SNHG16 in HCC progression was investigated by using mechanistic experiments and rescue assays. The results revealed that SNHG16 was highly expressed in HCC tissues and cell lines, which predicted poor prognosis of HCC patients. On one hand, the downregulation of SNHG16 induced G2/M cell cycle arrest, inducing cell apoptosis and suppression of cell proliferation. On the other hand, it inhibited cell metastasis and EMT progression demonstrated by in vitro loss-of-function cell experiments. Besides, knockdown of SNHG16 increased the sensitivity of HCC cells to cisplatin. For the detailed mechanism, SNHG16 was demonstrated to act as a let-7b-5p sponge in HCC. SNHG16 facilitated the G2/M cell cycle transition by directly acting on the let-7b-5p/CDC25B/CDK1 axis, and promoted cell metastasis and EMT progression by regulating the let-7b-5p/HMGA2 axis in HCC. In addition, the mechanism of SNHG16 for regulating HCC cell proliferation and metastasis was further confirmed in vivo by mouse experiments. Furthermore, these results can provide new insights into HCC treatment and its molecular pathogenesis, which may enlighten the further research of the molecular pathogenesis of HCC.     

G Protein-Coupled Receptor 87 (GPR87) Promotes the Growth and Metastasis of CD133+ Cancer Stem-Like Cells in Hepatocellular Carcinoma

Hepatocellular carcinoma (HCC) is a prevalent disease worldwide, and the majority of HCC-related deaths occur due to local invasion and distant metastasis. Cancer stem cells (CSCs) are a small subpopulation of cancer cells that have been hypothesized to be responsible for metastatic disease. Recently, we and others have identified a CSC population from human HCC cell lines and xenograft tumors characterized by their expression of CD133. However, the precise molecular mechanisms by which CD133⁺ cancer stem-like cells mediate HCC metastasis have not been sufficiently analyzed. Here, we have sorted HCC cells using CD133 as a cancer stem cell (CSC) marker by magnetic-activated cell sorting (MACS) and demonstrated that the CD133⁺ HCC cells not only possess greater migratory and invasive capacity in vitro but are also endowed with enhanced metastatic capacity in vivo and in human HCC specimens when compared to CD133⁻ HCC cells. Gene expression analysis of the CD133⁺ and CD133⁻ cells of the HCC line SMMC-7721 revealed that G protein-coupled receptor 87 (GPR87) is highly expressed in CD133⁺ HCC cells. In this study, we explored the role of GPR87 in the regulation of CD133 expression. We demonstrated that the overexpression of GPR87 up-regulated CD133 expression, promoted CSC-associated migratory and invasive properties in vitro, and increased tumor initiation in vivo. Conversely, silencing of GPR87 expression reduced the levels of CD133 expression. Conclusion: GPR87 promotes the growth and metastasis of CD133⁺ cancer stem-like cells, and our findings may reveal new targets for HCC prevention or therapy.     

The role of proline rich tyrosine kinase 2 (Pyk2) on cisplatin resistance in hepatocellular carcinoma

Aims

We previously demonstrated Proline rich tyrosine kinase 2 (Pyk2) plays important roles in regulating tumor progression, migration and invasion in hepatocellular carcinoma (HCC). In this study, we aimed to examine the role of proline rich tyrosine kinase 2 (Pyk2) on cisplatin resistance in HCC and to explore its underlying molecular mechanism.

Methodology/Principal Findings

Stable transfectants either overexpressing or suppressing Pyk2 were established in different HCC cell lines. MTT, colony formation and Annexin-V assays were employed to examine their in vitro responses to cisplatin. Xenograft ectopic and orthotopic nude mice models were generated to investigate the in vivo responses of them to cisplatin treatment. cDNA microarray was performed to identify Pyk2-induced genes which were further validated by quantitative real-time RT-PCR using clinical HCC samples. In vitro functional study demonstrated that Pyk2-overexpressing HCC transfectants exhibited relatively lower cytotoxicity, higher colony-forming ability and lower apoptosis to cisplatin compared with the control transfectants. Moreover, Pyk2 overexpressing HCC transfectants had a higher survival rate under cisplatin treatment by up-regulation of AKT phosphorylation. In vivo xenograft nude mice model demonstrated that Pyk2-overexpressing transfectants developed higher tolerance to cisplatin treatment together with less tumor necrosis and apoptosis. cDNA microarray analysis revealed that there were more than 4,000 genes differentially expressed upon overexpression of Pyk2. Several upregulated genes were found to be involved in drug resistance and invasion in cancers. Among them, the expression profiles of MDR1, GAGE1, STAT1 and MAP7 were significantly associated with the expression of Pyk2 in clinical HCC samples.

Conclusions

Our results may suggest a new evidence of Pyk2 on promoting cisplatin resistance of HCC cells through preventing cell apoptosis, activation of AKT pathway and upregulation of drug resistant genes.     

Fn14?Trail Effectively Inhibits Hepatocellular Carcinoma Growth

Background

New strategies for the treatment of hepatocellular carcinoma (HCC) are needed, given that currently available chemotherapeutics are inefficient. Since tumor growth reflects the net balance between pro-proliferative and death signaling, agents shifting the equilibrium toward the latter are of considerable interest. The TWEAK:Fn14 signaling axis promotes tumor cell proliferation and tumor angiogenesis, while TRAIL:TRAIL-receptor (TRAIL-R) interactions selectively induce apoptosis in malignant cells. Fn14•TRAIL, a fusion protein bridging these two pathways, has the potential to inhibit tumor growth, by interfering with TWEAK:Fn14 signaling, while at the same time enforcing TRAIL:TRAIL-R-mediated apoptosis. Consequently, Fn14•TRAIL''s capacity to inhibit HCC growth was tested.

Results

Fn14•TRAIL induced robust apoptosis of multiple HCC cell lines, while sparing non-malignant hepatocyte cell lines. Differential susceptibility to this agent did not correlate with expression levels of TRAIL, TRAIL-R, TWEAK and Fn14 by these lines. Fn14•TRAIL was more potent than soluble TRAIL, soluble Fn14, or a combination of the two. The requirement of both of Fn14•TRAIL''s molecular domains for function was established using blocking antibodies directed against each of them. Subcutaneous injection of Fn14•TRAIL abrogated HCC growth in a xenograft model, and was well tolerated by the mice.

Conclusions

In this study, Fn14•TRAIL, a multifunctional fusion protein originally designed to treat autoimmunity, was shown to inhibit the growth of HCC, both in vitro and in vivo. The demonstration of this fusion protein’s potent anti-tumor activity suggests that simultaneous targeting of two signaling axes by a single fusion can serve as a basis for highly effective anti-cancer therapies.     

Characterization of 9-nitrocamptothecin liposomes: anticancer properties and mechanisms on hepatocellular carcinoma in vitro and in vivo

Background

Hepatocellular carcinoma (HCC) is the third most common cause of cancer related mortality worldwide. 9-Nitrocamptothecin (9NC) is a potent topoisomerase-I inhibitor with strong anticancer effect. To increase the solubility and stability, we synthesized a novel 9NC loaded liposomes (9NC-LP) via incorporating 9NC into liposomes. In the present study, we determined the effects of 9NC and 9NC-LP on in vitro and in vivo, and the underlying mechanisms.

Methodology/Principal Findings

We first analyzed the characteristics of 9NC-LP. Then we compared the effects of 9NC and 9NC-LP on the proliferation and apoptosis of HepG2, Bel-7402, Hep3B and L02 cells in vitro. We also investigated their anticancer properties in nude mice bearing HCC xenograft in vivo. 9NC-LP has a uniform size (around 190 nm) and zeta potential (∼−11 mV), and exhibited a steady sustained-release pattern profile in vitro. Both 9NC and 9NC-LP could cause cell cycle arrest and apoptosis in a dose-dependent and p53-dependent manner. However, this effect was not ubiquitous in all cell lines. Exposure to 9NC-LP led to increased expression of p53, p21, p27, Bax, caspase-3, caspase-8, caspase-9 and apoptosis-inducing factor, mitochondrion-associated 1 and decreased expression of Bcl-2, cyclin E, cyclin A, Cdk2 and cyclin D1. Furthermore, 9NC-LP exhibited a more potent antiproliferative effect and less side effects in vivo. Western blot analysis of the xenograft tumors in nude mice showed similar changes in protein expression in vivo.

Conclusions/Significance

In conclusion, 9NC and 9NC-LP can inhibit HCC growth via cell cycle arrest and induction of apoptosis. 9NC-LP has a more potent anti-tumor effect and fewer side effects in vivo, which means it is a promising reagent for cancer therapy via intravenous administration.     

The role of daurisoline treatment in hepatocellular carcinoma: Inhibiting vasculogenic mimicry formation and enhancing sensitivity to sorafenib

BackgroundVasculogenic mimicry (VM) is a newly described tumor vascular phenomenon that is independent of traditional angiogenesis and provides an adequate blood supply for tumor growth. VM has been consistently observed in different cancer types. Hence, inhibition of VM may be considered a new anticancer therapeutic strategy.PurposeThis study aimed to elucidate the potential anticancer effect of daurisoline (DS) on hepatocellular carcinoma (HCC) and the potential molecular mechanism by which DS inhibits VM. We also verified whether combination treatment with sorafenib and DS constitutes a novel therapeutic approach to prevent HCC progression.MethodsThe effects of DS on proliferation were evaluated by Cell Counting Kit-8 (CCK-8), colony formation, and 5-ethynyl-2′-deoxyuridine (EdU) incorporation assays. 4′,6-Diamidino-2-phenylindole (DAPI) staining and flow cytometric analysis were employed to investigate its effects on apoptosis. Western blot analysis, Matrigel tube formation assays, pulldown assays and immunofluorescence staining were applied to validate the potential mechanism by which DS inhibits VM. Mouse xenograft models were used to evaluate anticancer activities.ResultsDS inhibited HCC cell proliferation, induced HCC cell apoptosis and repressed VM formation by inactivating RhoA/ROCK2-mediated AKT and ERK-p38 MAPK signaling. Additionally, DS dramatically sensitized HCC cell lines to sorafenib, a curative anticancer drug for patients with advanced HCC.ConclusionsOur study provides insights into the molecular mechanisms underlying DS-induced inhibition of VM, which may facilitate the development of a novel clinical anti-HCC drug. Moreover, our findings suggest that the combination of DS and sorafenib constitutes a potential therapeutic strategy for HCC.