Effect of Bcl—2 and caspase—3 on calcium distribution in apoptosis of HL—60 cells

Apoptosis manifests in two major execution programs downstream of the death signal:the caspase pathway and organelle dysfunction.An important antiapoptosis factor,Bcl-2 protein,contributes in caspase pathway of apoptosis.Calcium,an important intracellular signal element in cells,is also observed to have changes during apoptosis,which maybe affected by Bcl-2 protein.We have previously reported that in Harringtonine (HT) induced apoptosis of HL-60 cells,there‘s change of intracellular calcium distribution,oving from cytoplast especially Golgi‘s apparatus to nucleus and accumulating there with the highest concentration.We report here that caspase-3 becomes activated in HT-induced apoptosis of HL-60 cells,which can be inhibited by overexpression of Bcl-2 protein.No sign of apoptosis or intracellular calcium movement from Golgi‘s apparatus to nucleus in HL-60 cells overexpressing Bcl-2 or treated with Ac-DEVD-CHO,a specific inhibitor of caspase-3.The results indicate that activated caspase-2 can promote the movement of intracellular calcium from Golgi‘s apparatus to nucleus,and the process is inhibited by Ac-DEVD-CHO(inhibitor of caspase-3),and that Bcl-2 can inhibit the movement and accumulation of intracellular calcium in nucleus through its inhibition on caspase-3.Calcium relocalization in apoptosis seems to be irreversible,which is different from the intracellular calcium changes caused by growth factor.     

Induction of mitochondrion-mediated apoptosis of CHO cells by tripchlorolide

Tripchlorolide (TC) is a potent antitumor reagent purified from a Chinese herb Tripterygium Wilfordii Hook. f.. However, its cellular effects and mechanism of action are unknown. We showed here that TC induced apoptosis of Chinese Hamster Ovary (CHO) cells in time- and dose-dependent manners. TC resulted in the degradation of Bcl-2, the translocation of Bax from the cytosol to mitochondria, and the release of cytochrome c from mitochondria. Stable overexpression of human Bcl-2 could reduce the apoptosis of TC-treated cells by blocking the translocation of Bax and the release of cytochrome c. These results indicate that TC induces apoptosis of CHO cell by activating the mitochondrion-mediated apoptotic pathway involving the proteins of Bcl-2 family and cytochrome c.     

Up-regulation of Bcl-2 is required for the progression of prostate cancer cells from an androgen-dependent to an androgen-independent growth stage

Bcl-2 is an anti-apoptotic oncoprotein and its protein levels are inversely correlated with prognosis in many cancers.However, the role of Bcl-2 in the progression of prostate cancer is not clear. Here we report that Bcl-2 is required for theprogression of LNCaP prostate cancer cells from an androgen-dependent to an androgen-independent growth stage. ThemRNA and protein levels of Bcl-2 are significantly increased in androgen-independent prostate cancer cells. shRNA-medi-ated gene silencing of Bcl-2 in androgen-independent prostate cancer cells promotes UV-induced apoptosis and suppressesthe growth of prostate tumors in vivo. Growing androgen-dependent cells under androgen-deprivation conditions resultsin formation of androgen-independent colonies; and the transition from androgen-dependent to androgen-independentgrowth is blocked by ectopic expression of the Bcl-2 antagonist Bax or Bcl-2 shRNA. Thus, our results demonstratethat Bcl-2 is not only critical for the survival of androgen-independent prostate cancer cells, but is also required for theprogression of prostate cancer cells from an androgen-dependent to an androgen-independent growth stage.     

Induction of apoptosis in purified animal and plant nuclei by Xenopus egg extracts

We have developed a cell-free system that can trigger the nuclei purified from mouse liver and suspensioncultured carrot cells to undergo apoptosis as defined by the formation of apoptotic bodies and nucleosomal DNA fragments.The effects of different divalent cations and cycloheximide on DNA cleavage in this system were assessed.The fact that nuclei of plant cells can be induced to undergo apoptosis in a cell-free animal system suggests that animals and plants share a common signal transduction pathway triggering in the initiation stage of apoptosis.     

Reversion of malignancy in human gastric cancer MKN—45 cells through the transfection of transforming growth factor—β type Ⅱ receptor gene

Human gastric cancer MKN-45 cells which are resistant to TGF-β growth inhibition and possess TGF-β type I and type Ⅲ receptors,but not type Ⅱ receptors,have been used as a model system to reconstitute these cancer cells with TGF-β RII cDNA.The results of these experiments indicated that the reexpression of TGF-β RII gene in MKN-45 cells can restore their sensitivity to TGF-β growth inhibition,decrease their growth rate,reduce their cloning efficiency in soft agar and tumorigenicity in nude mice in stable transfectants,in comparison with their control MKN-45 cells.Among different RII transfectants,their difference in the changes of these parameters,as a result of the regain of autocrine negative growth control by TGF-β,is roughly proportional to their level of expression of transfected RII mRNA.From these data,it is concluded that the inactivation of TGF-β RII gene is related to the escape of growth control by TGF-β in MKN-45 cells.The importance of the study of the interplay of TGF-β and its receptor system in the negative growth control of gastric cancer,and possibly also of other cancers,is discussed.     

Fucoxanthin induces growth arrest and apoptosis in human bladder cancer T24 cells by up-regulation of p21 and down-regulation of mortalin

Fucoxanthin, a natural carotenoid, has been reported to have anti-cancer activity in human colon cancer cells, human prostate cancer cells, human leukemia cells, and human epithelial cervical cancer cells. This study was undertaken to evaluate the molecular mechanisms of fuco- xanthin against human bladder cancer T24 cell line. MTT analysis results showed that 5 and 10 ixM fucoxanthin inhibited the proliferation of T24 cells in a dose- and time- dependent manner accompanied by the growth arrest at Go/G1 phase of cell cycle, which is mediated by the up-regu- lation of p21, a cyclin-dependent kinase （CDK）-inhibitory protein and the down-regulation of CDK-2, CDK-4, cyclin D1, and cyclin E. In addition, 20 and 40 μM fucoxanthin induced apoptosis of T24 cells by the abrogation of morta- lin-p53 complex and the reactivation of nuclear mutant- type p53, which also had tumor suppressor function as wild-type p53. All these results demonstrated that the anti- cancer activity of fucoxanthin on T24 cells was associated with cell cycle arrest at Go/G1 phase by up-regulation of p21 at low doses and apoptosis via decrease in the expres- sion level of mortalin, which is a stress regulator and a mem- ber of heat shock protein 70, followed by up-regulation of cleaved caspase-3 at high doses.     

Expression of Bcl—2 inhibited Fas—mediated apoptosis in human hepatocellular carcinoma BEL—7404 cells

Apoptosis plays an important role in embryonic development,tissue remodeling,immune regulation and tumor regression.Two groups of molecules(Bcl-2 family and “Death factor” family) are involved in regulating apoptosis.In order to know about the effect of Bcl-2 on apoptosis induced by Fas,a typical member of “Death factor” family,the transfection experiments with expression vectors pcDNA3-fl and pcDNA3-bcl-2 were performed in BEL-7404 cells,a human hepatocellular carcinoma cell line which expresses endogenous Fas,but not FasL and Bcl-2.The data showed that the expression of FasL in pcDNA3-fl transfected hepatoma cells obviously induced the apoptosis of the cells.However,the overexpression of Bcl-2 in pcDNA3-bcl-2 transfected 7404/b-16 cells counteracted pcDNA3-fl transient transfection mediated apoptosis.Further study by cotransfection experiments indicated that Bid but not Bax (both were pro-apoptotic proteins of Bcl-2 family) blocked the inhibitory effect of Bcl-2 on Fas-mediated apoptosis.These results suggested that Fas-mediated apoptosis in human hepatoma cells is possibly regulated by Bcl-2 family proteins via mitochondria pathway.     

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     

Different roles of TGF-β in the multi-lineage differentiation of stem cells

Stem cells are a population of cells that has infinite or long-term self-renewal ability and can produce various kinds of descendent cells.Transforming growth factor β(TGF-β) family is a superfamily of growth factors,including TGF-β1,TGF-β2 and TGF-β3,bone morphogenetic proteins,activin/inhibin,and some other cytokines such as nodal,which plays very important roles in regulating a wide variety of biological processes,such as cell growth,differentiation,cell death.TGF-β,a pleiotropic cytokine,has been proved to be differentially involved in the regulation of multi-lineage differentiation of stem cells,through the Smad pathway,non-Smad pathways including mitogen-activated protein kinase pathways,phosphatidylinositol-3-kinase/AKT pathways and Rholike GTPase signaling pathways,and their cross-talks.For instance,it is generally known that TGF-β promotes the differentiation of stem cells into smooth muscle cells,immature cardiomyocytes,chondrocytes,neurocytes,hepatic stellate cells,Th17 cells,and dendritic cells.However,TGF-β inhibits the differentiation of stem cells into myotubes,adipocytes,endothelial cells,and natural killer cells.Additionally,TGF-β can provide competence for early stages of osteoblastic differentiation,but at late stages TGF-β acts as an inhibitor.The three mammalian isoforms(TGF-β1,2 and 3) have distinct but overlapping effects on hematopoiesis.Understanding the mechanisms underlying the regulatory effect of TGF-β in the stem cell multi-lineage differentiation is of importance in stem cell biology,and will facilitate both basic research and clinical applications of stem cells.In this article,we discuss the current status and progress in our understanding of different mechanisms by which TGF-β controls multi-lineage differentiation of stem cells.     

Overexpression of Bax induces down-regulation of store-operated calcium entry in prostate cancer cells

Store-operated Ca2+ channels control homeostasis between extracellular Ca2+ reservoir and intracellular Ca2+ storage and play important roles in apoptosis in a wide variety of cells, including prostate epithelia. Recent studies have shown that the acquired apoptosis-resistant nature of androgen-independent prostate cancer is associated with reduced function of store-operated Ca2+ entry (SOCE). This study investigates the functional interaction between Bax and SOCE in the apoptosis signaling cascade in prostate cancer. Our previous findings show that NRP-154, an androgen-independent prostate cancer cell line, could sustain overexpression of exogenous Bax without undergoing apoptosis. Here we show that sustained overexpression of Bax in NRP-154 cells leads to down-regulation of SOCE and reduced Ca2+ storage inside the endoplasmic reticulum. While reduced SOCE may represent an adaptive mechanism for cell survival, increased levels of Bax in the latent state enhances the sensitivity of NRP-154 cells to TGF-beta and thapsigargin-induced apoptosis. This enhanced apoptosis can be reduced by 2-aminoethoxydiphenyl borate (2-APB), an inhibitor of SOCE, or reversed under conditions where SOCE is only partially activated. Our results demonstrate a functional interaction between Bax and SOCE in apoptosis of prostate cancer, and support the concept that improving this interaction has therapeutic implications for prostate cancer.     

Regulation of trespin expression by modulators of cell growth,differentiation, and apoptosis in prostatic epithelial cells

We recently identified a novel rat ov-serpin, Trespin, which inhibits the trypsin-like serine proteinase plasmin and is expressed in several tissues, including prostate. In this report Trespin expression was studied in prostatic cell lines, NRP-152, NRP-154, and DP-153, derived from the Lobund-Wistar rat. Northern blots revealed Trespin mRNA is expressed in NRP-152 and DP-153 basal epithelial cell lines but not in the luminal line, NRP-154. Similarly, Trespin levels drop >30-fold following transdifferentiation of NRP-152 cells toward a luminal variant, further suggesting Trespin expression is specific for basal prostatic epithelial cells. Trespin expression in NRP-152 cells is up-regulated by dexamethasone (Dex) and insulin-like growth factor-I (IGF-I), each of which stimulate growth and prevent differentiation and apoptosis. However, Dex (alone) facilitates loss of Trespin by TGF-beta, yet enhances the ability of LR(3)-IGF-I to reverse such loss, similar to the pattern of apoptosis induced by TGF-beta. Likewise, several apoptosis inducers markedly decrease Trespin mRNA levels. HEK293 cells stably overexpressing Trespin display increased cell proliferation and partial resistance to growth inhibition and phosphorylation of c-Jun induced by the phorbol ester 12-O-tetradecanoylphorbol 13-acetate (TPA). Together these data strongly suggest that Trespin has critical functions tied to the regulation of growth, differentiation, and apoptosis of prostatic epithelial cells.     

Bcl-xL blocks transforming growth factor-beta 1-induced apoptosis by inhibiting cytochrome c release and not by directly antagonizing Apaf-1-dependent caspase activation in prostate epithelial cells

The mechanism by which transforming growth factor-beta1 (TGF-beta1) induces apoptosis of prostate epithelial cells was studied in the NRP-154 rat prostate epithelial cell line. TGF-beta 1 down-regulates expression of Bcl-xL and poly(ADP-ribosyl)polymerase (PARP), promotes cytochrome c release, up-regulates expression of latent caspase-3, and activates caspases 3 and 9. We tested the role of Bcl-xL in this cascade by stably overexpressing Bcl-xL to prevent loss by TGF-beta 1. Clones overexpressing Bcl-xL are resistant to TGF-beta 1 with respect to induction of apoptosis, cytochrome c release, activation of caspases 9 and 3, and cleavage of PARP; yet they remain sensitive to TGF-beta 1 by cell cycle arrest, induction of both fibronectin and latent caspase-3 expression, and loss of PARP expression. We show that Bcl-xL associates with Apaf-1 in NRP-154 cells; but this association does not inhibit the activation of caspases 9 and 3 by cytochrome c. Together, our data suggest that TGF-beta1 induces apoptosis through loss of Bcl-xL, leading to cytochrome c release and the subsequent activation of caspases 9 and 3. Moreover, our data demonstrate that the antiapoptotic effect of Bcl-xL occurs by inhibition of mitochondrial cytochrome c release and not through antagonizing Apaf-1-dependent processing of caspases 9 and 3.     

Transforming growth factor-beta 1 induces apoptosis independently of p53 and selectively reduces expression of Bcl-2 in multipotent hematopoietic cells

Transforming growth factor-beta1 (TGF-beta1) can inhibit cell proliferation or induce apoptosis in multipotent hematopoietic cells. To study the mechanisms of TGF-beta1 action on primitive hematopoietic cells, we used the interleukin-3 (IL-3)-dependent, multipotent FDCP-Mix cell line. TGF-beta1-mediated growth inhibition was observed in high concentrations of IL-3, while at lower IL-3 concentrations TGF-beta1 induced apoptosis. The proapoptotic effects of TGF-beta1 occur via a p53-independent pathway, since p53(null) FDCP-Mix demonstrated the same responses to TGF-beta1. IL-3 has been suggested to enhance survival via an increase in (antiapoptotic) Bcl-x(L) expression. In FDCP-Mix cells, neither IL-3 nor TGF-beta1 induced any change in Bcl-x(L) protein levels or the proapoptotic proteins Bad or Bax. However, TGF-beta1 had a major effect on Bcl-2 levels, reducing them in the presence of high and low concentrations of IL-3. Overexpression of Bcl-2 in FDCP-Mix cells rescued them from TGF-beta1-induced apoptosis but was incapable of inhibiting TGF-beta1-mediated growth arrest. We conclude that TGF-beta1-induced cell death is independent of p53 and inhibited by Bcl-2, with no effect on Bcl-x(L). The significance of these results for stem cell survival in bone marrow are discussed.     

FLICE-like inhibitory protein blocks transforming growth factor beta 1-induced caspase activation and apoptosis in prostate epithelial cells

Androgen withdrawal induces the regression of human prostate cancers, but such cancers eventually become androgen-independent and metastasize. Thus, deciphering the mechanism of androgen withdrawal-induced apoptosis is critical to designing new therapies for prostate cancer. Previously, we showed that in the rat, castration-induced apoptosis is accompanied by a reduction in the expression of the apical caspase inhibitor FLICE-like inhibitory protein (FLIP). To test the functional role of FLIP in inhibiting prostate epithelial cell apoptosis, we employed the rat prostate epithelial cell line NRP-152, which differentiates to a secretory phenotype in a low-mitogen medium and then undergoes apoptosis following the addition of transforming growth factor beta1 (TGFbeta1), mimicking androgen withdrawal-induced apoptosis. FLIP levels decline with TGFbeta1 treatment, suggesting that apoptosis is mediated by caspase-8 and indeed the caspase inhibitor crmA blocks TGFbeta1-induced apoptosis. Small interfering RNA-mediated knockdown of FLIP recapitulates and enhances TGFbeta1-induced cell death. NRP-152 cells stably transfected with constitutively expressed FLIP were refractory to TGFbeta1-induced apoptosis. TGFbeta1-induced caspase-3 activity is proportional to the level of cell death and inversely proportional to the level of FLIP expression in various clones. Moreover, neither caspase-3 nor PARP is cleaved in clones expressing high levels of FLIP. Furthermore, insulin, which inhibits differentiation, increases FLIP and inhibits TGFbeta-induced death in a FLIP-dependent manner. Although neither Fas-Fc, sTNFRII-Fc, nor DR5-Fc blocked TGFbeta1-induced cell death, there is a significant increase in tumor necrosis factor mRNA following TGFbeta stimulation, suggesting both an unexpected role for tumor necrosis factor in this model system and the possibility that FLIP blocks another unknown caspase-dependent mediator of apoptosis.     

Transforming growth factor beta 1 induces apoptosis through cleavage of BAD in a Smad3-dependent mechanism in FaO hepatoma cells

Transforming growth factor beta (TGF-beta) induces apoptosis in a variety of cells. We have previously shown that TGF-beta 1 rapidly induces apoptosis in the FaO rat hepatoma cell line. We have now studied the effect of TGF-beta 1 on the expression of different members of the Bcl-2 family in these cells. We observed no detectable changes in the steady-state levels of Bcl-2, Bcl-X(L), and Bax. However, TGF-beta 1 induced caspase-dependent cleavage of BAD at its N terminus to generate a 15-kDa truncated protein. Overexpression of the 15-kDa truncated BAD protein enhanced TGF-beta 1-induced apoptosis, whereas a mutant BAD resistant to caspase 3 cleavage blocked TGF-beta 1-induced apoptosis. Overexpression of Smad3 dramatically enhanced TGF-beta 1-induced cleavage of BAD and apoptosis, whereas antisense Smad3 blocked TGF-beta 1-induced apoptosis and BAD cleavage. These results suggest that TGF-beta 1 induces apoptosis through the cleavage of BAD in a Smad3-dependent mechanism.     

Proteome profile changes during transdifferentiation of NRP-152 rat prostatic basal epithelial cells

NRP-152 is an androgen responsive, non-tumorigenic cell line, which shows basal epithelial cell characteristics under normal growth conditions. It has been noted that NRP-152 undergoes morphological and cytoskeletal changes toward its luminal counterpart NRP-154 when it is grown under growth restrictive conditions. We have extensively investigated the details of protein change of NRP-152 during transdifferentiation using proteomic techniques. NRP-152 cells were cultured under normal and growth restrictive media conditions for 3, 5 days. NRP-154 cells were normally cultured. Protein samples were submitted to 2D gel electrophoresis and silver stained. Protein patterns on the gels were comparatively analysed using Melanie III software. Protein spots exhibiting significant changes in NRP-152 cells during the time course were excised and subjected to in-gel tryptic digestion. After 6 days of growth restrictive conditions in NRP-152, the cells were morphologically changed resembling luminal phenotype. Of the 35 protein spots that were up-reglated, 20 proteins from 21 spots were identified by peptide mass fingerprinting and, of 21 proteins spots that were down-regulated, 10 proteins from 12 spots were identified as landmark proteins. Our study confirmed that basal NRP-152 cells were proportionally transdifferentiated into luminal featuring cells according to the duration of growth restrictive culture conditions. This suggests that human prostatic basal epithelial cells may be changed into luminal cells under certain conditions. Proteomic approach enabled us to identify 30 proteins involved in this differentiation with a single experiment. These proteins will be subjected to further functional studies to evaluate their possible roles related to cellular differentiation. These data strongly support that proteomics is a very powerful approach for studying physiologic and pathologic cellular changes such as differentiation and carcinogenesis.     

Molecular mechanisms of resveratrol (3,4,5-trihydroxy-trans-stilbene) and its interaction with TNF-related apoptosis inducing ligand (TRAIL) in androgen-insensitive prostate cancer cells

Although resveratrol, an active ingredient derived from grapes and red wine, possesses chemopreventive properties against several cancers, the molecular mechanisms by which it inhibits cell growth and induces apoptosis have not been clearly understood. Here, we examined the molecular mechanisms of resveratrol and its interactive effects with TRAIL on apoptosis in prostate cancer PC-3 and DU-145 cells. Resveratrol inhibited cell viability and colony formation, and induced apoptosis in prostate cancer cells. Resveratrol downregulated the expression of Bcl-2, Bcl-X_L and survivin and upregulated the expression of Bax, Bak, PUMA, Noxa, and Bim, and death receptors (TRAIL-R1/DR4 and TRAIL-R2/DR5). Treatment of prostate cancer cells with resveratrol resulted in generation of reactive oxygen species (ROS), translocation of Bax to mitochondria and subsequent drop in mitochondrial membrane potential, release of mitochondrial proteins (cytochrome c, Smac/DIABLO, and AIF) to cytosol, activation of effector caspase-3 and caspase-9, and induction of apoptosis. Resveratrol-induced ROS production, caspase-3 activity and apoptosis were inhibited by N-acetylcysteine. Bax was a major proapoptotic gene mediating the effects of resveratrol as Bax siRNA inhibited resveratrol-induced apoptosis. Resveratrol enhanced the apoptosis-inducing potential of TRAIL, and these effects were inhibited by either dominant negative FADD or caspase-8 siRNA. The combination of resveratrol and TRAIL enhanced the mitochondrial dysfunctions during apoptosis. These properties of resveratrol strongly suggest that it could be used either alone or in combination with TRAIL for the prevention and/or treatment of prostate cancer.