In vivo and In vitro effect of a nutrient mixture on human hepatocarcinoma cell line SK-HEP-1

Long-term survival of patients with hepatocellular carcinoma (HCC), a common cancer worldwide, remains poor, due to metastasis and recurrence. Aim: To investigate the effect of a novel nutrient mixture (NM) containing ascorbic acid, lysine, proline, and green tea extract on human HCC cell line Sk-Hep-1 In vivo and In vitro. Methods: After one week of isolation, 5–6 week old male athymic nude mice were inoculated with 3 x 106 SK-Hep-1 cells subcutaneously and randomly divided into two groups; group A was fed a regular diet and group B a regular diet supplemented with 0.5% NM. Four weeks later, the mice were sacrificed and their tumors were excised, weighed and processed for histology. We also tested the effect of NM In vitro on SK-Hep-1 cells, measuring cell proliferation by MTT assay, invasion through Matrigel, apoptosis by green caspase detection kit, MMP secretion by zymography, and morphology by H&E staining. Results: NM inhibited tumor weight and burden of SK-Hep-1 xenografts by 42% and 33% respectively. In vitro, NM exhibited 33% toxicity over the control at 500 and 1000 μg/ml concentration. Zymography demonstrated MMP-2 and MMP-9 secretion which was inhibited by NM in a dose dependent fashion, with virtual total inhibition at 1000 μg/ml. Invasion through Matrigel was inhibited at 100, 500 and 1000 μg/ml by 53%, 83% and 100% respectively. NM induced slight apoptosis at 100 μg/ml, and profound apoptosis at 500 μg/ml and 1000 μg/ml concentration. Conclusions: These results suggest that NM has therapeutic potential in treatment of HCC

Human bone marrow stromal cells have mitogenic activity on SK-Hep-1 cells.

Siu, Yeung Tung.Thesis (M.Phil.)--Chinese University of Hong Kong, 2001.Includes bibliographical references (leaves 65-75).Abstracts in English and Chinese.Title Page --- p.iAbstract in English --- p.iiAbstract in Chinese --- p.iiiAcknowledgement --- p.ivTable of Contents --- p.v-viiiList of Figures --- p.ixList of Tables --- p.xAbbreviations --- p.xi-xiiChapter Chapter 1 --- IntroductionChapter 1.1 --- Growth factors involved in hepatocytes proliferation --- p.1-6Chapter 1.1.1 --- Hepatocyte growth factor (HGF) --- p.1Chapter 1.1.2 --- Tumor necrosis factor-a (TNF-α) and interleukin-6 (IL-6) --- p.2Chapter 1.1.3 --- Epidermal growth factor (EGF) and transforming growth factor-α (TGF-α) --- p.3Chapter 1.1.4 --- Other comitogens --- p.4Chapter 1.1.5 --- Transforming growth factor-β (TGF-β) --- p.5Chapter 1.2 --- Bone marrow stromal cells and hepatocytes proliferation --- p.7-12Chapter 1.2.1 --- Role of bone marrow stromal cells in bone marrow --- p.7Chapter 1.2.2 --- Bone marrow as a source of hepatic oval cells --- p.8Chapter 1.2.3 --- Growth factors secreted by bone marrow stromal cells involved in hepatocytes proliferation --- p.9Chapter 1.2.4 --- Endocrine in hepatocytes proliferation --- p.12Chapter 1.3 --- Objective of this study --- p.13-15Chapter Chapter 2 --- Materials and MethodsChapter 2.1 --- Cell cultures --- p.16Chapter 2.2 --- Selection of human hepatic cell line for the detection of mitogenic activity --- p.17-18Chapter 2.2.1 --- "Enrichment of human hepatic cell lines, Hep 3B, Hep G2, C3A, SK-Hep-1 and Chang cells at G0-G1 phases by serum deprivation" --- p.17Chapter 2.2.2 --- "Incubation of serum deprived Hep 3B, Hep G2, C3A, SK- Hep-1 and Chang cells with mitogenic stimuli" --- p.17Chapter 2.2.3 --- Cell cycle analysis by flow cytometry using propidium iodide staining --- p.17Chapter 2.3 --- "Detection of mitogenic activity of human bone marrow stromal cells on the selected cell line, SK-Hep-1 cells" --- p.18-20Chapter 2.3.1 --- Partially growth arrested human SK-Hep-1 cells --- p.18Chapter 2.3.2 --- Human bone marrow stromal cells --- p.19Chapter 2.3.2.1 --- Bone marrow stromal cellular extract --- p.19Chapter 2.3.2.2 --- Total protein assay --- p.19Chapter 2.3.3 --- Incubation of SK-Hep-1 cells with bone marrow stromal cellular extracts --- p.20Chapter 2.4 --- Characterization of hepatocyte mitogenic activity of bone marrow stromal cellular extract --- p.21-22Chapter 2.4.1 --- Dialysis --- p.21Chapter 2.4.2 --- Temperature treatment --- p.21Chapter 2.4.3 --- Proteolysis --- p.22Chapter 2.5 --- Performing a preliminary test on the difference between bone marrow stromal cellular extract and other growth factors --- p.22-26Chapter 2.5.1 --- Incubation of SK-Hep-1 cells with bone marrow stromal cellular extract or other growth factors --- p.22Chapter 2.5.2 --- Metabolic labeling of SK-Hep-1 cells with [32P]orthophosphate --- p.23Chapter 2.5.3 --- Incubation of labeled SK-Hep-1 cells with bone marrow stromal cellular extract or other growth factors --- p.23Chapter 2.5.4 --- SK-Hep-1 cells lysate extraction --- p.23Chapter 2.5.5 --- Two-dimensional electrophoresis --- p.24Chapter 2.5.5.1 --- First dimension isoelectric focusing --- p.24Chapter 2.5.5.2 --- Second dimension sodium dodecyl sulfate-polyacrylamide gel electrophoresis --- p.25Chapter 2.5.6 --- Amplification of radiolabeled signal by EN3HANCE --- p.25Chapter 2.5.7 --- Visualization of autoradiography --- p.26Chapter 2.5.8 --- Visualization by silver staining --- p.26Chapter Chapter 3 --- ResultsChapter 3.1 --- Selection of human hepatic cell line for the detection of mitogenic activity --- p.27-30Chapter 3.1.1 --- "Enrichment of human hepatic cell lines, Hep 3B, Hep G2, C3A, SK-Hep-1 and Chang cells at G0-G1 phases by serum deprivation" --- p.27Chapter 3.1.2 --- DNA synthesis of hepatic cell lines in response to 10 % FBS after serum deprivation --- p.29Chapter 3.2 --- "Detection of mitogenic activity of human bone marrow stromal cells on the selected cell line, SK-Hep-1 cells" --- p.31-39Chapter 3.2.1 --- Cell cycle distribution of partially growth arrested SK-Hep-1 cells in response to mitogens --- p.31Chapter 3.2.2 --- Time course on DNA synthesis of partially growth arrested SK-Hep-1 cells in response to FBS and bone marrow stromal cellular extract --- p.36Chapter 3.2.3 --- Dose response on DNA synthesis of partially growth arrested SK-Hep-1 cells in response to bone marrow stromal cellular extracts --- p.38Chapter 3.3 --- Characterization of hepatocyte mitogenic activity of bone marrow stromal cellular extract --- p.40-44Chapter 3.4 --- Performing a preliminary test on the difference between bone marrow stromal cellular extract and other growth factors --- p.45-49Chapter 3.4.1 --- Mitogenic response of SK-Hep-1 cells in response to bone marrow stromal cellular extract and other growth factors --- p.45Chapter 3.4.2 --- Early intracellular signaling of SK-Hep-1 cells in response to bone marrow stromal cellular extract and other growth factors --- p.47Chapter Chapter 4 --- DiscussionChapter 4.1 --- Selection of human hepatic cell line for the detection of mitogenic activity --- p.50Chapter 4.2 --- "Mitogenic activity of human bone marrow stromal cells on the selected cell line, SK-Hep-1 cells" --- p.51Chapter 4.3 --- Characterization of hepatocyte mitogenic activity of bone marrow stromal cellular extract --- p.52Chapter 4.4 --- Performing a preliminary test on the difference between bone marrow stromal cellular extract and other growth factors --- p.53Chapter 4.5 --- Possible directions for future investigation --- p.55Chapter 4.6 --- Conclusions --- p.56Chapter Chapter 5 --- AppendicesChapter 5.1 --- Reagents and solutiuons --- p.57-64Chapter 5.1.1 --- Selection of human hepatic cell line for the detection of mitogenic activity --- p.57Chapter 5.1.2 --- "Detection of mitogenic activity of human bone marrow stromal cells on the selected cell line, SK-Hep-1 cells" --- p.59Chapter 5.1.3 --- Characterization of hepatocyte mitogenic activity of bone marrow stromal cellular extract --- p.60Chapter 5.1.4 --- Performing a preliminary test on the difference between bone marrow stromal cellular extract and other growth factors --- p.61Chapter Chapter 6 --- References --- p.65-7

Untersuchungen zur PAR1-Typ-Thrombinrezeptrovermittelten EGFR-Transaktivierung in humanen SK-HEP-1-Leberkarzinomzellen

Aufgrund unzureichender Behandlungsmöglichkeiten für das hepatozelluläre Karzinom ist man gegenwärtig mit der Aufklärung der molekularen Mechanismen der Hepatokarzinogenese befasst. Durch vorhergehende Ergebnisse der eigenen Arbeitsgruppe konnte auf eine mögliche Wirkung von PAR1 im Prozess der HCC-Progression geschlossen werden. Mit Untersu­chungen an der humanen Leberkarzinomzelllinie SK‑HEP‑1 sollte im Rahmen der vorliegenden Arbeit die Charakterisierung der Funk­tion von PAR1 in HCC-Zellen fortgesetzt werden. Nachdem PAR1 auf RNA-Ebene auf SK-HEP-1-Zellen nachgewiesen werden konnte, wurde der Effekt einer PAR1-Aktivierung auf die Transaktivierung des Rezeptors für den epidermalen Wachstumsfaktor (EGFR) charakterisiert. Es wurde die mit der Aktivierung des EGFRs ver­bundene Tyrosinphosphorylierung mit Hilfe von Immunpräzipitation und Western Blot mit einem anti-EGFR-Antikörper sowie einem gegen phosphoryliertes Tyrosin gerichteten Antikörper bestimmt. Es zeigte sich, dass PAR1 der entscheidende Rezeptor für die Vermittlung des Effekts von Thrombin auf die EGFR-Aktivierung in SK‑HEP‑1-Zellen ist. Auf zellulärer Ebene wurden Untersuchungen zur Zell­migration von SK-Hep-1-Zellen durch eine Kollagen-beschichtete Polycarbonatmembranbarriere in einer modifizierten Boydenkammer durchgeführt. Die Ergebnisse zeigten, dass PAR1 eine Steigerung der chemotaktischen Migration von SK‑HEP‑1-Zellen vermittelt, wobei eine EGFR-Transaktivierung am migratorischen PAR1-Signaling beteiligt ist. Die im Rahmen der Arbeit durchgeführten Untersuchungen zeigen, dass der Proteinase-aktivierte Rezeptor 1 in Zellen der Leberkarzinomzelllinie SK­-HEP‑1 über eine Wechselwirkung mit dem EGFR in die Regulation zellulärer Eigenschaften eingreift

Tumor necrosis factor receptor I blockade shows that TNF-dependent and independent mechanisms synergise in TNF receptor associated periodic syndrome

TNF receptor associated periodic syndrome (TRAPS) is an autoinflammatory disease involving recurrent episodes of fever and inflammation. It is associated with autosomal dominant mutations in TNF receptor superfamily 1A gene localised to exons encoding the ectodomain of the p55 TNF receptor, TNF receptor-1 (TNFR1). The aim of this study was to investigate the role of cell surface TNFR1 in TRAPS, and the contribution of TNF-dependent and TNF-independent mechanisms to the production of cytokines. HEK-293 and SK-HEP-1 cell lines were stably transfected with WT or TRAPS-associated variants of human TNF receptor superfamily 1A gene. An anti-TNFR1 single domain antibody (dAb), and an anti-TNFR1 mAb, bound to cell surface WT and variant TNFR1s. In HEK-293 cells transfected with death domain-inactivated (R347A) TNFR1, and in SK-HEP-1 cells transfected with normal (full-length) TNFR1, cytokine production stimulated in the absence of exogenous TNF by the presence of certain TNFR1 variants was not inhibited by the anti-TNFR1 dAb. In SK-Hep-1 cells, specific TRAPS mutations increased the level of cytokine response to TNF, compared to WT, and this augmented cytokine production was suppressed by the anti-TNFR1 dAb. Thus, TRAPS-associated variants of TNFR1 enhance cytokine production by a TNF-independent mechanism and by sensitising cells to a TNF-dependent stimulation. The TNF-dependent mechanism requires cell surface expression of TNFR1, as this is blocked by TNFR1-specific dAb

Diverse Effects of beta-Carotene on Secretion and Expression of VEGF in Human Hepatocarcinoma and Prostate Tumor Cells

Oral administration of beta-carotene (BC) was found to exert opposite effects on plasma levels of vascular endothelial growth factor (VEGF) in two animal models. One study in nude mice injected via tail vein with hepatocarcinoma SK-Hep-1 cells showed that BC decreases the plasma VEGF level, whereas the other study in nude mice injected subcutaneously with prostate tumor PC-3 cells showed that BC increases the plasma VEGF level. Herein we investigated whether BC (0.5-20 mu M) possesses diverse effects on VEGF secretion in SK-Hep-1, PC-3 and melanoma B16F10 cells. We found that incubation of SK-Hep-1 cells with BC (1-20 mu M) for 6 h significantly decreased VEGF secretion, whereas BC (1-10 mu M) significantly increased the VEGF secretion in PC-3 cells. However, these effects disappeared at 12 h of incubation. Similar effects occurred in VEGF mRNA and protein expression after treatment of SK-Hep-1 and PC-3 cells with BC for 6 h. In contrast, BC (0.5-20 mu M) did not affect mRNA and protein expression and secretion of VEGF in B16F10 cells. We also found that the proliferation of SK-Hep-1 and B16F10 cells was significantly inhibited by 20 mu M BC at 6 and 12 h of incubation, whereas the proliferation of PC-3 cells was significantly inhibited by 20 mu M BC at 12 h of incubation. In summary, the present study demonstrated the tumor-specific effect of BC on VEGF secretion in different cancer cell lines

Ethanol Increases NADPH Oxidase-derived Oxidative Stress and Induces Apoptosis in Human Liver Adenocarcinoma Cells (SK-HEP-1)

Alcohol-induced liver injury is linked to oxidative stress and increased production of reactive oxygen species (ROS). Oxidative stress is an early event in the process of apoptosis. However, it is not completely understood how ethanol-induced oxidative stress induces apoptosis. In contrast, nicotinamide adenine dinucleotide phosphate oxidase (NOX) is known to generate ROS in hepatocytes. The purpose of the present study was to determine whether or not ethanol-induced ROS generation stimulates the death receptor or mitochondrial pathways of apoptosis in alcohol dehydrogenase containing human liver adenocarcinoma (SK-HEP-1) cells. Treatment with ethanol increased the generation of ROS and expression of NOX4 mRNA, and also induced mitochondrial dysfunction in SK-HEP-1 cells. Moreover, ethanol induced the activation of caspase-8 and -3 in hepatocytes. These activities were suppressed by pretreatment with N-acetyl-cysteine, an antioxidant, or apocynin, an inhibitor of NOX activity. These results suggested that ethanol induces an increase in NOX-derived ROS generation upstream of caspase-8 activation and in the mitochondria in SK-HEP-1 cells. In conclusion, this study demonstrated that ethanol increases the generation of ROS and subsequently induces apoptosis using a mechanism involving mitochondrial dysfunction and caspase activation in SK-HEP-1 cells

WIP1 inhibition by GSK2830371 potentiates HDM201 through enhanced p53 phosphorylation and activation in liver adenocarcinoma cells

\ua9 2021 by the authors. Licensee MDPI, Basel, Switzerland. Background: Intrahepatic cholangiocarcinoma (iCCA) is an adenocarcinoma arising from the intrahepatic bile duct. It is the second most common primary liver cancer and has a poor prog-nosis. Activation of p53 by targeting its negative regulators, MDM2 and WIP1, is a potential therapy for wild-type p53 cancers, but few reports for iCCA or liver adenocarcinoma exist. Methods: Both RBE and SK-Hep-1 liver adenocarcinoma cell lines were treated with the HDM201 (Siremadlin) MDM2-p53 binding antagonist alone or in combination with the GSK2830371 WIP1 phosphatase inhibitor. Cell proliferation, clonogenicity, protein and mRNA expression, cell cycle distribution, and RNA sequencing were performed to investigate the effect and mechanism of this combination. Results: GSK2830371 alone demonstrated minimal activity on proliferation and colony formation, but potentiated growth inhibition (two-fold decrease in GI50) and cytotoxicity (four-fold decrease in IC50) by HDM201 on RBE and SK-Hep-1 cells. HDM201 increased p53 protein expression, leading to transactivation of downstream targets (p21 and MDM2). Combination with GSK2830371 increased p53 phosphorylation, resulting in an increase in both p53 accumulation and p53-dependent trans-activation. G2/M arrest was observed by flow cytometry after this treatment combination. RNA sequencing identified 21 significantly up-regulated genes and five downregulated genes following p53 reactivation by HDM201 in combination with GSK2830371 at 6 h and 24 h time points compared with untreated controls. These genes were predominantly known transcriptional targets regulated by the p53 signaling pathway, indicating enhanced p53 activation as the predominant effect of this combination. Conclusion: The current study demonstrated that GSK2830371 enhanced the p53-dependent antiproliferative and cytotoxic effect of HDM201 on RBE and SK-Hep-1 cells, providing a novel strategy for potentiating the efficacy of targeting the p53 pathway in iCCA

Synergism of Rana catesbeiana ribonuclease and IFN-γ triggers distinct death machineries in different human cancer cells

AbstractRana catesbeiana ribonuclease (RC-RNase) possesses tumor-specific cytotoxicity, which can be synergized by IFN-γ. However, it is unclear how RC-RNase and RC-RNase/IFN-γ induce cell death. In this study, we use substrate cleavage assays to systematically investigate RC-RNase- and RC-RNase/IFN-γ-induced caspase activation in HL-60, MCF-7, and SK-Hep-1 cells. We find that RC-RNase and RC-RNase/IFN-γ induce mitochondria-mediated caspase activation in HL-60 and MCF-7 cells but not in SK-Hep-1 cells, although death of SK-Hep-1 cells is closely related to mitochondrial disruptions. Our findings provide evidence that RC-RNase and RC-RNase/IFN-γ can kill different cancer cells by distinct mechanisms. Compared with onconase, RC-RNase seems to harbor a more specific anti-cancer activity

A pro-inflammatory signalome is constitutively activated by C33Y mutant TNF receptor 1 in TNF receptor-associated periodic syndrome (TRAPS)

Mutations in TNFRSF1A encoding TNF receptor 1 (TNFR1) cause the autosomal dominant TNF receptor-associated periodic syndrome (TRAPS): a systemic autoinflammatory disorder. Misfolding, intracellular aggregation, and ligand-independent signaling by mutant TNFR1 are central to disease pathophysiology. Our aim was to understand the extent of signaling pathway perturbation in TRAPS. A prototypic mutant TNFR1 (C33Y), and wild-type TNFR1 (WT), were expressed at near physiological levels in an SK-Hep-1 cell model. TNFR1-associated signaling pathway intermediates were examined in this model, and in PBMCs from C33Y TRAPS patients and healthy controls. In C33Y-TNFR1-expressing SK-Hep-1 cells and TRAPS patients' PBMCs, a subtle, constitutive upregulation of a wide spectrum of signaling intermediates and their phosphorylated forms was observed; these were associated with a proinflammatory/antiapoptotic phenotype. In TRAPS patients' PBMCs, this upregulation of proinflammatory signaling pathways was observed irrespective of concurrent treatment with glucocorticoids, anakinra or etanercept, and the absence of overt clinical symptoms at the time that the blood samples were taken. This study reveals the pleiotropic effect of a TRAPS-associated mutant form of TNFR1 on inflammatory signaling pathways (a proinflammatory signalome), which is consistent with the variable and limited efficacy of cytokine-blocking therapies in TRAPS. It highlights new potential target pathways for therapeutic intervention