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Small interfering RNAs (siRNA) therapeutics have developed rapidly in recent years, despite the challenges associated with delivery of large, highly charged nucleic acids. Delivery of siRNA therapeutics to the liver has been established, with conjugation of siRNA to N-acetylgalactosamine (GalNAc) providing durable gene knockdown in hepatocytes following subcutaneous injection. GalNAc binds the asialoglycoprotein receptor (ASGPR) that is highly expressed on hepatocytes and exploits this scavenger receptor to deliver siRNA across the plasma membrane by endocytosis. However, siRNA needs to access the RNA-induced silencing complex (RISC) in the cytoplasm to provide effective gene knockdown and the entire siRNA delivery process is very inefficient, likely due to steps required for endosomal escape, intracellular trafficking, and stability of siRNA. To reveal the cellular factors limiting delivery of siRNA therapeutics, we performed a pooled, genome wide knockout screen based on delivery of GalNAc conjugated siRNA targeting the HPRT1 gene in the human hepatocellular carcinoma line Hep3B. Our primary pooled genome wide knockout screen identified candidate genes that when knocked out significantly enhanced siRNA efficacy in Hep3B cells. Follow-up studies indicate that knockout of one gene in particular, RAB18, improved siRNA efficacy.

The edible leaves of the mulberry (Morus alba L.) plant are used worldwide. They contain abundant polyphenolic compounds with strong anticancer properties. We previously revealed that apoptosis was mediated in p53-negative Hep3B cells, and mulberry leaf polyphenol extract (MLPE) induced autophagy in p53-transfected Hep3B cells. However, how this autophagy is induced by p53 in human hepatoma HepG2 (p53 wild type) cells remains unclear. In the current study, MLPE induced autophagy, as demonstrated by enhanced acidic vesicular organelle staining, by upregulating beclin-1, increasing LC3-II conversion, and phosphorylating AMPK. In HepG2 cells, these processes were associated with p53. Western blot also revealed phosphatidylinositol-3 kinase (PI3K), p-AKT, and fatty acid synthase (FASN) suppression in MLPE-treated cells. Moreover, treatment with the p53 inhibitor pifithrin-α (PFT-α) inhibited autophagy and increased apoptotic response in MLPE-treated HepG2 cells. PFT-α treatment also reversed MLPE-induced PI3K, p-AKT, and FASN suppression. Thus, co-treatment with MLPE and PFT-α significantly increased caspase-3, caspase-8, and cytochrome c release, indicating that p53 deficiency caused the apoptosis. In addition, rutin, a bioactive polyphenol in MLPE, may affect autophagy in HepG2 cells. This study demonstrates that MLPE is a potential anticancer agent targeting autophagy and apoptosis in cells with p53 status. Moreover, this work provides insight into the mechanism of p53 action in MLPE-induced cytotoxicity in hepatocellular carcinoma.

Abstract Background and aim: Lysyl oxidase-like 2 (LOXL2) plays a role in tumor microenvironment formation and metastasis of hepatocellular carcinoma (HCC), which has a high mortality burden. Liver cancer stem cells (LCSCs) are related with the major malignant phenotypes of HCC. The function of LOXL2 in regulation of LCSCs remains unknown.Methods: CD133+HepG2 and CD133+Hep3B cells were sorted by fluorescence-activated cell sorting (FACS) from two human hepatoblastoma cell lines. Spheroid formation, apoptosis, cell cycle, as well as transwell assays were performed upon LOXL2 knock down in CD133+HepG2 and CD133+Hep3B cells. Protein and mRNA levels were quantified by Western blotting, Immunofluorescence and real-time PCR. Results: Knockdown of LOXL2 decreased spheroid formation, migration and invasion (p < 0.05), also induced apoptosis (p < 0.05) and cell cycle arrest (p < 0.05) in CD133+HepG2 and CD133+Hep3B cells. Knockdown of LOXL2 effectively inhibited expression of the anti-apoptosis proteins baculoviral IAP repeat-containing 3 (BIRC3) and murine double minute 2 (MDM2) (p < 0.01), as well as autophagy marker microtubule-associated protein 1 light chain 3 B (LC3) and autophagy gene ATG5 in CD133+HepG2 and CD133+Hep3B cells (p < 0.01). Conclusions: The results revealed that LOXL2 inhibition could reduce the proliferation and expansion of LCSCs, making LOXL2 inhibitors an attractive and novel therapeutic strategy of HCC.

BackgroundSmall ubiquitin-like modifier specific peptidase 2 (SENP2) suppresses the progression and chemoresistance of several cancers, while few studies report its role in hepatocellular carcinoma (HCC). This study aimed to evaluate the effect of SENP2 on stemness, sorafenib sensitivity, and downstream pathway in HCC, with validation of its molecular mechanisms by compensation experiment.MethodsSENP2 was regulated by plasmid transfection; meanwhile, in a compensation experiment, protein kinase B (AKT) was activated by SC79 treatment and β-catenin (CTNNB1) was overexpressed by plasmid transfection. After modification, sorafenib sensitivity was detected by cell counting kit-8 assay; stemness was evaluated by CD133+ cell proportion and sphere formation assay.ResultsSENP2 was decreased in HCC cell lines (including Hep3B, Li7, and Huh7) compared with normal human liver epithelial cell lines, which was further reduced in HCC stem cells than in normal HCC cells. Subsequently, SENP2 overexpression inhibited CD133+ cell proportion, decreased sphere formation ability, promoted sorafenib sensitivity, suppressed AKT and glycogen synthase kinase-3β (GSK3β) phosphorylation, and reduced CTNNB1 expression in Huh7 and Hep3B cells, while SENP2 knockdown showed the reverse effects. The following compensation experiment revealed that activating AKT or overexpressing CTNNB1 promoted CD133+ cell proportion and sphere formation ability but suppressed sorafenib sensitivity in Huh7 and Hep3B cells. Moreover, activating AKT or overexpressing CTNNB1 attenuated the effect of SENP2 overexpression on stemness and sorafenib sensitivity in Huh7 and Hep3B cells.ConclusionSENP2 suppresses HCC stemness and increases sorafenib sensitivity through inactivating the AKT/GSK3β/CTNNB1 signaling pathway.

Isoalantolactone (IALT) is one of the isomeric sesquiterpene lactones isolated from the roots of Inula helenium L. IALT is known to possess various biological and pharmacological activities, but its anti-cancer mechanisms are not well understood. The aim of the present study was to investigate the anti-proliferative effects of IALT in human hepatocellular carcinoma (HCC) cells and to evaluate the potential anti-cancer mechanisms. Our results demonstrated that IALT treatment concentration-dependently suppressed the cell survival of HCC Hep3B cells, which was associated with the induction of apoptosis. IALT increased the expression of death-receptor-related proteins, activated caspases, and induced Bid truncation, subsequently leading to cleavage of poly (ADP-ribose) polymerase. In addition, IALT contributed to the cytosolic release of cytochrome c by destroying mitochondrial integrity, following an increase in the Bax/Bcl-2 expression ratio. However, IALT-mediated growth inhibition and apoptosis were significantly attenuated in the presence of a pan-caspase inhibitor, suggesting that IALT induced caspase-dependent apoptosis in Hep3B cells. Moreover, IALT activated the mitogen-activated protein kinases signaling pathway, and the anti-cancer effect of IALT was significantly diminished in the presence of a potent c-Jun N-terminal kinase (JNK) inhibitor. IALT also improved the generation of intracellular reactive oxygen species (ROS), whereas the ROS inhibitor significantly abrogated IALT-induced growth reduction, apoptosis, and JNK activation. Furthermore, ROS-dependent apoptosis was revealed as a mechanism involved in the anti-cancer activity of IALT in a 3D multicellular tumor spheroid model of Hep3B cells. Taken together, our findings indicate that IALT exhibited anti-cancer activity in HCC Hep3B cells by inducing ROS-dependent activation of the JNK signaling pathway.

A new paeoniflorin Ag(I) complex was synthesized using the hydrothermal method, which was characterized by some spectral techniques like elemental analysis, thermal analysis, 1H nuclear magnetic resonance (1H NMR), Fourier-transform infrared spectroscopy (FTIR), electrospray ionization mass spectrometry (ESI-MS) and geometry optimization. Cell Counting Kit-8 (CCK-8) was used to test the in vitro cytotoxicity of the paeoniflorin Ag(I) complexes in various cells (such as A549, Caco2, HepG2, Hep3B, LO2, H9C2 and PC12 cells). It is shown that the paeoniflorin Ag(I) complexes possessed a remarkable cytotoxicity against the Hep3B cell line, but a low cytotoxicity against the LO2 cell line. Cell cycle progression and membrane potential of Hep3B cells were studied using flow cytometry, and the cell examination of apoptosis was made by Annexin V-FITC/PI staining. Moreover, expressions levels of caspase family proteins and Bcl-2 were also investigated. Our findings demonstrated that the paeoniflorin Ag(I) complexes induced apoptosis of Hep3B cells via the mitochondrial membrane potential access by regulating Bcl-2 and caspase family proteins.

Abstract Liver cancer is the growth and spread of cells in the liver that are abnormal. In this research, we examined Curcumin's effects on the growth of Hep3B cells liver in Molecular and Biology Research Laboratory at An-Najah National University. Curcumin regarding several theoretical and practical studies starves cancer cells to death. The life cycle of Hep3B cell has been analyzed by MTS assay, which is used to assess cell proliferation, cell viability, cytotoxicity, and flow cytometry. Our results agreed that Curcumin could a potentially efficient medication in the treatment of hepatocellular carcinoma, according to the findings of this study which confirms that curcumin treatment inhibited the growth of Hep3B. Thus, taking curcumin in our food is recommended.

Abstract Background Hepatocellular carcinoma (HCC) represents a serious public health problem worldwide and has high morbidity and mortality. Dihydromyricetin (DHM) exhibits anticancer effect on a variety of malignancies, but its anticancer function of DHM in HCC has been unclear. The aim of this study was designed to investigate the anticancer effect of DHM on cell apoptosis, proliferation, migration and invasion of hepatoma carcinoma cells. Methods Cultured Hep3B cells were treated with different DHM concentrations, followed by cell apoptosis, proliferation, migration and invasion were examined by CCK-8, colony formation assay, wound healing, Transwell and flow cytometry, respectively. The mRNA and protein expression of BCL-2, Cleaved-caspase 3, Cleaved-caspase 9, BAK, BAX and BAD were validated by western blot. Results DHM markedly suppressed proliferation, migration, invasion and facilitated apoptosis in Hep3B cells. Mechanistically, DHM significantly downregulated the Bcl-2 expression, and upregulated the mRNA and protein levels of Cleaved-Caspase 3, Cleaved- Caspase 9, Bak, Bax and Bad. Furthermore, in the nude mice tumorigenic model, DHM treatment greatly decreased the weight of the HCC cancers compared to the weights in control and NDP group. Conclusions DHM could suppress cell proliferation, migration, invasion, and facilitated apoptosis in Hep3B cells. These findings could provide novel insights to develop potential therapeutic strategy for the clinical treatment of HCC.

AbstractA subset of hepatocellular carcinoma (HCC) overexpresses the chromosome 19 miRNA cluster (C19MC) and is associated with an undifferentiated phenotype marked by overexpression of cancer testis antigens (CTAs) including anti-apoptotic melanoma-A antigens (MAGEAs). However, the regulation of C19MC miRNA and MAGEA expression in HCCs are not understood. Here we show that, C19MC overexpression is tightly linked to a sub-set of HCCs with transcription-incompetent p53. Using next-generation and Sanger sequencing we found that, p53 in Hep3B cells is impaired by TP53-FXR2 fusion, and that overexpression of the C19MC miRNA-520G in Hep3B cells promotes the expression of MAGEA-3, 6 and 12 mRNAs. Furthermore, overexpression of p53-R175H and p53-R273H mutants promote miR-520G and MAGEA RNA expression and cellular transformation. Moreover, IFN-γ co-operates with miR-520G to promote MAGEA expression. On the other hand, metals such as nickel and zinc promote miR-526B but not miR-520G, to result in the suppression of MAGEA mRNA expression, and evoke cell death through mitochondrial membrane depolarization. Therefore our study demonstrates that a MAGEA-promoting network involving miR-520G, p53-defects and IFN-γ that govern cellular transformation and cell survival pathways, but MAGEA expression and survival are counteracted by nickel and zinc combination.