Activity In Hepatocellular Carcinoma Cells Research Articles

Abstract 5145: To explore the tumorigenic regulation mechanism of SPZ1 in hepatocellular carcinoma

Abstract Cancer directly affects at least one-third of the human population. Despite this extensively, the genetic determinants of cancer risk remain unknown. Spermatogenic leucine zipper 1 (SPZ1), a basic helix loop helix leucine zipper (bHLH-Zip) transcription factor, acted as a proto-oncogene in mouse and human. It has been demonstrated that SPZ1 was involved in early embryonic development, cell growth and differentiation. In this study, we compared the expression of SPZ1 in 168 paired specimens of HCCs and adjacent normal tissues and also in paired specimens from 140 patients with non-HCCs. In pathologic analysis, SPZ1 exhibited a tumor-specific expression pattern and a high correlation to patient survival time, tumor size, tumor number, and progression stage. Enforced expression of SPZ1 accelerated cell proliferation and tumorigenic activity in hepatoma cells. In contrast, short hairpin RNA-mediated attenuation of SPZ1 in hepatoma cells decreased cell proliferation and malignant transformation in vitro and in vivo. Together, we speculated that SPZ1 as a potential therapeutic target of HCCs Citation Format: LI-TING WANG, SHIH-HSIEN HSU. To explore the tumorigenic regulation mechanism of SPZ1 in hepatocellular carcinoma. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 5145.

The asialoglycoprotein receptor suppresses the metastasis of hepatocellular carcinoma via LASS2-mediated inhibition of V-ATPase activity

The asialoglycoprotein receptor (ASGR), which is expressed mainly in hepatocytes, is downregulated in poorly differentiated hepatocellular carcinoma (HCC). Here we investigated the role of ASGR1 in HCC metastasis as well as the possible underlying molecular mechanisms. We found that ASGR1 was downregulated in HCC tissue compared with adjacent non-tumorous liver tissue and that lower ASGR1 expression was associated with higher TNM stage and poorer prognosis in HCC patients. ASGR1 overexpression inhibited hepatoma cell migration and invasion in vitro and in vivo, while ASGR1 knockdown had the opposite effects. Furthermore, ASGR1 interacted directly with human longevity assurance homolog 2 of yeast LAG1 (LASS2). Knockdown of LASS2 attenuated the inhibitory effects of ASGR1 on hepatoma cell migration and invasion in vitro. ASGR1 decreased V-ATPase activity in hepatoma cells, and this was reversed by LASS2 knockdown. Finally, HCC patients with low LASS2 levels had poor prognosis, while those with high ASGR1 and LASS2 levels had better prognosis. Thus, ASGR1 may act as a potential metastasis suppressor in HCC, and the combination of ASGR1 and LASS2 may help predict the prognosis of HCC patients.

Vitamin C enhances epigenetic modifications induced by 5-azacytidine and cell cycle arrest in the hepatocellular carcinoma cell lines HLE and Huh7

Background5-Azacytidine (5-AZA), a DNA methyl transferase inhibitor, is a clinically used epigenetic drug for cancer therapy. Recently, we have shown that 5-AZA upregulates ten-eleven translocation (TET) protein expression in hepatocellular carcinoma (HCC) cells, which induce active demethylation. Vitamin C facilitates TET activity and enhances active demethylation. The aim of this study is to investigate whether vitamin C is able to enhance the effect of 5-AZA on active demethylation and to evaluate its consequence in HCC cell lines.MethodsHCC cell lines (Huh7 and HLE) were treated with 5-AZA and vitamin C. After 48 h of treatment, viability (resazurin conversion), toxicity (lactose dehydrogenase (LDH) release), and proliferation ((proliferating cell nuclear antigen (PCNA)) of single- and combined-treated cells were assessed. The effect of the treatment on 5-hydroxymethylcytosine (5hmC) intensity (immunofluorescence (IF) staining), TET, Snail, GADD45B, and P21 mRNA (real-time PCR) and protein expression (Western blot) were investigated.ResultsOur results indicated that vitamin C enhances the anti-proliferative and apoptotic effect of 5-AZA in HCC cell lines. By further analyzing the events leading to cell cycle arrest, we have shown for the first time in HCC that the combination of 5-AZA and vitamin C leads to an enhanced downregulation of Snail expression, a key transcription factor governing epithelial-mesenchymal transition (EMT) process, and cell cycle arrest.ConclusionsWe conclude that when combined with 5-AZA, vitamin C enhances TET activity in HCC cells, leading to induction of active demethylation. An increase in P21 expression as a consequence of downregulation of Snail accompanied by the induction of GADD45B expression is the main mechanism leading to cell cycle arrest in HCCs.

CIZ1 interacts with YAP and activates its transcriptional activity in hepatocellular carcinoma cells.

Dysregulation of Hippo-Yes-associate protein (YAP) signaling has important roles in the tumorigenesis of hepatocellular carcinoma (HCC). Our previous studies have shown that Cip1 interacting zinc finger protein 1 (CIZ1) activated YAP signaling in the HCC cells and promoted the growth and migration of cancer cells. However, the mechanisms for the activation of YAP signaling by CIZ1 are unknown. In this study, it was found that CIZ1 interacted with the transcriptional factor YAP in HCC cells. The nuclear matrix anchor domain of CIZ1 is responsible for its interaction with YAP. Moreover, CIZ1 enhanced the interaction between YAP and TEAD. Knocking down the expression of CIZ1 impaired the transcriptional activity as well as the biological functions of YAP. Taken together, our study demonstrated that CIZ1 is a positive regulator of YAP signaling, and CIZ1 might be a therapeutic target for HCC.

MiR-103 regulates hepatocellular carcinoma growth by targeting AKAP12

AKAP12/Gravin (A kinase anchor protein 12) belongs to the group of A-kinase scaffold proteins and functions as a tumor suppressor in some human primary cancers. While AKAP12 is found consistently downregulated in hepatocellular carcinoma (HCC), its involvement in hepatocarcinogenesis has not been fully elucidated. We identified targeting sites for miR-103 in the 3′-untranslated region (3′-UTR) of AKAP12 by bioinformatic analysis and confirm their function by a luciferase reporter gene assay. We reveal miR-103 expression to be inversely correlated with AKAP12 in HCC tissue samples and show that overexpressed miR-103 promotes cell proliferation and inhibits apoptosis by downregulating AKAP12 expression in HCC cell lines. On the other hand, repression of miR-103 suppresses proliferation and promotes apoptosis in HCC cells by increasing AKAP12. In xenografted HCC tumors, overexpression of AKAP12 suppresses tumor growth whereas overexpression of miR-103 enhances tumor growth while repressing AKAP12. Since the activation of telomerase is crucial for cells to gain immortality and proliferation ability, we investigated whether AKAP12 expression affected telomerase activity in HCC cells. Both AKAP12 overexpression and protein kinase Cα (PKCα) inhibition prevent nuclear translocation and phosphorylation of TERT and reduce telomerase activity in HCC cells. These findings indicate that miR-103 potentially acts as an oncogene in HCC by inhibiting AKAP12 expression and raise the possibility that miR-103 increases telomerase activity by increasing PKCα activity. Thus, miR-103 may represent a new potential diagnostic and therapeutic target for HCC treatment.

Abstract LB-030: Hepatocellular carcinoma-derived exosomes promote motility of immortalized hepatocyte through transfer of oncogenic proteins and RNAs

Abstract Exosomes are increasingly recognized as important mediators of cell-cell communication in cancer progression through the horizontal transfer of RNAs and proteins to neighboring or distant cells. Hepatocellular carcinoma (HCC) is a highly malignant cancer, whose metastasis is largely influenced by the tumor microenvironment. The possible role of exosomes in the interactions between HCC tumor cell and its surrounding hepatic milieu are however largely unknown. In this study, we comprehensively characterized the exosomal RNA and proteome contents derived from three HCC cell lines (HKCI-C3, HKCI-8 and MHCC97L) and an immortalized hepatocyte line (MIHA) using Ion Torrent sequencing and mass spectrometry, respectively. RNA deep sequencing and proteomic analysis revealed exosomes derived from metastatic HCC cell lines carried a large number of pro-tumorigenic RNAs and proteins, such as MET proto-oncogene, S100 family members and the caveolins. Of interest, we found exosomes from motile HCC cell lines could significantly enhance the migratory and invasive abilities of non-motile MIHA cell. We further demonstrated that uptake of these shuttled molecules could trigger PI3K/AKT and MAPK signaling pathways in MIHA with increased secretion of active MMP-2 and MMP-9. Our study showed for the first time that HCC-derived exosomes could mobilize normal hepatocyte, which may have implication in facilitating the protrusive activity of HCC cells through liver parenchyma during the process of metastasis. Citation Format: Nathalie Wong, Mian He. Hepatocellular carcinoma-derived exosomes promote motility of immortalized hepatocyte through transfer of oncogenic proteins and RNAs. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr LB-030. doi:10.1158/1538-7445.AM2015-LB-030

Tankyrase inhibitors attenuate WNT/β-catenin signaling and inhibit growth of hepatocellular carcinoma cells.

Deregulated WNT/β-catenin signaling contributes to the development of a subgroup of hepatocellular carcinoma (HCC), the second leading cause of cancer deaths worldwide. Within this pathway, the tankyrase enzymes (TNKS1 and TNKS2) degrade AXIN and thereby enhance β-catenin activity. We evaluate TNKS enzymes as potential therapeutic targets in HCC, and the anti-tumor efficacy of tankyrase inhibitors (XAV939, and its novel nitro-substituted derivative WXL-8) in HCC cells. Using semi-quantitative RT-PCR, we found significantly elevated levels of TNKS1/2 mRNA in tumor liver tissues compared to adjacent non-tumor livers, at protein levels only TNKS1 is increased. In HepG2, Huh7cells, siRNA-mediated knockdown suppression of endogenous TNKS1 and TNKS2 reduced cell proliferation, together with decreased nuclear β-catenin levels. XAV939 and WXL-8 inhibited cell proliferation and colony formation in HepG2, Huh7, and Hep40 cells (p < 0.05), with stabilization of AXIN1 and AXIN2, and decreased β-catenin protein levels. XAV939 and WXL-8 also attenuated rhWNT3A-induced TOPflash luciferase reporter activity in HCC cells, indicating reduced β-catenin transcriptional activity, consistent with decreased nuclear β-catenin levels. In vivo, intra-tumor injections of XAV939 or WXL-8 significantly inhibited the growth of subcutaneous HepG2 xenografts (P < 0.05). We suggest that tankyrase inhibition is a potential therapeutic approach for treating a subgroup HCC with aberrant WNT/β-catenin signaling pathway.

The neuroleptic drug pimozide inhibits stem-like cell maintenance and tumorigenicity in hepatocellular carcinoma.

Drug repurposing is currently an important approach for accelerating drug discovery and development for clinical use. Hepatocellular carcinoma (HCC) presents drug resistance to chemotherapy, and the prognosis is poor due to the existence of liver cancer stem-like cells. In this study, we investigated the effect of the neuroleptic agent pimozide to inhibit stem-like cell maintenance and tumorigenicity in HCC. Our results showed that pimozide functioned as an anti-cancer drug in HCC cells or stem-like cells. Pimozide inhibited cell proliferation and sphere formation capacities in HCC cells by inducing G0/G1 phase cell cycle arrest, as well as inhibited HCC cell migration. Surprisingly, pimozide inhibited the maintenance and tumorigenicity of HCC stem-like cells, particularly the side population (SP) or CD133-positive cells, as evaluated by colony formation, sphere formation and transwell migration assays. Furthermore, pimozide was found to suppress STAT3 activity in HCC cells by attenuating STAT3-dependent luciferase activity and down-regulating the transcription levels of downstream genes of STAT3 signaling. Moreover, pimozide reversed the stem-like cell tumorigenic phenotypes induced by IL-6 treatment in HCC cells. Further, the antitumor effect of pimozide was also proved in the nude mice HCC xenograft model. In short, the anti-psychotic agent pimozide may act as a novel potential anti-tumor agent in treating advanced HCC.

URG4/URGCP enhances the angiogenic capacity of human hepatocellular carcinoma cells in vitro via activation of the NF-κB signaling pathway.

BackgroundAngiogenesis is essential for tumor growth. Hepatocellular carcinoma (HCC) is characterized by hypervascularity; high levels of angiogenesis are associated with poor prognosis and a highly invasive phenotype in HCC. Up-regulated gene-4 (URG4), also known as upregulator of cell proliferation (URGCP), is overexpressed in multiple tumor types and has been suggested to act as an oncogene. This study aimed to elucidate the effect of URG4/URGCP on the angiogenic capacity of HCC cells in vitro.MethodsExpression of URG4/URGCP in HCC cell lines and normal liver epithelial cell lines was examined by Western blotting and quantitative real-time PCR. URG4/URGCP was stably overexpressed or transiently knocked down using a shRNA in two HCC cell lines. The human umbilical vein endothelial cell (HUVEC) tubule formation and Transwell migration assays and chicken chorioallantoic membrane (CAM) assay were used to examine the angiogenic capacity of conditioned media from URG4/URGCP-overexpressing and knockdown cells. A luciferase reporter assay was used to examine the transcriptional activity of nuclear factor kappa – light – chain - enhancer of activated B cells (NF-κB). NF-κB was inhibited by overexpressing degradation-resistant mutant inhibitor of κB (IκB)-α. Expression of vascular endothelial growth factor C (VEGFC), tumor necrosis factor-α (TNFα), interleukin (IL)-6, IL-8 and v-myc avian myelocytomatosis viral oncogene homolog (MYC) were examined by quantitative real-time PCR; VEGFC protein expression was analyzed using an ELISA.ResultsURG4/URGCP protein and mRNA expression were significantly upregulated in HCC cell lines. Overexpressing URG4/URGCP enhanced - while silencing URG4/URGCP decreased - the capacity of HCC cell conditioned media to induce HUVEC tubule formation and migration and neovascularization in the CAM assay. Furthermore, overexpressing URG4/URGCP increased - whereas knockdown of URG4/URGCP decreased - VEGFC expression, NF-κB transcriptional activity, the levels of phosphorylated (but not total) IκB kinase (IKK) and IκB-α, and expression of TNFα, IL-6, IL-8 and MYC in HCC cells. Additionally, inhibition of NF-κB activity in HCC cells abrogated URG4/URGCP-induced NF-κB activation and angiogenic capacity.ConclusionsThis study suggests that URG4/URGCP plays an important pro-angiogenic role in HCC via a mechanism linked to activation of the NF-κB pathway; URG4/URGCP may represent a potential target for anti-angiogenic therapy in HCC.Electronic supplementary materialThe online version of this article (doi:10.1186/s12885-015-1378-7) contains supplementary material, which is available to authorized users.

RACK1 expression contributes to JNK activity, but JNK activity does not enhance RACK1 expression in hepatocellular carcinoma SMMC-7721 cells.

Receptor for activated C kinase 1 (RACK1) is up-regulated in hepatocellular carcinoma (HCC) and has been reported to augment c-Jun N-terminal protein kinase (JNK) activity in HCC SMMC-7721 cells. By contrast, activator protein-1, a downstream JNK transcription factor, has been revealed to mediate the overexpression of RACK1 in melanoma cells. Therefore, the association between RACK1 and JNK activity in HCC cells has yet to be completely elucidated. The present study analyzed the effects of RACK1 or JNK loss of function on the levels of RACK1 protein, JNK activity, cell proliferation and apoptosis induced by tumor necrosis factor-related apoptosis inducing ligand in HCC SMMC-7721 cells. It was found that JNK loss of function exhibited no effect on RACK1 expression, whereas a loss of RACK1 function led to reduced JNK activity in SMMC-7721 cells. RACK1 and JNK loss of function resulted in the impaired oncogenic growth of SMMC-7721 cells. The present data further support a pivotal role of RACK1 in mediating enhanced JNK activity in HCC cells and also indicate that a novel mechanism exists for RACK1 overexpression in HCC SMMC-7721 cells.

Hepatoma-derived growth factor/nucleolin axis as a novel oncogenic pathway in liver carcinogenesis.

Hepatoma-derived growth factor (HDGF) overexpression is involved in liver fibrosis and carcinogenesis. However, the receptor(s) and signaling for HDGF remain unclear. By using affinity chromatography and proteomic techniques, nucleolin (NCL) was identified and validated as a HDGF-interacting membrane protein in hepatoma cells. Exogenous HDGF elicited the membrane NCL accumulation within 0.5 hour by protein stabilization and transcriptional NCL upregulation within 24 hours. Blockade of surface NCL by antibodies neutralization potently suppressed HDGF uptake and HDGF-stimulated phosphatidylinositol 3-kinase (PI3K)/Akt signaling in hepatoma cells. By using rescectd hepatocellular carcinoma (HCC) tissues, immunohistochemical analysis revealed NCL overexpression was correlated with tumour grades, vascular invasion, serum alpha-fetoprotein levels and the poor survival in HCC patients. Multivariate analysis showed NCL was an independent prognostic factor for survival outcome of HCC patients after surgery. To delineate the role of NCL in liver carcinogenesis, ectopic NCL overexpression promoted the oncogenic behaviours and induced PI3K/Akt activation in hepatoma cells. Conversely, NCL knockdown by RNA interference attenuated the oncogenic behaviours and PI3K/Akt signaling, which could be partially rescued by exogenous HDGF supply. In summary, this study provides the first evidence that surface NCL transmits the oncogenic signaling of HDGF and facilitates a novel diagnostic and therapeutic target for HCC.

The cytotoxic and mechanistic effects of aaptamine on hepatocellular carcinoma.

Hepatocellular carcinoma (HCC) is the fifth most common form of cancer and the third most frequent cause of cancer-associated mortality worldwide. We isolated aaptamine from the marine sponge Aaptos, and synthesized derivatives of this compound. Aaptamine and synthetic derivatives displayed various biological activities. This represents the first account of studies on the effects of aaptamine and its derivatives in hepatocarcinogenesis. In this study, Cell Counting Kit (CCK8) was used to evaluate the anti-proliferative effect of aaptamine on HCC in vitro. Additionally, a subcutaneous xenograft model was used to determine if aaptamine could inhibit hepatocellular carcinoma in vivo. We also used RT-PCR and Western blot to analyze the mechanisms behind these effects. Our results showed that aaptamine has anti-proliferation effects on the cell lines LM3 and HepG2. Aaptamine also suppressed the colony-formation ability of HCC cells. We found that aaptamine treatment led to cell cycle arrest in HCC cells, reduced the expression of SOX9 and CDK2. Significant anti-tumor effects were observed in aaptamine-administered tumor-bearing mice as compared to controls. However, structural changes made to aaptamine yielded two derivatives for which all the effects listed above were considerably reduced as compared to the original compound aaptamine. In conclusion, aaptamine is demonstrated for the first time to inhibit liver cancer progression. The aaptamine-induced cell cycle arrest was associated with the increased binding of p21 to Cdk2-cyclin D/E complexes, inhibition of Cdk2 kinase activity in HCC cells. Furthermore, aaptamine appears to be a promising and efficient treatment of liver cancer HCC-LM3 in vivo. We have also uncovered structural changes that might affect the biological activity. The work provides a promising drug candidate for HCC treatment.

4,5-Diaryl imidazoles with hydroxamic acid appendages as anti-hepatoma agents.

Hepatocellular carcinoma (HCC) is the most abundant tumour of the liver with rising patient numbers in the Western world countries. Despite newly approved drugs like protein kinase inhibitors the survival rate is still poor. In order to identify potential new drugs for the treatment of HCC we investigated the real-time cell viability, apoptosis induction (sub-G1 cells), and HDAC (histone deacetylase) activity of two hepatocellular cancer cell lines HepG2 and Hep3B treated with new imidazole-tethered hydroxamates. The tested cinnamyl hydroxamates exhibited significant antiproliferative and cytotoxic activity in HCC cells as apparent from high sub-G1 cell levels in flow cytometric cell cycle analyses. In Hep3B cells HDAC inhibition was observed comparable in magnitude to that induced by the clinically applied HDAC inhibitor SAHA (Zolinza, Vorinostat). The new imidazolyl hydroxamic acids lend themselves as a possible alternative to SAHA in the therapy of HCC. Even more so since similar 4,5-diarylimidazoles lacking only the hydroxamate functionality were previously shown in animal studies to be well tolerated and orally applicable.

Autophagy inhibition sensitizes hepatocellular carcinoma to the multikinase inhibitor linifanib.

Autophagy is a critical survival pathway for cancer cells under conditions of stress. Thus, induction of autophagy has emerged as a drug resistance mechanism. This study is to determine whether autophagy is activated by a novel multikinase inhibitor linifanib, thereby impairing the sensitivity of hepatocellular carcinoma (HCC) cells to this targeted therapy. Here, we found that linifanib induced a high level of autophagy in HCC cells, which was accompanied by suppression of phosphorylation of PDGFR-β and its downstream Akt/mTOR and Mek/Erk signaling pathways. Cell death induced by linifanib was greatly enhanced after autophagy inhibition by the pharmacological inhibitors or siRNAs against autophagy related genes, ATG5 and ATG7, in vitro. Moreover, HCQ, an FDA-approved drug used to inhibit autophagy, could significantly augment the anti-HCC effect of linifanib in a mouse xenograft model. In conclusion, linifanib can induce cytoprotective autophagy by suppression of PDGFR-β activities in HCC cells. Thus, autophagy inhibition represents a promising approach to improve the efficacy of linifanib in the treatment of HCC patients.

Abstract 3352: miR-122 targets pyruvate kinase M2 and affects metabolism of hepatocellular carcinoma

Abstract In contrast to normal differentiated cells that depend on mitochondrial oxidative phosphorylation for energy production, cancer cells have evolved to utilize aerobic glycolysis (Warburg's effect), with benefit of providing intermediates for biomass production. MicroRNA-122 (miR-122) is highly expressed in normal liver tissue regulating a wide variety of biological processes including cellular metabolism, but is reduced in hepatocellular carcinoma (HCC). Overexpression of miR-122 was shown to inhibit cancer cell proliferation, metastasis, and increase chemosensitivity, but its functions in cancer metabolism remains unknown. The present study aims to identify the miR-122 targeted genes and to investigate the associated regulatory mechanisms in HCC metabolism. We found the ectopic overexpression of miR-122 affected metabolic activities of HCC cells, evidenced by the reduced lactate production and increased oxygen consumption. Integrated gene expression analysis in a cohort of 94 HCC tissues revealed miR-122 level tightly associated with a battery of glycolytic genes, in which pyruvate kinase (PK) gene showed the strongest anti-correlation coefficient (Pearson r = -0.6938, p = &amp;lt;0.0001). In addition, reduced PK level was significantly associated with poor clinical outcomes of HCC patients. We found isoform M2 (PKM2) is the dominant form highly expressed in HCC and is a direct target of miR-122, as overexpression of miR-122 reduced both the mRNA and protein levels of PKM2, whereas PKM2 re-expression abrogated the miR-122-mediated glycolytic activities. The present study demonstrated the regulatory role of miR-122 on PKM2 in HCC, having an implication of therapeutic intervention targeting cancer metabolic pathways. Citation Format: Zhi Xu, Angela Liu, Nikki Lee, Jinfei Chen, John M. Luk. miR-122 targets pyruvate kinase M2 and affects metabolism of hepatocellular carcinoma. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3352. doi:10.1158/1538-7445.AM2014-3352

Specific and efficient gene delivery mediated by an asialofetuin-associated nanosystem

Gene therapy is considered a promising approach for the treatment of hepatocellular carcinoma (HCC). In this regard, the main goal of this work was to develop a specific and efficient gene delivery nanosystem to HCC based on 1-palmitoyl-2-oleoyl-sn-glycero-3-ethylphosphocholine:cholesterol cationic liposomes and asialofetuin (ASF), a specific ligand to the asialoglycoprotein receptor (ASGP-R) that is overexpressed in HCC. Our results show that association of ASF to lipoplexes promotes a substantial increase in their biological activity in HCC cells, not only in vitro, but also in an animal model. The transfection activity obtained with this novel nanosystem (ASF-lipoplexes) was much higher than that observed with a highly efficient commercial formulation. On the other hand, the presence of high concentrations of galactose substantially reduced the cell uptake and biological activity of the ASF-lipoplexes. These results, together with those obtained in the presence of inhibitors of endocytosis, show that the potentiation induced by the association of ASF to lipoplexes is due to its specific interaction with the ASGP-R. The physicochemical properties of the generated nanosystem also reinforce this observation. Overall, our results demonstrate for the first time that the novel ASF-lipoplexes present a noticeable ability to specifically and efficiently deliver genetic material into HCC cells.

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

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

Synthesis and anti-tumor activity evaluation of Matijin-Su derivatives

A series of Matijin-Su (MTS, N-(N-benzoyl-l-phenylalanyl)-O-acetyl-l-phenylalanol) derivatives was synthesized and evaluated for their anti-tumor activities in hepatocellular carcinoma cells. The IC50 of compounds 1, 3, 4, 11, 13 were less than 20μM, and compound 1 and 3 showed an IC50 value of less than 9μM. Expansion inhibition could be found significantly in compound 1 and 3-treated human hepatoma cell HepG2 and PLC/PRF/5, while both compounds exhibit lower toxicity to human hepatocyte cell line L-02. Compound 1 and 3 could induce cell cycle arrest at G1/S phase. This may be attributed to increase level of intracellular reactive oxygen species (ROS). Up-regulation of p38 MAPK activity in responding the ROS stabilize p53 and activate p21 transcription, the critical regulatory in G1/S checkpoint. Observations in this study shed light on the potential of MTS derivatives compound 1 and 3 as novel suppressors to human liver cancer.

Phosphorothioate-modified CpG oligodeoxynucleotide (CpG ODN) induces apoptosis of human hepatocellular carcinoma cells independent of TLR9.

Toll-like receptors (TLRs) expressed on cancer cells are closely associated with tumor development. In this study, we investigated the biological functions of the TLR9 ligand, CpG oligodeoxynucleotide (CpG ODN), on TLR9 expressed in the cytoplasm of hepatocellular carcinoma (HCC) cells. In vitro, human HCC cell lines were transfected with phosphorothioate-modified oligodeoxynucleotides TLR9 agonist OND M362 and its negative control ODN M362 ctrl, which inhibited the proliferation of HCC cells by inducing apoptosis without altering the cell cycle. Interestingly, ODN M362 and ODN M362 Ctrl displayed a similar proapoptotic effect on HCC, possibly related to phosphorothioate modification of the structure of CpG ODN. Although both of them resulted in the upregulation of the TLR9 receptor, their effect on HCC apoptosis was independent of TLR9. They also upregulated inflammatory cytokines, but did not activate the NF-κB signaling pathway. Finally, the activities of ODN M362 and ODN M362 Ctrl were demonstrated in nude mice inoculated with HCC cells. These findings suggest that the phosphorothioate-modified TLR9 agonist ODN M362, and its control, elicit antitumor activity in HCC cells and may serve as a novel therapeutic target for HCC therapy.

Inhibiting the oncogenic mir-221 by microRNA sponge: toward microRNA-based therapeutics for hepatocellular carcinoma.

We evaluated the capability of "microRNA sponges" in sequestering and inhibiting the over-expressed miR-221 in HCC cell lines. Advanced hepatocellular carcinoma (HCC) is a serious public health problem, with no effective cure at present. It has been demonstrated that the deregulation of microRNAs expression contributes to tumorigenesis. In HCC, miR-221 was shown to be up-regulated in more than 70% of the cases and was associated with higher tumor stage, metastasis and a shorter time to recurrence after surgery, suggesting an important pathogenic role. A tumor promoting function of miR-221 was proved in a transgenic mouse model, which was predisposed to the development of liver cancers. These findings suggested that miR-221 could represent a potential target for anti-tumor approaches. Novel adeno and adeno-associated viral vectors (AAVs) were developed: they were genetically modified to drive the expression of multiple binding sites for miR-221, the "miR-221 sponge", which was designed to sequester miR-221 cellular molecules. Analysis of viral vectors activity in HCC cells revealed their capability to reduce miR-221 endogenous levels, which was accompanied by the increase in CDKN1B/ p27 protein, a known target of miR-221. An increase in apoptosis was also measured in Hep3B cells after infection with any of the two viral vectors in comparison with control vectors, with stronger effects induced by adenovirus compared to AAV vectors. The depletion of oncogenic microRNAs represents a potential anti-cancer approach that needs to be tested for safety and efficacy. Here, we describe the development of novel "miR-221 sponge" vectors, which can reduce miR-221 activity in vitro and may be used for in vivo delivery.