A ligand for peroxisome proliferator activated receptor gamma inhibits cell growth and induces apoptosis in human liver cancer cells

BACKGROUND AND AIMS: Induction of apoptosis of cancer cells through ligands of nuclear hormone receptors (NHRs) is a new approach in cancer therapy. Recently, one of the NHRs, peroxisome proliferator activated receptor gamma (PPARgamma), has been shown to influence cell growth in certain cancer cells although its effect on hepatocellular carcinoma (HCC) has not been analysed. METHODS: Experiments were conducted using three human liver cancer cell lines, PLC/PRF/5, Hep G2 and HuH-7, in vitro. These cells were exposed to troglitazone, a synthetic ligand for PPARgamma, and the effects on cell growth were analysed. RESULTS: Expression of PPARgamma mRNA was detected in all three liver cancer cell lines. Activation of PPARgamma by troglitazone caused a marked growth inhibition in a dose dependent manner in three hepatoma cell lines. The DNA fragmentation ELISA assay and Hoechst 33258 staining revealed that the growth inhibitory effect by adding troglitazone was due to apoptosis of PLC/PRF/5, which strongly expressed PPARgamma. Troglitazone also induced activation of the cell death protease, caspase 3, but not caspase 8, in PLC/PRF/5 cells. However, expression levels of antiapoptotic factor bcl-2 and apoptosis inducing factor bax were not affected. CONCLUSION: Our study showed that PPARgamma was expressed in human liver cancer cells and that the ligand for PPARgamma, troglitazone, inhibited the growth of these cells by inducing apoptosis through caspase 3 activation, indicating that troglitazone could be potentially useful as an apoptosis inducer for the treatment of HCC.     

Selective COX-2 inhibitor, NS-398, suppresses cellular proliferation in human hepatocellular carcinoma cell lines via cell cycle arrest

AIM: To investigate the growth inhibitory mechanism of NS-398, a selective cyclooxygenase-2 (COX-2) inhibitor, in two hepatocellular carcinoma (HCC) cell lines (HepG2 and Huh7). METHODS: HepG2 and Huh7 cells were treated with NS-398. Its effects on cell viability, cell proliferation, cell cycles, and gene expression were respectively evaluated by water-soluble tetrazolium salt (WST-1) assay, 4’-6-diamidino-2-phenylindole (DAPI) staining, flow cytometer analysis, and Western blotting, with dimethyl sulfoxide (DMSO) as positive control. RESULTS: NS-398 showed dose- and time-dependent growth-inhibitory effects on the two cell lines. Proliferating cell nuclear antigen (PCNA) expressions in HepG2 and Huh7 cells, particularly in Huh7 cells were inhibited in a time- and dose-independent manner. NS-398 caused cell cycle arrest in the G1 phase with cell accumulation in the sub-G1 phase in HepG2 and Huh7 cell lines. No evidence of apoptosis was observed in two cell lines. CONCLUSION: NS-398 reduces cell proliferation by inducing cell cycle arrest in HepG2 and Huh7 cell lines, and COX-2 inhibitors may have potent chemoprevention effects on human hepatocellular carcinoma.     

Specific COX-2 inhibitor, meloxicam, suppresses proliferation and induces apoptosis in human HepG2 hepatocellular carcinoma cells

BACKGROUND AND AIMS: Cyclooxygenase-2 (COX-2) is associated with carcinogenesis. The aim of this study was to investigate the expression of COX-2 in four hepatocellular carcinoma (HCC) cell lines, and evaluate the effect of a selective COX-2 inhibitor, meloxicam, in HepG2, a high COX-2 expressing cell line. METHODS: Expression of COX-2 was detected using RT-PCR, Western blotting and immunohistochemical analysis. Cell proliferation was measured using MTT assay. Cell cycle distribution was determined by flow cytometry. Apoptosis was detected with TUNEL method. Expression of proliferating cell nuclear antigen (PCNA), cell cycle regulatory proteins including cyclins A, B1, D1 and E, and apoptosis-related proteins including Fas, Fas ligand and Bcl-2 were examined using Western blotting. RESULTS: Cyclooxygenase-2 was intensely expressed in HepG2, HLE and BEL7402 cells, but weakly expressed in SMMC-7402 cells. Meloxicam suppressed proliferation of HepG2 cells in a dose- and time-dependent manner, resulting in cell cycle arrest in S phase and cell accumulation in G0/G1 phase. Expression of PCNA, cyclin A but not cyclin B1, cyclin D1 or cyclin E was down-regulated by meloxicam. Meloxicam also induced apoptosis of HepG2 cells, with increased expression of Fas ligand, but the expression of Fas and Bcl-2 was not affected by meloxicam treatment. CONCLUSIONS: The present study demonstrates that the specific COX-2 inhibitor meloxicam suppresses proliferation and induces apoptosis in HCC cells that express COX-2, suggesting that COX-2 inhibition may offer a novel chemopreventive and therapeutic approach for HCC.     

Flavopiridol通过调节CDK4/CDK6/cyclinD1复合物表达抑制肝癌细胞增殖

目的研究flavopiridol对肝癌细胞Huh7增殖、凋亡和细胞周期的影响。方法Flavopiridol处理Huh7细胞后,检测细胞增殖和凋亡,进行细胞周期分析。Westernblot检测flavopiridol对cDK4／cDK6／cyclinD1复合物表达的影响。结果Flavopiridol可显著抑制Huh7细胞增殖;Flavopiridol可剂量依赖性导致Huh7细胞发生凋亡,空白对照组凋亡细胞2．65％,550nmol／L剂量处理组凋亡细胞14．17％;Flavopiridol可剂量依赖性引起Huh7细胞G1期阻滞,空白对照组G1期细胞51．06％,550nmol／L剂量处理组G1期细胞57．53％;Flavopiridol可抑制Huh7细胞Gl期相关的蛋白细胞周期素依赖激酶（CDK）4,CDK6,细胞周期素D1（cyclinD1）的表达。结论Flavopiridol通过抑制肝癌细胞G1期cDK4／cDK6／cyclinD1蛋白复合物表达,引起细胞G1期阻滞并发生凋亡,从而抑制细胞增殖。     

瘦素促进人肝癌细胞Huh 7增殖活性的实验研究

目的观察瘦素对人肝癌细胞Huh7增殖活性的影响并初步探讨其可能的机制。方法在培养液中加入不同浓度的瘦素后，细胞计数并绘制生长曲线，MTT法检测细胞增殖，流式细胞仪检测细胞周期及凋亡情况，Western blot法观察Cyclin D1蛋白的表达。结果细胞计数发现处理组的细胞数明显高于对照组；MTT法检测细胞增殖显示瘦素可以促进Huh7细胞的增殖。有一定的时间和剂量依耐性；流式细胞仪检测结果显示瘦素能明显降低G0／G1期细胞比例并提高s期细胞比例，细胞凋亡率也有明显降低；Western blot法显示瘦素（100ng／ml）处理24h后，Cyclin D1蛋白的表达明显增高。结论瘦素可以明显的促进人肝癌细胞Huh7的增殖，可能是通过提高Cyclin D1蛋白的表达来实现其作用。     

Role of autophagy in differential sensitivity of hepatocarcinoma cells to sorafenib

AIM: To investigate the role of sorafenib(SFN) in autophagy of hepatocellular carcinoma(HCC). We evaluated how SFN affects autophagy signaling pathway in human HCC cell lines. METHODS: Two different human HCC cell lines, Hep3 B and Huh7, were subjected to different concentrations of SFN. Cell viability and onset of apoptosis were determined with colorimetric assay and immunoblotting analysis, respectively. The changes in autophagy-related proteins, including LC3, ULK1, AMPK, and LKB, were determined with immunoblotting analysis in the presence or absence of SFN. To assess autophagic dynamics, autophagic flux was measured with chloroquine, a lysosomal inhibitor. The autophagic responsiveness between different HCC cell lines was compared under the autophagy enhancing conditions.RESULTS: Hep3 B cells were significantly more resistant to SFN than Huh7 cells. Immunoblotting analysis revealed a marked increase in SFN-mediated autophagy flux in Huh7 cells, which was, however, absent in Hep3 B cells. While both starvation and rapamycin enhanced autophagy in Huh7 cells, only rapamycin increased autophagy in Hep3 B cells. Immunoblotting analysis of autophagy initiation proteins showed that SFN substantially increased phosphorylation of AMPK and consequently autophagy in Huh7, but not in Hep3 B cells.CONCLUSION: The autophagic responsiveness to SFN is distinct between Hep3 B and Huh7 cells. Resistance of Hep3 B cells to SFN may be associated with altered autophagy signaling pathways.     

MDM2 antagonist can inhibit tumor growth in hepatocellular carcinoma with different types of p53 in vitro

Background and Aims: Nutlin‐3, a selective small‐molecule inhibitor of the p53‐MDM2 interaction, has been shown to have antitumor activities in various tumors with wild‐type p53. However, its effect on hepatocellular carcinoma (HCC) with different types of p53 remains unclear. This study is designed to determine nutlin‐3′s antitumor efficacy and underlying mechanisms of action in human HCC cells. Methods: Cell viability assay, cell cycle analysis, apoptosis assay, western blot, co‐ immunoprecipitation and siRNA experiments were analyzed in three human HCC cells. Anti‐tumoral effects of nutlin‐3 targeting the p53 and p73 pathways were evaluated in HCC cell lines. Results: Nutlin‐3 exerted the greatest anti‐tumoral effect to three human HCC cells with wild‐type p53, mutant p53 and p53‐null. Nutlin‐3 not only upregulated p53 in HepG2 cells, but also p73 in Huh7 and Hep3B cells, and disrupted p53‐MDM2 and p73‐MDM2 complexes in HCC cells. The compound inhibited cell proliferation, induced G0/G1 phase arrest, decreased the levels of CyclinD1, CyclinE, CDK2, CDK4, PCNA and E2F‐1, and increased the levels of p21 and p27. It also induced apoptosis, increased the Bax/Bcl‐2 ratio, then activated caspase‐9 and caspase‐3. Conclusions: Nutlin‐3 has significant anticancer effects against human HCC cells, regardless of p53 status, indicating that it is a promising therapy for human hepatocellular carcinoma.     

Cyclooxygenase-2 promotes hepatocellular carcinoma cell growth through Akt activation: evidence for Akt inhibition in celecoxib-induced apoptosis

Cyclooxygenase-2 (COX-2)-controlled prostaglandin (PG) metabolism recently has been implicated in the pathogenesis of hepatocellular carcinoma (HCC). However, the biologic role and molecular mechanism of COX-2-mediated PGs in the control of liver cancer growth have not been established. This study was designed to examine the direct effect of COX-2 and its inhibitor celecoxib on the growth control of liver cancer cells. Human HCC cell lines Hep3B and HepG2 transfected with COX-2 expression vector showed increased cell growth and enhanced phosphorylation of serine/threonine protein kinase B (Akt). The level of COX-2 expression and Akt phosphorylation is correlated positively in cultured HCC cells and human liver cancer tissues. Inhibition of Akt activation by phosphatidylinositol 3-kinase (PI3-kinase) inhibitor LY294002 significantly decreased the viability of Hep3B and HepG2 cells (P <.01). These results reveal a novel role of Akt activation in COX-2-induced HCC cell survival. Furthermore, HCC cells treated with the COX-2 inhibitor celecoxib showed significant reduction of Akt phosphorylation and marked morphologic and biochemical characteristics of apoptosis. Overexpression of COX-2 or addition of exogenous PGE(2) partially prevented celecoxib-induced apoptosis (P <.01). In conclusion, our results suggest the involvement of COX-2-dependent and -independent mechanisms in celecoxib-mediated HCC cell apoptosis.     

Imbalance between cell proliferation and cellular DNA fragmentation in hepatocellular carcinoma.

AIM/BACKGROUND: Hepatocellular carcinoma (HCC) is known for its rapid growth. This study was undertaken to determine the expression of proliferative markers, apoptosis (DNA fragmentation) and oncogene products known to regulate apoptosis (p53, bcl-2) in HCC. METHODS: 150 Chinese patients with HCC were studied (M:F 128:22, age 14-88 years). Immunohistochemistry was employed to detect cell proliferative markers (PCNA, Ki67), and oncogene products known to regulate apoptosis (p53, bcl-2). DNA fragmentation was determined by terminal dUTP nick end labeling (TUNEL). RESULTS: 98% and 95% of HCC had PCNA (median 2+) and Ki67 (median 2+) detected respectively. TUNEL labeling was detected in only a small number of tumor cells (no labeling in 11%, median 1/1000 cell labeled, range: 0-70/1000 cells). There was no correlation between TUNEL labeling and the clinical parameters (sex, age, cirrhosis, and survival) and the expression of cell proliferative markers. p53 was detected in 53% of the patients (median 1+, range: 0-4+) and bcl-2 was detected in a small proportion of tumor cells in only 13% of the HCCs (range: 0-1 +). The expression of p53 and Bcl-2 did not correlate with TUNEL labeling or the natural survival. CONCLUSIONS: Cell proliferation in HCC is unmatched by apoptosis, accounting for the rapid growth of this tumor. This lack of apoptosis in HCC is unrelated to the expression of p53 or bcl-2 over-expression.     

c-Met represents a potential therapeutic target for personalized treatment in hepatocellular carcinoma

c-Met, a high-affinity receptor for hepatocyte growth factor (HGF), plays a critical role in cancer growth, invasion, and metastasis. Hepatocellular carcinoma (HCC) patients with an active HGF/c-Met signaling pathway have a significantly worse prognosis. Although targeting the HGF/c-Met pathway has been proposed for the treatment of multiple cancers, the effect of c-Met inhibition in HCC remains unclear. The human HCC cell lines Huh7, Hep3B, MHCC97-L, and MHCC97-H were used in this study to investigate the effect of c-Met inhibition using the small molecule selective c-Met tyrosine kinase inhibitor PHA665752. MHCC97-L and MHCC97-H cells demonstrate a mesenchymal phenotype with decreased expression of E-cadherin and increased expression of c-Met, fibronectin, and Zeb2 compared with Huh7 and Hep3B cells, which have an epithelial phenotype. PHA665752 treatment blocked phosphorylation of c-Met and downstream phosphoinositide 3-kinase/Akt and mitogen-activated protein kinase/Erk pathways, inhibited cell proliferation, and induced apoptosis in c-Met-positive MHCC97-L and MHCC97-H cells. In xenograft models, administration of PHA665752 significantly inhibited c-Met-positive MHCC97-L and MHCC97-H tumor growth, and PHA665752-treated tumors demonstrated marked reduction of both c-Met phosphorylation and cell proliferation. c-Met-negative Huh7 and Hep3B cells were not affected by c-Met inhibitor treatment in vitro or in vivo. In addition, c-Met-positive MHCC97-L and MHCC97-H cells demonstrated cancer stem cell-like characteristics, such as resistance to chemotherapy, tumor sphere formation, and increased expression of CD44 and ABCG2, and PHA665752 treatment suppressed tumor sphere formation and inhibited CD44 expression. Conclusion: c-Met represents a potential target of personalized treatment for HCC with an active HGF/c-Met pathway.     

Gli-1 siRNA induced apoptosis in Huh7 cells

AIM： To investigate the effects of Gli-1 small interference RNA （siRNA） on Huh7 cells, and the change of Bcl-2 expression in Huh7 cells. METHODS： Human hepatocellular carcinoma cells Huh7 were used. Cell viability was analyzed by 3-（4, 5-Dimethylthiazol-2-yl）-2, 5-diphenyl tetrazolium bromide （MTT） assay. The expressions of Gli-1 and Bcl-2 family members were detected by RT-PCR and Western blot. Apoptosis was detected by Flow cytometry using propidium iodide, measured by Hoechst 33258 staining using Advanced Fluorescence Microscopy and caspase-3 enzymatic assay. Cell growth was analyzed after treatment with Gli-1 siRNA and 5-fluorouracil （5-Fu）. RESULTS： Inhibition of Gli-1 mRNA in Huh7 cells through Gli-1 siRNA reduced cell viability. Gli-1 siRNA treatment also induced apoptosis by three criteria, increase in the sub-G1 cell cycle fraction, nuclear condensation, a morphologic change typical of apoptosis, and activation of caspase-3. Gli-1 siRNA was also able to down-regulate Bcl-2. However, Gli-1 siRNA resulted in no significant changes in Bcl-xl, Bax, Bad, and Bid. Furthermore, Gli-1 siRNA increased the cytotoxic effect of 5-Fu on Huh7 cell. CONCLUSION： Down-regulation of Bcl-2 plays an important role in apoptosis induced by Gli-1 siRNA in HCC cells. Combination Gli-1 siRNA with chemotherapeutic drug could represent a more promising strategy against HCC. The effects of the strategies need further investigation in vivo and may have potential clinical application.     

Imbalance between cell proliferation and cellular DNA fragmentation in hepatocellular carcinoma

Abstract: Aim/Background: Hepatocellular carcinoma (HCC) is known for its rapid growth. This study was undertaken to determine the expression of proliferative markers, apoptosis (DNA fragmentation) and oncogene products known to regulate apoptosis (p53, bcl-2) in HCC. Methods: 150 Chinese patients with HCC were studied (M:F 128:22, age 14–88 years), Immunohistochemistry was employed to detect cell proliferative markers (PCNA, Ki67), and oncogene products known to regulate apoptosis (p53, bcl-2). DNA fragmentation was determined by terminal dUTP nick end labeling (TUNEL). Results: 98% and 95% of HCC had PCNA (median 2+) and Ki67 (median 2+) detected respectively. TUNEL labeling was detected in only a small number of tumor cells (no labeling in 11%, median 1/1000 cell labeled, range: 0–70/1000 cells). There was no correlation between TUNEL labeling and the clinical parameters (sex, age, cirrhosis, and survival) and the expression of cell proliferative markers. p53 was detected in 53% of the patients (median 1+, range: 0–4+) and bcl-2 was detected in a small proportion of tumor cells in only 13% of the HCCs (range: 0–1+). The expression of p53 and Bcl-2 did not correlate with TUNEL labeling or the natural survival. Conclusions: Cell proliferation in HCC is unmatched by apoptosis, accounting for the rapid growth of this tumor. This lack of apoptosis in HCC is unrelated to the expression of p53 or bcl-2 over-expression.     

Cyclooxygenase-2 (COX-2) is directly involved but not decisive in proliferation of human hepatocellular carcinoma cells

Expression of cyclooxygenase-2 (COX-2) is involved in the chronic inflammation-related development of hepatocellular carcinoma (HCC), and the use of selective COX-2 inhibitors might provide new chemoprevention strategies for HCC. However, the role of the COX-2 in hepatocarcinogenesis remains obscure, particularly as it has been primarily studied with selective COX-2 inhibitors that may affect other cellular proteins involved in cell proliferation. Therefore, we investigated the effects of the inhibition of COX-2 by the selective COX-2 inhibitor NS-398 as well as by COX-2 specific small interfering RNA (siRNA) in the human HCC cell lines Hep3B and SNU-387. These cell lines expressed COX-2, and NS-398 induced apoptosis of these cells. NS-398 inhibited more than 60% of prostaglandin E₂ (PGE2) production and cell proliferation in a concentration-dependent manner in these cells. The inhibition of proliferation was almost restored with PGE2 supplement, suggesting that NS-398 may inhibit cell growth partially through inhibition of COX-2 and PGE2 production in human HCC cells. However, treatment with NS-398 led to increased expression of COX-2 in Hep3B and SNU-387 cells. To examine the effect of COX-2 depletion on these cells, we electroporated COX-2-specific siRNAs into SNU-387 cells. We observed significant, sequence-specific reductions in COX-2 expression, PGE2 production, and cell proliferation, though the reduction in cell proliferation was less than that induced by NS-398. In conclusion, these data suggest that COX-2 itself is directly involved, though not decisively, in proliferation of human HCC cells. RNA interference may provide a useful tool for manipulating COX-2-related hepatocarcinogenesis in research and therapeutic settings. Supported by National Cancer Center, Korea grant 02101203