Hepatocellular Carcinoma Hep3B Cells Research Articles

Scutellarein Induces Fas-Mediated Extrinsic Apoptosis and G2/M Cell Cycle Arrest in Hep3B Hepatocellular Carcinoma Cells

Scutellarein (SCU), a flavone found in the perennial herb Scutellaria baicalensis, is known for a wide range of biological activities. In the present study, we investigated the effects of treatment with SCU flavonoids on inducing apoptosis via the extrinsic pathway in Hep3B cells. SCU treatment significantly inhibited Hep3B cell proliferation and induced G2/M phase cell cycle arrest by inhibiting the expression level of the proteins Cdc25C, cdk1 and Cyclin B1. Allophycocyanin (APC)/Annexin V and propidium iodide (PI) double-staining showed upregulation of apoptotic cell death fraction. We further confirmed apoptosis by 4′-6-diamidino-2-phenylindole (DAPI) fluorescent staining and observed DNA fragmentation with agarose gel electrophoresis. Further, immunoblotting results showed that treatment with SCU showed no changes in Bax and Bcl-xL protein levels. In addition, SCU treatment did not affect the mitochondrial membrane potential (MMP) in Hep3B cells. On the contrary, treatment with SCU increased the expression of Fas and Fas ligand (FasL), which activated cleaved caspase-8, caspase-3, and polymeric adenosine diphosphate ribose (PARP), whereas the expression level of death receptor 4 (DR4) decreased. We confirmed that the proteins expressed upon treatment with SCU were involved in the Fas-mediated pathway of apoptosis in Hep3B cells. Thus, our findings in the current study strongly imply that SCU can be a basic natural source for developing potent anti-cancer agents for hepatocellular carcinoma (HCC) treatment.

Orlistat Displays Antitumor Activity and Enhances the Efficacy of Paclitaxel in Human Hepatoma Hep3B Cells.

Orlistat has been proved to be an effective fatty acid synthase inhibitor that is able to inhibit the proliferation and induce apoptosis in many cancer cell types. However, the anticancer effects of orlistat on hepatocellular carcinoma are undefined. We found that orlistat inhibited cell growth and induced G0/G1 cell cycle arrest with increased cyclin D, cyclin E, and p21 expression in human hepatoma Hep3B cells. Furthermore, protein expression of cyclin A, cyclin B, Cdk1, Cdk2, and Cdk4 was reduced by orlistat. This study investigated the role of lipid metabolism on orlistat-induced human hepatoma Hep3B cell death. The decrease in the expression of key enzymes in fatty acid metabolism, including FASN, ACOT8, PPT1, FABP1, CPT1 and CPT2, was observed after orlistat treatment. We also demonstrated that peroxisomal activity was involved in the orlistat-induced Hep3B cell death. In this study, we established an in vitro model to investigate the effect of orlistat on lipid accumulation. We found that orlistat significantly inhibited the cellular lipid content when administered in fatty acid overload conditions in Hep3B cells. Combination treatment of orlistat and paclitaxel was able to induce a synergistic effect on growth inhibition and cell apoptosis in Hep3B cells. Our data suggested that orlistat displays antitumor activity and enhances the efficacy of paclitaxel in Hep3B cells.

Epigenetic modulation enhances immunotherapy for hepatocellular carcinoma

BackgroundAnti-PDL-1 immunotherapy for Hepatocellular Carcinoma (HCC) demonstrated a mixed response. Polycomb Repressor Complex 2(PRC2) contributes to the initiation and progression of HCC by suppressing tumor antigens and inhibiting an immune response. Two components of epigenetic modulation are Enhancer of Zeste Homolog 2 (EZH2, the catalytic component of PRC2) and DNA Methyltransferase 1 (DNMT1). We aim to investigate the potential role of epigenetic therapy targeting EZH2 and DNMT1 as a novel strategy to modulate immunotherapy response in HCC. MethodsHepG2, Hep3B, and Hepa1-6 HCC cell lines were treated with EZH2 inhibitor (DZNep) and DNMT1 inhibitor (5-Azacytidine) with and without anti-PDL-1. Quantitative RT-PCR and immunohistochemistry were performed to evaluate the expression of tumor suppressors, tumor antigens, and Th1 chemokines. In-vivo C57/LJ immunocompetent mice model with subcutaneous tumor inoculation was performed with intraperitoneal drug injections. ResultsThere was a significant upregulation of Th1 chemokines in HepG2 (CXCL9 5.5 ± 0.2 relative fold change; CXCL10 1.44 × 103 ± 37 relative fold change) and Hep3B (CXCL 9 6.85 × 103 ± 1.3 × 103 relative fold change; CXCL 10 2.15 × 103 ± 3.1 × 102 relative fold change). Additionally, there was a significant induction of cancer testis antigens NY-ESO-1 (3.6–3.7 ± 0.3 relative fold change) and LAGE (8.3–11.7 ± 1.9 relative fold change). In vivo model demonstrated statistically significant tumor regression in the combination treatment group (0.02 g ± 0.02) compared to epigenetic therapy (0.63 g ± 0.61) or immunotherapy alone (0.15 g ± 0.21) with untreated control (2.4 g ± 0.71). There was significantly increased trafficking of cytotoxic T- lymphocytes and associated apoptosis for the combination treatment group compared to epigenetic or immunotherapy alone. ConclusionsThis study demonstrates that epigenetic modulation could be a novel potential strategy to augment immunotherapy for HCC by stimulating T cell trafficking into tumor microenvironment via activation of transcriptionally repressed chemokine genes responsible for T-cell trafficking, inducing previously silent neoantigens for immune targets, and allowing tumor regression as a result. A clinical trial of this feasible combination therapy of these clinically available agents is warranted.

NKAP plays an oncogenic function partly through AKT signaling pathway in hepatocellular carcinoma.

NF-kB activating protein (NKAP) is a highly conserved protein involved in transcriptional repression, immune cell development, maturation, T cell acquisition of functional competency and maintenance of hematopoiesis. Here we first explore the function of NKAP in hepatocellular carcinoma (HCC). We found that NKAP was highly expressed in HCC tissues and associated with a poor patient survival. CCK8 assay showed that NKAP knockdown significantly decreased cell viability of HuH7 and Hep3B HCC cell lines. Cell invasion, tested by transwell assays, was significantly inhibited by NKAP knockdown in HuH7 and Hep3B cells (P<0.05). Percentage of cell apoptosis was significantly increased by NKAP knockdown in HuH7 cells (6.5% to 12.5%) and in Hep3B cells (8.3% to 27.3%). Furthermore, western blot results indicated that NKAP silence upregulated the expression of pro-apoptotic proteins Bax and Caspase3-P17 while downregulated anti-apoptotic protein Bcl2. Finaly, AKT signaling pathway was evaluated to reveal the underlying mechanism of NKAP in HCC cells. It was suggested that NKAP knockdown decreased the phosphorylation level of AKT and the expression of its downstream members p70S6K and Cyclin D1. Furthermore, we demonstrated that NKAP knockdown also played an oncogenic role in human gastric cancer AGS and MKN45 cells. In conclusion, for the first time our study reveals that NKAP promotes the proliferation and invasion in HCC cell lines at least partly through AKT signaling pathway.

Isorhamnetin induces ROS-dependent cycle arrest at G2/M phase and apoptosis in human hepatocarcinoma Hep3B cells.

Isorhamnetin is a 3'-O-methylated metabolite of quercetin that is found predominantly in a variety of medicinal plants. Although many previous studies have reported that this flavonol has diverse health-promoting effects, evidence for the underlying molecular mechanism of anti-cancer efficacy is still lacking. In this study, it was examined the anti-proliferative effect of isorhamnetin on human hepatocarcinoma Hep3B cells, and found that isorhamnetin induced cell cycle arrest at G2/M phase and apoptosis. Isorhamnetin-induced G2/M arrest was associated with decreased expression of proliferating cell nuclear antigen as well as cyclin A and cyclin B1. However, isorhamnetin increased expression of p21WAF1/CIP1, a cyclin-dependent kinase (Cdk) inhibitor, and increased p21 complexed with Cdk2 and Cdc2. In addition, Isorhamnetin-induced apoptosis was associated with increased expression of Fas/Fas ligand, reduced ratio of Bcl-2/Bax expression, truncation of Bid, cytosolic release of cytochrome c, and activation of caspase-8, -9 and -3. Isorhamnetin also enhanced intracellular levels of reactive oxygen species (ROS), while the addition of N-acetyl cysteine (NAC), a ROS inhibitor, significantly diminished isorhamnetin-induced mitochondrial dysfunction. Furthermore, the interruption of ROS generation using NAC significantly attenuated isorhamnetin-mediated G2/M arrest and apoptosis. Collectively, this is the first report to show that isorhamnetin inhibited the proliferation of human hepatocarcinoma cells by ROS-dependent arrest of the cell cycle at the G2/M phase and induction of apoptosis.

Enantiomeric β-carboline dimers from Picrasma quassioides and their anti-hepatoma potential

Four pairs of enantiomeric β-carboline alkaloids, (+/−)-kumudine A-D, along with their biosynthesis-related compound kumudine E, were obtained from the stems of Picrasma quassioides. Their structures, including the absolute configurations, were determined via extensive spectroscopic data combined with electronic circular dichroism (ECD) spectroscopic analyses and quantum mechanical ECD calculations. (+/−)-Kumudine A possessed a scaffold of β-carboline-phenylpropanoid adduct, which were the first examples of this type of β-carboline alkaloid from nature. The cytotoxicity assay against hepatocellular carcinoma Hep3B and HepG2 cells was evaluated by SRB assay, which showed that (−)-Kumudine B had stronger effect than its enantiomer (+)-Kumudine B in Hep3B cells. Moreover, further flow cytometry analysis also supported the enantioselectivity between (+)-Kumudine B and (−)-Kumudine B, suggesting that the compounds caused death of hepatoma cells through apoptosis induction.

Forcing ATGL expression in hepatocarcinoma cells imposes glycolytic rewiring through PPAR-\u03b1/p300-mediated acetylation of p53

Metabolic reprogramming is a typical feature of cancer cells aimed at sustaining high-energetic demand and proliferation rate. Here, we report clear-cut evidence for decreased expression of the adipose triglyceride lipase (ATGL), the first and rate-limiting enzyme of triglyceride hydrolysis, in both human and mouse-induced hepatocellular carcinoma (HCC). We identified metabolic rewiring as major outcome of ATGL overexpression in HCC-derived cell lines. Indeed, ATGL slackened both glucose uptake/utilization and cell proliferation in parallel with increased oxidative metabolism of fatty acids and enhanced mitochondria capacity. We ascribed these ATGL—downstream events to the activity of the tumor-suppressor p53, whose protein levels—but not transcript—were upregulated upon ATGL overexpression. The role of p53 was further assessed by abrogation of the ATGL-mediated effects upon p53 silencing or in p53-null hepatocarcinoma Hep3B cells. Furthermore, we provided insights on the molecular mechanisms governed by ATGL in HCC cells, identifying a new PPAR-α/p300 axis responsible for p53 acetylation/accumulation. Finally, we highlighted that ATGL levels confer different susceptibility of HCC cells to common therapeutic drugs, with ATGL overexpressing cells being more resistant to glycolysis inhibitors (e.g., 2-deoxyglucose and 3-bromopyruvate), compared to genotoxic compounds. Collectively, our data provide evidence for a previously uncovered tumor-suppressor function of ATGL in HCC, with the outlined molecular mechanisms shedding light on new potential targets for anticancer therapy.

14-3-3ζ binds to hepatitis B virus protein X and maintains its protein stability in hepatocellular carcinoma cells.

14‐3‐3ζ, a phosphopeptide‐binding molecule, is reportedly overexpressed in the cancerous tissues of patients with hepatocellular carcinoma (HCC). Hepatitis B virus (HBV) protein X (HBx) draws intensive attention in HBV‐related HCC because it not only regulates HBV replication, but also promotes carcinogenesis by interacting with various tumor or antitumor molecules. This study is performed to investigate whether and how 14‐3‐3ζ interacts with HBx. The coimmunoprecipitation (Co‐IP) results showed that 14‐3‐3ζ bond to HBx in HBV‐infected Hep3B HCC cells and CSQT‐2 portal vein tumor thrombosis (PVTT) cells. By performing Co‐IP assay in HBV‐free Huh7 cells expressing wild‐type HBx, mutant HBx‐S31A, or HBx‐S31D (serine31 was mutated into alanine31 or aspartic acid31), we found that the phosphorylated serine31 with its near amino acid residues constituted a RPLphosphoS31 GP (R, arginine; P, proline; L, leucine; S, serine; G, glycine) motif in HBx for 14‐3‐3ζ docking. This 14‐3‐3ζ‐HBx interaction was partly impaired when Akt signaling transduction was blocked by LY294002. Furthermore, 14‐3‐3ζ silencing augmented HBx ubiquitination and decreased its expression in cancer cells and xenograft tumor. The migratory and invasive abilities of CSQT‐2 cells were inhibited upon 14‐3‐3ζ silencing, whereas partly restored by HBx overexpression. Additionally, 14‐3‐3ζ positively correlated with HBx to be overexpressed in the primary HCC tissues (r = 0.344) and metastatic PVTT (r = 0.348). In summary, findings of this study reveal a novel 14‐3‐3ζ‐HBx interaction in HCC cells and suggest 14‐3‐3ζ as a candidate target for treating HBV‐related HCC.

Dual-vector prodrug activator gene therapy using retroviral replicating vectors

Retroviral replicating vectors (RRVs) have been shown to achieve efficient tumor transduction and enhanced therapeutic benefits in a variety of cancer models. In the present study, we evaluated a possible combinatorial effect of prodrug activator genes delivered by two different RRVs derived from amphotropic murine leukemia virus (AMLV) and gibbon ape leukemia virus (GALV) on human hepatocellular carcinoma Hep3B cells. Both RRVs showed efficient replicative spread in culture and can overcame superinfection resistance each other. Notably, the replication and spread of each RRV in culture remained unaffected by pretransduction with the counterpart RRV. We further transduced cells with RRVs which individually possessed the prodrug activator genes yeast cytosine deaminase (CD) and herpes simplex virus thymidine kinase (TK) alone or in combination, and evaluated the cytotoxic effects of RRV-mediated gene therapy with CD and TK in the presence of the respective prodrugs, 5-fluorocytosine and ganciclovir. All combinations of the two prodrug activator genes produced synergistic cytocidal effects, but the combined effects of the different genes were significantly greater than those of the same genes when delivered by two different vectors. The present findings indicate the potential utility of dual-vector gene therapy using two different RRVs carrying different prodrug activator genes.

LncRNA DGCR5 represses the development of hepatocellular carcinoma by targeting the miR-346/KLF14 axis.

Long non-coding RNAs (lncRNAs) are a class of regulatory noncoding RNAs. Emerging evidence highlights the critical roles of lncRNAs in the progression of hepatocellular carcinoma (HCC). Although many lncRNAs have been identified in the development of HCC, the association between DiGeorge syndrome critical region gene 5 (DGCR5) and HCC remains unclear. In the current study, we focused on the biological role of DGCR5 in HCC. We observed that DGCR5 was decreased in HCC cells, including SMCC7721, Hep3B, HepG2, MHCC-97L, MHCC-97H, and SNU449 hepatocellular carcinoma cells, compared with the normal human liver cell line THLE-3 normal human liver cells. In addition, DGCR5 overexpression could repress HCC cell growth, migration, and invasion considerably. Increasing studies have indicated the interactions between lncRNAs and microRNAs. MicroRNAs are endogenous small noncoding RNAs and they can play important roles in tumorigenesis. MicroRNA 346 (miR-346) has been demonstrated in various human cancer types, including HCC. MiR-346 was found to be increased in HCC cells and DGCR5 can act as a sponge of miR-346 to modulate the progression of HCC. The binding correlation between DGCR5 and miR-346 was validated in our research. Subsequently, Krüppel-like factor 14 (KLF14) was predicted as a downstream target of miR-346 and miR-346 can induce the development of HCC by inhibiting KLF14. Finally, we proved that DGCR5 can rescue the inhibited levels of KLF14 repressed by miR-346 mimics in MHCC-97H and Hep3B cells. Taken together, it was indicated in our study that DGCR5 can restrain the progression of HCC through sponging miR-346 and modulating KLF14 in vitro.

Rab23 Promotes Hepatocellular Carcinoma Cell Migration Via Rac1/TGF-β Signaling.

Rab23 is a member of Ras-related small GTPase family, which plays a critical role in the progression of wide range of tumors. However, its biological function in hepatocellular carcinoma still remains unclear. Here, we investigated the effects of Rab23 on proliferation and migration in hepatocellular carcinoma cell and its potential mechanisms. We found over-expression of Rab23 promoted the Hep3B hepatocellular carcinoma cell migration, which could be reversed by Rab23 silencing. Rab23 induced Rac1 activation and followed progression of epithelial-mesenchymal transition (EMT) along with upregulation of N-cadherin, snail as well as vimentin and downregulation of E-cadherin via upregulating Transforming Growth Factor-β (TGF-β). Silencing Rac1 significantly attenuated Rab23-induced HepG2 migration and TGF-β. Moreover, knockdown of TGF-β effectively attenuated Rab23-induced EMT. Taken together, we demonstrated a mechanistic cascade of Rab23 enhangcing Rac1 activation and subsequent TGF-β expression, leading to hepatocellular carcinoma cell migration.

Stereoisomeric guaiacylglycerol-β-coniferyl aldehyde ether induces distinctive apoptosis by downregulation of MEK/ERK pathway in hepatocellular carcinoma cells

Two 8-O-4′-type neolignan epimers erythro-guaiacylglycerol-β-coniferyl aldehyde ether (1) and threo-guaiacylglycerol-β-coniferyl aldehyde ether (2) were isolated from the stems of Picrasma quassioides. Further chiral separation gave two pairs of enantiomers 1a/1b and 2a/2b. The cytotoxicity assay against hepatocellular carcinoma Hep3B and HepG2 cells was evaluated by MTT assay. The results showed that 1b (IC50 = 45.56 μM) and 2b (IC50 = 39.02 μM) had more cytotoxic effect than its enantiomers 1a (IC50 = 82.66 μM) and 2a (IC50 = 67.97 μM) in Hep3B cells, respectively. Moreover, 1b and 2b could induce more apoptotic cells as well as higher reactive oxygen species (ROS) generation than 1a and 2a at 50 μM. In addition, a further study on the phosphoinositide 3-kinase (PI3K)/AKT and mitogen-activated protein kinase kinase (MEK)/extracellular signal-regulated kinase (ERK) signaling pathways was investigated. The results revealed that all compounds had no significant effect on PI3K/AKT pathway, however, 1b and 2b attenuated the relative levels of p-MEK and p-ERK when compared with 1a and 2a. Taken together, the absolute configurations of guaiacylglycerol-β-coniferyl aldehyde ether had an impact on the inhibitory effect on Hep3B cells. The inactivation of MEK/ERK signaling pathway might contribute to apoptosis induction and ROS generation in 1b- and 2b-treated cells.

Daphnegiravone D from Daphne giraldii Nitsche induces p38-dependent apoptosis via oxidative and nitrosative stress in hepatocellular carcinoma cells

Daphnegiravone D (DGD), a prenylated flavonoid from Daphne giraldii Nitsche, significantly inhibited cell growth of several cancer cell lines without cytotoxicity on human normal cells. Our previous study showed that DGD could induce apoptosis in hepatocellular carcinoma Hep3B and HepG2 cells, but the detailed mechanism was still unclear. The present study provides that DGD-induced oxidative and nitrosative stress contribute to apoptotic cell death in Hep3B and HepG2 cells. Furthermore, there is a positive loop between oxidative stress and p38 activation, similar result is observed between nitrosative stress and p38. N-Acetylcysteine (NAC), a reactive oxygen species scavenger, could relieve DGD-induced oxidative stress, but exerts little effect on nitrosative stress. In addition, carboxy-PTIO (PTIO, a well-known scavenger of reactive nitrogen species) down-regulates the induction of nitrosative stress without obvious effect on oxidative stress in DGD-treated cells. In conclusion, the induction of oxidative and nitrosative stress could enhance p38-mediated apoptosis in DGD-treated Hep3B and HepG2 cells. Moreover, we speculated that OS and NS could not ultimately affect each other in DGD-treated HCC cells. This study gives a new insight on the mechanism of DGD-induced apoptotic cell death via oxidative and nitrosative stress in HCC cells.

Cytokine-mediated modulation of the hepatic miRNome: miR-146b-5p is an IL-6-inducible miRNA with multiple targets.

Interleukin-6 (IL-6)-type cytokines play important roles in liver (patho-)biology. For instance, they regulate the acute phase response to inflammatory signals and are involved in hepatocarcinogenesis. Much is known about the regulation of protein-coding genes by cytokines whereas their effects on the miRNome is less well understood. We performed a microarray screen to identify microRNAs (miRNAs) in human hepatocytes which are modulated by IL-6-type cytokines. Using samples of 2 donors, 27 and 68 miRNAs (out of 1,733) were found to be differentially expressed upon stimulation with hyper-IL-6 (HIL-6) for up to 72 h, with an overlap of 15 commonly regulated miRNAs. qPCR validation revealed that miR-146b-5p was also consistently up-regulated in hepatocytes derived from 2 other donors. Interestingly, miR-146b-5p (but not miR-146a-5p) was induced by IL-6-type cytokines (HIL-6 and OSM) in non-transformed liver-derived PH5CH8 and THLE2 cells and in Huh-7 hepatoma cells, but not in HepG2 or Hep3B hepatoma cells. We did not find evidence for a differential regulation of miR-146b-5p expression by promoter methylation, also when analyzing the TCGA data set on liver cancer samples. Inducible overexpression of miR-146b-5p in PH5CH8 cells followed by RNA-Seq analysis revealed effects on multiple mRNAs, including those encoding IRAK1 and TRAF6 crucial for Toll-like receptor signaling. Indeed, LPS-mediated signaling was attenuated upon overexpression of miR-146b-5p, suggesting a regulatory loop to modulate inflammatory signaling in hepatocytes. Further validation experiments suggest DNAJC6, MAGEE1, MPHOSPH6, PPP2R1B, SLC10A3, SNRNP27, and TIMM17B to be novel targets for miR-146b-5p (and miR-146a-5p).

Bixin Triggers Apoptosis of Human Hep3B Hepatocellular Carcinoma Cells: An Insight to Molecular and IN SILICO Approach

Hepatocellular carcinoma (HCC) is the most common liver cancer and is known to be resistant to conventional chemotherapy. The use of herbal medicine and supplements has increased over recent decades following side effects and resistant to conventional chemotherapy. The seeds of Bixa orellana L. commonly known as annatto have recently gained scientific attention due to presence of a carotenoid bixin for its substantial anticancer properties. However, molecular mechanisms underlying bixin-induced apoptosis are still unclear. Treatment of bixin significantly decreased the number of Hep3B cells and morphological study revealed the change in cellular and nuclear morphology that trigger the events of apoptosis confirmed by annexin V/PI staining. Further DCFDA and rhodamine 123 spectrofluorimetry study showed elevation in reactive oxygen species (ROS) production and loss of mitochondrial membrane potential (MMP), respectively. ROS production caused DNA damage and apoptosis was marked by cell cycle arrest, up-regulation of Bax and FasL protein as well as cleavage of caspase-9, caspase-8 and caspase-3 protein. Docking study with pro-apoptotic molecule Bax and surface Fas ligand exhibited energetically favourable binding interaction. Collectively, these results suggest that bixin capable of modulating the extrinsic and intrinsic molecules of apoptosis indicating its potential for development of promising candidate for hepatocellular carcinoma.

Oct3/4 is potentially useful for the suppression of the proliferation and motility of hepatocellular carcinoma cells

Hepatocellular carcinoma (HCC) cells are immature compared with healthy mature hepatocytes. Transcription factors serve a role in hepatocyte differentiation. The expression levels of transcription factors in HCC cell lines have been investigated to determine potential therapeutic targets. In the present study, the HLE, HLF, PLC/PRF/5, Huh-7, Hep3B, Huh-6 and HepG2 HCC cell lines were subjected to reverse-transcription polymerase chain reaction (RT-PCR) of transcription factors, including NANOG, Oct3/4, GATA binding protein 4 (GATA4), GATA6 and hematopoietically expressed homeobox (HHEX). In addition, these cell lines were analyzed using RT-quantitative PCR (RT-qPCR) of NANOG and Oct3/4. The 201B7 human induced pluripotent stem cells were evaluated as a model of pluripotent cells. The HLF cells were transfected with Oct3/4 small interfering RNA (siRNA) and used in an MTS colorimetric assay and a scratch assay. NANOG was not expressed in any of the cell lines. However, GATA4, GATA6 and HHEX were expressed in the majority of the HCC cell lines. In addition, NANOG and Oct3/4 were expressed in 201B7 cells. Oct3/4 was expressed in HLE, HLF and Hep3B cells; however, its expression levels were significantly reduced compared with those in 201B7 cells. RT-qPCR demonstrated that the expression of Oct3/4 siRNA suppressed the proliferation and motility of HLF cells. Oct3/4 siRNA may be a potentially effective therapy for the suppression of the proliferation and motility of HCC cells.

Ginnalin A and SB203580 show additive effect on Hep-3B hepatocellular carcinoma cell line

Abstract Objective Investigation of the anticarcinogenic effects of natural products with low toxicity is very important in the development of new therapeutic strategies against cancer. Ginnalin A (GA) is one of the most important phenolic compounds of Acer genus and its anticancer effect has been shown that in various cancer cell lines. SB203580, a p38 MAPK inhibitor, can inhibit cell proliferation independently of p38 MAPK. The objective of this study was to investigate combination effect of GA and SB203580 on Hep-3B cell line. Material and methods Cell viability was determined by using XTT method after the treatment with GA, SB203580 and combination of both. Anticarcinogenic effects of GA and SB203580 both in single and in combination have been analyzed with Caspase-3 activity assay and expression levels of important genes involved in cell cycle and apoptosis were evaluated by qPCR. Results GA and SB203580 have shown additive effect on Hep-3B cells in the combination inhibited 50% of cell viability. And, SB203580 increased the effect of GA on activation of Caspase-3 and expressions of genes important in apoptosis and cell cycle. Conclusion This study indicates that GA and SB203580 can be an effective for development of new therapeutic strategies in hepatocellular carcinoma.

Biostatistics mining associated method identifies AKR1B10 enhancing hepatocellular carcinoma cell growth and degenerated by miR-383-5p

Previous studies have reported that the aberrantly expressed AKR1B10 is associated with many cancer development, however the functional roles of AKR1B10 and its regulatory mechanisms in hepatocellular carcinoma (HCC) have been limited studied. In this project, we identified AKR1B10 functional as an oncogene in HCC through tumor/normal human tissue comparison from both GEO microarray and TCGA RNAseq dataset. Further experimental validations from three HCC cell lines (SMMC-7721, HePG2 and HeP3B) also suggested the ontogenetic functions of AKR1B10 in HCC tumor growth. By knocking down AKR1B10 through shRNA in HCC HeP3B cells, we showed it significantly induced cell cycle arrest and inhibited cell growth. Interestingly, integrative analysis of TCGA RNAseq data and miRNA-seq data predicted that miR-383-5p, a novel post-transcriptional tumor suppressor, is negatively associated with AKR1B10 expression. To further investigate the role of miR-383-5p in regulating AKR1B10 in HCC, we performed Dual-luciferase reporter assay experiments. Results showed that miR-383-5p is an upstream modulator targeting AKR1B10 in the post-transcriptional stage. Thus, we report AKR1B10 modulated regulated by miR-383-5p, promotes HCC tumor progress, and could be potentially a therapeutic target for precision medicine in HCC.

Abstract 1120: Wnt/β-catenin mediated enrichment of EpCAM positive cancer stem cells promote drug resistance in hepatocellular carcinoma

Abstract Introduction: Hepatocellular carcinoma (HCC) attains resistance to anti-cancer drugs which accounts for most of the therapeutic failures, and is one of the major factors for poor prognosis and poor overall survival of HCC patients. Cancer stem cells (CSCs) are sub-population of cells that bear stem-like properties, and are believed to contribute in tumor initiation, drug resistance and recurrence in tumor microenvironment. We hypothesize that canonical Wnt/β-catenin mediated CSC enrichment is responsible for acquired drug resistance in HCC. We aimed to study HCC CSCs for aberrant Wnt/β-catenin signaling, possible dedifferentiation, and drug resistance property. Methods: Total n=26 human HCC specimens with adjacent controls were analyzed for EpCAM and β-catenin expression by histology and western blot. In vitro CSC enrichment was achieved by treating murine (Hepa1-6) and human (HepG2, Hep3B) HCC cells in serum-free condition. CSC enrichment was confirmed by analyzing for established surface markers (EpCAM, CD44, CD133, CD90) and functional markers (Aldeflour assay and Hoechst-33342 efflux). For possible dedifferentiation, we analyzed gene expression profile during enrichment process using qRT-PCR for dedifferentiation and stem cell specific genes. Drug resistance property of CSCs was studied for Doxorubicin and Sorafenib by XTT assay. To investigate CSC activation, Wnt/β-catenin signaling was studied by analyzing expression of β-catenin, GSK3β, p-GSK3β, EpCAM, ABCG2; and downstream targets (C-Myc,Cyclin-D1,TCF1) and findings were confirmed by β-catenin knockdown, inhibitors of Wnt pathways (LiCl, XAV939, FH535) and TOP flash reporter assay. To study in vivo tumorigenesis of HCC CSCs, immunocompetent mouse model was established using C57L/J mouse and copGFP expressing Hepa1-6 cells for possible lineage tracking. Tumor growth was monitored using high-frequency ultrasound and animals were euthanized after 18 days. Histology and ICC analysis was performed to confirm the tumors and findings. Results: Human specimens showed concomitant EpCAM and beta-catenin expression. Spheroid forming HCC CSCs showed significant higher β-catenin and EpCAM expression, and acquired drug resistance compared with control HCC cells. Silencing β-catenin by knockdown abolish drug resistance property of CSCs for Doxorubicin and reverse spheroids to adherent phenotype. Our in vivo findings confirmed EpCAM+/+ CSCs have shown significant tumorigenesis compared with no tumors in EpCAM-/- (n=9, p&amp;lt;0.005). Conclusions: Our findings suggest that possible dedifferentiation by canonical β-catenin confer drug resistance and CSC properties to HCC cells. Sorted EpCAM+ CSCs showed very aggressive in vivo tumor initiation and growth in immunocompetent orthotopic mouse model. This EpCAM+ CSCs population could potentially responsible for HCC recurrence and therapeutic failure. Citation Format: Harshul Pandit, Yan Li, Gouzhen Cui, Qianqian Zheng, Suping Li, Robert Martin. Wnt/β-catenin mediated enrichment of EpCAM positive cancer stem cells promote drug resistance in hepatocellular carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1120.

Abstract 4563: PD-L1 expression associated with treatment responses in colorectal cancer patients with XELOX/FOLFOX chemotherapy: Potential of checkpoint blockage and natural killer cell-based immunotherapy

Abstract Chemo-resistance is a major obstacle in human cancer treatment. Immunotherapies including checkpoint blockage and cell-based therapy have been clinically validated for cancer treatment. The present study aimed to investigate the role of PD-L1 and potential of immunotherapy for chemo-resistant cancers. In a pilot study, we have examined the expression of PD-L1 in colorectal cancer (CRC) patients who received XELOX (capecitabine plus oxaliplatin) or FOLFOX (fluorouracil plus oxaliplatin) chemotherapy. Detection rate of PD-L1 expression by immunohistochemistry was higher in CRC patients who demonstrated poor response (5/10, 50%) compared to good responders (0/5, 0%) on XELOX/FOLFOX chemotherapy. Chemo-resistant CRC cell lines (HT29 and HCT116) were used to examine the role of PD-L1. Cells were plated out, selected under fluorouracil and oxaliplatin in serial, and referred as fluorouracil-oxaliplatin-resistant (FOR) cells. Half maximal inhibitory concentration (IC50) for respective drug was determined by MTT assay. HT29 FOR, compared to parental cells, showed chemo-resistance (359.4 and 10.1 fold increase to fluorouracil and oxaliplatin respectively) and similarly for HCT116 FOR (8.0 and 9.0 fold increase respectively). Hepatocellular carcinoma Hep3B and nasopharyngeal carcinoma C666-1 cells that have been selected for fluorouracil chemo-resistant (FR) were included. All the chemo-resistant cell lines showed increased expression of PD-L1 by real-time qPCR compared to their respective parental cells (HT29 FOR: 6.1 fold; HCT116 FOR: 1.9 fold; Hep3B FR: 3.1 fold; C666-1 FR: 4.4 fold). To explore the potential of immunotherapy, natural killer (NK) cells were isolated from peripheral blood PBMC of healthy donors. NK cells were expanded and activated ex vivo in stem cell growth medium supplemented with interleukins. NK co-culture assay was performed and apoptosis of cancer cells examined by flow cytometry. Parental cancer cells were sensitive to cytotoxic killing by NK cells compared to control no NK treatment (36.0% vs 2.8% apoptotic cells, P=0.001), and PD1 antibody (Nivolumab) treatment promoted NK-mediated cytotoxicity (52.9%, P=0.034). Notably, chemo-resistant cancer cells were similarly sensitive to cytotoxic killing by NK cells compared to control (27.1% vs 3.4%, P=0.002), and PD1 antibody (Nivolumab) treatment further enhanced NK-mediated cytotoxicity (52.8%, P=0.013). In summary, PD-L1 expression was associated with poor response for XELOX/FOLFOX chemotherapy in CRC patients. Chemo-resistant cancer cells showed elevated PD-L1 expression, nonetheless, susceptible to NK-mediated cytotoxicity and checkpoint blockage by PD1 antibody augmented the treatment response. Further studies are warranted to examine immunotherapy for chemo-resistant cancers. Citation Format: Elaine H.L. SIU, Simon S.M. NG, Anthony W.H. CHAN, James Y.W. LAU, Kwok Wai LO, Charing C.N. CHONG, Paul B.S. LAI, Stephen L. CHAN, Marcus H.N. LAW, Linda W.C. NG, S.T. CHEUNG. PD-L1 expression associated with treatment responses in colorectal cancer patients with XELOX/FOLFOX chemotherapy: Potential of checkpoint blockage and natural killer cell-based immunotherapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4563.