Dual apoptotic effect of Xrel3 c-Rel/NF-kappaB homolog in human cervical cancer cells

Cervical cancer is one of the most common cancers affecting a woman's reproductive organs. Despite its frequency and recurrence, the death rate has been declining over the past 40 years, due to early detection and treatment. In a previous report [Shehata Marlene, Shehata Marian, Shehata Fady, Pater Alan. Apoptosis effects of Xrel3 c-Rel/Nuclear factor-kappa B homolog in human cervical cancer cells. Cell Biology International, in press], we studied the role of the NF-kappaB gene family in HeLa human cervical cancer cells, using the Xrel3 c-Rel homologue of Xenopus laevis. These results showed that the expression of Xrel3/c-Rel slowed cell growth, consistent with an upregulated expression of the cell cycle inhibitor p21 and the activated poly(ADP-ribose) polymerase (PARP) apoptosis effector. However, in this report, we examined more apoptotic and anti-apoptotic factors acting upstream and downstream in apoptosis pathways after cisplatin treatment of HeLa cervical cancer cells. After 1 microM cisplatin treatment, Xrel3 had an anti-apoptotic effect, based on significantly lower levels of apoptotic proteins, including caspase-8, caspase-3 and p21. Anti-apoptotic BAG-1 isoforms were upregulated. After 5 microM cisplatin treatment, expression of HeLa Xrel3 had an apoptotic effect, based on significantly increased expression of the cell cycle inhibitor p21 and apoptotic proteins, including cleaved PARP, caspase-8, and caspase-3. However, anti-apoptotic Bcl-2 and Bcl-X(L) were elevated and the cell cycle regulator cyclin D1 was slightly upregulated with both 1 and 5 microM cisplatin treatment. The HPV E6 oncoprotein showed no significant changes. These results support previous conclusions on the potential anti-apoptotic effects of c-Rel/NF-kappaB in mild stress environments, as opposed to the apoptotic effects associated with high stress conditions [Lake BB, Ford R, Kao KR. Xrel3 is required for head development in Xenopus laevis. Development 2001; 128(2), 263-73.]. Thus, c-Rel/NF-kappaB may potentially be of clinical significance in chemotherapy.     

Gamma interferon-inducible protein 10 induces HeLa cell apoptosis through a p53-dependent pathway initiated by suppression of human papillomavirus type 18 E6 and E7 expression

Gamma interferon-inducible protein 10 (IP10) is a member of the CXC family of chemokines. By differential mRNA display, we have demonstrated the upregulation of IP10 in coxsackievirus B3 (CVB3)-infected mouse hearts. Functional characterization of the IP10 gene in IP10-transfected Tet-On HeLa cells has found that IP10 induced cell apoptosis and inhibited viral replication. In the characterization of the IP10-induced apoptotic pathway, we found that overexpression of IP10 upregulated p53 and resulted in altered expression of p53-responsive genes such as the p21Cip1, p27kip1, NF-kappaB, Bax, and PUMA genes and the mitochondrial translocation of Bax. However, transduction of the IP10 cells with adenovirus expressing dominant negative p53 not only ablated p53-triggered gene expression but also abolished IP10-induced apoptosis and restored CVB3 replication to the control levels. These data suggest a novel mechanism by which IP10 inhibits viral replication through the induction of host cell death via a p53-mediated apoptotic pathway. We also found that constantly high-level expression of p53 in these tumor cells is attributed to the IP10-induced suppression of human papillomavirus E6 and E7 oncogene expression. Taken together, these data reveal not only a previously unrecognized link between chemokine IP10 and p53 in antiviral defense but also a mechanism by which IP10 inhibits tumor cell growth.     

Smac调控Caspase-3对耐药肺腺癌细胞株生物活性及相关信号的研究

摘要 目的：探讨Smac基因调控Caspase-3表达对紫杉醇耐药肺腺癌细胞株生物活性及经典凋亡信号通路的作用机制。方法：取构建好的耐药A549细胞,将其分为A549细胞（LC）组、A549细胞+Smac-NC（SN）组、A549细胞+Smac抑制剂（SI）组、A549细胞+Smac激动剂（SM）组、A549细胞+Caspase-3-NC（CN）组、A549细胞+Caspase-3抑制剂（CI）组、A549细胞+Caspase-3激动剂（CM）组、A549细胞+Smac激动剂+Caspase-3激动剂（MM）组;Real-time PCR法检测正常肺上皮细胞及4种肺腺癌细胞系中Smac、Caspase-3表达水平,将阴性对照、Smac、Caspase-3类似物转染至紫杉醇耐药肺腺癌细胞株,MTT法检测细胞增殖,流式细胞仪检测细胞凋亡,免疫印迹法检测经典凋亡信号通路表达,并分析Smac与Caspase-3的相关性。结果：肺腺癌细胞系中的Smac、Caspase-3 mRNA表达量显著低于正常肺上皮细胞系BEAS-2B（P＜0.05）,其中A549的Smac、Caspase-3 mRNA值最小（P＜0.05）,因此选取其作为此次实验细胞;LC组与SN组相比,细胞增殖率、凋亡率及Caspase-3、Bcl-2、Bax、Cyto-C蛋白表达基本无差异（P＞0.05）,与SN组相比,SI组细胞凋亡率及Caspase-3、Bax、Cyto-C蛋白表达明显降低（P＜0.05）,增殖率、Bcl-2表达明显升高（P＜0.05）,与SI组相比,SM组细胞凋亡率及Caspase-3、Bax、Cyto-C蛋白表达明显升高（P＜0.05）,增殖率、Bcl-2表达明显降低（P＜0.05）;LC组与CN组相比,细胞增殖率、凋亡率及Caspase-3、Bcl-2、Bax、Cyto-C蛋白表达基本无差异（P＞0.05）,与CN组相比,CI组细胞凋亡率及Caspase-3、Bax、Cyto-C蛋白表达明显降低（P＜0.05）,增殖率、Bcl-2表达明显升高（P＜0.05）,与CI组相比,CM组细胞凋亡率及Caspase-3、Bax、Cyto-C蛋白表达明显升高（P＜0.05）,增殖率、Bcl-2表达明显降低（P＜0.05）;SM组与CM组相比,细胞增殖率、凋亡率及Caspase-3、Bcl-2、Bax、Cyto-C蛋白表达基本无差异（P＞0.05）,与CM组相比,MM组细胞凋亡率及Caspase-3、Bax、Cyto-C蛋白表达明显升高（P＜0.05）,增殖率、Bcl-2表达明显降低（P＜0.05）;Smac与Caspase-3呈现正相关（r=0.470,P=0.002）,组间具有显著差异。结论：Smac基因可显著改善紫杉醇耐药肺腺癌细胞株细胞生物活性,并激活经典凋亡信号通路,其作用机制可能与调控Caspase-3表达有关。     

PHTS, a novel putative tumor suppressor, is involved in the transformation reversion of HeLaHF cells independently of the p53 pathway

HeLaHF is a non-transformed revertant of HeLa cells, likely resulting from the activation of a putative tumor suppressor(s). p53 protein was stabilized in this revertant and reactivated for certain transactivation functions. Although p53 stabilization has not conclusively been linked to the reversion, it is clear that the genes in p53 pathway are involved. The present study confirms the direct role of p53 in HeLaHF reversion by demonstrating that RNAi-mediated p53 silencing partially restores anchorage-independent growth potential of the revertant through the suppression of anoikis. In addition, we identified a novel gene, named PHTS, with putative tumor suppressor properties, and showed that this gene is also involved in HeLaHF reversion independently of the p53 pathway. Expression profiling revealed that PHTS is one of the genes that is up-regulated in HeLaHF but not in HeLa. It encodes a putative protein with CD59-like domains. RNAi-mediated PHTS silencing resulted in the partial restoration of transformation (anchorage-independent growth) in HeLaHF cells, similar to that of p53 gene silencing, implying its tumor suppressor effect. However, the observed increased transformation potential by PHTS silencing appears to be due to an increased anchorage-independent proliferation rate rather than suppression of anoikis, unlike the effect of p53 silencing. p53 silencing did not affect PHTS gene expression, and vice versa, suggesting PHTS may function in a new and p53-independent tumor suppressor pathway. Furthermore, over-expression of PHTS in different cancer cell lines, in addition to HeLa, reduces cell growth likely via induced apoptosis, confirming the broad PHTS tumor suppressor properties.     

Persistent and remodeling hepatic preneoplastic lesions present differences in cell proliferation and apoptosis, as well as in p53, Bcl-2 and NF-kappaB pathways

During rat hepatocarcinogenesis preneoplastic lesions (PNL) emerge which may persist (pPNL) and be sites of progress to cancer or suffer remodeling (rPNL) tending to disappear. Cellular and molecular mechanisms involved in both phenotypes are not sufficiently elucidated. pPNL and rPNL cellular proliferation and apoptosis were evaluated in rats submitted to the resistant hepatocyte (RH) model, and an adjusted growth index (AGI) was established. p53, Bcl-2, and NF-kappaB p65 subunit expression was evaluated by immunohistochemistry in pPNL and rPNL. p65 expression and NF-kappaB activation was evaluated by Western blot assays in whole livers. A lower number of BrdU-stained hepatocyte nuclei/mm(2) and higher number of apoptotic bodies (AB) per mm(2) were observed in remodeling compared to pPNL. Cytoplasmic p53 accumulation is related to increased hepatocarcinoma malignancy. We observed that 71.3% pPNL and 25.4% rPNL (P < 0.05) presented p53 staining in the cytoplasm. Similarly, 67.7% pPNL and 23.1 % rPNL (P < 0.05) presented increased Bcl-2 staining. Thirty-two percent pPNL and 15.6% rPNL (P < 0.05) presented p65 staining. Compared to normal rats, increase (P < 0.05) of hepatic p65 expression and NF-kappaB activation in rats submitted to the RH model was observed. In agreement to previous studies hepatic pPNL and rPNL differ regarding cell proliferation and apoptosis. Moreover, persistence and remodeling involve differences in p53, Bcl-2, and NF-kappaB pathways. These data point to molecular pathways that may direct preneoplastic lesions to spontaneously regress or to progress to cancer. J. Cell. Biochem. 103: 538-546, 2008. (c) 2007 Wiley-Liss, Inc.     

Positive correlation between the expression of X-chromosome RBM genes (RBMX, RBM3, RBM10) and the proapoptotic Bax gene in human breast cancer

In a recent report, it has been postulated that the ubiquitous RBM proteins might constitute a novel family of apoptosis modulators. We measured the expression of the X-chromosome RBM genes (RBMX, RBM3, and RBM10) in 122 breast cancers by means of differential RT-PCR. Using the same method, we also studied the expression of the apoptosis-related genes Bcl-2 and Bax. Markers of hormone dependence (estrogen and progesterone receptors), proliferation (Ki67 and DNA-ploidy), angiogenesis (VEGF and CD105), as well as oncogene (c-erb-B2), and tumor suppressor gene (p53) expression were also analyzed. The expression of all X-chromosome RBM genes was significantly associated with the expression of the proapoptotic Bax gene (RBMX, P=0.039; RBM3, P<0.001; RBM10 large variant, P<0.001; RBM10 small variant, P<0.001). Furthermore, the expression of both RBM10 variants was significantly associated with the expression of the VEGF gene (large variant, P=0.004; small variant, P=0.003). We also found an association of borderline significance (P=0.05) between the expression of RBM3, the large variant of RBM10 and wild-type p53. Expression of the small RBM10 variant, finally, was associated with high proliferation of the tumors (Ki67>or=20%; P=0.037). The expression of both RBM10 variants seems to be interdependent to a significant degree (r=0.26, P=0.006). From these results, it seems that the X-chromosome, through its RBM genes, plays a formerly unknown role in the regulation of programmed cell death (apoptosis) in breast cancer.     

Inhibition of Fas expression by RNAi modulates 5-fluorouracil-induced apoptosis in HCT116 cells expressing wild-type p53

Drug resistance to 5-fluorouracil (5-FU) is still a major limitation to its clinical use. In addition, the clinical value of p53 as a predictive marker for 5-FU-based chemotherapy remains a matter of debate. Here, we used HCT116 human colorectal cancer cells expressing wild-type p53 and investigated whether inhibition of Fas expression by interference RNA modulates 5-FU-induced apoptosis. Cells were treated with 5-FU (1, 4 or 8 microM) for 8-48 h. Cell viability was evaluated by trypan blue dye exclusion. Apoptosis was assessed by changes in nuclear morphology and caspase activity. The interference RNA technology was used to silence Fas expression. Caspase activation, p53, Fas, cytochrome c, and Bcl-2 family protein expression was evaluated by immunoblotting. 5-FU was cytotoxic in HCT116 cells (p<0.001). Nuclear fragmentation and caspase-3, -8 and -9 activities were also markedly increased in HCT116 cells after 5-FU (p<0.001). In addition, wild-type p53 and Fas expression were 25- and 4-fold increased (p<0.05). Notably, when interference RNA was used to inhibit Fas, 5-FU-mediated nuclear fragmentation and caspase activity were markedly reduced in HCT116 cells. Finally, western blot analysis of mitochondrial extracts from HCT116 cells exposed to 5-FU showed a 6-fold increase in Bax, together with a 3-fold decrease in cytochrome c (p<0.001). In conclusion, 5-FU exerts its cytotoxic effects, in part, through a p53/Fas-dependent apoptotic pathway that involves Bax translocation and mitochondrial permeabilization.     

白藜芦醇对猪前体脂肪细胞凋亡的作用及机理

旨在研究白藜芦醇对猪前体脂肪细胞凋亡的作用,探讨其分子机制。以50 μmol/L、100 μmol/L、200 μmol/L、400 μmol/L白藜芦醇处理猪前体脂肪细胞,采用Hoechst 33258染色剂染色,光学和荧光显微镜分别观察细胞的形态学变化。semi-qRT-PCR和Western blotting方法检测凋亡相关基因sirt1、caspase-3、bcl-2、bax、p53、NF-κB的mRNA和蛋白的表达变化。结果表明,白藜芦醇处理后,前体脂肪细胞出现明显的细胞凋亡,伴随细胞体积缩小,染色质凝集,核质固缩等特征显现,与对照组相比200 μmol/L处理组细胞的凋亡率显著升高 (P＜0.05)。凋亡相关基因sirt1、caspase-3和bax的mRNA和蛋白表达水平显著上调 (P＜0.05),而bcl-2、p53、NF-κB等基因的表达水平明显下调 (P＜0.05)。进一步证实白藜芦醇特异性地增加sirt1的表达活性,而sirt1的上调影响caspase-3和bcl-2家族因子的活性,同时参与调控p53和NF-κB的转录表达。因此,推测sirt1调控凋亡相关因子表达是白藜芦醇诱导前体脂肪细胞凋亡的关键原因。     

Cisplatin restores p53 function and enhances the radiosensitivity in HPV16 E6 containing SiHa cells

Most HPV-positive cervical cancer cells possess wild type p53 gene, but its normal p53 functions are disrupted by expression of HPVs E6. Treatment with 0-20 microM cisplatin for 24 h in HPV16 E6 containing SiHa cells suppressed E6 mRNA, reduced E6 protein, and restored p53 expression in dose-dependent manners. Dual-parameter flow cytometric analysis indicated that sub-G(1) apoptotic cells, but not necrotic cells were the major species for cisplatin-induced cytotoxicity in SiHa cells. After 0-10 microM cisplatin treatment, slightly more apoptotic cells appeared from SiHa cells than those from dominant negative p53-transfected SiHa cells. There was no different ionizing radiation (IR)-induced apoptosis in these two different cells. On the other hand, cisplatin enhanced more IR-induced sub-G(1) apoptosis in SiHa than mp53-SiHa cells. These accompanied with prolonged p53 restoration in irradiated-SiHa cells after 24 h cisplatin treatment and thereafter. In contrast, it was not found in cells after irradiation alone. Similar results were also shown in Mdm2 expression in SiHa cells after combined treatment. Therefore, cisplatin restored p53 expression and prolonged IR-induced p53 restoration would be possible candidates to response more sub-G(1) apoptosis in irradiated SiHa cells. These results provided another new explanation on cisplatin sensitizing radiotherapy for HPV16 E6 containing cancer cells.     

Spatiotemporal activation of caspase‐dependent and ‐independent pathways in staurosporine‐induced apoptosis of p53wt and p53mt human cervical carcinoma cells

Background information. Caspase‐dependent and ‐independent death mechanisms are involved in apoptosis in a variety of human carcinoma cells treated with antineoplastic compounds. Our laboratory has reported that p53 is a key contributor of mitochondrial apoptosis in cervical carcinoma cells after staurosporine exposure. However, higher mitochondrial membrane potential dissipation and greater DNA fragmentation were observed in p53^wt (wild‐type p53) HeLa cells compared with p53^mt (mutated p53) C‐33A cells. Here, we have studied events linked to the mitochondrial apoptotic pathway. Results. Staurosporine can induce death of HeLa cells via a cytochrome c/caspase‐9/caspase‐3 mitochondrial‐dependent apoptotic pathway and via a delayed caspase‐independent pathway. In contrast with p53^wt cells, p53^mt C‐33A cells exhibit firstly caspase‐8 activation leading to caspase‐3 activation and Bid cleavage followed by cytochrome c release. Attenuation of PARP‐1 [poly(ADP‐ribose) polymerase‐1] cleavage as well as oligonucleosomal DNA fragmentation in the presence of z‐VAD‐fmk points toward a major involvement of a caspase‐dependent pathway in staurosporine‐induced apoptosis in p53^wt HeLa cells, which is not the case in p53^mt C‐33A cells. Meanwhile, the use of 3‐aminobenzamide, a PARP‐1 inhibitor known to prevent AIF (apoptosis‐inducing factor) release, significantly decreases staurosporine‐induced death in these p53^mt carcinoma cells, suggesting a preferential implication of caspase‐independent apoptosis. On the other hand, we show that p53, whose activity is modulated by pifithrin‐α, isolated as a suppressor of p53‐mediated transactivation, or by PRIMA‐1 (p53 reactivation and induction of massive apoptosis), that reactivates mutant p53, causes cytochrome c release as well as mitochondrio—nuclear AIF translocation in staurosporine‐induced apoptosis of cervical carcinoma cells. Conclusions. The present paper highlights that staurosporine engages the intrinsic mitochondrial apoptotic pathway via caspase‐8 or caspase‐9 signalling cascades and via caspase‐independent cell death, as well as through p53 activity.