Absence of cytokeratin in human hepatoma cell lines

The immunofluorescence study revealed that both our established human hepatoma cell lines, HA22T/VGH and HA47T/VGH, were absent of cytokeratin. This observation was further confirmed by a western blot study. However, they as well as the other human hepatoma cells, Hep G2, Hep 3B, and SK-Hep-1 expressed vimentin.     

The Chinese medicine Bu-Zhong-Yi-Qi-Tang inhibited proliferation of hepatoma cell lines by inducing apoptosis via G0/G1 arrest

Bu-Zhong-Yi-Qi-Tang (BZYQT), a Chinese herbal medicine, inhibited the proliferation of human hepatoma cell lines (Hep3B, HepG2 and HA22T) dose-dependently. The IC50s of BZYQT on the proliferation of Hep3B, HepG2 and HA22T were 432.5+/-31.8 microg/ml, 455.4+/-24.2 microg/ml, and 2284.3+/-77.2 microg/ml respectively on day 3. However, BZYQT did not significantly inhibit the proliferation of normal human hepatocytes (Chang liver, CCL-13) at the concentration under 5,000 microg/ml. Major compounds of BZYQT, including astragaloside IV, ginsenoside Rb1 and Rg1, saikosaponin a and c, and glycyrrhizin, have been identified. To investigate the key inhibitors of BZYQT. Hep3B cells were treated with BZYQT, individual major compounds of BZYQT, and mixture of major compounds in the same ratio as present in BZYQT. Significant inhibition of proliferation was detected in BZYQT and its major compounds mixture in a comparable level. Not any individual major compound examined could suppress the proliferation of Hep3B cells. This data indicated that there could be synergistic or additive effects of the ingredients in BZYQT. BrdU incorporation, cell cycle analysis and DNA fragmentation assay revealed that BZYQT suppressed the proliferation of hepatoma cells via G0/G1 cell cycle arrest and inhibition of DNA synthesis followed by apoptosis.     

Increase of Bax/ Bcl-XL ratio and arrest of cell cycle by luteolin in immortalized human hepatoma cell line

Luteolin is a common constituent of many kinds of fruits and vegetables. It possesses the anti-neoplastic activities against several human cancers, but its activity against hepatocellular carcinoma (HCC) is seldom mentioned. To evaluate the activity against HCC and to provide information about the mechanism, we tested luteolin against five human hepatoma cell lines, namely HepG2, SK-Hep-1, PLC/PRF/5, Hep3B, and HA22T/VGH, with XTT assay and flow cytometry. The results showed that luteolin inhibited PLC/PRF/5, Hep3B and HA22T/VGH at a concentration of 1 microg/ml, but it needed 5 microg/ml to inhibit HepG2 and 10 microg/ml for SK-Hep1 (P <0.05). The inhibitive concentrations of 50% (IC50) of luteolin were between 7.29 microg/ml and 32.59 microg/ml, which were comparable with those of 5-FU (15.35 microg/ml to 32.84 microg/ml). The least effective cell line as affected by luteolin (SK-Hep1) was the most effective one when treating with 5-FU. The least effective cell line as affected by 5-FU (HA22T/VGH) was effectively affected by luteolin. It seemed that luteolin had some complementary activity to 5-FU against these HCC cell lines. The luteolin-treated PLC/PRF/5 cells exhibited typical changes of apoptosis with a characteristic DNA laddering pattern on gel electrophoresis. Luteolin also activated casepase-3, increased Bax protein with a concomitant decrease in Bcl-XL level. Increase in Bax/ Bcl-XL ratio and activation of caspase-3 supported the apoptotic finding on gel electrophoresis. Luteolin also induced cell cycle arrest at G0/G1 phase. We suggested that luteolin might exhibit anti-HCC activity as efficient as 5-FU by the mechanism of not only cell cycle arrest but also apoptosis.     

Reduction of PKC alpha decreases cell proliferation, migration, and invasion of human malignant hepatocellular carcinoma

Protein kinase C (PKC) superfamily play key regulatory roles on the development of cancer. However, the exact role of these enzymes in human hepatocellular carcinoma (HCC) has not been well established. Using the RT-PCR and Western blotting to analyze the levels of PKC isoforms mRNA and protein in the five different differentiated hepatoma cell lines, we found that PKC alpha was highly expressed in the poor-differentiated HCC cell lines (SK-Hep-1 and HA22T/VGH) as compared with that in the well-differentiated HCC cell lines (PLC/PRF/5, Hep3B, and HepG2). When treated with PKC alpha antisense oligonucleotides (ODN), both HA22T/VGH and SK-Hep-1 cells lines showed the reduction of PKC alpha expression, as well as a deceleration in the growth rate and in the level of cyclin D1, but the increase in the levels of p53 and p21(WAF1/CIP1). Moreover, the reduction of PKC alpha expression also inhibited the migratory and invasive potential of both HA22T/VGH and SK-Hep-1 cells lines, and revealed a down-regulation of several migration/invasion-related genes (MMP-1, u-PA, u-PAR, and FAK). These phenomenon were also confirmed by DNA-based small interfering RNA (siRNA) PKC alpha and PKC alpha/beta specific inhibitor Go6976. Thus, the results indicated that PKC alpha may be associated with regulation of cell proliferation/migration/invasion in human poorly differentiated HCC cells, suggesting a role for the PKC alpha in the malignant progression of human HCC.     

Anion exchanger inhibitor DIDS induces human poorly-differentiated malignant hepatocellular carcinoma HA22T cell apoptosis

Anion exchangers (AEs) of the Cl^-/HCO3^- exchanger family contribute to the regulation of intracellular acid-base balance. Recently, we found that anion exchanger 2 (AE2) was significantly expressed in human hepatocellular carcinoma (HCC) and in poorly-differentiated human HCC HA22T/VGH cells. In the present study, we further explored the pharmacological function of AE in four human HCC cell lines (SK-Hep-1, HA22T/VGH, HepG2, and Hep3B) following the treatment of 4,4’-diisothiocyanatostilbene-2,2′-disulfonic acid (DIDS), an AEs specific inhibitor. After administrations with 400–1000 μM of DIDS, cell proliferation was greatly inhibited in a dose-dependent manner from 47.5 to 65.0% in higher malignant HA22T/VGH cells, but not in other cell lines. The results of 4,6-diamidino-2-phenylindole (DAPI) staining, DNA fragmentation and flow cytometric analysis further revealed that cell apoptosis induced by DIDS was also observed in HA22T/VGH cells. Therefore, these findings suggested that AE may be involved, in part, in the proliferation and survival of HA22T cells and could be a new potential therapeutic target against specific human HCC. The authors Chih-Yang Huang and Jer-Yuh Lin contributed equally to this article.     

Involvement of the ornithine decarboxylase/polyamine system in precondition-induced cardioprotection through an interaction with PKC in rat hearts

Anion exchanger (AE) 2, belonging to the chloride–bicarbonate transporter family, has been reported to involve cell survival for hepatocellular carcinoma (HCC) cells. Our previous findings showed that AE2 gene was highly expressed in a poorly differentiated HCC cell line, HA22T/VGH. Additionally, treatment with 4,4′-diisothiocyanatostilbene-2,20-disulfonic acid (DIDS), an AE-specific inhibitor, significantly inhibited cell proliferation and induced cell apoptosis for the HA22T/VGH. To further investigate the biological functions of AE2 in human HCC, suppression of AE2 expression by the antisense oligonucleotide-AE2 (AS-AE2) was performed, and the cell viability, cell cycle regulation, and cell apoptosis for HCC cell lines were monitored. The results showed that AS-AE2 treatment could efficiently suppress the mRNA expression of AE2 for various differentiated HCC cells, including HA22T/VGH, SK-Hep-1, PLC/PRF/5, Hep3B, and HepG2. Moreover, AS-AE2 treatment significantly reduced cell viability, arrested cell cycle at sub-G1 phase, and induced cell apoptosis for the poorly differentiated HA22T/VGH, but not for other moderately or well-differentiated HCC cell lines. The findings indicated that AE2 may play an important role in the progression of HCC cells, and provide a new strategy for the development of therapeutic treatment against human HCC.     

Paeoniae Radix,a Chinese herbal extract,inhibit hepatoma cells growth by inducing apoptosis in a p53 independent pathway

Paeoniae Radix (PR) is the root of traditional Chinese Herb named Paeonia lactiflora Pallas, which is commonly used to treat liver diseases in China for centuries. Several earlier studies have indicated that PR has anticancer growth activities, however the mechanism underlying these activities was unclear and remained to be elucidated. In this study, we evaluated the molecular mechanism of the effect of PR on human hepatoma cell lines, HepG2 and Hep3B. Our results showed that the water-extract of Paeoniae Radix (PRE) had inhibitory effect on the growth of both HepG2 and Hep3B cell lines. The induction of internucleosomal DNA fragmentation and chromatin condensation appearance, and accumulation of sub-G1 phase of cell cycle profile in PRE treated hepatoma cells evidenced that the cytotoxicity of PRE to the hepatoma cells is through activation of the cell death program, apoptosis. The activation of apoptosis by PRE is independent of the p53 pathway as Hep3B cell is p53-deficient. In addition, the differential gene expression of PRE treated HepG2 was examined by cDNA microarray technology and RT-PCR analysis. We found that the gene expression of BNIP3 was up-regulated while ZK1, RAD23B, and HSPD1 were down-regulated during early apoptosis of the hepatoma cell mediated by PRE. The elucidation of the drug targets of PR on inhibition of tumor cells growth should enable further development of PR for liver cancer therapy.     

Activation of heme oxygenase and heat shock protein 70 genes by stress in human hepatoma cells

Effects of various stresses were examined on the accumulation of mRNA for microsomal heme oxygenase and a heat shock protein, hsp70, in three human hepatoma cell lines. By heat shock, hsp70 mRNA was induced in all three hepatoma lines, Hep G2, Hep 3B and Hep G2f, while heme oxygenase mRNA was increased only in Hep 3B. Time-courses of the heat shock induction of both mRNAs in Hep 3B were similar. Arsenite caused induction of both mRNAs in all three cell lines, while cadmium increased them in Hep G2 and Hep 3B, but not in Hep G2f cells. These findings suggest that, although both hsp70 and heme oxygenase are heat shock proteins, the mode of induction of mRNAs for these proteins is different.     

Induction of apoptosis in p53-deficient human hepatoma cell line by wild-type p53 gene transduction: inhibition by antioxidant

We investigated the role of wild-type (wt)-p53 as an inducer of apoptotic cell death in human hepatoma cell lines. Following the retrovirus-mediated transduction of the wt-p53 gene, Hep3B cells lacking the endogenous p53 expression began to die through apoptosis in 4 h. They showed a maximal apoptotic death at 12 h, whereas HepG2 cells expressing endogenous p53 did not. However, the transduction of the wt-p53 gene elicited growth suppression of both Hep3B and HepG2 cells. P21(WAF1/CIP1), a p53-inducible cell cycle inhibitor, was induced, not only in Hep3B cells undergoing apoptosis, but also in HepG2 cells. The kinetics of the p21(WAF1/CIP1) induction, DNA fragmentation, and growth suppression of the Hep3B cells showed that DNA fragmentation and growth suppression progressed rapidly following p21(WAF1/CIP1) accumulation. N-acetyl-cysteine or glutathione, potent antioxidants, strongly inhibited the DNA fragmentation, but did not reduce the elevated level of p21(WAF1/CIP1). These findings suggested that p21(WAF1/CIP1) was not a critical mediator for the execution of p53-mediated apoptosis, although it contributed to the growth inhibition of cells undergoing apoptosis. Furthermore, p53-mediated apoptosis could be repressed by antioxidants.     

Increased expression of N-acetylglucosaminyltransferase-V in human hepatoma cells by retinoic acid and 1alpha,25-dihydroxyvitamin D3

UDP-N-acetylglucosamine: alpha-6-D-mannoside beta-1,6N-acetylglucosaminyltransferase-V activities were determined in human hepatoma cell lines of Hep3B and HepG2, and also compared with those of normal liver tissues and primary hepatocytes. When GlcNAcbeta1-2Manalpha1-3(GlcNAcbeta1-2Manalpha1-4)(Manbeta1-4GlcNAc-2-amino pyridine (GlcN,GlcN-biant-PA) and UDP-GlcNAc were used as substrates, the enzymes displayed optimum temperatures of 50 degrees C, optimum pHs of 6.5 in each case, K(m) values for UDP-GlcNAc to be 5.8 (Hep3B) and 4.5 mM (HepG2) and K(m) values for GlcN,GlcN-biant-PA (mM) to be 1.28 (Hep3B) and 2.4 (HepG2). This indicates that values of Hep3B GlcNAc-transferase-V were distinguishable with HepG2 enzyme. Furthermore, Hep3B enzyme in membrane fraction showed about 1.5-fold higher specific activity (1.423 pmol/(h mg) than that (1.066 pmol/(h mg)) of HepG2. Normal hepatocytes are characterized by very low level of GlcNAc-transferase-V activity whereas hepatoma cells contained high activities. Treatment of hepatoma cells with retinoic acid and 1alpha,2,5-dihydroxyvitamin D(3) (Vit-D(3)) resulted in an increase in GlcNAc-transferase-V activity, while treatment with dimethyl sulfoxide and cytosine-arabinoside resulted in decrease in the enzyme activity. Although retinoic acid (RA) treated cells shows a changed GlcNAc-transferase-V mRNA expression, expression of marker proteins such as alpha-fetoprotein and albumin was not changed. This is the first demonstration of GlcNAc-transferase-V activity in RA and Vit-D(3)-treated hepatoma cell lines.     

Apoptosis induction by gamma-tocotrienol in human hepatoma Hep3B cells

We evaluated the antitumor activity of tocotrienol (T3) on human hepatoma Hep3B cells. At first, we examined the effect of T3 on the proliferation of human hepatoma Hep3B cells and found that gamma-T3 inhibited cell proliferation at lower concentrations and shorter treatment times than alpha-T3. Then, we examined the effect of gamma-T3 apoptosis induction and found that gamma-T3 induced poly (ADP-ribose) polymerase (PARP) cleavage and stimulated a rise in caspase-3 activity. In addition, gamma-T3 stimulated a rise in caspase-8 and caspase-9 activities. We also found that gamma-T3-induced apoptotic cell death was accompanied by up-regulation of Bax and a rise in the fragments of Bid and caspase-8. These data indicate that gamma-T3 induced apoptosis in Hep3B cells and that caspase-8 and caspase-9 were involved in apoptosis induction. Moreover, these results suggest that Bax and Bid regulated apoptosis induction by gamma-T3.     

Induction of plasma protein secretion in a newly established human hepatoma cell line.

To study the expression and the regulation of hepatocyte markers, we have undertaken to establish human hepatoma cell lines of various phenotypes. We now report the establishment of a new human hepatoma cell line, HA22T/VGH. This cell line has many of the properties of human hepatocellular carcinoma. Only 5 of 15 plasma proteins investigated were detected in the medium of a 10-day-old HA22T/VGH culture. However, when the HA22T/VGH cells and a clonal derivative, C5, were cultured in an aggregated form, all 15 plasma proteins were found in the culture medium. These results indicate that hepatoma cell lines with different phenotypes can be established, and they provide a good experimental framework to investigate differentiation of human hepatocytes.     

Mutational analysis on the function of the SWAP-70 PH domain

Hepatocellular carcinoma (HCC), the major manifestation of primary liver cancer, is one of the most frequent and malignant cancers worldwide, especially in Taiwan. Estrogen receptors (ERs) have been reported to play either a proliferation- or apoptosis-enhancing role in the differentiation of cancers, including HCC. In a previous experiment, we showed that transient overexpressed estrogen receptor-α induced early stage HCC cell line Hep 3B cell apoptosis by increasing the hTNF-α gene expression in a ligand-independent manner. To further clarify if the apoptotic effect occurs in poorly differentiated HCC cell line, HA22T, and elucidate the roles of ERs and TNF-α, DNA fragmentation and caspase activity were measured in late stage HCC cell line, HA22T, by measuring the expression of hER-α and hER-β using a Tetracycline-induciable system (Tet-on). Increased DNA fragmentation and caspase-3 activity were found in hERβ-overexpressed HA22T cells treated with estrogen (10⁻⁸ M) but not in hERα-overexpressed HA22T cells. Using RT-PCR/PCR and western blotting in HA22T cells, overexpressed hER-β was also found to increase the expression of hTNF-α mRNA and induce hTNF-α-dependent luciferase activity in a ligand-dependent manner. Additionally, LPS treatment and hER-β overexpression both enhance caspase-8 activities, whereas neither hER-β nor E₂ treatment affected caspase-9 activities. In addition, the overexpressed hER-β plus E₂ enhanced DNA fragmentation and caspase-8 activities were only partially reduced by anti-hTNF-α (0.1ng/ml), which was possibly due to the involvement of P53 and TGF-β. Taken together, our data indicates that overexpressed hER-β but not hER-α may induce caspase-8-mediated apoptosis by increasing the hTNF-α gene expression in a ligand-dependent manner in poorly differentiated HA22T cells. (Mol Cell Biochem xxx: 1–9, 2005)Shares equally contribution Contract grant sponsor: National Science Council; Contract grant number: NSC 91-2314-B-075A-006, NSC 92-2314-B-075A-014.     

The effect of lycopene on cell growth and oxidative DNA damage of Hep3B human hepatoma cells

Lycopene, the predominant carotenoid in tomatoes and tomato-based foods, is reported to protect against various cancers, especially prostate cancer. We investigated the effect of lycopene on DNA damage and cell growth inhibition in the Hep3B human hepatoma cell line. Lycopene was analyzed by HPLC, and cell proliferation was determined by 3-(4,5-dimethylthiazolyl-2)-2,5-diphenyltetrazolium bromide (MTT) assay. A final lycopene concentration of 0.1-50 microM was added to cells plated in 96-well plates. After a 24-hr incubation, cell viability was measured as absorbance at 570 nm after the MTT assay. The effects of lycopene on cell cycle progression were investigated with flow cytometry. Lycopene induced G0/G1 arrest and S phase block. Oxidative DNA damage was determined by the Comet (single-cell gel electrophoresis) assay. Lycopene inhibited cell growth in a dose-dependent manner. Cell growth was inhibited 20% at 0.2 microM lycopene and 40% at 50 microM lycopene after a 24-hr incubation. In the Comet assay, lycopene-treated cells showed less DNA damage than did placebo-treated cells. The inhibition of Hep3B cell growth in this study demonstrates the antitumor properties of lycopene.     

Anti-proliferative and pro-apoptotic effect of Smilax glabra Roxb. extract on hepatoma cell lines

Smilax glabra Roxb. (SGR) is the root of a traditional Chinese herb, referred to as tu fu ling in Chinese medicine. It is an inexpensive traditional Chinese medicine commonly used for the treatment of liver diseases, and a few studies have indicated that SGR has anti-hepatocarcinogenic and anti-cancer growth activities. In the current study, raw SGR plant was extracted with Accelerate Solvent Extractor, and the molecular mechanism by which S. glabra Roxb. extract (SGRE) has an anti-proliferative effect on the human hepatoma cell lines, HepG2 and Hep3B, was determined. We showed that SGRE inhibited HepG2 and Hep3B cell growth by causing cell-cycle arrest at either S phase or S/G2 transition and induced apoptosis, as evidenced by a DNA fragmentation assay. SGRE-induced apoptosis by alternation of mitochondrial transmembrane depolarization, release of mitochondrial cytochrome c, activation of caspase-3, and cleavage of poly(ADP-ribose) polymerase. The SGRE-mediated mitochondria-caspase dependent apoptotic pathway also involved activation of p38, JNK, and ERK mitogen-activated protein kinase signaling. Isometric compounds of astilbin (flavonoids) and smilagenin (saponin) have been identified as the main chemical constituents in SGRE by HPLC-MS/MS. These results have identified, for the first time, the biological activity of SGRE in HepG2 and Hep3B cells and should lead to further development of SGR for liver disease therapy.     

Ganoderiol F, a ganoderma triterpene, induces senescence in hepatoma HepG2 cells

Ganoderiol F (GolF), a tetracyclic triterpene, was isolated from Ganoderma amboinense and found to induce senescence of cancer cell lines. GolF induced growth arrest of cancer cell lines HepG2, Huh7 and K562, but exerted much less effect in hepatoma Hep3B cells and normal lung fibroblast MRC5 cells, and no effect on peripheral blood mononuclear cells. GolF treatment of the cancer cells, with the exception of Hep3B, resulted in prompt inhibition of DNA synthesis and arrest of cell progression cycle in G1 phase. Short-term exposure of HepG2 cells to GolF temporarily arrested progression of the cell cycle; cell growth was recovered if the drug was withdrawn from the medium after a 24-h exposure. After 18 days of continuous treatment of HepG2 cells with 30 muM GolF, over 50% of cells were found to be enlarged and flattened, and were beta-galactosidase positive phenotypes of senescent cells. GolF was found to inhibit activity of topoisomerases in vitro, which may contribute to the inhibition of cellular DNA synthesis. Activation of the mitogen-activated protein kinase EKR and up-regulation of cyclin-dependent kinase inhibitor p16 were found in early stages of GolF treatment and were presumed to cause cell-cycle arrest and trigger premature senescence of HepG2 cells. The growth-arrest and senescence induction capability on cancer cells suggest anticancer potential of GolF.     

A solution hybridization/RNase protection assay with riboprobes to determine absolute levels of apoB, A-I, and E mRNA in human hepatoma cell lines

A solution hybridization/RNase protection assay with riboprobes was developed to quantitate apolipoprotein mRNA concentrations. Previously, radiolabeled DNA probes have been used in solution hybridization/S1 nuclease protection assays for this purpose. The new assay requires less time for probe preparation and hybridization compared to previous assays. In addition, the vector used for riboprobe preparation can also be used to conveniently produce cRNA required to generate the standard curve to quantitate absolute apolipoprotein mRNA levels. The solution hybridization RNase protection assay was used to quantitate apoB, A-I, and E mRNA levels in four human hepatoma cell lines, HepG2, Hep3B, WRL-68, SK-Hep2. HepG2 and Hep3B, but not WRL-68 and SK-Hep2 cells had concentrations of all three apolipoprotein mRNAs comparable to liver in vivo. These data suggest that HepG2 and Hep3B are suitable models to study liver specific apolipoprotein gene expression.     

Unique structural properties of apolipoprotein B in low-density lipoproteins produced by several human hepatoma-derived cell lines

Previous work has shown that low-density lipoproteins (LDL) secreted by hepatoma-derived cell lines have an unusual composition compared to plasma LDL; rather than cholesteryl ester, the hepatoma cell-secreted LDL have a triacylglycerol core. We have found that they also have an increased negative charge, as judged by agarose electrophoresis. Since apolipoprotein B is a glycoprotein containing carbohydrate chains terminated with negatively charged sialic acid residues, we examined whether increased glycosylation of the apolipoprotein B from three hepatoma cell lines (Hep G2, Hep 3B and Huh 7) might account for the differences in LDL charge. The weight percent carbohydrate for Hep G2, Hep 3B and Huh 7 LDL-protein (1.1 +/- 0.2; 1.7 +/- 0.8; 0.4 +/- 0.1) was found to be extremely low compared with the 2.8-9% range we found for plasma LDL-protein, while the amount of LDL-lipid associated carbohydrate from hepatoma LDL was similar to that we found in plasma LDL. Furthermore, desialation of hepatoma cell-secreted LDL with neuraminidase did not normalize the negative charge to that of neuraminidase-treated plasma LDL. Western blots of thrombin proteolytic fragments indicated that, in addition to the T1-T4 fragments seen in plasma apolipoprotein B, apolipoprotein B of hepatoma-derived LDL produced four to five new fragments (T5-T9), suggesting increased exposure of proteolytic sites. Western blotting of the new fragments with antibodies specific for known apolipoprotein B sequences suggests that many of the new cleavage sites cluster in or near the putative LDL receptor recognition site.