Effect of DNA Methyltransferase in Comparison to and in Combination with Histone Deacetylase Inhibitors on Hepatocellular Carcinoma HepG2 Cell Line

Background: DNA demethylating agents and histone deacetylase inhibitors can affect reactivation of geneexpression and apoptosis induction by DNA acetylation and demethylation. The aim of the present study was to analyzethe effects of DNA demethylating agent genistein (GE) and histone deacetylase inhibitor valproic acid VPA), aloneand combined, on hepatocellular carcinoma Hep G2 cell line. Methods: The cells were treated with various doses ofgenistein and valproic acid (alone and combined) and the MTT assay and flow cytometry were used to determine cellviability and apoptosis. Results: Genistein and valproic acid inhibited the growth of HepG 2 cells significantly. Resultof flow cytometry demonstrated that genistein and valproic acid (alone and combined) induce apoptosis significantly ina time‑dependent manner. Conclusions: Genistein and valproic acid can significantly inhibit proliferation and induceapoptosis in HepG2 cell line. The apoptotic effects of GE in combination with VPA were more significant that of eachcompound alone.     

生长抑素增强三苯氧胺抗乳腺癌作用的体外研究

Objective:To study the antineoplastic effects of tamoxifen(TAM) in combination with a somatostain analogue(octreotide,OCT) on breast cancer.Methods:Estrogen receptor(ER)-positive(MCF-7) and ER-negative(MDA-MB-435S)human breast carcinoma cell lines were treated with TAM or OCT,or combination of both agents in vitro.Cell proliferation was evaluated by MTT assay,distribu-tion of cell cycle and rate of apoptosis were detemined by flow cytometry.Results:The inhibitory effect of OCT or TAM on proliferation of MCF-7 cells was associated with cell arrest in G0/G1 phase and induction of apoptosis.The inhibitory effect on proliferation of MCF-7 cells enhanced when treatment of TAM combined with OCT.The increased rate of apoptosis induced by combination of TAM and OCT was much higher than use of either TAM or OCT alone.TAM or OCT also had weak inhibitory effect on MDA-MB435S cell.The cells were arrested at S phase by TAM and at G0/G1 phase by OCT, but the induction of apoptosis was not identified.However,the rate of apoptosis was 22.7% if combined treatment of TAM and OCT applied.Conclusion:TAM and OCT can synergistically inhibit proliferation and induce apoptosis of ER-positive and ER-negative breast cancer cells.The synergism of TAM and OCT may be of interest in the clinical treatment of breast carcinoma.     

Dentatin from Clausena excavata Induces Apoptosis in HepG2 Cells via Mitochondrial Mediated Signaling

Hepatocellular carcinoma (HCC) is a primary liver cancer with high global incidence and mortality rates.Current candidate drugs to treat HCC remain lacking and those in use possess undesirable side effects. In thisinvestigation, the antiproliferative effects of dentatin (DTN), a natural coumarin, were evaluated on HepG2 cellsand DTN’s probable preliminary molecular mechanisms in apoptosis induction were further investigated. DTNsignificantly (p<0.05) suppressed proliferation of HepG2 cells with an IC50 value of 12.0 μg/mL, without affectinghuman normal liver cells, WRL-68 (IC50> 50 μg/mL) causing G0/G1 cell cycle arrest via apoptosis induction.Caspase colorimetric assays showed markedly increased levels of caspase-3 and caspase-9 activities throughoutthe treatment period. Western blotting of treated HepG2 cells revealed inhibition of NF-κB that triggers themitochondrial-mediated apoptotic signaling pathway by up-regulating cytoplasmic cytochrome c and Bax, anddown-regulating Bcl-2 and Bcl-xL. The current findings suggest DTN has the potential to be developed furtheras an anticancer compound targeting human HCC.     

RNA干扰SMYD3基因表达对诱导肝癌细胞凋亡的影响

背景与目的:SMYD3(SETandMYNDdomain-containingprotein3)基因的表达蛋白是一种组蛋白甲基转移酶,参与肿瘤细胞增殖与凋亡的调控。本研究旨在探讨利用RNA干扰(RNAinterference,RNAi)抑制SMYD3基因表达对肝癌细胞增殖与凋亡的影响。方法:RT-PCR、免疫组织化学法分别检测SMYD3在肝癌细胞和肝癌组织中的表达。构建小发夹状RNA(smallhairpinRNA,shRNA)干扰质粒Pgenesil-1-s1、Pgenesil-1-s2及无干扰效应质粒Pgenesil-1-hk并转染入肝癌细胞HepG2,阻抑其表达SMYD3,以空质粒Pgenesil-1转染组为对照。Westernblot检测阻抑效应;MTT检测细胞增殖抑制率,流式细胞术及TUNEL检测细胞凋亡。结果:SMYD3在肝癌组织和多种肝癌细胞中表达明显增强。shRNA转染HepG2细胞后:SMYD3蛋白表达下调75%～85%;细胞增殖明显受抑制,抑制率高达60.95%～72.14%;流式细胞术结果显示Pgenesil-1-s1组HepG2细胞凋亡率(17.68±2.36)%、Pgenesil-1-s2组(19.07±1.78)%,均显著高于Pgenesil-1-hk组[(1.44±0.28)%]及Pgenesil-1组[(0.47±0.12)%](P<0.01);TUNEL检测的凋亡指数结果与流式细胞术检测结果类似。结论:SMYD3高表达于多种肝癌细胞及肝癌组织;RNAi能特异性下调SMYD3的表达,抑制肝癌细胞增殖并促进细胞凋亡,提示其可能为治疗肝癌提供新的途径。     

PJ34, an inhibitor of PARP-1, suppresses cell growth and enhances the suppressive effects of cisplatin in liver cancer cells

It has been suggested that poly(ADP-ribose) polymerase-l (PARP-l) plays an important role in DNA repair, cell death and proliferation, as well as in the stabilization of the genome. Pharmacological inhibition or genetic ablation of PARP-1 had a beneficial outcome in cancer chemotherapy since the cancer cells lacked PARP-1 and were sensitive to chemotherapeutic DNA damage. As a novel potent specific inhibitor of PARP-l, PJ34 has been reported to enhance chemotherapeutic effects in certain types of tumors. In a previous study, we found that PARP-1 expression was significantly increased in human hepatocellular carcinoma (HCC) compared to its surrounding liver tissue. This study investigated whether or not the inhibition of PARP-1 activity by PJ34 produces suppressive effects on human liver cancer cells and sensitizes the tumor cells to chemotherapeutic agents. We conclude that PJ34 significantly suppresses HepG2 cell growth in a dose-dependent manner, and inhibits HepG2 cell-derived tumor growth in nude mice. The suppressive effects of PJ34 are associated with increased cell apoptosis. Furthermore, PJ34 enhances suppressive effects of cisplatin in HepG2 cells. These results suggest that PJ34 may be developed into an effective agent for the treatment of human HCC.     

Tripartite motif-containing 3 (TRIM3) inhibits tumor growth and metastasis of liver cancer

Background:Reduced expression of tripartite motif-containing 3 (TRIM3) has been reported to be involved in the pathogenesis of human glioblastoma.In our previous research,we found that TRIM3 expression was markedly reduced in human primary hepatocellular carcinoma (HCC) tissues and that low TRIM3 expression was associated with short survival of HCC patients.However,the role of TRIM3 in liver cancer remains unknown.This study aimed to investigate the function of TRIM3 in liver cancer cells.Methods:The protein levels of TRIM3 in five liver cancer cell lines (SK-Hep1,Hep3B,Huh7,HepG2,Bel-7402) and one normal liver cell line (L02) were detected with Western blotting.HepG2 and Bel-7402 cells with IowTRIM3 expression were infected with recombinant lentiviruses overexpressing TRIM3 (LV-TRIM3),whereas Huh7 and Hep3B cells with high TRIM3 expression were transfected with TRIM3-targeted small interfering RNA (siTRIM3).The functions of TRIM3 in the proliferation,colony formation,cell cycle,migration,invasion,and apoptosis of the above cell lines were examined.The effect of TRIM3 on tumor growth and metastases in nude mice was also investigated.Results:TRIM3 was overexpressed in HepG2 and Bel-7402 cells with LV-TRIM3 infection,which further reduced proliferation,colony formation,migration,and invasion of both cell lines.Cell cycle analysis showed thatTRIM3 overexpression induced G0/G1 phase arrest in HepG2 and Bel-7402 cells.Moreover,apoptosis was not increased in HepG2 or Bel-7402 cells overexpressing TRIM3.Contrarily,silencing TRIM3 expression in Huh7 and Hep3B cells by siTRIM3 led to significantly decreased percentages of both cells in the G0/G1 phase and promoted cell proliferation,colony formation,migration,and invasion.In vivo experiment results confirmed thatTRIM3 overexpression suppressed tumor growth and metastasis.Conclusions:TRIM3 plays a tumor-suppressing role in the regulation of liver cancer development by reducing cell proliferation through cell cycle arrest at the G0/G1 phase.     

Combined in vitro anti-tumoral action of tamoxifen and retinoic acid derivatives in hepatoma cells.

Chemotherapy does not have a prominent role in the treatment of hepatoma. However, an acyclic retinoid prevented tumor recurrence post-hepatectomy, and tamoxifen (TAM) induced apoptosis in tumor cells. Combination therapy of these agents on proliferation and apoptosis of hepatoma cells has not been explored. HepG2, Hep1B, Hepa1-6 and MH1C1 hepatoma cells were incubated with TAM, 9-cis- and all-trans retinoic acid (CRA, ATRA, respectively) alone or in combination. Proliferation rate was assessed and apoptosis was analyzed by flow cytometry, immunostaining, caspase activity assays and the expression of apoptosis- and/or cell cycle-related molecules. CRA and TAM, but not ATRA monotherapy were moderately effective. Apoptosis was accompanied by upregulation of caspase 3 and 8 activity, and increased p27, bax, caspase 3 expression, while the levels of p21cip/waf and bcl-2 were unchanged or decreased. Combination therapy enhanced apoptosis from a maximum of 60% after monotherapy to more than 90% after 96 h in all cell types. Pro-apoptotic effects were paralleled by inhibition of proliferation. Combination of TAM and CRA, but not ATRA, have an additive to synergistic anti-proliferative and pro-apoptotic effect on HCC cells. This justifies trials for HCC using combinations of these biological response modifiers.     

Naringenin (Citrus Flavonone) Induces Growth Inhibition, Cell Cycle Arrest and Apoptosis in Human Hepatocellular Carcinoma Cells

Search for new substances with antiproliferative activity and apoptosis inducing potential towards HepG2 cells is important since HCC is notoriously resistant to conventional chemotherapy. Dietary phytochemicals with significant anti-proliferative and apoptosis inducing potential are considered as agents promising for cancer therapy. Naringenin, a common dietary flavonoid abundantly present in fruits and vegetables, is believed to possess strong cytotoxic activity in numerous types of cancer cells. However, the detailed molecular mechanisms of its antiproliferative effects and apoptosis induction are still unclear. In this study, we investigated antiproliferative and apoptosis-inducing effect of naringenin in human hepatocellular carcinoma HepG2 cells. Naringenin was shown to inhibit the proliferation of HepG2 cells resulted partly from an accumulation of cells in the G0_/G1 and G2/M phase of the cell cycle. Naringenin induced a rapid accumulation of p53, which might account for the naringenin-induced G0_/G1 and G2/M phase arrests in Hep G2 cells. In addition, naringenin have been shown to induce apoptosis as evidenced by nuclei damage and increased proportion of apoptotic cells detected by flow cytometry analysis. Naringenin triggered the mitochondrial-mediated apoptosis pathway as shown by an increased ratio of Bax/Bcl-2, subsequent release of cytochrome C, and sequential activation of caspase-3. Our results showed that naringenin had inhibitory effect on the growth of HepG2 cell line through inhibition of cell proliferation and apoptosis induction. The elucidation of the drug targets of naringenin on inhibition of tumor cells growth should enable further development of naringenin for liver cancer therapy.     

Recombinant adenovirus-mediated overexpression of TIMP-1 effectively suppresses hepatocellular carcinoma cell line HepG2 in vitro and in vivo

OBJECTIVE: To explore the effects of overexpression of human tissue inhibitors of metalloproteinase-1 (hTIMP-1) on proliferation, invasion, metastasis, angiogenesis, and apoptosis in human hepatocellular carcinoma (HCC) cells in vitro and in vivo. METHODS: Recombinant adenoviral vector containing hTIMP-1 (AdhTIMP-1) was constructed previously. HepG2 cells were infected by AdhTIMP-1 and the changes of cell proliferation and invasion were detected in vitro. The anticancer activity of AdhTIMP-1 was evaluated in BAL B/c mice bearing HCC. Tumor volume and pulmonary metastases were observed. The mechanisms underlying the antitumor effect in vivo were investigated based on detection of microvessel density and apoptosis in tumor tissues. RESULTS: The resultant AdhTIMP-1 was successfully constructed and the expression of hTIMP-1 was detected by Western blot and RT-PCR. AdTIMP-1 could effectively infect HepG2 cells and significantly inhibit the proliferative activity and invasive ability of the tumor cells. Compared with the controls, pre-infection of HepG2 cells by AdhTIMP-1 resulted in a significant inhibition of tumor formation by 75. 8%. A single local injection of AdhTIMP-1 into pre-established tumors significantly reduced the tumor growth rate by 45.4%, tumor-associated angiogenesis index by 47.8%, lung metastases by 70.4%, and showed a 3-fold increase of apoptotic tumor cells. CONCLUSION: Our data indicated that AdhTIMP-1 can significantly attenuate tumor proliferation and invasion, reduce metastasis, inhibit angiogenesis, and induce apoptosis in HCC-bearing mice and may pave the way for further liver cancer gene therapy.     

Inhibition of cell proliferation by nobiletin, a dietary phytochemical, associated with apoptosis and characteristic gene expression, but lack of effect on early rat hepatocarcinogenesis in vivo

Dietary phytochemicals can inhibit the development of certain types of tumors. We here investigated the effects of nobiletin (Nob), garcinol (Gar), auraptene (Aur), beta-cryptoxanthin- and hesperidine-rich pulp (CHRP) and 1,1'-acetoxychavicol acetate (ACA) on hepatocarcinogenesis in a rat medium-term liver bioassay, and also examined their influence on cell proliferation, cell cycle kinetics, apoptosis and cell invasion of rat and human hepatocellular carcinoma (HCC) cells, MH1C1 and HepG2, respectively. While there were no obvious suppressive effects on the development of putative preneoplastic liver lesions, inhibition of hepatocarcinoma cell proliferation was evident in the Nob group. Nob also caused G2/M cell cycle arrest and apoptosis. Microarray analysis identified a set of genes specifically regulated by Nob, and these are likely to be involved in the observed growth suppression of HCC cells. These results suggest that phytochemicals might have chemopreventive potential in late stages of hepatocarcinogenesis.     

Effect of anti-estrogen combined with roscovitine, a selective CDK inhibitor, on human breast cancer cells differing in expression of ER

Roscovitine (ROSC), a selective inhibitor of cyclin-dependent kinases (CDKs) reduces numbers of cancer cells in a concentration-dependent manner. At low doses ROSC arrests cell cycle progression and at higher doses it induces apoptosis. ROSC efficiently inhibits proliferation of human ER-alpha positive MCF-7 breast cancer cells by inducing G/M arrest and concomitantly initiates apoptosis by a p53-dependent pathway. However, the effect of ROSC is much weaker on MCF-7 cells maintained in the presence of estrogen-mimicking compounds. Therefore, we have examined the action of ROSC on other breast cancer cell lines differing in ER status and confirmed that tamoxifen (TAM) affects the efficacy of this CDK inhibitor. ROSC was effective against all tested breast cancer cell lines, arresting them at G1/S or G2/M transition and inducing apoptosis in SKBR-3 cells. Interestingly, TAM affected all tested cell lines, irrespective of their ER-a status, and in combination with ROSC it enhanced G1 or G2 arrest. Our results provide evidence that ROSC can be combined with antiestrogen therapy and that the mode of ROSC action strongly depends on the cellular context. The effect of TAM on ER-negative cancer cells indicates that TAM also crosstalks with other steroid hormone receptors.     

LAMP2抑制肝癌细胞增殖的机制

目的:探索LAMP2分子对肝癌细胞增殖的影响及其机制.方法:首先利用Western blot及QT-PCR技术检测在不同肝癌细胞系中LAMP2分子的表达水平,然后选择一种肝癌细胞系HepG2进行慢病毒转染以建立LAMP2沉默的稳定细胞系HepG2-9455.利用MTT法对比LAMP2沉默细胞系和未沉默细胞系细胞的增殖情况.检测其可能影响的几个关键信号通路分子如PI3K、AKT、NF-κB、ERK等以明确其作用机制.结果:LAMP2在各肝癌细胞系中普遍表达较高,其在正常肝组织中主要分布于胆管周围并且呈极性分布,然而在肝癌组织中LAMP2的定位却发生了紊乱.通过慢病毒构建的稳定细胞系HepG2-9455其LAMP2表达下降了90%,而在下调LAMP2后可以显著抑制肝癌细胞HepG2的增殖.并且在下调LAMP2分子后HepG2细胞的PI3K、AKT和NF-κB分子发生明显下调.结论:LAMP2分子可以通过调节下游的PI3K、NF-κB(p-p65)分子而影响肿瘤细胞的增殖.提示LAMP2分子在肝癌的恶性进展中可能发挥了重要的作用.     

人参皂苷下调IL-1受体相关激酶1基因表达抑制肝癌细胞增殖及促进其凋亡作用及机制

目的:探索人参皂苷F11对肝脏恶性肿瘤细胞增殖及凋亡的影响.方法:选择四种癌细胞系HepG2、Hep3B、SMMC-7721、MHCC-97H利用Western blot技术检测IRAK1在这些细胞中的基础表达量.然后选择一种IRAK1高表达的肝癌细胞系对其进行人参皂苷处理以观察该药物对IRAK1分子的影响,分别利用WST-1法检测肿瘤细胞的增殖及流式技术检测细胞凋亡是否发生改变.接着在HepG2细胞中转染si-IRAK1来从RNA及蛋白水平降低该分子后检测肿瘤细胞的增殖.结果:使用人参皂苷或者siIRAK1处理HepG2细胞可以明显降低IRAK1分子的表达,且它们对肿瘤细胞增殖的抑制率分别是49.6％ (P ＜0.000 1)和45.3％ (P ＜0.05).未进行人参皂苷处理的HepG2的凋亡率为2.9％左右,加入10μg/ml的人参皂苷后可以使肝癌细胞的凋亡率增加3倍左右达到12.0％左右(P＜0.01).结论:人参皂苷可以通过降低IRAK1及其活化后的p-IRAK1分子降低肝癌细胞的增殖及促进其凋亡,从而抑制肿瘤的恶行进展.     

长链非编码RNA SNHG5在肝癌中表达及其对HepG2细胞增殖、凋亡的影响

目的 检测长链非编码RNA SNHG5在肝癌组织中的表达,并探讨其对肝癌HepG2细胞增殖、凋亡等生物学行为的调控作用与相关机制。方法 采用荧光定量PCR（QPCR）检测30例肝癌组织和对应癌旁组织中SNHG5的表达水平;采用小干扰RNA（siRNA）技术敲低肝癌HepG2细胞SNHG5的表达量,采用CCK-8法、流式细胞术、QPCR以及Western blotting检测HepG2细胞的增殖、凋亡情况以及凋亡通路相关标志物表达水平的变化。结果 肝癌组织中SNHG5的表达水平较其配对癌旁组织明显上调（P＜0.05）。敲低SNHG5表达后HepG2细胞的增殖、抗凋亡能力均受到抑制,细胞凋亡蛋白cleaved caspase-3、BAX表达上调。结论 相较于正常组织,肝癌组织中长链非编码SNHG5呈高表达,SNHG5可能通过调节肝癌细胞的增殖、凋亡能力以及抑制凋亡通路激活进程而发挥促癌作用,可能是肝癌治疗的潜在靶点。     

重组腺病毒介导基质金属蛋白酶组织抑制因子-1对人肝癌HepG2细胞的影响与作用机制

目的探讨携带人基质金属蛋白酶组织抑制因子-1(hTIMP-1)的重组腺病毒(AdhTIMP-1)对人肝癌HepG2细胞增殖、侵袭、转移、凋亡以及瘤内血管生成的作用。方法构建AdhTIMP-1并感染人肝癌HepG2细胞,采用Boyden chamber检测细胞体外侵袭能力;感染AdhTIMP-1的HepG2细胞接种于裸鼠皮下观察其成瘤情况;建立荷瘤鼠模型,瘤内注射AdhTIMP-1,观察其对肿瘤生长的作用;处死荷瘤鼠,计数肺转移结节;CD34免疫组化观察肿瘤血管生成;原位末端标记(TUNEL)法检测肝癌细胞凋亡。结果AdhTIMP-1感染的HepG2细胞体外侵袭力下降91.4%,成瘤量下降75.8%,荷瘤鼠瘤体内注射AdhTIMP-1使肿瘤生长减少45.4%,组织血管密度减少47.8%,肺转移结节减少70.4%,肿瘤组织中细胞凋亡则增加了3倍。结论AdhTIMP-1能抑制肝癌HepG2细胞的侵袭、转移及瘤内血管形成,诱导肝癌细胞凋亡,可用于肝癌基因治疗的研究。     

Inhibition of human tumor xenograft growth in nude mice by a novel monoclonal anti-HSPG isolated from human liver

Heparan sulfate proteoglycans (HSPGs) were isolated from normal human liver and α monoclonal antibody (MAb) was raised against them. Preliminary studies showed that MAb clone 1E4-1C2 was able to react with many cell lines tested, including hematopoietic cells and solid tumors. MAb1E4-1C2 was used to study whether HSPG was involved in growth and proliferation of human liver cancer using hepatocellular carcinoma (HCC) cell line (HepG2) as a model. Inhibition by MAb1E4-1C2 of HepG2 cell proliferation was studied in vitro by MTT assay. For in vivo assay, xenograft induction in athymic mice was performed. The results showed that MAb1E4-1C2 inhibited proliferation of HepG2 cells significantly, compared to isotype and medium control. MAb1E4-1C2 also suppressed the growth of tumor, resulting in smaller tumor size and weight. The investigation also showed that MAb1E4-1C2 inhibited proliferation and restricted tumor growth through the induction of apoptosis. The results suggest that HSPG might be involved in liver cancer cell proliferation. Therefore, a specific MAb that was raised against liver HSPG might be an alternative therapeutic agent for the treatment of human liver cancer.     

Aes对肝癌细胞恶性生物学行为的影响

背景与目的:Aes(amino-terminal enhancer of split,Aes)是一种能够与转录因子结合调节转录活性的蛋白,以往的研究显示其在发育过程中起重要作用,近期研究发现Aes可参与结肠癌转移。本研究旨在观察外源Aes在肝癌细胞中的定位以及其对肝癌细胞增殖、侵袭和迁移能力的影响。方法:构建pEGFP-Aes表达载体,并通过LipofectamineTM2000将其转入Aes低表达的肝癌细胞系HepG2中,蛋白质印迹法(Westernblot)检测外源Aes的表达,荧光显微镜观察pEGFP-Aes在细胞中的定位,应用细胞计数盒(Cell CountingKit)-8检测转染Aes后细胞增殖活力变化;划痕实验检测转染前后细胞迁移能力的变化;Transwell实验检测Aes对细胞侵袭能力的影响。结果:Aes在HepG2中表达较低,将Aes转入肝癌细胞系HepG2,Aes在细胞核和细胞质中都有表达,并且在细胞核中形成点状浓积,Aes对肝癌细胞HepG2的增殖无显著影响,但能抑制HepG2的侵袭和迁移能力(P<0.05)。结论:Aes对肝癌细胞恶性表型的影响主要是抑制其侵袭和迁移能力。