EPA诱导人肝癌细胞SMMC-7721凋亡及端粒酶活性调控机制研究

目的:探究二十碳五烯酸(Eicosa Pentaenoic Acid.EPA)对SMMC-7721人肝癌细胞的凋亡、端粒逆转录酶h TERT的调控作用及端粒酶表达活性的影响。方法:体外培养SMMC-7721人肝癌细胞,用不同浓度的EPA(0μM、25μM、50μM、100μM、200μM)作用于SMMC-7721肝癌细胞(24 h、48 h、72 h)后,显微镜下观察其形态学变化;应用MTT法检测SMMC-7721肝癌细胞细胞增殖变化情况;Western-blot法检测h TERT、Bax、Bcl-2蛋白表达水平变化;Real Time-PCR检测h TERTm RNA的表达变化;ELISA法检测SMMC-7721肝癌细胞端粒酶活性的表达水平。结果:EPA可诱导肝癌细胞SMMC-7721发生细胞凋亡,具有明显的时间计量依赖关系。在此过程中Bcl-2蛋白表达的降低和Bax蛋白表达上调,同时端粒酶逆转录酶h TERT蛋白及其m RNA的表达水平和端粒酶活性均明显降低。结论:抑制端粒酶逆转录酶基因(h TERTm RNA)表达而抑制端粒酶的活性、诱导癌细胞凋亡,可能是EPA的抗癌作用机制之一。     

沉默EVL表达抑制肝癌细胞SMMC-7721的增殖和迁移能力

Ena/VASP 样蛋白（Ena/VASP like protein,EVL）是Ena/VASP家族成员之一,它参与肌动蛋白细胞骨架重组,以及细胞迁移、收缩环形成和细胞间附着.EVL在肝癌SMMC-7721细胞中高表达. 抑制EVL蛋白表达后,SMMC-7721细胞的增殖与迁移能力降低.为研究EVL在肝癌细胞的功能,构建了靶向shRNA干扰表达载体,稳定转染肝癌SMMC-7721细胞. MTT实验和细胞集落形成实验显示,与转染对照比较,沉默EVL蛋白表达可明显抑制SMMC-7721肝癌细胞的增殖、集落形成能力. Transwell实验证明,沉默EVL表达导致SMMC-7721细胞迁移能力降低. 进而,流式细胞术揭示,沉默EVL表达的SMMC-7721细胞G0/G1期细胞比例增多.研究结果提示,EVL蛋白可促进肝癌细胞的增殖与迁移;该结果可解释EVL在肝癌细胞中高表达的意义.     

6-Shogaol induces apoptosis in human hepatocellular carcinoma cells and exhibits anti-tumor activity in vivo through endoplasmic reticulum stress

6-Shogaol is an active compound isolated from Ginger (Zingiber officinale Rosc). In this work, we demonstrated that 6-shogaol induces apoptosis in human hepatocellular carcinoma cells in relation to caspase activation and endoplasmic reticulum (ER) stress signaling. Proteomic analysis revealed that ER stress was accompanied by 6-shogaol-induced apoptosis in hepatocellular carcinoma cells. 6-shogaol affected the ER stress signaling by regulating unfolded protein response (UPR) sensor PERK and its downstream target eIF2α. However, the effect on the other two UPR sensors IRE1 and ATF6 was not obvious. In prolonged ER stress, 6-shogaol inhibited the phosphorylation of eIF2α and triggered apoptosis in SMMC-7721 cells. Salubrinal, an activator of the PERK/eIF2α pathway, strikingly enhanced the phosphorylation of eIF2α in SMMC-7721 cells with no toxicity. However, combined treatment with 6-shogaol and salubrinal resulted in significantly increase of apoptosis and dephosphorylation of eIF2α. Overexpression of eIF2α prevented 6-shogaol-mediated apoptosis in SMMC-7721 cells, whereas inhibition of eIF2α by small interfering RNA markedly enhanced 6-shogaol-mediated cell death. Furthermore, 6-shogaol-mediated inhibition of tumor growth of mouse SMMC-7721 xenograft was associated with induction of apoptosis, activation of caspase-3, and inactivation of eIF2α. Altogether our results indicate that the PERK/eIF2α pathway plays an important role in 6-shogaol-mediated ER stress and apoptosis in SMMC-7721 cells in vitro and in vivo.     

Fluvastatin, a lipophilic statin, induces apoptosis in human hepatocellular carcinoma cells through mitochondria-operated pathway

Fluvastatin, a lipophilic statin, was known to inhibit proliferation and induce apoptosis in many cancer cells. Its potential anticancer was evaluated in three hepatocellular carcinoma (HCC) cell lines (HepG2, SMMC-7721 and MHCC-97H). Cells were treated with fluvastatin in vitro and its effect on cell proliferation, cell cycle, invasion and apoptosis was determined. Mechanism of apoptosis induced by fluvastatin on HCC cell lines was also investigated through western blotting and mitochondrial membrane potential (MMP) analysis. It was observed that fluvastatin inhibited proliferation of HCC cells by inducing apoptosis and G2/M phase arrest in a dose-dependent manner. The results of cell invasion assay revealed that fluvastatin significantly decreased the invasion potency of HCC cells. A mitochondria-operated mechanism for fluvastatin induced apoptosis might be involved and was supported by Western blotting and MMP analysis. After fluvastatin treatment, expression of Bcl-2 and procaspase-9 were downregulated, cytochrome c (cytosolic extract), Bax and cleaved-caspase-3 protein expression were increased. Furthermore, a breakdown of MMP in HCC cells was observed. To conclude, these results have provided a rationale for clinical investigations of fluvastatin in future as a potential anticancer reagent for growth control of HCC.     

蛋白激酶PRKX对人肝癌细胞粘附和迁移能力的影响

目的观察蛋白激酶PRKX对人肝癌细胞SMMC-7721粘附和迁移能力的影响。方法采用脂质体转染的方法,将PRKX表达质粒转染到SMMC-7721细胞中,蛋白印迹方法鉴定转染前后PRKX蛋白的表达。细胞-基质粘附实验测定对照组和PRKX转染组SMMC-7721细胞的粘附能力。细胞迁移实验测定对照组和PRKX转染组SMMC-7721细胞的迁移能力。结果 SMMC-7721细胞转染组PRKX蛋白的表达增加,SMMC-7721细胞转染组的粘附能力和迁移能力均较对照组增加。结论 PRKX可增加人肝癌细胞SMMC-7721的粘附和迁移能力。     

二氢青蒿素通过诱导铁死亡抑制肝癌细胞生长

二氢青蒿素（dihydroartemisinin,DHA）是青蒿素的一种衍生物,在多种肿瘤中表现出明显的抗肿瘤活性,但其具体机制不详。本文探讨了DHA对肝癌细胞的毒性作用机制。利用CCK-8试剂检测DHA对肝癌细胞株活力的影响,通过荧光探针染色及流式细胞术分析细胞内ROS及脂质过氧化物水平的变化;通过谷胱甘肽测定试剂盒检测细胞内还原型谷胱甘肽含量的变化,并通过免疫印迹分析DHA作用下细胞内铁死亡通路蛋白质中GPX4的变化。结果发现,DHA能显著抑制SMMC-7721及Huh-7细胞活力,其半数抑制浓度分别为23.74 μmol/L及26.92 μmol/L。 在35 μmol/L DHA 处理下,SMMC-7721及Huh-7细胞内ROS分别升高2.6倍和2.1倍,脂质过氧化物升高2.3倍和1.7倍。DHA可诱导细胞内GSH含量下降,并能下调铁死亡相关蛋白质GPX4蛋白水平。通过利用小分子抑制剂进行功能恢复实验发现,ROS抑制剂、铁螯合剂及铁死亡抑制剂都可不同程度恢复DHA引起的细胞活力下降。进一步检测发现,铁死亡抑制剂可抑制DHA诱导的脂质过氧化,并恢复GSH含量及GPX4蛋白水平。结果表明,在肝癌细胞中,DHA可通过诱导细胞发生铁死亡抑制肝癌细胞生长。     

二氢青蒿素通过诱导铁死亡抑制肝癌细胞生长

二氢青蒿素（dihydroartemisinin,DHA）是青蒿素的一种衍生物,在多种肿瘤中表现出明显的抗肿瘤活性,但其具体机制不详。本文探讨了DHA对肝癌细胞的毒性作用机制。利用CCK-8试剂检测DHA对肝癌细胞株活力的影响,通过荧光探针染色及流式细胞术分析细胞内ROS及脂质过氧化物水平的变化;通过谷胱甘肽测定试剂盒检测细胞内还原型谷胱甘肽含量的变化,并通过免疫印迹分析DHA作用下细胞内铁死亡通路蛋白质中GPX4的变化。结果发现,DHA能显著抑制SMMC-7721及Huh-7细胞活力,其半数抑制浓度分别为23.74 μmol/L及26.92 μmol/L。 在35 μmol/L DHA 处理下,SMMC-7721及Huh-7细胞内ROS分别升高2.6倍和2.1倍,脂质过氧化物升高2.3倍和1.7倍。DHA可诱导细胞内GSH含量下降,并能下调铁死亡相关蛋白质GPX4蛋白水平。通过利用小分子抑制剂进行功能恢复实验发现,ROS抑制剂、铁螯合剂及铁死亡抑制剂都可不同程度恢复DHA引起的细胞活力下降。进一步检测发现,铁死亡抑制剂可抑制DHA诱导的脂质过氧化,并恢复GSH含量及GPX4蛋白水平。结果表明,在肝癌细胞中,DHA可通过诱导细胞发生铁死亡抑制肝癌细胞生长。     

Longikaurin A,a natural ent-kaurane,induces G2/M phase arrest via downregulation of Skp2 and apoptosis induction through ROS/JNK/c-Jun pathway in hepatocellular carcinoma cells

Hepatocellular carcinoma (HCC) is the most common form of primary liver cancer, and is also highly resistant to conventional chemotherapy treatments. In this study, we report that Longikaurin A (LK-A), an ent-kaurane diterpenoid isolated from the plant Isodon ternifolius, induced cell cycle arrest and apoptosis in human HCC cell lines. LK-A also suppressed tumor growth in SMMC-7721 xenograft models, without inducing any notable major organ-related toxicity. LK-A treatment led to reduced expression of the proto-oncogene S phase kinase-associated protein 2 (Skp2) in SMMC-7721 cells. Lower Skp2 levels correlated with increased expression of p21 and p-cdc2 (Try15), and a corresponding decrease in protein levels of Cyclin B1 and cdc2. Overexpression of Skp2 significantly inhibited LK-A-induced cell cycle arrest in SMMC-7721 cells, suggesting that LK-A may target Skp2 to arrest cells at the G2/M phase. LK-A also induced reactive oxygen species (ROS) production and apoptosis in SMMC-7721 cells. LK-A induced phosphorylation of c-Jun N-terminal kinase (JNK), but not extracellular signal-regulated kinase and P38 MAP kinase. Treatment with, the JNK inhibitor SP600125 prevented LK-A-induced apoptosis in SMMC-7721 cells. Moreover, the antioxidant N-acetylcysteine prevented phosphorylation of both JNK and c-Jun. Taken together, these data indicate that LK-A induces cell cycle arrest and apoptosis in cancer cells by dampening Skp2 expression, and thereby activating the ROS/JNK/c-Jun signaling pathways. LK-A is therefore a potential lead compound for development of antitumor drugs targeting HCC.     

Plumbagin induces autophagy and apoptosis of SMMC-7721 cells in vitro and in vivo

Plumbagin (PL), an active naphthoquinone compound, has been demonstrated to be a potential anticancer agent. However, the underlying anticancer mechanism is not fully understood. In this study, the human hepatocellular carcinoma (HCC) SMMC-7721 cell line was studied in an in vitro model. The cell proliferation was inhibited by PL in a dose- and time-dependent manner. Electron microscopy, acridine orange staining, and immunofluorescence were used to evaluate autophagosome formation and LC3 protein expression in PL-treated SMMC-7721 cells. Real-time polymerase chain reaction and Western blot showed that PL treatment suppressed the expression of apoptosis and autophagy factors (LC3, Beclin1, Atg7, and Atg5), which are associated with tumor apoptosis and autophagy in SMMC-7721 cells. In the study of in vitro tumor nude mouse models, PL can inhibit tumor growth. Cell apoptosis and autophagy of the transplanted tumors were evaluated by hematoxylin and eosin staining, terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling staining, and Western blot. In addition, in the in vivo studies of HCC cells, we found that pretreatment with the autophagy inhibitor 3-methyladenine blocked the formation of apoptosis induced by PL. In contrast, administration of the apoptosis inhibitor Z-VAD did not affect PL-induced autophagy. Taken together, our findings strongly suggest that PL is a promising drug with significant antitumor activity in HCC.     

三氧化二砷对肝癌细胞系SMMC-7721凋亡及 Smac caspase-9、caspase-3 蛋白表达的影响

目的：研究三氧化二砷（As203）对人肝癌细胞SMMC-7721的促凋亡作用及对Smac、caspase-9、caspase-3表达的影响。方法：人肝癌细胞SMMC-7721经As20,处理,共分为四组,分别为空白对照组、低剂量组、中等剂量组、高剂量组。分别采用MTT、Hoechst33258染色法、Annexin V-FITC／PI双染法观察其对SMMC．7721细胞增殖的抑制,凋亡细胞核的形态学变化,以及诱导凋亡作用;采用Westemblot法检测凋亡相关蛋白Smac、caspase-9、caspase-3表达的变化。结果：MTT显示：As203在体外能明显抑制SMMC-7721的生长,具有时间剂量依赖关系,与空白对照组相比,其余三组细胞生存率明显下降,差异均有统计学意义（P〈0．05）;Hoechst33258显示细胞呈明显的凋亡细胞形态学特征,具有剂量依赖性;AnnexinV-FITC／PI双染法显示：As203作用24小时可诱导SMMC-7721细胞凋亡,且呈剂量依赖性,与空白对照组相比（2．69±0．58）,其余三组（4．01±0．58）、（5．99±1．69）、（9．26±2．34）差异均有统计学意义（P〈0．05）;Westernblot显示：As2O3作用SMMC-7721细胞24小时,Smac、caspase-9、caspase-3表达上升,呈剂量依赖性,与空白对照组相比,其余三组蛋白表达量明显增加,差异均有统计学意义（P〈0．05）。结论：-定量的As203能抑制SMMC-7721细胞增殖,促进其凋亡,其机制可能与调控Smac、caspase-9、caspase-3表达有关。     

独角莲对肝癌细胞SMMC-7721细胞增殖抑制作用机理的研究

临床上独角莲对包括肝癌在内的多种肿瘤有一定的治疗作用。为揭示其抑癌机理,我们用独角莲根茎水提物(the aqueous extract from dried powdered rhizomes of Typhonium giganteum Engl.,AEoTGE)作用肝癌细胞株SMMC-7721,研究独角莲对SMMC-7721细胞增殖;细胞周期和细胞凋亡的影响。噻唑氮蓝(MTT)比色实验和流式细胞仪(flow cytometry,FCM)测定表明：AEoTGE能较强抑制SMMC-7721细胞的生长,SMMC-7721细胞被阻滞在S期,井诱导细胞凋亡。提示独角莲是一种在肝癌治疗上有前景的中草药。     

Identification and expression profiles of genes and protens in SMMC-7721 cells

In the study presented here, we first evaluated effect of CDDP on liver cancer cells SMMC-7721 apoptosis and motility capacity. Then, we evaluate inhibitory effect of CDDP on tumour growth and its possible molecular mechanism in liver cancer mice model. Results showed that the apoptosis rate of cells decreased with increasing CDDP. Analysis of the effect of the CDDP on cell cycle was performed by flow cytometry and results show a dose-dependent increase in the percentage of cells in the S-phase of the cell cycle, with a decrease in the percentage of cells in the G1 and G2/M phases. CDDP did not close the wound even after 48 h, as opposed to untreated cells (0 mg/l). Similarly, the migratory and invasion capacity of SMMC-7721 cells was also reduced after treatment with CDDP, as evaluated by a transwell assay. Animal experiment indicated that CDDP administration could increase blood WBC, total protein, albumin and A/G, decrease blood alanine aminotransferase, aspartate aminotransferase and alkaline phosphatase levels in hepatocellular carcinomas mice. Immunohistochemistry analysis showed that positive expression of Fas and Bax proteins in the medicine-treated (II, III) group was significantly higher, whereas the expression of NF-κB, P53, Bcl-2 proteins was significantly lower than those of the control group. Gene expression analysis using Real time PCR methods revealed a significant up-regulation in the expression levels of Bax mRNA in the medicne-treated (II, III) group when compared to untreated control. In contrast, CDDP-treated group showed a significant down regulation in the expression levels of Bcl-2 mRNA as compared to untreated control group. These results are in agreement with immunohistochemistry data. Our observations indicate that CDDP has damaged effects on liver tumour cells SMMC-7721 including apoptosis, motility and cell cycle under in vitro. CDDP can enhance pro-apoptosis gene Fas, Bax expression, decrease anti-apoptosis genes Bcl-2 expression, and mutant genes P53, NF-κB proteins expression.     

Effects of matrine against the growth of human lung cancer and hepatoma cells as well as lung cancer cell migration

The purpose of this study is to investigate in vitro and ex vivo effects of matrine on the growth of human lung cancer and hepatoma cells and the cancer cell migration as well as the expressions of related proteins in the cancer cells. Matrine significantly inhibited the in vitro and ex vivo growth of human non-small cell lung cancer A549 and hepatoma SMMC-7721 cells. Matrine induced the apoptosis in A549 and SMMC-7721 cells. Western blot analysis indicated that matrine dose-dependently down-regulated the expression of anti-apoptotic protein Bcl-2 and up-regulated the level of pro-apoptotic protein bax, eventually leading the reduction of ratios of Bcl-2/Bax proteins in A549 and SMMC-7721 cells. Furthermore, matrine significantly suppressed the A549 cell migration without reducing the cell viability. In addition, matrine dramatically reduced the secretion of vascular endothelial growth factor A in A549 cells. More importantly, matrine markedly enhanced the anticancer activity of anticancer agent trichostatin A (the histone deacetylase inhibitor) by strongly reducing the viability and/or the ratio of Bcl-2/Bax protein in A549 cells. Our findings suggest that matrine may have the broad therapeutic and/or adjuvant therapeutic application in the treatment of human non-small cell lung cancer and hepatoma.     

TGF-β1-promoted epithelial-to-mesenchymal transfor mation and cell adhesion contribute to TGF-β1-enhanced cell migration in SMMC-7721 cells

Transforming growth factor-bl (TGF-β1), a multi-function polypeptide, is a double-edged sword in cancer. For some tumor cells, TGF-β1 is a potent growth inhibitor and apoptosis inducer. More commonly, TGF-β1 losesits growth-inhibitory and apoptosis-inducing effects, but stimulates the metastatic capacity of tumor cells. It is currently little known about TGF-β1-promoted cell migration in hepatocellular carcinoma (HCC) cells, let alone its mechanism. In this study, we found that TGF-β1 lost its tumor-suppressive effects, but significantly stimulated cellmigration in SMMC-7721 human HCC cells. By FACS and Western blot analysis, we observed that TGF-β1 enhanced the expression of ct5131 integrin obviously, and subsequently stimulated cell adhesion onto fibronectin(Fn). Furthermore, we observed that TGF-β1 could also promote SMMC-7721 cells adhesion onto laminin (Ln).Our data also provided evidences that TGF-β1 induced epithelial-to-mesenchymal transformation (EMT) in SMMC-7721 cells. First, SMMC-7721 cells clearly switched to the spindle shape morphology after TGF-β1 treatment.Furthermore, TGF-β1 induced the down-regulation of E-cadherin and the nuclear translocation of β1-catenin. These results indicated that TGF-β1-promoted cell adhesion and TGF-β1-induced epithelial-to-mesenchymal transfor-mation might be both responsible for TGF-β1-enhanced cell migration.     

Mechanism of Arctigenin-Induced Specific Cytotoxicity against Human Hepatocellular Carcinoma Cell Lines: Hep G2 and SMMC7721

Arctigenin (ARG) has been previously reported to exert high biological activities including anti-inflammatory, antiviral and anticancer. In this study, the anti-tumor mechanism of ARG towards human hepatocellular carcinoma (HCC) was firstly investigated. We demonstrated that ARG could induce apoptosis in Hep G2 and SMMC7721 cells but not in normal hepatic cells, and its apoptotic effect on Hep G2 was stronger than that on SMMC7721. Furthermore, the following study showed that ARG treatment led to a loss in the mitochondrial out membrane potential, up-regulation of Bax, down-regulation of Bcl-2, a release of cytochrome c, caspase-9 and caspase-3 activation and a cleavage of poly (ADP-ribose) polymerase in both Hep G2 and SMMC7721 cells, suggesting ARG-induced apoptosis was associated with the mitochondria mediated pathway. Moreover, the activation of caspase-8 and the increased expression levels of Fas/FasL and TNF-α revealed that the Fas/FasL-related pathway was also involved in this process. Additionally, ARG induced apoptosis was accompanied by a deactivation of PI3K/p-Akt pathway, an accumulation of p53 protein and an inhibition of NF-κB nuclear translocation especially in Hep G2 cells, which might be the reason that Hep G2 was more sensitive than SMMC7721 cells to ARG treatment.     

Oroxylin A induced apoptosis of human hepatocellular carcinoma cell line HepG2 was involved in its antitumor activity

We previously reported that wogonin, a flavonoid compound, was a potent apoptosis inducer of human hepatoma SMMC-7721 cells and murine sarcoma S180 cells. In the present study, the effect of oroxylin A, one wogonin structurally related flavonoid isolated from Scutellariae radix, on human hepatocellular carcinoma cell line HepG2 was examined and molecular mechanisms were also investigated. Oroxylin A inhibited HepG2 cell proliferation in a concentration- and time-dependent manner measured by MTT-assay. Treatment with an apoptosis-inducing concentration of oroxylin A caused typical morphological changes and apoptotic blebbing in HepG2 cells. DNA fragmentation assay was used to examine later apoptosis induced by oroxylin A. FACScan analysis revealed a dramatic increase in the number of apoptotic and G(2)/M phase arrest cells after oroxylin A treatment. The pro-apoptotic activity of oroxylin A was attributed to its ability to modulate the concerted expression of Bcl-2, Bax, and pro-caspase-3 proteins. The expression of Bcl-2 protein and pro-caspase-3 protein was dramatically decreased after treatment with oroxylin A. These results demonstrated that oroxylin A could effectively induce programmed cell death and suggested that it could be a promising antitumor drug.     

2′,4′-Dihydroxy-6′-methoxy-3′,5′-dimethylchalcone,from buds of Cleistocalyx operculatus,induces apoptosis in human hepatoma SMMC-7721 cells through a reactive oxygen species-dependent mechanism

Nowadays, much effort is being devoted to detect new substances that not only significantly induce the death of tumor cells, but also have little side effect on normal cells. Our previous study showed that 2′,4′-dihydroxy-6′-methoxy-3′,5′-dimethylchalcone (DMC) exhibited significant cytotoxic potential with an IC₅₀ value of 32.3 ± 1.13 μM against SMMC-7721 cells and could induce SMMC-7721 cells apoptosis. In the present study, we found that DMC was almost nontoxic to human normal liver L-02 and human normal fetal lung fibroblast HFL-1 cells as their IC₅₀ values (111.0 ± 4.57 and 152.0 ± 4.83 µM for L-02 and HFL-1 cells, respectively) were much higher. To further explore the apoptotic mechanism of DMC, we investigated the role of the reactive oxygen species (ROS) in the apoptosis induced by DMC in SMMC-7721 cells. Our results suggested that the cytotoxicity and the generation of intracellular ROS were inhibited by N-acetylcysteine (NAC). Reversal of apoptosis in NAC pretreated cells indicated the involvement of ROS in DMC-induced apoptosis. The loss of mitochondrial membrane potential (ΔΨm) induced by DMC was significantly blocked by NAC. NAC also prevented the decrease of Caspase-3 and -9 activities, the increase of Bcl-2 protein expression and the decrease of p53 and PUMA protein expressions. Together, these results indicated that ROS played a key role in the apoptosis induced by DMC in human hepatoma SMMC-7721 cells.     

Two specific inhibitors of the phosphatidylinositol 3-kinase LY294002 and wortmannin up-regulate β1,4-galactosyltransferase I and thus sensitize SMMC-7721 human hepatocarcinoma cells to cycloheximide-induced apoptosis

Previous study indicated that β1,4-galactosyltransferase I (β1,4GT1) was up-regulated by cycloheximide (CHX) and thus enhanced apoptosis induced by CHX in SMMC-7721 cells. In this study, we reported that constitutively active Akt protein (myr-Akt) inhibited CHX-induced apoptosis in SMMC-7721 cells through down-regulating β1,4GT1. However, the two PI3K inhibitors LY294002 and wortmannin treatment up-regulated β1,4GT1 through enhancing Sp1 protein expression and consequently increased CHX-induced SMMC-7721 cells apoptosis. Besides, our results suggested that β1,4GT1 and cell surface galactose residues synthesized by elevated β1,4GT1 played an important role in SMMC-7721 cells apoptosis treated with CHX and PI3K inhibitor together. Moreover, we found that CHX accentuated β1,4GT1 through down-regulating Akt expression to mediate SMMC-7721 cells apoptosis. Taken together, PI3K inhibitors LY294002 and wortmannin up-regulated β1,4GT1 and enhanced CHX-induced apoptosis in SMMC-7721 cells, which suggested that PI3K inhibitors might have therapeutic potential when combined with CHX in the treatment of hepatoma.     

PI3K信号通路通过Skp2、p27调节肝癌细胞的增殖

探讨磷脂酰肌醇3-激酶(PI3K)信号通路调节肝癌细胞增殖的机制.用LY294002特异性阻断PI3K信号通路后,人肝癌细胞(SMMC-7721)的增殖明显被抑制.RT-PCR及蛋白质印迹结果显示,LY294002增加了p27蛋白的表达,但不影响p27的mRNA表达.在LY294002处理的细胞中转入p27的RNAi质粒以干扰p27蛋白的表达后,肝癌细胞的增殖能力可部分恢复.放线菌酮(Chx)处理实验表明,阻断PI3K信号通路使p27蛋白的半衰期延长,稳定性增加.进一步研究发现,LY294002可抑制介导p27蛋白降解的关键分子Skp2的mRNA表达,还可缩短Skp2蛋白的半衰期,降低Skp2蛋白的稳定性.但在SMMC-7721中分别转染PI3K下游重要靶分子Akt的持续激活和失活突变体,却并不影响p27蛋白的表达.这些结果表明,PI3K信号通路在转录及翻译后水平调节Skp2的表达而影响p27蛋白的降解,从而调节肝癌细胞的增殖,但Akt并没有参与这种调节.     

Downregulation of NIN/RPN12 binding protein inhibit the growth of human hepatocellular carcinoma cells

The ribosome assembly factor NIN/RPN12 binding protein (Nob1) has been suggested to be essential for processing of the 20S pre-rRNA to the mature 18S rRNA, and is also reported to participate in proteasome biogenesis. However, it is unclear whether Nob1 is involved in tumor cells growth. The aim of this study was to determine whether the suppression of Nob1 by short hairpin RNA (shRNA) inhibits the growth of human hepatocellular carcinoma (HCC) cells. Recombinant lentiviral shRNA expression vector carrying Nob1 was constructed and then infected into human HCC cell line SMMC-7721. The growth properties of SMMC-7721/pGCSIL-GFP-shNC and pGCSIL-GFP-shNob1 cells were determined by MTT, BrdU incorporation assay, and flow cytometric analysis. In addition, the colony formation and tumor growth ability in nude mice were detected to define the function of Nob1 in cell transformation and tumorigenesis. Our data showed that the growth and proliferation of SMMC-7721/pGCSIL-GFP-shNob1 cells were significantly reduced compared with the SMMC-7721/pGCSIL-GFP-shNC. In addition, the colony formation was impaired after the suppression of Nob1 in SMMC-7721 cells. And in vivo, the tumor formation ability of the SMMC-7721/pGCSIL-GFP-shNob1 cells was significantly reduced compared with the control cells. Our data support that Nob1 is an important regulator of the tumorigenic properties of human HCC and could be used as a candidate therapeutic target in human HCC.