Hepatitis B virus X protein modulates the apoptosis of hepatoma cell line induced by TRAIL

TRAIL (TNF-related apoptosis-inducing ligand) is one member of TNF superfamily[1]. It is unique, for it could specifically induce the apoptosis of tumor cells or virus-infected cells but have no cytotoxic effects onnormal cells[1,2]. Owing to this characteristic, it has become a promising candidate molecule for biological therapy for tumor. Many factors could affect the sensitivity towardsTRAIL-induced apoptosis, including cytokines, virus infection, drugs, radials, etc. Studies show tha…     

Blockade of preS2 down-regulates the apoptosis of HepG2.2.15 cells induced by TRAIL

The TNF-related apoptosis-inducing ligand (TRAIL) has recently been implicated in the death of hepatocytes under infectious but not normal conditions. Our previous studies showed that both the whole HBV genome and its HBx protein enhanced TRAIL-induced hepatocyte apoptosis. We report here that preS2-containing viral proteins are also potent regulators of TRAIL-induced apoptosis. HBV-transfected hepatoma cell line, HepG2.2.15, pretreated with antisense oligonucleotide against preS2 gene, showed a lower sensitivity towards TRAIL-induced apoptosis. However, this effect might not be related with the expression level of TRAIL receptors. These results establish that besides HBx, preS2 viral proteins are also involved in enhancing TRAIL-induced hepatocyte apoptosis. The novel role of preS2 would be useful to further unravel the mechanisms of imbalanced apoptosis during HBV infection and provides a potential therapeutic target for HBV-related diseases.     

Hepatitis B virus sensitizes hepatocytes to TRAIL-induced apoptosis through Bax

Hepatitis B virus (HBV) infection afflicts >300 million people worldwide and is a leading cause of hepatocyte death, cirrhosis, and hepatocellular carcinoma. While the morphological characteristics of dying hepatocytes are well documented, the molecular mechanisms leading to the death of hepatocytes during HBV infection are not well understood. TRAIL, the TNF-related apoptosis-inducing ligand, has recently been implicated in the death of hepatocytes under certain inflammatory but not normal conditions. To determine the potential roles of TRAIL in HBV-induced hepatitis, we examined the effects of HBV and its X protein (HBx) on TRAIL-induced hepatocyte apoptosis both in vivo and in vitro. We found that hepatitis and hepatic cell death in HBV transgenic mice were significantly inhibited by a soluble TRAIL receptor that blocks TRAIL function. We also found that HBV or HBx transfection of a hepatoma cell line significantly increased its sensitivity to TRAIL-induced apoptosis. The increase in TRAIL sensitivity were associated with a dramatic up-regulation of Bax protein expression. Knocking down Bax expression using Bax-specific small interference RNA blocked HBV-induced hepatitis and hepatocyte apoptosis. The degradation of caspases 3 and 9, but not that of Bid or caspase-8, was preferentially affected by Bax knockdown. These results establish that HBV sensitizes hepatocytes to TRAIL-induced apoptosis through Bax and that Bax-specific small interference RNA can be used to inhibit HBV-induced hepatic cell death.     

The hepatitis B virus protein MHBs(t) sensitizes hepatoma cells to TRAIL-induced apoptosis through ERK2

The TNF-related apoptosis-inducing ligand (TRAIL) has recently been implicated in the death of hepatocytes under infectious but not normal conditions. Infectious agents, such as hepatitis B virus (HBV), may play important roles in regulating the sensitivity of hepatocytes to TRAIL. Our previous studies showed that HBx, a protein encoded by the HBV genome, enhanced TRAIL-induced apoptosis through upregulating Bax. We report here that another HBV protein called MHBs(t) (C-terminally truncated middle hepatitis B surface protein) is also a potent regulator of TRAIL-induced apoptosis. Overexpressing MHBs(t) in hepatoma cells enhanced TRAIL-induced apoptosis. Mechanistic studies reveal that MHBs(t) had no effect on Bax or TRAIL receptor expression or procaspase-8 activation, but selectively enhanced the activation of ERK2 (extracellular signal-regulated kinase 2) and the degradation of procaspases-3 and 9. ERK2 activation is required for the MHBs(t) effect because ERK2 inhibition by its inhibitor PD98059 significantly reversed TRAIL-induced apoptosis of MHBs(t)-transfected cells. These results establish that unlike HBx, MHBs(t) enhances TRAIL-induced hepatocyte apoptosis through a novel mechanism that involves ERK2. Therefore, manipulating the ERK2 signaling pathway may provide new therapeutic opportunities to contain hepatic cell death during HBV infection. Xiaohong Liang and Juan Du contributed equally to this work.     

HBXIP基因对乙肝病毒X蛋白诱导细胞凋亡的影响

探讨乙型肝炎病毒X蛋白结合蛋白(hepatitisBXinteractingprotein ,HBXIP)基因在乙型肝炎病毒X蛋白(HBX)诱导肝癌细胞凋亡时对细胞周期的影响.构建HBXIP基因真核表达载体pcDNA3 hbxip ,进行瞬时基因转染,将克隆有HBx基因的pCMV X (分别为1μg、2 μg和3μg)和pcDNA3 hbxip质粒分别和共转染至人H74 0 2肝癌细胞中(总体积分别为5 0 μl) .发现瞬时转染3μgpCMV X质粒后,肝癌细胞凋亡发生率为34 4 % ,肝癌细胞的细胞周期相关蛋白p2 7表达水平发生明显上调;与对照组相比,瞬时转染1μg、2 μg和3μg时,细胞周期蛋白D和细胞周期蛋白E的表达水平均发生明显上调,但随着HBX水平的增加细胞周期蛋白D和细胞周期蛋白E的表达水平发生明显下降;在稳定转染pCMV X质粒的H74 0 2 X肝癌细胞中无明显的细胞凋亡发生,研究发现p2 7的表达水平发生了明显下调,而细胞周期蛋白D和细胞周期蛋白E的表达水平发生了明显上调;当pcDNA3 hbxip质粒与pCMV X质粒进行共瞬时转染时,细胞凋亡发生率由pcDNA3质粒与pCMV X质粒共转染时的2 9 2 %下降为13 3% ,p2 7的表达水平发生了下调,但细胞周期蛋白D和细胞周期蛋白E的表达水平无明显变化.研究结果表明,瞬时转染一定剂量的x基因可导致肝癌细胞发生凋亡,细胞周期相关蛋白p2 7、细胞周期蛋白D和     

Hepatitis B virus core protein inhibits TRAIL-induced apoptosis of hepatocytes by blocking DR5 expression

Hepatitis B virus (HBV) causes chronic hepatitis in hundreds of millions of people worldwide, which can eventually lead to hepatocellular carcinoma (HCC). The molecular mechanisms underlying HBV persistence are not well understood. TRAIL, the TNF-related apoptosis-inducing ligand, has recently been implicated in hepatocyte death during HBV infection. We report here that the HBV core protein (HBc) is a potent inhibitor of TRAIL-induced apoptosis. Overexpressing HBc significantly decreased TRAIL-induced apoptosis of human hepatoma cells, whereas knocking-down HBc expression in hepatoma cells transfected with HBV genome enhanced it. When present in the same cell, HBc blocked the pro-apoptotic effect of the HBV X protein (HBx). The resistance of HBc-expressing cells to TRAIL-induced apoptosis was associated with a significant reduction in death receptor 5 (DR5) expression. Upon transfection, HBc significantly repressed the promoter activity of the human DR5 gene. Importantly, HBc gene transfer inhibited hepatocyte death in a mouse model of HBV-induced hepatitis; and in patients with chronic hepatitis, DR5 expression in the liver was significantly reduced. These results indicate that HBc may prevent hepatocytes from TRAIL-induced apoptosis by blocking DR5 expression, which in turn contributes to the development of chronic hepatitis and HCC. They also call into question the potential side effects of HBc-based vaccines.     

Upregulated microRNA-29a by hepatitis B virus X protein enhances hepatoma cell migration by targeting PTEN in cell culture model

Hepatitis B virus X protein (HBx) plays important roles in the development of hepatocellular carcinoma (HCC). MicroRNAs (miRNAs) contribute to cancer development by acting as oncogenes or tumor suppressors. Previously, we reported that HBx was able to promote the migration of hepatoma HepG2 cells. However, the regulation of miRNAs in the development of HBV-related HCC is poorly understood. In the present study, we reported that miR-29a was a novel regulator of migration of hepatoma cells mediated by HBx. Our data showed that the expression of miR-29a was dramatically increased in p21-HBx transgenic mice, HBx-transfected hepatoma HepG2-X (or H7402-X) cells and HepG2.2.15 cells that constitutively replicate HBV. However, our data showed that miR-29a was upregulated in 4 of the 11 clinical HCC samples. We found that the overexpression of miR-29a promoted the migration of HepG2 cells, while a specific miR-29a inhibitor could partially abolish the enhanced migration of HepG2-X cells. Moreover, we identified PTEN was one of the target genes of miR-29a in HepG2 cells. The deletion of the miR-29a-binding site was able to abolish the role of miR-29a in suppression of luciferase activity of the PTEN 3'UTR reporter. Meanwhile, the overexpression of PTEN was able to reverse the promoted migration of HepG2 cells mediated by miR-29a. Moreover, our data showed that the modulation of Akt phosphorylation, a downstream factor of PTEN, was involved in the cell migration enhanced by miR-29a, suggesting that miR-29a is responsible for the cell migration through its target gene PTEN. Thus, we conclude that miR-29a is involved in the regulation of migration of hepatoma cells mediated by HBx through PTEN in cell culture model.     

Inhibition of eIF2α dephosphorylation enhances TRAIL-induced apoptosis in hepatoma cells

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is an inducer of cancer cell death that holds promise in cancer therapy. Cancer cells are more susceptible than normal cells to the cell-death-inducing effects of TRAIL. However, a variety of cancer cells are resistant to TRAIL through complex mechanisms. Here, we investigate the effects of inhibition of eukaryotic initiation factor 2 subunit α (eIF2α) dephosphorylation on TRAIL-induced apoptosis in hepatoma cells. Treatment of hepatoma cells with salubrinal, an inhibitor of eIF2α dephosphorylation, enhances TRAIL-induced eIF2α phosphorylation, CCAAT/enhancer-binding protein homologous protein (CHOP) expression and caspase activation. Salubrinal enhances TRAIL-induced apoptosis, which could be abrogated by caspase inhibitor. Overexpression of phosphomimetic eIF2α (S51D) enhances TRAIL-induced CHOP expression, caspase 7 and PARP cleavage and apoptosis. By contrast, overexpression of phosphodeficient eIF2α (S51A) abrogates the stimulation of TRAIL-induced apoptosis by salubrinal. Moreover, knockdown of growth arrest and DNA damage-inducible protein 34 (GADD34), which recruits protein phosphatase 1 to dephosphorylate eIF2α, enhances TRAIL-induced eIF2α phosphorylation, CHOP expression, caspase activation and apoptosis. Furthermore, the sensitization of hepatoma cells to TRAIL by salubrinal is dependent on CHOP. Knockdown of CHOP abrogates the stimulation of TRAIL-induced caspase activation and apoptosis by salubrinal. Combination of salubrinal and TRAIL leads to increased expression of Bim, a CHOP-regulated proapoptotic protein. Bim knockdown blunts the stimulatory effect of salubrinal on TRAIL-induced apoptosis. Collectively, these findings suggest that inhibition of eIF2α dephosphorylation may lead to synthetic lethality in TRAIL-treated hepatoma cells.     

Celastrol, a Triterpene, Enhances TRAIL-induced Apoptosis through the Down-regulation of Cell Survival Proteins and Up-regulation of Death Receptors

Whether celastrol, a triterpene from traditional Chinese medicine, can modulate the anticancer effects of TRAIL, the cytokine that is currently in clinical trial, was investigated. As indicated by assays that measure plasma membrane integrity, phosphatidylserine exposure, mitochondrial activity, and activation of caspase-8, caspase-9, and caspase-3, celastrol potentiated the TRAIL-induced apoptosis in human breast cancer cells, and converted TRAIL-resistant cells to TRAIL-sensitive cells. When examined for its mechanism, we found that the triterpene down-regulated the expression of cell survival proteins including cFLIP, IAP-1, Bcl-2, Bcl-xL, survivin, and XIAP and up-regulated Bax expression. In addition, we found that celastrol induced the cell surface expression of both the TRAIL receptors DR4 and DR5. This increase in receptors was noted in a wide variety of cancer cells including breast, lung, colorectal, prostate, esophageal, and pancreatic cancer cells, and myeloid and leukemia cells. Gene silencing of the death receptor abolished the effect of celastrol on TRAIL-induced apoptosis. Induction of the death receptor by the triterpenoid was found to be p53-independent but required the induction of CAAT/enhancer-binding protein homologous protein (CHOP), inasmuch as gene silencing of CHOP abolished the induction of DR5 expression by celastrol and associated enhancement of TRAIL-induced apoptosis. We found that celastrol also induced reactive oxygen species (ROS) generation, and ROS sequestration inhibited celastrol-induced expression of CHOP and DR5, and consequent sensitization to TRAIL. Overall, our results demonstrate that celastrol can potentiate the apoptotic effects of TRAIL through down-regulation of cell survival proteins and up-regulation of death receptors via the ROS-mediated up-regulation of CHOP pathway.     

Phenethyl isothiocyanate sensitizes glioma cells to TRAIL-induced apoptosis

Tumor necrosis factor-related apoptosis-induced ligand (TRAIL) is a promising antitumor therapy. However, many cancer cells, including malignant glioma cells, tend to be resistant to TRAIL, highlighting the need for strategies to overcome TRAIL resistance. Here we show that in combination with phenethyl isothiocyanate (PEITC), exposure to TRAIL induced apoptosis in TRAIL-resistant glioma cells. Subtoxic concentrations of PEITC significantly potentiated TRAIL-induced cytotoxicity and apoptosis in glioma cells. PEITC dramatically upregulated DR5 receptor expression but had no effects on DR4 receptor. PEITC enhances TRAIL-induced apoptosis through the downregulation of cell survival proteins and the upregulation of DR5 receptors through actions on the ROS-induced-p53.     

乙肝病毒X 蛋白通过CREB 上调HBx结合蛋白促进肝癌细胞迁移

乙型肝炎病毒X蛋白（hepatitis B virus X protein,HBx）对肝癌的发生发展具有十分重要的作用. HBx 具有促进肝癌迁移的作用,但其作用的分子机制不清. 本研究对 HBx 促进肝癌细胞迁移的分子机制进行了探讨. 伤口愈合和 Boyden’s chamber结果表明,HBx 可明显促进肝癌 HepG2 细胞迁移. 在稳定转染 HBx 的 HepG2（HepG2-X）细胞中转染 HBx 结合蛋白（hepatitis B X-interacting protein,HBXIP）的 RNA 干扰片段,可明显抑制 HBx 的促迁移作用. 免疫组化和实时定量 PCR 结果表明,HBXIP 在肝癌组织中显著高表达,并且与 HBx 表达成正相关. 荧光素酶报告基因和免疫印迹结果表明,HBx 显著增强 HBXIP 的启动子活性和蛋白质表达水平. 应用 HBx 的 RNA 干扰处理 HepG2-X 细胞,HBXIP 的启动子活性和蛋白质表达水平明显下降.将 HBXIP 启动子区的cAMP效应元件结合因子（CREB）结合位点突变后,HBx 上调 HBXIP 的作用消失. 应用 CREB 的 RNA 干扰处理肝癌细胞,在启动子水平和蛋白质水平上, HBx 对 HBXIP 的上调作用被显著抑制. 染色质免疫共沉淀结果表明,HBx 能够通过 CREB 结合到 HBXIP 的启动子上,进而发挥激活 HBXIP 的功能. 本研究结果表明,HBx 促进肝癌细胞迁移的作用是通过 CREB 上调 HBXIP 实现的. 这一发现对进一步揭示 HBx 促进肝癌细胞迁移的分子机制具有重要意义.     

pVAX1 plasmid vector-mediated gene transfer of soluble TRAIL suppresses human hepatocellular carcinoma growth in nude mice

The extracellular domain of the tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) may function as a soluble cytokine to selectively kill various cancer cells without toxicity to most normal cells. We used a high-biosafety plasmid pVAX1 as a vector and constructed a recombinant plasmid expressing the extracellular domain (95-281 aa) of human TRAIL fused with signal peptides of human IgGgamma, designated as pVAX-sT. Transduction of human BEL7402 liver cancer cells with pVAX-sT led to high levels of sTRAIL protein in the cell culture media and induced apoptosis. The therapeutic potential of pVAX-sT was then evaluated in the BEL7402 transplanted naked mouse model. Subsequent intratumoral administration of naked pVAX-sT resulted in the expression of soluble TRAIL in the sera and the tumor site, as well as effective suppression of tumor growth, with no toxicity to liver. In conclusion, the successful inhibition of liver cancer growth and the absence of detectable toxicity suggest that pVAX-sT could be useful in the gene therapy of liver cancer.     

Chemotherapy overcomes TRAIL-R4-mediated TRAIL resistance at the DISC level

TNF-related apoptosis-inducing ligand or Apo2L (Apo2L/TRAIL) is a promising anti-cancer drug owing to its ability to trigger apoptosis by binding to TRAIL-R1 or TRAIL-R2, two membrane-bound receptors that are often expressed by tumor cells. TRAIL can also bind non-functional receptors such as TRAIL-R4, but controversies still exist regarding their potential to inhibit TRAIL-induced apoptosis. We show here that TRAIL-R4, expressed either endogenously or ectopically, inhibits TRAIL-induced apoptosis. Interestingly, the combination of chemotherapeutic drugs with TRAIL restores tumor cell sensitivity to apoptosis in TRAIL-R4-expressing cells. This sensitization, which mainly occurs at the death-inducing signaling complex (DISC) level, through enhanced caspase-8 recruitment and activation, is compromised by c-FLIP expression and is independent of the mitochondria. Importantly, TRAIL-R4 expression prevents TRAIL-induced tumor regression in nude mice, but tumor regression induced by TRAIL can be restored with chemotherapy. Our results clearly support a negative regulatory function for TRAIL-R4 in controlling TRAIL signaling, and unveil the ability of TRAIL-R4 to cooperate with c-FLIP to inhibit TRAIL-induced cell death.     

PTHrP Overexpression Increases Sensitivity of Breast Cancer Cells to Apo2L/TRAIL

Parathyroid hormone-related protein (PTHrP) is a key component in breast development and breast tumour biology. PTHrP has been discovered as a causative agent of hypercalcaemia of malignancy and is also one of the main factors implicated in breast cancer mediated osteolysis. Clinical studies have determined that PTHrP expression by primary breast cancers was an independent predictor of improved prognosis. Furthermore, PTHrP has been demonstrated to cause tumour cell death both in vitro and in vivo. Apo2L/TRAIL is a promising new anti-cancer agent, due to its ability to selectively induce apoptosis in cancer cells whilst sparing most normal cells. However, some cancer cells are resistant to Apo2L/TRAIL-induced apoptosis thus limiting its therapeutic efficacy. The effects of PTHrP on cell death signalling pathways initiated by Apo2L/TRAIL were investigated in breast cancer cells. Expression of PTHrP in Apo2L/TRAIL resistant cell line MCF-7 sensitised these cells to Apo2L/TRAIL-induced apoptosis. The actions of PTHrP resulted from intracellular effects, since exogenous treatment of PTHrP had no effect on Apo2L/TRAIL-induced apoptosis. Apo2L/TRAIL-induced apoptosis in PTHrP expressing cells occurred through the activation of caspase-10 resulting in caspase-9 activation and induction of apoptosis through the effector caspases, caspase-6 and -7. PTHrP increased cell surface expression of Apo2L/TRAIL death receptors, TRAIL-R1 and TRAIL-R2. Antagonistic antibodies against the death receptors demonstrated that Apo2L/TRAIL mediated its apoptotic signals through activation of the TRAIL-R2 in PTHrP expressing breast cancer cells. These studies reveal a novel role for PTHrP with Apo2L/TRAIL that maybe important for future diagnosis and treatment of breast cancer.     

Lithium enhances TRAIL-induced apoptosis in human lung carcinoma A549 cells

Non-small cell lung cancer (NSCLC) A549 cells are resistant to tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-induced apoptosis. Therefore, combination therapy using sensitizing agents to overcome TRAIL resistance may provide new strategies for treatment of NSCLC. Here, we investigated whether lithium chloride (LiCl), a drug for mental illness, could sensitize A549 cells to TRAIL-induced apoptosis. We observed that LiCl significantly enhanced A549 cells apoptosis through up-regulation of death receptors DR4 and DR5 and activation of caspase cascades. In addition, G2/M arrest induced by LiCl also contributed to TRAIL-induced apoptosis. Concomitantly, LiCl strongly inhibited the activity of c-Jun N-terminal kinases (JNKs), and the inhibition of JNKs by SP600125 also induced G2/M arrest and augmented cell death caused by TRAIL or TRAIL plus LiCl. However, glycogen synthase kinase-3β (GSK3β) inhibition was not involved in TRAIL sensitization induced by LiCl. Collectively, these findings indicated that LiCl sensitized A549 cells to TRAIL-induced apoptosis through caspases-dependent apoptotic pathway via death receptors signaling and G2/M arrest induced by inhibition of JNK activation, but independent of GSK3β.     

COX-2在乙肝病毒X蛋白（HBx）促进肝癌细胞和肝细胞增殖中的作用

乙型肝炎病毒（hepatitis B virus, HBV）X蛋白（HBx）对肝癌的发生发展具有十分重要的作用．大量研究结果表明,与花生四烯酸代谢相关的磷脂酶COX-2与肿瘤细胞的增殖密切相关．为了阐明COX-2在HBx促进肝细胞增殖中的作用,在前期应用基因芯片方法检测发现稳定转染HBx 基因的肝癌细胞H7402-X中COX-2基因表达明显上调的基础上,本研究应用免疫印迹技术在蛋白表达水平上获得了相同的结果．进而,应用COX-2的特异性抑制剂Indo分别作用H7402-X细胞和L-O2-X细胞（稳定转染HBx 基因的人永生化L-O2肝细胞）,观察HBx蛋白是否通过COX-2促进肝癌细胞或肝细胞增殖．BrdU掺入实验和流式细胞仪检测结果显示,50 μmol/L的Indo可明显抑制H7402-X细胞和L-O2-X细胞的增殖．本研究结果提示,HBx可通过COX-2所介导的花生四烯酸代谢促进肝癌细胞和肝细胞增殖．     

XPD could suppress growth of HepG2.2.15 and down-regulate the expression of hepatitis B virus x protein through P53 pathway

ObjectivesWe investigated the effects of xeroderma pigmentosum D (XPD) on the growth of hepatoma cells and the expressions of P21, Bax, Bcl-2 and Hepatitis B virus X protein (HBx). In addition, we examined whether XPD affected the aforementioned genes via the P53 pathway.MethodsHuman hepatoma cells (HepG2.2.15) were transfected with XPD expression vector, followed by incubation with Pifithrin-α (P53 inhibitor). By using RT-PCR and Western blotting, the expression levels of XPD, P53, phospho-P53 (ser-15), P21, Bax, Bcl-2 and HBx were detected. The cell cycle and the apoptosis rate were examined with flow cytometry, and the cell viability was detected by MTT.ResultsOver-expression of XPD up-regulated the expressions of P53, phospho-P53 (ser-15), P21 and Bax but down-regulated the expressions of Bcl-2 and HBx. XPD inhibited the viability of HepG2.2.15 and exacerbated the apoptosis. However, the inhibition of P53 by Pifithrin-α abolished the above-mentioned effects of XPD.ConclusionXPD could suppress growth of hepatoma cells, up-regulate the expressions of P21 and Bax, and down-regulate the expressions of Bcl-2 and HBx through the P53 pathway. There may be mutual influences among XPD, P53 and HBx that co-regulate hepatocarcinogenesis.     

基于肝癌细胞线粒体功能受损和caspase-3信号通路探讨罗哌卡因促进肝癌细胞凋亡的作用机制研究

摘要 目的：基于肝癌细胞线粒体功能受损和天冬氨酸蛋白水解酶3（caspase-3）信号通路探讨罗哌卡因促进肝癌细胞凋亡的作用机制。方法：选用细胞株人肝癌细胞BEL-7402进行实验研究。用不同浓度罗哌卡因处理BEL-7402细胞后,采用溴化噻唑蓝四氮唑（MTT）法检测肝癌细胞的增殖情况,光镜及4,6-二苯胺-2-苯吲哚二盐酸盐（DAPI）溶液染色观察细胞形态,台盼蓝染色法测定细胞活力,流式细胞术分析BEL-7402细胞的凋亡情况,电子显微镜下观察细胞线粒体,激光共聚焦显微镜观察caspase-3在BEL-7402细胞中的细胞核迁移情况,蛋白免疫印迹试验评价罗哌卡因对细胞质凋亡相关蛋白、线粒体凋亡相关蛋白、BEL-7402细胞和线粒体凋亡相关蛋白表达的影响。结果：罗哌卡因能够抑制肝癌细胞的生长,并呈剂量依赖性和时间依赖性。罗哌卡因可诱导BEL-7402细胞发生凋亡,显著增加BEL-7402细胞的凋亡率。罗哌卡因能够损伤肝癌细胞线粒体功能。激光共聚焦显微镜观察显示caspase-3分子迁移到细胞核。罗哌卡因与caspase-3相互作用,促进caspase-3向细胞核内迁移,刺激caspase-3和聚腺苷二磷酸核糖聚合酶（PARP-1）、天冬氨酸蛋白水解酶9（caspase-9）蛋白的表达,抑制B细胞淋巴瘤-2基因（Bcl-2）的表达,促进凋亡酶激活因子（Apaf-1）的表达,促进线粒体释放细胞色素C（Cytochrome C）,激活caspase-3活性。结论：罗哌卡因具有促进肝癌细胞凋亡的作用,其作用机制可能与破坏肝癌细胞线粒体功能和激活caspase-3信号通路有关。     

Distinct intracellular signaling in tumor necrosis factor-related apoptosis-inducing ligand- and CD95 ligand-mediated apoptosis

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is a potent inducer of apoptosis in tumor cells but not in healthy cells. Similar to CD95 ligand (CD95L), TRAIL signaling requires ligand-receptor interaction; the downstream signaling molecules, such as Fas-associated death domain and caspase-8, also seem similar. Using cells stably expressing TRAIL and CD95L, we show that both TRAIL and CD95L induce apoptosis in the rat colon carcinoma cell line CC531. The mitochondrial damage (loss of mitochondrial membrane potential (MMP) and release of cytochrome c) observed after co-incubation with TRAIL-expressing cells occurs much earlier than that observed with CD95L-expressing cells. The decrease in MMP induced by both ligands was caspase-8-mediated; no difference in caspase-8 activation by TRAIL and CD95L was found. TRAIL, but not CD95L, induced activation of caspase-10. bcl-2 overexpression could not prevent TRAIL-induced mitochondrial dysfunction, whereas it completely prevented CD95L-mediated loss of MMP and cytochrome c release. The selective effect of TRAIL on tumor cells and the apparent inability of bcl-2 to block TRAIL-induced apoptosis suggest that TRAIL may offer a lead for cancer therapy in the future.