Interferon-alpha-2b induces autophagy in hepatocellular carcinoma cells through Beclinl pathway

Objeαive： To determine whether Interferon-alpha-2b （IFN-α2b） can modulate the autophagic response in hepatocellular carcinoma cells. Methods： Hepatocellular carcinoma cells were treated with IFN-α2b. Autophagy was assessed by acridine orange staining, GFP-LC3 dotted assay, transmission eleαron microscopy and immunoblotting. Results： Acridine orange staining showed that IFN-α2b triggered the accumulation of acidic vesicular and autolysosomes in HepG2 cells. The acridine orange HepG2 cell ratios were （4.3±1.0）%, （6.9±1.4）%, and （13.1±2.3）%, respeαively, after treatment with 100, 1,000, and 10,000 IU/mL IFN-α2b for 48 h. A markedly punαate pattern was observed in HepG2 cells treated with 10,000 IU/mL [FN-α2b for 48 h, but only diffuse and weakly fluorescent GFP-LC3 punαa was observed in control cells. HepG2 cells treated with 10,000 IU/mL IFN-α2b for 48 h developed autophagosome-like charaαeristics, including single- or double-membrane vacuoles containing intaα and degraded cellular debris. The Beclinl and LC3-II protein expression was up-regulated by IFN-α2b treatment. Conclusion： Autophagy can be induced in a dose-dependent manner by treatment with IFN-α2b in HepG2 cells, and the Beclinl signaling pathway was stimulated by IFN-α2b.     

Giganteaside D induces ROS-mediated apoptosis in human hepatocellular carcinoma cells through the MAPK pathway

Purpose

Every year, almost one million individuals are diagnosed with hepatocellular carcinoma (HCC) worldwide and more than 690,000 patients die of it. At present, most therapeutic anti-HCC agents are not effective, which is due to the appearance of chemo-resistance and/or toxic side effects. Therefore, it is imperative to find novel more effective anti-HCC agents. Here, we evaluated the effect of giganteaside D (GD), an oleanolic acid saponin from P. scabiosaefolia, on the growth and apoptosis of HCC cells.

Methods and results

Using MTT and clonogenic assays, we found that GD exhibited a significant growth inhibitory effect on the HCC-derived cell lines HepG2 and Bel-7402. In addition, we found that GD induced mitochondria-mediated apoptosis in these HCC-derived cells, as indicated by a decreased mitochondrial potential, activation of Caspase-9 and Caspase-3, cleavage of PARP and release of Cytochrome C from the mitochondria. Besides, we found that GD stimulated the generation of reactive oxygen species (ROS) and that blockage of ROS attenuated the GD-induced mitochondria-mediated apoptosis. Additionally, we found that GD treatment led to a decrease in phosphorylated Erk (p-Erk) and triggered the generation of p-JNK, both components of the mitogen-activated protein kinase (MAPK) signaling pathway. Inhibition of Erk or JNK by specific inhibitors or siRNAs augmented or attenuated the cytotoxic and apoptotic effects of GD.

Conclusions

From our results we conclude that GD can induce ROS-mediated apoptosis in HCC-derived cells through the MAPK pathway. This observation may open up avenues to explore the future use of GD as a HCC chemotherapeutic agent.

     

Profilin 1 induces drug resistance through Beclin1 complex‐mediated autophagy in multiple myeloma

Autophagy plays an important role in multiple myeloma (MM) for homeostasis, survival and drug resistance, but which genes participate in this process is unclear. We identified several cytoskeleton genes upregulated in MM patients by gene expression profiling (GEP) datasets; in particular, patients with high profilin 1 (PFN1) expression had poor prognosis in MM. In vitro, overexpressed PFN1 promotes proliferation and bortezomib (BTZ) resistance in MM cells. Further study indicated overexpression of PFN1 significantly promoted the process of autophagy and induced BTZ resistance in MM. Otherwise, knockdown of PFN1 blocked autophagy and sensitized MM to BTZ. Co‐immunoprecipitation in MM cells indicated that PFN1 could bind Beclin1 complex and promote the initiation of autophagy. Inhibition of autophagy by blocking the formation of Beclin1 complex could reverse the phenotype of BTZ resistance in MM. Our findings suggested that PFN1 could promote autophagy through taking part in Beclin1 complex and contribute to BTZ resistance, which may become a novel molecular target in the therapy of MM.     

Restoration of klotho expression induces apoptosis and autophagy in hepatocellular carcinoma cells

Purpose

Klotho has been identified as a tumor suppressor in several human malignancies including hepatocellular carcinoma (HCC). However, the signaling pathways involved in the tumor suppressive role of klotho in HCC have not been reported. Here, we investigated the role of klotho in HCC cell proliferation, apoptosis, autophagy, and invasion, as well as its associated signal transduction pathways.

Methods

Restoration of klotho gene expression was established by delivering a klotho gene expression vector into the human HCC cell lines HepG2 and MHCC-97-H. Cell viability was measured using a cell counting (CCK-8) assay and apoptosis was analyzed through flow cytometry. Autophagy was measured via LC3-I and LC3-II protein expression levels and tumor cell invasion was assessed using a Matrigel invasion chamber assay. Expression and phosphorylation of several apoptosis and survival related proteins were assessed using Western blot assays.

Results

Exogenous klotho gene expression significantly inhibited HCC cell proliferation, induced HCC cell apoptosis, increased LC3-I and LC3-II protein expression in HCC cells, and decreased migration of HCC cells in a Matrigel invasion chamber assay. Exogenous klotho gene expression also down-regulated the phosphorylation levels of the IGF-1 receptor, and the downstream Akt, ERK, and p70S6K proteins. Both apoptosis and autophagy inhibitors decreased klotho-induced apoptosis and autophagy.

Conclusion

Klotho is a tumor suppressor that, through the regulation of IGF-1R phosphorylation and subsequent activation of downstream Akt-p70S6K and ERK signaling, regulates HCC tumor cell proliferation, apoptosis, autophagy and invasion.     

新补骨脂异黄酮通过caspase-3/GSDME通路诱导肝细胞癌Huh-7细胞焦亡

目的：探讨新补骨脂异黄酮（NBIF）对肝细胞癌（HCC）Huh-7细胞焦亡的影响及其分子机制。方法：体外培养Huh-7细胞,用CCK-8法检测不同浓度的NBIF处理48 h时对细胞存活率的影响,光学显微镜下观察NBIF处理后Huh-7细胞的形态变化,乳酸脱氢酶（LDH）释放实验检测细胞的LDH释放量,WB实验检测细胞中GSDME、caspase-3的蛋白水平变化。采用si RNA干扰Huh-7细胞中caspase-3、GSDME表达后,CCK-8法检测NBIF处理对细胞存活率的影响,WB实验检测GSDME蛋白表达水平,观察NBIF处理对细胞形态的影响,并检测细胞LDH释放量。结果：60μmol/L以上的NBIF均能显著抑制Huh-7细胞的增殖（均P<0.01）,光学显微镜下观察到NBIF处理后的细胞出现肿胀、吐泡现象,且LDH释放增加（P<0.01）;WB实验结果表明,NBIF能够激活caspase-3蛋白并切割GSDME蛋白,增加GSDME-N的表达（均P<0.01）。干扰caspase-3、GSDME表达后,NBIF对细胞的抑制作用减弱（均P<0.01）,G...     

The BH3 mimetic S1 induces autophagy through ER stress and disruption of Bcl-2/Beclin 1 interaction in human glioma U251 cells

Previous results showed that a novel BH3 mimetic S1 could induce cell death in a wide range of cancer types in vitro through Bax/Bak-dependent apoptosis. We demonstrated that in addition to mitochondrial pathway apoptosis, endoplasmic reticulum (ER) stress-associated apoptosis was also induced by S1. Moreover, S1 can induce autophagy in U251 cells, which may occur through ER stress and disruption of the association of Bcl-2 and Beclin 1. Inhibition of autophagy by the autophagic inhibitors 3-methyladenine (3-MA) or chloroquine (CQ) increased S1-induced apoptosis. In conclusion, autophagy plays an important role in S1-induced U251 cell death.     

PTEN依赖其磷酸酶活性诱导肝癌细胞发生失巢凋亡机制的探讨

目的 研究抑癌基因PTEN对人肝癌SMMC-7721细胞失巢凋亡的影响并探讨其可能的机制。方法 将携有野生型PTEN基因及无磷酸活性的突变型C124A-PTEN基因的真核表达载体转染SMMC-7721细胞,利用Western印迹杂交法,检测PTEN蛋白表达与蛋白激酶B(PKB／Akt)、焦点黏附激酶(FAK)磷酸化水平的变化,并应用流式细胞仪技术和激光共聚焦显微镜技术,分析各种细胞在黏附与失黏附状态下的凋亡。结果 与对照细胞相比,转染野生型PTEN的SMMC-7721细胞中,FAK和Akt的磷酸化水平分别降低了65．2％和89．1％,且失巢凋亡率由9．5％增至31．3％;而转染C124A-PTEN的SMMC-7721细胞中,FAK和Akt的磷酸化水平及失巢凋亡率均无明显变化。结论 抑癌基因PTEN可依赖其磷酸酶活性抑制FAK和Akt的磷酸化,并诱导肝癌细胞发生失巢调亡。     

过表达PRRX1 通过p53 介导的线粒体凋亡途径诱导肝癌SMMC7721 细胞凋亡

[摘要] 目的：探讨配对相关同源框1 蛋白（PRRX1）过表达对肝癌SMMC7721 细胞凋亡的影响及其分子机制。方法：分别用慢病毒介导PRRX1 过表达载体（pGMLV-PRRX1）、空载质粒（Vector）感染人肝癌SMMC7721 细胞,用qPCR和WB实验检测慢病毒感染后细胞中PRRX1 mRNA和蛋白的表达变化,用CCK-8 法、Annexin-V FITC/PI 染色流式细胞术分别检测PRRX1 过表达对SMMC7721 细胞增殖、凋亡的影响,用线粒体膜电位检测试剂盒（JC-10 染色法）检测细胞线粒体膜电位变化,用caspase 活性检测试剂盒（分光光度法）测定细胞中caspase-8 和caspase-9 酶活性,用WB实验检测细胞中p53、Bcl-2、Bax、Fas、Cleaved-caspase-3以及线粒体和细胞质中细胞色素C（Cty C）蛋白的表达。结果：成功构建PRRX1 过表达的SMMC7721 细胞株,感染细胞中PRRX1 mRNA和蛋白的表达水平显著升高（均P<0.01）。与对照组和空载组比较,PRRX1 过表达组SMMC7721 细胞的增殖能力显著下降、细胞凋亡率显著增高、Cleaved-caspase-3 剪切水平显著升高、线粒体膜电位显著下降、线粒体中Cty C蛋白表达下调、胞质中Cty C蛋白表达上调以及caspase-9 酶活性升高（P<0.05 或P<0.01）,同时p53 和Bax 蛋白表达增加而Bcl-2 蛋白表达降低（均P<0.05）,但Fas 蛋白表达及caspase-8 酶活性无显著变化（均P>0.05）。结论: PRRX1 过表达可诱导肝癌SMMC7721 细胞凋亡,其机制可能与p53介导的线粒体凋亡途径被激活有关。     

MAC related mitochondrial pathway in oroxylin A induces apoptosis in human hepatocellular carcinoma HepG2 cells

Oroxylin A is a flavonoid isolated from the root of Scutellaria baicalensis Georgi. Our previous work demonstrated that the anti-tumor activity of oroxylin A was mainly attributed to its apoptosis inducing effect in cells. The present study explores the exact molecular mechanism of oroxylin A-induced apoptosis in tumor cells. We showed that oroxylin A-induced apoptosis in HepG2 cells was achieved through mitochondrial pathway. We also investigated which mitochondrial channels, PTP or MAC or both, were involved in the permeabilization of the mitochondrial outer membrane after treatment with oroxylin A. The results showed that oroxylin A-induced apoptosis in a PTP-independent manner; therefore, we focused our attention on MAC. As Bax is an essential constituent of MAC in certain systems, we examined the activation, subcellular location, oligomeric structure of Bax in HepG2 cells treated with oroxylin A. Moreover, our results showed that overexpression of Bcl-2 inhibited oroxylin A-induced apoptosis. In summary, we have demonstrated that opening of MAC, but not PTP, played a key role in oroxylin A-induced activation of mitochondrial apoptotic pathway in HepG2 cells.     

Lamc1 promotes the Warburg effect in hepatocellular carcinoma cells by regulating PKM2 expression through AKT pathway

Hepatocellular carcinoma (HCC), the most common aggressive malignancy of liver, is the third leading cause of cancer death across the world. Laminin gamma 1 (Lamc1), encodes laminin-γ1, an extracellular matrix protein involved in various progresses such as tumor cell proliferation and metabolism. In the present study, high expression of Lamc1 and PKM2 was observed in tumor tissues of HCC patients. In vitro, down-regulation of Lamc1 inhibited proliferation of HCC cells by promoting cell death, reduced glucose consumption and lactate production, accompanied by a decrease in the expression of glucose transporter 1 (GLUT1) and lactate dehydrogenase A (LDHA), and PTEN increased, as well as PTEN S380 and AKT S473/T308 phosphorylation decreased, while Lamc1 up-regulation had the opposite effect. The effects of PKM2 were similar to that of Lamc1 and markedly counteracted the effects of Lamc1 down-regulation. In addition, Lamc1-induced increase in PKM2 expression was strongly attenuated by a PI3K inhibitor, LY294002 or a si-p110 PI3K, with a significant decrease in GLUT1 and LDHA expression, as well as decreased AKT T308 phosphorylation. Thus, we speculated that Lamc1 was implicated in the progression of HCC probably by regulating PKM2 expression through PTEN/AKT pathway.     

人参皂苷Rb1 通过KEAP1/PGAM5/AIFM1 通路促进肝细胞癌HepG2细胞发生氧死亡

目的：探讨人参皂苷Rb1（Gn-Rb1）对肝细胞癌（HCC）HepG2细胞氧死亡的影响及其可能的分子机制。方法：采用生物信息学方法分析氧死亡的关键基因PGAM5表达对HCC患者生存期的影响。选取辽宁省肿瘤医院收治的8例HCC患者的HCC 组织与癌旁组织,通过WB法及qPCR 检测氧死亡相关基因蛋白与mRNA的表达情况。将HepG2 细胞随机分为对照组与Gn-Rb1组（予以200 μmol/L Gn-Rb1干预）,采用细胞克隆形成实验、划痕愈合实验分别检测Gn-Rb1对HepG2细胞的集落形成能力、迁移能力的影响,ELISA 检测对细胞ROS 生成水平的影响,微板法检测对细胞LDH 释放水平的影响;WB法、qPCR 法检测Gn-Rb1对HepG2氧死亡关键基因蛋白质与mRNA水平表达的影响。结果：生物信息学分析发现,PGAM5高表达肝癌患者总生存时间较低表达患者更长（P<0.05）。在临床HCC组织与癌旁组织样本中发现,相较于癌旁组织,在蛋白质与mRNA水平上,肿瘤组织KEAP1与PGAM5表达显著降低,NRF2表达显著升高（均P<0.01）,p-AIFM1蛋白水平显著升高（P<0.05）。对HepG2细胞予以200 μmol/L Gn-Rb1干预后,相较于对照组,Gn-Rb1组HepG2细胞的迁移能力与集落形成能力显著降低（均P<0.01）,而LDH水平显著升高（P<0.05）;相比于对照组,在mRNA和蛋白质水平上,Gn-Rb1 组细胞中KEAP1、PGAM5 表达均显著升高而NRF2表达均显著降低（均P<0.05）,p-AIFM1蛋白表达显著降低（P<0.01）。结论：HCC组织中氧死亡被抑制,而Gn-Rb1能够通过调控KEAP1/PGAM5/AIFM1通路促进HepG2细胞氧死亡的发生,抑制细胞增殖和迁移能力。     

MicroRNA-10b promotes migration and invasion through CADM1 in human hepatocellular carcinoma cells

MicroRNA-10b (miR-10b) was recently reported to be dysregulated in some types of cancer and to play a role in invasion and metastasis. However, effects and potential mechanisms of action of miR-10b in the metastasis of hepatocellular carcinoma (HCC) have not been explored. In this study, we confirmed that miR-10b is highly expressed in metastatic HCC tissues and in metastatic HCC cell lines by qRT-PCR. Moreover, patients with higher miR-10b expression had significantly poorer overall survival, and high miR-10b expression was an independent predictor of poor prognosis. Inhibition of miR-10b reduced cell migration and invasion in MHCC97H cells, whereas over-expression of miR-10b in HepG2 cells increased cell migration and invasion. Bioinformatics and luciferase reporter assays revealed that miR-10b binds the 3??-UTR of CADM1 mRNA and represses its translation. Western blot and qRT-PCR showed that CADM1 is inhibited by miR-10b over-expression. Silencing of CADM1 resulted in substantially increased cell motility and invasion similar to that observed with over-expression of miR-10b in HepG2 cells. These results suggest that miR-10b may positively regulate the invasion and metastasis of HCC through targeting CADM1.     

Furanodiene induces G2/M cell cycle arrest and apoptosis through MAPK signaling and mitochondria-caspase pathway in human hepatocellular carcinoma cells

Furanodiene (C15H20O), a pure compound isolated from Traditional Chinese medicine, Curcuma wenyujin, named Ezhu in Chinese, which structure was determined on the basis of NMR, MS and UV spectrum. In this study, we attempted to characterize in detail the signaling cascades resulted from furanodiene-induced apoptosis in human hepatoma HepG2 cells. Furanodiene inhibited HepG2 cell growth by causing cell cycle arrest at G2/M and inducing apoptosis as evidenced by DNA fragmentation assay. We found that furanodiene induced mitochondrial transmembrane depolarization, release of mitochondrial cytochrome c, activation of caspases-3 and the cleavage of PARP. The furanodiene mediated mitochondria-caspase apoptotic pathway also involved activation of p38 and inhibition of ERK mitogen-activated protein kinase (MAPK) signaling. These results for the first time have identified the biological activity of furanodiene against HepG2 cells and provide rationales for further development of essential oil of Ezhu and its ingredients such as furanodiene on treatment of liver diseases.     

表没食子儿茶素没食子酸酯诱导人肝癌细胞HepG2凋亡与TGF-β1-Smad通路激活有关

目的 探讨表没食子儿茶素没食子酸酯(EGCG)对人肝癌细胞HepG2细胞周期和凋亡的影响及其机制.方法 四甲基偶氮唑蓝(MTT)法检测EGCG对HepG2细胞增殖的影响,流式细胞术检测EGCG对HepG2细胞周期的影响以及细胞凋亡情况,逆转录聚合酶链反应(RT-PCR)和萤光素酶报告基因系统检测验证HepG2细胞中TGF-β1-Smad通路的完整性,实时聚合酶链反应(realtime PCR)检测EGCG对HepG2细胞中Smad2、Smad3、Smad4和Smad7 mRNA表达的影响.结果 高浓度EGCG干预时,HepG2细胞的增殖能力随着EGCG浓度和作用时间的增加而下降,呈明显的时效和量效关系,作用72 h,HepG2细胞的IC50为111.76 μmol/L.随EGCG剂量增大,HepG2细胞中G0/G1期细胞的比例逐渐增高,80、120和160 μmol/L EGCG处理组分别为44.9%、54.8%和91.3%;相反,S期细胞比例逐渐降低,80、120和160 μmol/L EGCG处理组依次为38.5%、29.2%和3.0%.随着EGCG剂量增大,HepG2细胞的早期凋亡增加,80、120和160 μmol/L EGCG处理组的早期凋亡率分别为1.82%、4.22%和6.83%;晚期凋亡也随之增加,80、120和160 μmol/L EGCG处理组的晚期凋亡率分别为7.92%、24.19%和27.92%.HepG2细胞中TGF-β1-Smad通路是完整的.EGCG对HepG2细胞中Smad2、Smad3和Smad4 mRNA的表达无明显影响,但下调Smad7 mRNA的表达.结论 EGCG能诱导人肝癌细胞HepG2的凋亡,其机制可能涉及TGF-β1-Smad通路的激活.     

表没食子儿茶素没食子酸酯诱导人肝癌细胞HepG2凋亡与TGF-β1-Smad通路激活有关

Objective To investigate the cytotoxic effect of epigallocatechin gallate(EGCG)on human hepatocellular carcinoma cell line HepG2 cells and corresponding changes of TGF-β1-Smad pathway.Methods The cytotoxic effect of EGCG on HepG2 cells was determined by MTT assay.Cell cycle and apoptosis rate were detected by flow cytometry.RT-PCR and luciferase assay were used to verify whether TGF-β1-Smad signaling pathway is intact in HepG2.The mRNA expression of Smad 2,Smad3,Smad4 and Smad7 was detected by real-time PCR.Results EGCG induced apoptosis in the HepG2 ceils in a time-and concentration-dependent manner.The proportion of G1 phase cells was increased gradually as the concentration increased.However,the percentage of cells in S phase was decreased gradually.Annexin V/PI assay demonstrated that early apoptosis increased as the concentration increased,and late apoptosis also increased,when treated with high-concentration EGCG.The intact TGF-β1-Smad pathway was verified by luciferase assay and RT-PCR.There was no significant effect of EGCG on mRNA level of Smad 2,Smad 3,and Smad 4 in HepG2 cells,but downregulated mRNA level of Smad 7.Conclusion EGCG can reduce apoptosis in human hepatocellular carcinoma cell line HepG2 cells.The activation of TGF-β1-Smad signaling pathway may be involved in its cytotoxicity mechanisms.     

SPINK1 contributes to proliferation and clonal formation of HT29 cells through Beclin1 associated enhanced autophagy

We aimed to explore the molecular mechanism that were involved in SPINK1-induced proliferation and clonogenic survival of human colorectal carcinoma (CRC) HT29 cells. Initially, we generated HT29 cells either permanently silencing or overexpressing SPINK1 protein. The results showed that SPINK1 overexpression (OE) significantly stimulated the proliferation and clonal formation of HT29 cells at the varied time points. Secondly, we found SPINK1 OE enhanced the ratio of LC3II/LC3I and the level of autophagy-related gene 5 (ATG5), whereas SPINK1 knockdown (Kd) reversed the above outcome under normal culturing and/or fasting condition in the cells, indicating its role in autophagy enhancement. Moreover, LC3-GFP-transfected SPINK1-OE HT29 cells strengthened the fluorescence intensity compared with the untransfected control. Chloroquine (CQ) significantly decreased the level of autophagy in both control and SPINK1-OE HT29 cells. The autophagy inhibitors, CQ and 3-Methyladenine (3-MA), remarkably inhibited the proliferation and colony formation of SPINK1-OE HT29 cells, while ATG5 upregulation resulted in the growth of the cells, suggesting the important function of autophagy in cell’s growth. Thirdly, SPINK1-induced autophagy was independently of mTOR signaling as p-RPS6 and p-4EBP1 were activated in SPINK1-OE HT29 cells. Instead, Beclin1 up and down regulation were clearly observed in SPINK1-OE and SPINK1 Kd HT29 cells, respectively. Moreover, Beclin1 silencing apparently reduced autophagy in SPINK1-OE HT29 cells, indicating that SPINK1-induced autophagy was closely associated with Beclin1. Collectively, SPINK1-promoted proliferation and clonal formation of HT29 cells were closely associated with Beclin1 associated enhanced autophagy. The above findings would open a new window for probing the role of SPINK1-related autophagic signaling in the pathogenesis of CRC.     

酸性环境诱导肝癌HepG2细胞EMT

目的 研究酸性环境对肝癌HepG2细胞上皮细胞间质转化(EMT)的影响。方法分别在酸性和碱性条件下培养HepG2细胞,观察细胞形态的区别。划痕实验检测两组细胞的迁移能力,Matrigel侵袭实验检测细胞的侵袭能力。RT-PCR检测两组细胞EMT相关基因mRNA的表达水平,Western blot检测两组细胞EMT相关基因蛋白的表达水平。结果 酸性条件下培养的HepG2细胞形态明显从上皮型转变为间质型;酸性条件下HepG2细胞的迁移能力明显强于碱性条件下;酸性条件下HepG2细胞穿越Matrigel的数量明显多于碱性条件下;酸性条件下HepG2细胞EMT相关基因Vimentin、Slug、Snail及Zeb1的mRNA表达以及EMT相关蛋白Vimentin和MMP9的表达强度均明显高于碱性条件下。结论 酸性环境能诱导肝癌HepG2细胞EMT的发生。     

Beclin 1 mRNA strongly correlates with Bcl-XLmRNA expression in human hepatocellular carcinoma

Beclin 1 physically associates with Bcl-x(L) and is considered as a haploinsufficient tumor suppressor. As the role of Beclin 1 in hepatocellular carcinoma (HCC) is unknown, we determined Beclin 1 mRNA expression in 27 pairs of tumoral/nontumoral (T/NT) liver samples. The Beclin 1 mRNA T/NT ratio was less than 0.5 in 2 tumors and more than 2 in 1 tumor, and was positively correlated with the Bcl-X(L) mRNA T/NT ratio (P < 0.001), but not with the proliferating cell nuclear antigen mRNA T/NT ratio. Coregulation of Beclin 1 and Bcl-X(L) expression in HCC may suggest cooperation in the regulation of apoptosis.     

缺氧诱导肝癌细胞获得干细胞特性的初步实验研究

目的:观察缺氧对肝癌干细胞特性的影响.方法:用肝癌细胞系HepG2、SMMC-7721建立细胞缺氧培养模型,利用倒置显微镜观察细胞形态学变化;克隆形成实验检测肿瘤细胞克隆形成能力;"肿瘤球"形成实验检测肿瘤细胞自我更新能力;实时定量RT-PCR和Western blot检测干细胞相关基因Oct-4、Sox-2、Nanog mRNA和蛋白表达;采用流式细胞术检测干细胞表面标志物CD133表达.结果:缺氧条件下HepG2、SMMC-7721细胞克隆形成率显著高于常氧组(P<0.05).缺氧条件下HepG2、SMMC-7721细胞肿瘤球形成率显著高于常氧组(P<0.05).常氧条件下HepG2、SMMC-7721细胞中干细胞相关基因Oct-4、Sox-2、Nanog mRNA及蛋白表达水平极低,缺氧处理48 h后,其mRNA及蛋白表达水平明显增高(P<0.05).缺氧条件下HepG2、SMMC-7721细胞CD133阳性表达率表达量显著增加(P<0.05).在缺氧36 h和48 h,CD133蛋白表达水平显著增强(P<0.05).结论:缺氧能诱导肝癌细胞干细胞特性的获得.