Inhibited Hepatocellular Carcinoma Cell Growth Research Articles

Pegylated interferon's anti‐hepatoma effect is mediated by β‐catenin inhibition

Aberrant β-catenin activation, a main component of the Wnt pathway, has been implicated in liver cancer. It also promotes cell proliferation and hence is a logical therapeutic target in cancers. Here, we investigate the effect of Ribavirin & Pegasys, alone or in combination, on β-catenin. Other reports have shown an anti-tumor role of these anti-HCV agents, however their effect on Wnt/β-catenin pathway is unknown. This is of particular relevance based on recent observations of decreased HCC incidence in HCV patients undergoing anti-HCV treatment. We examined the effect of these drugs on morphology of hepatoma cells & on the Wnt pathway components. Short-term experiments failed to detect any effect of Ribavirin on morphology or total & nuclear β-catenin. Administration of 3 doses of pegylated interferon demonstrated a 50% decrease in nuclear β-catenin protein. Based on these results, Hep3B and HepG2 cells were treated long-term (10–12 doses) with Pegasys (+/− Ribavirin). A loss of cell viability was observed at 6–8 doses. Western blot exhibited a dramatic decrease in total & active β-catenin protein. GSK3β, a kinase that phosphorylates β-catenin to induce its degradation, underwent a significant activation following the combination therapy only. Also, Pegasys significantly affected β-catenin gene expression to 20% of the control by real time PCR. Thus Pegasys alone or in combination with Ribavirin significantly inhibits hepatoma cell growth and viability and appears to do so at least in part by β-catenin inhibition via multi-factorial mechanisms.

Use of Adenovirus-Delivered siRNA to Target Oncoprotein p28 GANK in Hepatocellular Carcinoma

RNA interference (RNAi) is a powerful tool to silence gene expression. The adenoviral vector expressing small interfering RNA (siRNA) is highly effective in mammalian cells. However, its potential use as a therapeutic tool to target an oncogene specifically remains to be seen. We applied the adenovirus-delivered siRNA (AdSiRNA) to inhibit a hepatocellular carcinoma (HCC) oncogene, p28GANK, in HCC cell lines and investigated its antitumor effects. The T7-RNA polymerase system was used to screen the specific target site. Double-strand oligonucleotide for transcription of short hairpin RNA was constructed into the adenoviral vector. Four HCC cell lines were infected with the RNAi-containing adenovirus. The RNAi effects on HCC were studied in cultured cells as well as in animal models. p28GANK expression was suppressed by up to 80% in HCC cells. Depletion of p28GANK inhibited HCC cell growth and tumorigenesis, enhanced dephosphorylation of RB1, and decreased transcription activity of E2F-1 in HuH-7 cells. Furthermore, depletion of p28GANK induced caspase-8- and caspase-9-mediated apoptosis of HCC cells. Finally, targeting p28GANK by adenovirus injection inhibited the growth of established tumors in nude mice. This study shows that the T7-system screening-based AdSiRNA can be used successfully to silence an oncogene. We proved the therapeutic potential of AdSiRNA on the treatment of HCC by targeting p28GANK. Our results indicate that p28GANK may serve as a novel therapeutic target for treating HCC.

Cell cycle arrest and apoptosis induction in hepatocellular carcinoma cells by HMG-CoA reductase inhibitors. Synergistic antiproliferative action with ligands of the peripheral benzodiazepine receptor

Hepatocellular carcinoma (HCC) is the fifth most common cause of cancer deaths worldwide. Inhibitors of cholesterol biosynthesis ('statins') have been proposed as promising adjunctive anticancer agents to treat HCC, but their mode of action is yet poorly characterized. We additionally investigated the potential benefit of a combination of peripheral benzodiazepine receptor (PBR) ligands and statins. We analyzed the growth inhibitory effects of PBR ligands, statins, and their combination in two human HCC cell lines. Moreover, we investigated the regulation of cellular cholesterol levels and the expression of 3-hydroxy-3-methylglutaryl coenzyme-A reductase (HMG-CoAR), the target of statins. Statins inhibited the proliferation of HCC cells by inducing apoptosis and G1/S cell cycle arrest. Statin-induced apoptosis was characterized by a breakdown of the mitochondrial membrane potential, caspase activation and nuclear degradation. Furthermore, activation of ERK1/2 was downregulated while p38MAPK was activated. Synergistic growth inhibition was obtained by the combination of the PBR ligand FGIN-1-27 with statins. PBR ligands induced a decrease of HMG-CoAR expression. This downregulation may be responsible for the enhanced sensitivity of HCC cells to statins. Our data shed light on the signaling cascades mediating statin-induced growth inhibition of HCC cells. Moreover, PBR ligands sensitized HCC cells to statins, suggesting a new strategy to treat HCC.

Laminin-5 offsets the efficacy of gefitinib (‘Iressa’) in hepatocellular carcinoma cells

Prognosis and survival of patients with hepatocellular carcinoma (HCC) is still very poor, and no therapies are currently available to inhibit tumour growth and metastases. Recently, we reported that the expression of an extracellular matrix component (ECM), namely Laminin-5 (Ln-5), is directly related to poor prognosis in HCC patients. The aim of our study is to investigate the preclinical effect of gefitinib in an in vitro HCC model. We found that the IC50 of gefitinib in HCC cells ranged from 0.7 to 10.0 μM, whereas Ln-5 inhibited the activity of gefitinib in a dose-dependent manner. Complete inhibition of phosphorylated (p)-EGFR (epidermal growth factor receptor) was obtained within 6 h exposure to gefitinib and complete restoration of the receptor status was obtained after 24 h. A downstream effect yields a decrease in p-Akt and p-Erk 1/2. The addition of exogenous Ln-5 has no effect on p-EGFR, whereas it restores p-Erk 1/2 and p-Akt. Consistently, Ln-5 induces recovery of HCC cells from Gefitinib-induced apoptosis. In conclusion, gefitinib inhibits HCC cell growth and we report for the first time that Ln-5, but not other ECM molecules, reduces the ability of gefitinib to inhibit cell growth via Akt. As patients with HCC have different Ln-5 expression levels, these results may help to better understand which patients might benefit from gefitinib treatment.

The combination of tamoxifen and 9cis retinoic acid exerts overadditive anti-tumoral efficacy in rat hepatocellular carcinoma

Background/Aims Medical treatment for hepatocellular carcinoma (HCC) remains elusive. While an acyclic retinoid improved tumor-free survival after hepatoma resection, tamoxifen finally proved ineffective. Combination therapy of both agents has not been investigated in vitro and in vivo. Methods MH7777A hepatoma cells were incubated with tamoxifen (TAM) and 9-cis retinoic acid (CRA) alone or in combination. Proliferation rate and apoptosis were assessed by BrdU incorporation and flow cytometry. In vivo efficacy was studied using the Morris hepatoma model in immunocompetent rats. End points were macroscopic tumor growth, metastasis and immunohistochemistry for proliferative and apoptotic tumor cells (PCNA and TUNEL staining). Results In vitro, CRA and TAM monotherapy was effective only in the highest concentration. Combination therapy significantly enhanced apoptosis rate and growth inhibition in hepatoma cells. While in vivo monotherapy did not reduce tumor growth or metastasis, their combination reduced tumor size after 28 days by 64.5±28%. This was paralleled by an increase in TUNEL positive and a decrease in PCNA positive cells. Conclusions The combination of TAM and CRA enhances their anti-tumoral efficacy in vitro as well as in vivo, while monotherapy is ineffective. This combination could be a promising adjunctive therapy of HCC.

Potentiated anticancer effects on hepatoma cells by the retinoid adapalene

Retinoids can block cell proliferation and induce apoptosis in tumor cells. The antitumoral effect of synthetic retinoids like Adapalene (ADA) on hepatoma cells (HepG2, Hep1B) was investigated. Cell proliferation was assessed by measuring DNA synthesis and apoptosis by flow cytometry and immunocytochemistry. Cell cycle- and apoptosis-associated proteins were semi-quantified by Western Blotting and breakdown of mitochondrial membrane potentials was detected by JC-1 staining. ADA at 10 −4 M efficiently induced apoptosis, reaching 61.7% in HepG2 and 79.1% in Hep1B after 72 h incubation. This was accompanied by up-regulation of pro-apoptotic bax and caspase 3, while bcl-2 was down-regulated, shifting the bax/bcl-2 ratio to >2.3 in hepatoma cells. ADA inhibits hepatoma cell growth in vitro and is a powerful inducer of hepatoma cell apoptosis.

The combination of tamoxifen and 9cis retinoic acid exerts overadditive anti-tumoral efficacy in rat hepatocellular carcinoma

Background/Aims Medical treatment for hepatocellular carcinoma (HCC) remains elusive. While an acyclic retinoid improved tumor-free survival after hepatoma resection, tamoxifen finally proved ineffective. Combination therapy of both agents has not been investigated in vitro and in vivo. Methods MH7777A hepatoma cells were incubated with tamoxifen (TAM) and 9-cis retinoic acid (CRA) alone or in combination. Proliferation rate and apoptosis were assessed by BrdU incorporation and flow cytometry. In vivo efficacy was studied using the Morris hepatoma model in immunocompetent rats. End points were macroscopic tumor growth, metastasis and immunohistochemistry for proliferative and apoptotic tumor cells (PCNA and TUNEL staining). Results In vitro, CRA and TAM monotherapy was effective only in the highest concentration. Combination therapy significantly enhanced apoptosis rate and growth inhibition in hepatoma cells. While in vivo monotherapy did not reduce tumor growth or metastasis, their combination reduced tumor size after 28 days by 64.5±28%. This was paralleled by an increase in TUNEL positive and a decrease in PCNA positive cells. Conclusions The combination of TAM and CRA enhances their anti-tumoral efficacy in vitro as well as in vivo, while monotherapy is ineffective. This combination could be a promising adjunctive therapy of HCC.

Down-regulation of c-myc and Cyclin D1 genes by antisense oligodeoxy nucleotides inhibits the expression of E2F1 and in vitro growth of HepG2 and Morris 5123 liver cancer cells.

A number of genetic interactions are involved in the control of cell cycle, but their role and nature have not been completely clarified. The knowledge of the behavior of these interactions in hepatocellular carcinoma, could optimize preventive and therapeutic strategies based on cell cycle restraint. We studied downstream events following c-MYC and CYCLIN D1 gene inhibition, by lipoplex-delivered MYC and CYCLIN D1 antisense oligodeoxy nucleotides (aODNM, aODND1), in in vitro cultured human HepG2 and rat Morris 5123 hepatoma cells. 0.5-20 micro M aODN(M) and aODND1 inhibited in vitro growth of both cell types. Scramble oligomer (SCR) and sense ODNs had no or relatively poor effect. Ten micromolar aODNM and aODND1, but not SCR, also induced a significant increase in the apoptotic index of HepG2 and 5123 cells, and inhibited colony formation in soft agar by HepG2 cells. Treatment of the cells with aODNM plus aODND1 had no additive effect on growth and apoptosis. aODNM and aODND1 induced >50% decrease in c-MYC and CYCLIN D1 gene expression, respectively, at both mRNA and protein level. The inhibition of gene expression by aODNs was highly specific, and SCR was without effect. The reduction in c-MYC and CYCLIN D1 expression by aODNs, was associated with a >50% decrease in E2F1 mRNA and protein production, without changes in CYCLIN A and CYCLIN E expression. These results suggest the involvement of both c-MYC and CYCLIN D1 on E2F1 gene function, and indicate that aODNM and aODND1 may inhibit hepatoma cell growth through down-regulation of the E2F1 gene. The inhibition of E2F1 gene expression by E2F1 aODN, was associated with strong growth restraint of HepG2 cells. Thus, interactions of c-MYC and CYCLIN D1 with E2F1 gene are essential for cell cycle activity in hepatoma cells, and their inhibition may have a therapeutic effect.

Antisense oligonucleotides against aldehyde dehydrogenase 3 inhibit hepatoma cell proliferation by affecting MAP kinases

The increased activity of enzymes that eliminate anti-tumour drugs or their metabolites is one of the important limiting factors in therapeutic protocols. Among these enzymes, aldehyde dehydrogenase 3 (ALDH3) is considered a mechanism by which tumour cells evade the cytotoxic effects exerted by cyclophosphamide and drugs acting by free radical generation. It is also important in metabolising cytostatic aldehydes derived from lipid peroxidation. Therefore, ALDH3 may play a role in regulating cell proliferation in tumour cells with high activity of this enzyme. We previously reported that antisense oligonucleotides (AS-ODN) against ALDH3 strongly inhibit hepatoma cell growth, suggesting that this effect could be due to the accumulation of cytostatic aldehydes in the cells. In this research we demonstrate that AS-ODN against ALDH3 increase the quantity of malondialdehyde in the cells, and inhibit cell proliferation by affecting the MAPK pathway: a reduction of pRaf-1 and pERK1,2 was observed. These results confirm the importance of aldehydes derived from lipid peroxidation and of ALDH3 in regulating hepatoma proliferation. Moreover, the results indicate the use of AS-ODN against ALDH3 as a possible strategy to reduce growth in tumours overexpressing this enzyme.

Oxidative Stress-mediated Apoptosis: THE ANTICANCER EFFECT OF THE SESQUITERPENE LACTONE PARTHENOLIDE

The sesquiterpene lactone parthenolide, the principal active component in medicinal plants, has been used conventionally to treat migraines, inflammation, and tumors. However, the antitumor effects of parthenolide and the mechanism(s) involved are poorly understood. We found that parthenolide effectively inhibits hepatoma cell growth in a tumor cell-specific manner and triggers apoptosis of hepatoma cells. Parthenolide triggered apoptosis in invasive sarcomatoid hepatocellular carcinoma cells (SH-J1) as well as in other ordinary hepatoma cells at 5-10 microm concentrations and arrested the cell growth (at G(2)/M) at sublethal concentrations (1-3 microm). During parthenolide-induced apoptosis, depletion of glutathione, generation of reactive oxygen species, reduction of mitochondrial transmembrane potential, activation of caspases (caspases-7, -8, and -9), overexpression of GADD153 (an oxidative stress or anticancer agent inducible gene), and subsequent apoptotic cell death was observed. This induced apoptosis could be effectively inhibited or abrogated by an antioxidant N-acetyl-l-cysteine, whereas l-buthionine-(S,R)-sulfoximine enhanced it. Furthermore, stable overexpression of GADD153 sensitized the cells to apoptosis induced by parthenolide, and this susceptibility could be reversed by transfection with an antisense to GADD153. Parthenolide did not alter the expression of Bcl-2 or Bcl-X(L) proteins during apoptosis in hepatoma cells. Oxidative stress may contribute to parthenolide-induced apoptosis and to GADD153 overexpression in a glutathione-sensitive manner. The sensitivity of tumor cells to parthenolide appears to result from the low expression level of the multifunctional detoxification enzyme glutathione S-transferase pi. Finally, parthenolide and its derivatives may be useful chemotherapeutic agents to treat these invasive cancers.

Induction of apoptosis in hepatocellular carcinoma cell lines by emodin.

Previous experiments have shown that emodin is highly active in suppressing the proliferation of several tumor cell lines. However, it is not clear that emodin can induce growth inhibition of hepatoma cells. We have found that emodin induces apoptotic responses in the human hepatocellular carcinoma cell lines (HCC) Mahlavu, PLC/PRF/5 and HepG2. The addition of emodin to these three cell lines led to inhibition of growth in a time‐and dose‐dependent manner. Emodin generated reactive oxygen species (ROS) in these cells which brought about a reduction of the intracellular mitochondrial transmembrane potential (ΔΨm), followed by the activation of caspase–9 and caspase–3, leading to DNA fragmentation and apoptosis. Our findings demonstrate that ROS and the resulting oxidative stress play a pivotal role in apoptosis. Preincubation of hepatoma cell lines with the hydrogen peroxide‐scavenging enzyme, catalase (CAT) and cyclosporin A (CsA), partially inhibited apoptosis. These results demonstrate that enhancement of generation of ROS, ΔΨmdisruption and caspase activation may be involved in the apoptotic pathway induced by emodin.

Assessment of growth inhibition and morphological changes in in vitro and in vivo hepatocellular carcinoma models post treatment with dl1520 adenovirus.

E1B-deleted virus dl1520 (ONYX-015) has been previously used in clinical trials mainly for treatment of head and neck tumors, and has been shown to have beneficial effects independent of p53 status. The main aim of this investigation was to carry out a preclinical study for assessment of the use of dl1520 in in vitro and in vivo hepatocellular carcinoma (HCC) models with various p53 status (deleted, mutant, and wild type), and study the ultrastructural changes in the carcinoma cells during and following treatment with dl1520. dl1520 (ONYX-015) virus was used for treatment of three HCC cell lines in culture, then for treatment of developed xenografts in SCID mice. The effects of dl1520 on HCC cell growth and accompanied morphological changes were assessed by various techniques including transmission electron microscopy. dl1520 infection was confirmed using polymerase chain reaction and immunolabeling at transmission electron microscopy level. dl1520 was effective in killing cells and inhibiting HCC cell growth both in vitro and in vivo. The cell killing was at higher levels in cells possessing abnormal p53. Survival rates in SCID mice treated with dl1520 were statistically significantly higher in HCC tumors with deleted and mutant p53, than in tumors with wild-type p53. The findings in this study suggest that dl1520 could be safely and effectively used for treatment of HCC dependent on the p53 status of the cells in vivo. Characteristic morphological changes that took place in the dl1520-treated HCC cells/tumors were distinct at transmission electron microscopy level and are the first of their kind to be reported.

The histone-deacetylase inhibitor Trichostatin A blocks proliferation and triggers apoptotic programs in hepatoma cells

Background/Aims : Effective treatment for hepatocellular carcinoma is urgently needed. The histone-deacetylase inhibitor Trichostatin A (TSA) was shown to induce apoptosis in non-hepatic cells at submicromolar concentrations. However, the effect of TSA on hepatoma cells is unknown. Methods : The hepatoma cells HepG2, MH1C1, Hepa1–6 and Hep1B as well as human fibroblasts (control cells) were exposed to TSA (10 −6 to 10 −9 M). Cell proliferation was assessed by measuring DNA-synthesis and cell numbers. Apoptosis was quantified by flow cytometry and by the TdT-mediated dUTP nick-end labeling method. Expression patterns of cell cycle- and/or apoptosis-associated p27, p21 cip/waf , bax, bcl-2, cyclin A and (pro)-caspase 3 were studied using quantitative Western blotting. Activation of caspase 3 was analyzed via a colorimetric assay. Results : 10 −6 M TSA inhibited DNA-synthesis by 46% (HepG2) to 64% (MH1C1) after 24 h, inducing a G 2 /M-phase arrest and apoptosis. TSA increased activation of caspase 3 and expression of cyclin A, p2l cip/waf , bax and (pro)-caspase 3, while bcl-2 was downregulated. Human fibroblasts remained unaffected. Conclusions : TSA inhibits hepatoma cell growth in vitro, which are otherwise particularly resistant to chemotherapy. Its anti-proliferative activity is paralleled by a comparable rate of apoptosis. TSA may be a promising agent for treatment of hepatocellular carcinoma in vivo.

Comparison of 2‐methoxyestradiol‐induced, docetaxel‐induced, and paclitaxel‐induced apoptosis in hepatoma cells and its correlation with reactive oxygen species

BACKGROUND Previously, the authors observed that paclitaxel treatment of hepatoma cells resulted in differential cytotoxicity. Whether other antimicrotubule agents (docetaxel and 2-methoxyestradiol) are more effective than paclitaxel is not clear. Moreover, whether the modulation of reactive oxygen species (ROS) is involved in the drug-induced growth inhibition of hepatoma cells is not known. METHODS The authors examined the effects of 2-methoxyestradiol, paclitaxel, and docetaxel on HepG2, Hep3B, HA22T/VGH, and Hepa1-6 hepatoma cell lines. The parameters examined included cell viability, cell membrane permeability, cell cycle distribution, DNA fragmentation, and ROS generation. RESULTS Docetaxel and paclitaxel inhibited the growth of hepatoma cells at submicromolar concentrations, whereas that of 2-methoxyestradiol was within a micromolar range. This drug-induced growth inhibition was cell cycle dependent. 2-Methoxyestradiol-treated (10–50 μM) cells resulted in G2/M block prior to apoptosis. High dose (0.1 μM) docetaxel- and paclitaxel-treated cells resulted in a G2/M arrest followed by generation of polyploidy or apoptosis; however, low dose (0.01 μM) treatment induced apoptosis without G2/M arrest. The low dose effect was more significant in docetaxel-treated cells than in paclitaxel-treated cells. Although these antimicrotubule agents increased the formation of ROS, antioxidant treatment did not block drug-induced cell cycle and growth inhibition effects. CONCLUSIONS The current results suggest that the growth inhibition of hepatoma cells induced by 2-methoxyestradiol, paclitaxel, and docetaxel was mediated through G2/M-phase arrest, caspase activation, and DNA fragmentation. The drug-induced apoptosis was independent of ROS formation. Docetaxel was more effective than paclitaxel in killing hepatoma cells. The potential of using 2-methoxyestradiol and docetaxel for the treatment of patients with hepatoma is worthy of further study. Cancer 2000;89:983–94. © 2000 American Cancer Society.

Comparison of 2-methoxyestradiol-induced, docetaxel-induced, and paclitaxel-induced apoptosis in hepatoma cells and its correlation with reactive oxygen species

Previously, the authors observed that paclitaxel treatment of hepatoma cells resulted in differential cytotoxicity. Whether other antimicrotubule agents (docetaxel and 2-methoxyestradiol) are more effective than paclitaxel is not clear. Moreover, whether the modulation of reactive oxygen species (ROS) is involved in the drug-induced growth inhibition of hepatoma cells is not known. The authors examined the effects of 2-methoxyestradiol, paclitaxel, and docetaxel on HepG2, Hep3B, HA22T/VGH, and Hepa1-6 hepatoma cell lines. The parameters examined included cell viability, cell membrane permeability, cell cycle distribution, DNA fragmentation, and ROS generation. Docetaxel and paclitaxel inhibited the growth of hepatoma cells at submicromolar concentrations, whereas that of 2-methoxyestradiol was within a micromolar range. This drug-induced growth inhibition was cell cycle dependent. 2-Methoxyestradiol-treated (10-50 microM) cells resulted in G2/M block prior to apoptosis. High dose (0.1 microM) docetaxel- and paclitaxel-treated cells resulted in a G2/M arrest followed by generation of polyploidy or apoptosis; however, low dose (0.01 microM) treatment induced apoptosis without G2/M arrest. The low dose effect was more significant in docetaxel-treated cells than in paclitaxel-treated cells. Although these antimicrotubule agents increased the formation of ROS, antioxidant treatment did not block drug-induced cell cycle and growth inhibition effects. The current results suggest that the growth inhibition of hepatoma cells induced by 2-methoxyestradiol, paclitaxel, and docetaxel was mediated through G2/M-phase arrest, caspase activation, and DNA fragmentation. The drug-induced apoptosis was independent of ROS formation. Docetaxel was more effective than paclitaxel in killing hepatoma cells. The potential of using 2-methoxyestradiol and docetaxel for the treatment of patients with hepatoma is worthy of further study.

Effects of a Hot-Water Extract of Trametes versicolor (L.: Fr.) Lloyd (Aphyllophoromycetideae) on the Recovery of Rat Liver Function

To investigate influences of Trametes versicolor on hepatocarcinoma and regenerated normal hepatocytes, hot-water extract (CVA) and ethanol-soluble (CVB) and ethanol-insoluble (CVC) fractions of its basidiocarps were examined for growth inhibition of hepatoma cells and primary culture of hepatocytes isolated from regenerated rat liver at 24 h after partial hepatectomy were studied for effects on DNA, RNA, protein, and collagen biosynthesis. Fractions CVA, CVB, and CVC inhibited the growth of the SK-Hep-1 cell line at a concentration of 100 μg/ml. The 50% inhibitory concentrations (IC50) of CVA, CVB, and CVC fractions against SK-Hep-1 were 36.9, 64.2, and 37.1 μg/ml, respectively. In DNA biosynthesis ability that was measured by incorporation of [3H]thymidine in the primary cultured hepatocytes, fractions CVA and CVC augmented DNA biosynthesis by 1.5- and 1.4-fold of that of the control, respectively. Fractions CVA and CVC also increased RNA biosynthesis ability to 1.5- and 1.8-fold of that of the control, respectively. Fractions CVA and CVC potentiated protein biosynthesis ability to 1.4- and 1.5-fold to that of the control, respectively. Stimulatory effects of both CVA and CVC fractions on collagen biosynthesis were 1.2-fold of that of the control, respectively. In summary, it was found that all three fractions significantly inhibited human hepatoma cell growth in vitro at a concentration of 100 μg/ml and fractions CVA and CVC significantly restored the function of the regenerated hepatocytes. Therefore, the high molecular weight fraction, CVC, showed inhibitory action on the hepatoma cell and stimulatory or recovery functions of normal hepatocytes. Therefore the high molecular weight fraction of T. versicolor has the potential to be developed as a hepatoprotective agent.

Inhibition of Hepatoma Cell Growth in Vitro by Arylating and Non-arylating K Vitamin Analogs: SIGNIFICANCE OF PROTEIN TYROSINE PHOSPHATASE INHIBITION

We recently found that a thioether analog of K vitamin (Cpd 5) inhibited the activity of protein-tyrosine phosphatases (PTPases) and induced protein-tyrosine phosphorylation in a human hepatoma cell line (Hep3B). We have now examined the structural requirements for induction of protein-tyrosine phosphorylation and PTPase inhibition by several K vitamin analogs. Thioether analogs with sulfhydryl arylation capacity, especially those with a hydroxy (Cpd 5) or a methoxy group at the end of the side chain, induced protein-tyrosine phosphorylation, but non-arylating analogs, such as those with an all-carbon or O-ether side chain, did not. Among the receptor-tyrosine kinases, epidermal growth factor receptors were tyrosine-phosphorylated by treatment with thioether analogs, whereas insulin and hepatocyte growth factor receptors were not. An increase in tyrosine-phosphorylated ERK2 mitogen-activated protein kinase was also observed. The activity of purified T cell PTPase was inhibited only by the thioether analogs, but not by non-arylating analogs. Furthermore, the epidermal growth factor receptor dephosphorylation activity of Hep3B cell lysates was inhibited by Cpd 5 treatment. A similar induction of protein-tyrosine phosphorylation by Cpd 5 was seen in other human hepatoma cell lines together with growth inhibition. However, one cell line (HepG2), which was relatively resistant to growth inhibition by Cpd 5, did not increase its phosphorylation levels upon Cpd 5 treatment. These results suggest that cell growth inhibition by thioether analogs is closely associated with inhibition of PTPases by sulfhydryl arylation and with tyrosine phosphorylation of selected proteins.

Tamoxifen inhibits hepatoma cell growth through an estrogen receptor independent mechanism

Abstract Background/Aims: Tamoxifen has previously been shown to prolong the survival of patients with advanced stages of hepatocellular carcinoma and it has been suggested that it inhibits the growth of hepatoma cells through an estrogen receptor-dependent mechanism. We have studied the effects of the synthetic estrogen, mestranol, and the antiestrogen, tamoxifen, on the growth regulation of hepatoma cells in vitro . Methods: Cells were maintained under fully estrogenized conditions and were deprived of estrogen shortly before conducting experiments. Results: In the human hepatoma cell line Hep 3B, tamoxifen inhibited cell growth in a concentration and time-dependent manner with effective concentrations ranging from 0.1 μM to 10 μM. Mestranol inhibited cell growth at a concentration of 10 μM and had an additive effect with tamoxifen on growth inhibition. Expression of estrogen receptors in hepatoma cells was not detected by enzyme immunoassay, Northern blot analysis or reporter gene expression assay. Furthermore, the introduction of estrogen receptors into Hep 3B cells did not alter the effect of tamoxifen and mestranol on cell growth. Conclusions: This study suggests that tamoxifen inhibits the growth of Hep 3B hepatoma cells through an estrogen receptor-independent mechanism.

Tamoxifen inhibits hepatoma cell growth through an estrogen receptor independent mechanism

Background/Aims: Tamoxifen has previously been shown to prolong the survival of patients with advanced stages of hepatocellular carcinoma and it has been suggested that it inhibits the growth of hepatoma cells through an estrogen receptor-dependent mechanism. We have studied the effects of the synthetic estrogen, mestranol, and the antiestrogen, tamoxifen, on the growth regulation of hepatoma cells in vitro. Methods: Cells were maintained under fully estrogenized conditions and were deprived of estrogen shortly before conducting experiments. Results: In the human hepatoma cell line Hep 3B, tamoxifen inhibited cell growth in a concentration and time-dependent manner with effective concentrations ranging from 0.1 μM to 10 μM. Mestranol inhibited cell growth at a concentration of 10 μM and had an additive effect with tamoxifen on growth inhibition. Expression of estrogen receptors in hepatoma cells was not detected by enzyme immunoassay, Northern blot analysis or reporter gene expression assay. Furthermore, the introduction of estrogen receptors into Hep 3B cells did not alter the effect of tamoxifen and mestranol on cell growth. Conclusions: This study suggests that tamoxifen inhibits the growth of Hep 3B hepatoma cells through an estrogen receptor-independent mechanism.

Growth Inhibition of Hepatoma Cells Induced by Vitamin K and Its Analogs

Congeners of vitamin K are known to inhibit cell growth, although the precise mechanisms of growth inhibition are not well understood. To investigate the mechanisms involved, we synthesized several vitamin K analogs and examined their growth inhibitory activities for a human hepatoma cell line (Hep3B). The analogs included 2-methyl-1,4-naphthoquinone and trimethyl-benzoquinone, with and without aliphatic side chains at position 3. The side chains were all-carbon, thioethers, or O-ethers. Growth inhibition was potent in the compounds with short chains. The presence of a sulfur (thioether) or oxygen atom (O-ether) at the site of attachment of the side chain to the ring potentiated the activity. Apoptotic cell death was induced by the potent growth inhibitory compounds at low concentrations (20-60 microM), whereas necrotic cell death followed treatment with the same compounds at high concentrations. Expression of c-myc, which is thought to be associated with apoptosis, was increased by most of the compounds tested. Both reduced glutathione and cysteine almost completely abrogated the growth inhibitory effects of the thioether analogs as well as of vitamin K3. The effect of glutathione was less prominent for the all-carbon and O-ether analogs, and cysteine had no effect on these analogs. Catalase and deferoxamine mesylate had no significant effect on the thioether analogs, although they showed partial antagonistic effects on the growth inhibition of vitamin K3 and the all-carbon and O-ether analogs. Other non-thiol antioxidants tested had no effect on any of the analogs. Our results indicated that vitamin K-related quinoid compounds cause growth inhibition and both apoptotic and necrotic cell death and that the effects may be mediated by interaction at position 3 of their quinoid nuclei with cellular thiols.