Differentiating effect of sodium butyrate on human hepatoma cell lines PLC/PRF/5, HCC-M and HCC-T.

The in vitro effect of sodium butyrate (SB) on human hepatoma cell lines PLC/PRF/5, HCC-M and HCC-T was investigated. SB was added at the non-toxic but cytostatic concentration of 1 mM. In all these cell lines, SB reduced cell proliferation and changed the morphology of the cells into a fibroblast-like shape. In PLC/PRF/5, alpha-fetoprotein production and c-myc expression were inhibited. In contrast, gene expression of albumin, one of the normal liver-cell products, and that of integrated hepatitis B virus genome, was increased. In HCC-M and HCC-T, c-myc expression, which was enhanced in the naive state, was reduced. In HCC-M, fos expression was inhibited but the expression of N- and K-ras genes did not change. SB seemed to induce normal or mature properties of hepatocytes in human hepatoma cell lines.     

不同p53状态的肝癌细胞系DNA损伤修复能力的比较

目的 观察不同p53状态的肝癌细胞在DNA损伤因素存在下，探讨p53在肝细胞癌发生过程中的作用。方法 选用内源性表达野生型p53、突变型p53、p53全基因缺失的HepG2、PLC／PRF／5、Hep3B肝癌细胞系，将含有p53结合序列的CAT报告基因质粒分别转染各细胞，通过ELISA方法检测报告基因CAT活化情况，观察p53功能状态；用细胞生长曲线比较不同p53状态的细胞生长速度的差异；给予一定剂量的紫外线照射，检测各细胞程序外DNA损伤修复(UDS)能力；观察紫外线照射后细胞克隆形成情况，反映不同细胞系在紫外线照射后细胞存活率的不同。结果 报告基因CAT在HepG2细胞表达最强，而在PLC／PRF／5、Hep3B肝癌细胞均处于较低水平，说明HepG2细胞具有功能性的p53表达；HepG2细胞生长速度明显慢于其他两种细胞；紫外线照射后，HepG2具有较好的DNA损伤修复能力以及较高的细胞生存率。结论 野生型p53具有抑制肝癌细胞生长、保持细胞良好的DNA损伤修复的功能。     

Radiation-induced intercellular signaling mediated by cytochrome-c via a p53-dependent pathway in hepatoma cells

The tumor suppressor p53 has a crucial role in cellular response to DNA damage caused by ionizing radiation, but it is still unclear whether p53 can modulate radiation-induced bystander effects (RIBE). In the present work, three different hepatoma cell lines, namely HepG2 (wild p53), PLC/PRF/5 (mutation p53) and Hep3B (p53 null), were irradiated with γ-rays and then co-cultured with normal Chang liver cell (wild p53) in order to elucidate the mechanisms of RIBE. Results showed that the radiosensitivity of HepG2 cells was higher than that of PLC/PRF/5 and Hep3B cells. Only irradiated HepG2 cells, rather than irradiated PLC/PRF/5 or Hep3B cells, could induce bystander effect of micronuclei (MN) formation in the neighboring Chang liver cells. When HepG2 cells were treated with 20?μM pifithrin-α, an inhibitor of p53 function, or 5?μM cyclosporin A (CsA), an inhibitor of cytochrome-c release from mitochondria, the MN induction in bystander Chang liver cells was diminished. In fact, it was found that after irradiation, cytochrome-c was released from mitochondria into the cytoplasm only in HepG2 cells in a p53-dependent manner, but not in PLC/PRF/5 and Hep3B cells. Interestingly, when 50?μg/ml exogenous cytochrome-c was added into cell co-culture medium, RIBE was significantly triggered by irradiated PLC/PRF/5 and Hep3B cells, which previously failed to provoke a bystander effect. In addition, this exogenous cytochrome-c also partly recovered the RIBE induced by irradiated HepG2 cells even with CsA treatment. Our results provide new evidence that the RIBE can be modulated by the p53 status of irradiated hepatoma cells and that a p53-dependent release of cytochrome-c may be involved in the RIBE.     

Effects of sodium n-butyrate on alpha-fetoprotein and albumin secretion in the human hepatoma cell line PLC/PRF/5

The in vitro effects of sodium n-butyrate (butyrate) on the growth, morphology and secretion of alpha-fetoprotein (AFP) and albumin by the human heptoma cell line PLC/PRF/5 were studied. Butyrate caused a marked reduction in the growth rate, colony forming efficiency in soft agar and de novo synthesis of DNA as well as remarkable morphological changes including cell enlargement, flattening and a decreased number of nucleoli. Secretion of AFP was reduced during culture with butyrate, while that of albumin was increased. The requirement of de novo protein synthesis for the increase in albumin and decrease of AFP by butyrate was demonstrated by inhibition studies with cycloheximide. These results suggest that butyrate caused the hepatoma cells to acquire in vitro properties that are considered to be more consistent with normal liver cells.     

Chromosomes of human hepatoma cell lines

The karyotypes of three human hepatoma cell lines Hep G2, Hep 3B and PLC/PRF/5 were investigated by G- and C-banding techniques. In addition to ploidy changes, typical for most carcinoma cell lines, certain markers were found that remained stable throughout passage of these cultures. Chromosome I is involved in multiple translocations, resulting in at least three copies of the chromosome I heterochromatin region in each cell line. Inversion in the 9qh region is also seen. In addition, each of the cell lines consistently contains trisomy of 17q. The rearrangements of chromosome I are most striking in the Hep 3B and PLC/PRF/5 cell lines, which are derived from human hepatocellular carcinomas and contain integrated copies of the hepatitis B viral genome. These two cell lines are characterized by the presence of at least five copies of the I (p13←q21) region that result from multiple deletions and/or translocations; by consistent trisomy and polymorphism of the 9qh region; and by trisomy of chromosome 10 (also involved in rearrangements). The Hep G2 and Hep 38 cell lines behave functionally as highly differentiated liver parenchymal cells and are karyologically distinguishable from PLC/PRF/5 both by the presence of trisomy of 6 (pter←q14) and by the finding that one of the homologues of chromosome 15 is 15q+.     

Modulation of EGF receptor expression by differentiating agents in human colon carcinoma cell lines

The existence of an autocrine loop for self-stimulation of growth in malignant cells has been proposed for transforming growth factor-alpha (TGF alpha) and its receptor, the epidermal growth factor (EGF) receptor, in a variety of malignant cell types. Expression of both has been described in colon carcinoma. In order to investigate whether there is a correlation between TGF alpha and EGF receptor mRNA expression and differentiation, we studied the effects of differentiating agents on seven human colon carcinoma cell lines. All of the lines responded to the differentiating agents. In four of the seven lines there was increased EGF receptor mRNA two to five days after treatment with 2 mM sodium butyrate. In three of these lines TGF alpha mRNA and protein were also increased. In the one cell line treated with the differentiating agents DMF and DMSO, EGF receptor mRNA was also increased. [125I]-EGF binding to the cells was measured before and after treatment with butyrate. In two of three cell lines, increased EGF receptor mRNA was accompanied by a 2.4-fold increase in the number of binding sites per cell. In SW620 cells, no EGF receptor binding was detected before or after butyrate treatment. In the two cell lines in which butyrate increased EGF receptor binding, simultaneous treatment with EGF did not enhance growth. These data demonstrate increased expression of the TGF alpha/EGF receptor system after differentiation of colon carcinoma cell lines and suggest that their expression may be characteristic of a differentiated phenotype.     

H32, a non-quinone sulfone analog of vitamin K3, inhibits human hepatoma cell growth by inhibiting Cdc25 and activating ERK

We previously synthesized a K-vitamin derivative, Cpd 5, which was a potent growth inhibitor of human tumor cells, including Hep3B hepatoma cells. However, being a quinone compound, Cpd 5 has the potential for generating toxic reactive oxygen species (ROS). We therefore synthesized a nonquinone sulfone derivative, H32, which has a sufone group substituting the quinone. The IC50 of H32 for Hep3B cells was found to be 2.5 microM, which was 2.5 and 3.2 times more potent than Cpd 5 and vitamin K3 respectively. It induced apoptosis in Hep3B cells but did not generate ROS when compared to Cpd 5. Interestingly, under similar culture conditions, normal rat hepatocytes were 14-fold more and 7-fold more resistant to the growth inhibitory effects of H32 than Hep3B and PLC/PRF5 cells respectively. H32 preferentially inhibited the activities of the cell cycle controlling Cdc25A phosphatase likely by binding to its catalytic cysteine. As a consequence, it induced inhibitory tyrosine phosphorylation of the Cdc25 substrate kinases Cdk2 and Cdk4 in Hep3B cells and the cells undergo an arrest in the G1 phase of the cell cycle. H32 also induced persistent phosphorylation of the MAPK protein ERK1/2, but marginal JNK1/2 and p38 phosphorylation. The ERK inhibitor U0126, added at least 30 min prior to H32, antagonized the growth inhibition induced by H32. However, the JNK and p38 inhibitors, JNKI-II and SB203580, were not able to antagonize H32 induced growth inhibition. Thus, H32 differentially inhibited growth of normal and liver tumor cells by preferentially inhibiting the actions of Cdc25 phosphatases and inducing persistent ERK phosphorylation.     

Enhanced expression of hepatitis B surface antigen by sodium butyrate in PLC/PRF/5 human hepatoma cells

The effect of butyric acid, a natural fermentation product of colonic bacterial flora, on hepatitis B surface antigen (HBsAg) expression was investigated in HBsAg-positive PLC/PRF/5 human hepatoma cells. By Northern blot analysis, the levels of HBsAg mRNA increased dose-dependently using sodium butyrate (0-2 mmol/l). In transient chloramphenicol acetyltransferase plasmid transfection experiments, the HBsAg-preS2 promoter activity as well as the HBV enhancer 1 activity was stimulated by sodium butyrate, whereas the HBsAg-preS1 promoter activity was not. These results indicate that butyric acid functions as a physiological regulator of HBsAg expression through the portal blood flow and possibly contributes to increased expression ratio of preS2/S to preS1 polypeptides recognized in persistant HBV infection.     

Cell growth inhibition and gene expression induced by the histone deacetylase inhibitor, trichostatin A, on human hepatoma cells

OBJECTIVE: Histone deacetylase (HDAC) inhibitors have been reported to induce cell growth arrest, apoptosis and differentiation in tumor cells. The effect of the HDAC inhibitor, trichostatin A (TSA), on hepatoma cells, however, has not been well studied. In this study, we examined cell viability and gene expression profile in hepatoma cell lines treated with TSA. METHODS: To study cell growth inhibition and induction of apoptosis by TSA on human hepatoma cell lines including HuH7, Hep3B, HepG2, and PLC/PRF/5, cells were treated with TSA at various concentrations and analyzed by the 3-(4, 5-dimethyl-2-thiazolyl)-2H-tetrazolium bromide (MTT) and TUNEL assays, respectively. Changes in gene expression profile after exposure to TSA were assessed using a cDNA microarray consisting of 557 distinct cDNA of cancer-related genes. The levels of acetylated histones were examined by the chromatin immunoprecipitation (ChIP) assay using anti-acetylated histone H3 or H4 antibody. RESULTS: The MTT assay demonstrated that TSA showed cell growth inhibition not only in a concentration-dependent but also a time-dependent manner on all cell lines studied. The TUNEL assay also revealed the potential of TSA to induce apoptosis. The microarray analysis revealed that 8 genes including collagen type 1, alpha2 (COL1A2), insulin-like growth factor binding protein 2 (IGFBP2), integrin, alpha7 (ITGA7), basigin (BSG), quiescin Q6 (QSCN6), superoxide dismutase 3, extracellular (SOD3), nerve growth factor receptor (NGFR), and p53-induced protein (PIG11) exhibited substantial induction (ratio >2.0) after TSA treatment in multiple cell lines. ChIP assay, in general, showed a good correlation between the expression level of mRNA and levels of acetylated histones in these upregulated genes. CONCLUSIONS: This study showed cell growth inhibition and the gene expression profile in hepatoma cell lines exposed to TSA. The alteration in levels of acetylated histones was closely associated with expression of specific cancer-related genes in hepatoma cells.     

Apoptotic effects of extract from Antrodia camphorata fruiting bodies in human hepatocellular carcinoma cell lines

The fruiting body of Antrodia camphorata is well known in Taiwan as a traditional medicine for treating cancer and inflammation. The purpose of this study was to evaluate the apoptotic effects of ethylacetate extract from A. camphorata (EAC) fruiting bodies in two human liver cancer cell lines, Hep G2 and PLC/PRF/5. Treatment with EAC decreased the cell growth of Hep G2 and PLC/PRF/5 cells in a dose dependent manner. In Fas/APO-1 positive-Hep G2 cells, EAC increased the expression level of Fas/APO-1 and its two forms of ligands, membrane-bound Fas ligand (mFasL) and soluble Fas ligand (sFasL), in a p53-indenpendent manner. In addition, EAC also initiated mitochondrial apoptotic pathway through regulation of Bcl-2 family proteins expression, release of cytochrome c, and activation of caspase-9 both in Hep G2 and PLC/PRF/5 cells. Furthermore, EAC also inhibited the cell survival signaling by enhancing the amount of IkappaBalpha in cytoplasm and reducing the level and activity of NF-kappaB in the nucleus, and subsequently attenuated the expression of Bcl-X(L) in Hep G2 and PLC/PRF/5 cells. EAC therefore decreased the cell growth and induced apoptosis both in Hep G2 and PLC/PRF/5 cells.     

Modulation of c-myc expression by transforming growth factor beta 1 in human hepatoma cell lines

The effects of transforming growth factor beta 1 (TGF-beta 1) on cell proliferation of human hepatoma cell lines, PLC/PRF/5 and Mahlavu, were investigated under serum-free conditions. DNA synthesis was strongly inhibited in the PLC/PRF/5 cells by addition of TGF-beta 1 (0.5 to 4.0 ng/ml), but remained unchanged in the Mahlavu cells. Also the expression of c-myc mRNA was suppressed by the addition of TGF-beta 1 in the PLC/PRF/5 cells but not in the Mahlavu cells. These results indicate that TGF-beta 1 might regulate cell growth, in part, by modulating c-myc expression, although there is no direct proof that c-myc expression is really relevant to DNA synthesis mediated by TGF-beta 1.     

Adenovirus-mediated tBid overexpression results in therapeutic effects on p53-resistant hepatocellular carcinoma

Hepatocellular carcinoma (HCC) is one of the most common malignancies worldwide with a very high mortality. Because the success of the conventional therapies is limited, gene therapy may represent an alternative for HCC management. Our earlier study has shown that Bid plays a role in the development of HCC. The aim of our study is to evaluate the possibility of using truncated Bid (tBid) as a novel therapy for HCC treatment. Two HCC cell lines, Hep3B and PLC/PRF/5, were used in the experiment. Hep3B was a p53-resistant while PLC/PRF/5 a p53-sensitive. A recombinant adenovirus-Ad/AFPtBid, which contained a tBid gene driven by an alpha-fetoprotein (AFP) promoter, was constructed. Both Hep3B and PLC/PRF/5 cells infected with Ad/AFPtBid showed a significant decrease in cell viability. The decrease in cell viability by Ad/AFPtBid resulted from apoptosis of HCC cells, evident by enhanced activity of caspases and increased release of cytochrome c. In vivo experiment was performed by the intratumor injection of Ad/AFPtBid in nude mice inoculated with Hep3B. Ad/AFPtBid injection significantly inhibited tumor growth, and tumor tissues showed a marked increase in TUNEL-positive cells. Our experiment also demonstrated that Ad/AFPtBid only targeted AFP-producing cells but not those non-AFP producing cells. In conclusion, these results indicate that the introduction of Ad/AFPtBid can not only significantly but specifically kill HCC cells that produce AFP. The cell death induced by Ad/AFPtBid in HCC cells is via an apoptotic pathway that can be independent of p53 status.     

Modulation of c-myc Expression by Transforming Growth Factor β1 in Human Hepatoma Cell Lines

The effects of transforming growth factor β1 (TGF-β1) on cell proliferation of human hepatoma cell lines, PLC/PRF/5 and Mahlavu, were investigated under serum-free conditions. DNA synthesis was strongly inhibited in the PLC/PRF/5 cells by addition of TGF-β1 (0.5 to 4.0 ng/ml), but remained unchanged in the Mahlavu cells. Also the expression of c- myc mRNA was suppressed by the addition of TGF-β1 in the PLC/PRF/5 cells but not in the Mahlavu cells. These results indicate that TGF-β1 might regulate cell growth, in part, by modulating c- myc expression, although there is no direct proof that c- myc expression is really relevant to DNA synthesis mediated by TGF-β1.     

Growth of human hepatoma cells lines with differentiated functions in chemically defined medium

A human hepatoma cell line, HuH-7, which was established from a hepatocellular carcinoma, was found to replicate continuously in a chemically defined medium when the medium was supplemented with Na2SeO3. The cells grew better in this medium than in serum-containing medium without any adaptation period. Other established human hepatoma and hepatoblastoma cell lines, HuH-6 cl-5, PLC/PRF/5, huH-1, and huH-4, also grew in the defined medium. Although HLEC-1 cells failed to proliferate continuously with Na2SeO3 alone, they grew if a cell-free conditioned medium from HuH-7 cells was added to the medium. These cell lines, except the HLEC-1 cell line, produced the following human plasma proteins among those examined: albumin, prealbumin, alpha 1-antitrypsin, ceruloplasmin, fibrinogen, fibronectin, haptoglobin, hemopexin, beta-lipoprotein, alpha 2-macroglobulin, beta 2-microglobulin, transferrin, lipoprotein, alpha 2-macroglobulin, beta 2-microglobulin, transferrin, Complement Components 3 and 4, and alpha 1-fetoprotein. Beside plasma proteins, the media from HuH-7, HuH-6 cl-5, PLC/PRF/5, and huH-1 contained anti-carcinoembryonic antigen-reactive proteins, and those from PLC/PRF/5, huH-1, and huH-4 medium contained hepatitis B surface antigen. These proteins were detected during periods of serial cultivation over 9 months under the above culture conditions. The hepatoma cell lines grown in the fully defined synthetic medium may provide a new approach for investigating the growth and metabolism of human hepatoma cells in vitro.     

Effects of alpha-difluoromethyl ornithine on alpha-fetoprotein and albumin secretion in human hepatoma cell line, PLC/PRF/5

The in vitro effects of alpha-difluoromethyl ornithine (DFMO) on the growth and the secretion of alpha-fetoprotein (AFP) and albumin in human hepatoma cell line PLC/PRF/5 were studied. DFMO caused a marked reduction in the growth rate and de novo synthesis of DNA, whereas secretion of AFP and albumin was not altered. These data indicate that AFP and albumin secretion are neither linked to polyamine synthesis nor to cellular proliferation in human hepatoma cells.     

Sorafenib blocks the RAF/MEK/ERK pathway, inhibits tumor angiogenesis, and induces tumor cell apoptosis in hepatocellular carcinoma model PLC/PRF/5

Angiogenesis and signaling through the RAF/mitogen-activated protein/extracellular signal-regulated kinase (ERK) kinase (MEK)/ERK cascade have been reported to play important roles in the development of hepatocellular carcinomas (HCC). Sorafenib (BAY 43-9006, Nexavar) is a multikinase inhibitor with activity against Raf kinase and several receptor tyrosine kinases, including vascular endothelial growth factor receptor 2 (VEGFR2), platelet-derived growth factor receptor (PDGFR), FLT3, Ret, and c-Kit. In this study, we investigated the in vitro effects of sorafenib on PLC/PRF/5 and HepG2 HCC cells and the in vivo antitumor efficacy and mechanism of action on PLC/PRF/5 human tumor xenografts in severe combined immunodeficient mice. Sorafenib inhibited the phosphorylation of MEK and ERK and down-regulated cyclin D1 levels in these two cell lines. Sorafenib also reduced the phosphorylation level of eIF4E and down-regulated the antiapoptotic protein Mcl-1 in a MEK/ERK-independent manner. Consistent with the effects on both MEK/ERK-dependent and MEK/ERK-independent signaling pathways, sorafenib inhibited proliferation and induced apoptosis in both HCC cell lines. In the PLC/PRF/5 xenograft model, sorafenib tosylate dosed at 10 mg/kg inhibited tumor growth by 49%. At 30 mg/kg, sorafenib tosylate produced complete tumor growth inhibition. A dose of 100 mg/kg produced partial tumor regressions in 50% of the mice. In mechanism of action studies, sorafenib inhibited the phosphorylation of both ERK and eIF4E, reduced the microvessel area (assessed by CD34 immunohistochemistry), and induced tumor cell apoptosis (assessed by terminal deoxynucleotidyl transferase-mediated nick end labeling) in PLC/PRF/5 tumor xenografts. These results suggest that the antitumor activity of sorafenib in HCC models may be attributed to inhibition of tumor angiogenesis (VEGFR and PDGFR) and direct effects on tumor cell proliferation/survival (Raf kinase signaling-dependent and signaling-independent mechanisms).     

Anti-proliferative effect of apigenin and its apoptotic induction in human Hep G2 cells

Apigenin, a common dietary flavonoid abundantly present in fruits and vegetables, is believed to possess preventive and therapeutic potential against cancers. In this study, the anti-hepatoma property of apigenin was evaluated on three different human hapatoma cells, namely Hep G2, Hep 3B, and PLC/PRF/5 cells. Results showed that apigenin exhibited a significant growth inhibition against the three selected hepatoma cell lines but not the normal murine liver BNL CL.2 cells. Interestingly, it was shown to possess a similar potency as a commercial anti-hepatoma agent 5-flurouracil (5-FU: positive control) against Hep G2 cells, with IC50 value of 8.02+/-1.30 microg/ml. Therefore, we conducted our study further to investigate the cellular mechanism of apigenin effect on Hep G2 cell death. Using DNA ladder and flow cytometric analysis, apigenin was found to induce apoptosis in Hep G2 cells. It also increased the accumulation of p53 and further enhanced the level of p21/WAF1. Together, it was shown that the apoptosis induced by apigenin in Hep G2 cells was possibly mediated through the p53-dependent pathway and the induction of p21 expression, which was probably associated with the cell cycle arrest in G2/M phase. The present study concludes that the anti-hepatoma activity of apigenin is as effective as 5-FU and its apoptotic mechanism might be mediated through the p53-dependent pathway and the induction of p21 expression.     

Secretion of extracellular matrix (fibronectin), growth factor (transforming growth factor beta) and protease (cathepsin D) by hepatoma cells

We investigated facilitation of invasion by growth factors and chemotactic factors in tumor cell lines, particularly hepatocellular carcinoma. Hepatoma cells (PLC/PRF/5 and Hep G2) showed strong chemotaxis toward their respective conditioned media while metastatic pancreatic cancer cells (SU.86.86) and colon cancer cells (LS 174T) did not migrate toward their respective conditioned media. Based on immunoblotting, PLC/PRF/5 cells secrete fibronectin (an extracellular matrix constituent), transforming growth factor-beta (TGFbeta; a growth factor), and cathepsin D (a protease). Fibronectin induced a migratory response in PLC/PRF/5 cells, and anti-fibronectin antibody abolished the migratory response of these cells to their conditioned medium. Anti-integrin-beta(1) antibody also impeded migration of these cells toward conditioned medium. Polyclonal anti-TGFbeta antibody and protease inhibitors (alpha(2)-macroglobulin and leupeptin) added to culture media-modulated secretion of fibronectin by PLC/PRF/5 cells. Although exogenous TGFbeta suppressed SU.86.86 cells, it enhanced PLC/PRF/5 cell adhesion to substrate, increasing viable cell numbers. These actions indicate that hepatocellular carcinoma may possess a forceful autocrine mechanism enabling cells to survive and proliferate under cirrhotic conditions.