Telmisartan induces apoptosis and regulates Bcl-2 in human renal cancer cells

It has been well-characterized that the renin-angiotensin system (RAS) physiologically regulates systemic arterial pressure. However, RAS signaling has also been shown to increase cell proliferation during malignancy, and angiotensin receptor blockers (ARBs) are able to decrease pro-survival signaling by inhibiting anti-apoptotic molecules and suppressing caspase activity. In this study, the apoptotic effects of telmisartan, a type of ARB, was evaluated using a non-cancerous human renal cell line (HEK) and a human renal cell carcinoma (RCC) cell line (786). Both types of cells were treated with telmisartan for 4 h, 24 h, and 48 h, and then were assayed for levels of apoptosis, caspase-3, and Bcl-2 using MTT assays, flow cytometry, and immunostaining studies. Analysis of variance was used to identify significant differences between these data (P < 0.05). Following the treatment of 786 cells with 100 µM and 200 µM telmisartan, a marked inhibition of cell proliferation was observed. 50 µM cisplatin also caused high inhibition of these cells. Moreover, these inhibitions were both concentration- and time-dependent (P < 0.05). Various apoptotic effects were also observed compared with control cells at the 24 h and 48 h timepoints assayed (P < 0.001). Furthermore, positive caspase-3 staining and down-regulation of Bcl-2 were detected, consistent with induction of cell death. In contrast, treatment of HEK cells with telmisartan did not produce an apoptotic effect compared with control cells at the 24 h timepoint (P > 0.05). Treatment with cisplatin promoted in HEK cells high index of apoptosis (P < 0.001). Taken together, these results suggest that telmisartan induces apoptosis via down-regulation of Bcl-2 and involvement of caspase-3 in human RCC cells.     

Lithium inhibits aluminum-induced apoptosis in rabbit hippocampus,by preventing cytochrome c translocation,Bcl-2 decrease,Bax elevation and caspase-3 activation

A variety of studies on neuronal death models suggest that lithium has neuroprotective properties. In the present investigation, we have examined the effect of chronic lithium treatment on hippocampus, as monitored by changes at the subcellular level of apoptosis-regulatory proteins which have been induced by the neurotoxin, aluminum maltolate. Intracisternal administration of aluminum into rabbit brain induces cytochrome c release, decreases levels of the anti-apoptotic proteins Bcl-2 and Bcl-X(L), increases levels of the pro-apoptotic Bax, activates caspase-3, and causes DNA fragmentation as measured by the TUNEL assay. Pretreatment for 14 days with 7 mm of lithium carbonate in drinking water prevents aluminum-induced translocation of cytochrome c, and up-regulates Bcl-2 and Bcl-X(L,) down-regulates Bax, abolishes caspase-3 activity and reduces DNA damage. The regulatory effect of lithium on the apoptosis-controlling proteins occurs in both the mitochondria and endoplasmic reticulum. We propose that the neuroprotective effect of lithium involves the modulation of apoptosis-regulatory proteins present in the subcellular organelles of rabbit brain.     

Bcl-2-regulated apoptosis and cytochrome c release can occur independently of both caspase-2 and caspase-9

Apoptosis in response to developmental cues and stress stimuli is mediated by caspases that are regulated by the Bcl-2 protein family. Although caspases 2 and 9 have each been proposed as the apical caspase in that pathway, neither is indispensable for the apoptosis of leukocytes or fibroblasts. To investigate whether these caspases share a redundant role in apoptosis initiation, we generated caspase-2(-/-)9(-/-) mice. Their overt phenotype, embryonic brain malformation and perinatal lethality mirrored that of caspase-9(-/-) mice but were not exacerbated. Analysis of adult mice reconstituted with caspase-2(-/-)9(-/-) hematopoietic cells revealed that the absence of both caspases did not influence hematopoietic development. Furthermore, lymphocytes and fibroblasts lacking both remained sensitive to diverse apoptotic stimuli. Dying caspase-2(-/-)9(-/-) lymphocytes displayed multiple hallmarks of caspase-dependent apoptosis, including the release of cytochrome c from mitochondria, and their demise was antagonized by several caspase inhibitors. These findings suggest that caspases other than caspases 2 and 9 can promote cytochrome c release and initiate Bcl-2-regulated apoptosis.     

How the Bcl-2 family of proteins interact to regulate apoptosis

Commitment of cells to apoptosis is governed largely by protein-protein interactions between members of the Bcl-2 protein family. Its three sub-families have distinct roles： the BH3-only proteins trigger apoptosis by binding via their BH3 domain to pro-survival relatives, while the pro-apoptotic Bax and Bak have an essential downstream role involving disruption of organellar membranes and induction of caspase activation. The BH3-only proteins act as damage sensors, held inert until their activation by stress signals. Once activated, they were thought to bind promiscuously to pro-survival protein targets but unexpected selectivity has recently emerged from analysis of their interactions. Some BH3-only proteins also bind to Bax and Bak. Whether Bax and Bak are activated directly by these BH3-only proteins, or indirectly as a consequence of BH3-only proteins neutralizing their pro-survival targets is the subject of intense debate. Regardless of this, a detailed understanding of the interactions between family members, which are often selective, has notable implications for designing anti-cancer drugs to target the Bcl-2 family.     

Small hairpin RNA targeting at Bcl-2 increases cytarabine-induced apoptosis in Raji cells

Bcl-2, a prominent member of the Bcl-2 family proteins, is responsible for the dysregulation of apoptosis and resistance to chemotherapy. In this study, we inves-tigated whether small hairpin RNA （shRNA） targeting at Bcl-2 mRNA could enhance cytarabine （Ara-C）- induced apoptosis in Raji cells. Bcl-2 shRNA was trans-fected into Raji cells and the expression levels of Bcl-2 mRNA and protein were assayed by RT-PCR and immunofluorescence. Cell proliferation was determined by MTT assay. Apoptosis was determined by morphological observation and flow cytometric analysis. Our results show that expression levels of Bcl-2 mRNA and protein from Raji cells transfected with Bcl-2 shRNA decreased, compared with either negative control shRNA group or untransfected cells group （P 〈 0.05）. Viability of cells transfected with Bcl-2 shRNA was less than the cells transfected with control shRNA and untransfected Raji cells, respectively （P〈 0.05）. Bcl-2 shRNA combined with Ara-C significantly inhibited the growth of cells （P 〈 0.05）. There was no difference in cell survival between control shRNA/Ara-C combination and cells treated with Ara-C alone. Using Giemsa staining, cells treated with Bcl-2 shRNA plus Ara-C at 48h displayed changes of apoptosis. Apoptotic rates of Raji cells treated with Bcl-2 shRNA combined with Ara-C significantly increased （P 〈 0.05）, compared with either control shRNA/Ara-C combi- nation or Ara-C-treated cells alone. Our results suggest that the shRNA against Bcl-2 mRNA could increase Ara-C-induced apoptosis in Raji cells.     

High-mobility group A1 protein inhibits p53-mediated intrinsic apoptosis by interacting with Bcl-2 at mitochondria

The high-mobility group A (HMGA) proteins are a family of non-histone chromatin factors, encoded by the HMGA1 and HMGA2 genes. Several studies demonstrate that HMGA proteins have a critical role in neoplastic transformation, and their overexpression is mainly associated with a highly malignant phenotype, also representing a poor prognostic index. Even though a cytoplasmic localization of these proteins has been previously reported in some highly malignant neoplasias, a clear role for this localization has not been defined. Here, we first confirm the localization of the HMGA1 proteins in the cytoplasm of cancer cells, and then we report a novel mechanism through which HMGA1 inhibits p53-mitochondrial apoptosis by counteracting the binding of p53 to the anti-apoptotic factor Bcl-2. Indeed, we demonstrate a physical and functional interaction between HMGA1 and Bcl-2 proteins. This interaction occurs at mitochondria interfering with the ability of p53 protein to bind Bcl-2, thus counteracting p53-mediated mitochondrial apoptosis. This effect is associated with the inhibition of cytochrome c release and activation of caspases. Consistent with this mechanism, a strong correlation between HMGA1 cytoplasmic localization and a more aggressive histotype of thyroid, breast and colon carcinomas has been observed. Therefore, cytoplasmic localization of HMGA1 proteins in malignant tissues is a novel mechanism of inactivation of p53 apoptotic function.     

Structural biology of the Bcl-2 family and its mimicry by viral proteins

Intrinsic apoptosis in mammals is regulated by protein–protein interactions among the B-cell lymphoma-2 (Bcl-2) family. The sequences, structures and binding specificity between pro-survival Bcl-2 proteins and their pro-apoptotic Bcl-2 homology 3 motif only (BH3-only) protein antagonists are now well understood. In contrast, our understanding of the mode of action of Bax and Bak, the two necessary proteins for apoptosis is incomplete. Bax and Bak are isostructural with pro-survival Bcl-2 proteins and also interact with BH3-only proteins, albeit weakly. Two sites have been identified; the in-groove interaction analogous to the pro-survival BH3-only interaction and a site on the opposite molecular face. Interaction of Bax or Bak with activator BH3-only proteins and mitochondrial membranes triggers a series of ill-defined conformational changes initiating their oligomerization and mitochondrial outer membrane permeabilization. Many actions of the mammalian pro-survival Bcl-2 family are mimicked by viruses. By expressing proteins mimicking mammalian pro-survival Bcl-2 family proteins, viruses neutralize death-inducing members of the Bcl-2 family and evade host cell apoptosis during replication. Remarkably, structural elements are preserved in viral Bcl-2 proteins even though there is in many cases little discernible sequence conservation with their mammalian counterparts. Some viral Bcl-2 proteins are dimeric, but they have distinct structures to those observed for mammalian Bcl-2 proteins. Furthermore, viral Bcl-2 proteins modulate innate immune responses regulated by NF-κB through an interface separate from the canonical BH3-binding groove. Our increasing structural understanding of the viral Bcl-2 proteins is leading to new insights in the cellular Bcl-2 network by exploring potential alternate functional modes in the cellular context. We compare the cellular and viral Bcl-2 proteins and discuss how alterations in their structure, sequence and binding specificity lead to differences in behavior, and together with the intrinsic structural plasticity in the Bcl-2 fold enable exquisite control over critical cellular signaling pathways.     

Mechanisms of arsenic trioxide induced apoptosis of human cervical cancer HeLa cells and protection by Bcl-2

It was recently reported that arsenic trioxide (As_2O_3) can induce complete remission in patients with acute promyelocytic leukemia (APL). In this present article, the biological effect of As_2O_3 on human cervical cancer HeLa cells and HeLa cells overexpressing Bcl-2 is studied. By MTT and colony forming ability assays, morphology alteration, flow cytometric analysis, DNA gel electrephoresis and in situ cell death detection (TUNEL), it was found that As_2O_3 inhibited the growth of HeLa cells and induced G2/M arrest and apoptosis of the cells. RT-PCR, Northern blot, Western blot analysis revealed that As_2O_3 induced HeLa cell apoptosis possibly via decreasing the expression of c-myc and viral genes. HeLa cells overexpressing Bcl-2 partly resist As_2O_3 induced apoptosis, which might be relative to preventing the cells from As_2O_3 caused G2/M block, downregulation of c-myc gene expression and inhibition of viral gene expression was also noted, However, it was found that As_2O_3 at a high concentratio     

A novel small molecule inhibitor targeted at Bcl-2

Recently, the heterocyclic compound 8-oxo-3-thiomorpholino-8H-acenaphtho[1,2-b]pyrrole-9-carboni-trile (S1) was synthesized and shown to induce apoptosis in both (H22) hematoma and (MCF-7) ade-nocarcinoma cells. The IC50 values of S1 against the two cell lines were 0.17 and 0.09 μmol/L, respec-tively. Furthermore, the apoptosis-inducing activity of this compound was highlighted both in vivo and in vitro. Subsequent experiments identified Bcl-2 as the primary target of S1, as a significant reduc-tion in Bcl-2 protein levels was observed in H22 cells following a two-hour treatment with 10 μmol/L S1. While rapid depolarization of mitochondrial membranes led immediately to caspase 9 activation, no changes were identified in either caspase 8 levels or levels in Bcl-2 mRNA. These data were consistent with the results of circular dichroism (CD) spectra analysis, revealing that S1 inactivated the Bcl-2 protein by destroying its critical alpha helices. Taken together, these results suggest the potential of S1 in the development of new therapeutic agents.     

A novel small molecule inhibitor targeted at Bcl-2

Recently, the heterocyclic compound 8-oxo-3-thiomorpholino-8H-acenaphtho[1,2-b]pyrrole-9-carbonitrile (S1) was synthesized and shown to induce apoptosis in both (H22) hematoma and (MCF-7) adenocarcinoma cells. The IC₅₀ values of S1 against the two cell lines were 0.17 and 0.09 μmol/L, respectively. Furthermore, the apoptosis-inducing activity of this compound was highlighted both in vivo and in vitro. Subsequent experiments identified Bcl-2 as the primary target of S1, as a significant reduction in Bcl-2 protein levels was observed in H22 cells following a two-hour treatment with 10 μmol/L S1. While rapid depolarization of mitochondrial membranes led immediately to caspase 9 activation, no changes were identified in either caspase 8 levels or levels in Bcl-2 mRNA. These data were consistent with the results of circular dichroism (CD) spectra analysis, revealing that S1 inactivated the Bcl-2 protein by destroying its critical alpha helices. Taken together, these results suggest the potential of S1 in the development of new therapeutic agents.     

顺铂对小鼠耳蜗螺旋神经节细胞凋亡及caspase-3表达的影响

目的:建立小鼠顺铂(CDDP)耳毒性模型,研究不同剂量顺铂对小鼠耳蜗螺旋神经节细胞凋亡及caspase-3表达的影响。方法:采用末端脱氧核苷酸转移酶介导的缺口末端标记(TUNEL)技术检测螺旋神经节细胞的凋亡;应用免疫组织化学Envision法检测caspase-3在螺旋神经节中的表达;同时结合听脑干反应(ABR)测试,观察用药前后小鼠听力的变化。结果:不同剂量顺铂组小鼠体重和听力明显下降,与对照组比较均有显著性差异(P0.05,P0.01);并且随着顺铂给药剂量的增加,小鼠耳蜗螺旋神经节中TUNEL阳性细胞数增多,以及caspase-3表达明显增强。结论:应用小鼠能建立可靠的顺铂耳毒性模型;顺铂可导致耳蜗螺旋神经节细胞凋亡,而且此凋亡过程中有caspase-3的参与,进一步证实了凋亡可能是顺铂耳毒性机制之一。     

Rosmarinic acid failed to suppress hydrogen peroxide-mediated apoptosis but induced apoptosis of Jurkat cells which was suppressed by Bcl-2

Rosmarinic acid (RosA), frequently found as a secondary metabolite in herbs and medicinal plants, has exhibited antioxidative and anti-inflammatory activities. RosA was shown to inhibit the proliferation and induce apoptosis of Jurkat T cells but the mechanism of action of RosA in apoptosis remains elusive. RosA inhibited the proliferation of Jurkat cells in a dose-dependent manner by suppressing the expression of cyclin D₃ and p21^Cip1/Waf1 and up-regulating p27^Kip1. RosA induced apoptosis of Jurkat cells in a dose-dependent manner and failed to protect them from hydrogen peroxide (H₂O₂)-mediated apoptosis. Induction of apoptosis by RosA correlated with suppression of Bcl-2 but not of Bak or PUMA. Overexpression of Bcl-2 protected Jurkat cells from both H₂O₂- and RosA-induced apoptosis by altering the ratio of anti- to pro-apoptotic members of the Bcl-2 family. In conclusion, RosA inhibited Jurkat cell proliferation by altering the expression of cyclins and cyclin-dependent kinase inhibitors and induced apoptosis most likely acting through the mitochondrial pathway and possessed no anti-oxidant properties.     

吗啡对脑缺血再灌注损伤后神经元凋亡及Bcl-2的影响

目的:探讨吗啡预处理对大鼠脑缺血再灌注损伤后神经元凋亡及Bcl-2蛋白表达的影响.方法:Wistar大鼠随机分为假手术组、模型组、吗啡组,各18只.四动脉阻断法建立脑缺血模型,吗啡组在脑缺血前60 min腹腔内注射吗啡1mg/kg.脑缺血8 min再灌注12h、72h及168h各取6只大鼠的脑组织,观察海马区病理学改变、神经元凋亡及Bcl-2表达.结果:吗啡预处理能使各灌注点海马神经元病理改变减轻、凋亡细胞数减少(P<0.01)、Bel-2表达增加(P<0.01).吗啡组细胞凋亡数减少趋势与Bcl-2表达上调趋势一致.结论:吗啡预处理可减轻缺血性脑损伤;吗啡抗凋亡作用机制与Bcl-2密切相关.     

Early events in Bcl-2-enhanced apoptosis

Transfection of PC12 pheochromocytoma cells with bcl-2 potentiates apoptosis induced by the antimitotic agent, neocarzinostatin (NCS). The mechanism of potentiation involves caspase 3-dependent cleavage of Bcl-2 to its pro-apoptotic counterpart, but the cellular events proximal to caspase 3 activation in this system are not known. Two min after initiation of NCS treatment, Bax begins to translocate from cytosol to the mitochondria; the mitochondrial localization of Bax persists for 30 min after NCS treatment. At the same time, cytochrome C is released from the mitochondria to cytosol. The mitochondrial membrane potential exhibits differential change in mock- and bcl-2-transfected PC12 cells. In mock-transfected PC12 cells, the mitochondrial membrane potential increases immediately, peaks at 15 min following initiation of NCS treatment, and drops thereafter. In contrast, in bcl-2-transfected PC12 cells, the membrane potential drops immediately following NCS treatment. Caspase 9 is activated and peaks at 10 min in both mock- and bcl-2 transfected PC12 cells, however, the peak activity of caspase 9 is higher and caspase 9 activation lasts longer (30 min) after the treatment in bcl-2 transfectants. Not until 30 min after initiation of a 1 h treatment with NCS is Bcl-2 protein cleaved in bcl-2-transfected cells. Thus, in bcl-2-transfected cells, the mitochondrial membrane potential drops and cytochrome C is released from the mitochondria despite the presence of large amounts of intact mitochondrial Bcl-2. This makes it unlikely that cleavage of Bcl-2 is the only factor involved in potentiation of NCS-induced apoptosis by Bcl-2.     

Heregulin-induced apoptosis

Heregulins (HRGs) are a group of polypeptide factors that are encoded by four different HRG genes that can express multiple isoforms through alternate RNA splicing. A number of HRG isoforms possess both growth stimulatory and growth inhibitory functions that are necessary for their important role in the development and maintenance of the heart, nervous system and epithelial cells in multiple organs including the breast. Growth inhibition by HRG relates to its ability to induce apoptosis, differentiation, and cell cycle G₂ arrest. Current studies suggest that HRGs can induce a unique form of apoptosis. In this article, we review recent progress in characterizing and understanding HRG-induced apoptosis. Particular attention has been given to: (1) the activation of caspases-7 and -9; (2) the role of the anti-apoptotic Bcl-2 protein; and (3) the signaling molecules and pathways that regulate HRG-induced apoptosis, including the p38, JNK, mTOR kinase, and PKC kinase.     

Regulation of apoptosis by Bcl-2 family proteins

For multicellular organisms, the rigorous control of programmed cell death is as important as that of cell proliferation. The mechanisms involved in the regulation of cell death are not yet understood, but a key component is the family of caspases which are activated in a cascade and are responsible for the apoptotic-specific changes and disassembly of the cell. Although the caspases represent a central point in apoptosis, their activation is regulated by a variety of other factors. Among these, Bcl-2 family plays a pivotal role in caspases activation, by this deciding whether a cell will live or die. Bcl-2 family members are known to focus much of their response to the mitochondria level, upstream the irreversible cellular damage, but their functions are not yet well defined. This review summarizes the recent data regarding the Bcl-2 proteins and the ways they regulate the apoptosis.     

Bcl-2 proteins regulate ER membrane permeability to luminal proteins during ER stress-induced apoptosis

Endoplasmic reticulum (ER) stress-induced apoptosis may arise from multiple environmental and pharmacological causes, but the precise mechanism(s) involved are not completely known. Members of Bcl-2 protein family are important regulators of apoptosis. In this study, we report that in a process dependent on the proapoptotic Bcl-2 members Bax and Bak, exogenously expressed fluorescent protein localized to the ER lumen is released into the cytosol in cells undergoing ER stress. Upon ER stress induction, endogenous ER luminal proteins are also released into the cytosol in a similar manner accompanied by translocation and anchorage of Bax to the ER membrane. In addition, Bax and truncated-Bid (tBid) mediate a global increase in ER membrane permeability to ER luminal proteins in vitro. Importantly, antiapoptotic Bcl-X_L antagonizes the effects of proapoptotic Bcl-2 proteins on ER membrane permeability. Consistent with Bax translocation to the ER membrane in whole apoptotic cells, there is also increased tight association of Bax with the ER membrane correlated with the increase in ER membrane permeability in vitro. Overall, these data suggest that the regulation of ER membrane permeability by Bcl-2 proteins could be an important molecular mechanism of ER stress-induced apoptosis.