SK-Hep1 Cells Research Articles

Extract of Saccharina japonica induces apoptosis companied by cell cycle arrest and endoplasmic reticulum stress in SK-Hep1 human hepatocellular carcinoma cells.

Saccharina japonica is a family member of Phaeophyceae (brown macro-alga) and extensively cultivated in China, Japan and Korea. Here, the potential anti-cancer effect of n-hexane fraction of S. japonica was evaluated in SK-Hep1 human hepatocellular carcinoma cells. The N-hexane fraction reduced cell viability and increased the numbers of apoptotic cells in a both dose- and time-dependent manner. Apoptosis was activated by both caspase-dependent and independent pathways. The caspase-dependent cell death pathway is mediated by cell surface death receptors and activated caspase-8 amplified the apoptotic signal either through direct activation of downstream caspase-3 or pro-apoptotic proteins (Bad, Bax and Bak) subsequently leading to the release of cytochrome c. On the other hand, caspase-independent apoptosis appeared mediated by disruption of mitochondrial membrane potential and translocation of AIF to the nucleus where they induced chromatin condensation and/or large-scale DNA fragmentation. In addition, the n-hexane fraction induced endoplasmic reticulum (ER)-stress and cell cycle arrest. The results suggested that potential anti-cancer effects of n-hexane extract from S. japonica on SK-Hep1 cells.

Alternative splicing variants of human Fbx4 disturb cyclin D1 proteolysis in human cancer

Fbx4 is a specific substrate recognition component of SCF ubiquitin ligases that catalyzes the ubiquitination and subsequent degradation of cyclin D1 and Trx1. Two isoforms of human Fbx4 protein, the full length Fbx4α and the C-terminal truncated Fbx4β have been identified, but their functions remain elusive. In this study, we demonstrated that the mRNA level of Fbx4 was significantly lower in hepatocellular carcinoma tissues than that in the corresponding non-tumor tissues. More importantly, we identified three novel splicing variants of Fbx4: Fbx4γ (missing 168–245nt of exon1), Fbx4δ (missing exon6) and a N-terminal reading frame shift variant (missing exon2). Using cloning sequencing and RT-PCR, we demonstrated these novel splice variants are much more abundant in human cancer tissues and cell lines than that in normal tissues. When expressed in Sk-Hep1 and NIH3T3 cell lines, Fbx4β, Fbx4γ and Fbx4δ could promote cell proliferation and migration in vitro. Concordantly, these isoforms could disrupt cyclin D1 degradation and therefore increase cyclin D1 expression. Moreover, unlike the full-length isoform Fbx4α that mainly exists in cytoplasm, Fbx4β, Fbx4γ, and Fbx4δ locate in both cytoplasm and nucleus. Since cyclin D1 degradation takes place in cytoplasm, the nuclear distribution of these Fbx4 isoforms may not be involved in the down-regulation of cytoplasmic cyclin D1. These results define the impact of alternative splicing on Fbx4 function, and suggest that the attenuated cyclin D1 degradation by these novel Fbx4 isoforms provides a new insight for aberrant cyclin D1 expression in human cancers.

D-glucose as a modifying agent in gelatin/collagen matrix and reservoir nanoparticles for Calendula officinalis delivery

Gelatin/Collagen-based matrix and reservoir nanoparticles require crosslinkers to stabilize the formed nanosuspensions, considering that physical instability is the main challenge of nanoparticulate systems. The use of crosslinkers improves the physical integrity of nanoformulations under the-host environment. Aldehyde-based fixatives, such as formaldehyde and glutaraldehyde, have been widely applied to the crosslinking process of polymeric nanoparticles. However, their potential toxicity towards human beings has been demonstrated in many previous studies. In order to tackle this problem, d-glucose was used during nanoparticle formation to stabilize the gelatin/collagen-based matrix wall and reservoir wall for the deliveries of Calendula officinalis powder and oil, respectively. In addition, therapeutic selectivity between malignant and normal cells could be observed. The C. officinalis powder loaded nanoparticles significantly strengthened the anti-cancer effect towards human breast adenocarcinoma MCF7 cells and human hepatoma SKHep1 cells when compared with the free powder. On the contrary, the nanoparticles did not show significant cytotoxicity towards normal esophageal epithelial NE3 cells and human skin keratinocyte HaCaT cells. On the basis of these evidences, d-glucose modified gelatin/collagen matrix nanoparticles containing C. officinalis powder might be proposed as a safer alternative vehicle for anti-cancer treatments.

Activation of AMP-activated protein kinase attenuates hepatocellular carcinoma cell adhesion stimulated by adipokine resistin

BackgroundResistin, adipocyte-secreting adipokine, may play critical role in modulating cancer pathogenesis. The aim of this study was to investigate the effects of resistin on HCC adhesion to the endothelium, and the mechanism underlying these resistin effects.MethodsHuman SK-Hep1 cells were used to study the effect of resistin on intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1) expressions as well as NF-κB activation, and hence cell adhesion to human umbilical vein endothelial cells (HUVECs). 5-Aminoimidazole-4-carboxamide 1-β-D-ribofuranoside (AICAR), an AMP-activated protein kinase (AMPK) activator, was used to determine the regulatory role of AMPK on HCC adhesion to the endothelium in regard to the resistin effects.ResultsTreatment with resistin increased the adhesion of SK-Hep1 cells to HUVECs and concomitantly induced NF-κB activation, as well as ICAM-1 and VCAM-1 expressions in SK-Hep1 cells. Using specific blocking antibodies and siRNAs, we found that resistin-induced SK-Hep1 cell adhesion to HUVECs was through NF-κB-regulated ICAM-1 and VCAM-1 expressions. Moreover, treatment with AICAR demonstrated that AMPK activation in SK-Hep1 cells significantly attenuates the resistin effect on SK-Hep1 cell adhesion to HUVECs.ConclusionsThese results clarify the role of resistin in inducing HCC adhesion to the endothelium and demonstrate the inhibitory effect of AMPK activation under the resistin stimulation. Our findings provide a notion that resistin play an important role to promote HCC metastasis and implicate AMPK may be a therapeutic target to against HCC metastasis.

Inhibition of TNF-α-Mediated NF-κB Transcriptional Activity by Dammarane-Type Ginsenosides from Steamed Flower Buds of Panax ginseng in HepG2 and SK-Hep1 Cells

Panax ginseng is a medicinal herb that is used worldwide. Its medicinal effects are primarily attributable to ginsenosides located in the root, leaf, seed, and flower. The flower buds of Panax ginseng (FBPG) are rich in various bioactive ginsenosides, which exert immunomodulatory and anti-inflammatory activities. The aim of the present study was to assess the effect of 18 ginsenosides isolated from steamed FBPG on the transcriptional activity of NF-κB and the expression of tumor necrosis factor-α (TNF-α)-stimulated target genes in liver-derived cell lines. Noticeably, the ginsenosides Rk3 and Rs4 exerted the strongest activity, inhibiting NF-κB in a dose-dependent manner. SF and Rg6 also showed moderately inhibitory effects. Furthermore, these four compounds inhibited the TNF-α-induced expression of IL8, CXCL1, iNOS, and ICAM1 genes. Consequently, ginsenosides purified from steamed FBPG have therapeutic potential in TNF-α-mediated diseases such as chronic hepatic inflammation.

Destruxin B inhibits hepatocellular carcinoma cell growth through modulation of the Wnt/β-catenin signaling pathway and epithelial–mesenchymal transition

The aberrant activation of Wnt/β-catenin signaling plays an important role in the carcinogenesis and progression of hepatocellular carcinoma (HCC). Therefore, the Wnt/β-catenin signaling molecules are attractive candidates for the development of targeted therapies for this disease. The present study showed that destruxin B (DB) inhibits the proliferation and induces the apoptosis of HCC cells by decreasing the protein expression of anti-apoptotic Bcl-2 and Bcl-xL and increasing the expression of the proapoptotic protein Bax. More importantly, DB also attenuates Wnt-signaling in HCC cells by downregulating β-catenin, Tcf4, and β-catenin/Tcf4 transcriptional activity, which results in the decreased expression of β-catenin target genes, such as cyclin D1, c-myc, and survivin. Furthermore, DB affects the migratory and invasive abilities of Sk-Hep1 cells through the suppression of markers of the epithelial–mesenchymal transition (EMT). A synergistic anti-proliferative and migratory effect was achieved using the combination of DB and sorafenib in Sk-Hep1 cells. In conclusion, DB acts as a novel Wnt/β-catenin inhibitor and reduces the aggressiveness and invasive potential of HCC by altering the cells’ EMT status and mobility. DB in combination with sorafenib may be considered for future clinical use for the management of metastatic HCC.

Epigallocatechin-3-gallate inhibits cell growth, induces apoptosis and causes S phase arrest in hepatocellular carcinoma by suppressing the AKT pathway

Epigallocatechin-3-gallate (EGCG) has been shown to inhibit the growth and induce apoptosis of certain cancer cells. The aim of this study was to determine the role of EGCG in hepatocellular carcinoma (HCC) and the underlying mechanism(s) thereof. MTT assay was used to determine the cell growth inhibition by EGCG. Apoptosis induced by EGCG was investigated by both AO/EB staining and flow cytometry. The cell cycle distribution was analyzed by flow cytometry. The mRNA levels of the AKT pathway were analyzed by quantitative PCR. The expression of AKT and its phosphorylation at Ser473 were detected by western blotting. The IC50 of EGCG at 48h for HepG2, SMMC7721 and SK-hep1 cells were 74.7, 59.6 and 61.3µg/ml, respectively. Significantly higher proportion of SMMC7721 cells entered the Sphase upon treatment with EGCG for 48h compared with control cells. EGCG decreased the mRNA levels of PI3K, AKT and NF-κB. The protein levels of AKT decreased and its phosphorylation at Ser473 was downregulated with EGCG treatment. EGCG inhibited growth by affecting the cell cycle and induced apoptosis in different HCC cells by downregulating PI3K/AKT activity. The results suggest the potential of EGCG as an anticancer agent in the prevention or treatment of HCC.

Role of NADPH oxidase-4 in saturated fatty acid-induced insulin resistance in SK-Hep-1 cells

We aimed to develop a cell culture model of type 2 diabetes by treating SK-Hep-1 cells with four free fatty acids [i.e., palmitic acid, stearic acid (SA), linoleic acid and oleic acid]. The results showed that Akt phosphorylation was increased in SK-Hep-1 cells treated with insulin in a time- and concentration-dependent manner, which was inhibited by saturated fatty acids, but not by unsaturated fatty acids. Moreover, protein levels of NADPH oxidase (NOX) 4 but not NOX2 were increased following SA treatment and, consequently, increased reactive oxygen species production and decreased cellular glutathione were observed. Apocynin, a NOX4 inhibitor, restored the SA-induced inhibition of Akt phosphorylation, suggesting the role of NOX4 in insulin resistance induced by SA. Neither phosphorylation level nor protein level of the stress signaling kinases, such as c-Jun N-terminal kinase or p38 mitogen activated protein kinase, was changed by SA treatment. Although binding immunoglobulin protein, a marker of endoplasmic reticulum stress, was transiently increased in SKHep-1 cells treated with SA, 4-phenyl butyric acid, a chemical chaperone, had no effect on the insulin-mediated Akt phosphorylation inhibited by SA. The present study provides a useful model for screening anti-insulin resistance drugs and finding new drug targets for treatment of diabetes.

Abstract C268: MET-inhibition using SU11274 and HER-inhibitions with lapatinib impair proliferation, motility and invasion in hepatocellular carcinoma (HCC) and exhibits an increased efficacy un sorafenib-tolerant model.

Abstract Introduction: Sorafenib significantly improved overall survival in advanced HCC. However patients may display primary resistance and a majority of them will develop acquired resistance over time when treated with sorafenib. Resistance to sorafenib has been correlated with upregulation of angiogenic pathways, including the c-MET pathway (Roskoski, 2007). In the SHARP study, patients exhibiting high HGF levels at baseline had a poor prognosis and appeared to benefit less from sorafenib in terms of survival than those with low levels of HGF (Pena, 2008). Therefore, MET appears to be a potential target to counteract sorafenib resistance. In this study we evaluate MET and EGFR expression and their sensitivity towards MET and EGFR inhibitors in terms of signaling, viability, and invasion in two HCC cell lines, one sensitive to sorafenib and with acquired sorafenib-tolerance. Methods: SU11274 is a specific inhibitor of overexpressed and activated MET. Antiproliferative effects of SU11274 (MET inhibitor) and lapatinib an (EGFR/HER2 inhibitor) (lapatinib) were evaluated in the human HCC cell lines, SK-HEP1 and its sorafenib-tolerant counterpart, SK-Sora, using MTT assays. mRNA expression and protein levels were assessed by qRT-PCR and Western blot, respectively. Cell motility was investigated by wound-healing and matrigel invasion assays. Results: SK-Sora was characterized by a 2-fold higher MET expression but a 3-fold lower EGFR expression respect to SK-HEP1. This differential expression of MET/EGFR between the two cell lines was associated with their sensitivity to SU11274 and lapatinib in proliferation assays. Whereas 5µM SU11274 or 20µM lapatinib induced 50 % growth inhibition of SK-HEP1 cells, SK-Sora cells displayed 86% and 27% of growth inhibition, respectively in the same conditions. These results indicated that baseline MET and EGFR expression levels may be good biomarkers to predict increased sensitivity or resistance to MET or EGFR inhibitors. Interestingly, SU11274 also more potently decreased HGF-dependent invasion in SK-Sora (80%) cells than in SK-HEP1 (50%) cells. To understand the differences observed regarding proliferation and invasion between SK-HEP1 and SK-Sora cells, we assessed MET-dependent signaling in both cell lines. HGF-induced MET activation was associated with differential pattern of p-METTyr1234/35, p-GAB1, p-ERK1/2, and p-AKTser473 between both cell lines, with higher phosphorylation of MET and GAB1 in SK-Sora than in SK-HEP1. Interestingly, we showed that SK-Sora cells were more sensitive to the inhibition of HGF-induced signaling by SU11274 than SK-HEP1 cells. Differential effects of lapatinib on cell signaling and invasion will be detailed. Conclusion: MET inhibition showed more potent effects in the sorafenib-tolerant cell line, which had increased MET expression levels, as compared to the parental cell line SK-HEP1. These results suggest that MET could be an attractive target in HCC patients particularly in the context of sorafenib resistance. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):C268. Citation Format: Annemilai Tijeras-Raballand, Eugenia Riveiro, Maria Serova, Cindy Neuzillet, Matthieu Martinet, Valérie Paradis, Mohamed Bouattour, Eric Raymond, Sandrine Faivre, Armand de Gramont. MET-inhibition using SU11274 and HER-inhibitions with lapatinib impair proliferation, motility and invasion in hepatocellular carcinoma (HCC) and exhibits an increased efficacy un sorafenib-tolerant model. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr C268.

Abstract A228: TGF-beta signaling inhibition using LY2157299 affects proliferation or invasion in hepatocarcinoma cells and patient samples.

Abstract Background: To date, the only FDA approved targeted agent in advanced hepatocarcinomas is sorafenib, indicating a critical need for innovative therapeutic options. LY2157299, a selective ATP-mimetic inhibitor of TGF-β receptor (TβR)-I activation, is currently under clinical investigation in HCC patients after sorafenib or in patients ineligible for sorafenib. Our study aimed at investigating the effects of LY2157299 in HCC cell lines and patient samples with various AFP expression levels. Materials and Methods: Antiproliferative effects of LY2157299 were evaluated in a panel of human HCC cells by MTT assay. Baseline and phosphorylated (p-) protein levels were assessed by Western blot analysis and mRNA expressions by qRT-PCR. Invasion assays were done on matrigel and in OptiCell systems. Tumor samples from HCC patients were surgically resected and cut in 300 µm thick slices using a Tissue Slicer. Each slice was randomly exposed to LY2157299 (1µM and 10µM) and sorafenib (5µM) for 48h. At the end of treatment, tumor samples were analyzed by immunohistochemistry (IHC) or immunofluorescence (IF). Results: LY2157299 was evaluated in HEPG2 and HUH-7 AFP-positive and HEP3B and SK-HEP1 AFP negative cells as well as “in lab-established” SK-HEP1-derived cell lines resistant to the tyrosine kinase inhibitors sorafenib (SK-Sora) and sunitinib (SK-Suni). Exogenous stimulation of all HCC cell lines with TGF-β yielded downstream activation of canonical Smad pathway that was potently inhibited with LY2157299 treatment at micromolar concentrations. In contrast, the non-canonical pathways such as MAPK and Akt/mTOR were not activated by TGF-β in most cell lines with the exception of SK-HEP1 cells where LY2157299 restored the basal level of p-ERK1/2, p-AKT and p-S6 kinase phosphorylation. Low concentrations of LY2157299 displayed antiproliferative effects in HEPG2 and HEP3B cells when stimulated by TGF-β but not in other cell lines. LY2157299 yielded potent anti-migratory and anti-invasive properties in invasive SK-HEP1, SK-Suni and SK-Sora cells. Tumor slices from surgically resected advanced HCC tumor from 5 different patients, were exposed ex vivo to LY2157299 or sorafenib for 48h. This method allows the evaluation of novel anticancer agents in whole tumors containing cancer cells and stromal cells. LY2157299 but not sorafenib decreased 2-5 fold the p-Smad2/3. LY2157299 treatment mainly affected PI3K/AKT rather than MAPK signalling pathway. In contrast, sorafenib treatment reduced both PI3K and MAPK pathway activation. IHC analysis of LY2157299 and sorafenib-exposed samples showed a significant decrease (>3 fold) of the proliferative marker Ki67 and increase of the apoptotic marker caspase-3 (3-5 fold). Interestingly, all molecular effects were independent of AFP expression. Conclusion: TGF-β/TβR-I inactivation using LY2157299 inhibits TGF-β-dependent cell signaling in HCC cell lines with either anti-proliferative or anti-invasive effects depending on the model. In tumor samples from patients, inhibition of TGF-β signaling was associated with inhibition of proliferation and apoptosis induction regardless AFP level. Our data suggest that LY2157299 may be useful for patients with HCC. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):A228. Citation Format: Marie Serova, Annemilaï Tijeras-Raballand, Celia Dos Santos, Karim A. Benhadji, Valerie Paradis, Eric Raymond, Sandrine Faivre, Armand de Gramont. TGF-beta signaling inhibition using LY2157299 affects proliferation or invasion in hepatocarcinoma cells and patient samples. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr A228.

Characterization of Niemann-Pick Type C2 Protein Expression in Multiple Cancers Using a Novel NPC2 Monoclonal Antibody

Niemann-Pick Type C2 (NPC2) plays an important role in the regulation of intracellular cholesterol homeostasis via direct binding with free cholesterol. However, little is known about the significance of NPC2 in cancer. In this study, we have pinpointed the impact of various different cancers on NPC2 expression. A series of anti-NPC2 monoclonal antibodies (mAbs) with the IgG2a isotype were generated and peptide screening demonstrated that the reactive epitope were amino acid residues 31-40 of the human NPC2 protein. The specificity of these mAbs was confirmed by Western blotting using shRNA mediated knock-down of NPC2 in human SK-Hep1 cells. By immunohistochemical staining, NPC2 is expressed in normal kidney, liver, breast, colon, lung, esophageal, uterine cervical, pancreatic and stomach tissue. Strong expression of NPC2 was found in the distal and proximal convoluted tubule of kidney and the hepatocytes of liver. Normal esophageal, uterine cervical, pancreatic, stomach, breast, colon and lung tissue stained moderately to weakly. When compared to their normal tissue equivalents, NPC2 overexpression was observed in cancers of the breast, colon and lung. Regarding to breast cancer, NPC2 up-regulation is associated with estrogen receptor (-), progesterone receptor (-) and human epidermal growth factor receptor (+). On the other hand, NPC2 was found to be down-regulated in renal cell carcinoma, liver cirrhosis and hepatoma tissues. By antigen-capture enzyme immunoassay ELISA, the serum NPC2 is increased in patients with cirrhosis and liver cancer. According to western blot data, the change of glycosylated pattern of NPC2 in serum is associated with cirrhosis and liver cancer. To the best of our knowledge, this is the first comprehensive immunohistochemical and serological study investigating the expression of NPC2 in a variety of different human cancers. These novel monoclonal antibodies should help with elucidating the roles of NPC2 in tumor development, especially in liver and breast cancers.

β-Catenin Knockdown in Liver Tumor Cells by a Cell Permeable Gamma Guanidine-based Peptide Nucleic Acid

Hepatocellular cancer (HCC) is the third cause of death by cancer worldwide. In the current study we target β- catenin, an oncogene mutated and constitutively active in 20-30% of HCCs, via a novel, cell permeable gamma guanidine-based peptide nucleic acid (γGPNA) antisense oligonucleotide designed against either the transcription or the translation start site of the human β-catenin gene. Using TOPflash, a luciferase reporter assay, we show that γGPNA targeting the transcription start site showed more robust activity against β-catenin activity in liver tumor cells that harbor β-catenin gene mutations (HepG2 & Snu-449). We identified concomitant suppression of β-catenin expression and of various Wnt targets including glutamine synthetase (GS) and cyclin-D1. Concurrently, γGPNA treatment reduced proliferation, survival and viability of HCC cells. Intriguingly, an angiogenesis quantitative Real-Time-PCR array identified decreased expression of several pro-angiogenic secreted factors such as EphrinA1, FGF-2, and VEGF-A upon β-catenin inhibition in liver tumor cells. Conversely, transfection of stabilized-β-catenin mutants enhanced the expression of angiogenic factors like VEGF-A. Conditioned media from HepG2 cells treated with β-catenin but not the mismatch γGPNA significantly diminished spheroid and tubule formation by SK-Hep1 cells, an HCC-associated endothelial cell line. Thus, we report a novel class of cell permeable and efficacious γGPNAs that effectively targets β-catenin, a known oncogene in the liver. Our study also identifies a novel role of β-catenin in liver tumor angiogenesis through paracrine mechanisms in addition to its roles in proliferation, survival, metabolism and cancer stem cell biology, thus further strengthening its effectiveness as a therapeutic target in HCC.

Anti-metastatic Potential of Ethanol Extract of Saussurea involucrata against Hepatic Cancer in vitro

The rates of morbidity and mortality of hepatocellular carcinoma (HCC) have not lessened because of difficulty in treating tumor metastasis. Mongolian Saussurea involucrata (SIE) possesses various anticancer activities, including apoptosis and cell cycle arrest. However, detailed effects and molecular mechanisms of SIE on metastasis are unclear. Thus, the present study was undertaken to investigate antimetastatic effects on HCC cells as well as possible mechanisms. Effects of SIE on the growth, adhesion, migration, aggregation and invasion of the SK-Hep1 human HCC cell line were investigated. SIE inhibited cell growth of metastatic cells in dose- and time-dependent manners. Incubation of SK-Hep1 cells with 200-400 μg/mL of SIE significantly inhibited cell adhesion to gelatin-coated substrate. In the migration (wound healing) and aggregation assays, SIE treated cells showed lower levels than untreated cells. Invasion assays revealed that SIE treatment inhibited cell invasion capacity of HCC cells substantially. Quantitative real time PCR showed inhibitory effects of SIE on MMP-2/-9 and MT1-MMP mRNA levels, and stimulatory effects on TIMP-1, an inhibitor of MMPs. The present study not only demonstrated that invasion and motility of cancer cells were inhibited by SIE, but also indicated that such effects were likely associated with the decrease in MMP-2/-9 expression of SK-Hep1 cells. From these results, it was suggested that SIE could be used as potential anti-tumor agent.

Downregulation of microRNA-214 and overexpression of FGFR-1 contribute to hepatocellular carcinoma metastasis

miR-214 is one of the most significantly downregulated microRNAs (miRNAs) in hepatocellular carcinoma (HCC). Fibroblast growth factor receptor 1 (FGFR-1) is a miR-214 target gene implicated in the progression of HCC. However, the roles of miR-214 and FGFR-1 in HCC are not fully understood. Here, we analyzed the expression of miR-214 and FGFR-1 in 65 cases of HCC and paired non-neoplastic tissue specimens using real-time PCR and Western blot (WB), respectively. Our data indicated that miR-214 was downregulated and FGFR-1 was overexpressed in HCC compared to the paired non-neoplastic tissues. The low miR-214 expression was correlated with portal vein invasion (p=0.016) and early recurrence (p=0.045) in HCC patients. Moreover, the low miR-214 expression was correlated with high positive rate of FGFR-1 in HCC cases (p=0.020). Our data further demonstrated that miR-214 overexpression in SK-HEP1 and HepG2 cells downregulated FGFR-1 expression and inhibited liver cancer cell invasion. The Luciferase assay results further demonstrated the targeted regulation of FGFR-1 by miR-214. In conclusion, our data indicate that the downregulation of miR-214 in HCC and the upregulation of its target gene FGFR-1 is associated with HCC progression. Therefore, miR-214 and FGFR-1 are potential prognostic markers and therapeutic targets in HCC.

Bufalin inhibits migration and invasion in human hepatocellular carcinoma SK-Hep1 cells through the inhibitions of NF-kB and matrix metalloproteinase-2/-9-signaling pathways

Metastasis plays an important role in mortality of cancer patients. Migration and invasion are the major characteristics of tumor metastasis. The induction of matrix metalloproteinases (MMPs) such as MMP-2 and -9 are particularly important for the invasiveness of various cancer cells. Bufalin, a class of toxic steroids, was purified from the skin glands of Bufo gargarizans or Bufo melanostictus; it is known to inhibit proliferation of human cancer cells. In this study, we investigated the antiinvasive mechanisms of bufalin in the human hepatocellular cancer cell line SK-Hep1. Bufalin significantly reduced serum-induced cell invasion and migration. Furthermore, bufalin markedly inhibited MMP-2 and -9 activity, mRNA expression and protein levels in SK-Hep1 cells. Bufalin attenuated phosphoinisitide-3-kinase (PI3K) and phosphorylation of AKT which was associated with reduced levels of nuclear factor kappa B (NF-κB). Bufalin also suppressed protein levels of FAK and Rho A, VEGF, MEKK3, MKK7, and uPA and it diminished NF-κB translocation. Based on these observations, we propose that bufalin is acts as an antiinvasive agent by inhibiting MMP-2 and -9 and involving PI3K/AKT and NF-κB pathways. Bufalin is a potential therapeutic agent that may have efficacy in preventing the invasion and metastasis of malignant liver tumors.

Daidzein causes cytochrome c-mediated apoptosis via the Bcl-2 family in human hepatic cancer cells

Daidzein, which belongs to the group of isoflavones from soybeans, has been extensively researched prostate, cervix, brain, breast, and colon cancer cell lines. However, daidzein has not been thoroughly investigated in human hepatic cancer cells; therefore, we investigated whether it inhibits hepatic cancer cell growth. Decreased cell proliferation was measured in daidzein-treated hepatic cancer cells (SK-HEP-1) upon real-time cell electronic sensing analysis however, it was not affected on normal human hepatocytes (Chang). Daidzein-induced apoptosis was demonstrated by comet and TUNEL assay. Moreover, we conducted two-dimensional electrophoresis to study the mechanism of daidzein-induced apoptosis in daidzein-treated SK-HEP-1 cells. Expression of peroxiredoxin-3 (Prdx-3), which modulates redox homeostasis of cells, was increased in protein analysis. Additionally, we measured the levels of reactive oxygen species and it was decreased in daidzein-treated SKHEP-1 cells. Daidzein-induced apoptosis in SK-HEP-1 cells was also associated with the up-regulation of Bak and down-regulation of Bcl-2 and Bcl-xL proteins. Moreover, daidzein treatment increased in the release of mitochondrial cytochrome c and activation of APAF-1, caspase 9 and caspase 3. Overall, these result indicate that daidzein is a potent inducer of apoptosis in hepatic cancer cells via mitochondrial pathway.

Darbepoetin inhibits proliferation of hepatic cancer cells in the presence of TGF-β

Darbepoetin (DPO), an erythropoietin (EPO) derivative, was licensed in 2002 to treat patients with solid tumors suffering from chemotherapy-dependent anemia, although various tumors express EPO to improve vascularization, thus favoring tumor growth and spreading. Therefore, we wanted to investigate direct effects of DPO on the liver tumor cell lines HepG2, SkHep1, Huh-7, AKN1, HCC-T and HCC-M, as well as on primary human hepatocytes (hHeps). DPO (0-40ng/ml) did not affect viability of hHeps, HepG2, SkHep1, AKN1, HCC-T and HCC-M cells, as determined by Resazurin conversion. However, Huh-7 cells' viability dose-dependently decreased from 5ng/ml DPO on. Lack of LDH release into culture medium and negative DNA laddering excluded apoptosis or necrosis as the cause for the reduced Resazurin conversion. In Huh-7 cells, DPO increased the expression of p53. Interestingly, Huh-7 cells showed the highest basal TGF-β1 expression as compared to the other cell lines. Upon inhibition of TGF-β1 signaling, DPO no longer reduced viability in Huh-7 cells. On the contrary, co-incubation with TGF-β1 made the other cell lines responsive to DPO. Summarizing our data, we show that DPO reduces the growth of Huh-7 cells by up-regulation of the tumor-suppressor gene p53. This mechanism seems to be dependent on a strong TGF-β expression and corresponding signaling in these cells, as other cell lines became responsive to DPO with TGF-β1 supplementation. The knowledge of this mechanism offers great perspectives for the understanding and treatment of solid liver tumors.

CAPE promotes TRAIL-induced apoptosis through the upregulation of TRAIL receptors via activation of p38 and suppression of JNK in SK-Hep1 hepatocellular carcinoma cells

Caffeic acid phenethyl ester (CAPE), a phenolic compound derived from honeybee propolis, has been reported to possess anticancer activities in several types of malignant cells. Here, we show that treatment with tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) in combination with CAPE significantly sensitized SK-Hep1 cells to TRAIL-induced apoptosis. The sensitization to TRAIL was accompanied by the activation of extrinsic and intrinsic apoptotic pathways, leading to the activation of caspases, mitochondrial disruption and PARP cleavage. Moreover, TRAIL receptors, such as DR4 and DR5 were significantly upregulated by CAPE treatment, and both DR4/Fc and DR5/Fc chimera markedly abrogated apoptosis induced by CAPE and TRAIL, demonstrating the critical role of these death receptors in combination-induced apoptosis. The effect of CAPE on mitogen-activated protein kinases (MAPKs) was further examined, where CAPE treatment resulted in the activation of p38 and the inhibition of JNK, without affecting levels of phospho-ERK. Our results showed that p38 and JNK exhibited the opposite role in SK-Hep1 cells. The inhibition of p38, using SB203580, blocked the CAPE-induced expression of death receptors and attenuated the combination‑induced apoptosis, suggesting the pro-apoptotic role of p38. In contrast, JNK-specific inhibition, by SP600125, triggered upregulation of DR4 and DR5, and sensitized SK-Hep1 cells to TRAIL, indicating that the CAPE-induced suppression of JNK may contribute to the sensitizing effect of CAPE through the upregulation of death receptors. Taken together, these results indicate that CAPE potentiated TRAIL-induced apoptosis in SK-Hep1 cells, through upregulation of TRAIL receptors via modulation of p38 and JNK signaling pathways.

Ethanol-induced apoptosis in human liver adenocarcinoma cells (SK-Hep1): Fas- and mitochondria-mediated pathways and interaction with MAPK signaling system

For studying molecular mechanisms regulating the fate of ethanol-treated hepatocytes, involvement of Fas in ethanol-induced apoptosis was examined in human liver adenocarcinoma (SK-Hep1) cells in which the function of Fas-associated death domain (FADD) protein was knocked down by transfection. In FADD-knocked down cells, while ethanol-induced increase in generation of reactive oxygen species (ROS) was unaffected, apoptosis was significantly suppressed, demonstrating the involvement of Fas in ethanol-induced hepatocyte apoptosis more directly than in the past reports. On the other hand, effects of mitogen-activated protein kinase (MAPK), which is well known to determine the fate of various cells, on ethanol-induced apoptosis have not been examined in SK-Hep1 cells. Of three major MAPKs, only p38 MAPK and JNK were found activated by 200mM ethanol treatment. When cells were incubated with inhibitors of p38 MAPK and JNK, ethanol-induced apoptosis was decreased while ROS generation was unaffected, and examination of pro-apoptotic Bax and anti-apoptotic Bcl-2 levels showed decrease of the former and increase of the latter. We concluded that oxidative stress inflicted by ROS triggered Fas-mediated and mitochondria-mediated apoptotic pathways in ethanol-treated SK-Hep1 cells, and that p38 MAPK and JNK were promoting mitochondrial pathway, suggesting interaction between apoptosis and MAPK signaling systems.

Abstract 2094: Effects of TGF-beta signaling inhibition with LY2157299 in hepatocarcinoma models and in ex vivo whole tumor tissue samples from patient specimen.

Abstract Background: Therapeutic options for patients with advanced hepatocarcinomas (HCC) are still limited. TGF-β1 and α-fetoprotein (AFP) have been shown to be independent prognostic factors for disease progression and recurrence. LY2157299, a selective ATP-mimetic inhibitor of TGF-β receptor (TβR)-I activation, is currently under clinical investigation in HCC patients. Our study aimed at exploring the effects of LY2157299 in HCC cell lines and patient samples with various AFP expression levels. Materials and Methods: Antiproliferative effects of LY2157299 were evaluated in a panel of human HCC cells by MTT assay. Baseline and phosphorylated protein levels were detected by Western blot analysis and mRNA expressions by qRT-PCR. Invasion assays were done on matrigel and in OptiCell devices. Tumor samples from HCC patients were surgically resected, cut in 300 μm thick slices using a Tissue Slicer. Each slice was randomly selected and exposed to LY2157299 (1μM and 10μM) and sorafenib (5μM) for 48h. Tumor samples were analyzed by immunohistochemistry (IHC) or immunofluorescence (IF). Results: LY2157299 was evaluated in HEPG2, HEP3B and SK-HEP1 cells as well as SK-HEP1-derived cells tolerant to sorafenib (SK-Sora) and sunitinib (SK-Suni). Exogenous stimulation of all HCC cell lines with TGF-β yielded downstream activation of p-Smad2 and p-Smad3 that was potently inhibited with LY2157299 treatment at micromolar concentrations. Low concentrations of LY2157299 displayed antiproliferative effects in HEPG2 cells when stimulated by TGF-β but not in SK-HEP1, SK-Sora, SK-Suni and HEP3B cells. Interestingly, HEPG2 cells were the only cell line displaying high levels of AFP as along with an epithelial phenotype. LY2157299 yielded potent anti-migratory and anti-invasive properties in invasive SK-HEP1, SK-Suni and SK-Sora cells. Tumor slices from surgically resected tumor samples from 3 patients with advanced HCC, were exposed ex vivo to 1 μM and 10 μM LY2157299 or 5 μM sorafenib for 48h. This method allows evaluating the effects of novel anticancer agents in whole tumors containing cancer cells and stroma cells. LY2157299 but not sorafenib decreased p-Smad2/3 downstream TGF-β signaling as well as AFP levels. IHC analysis of LY2157299 and sorafenib-exposed samples showed a significant decrease of the proliferative marker Ki67 and increase of the apoptotic marker caspase-3. Interestingly these effects were independent of AFP expression. Conclusion: TGF-β/TβR-I inactivation using LY2157299 inhibits TGF-β-dependent cell signaling in HCC cell lines with either anti-proliferative or anti-invasive effects depending on the model. In tumor samples from patients, inhibition of TGF-β signaling was associated with decreased AFP levels, inhibition of proliferation and apoptosis induction. Our data suggest that LY2157299 may be useful for patients with HCC. Citation Format: Marie Serova, Annemilaï Tijeras-Raballand, Celia Dos Santos, Nelly Muller, Karim A. Benhadji, Valerie Paradis, Sandrine Faivre, Eric Raymond, Armand de Gramont. Effects of TGF-beta signaling inhibition with LY2157299 in hepatocarcinoma models and in ex vivo whole tumor tissue samples from patient specimen. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2094. doi:10.1158/1538-7445.AM2013-2094