Expression of Lysophosphatidic Acid Receptor 1 and Relation with Cell Proliferation,Apoptosis, and Angiogenesis on Preneoplastic Changes Induced by Cadmium Chloride in the Rat Ventral Prostate

Background

Lysophosphatidic acid (LPA) is a phospholipid growth factor involved in cell proliferation, differentiation, migration, inflammation, angiogenesis, wound healing, cancer invasion, and survival. This study was directed to evaluate the immunoexpression of LPA-1, cell proliferation, apoptosis, and angiogenesis markers in preneoplastic lesions induced with cadmium chloride in rat prostate.

Methods

The following parameters were calculated in ventral prostate of normal rats and rats that received Cd in drinking water during 24 months: percentages of cells immunoreactive to LPA-1 (LI_LPA1), PCNA (LI_PCNA), MCM7 (LI_MCM7), ubiquitin (LI_UBI), apoptotic cells (LI_APO), and p53 (LI_p53); volume fraction of Bcl-2 (V_FBcl-2); and length of microvessels per unit of volume (L_VMV/mm3). Data were analyzed using Student''s t-test and Pearson correlation test.

Results

The LI_LPA1 in dysplastic lesions and normal epithelium of Cd-treated rats was significantly higher than those in the control group. Markers of proliferation were significantly increased in dysplastic lesions, whereas some apoptotic markers were significantly decreased. No significant differences between groups were found in V_FBcl-2. Dysplastic lesions showed a significant increase of LI_p53. The length of microvessels per unit of volume was elevated in dysplastic acini. Statistically significant correlations were found only between LI_LPA1 and LI_UBI.

Conclusions

Our results suggest that LPA-1 might be implicated in dysplastic lesions induced by cadmium chloride development. More studies are needed to confirm its potential contribution to the disease.     

Inhibition of cell proliferation and induction of apoptosis by genistein in experimental hepatocellular carcinoma

Hepatocellular carcinoma (HCC) is the leading cause of cancer related deaths in the world, with increasing incidence in many developed countries. Epidemiological data suggest that consumption of soy products may be associated with a decreased risk of cancer. We investigate the effects of genistein on cell proliferation, apoptosis and caspase-3 in DEN induced (200 mg/kg body weight; by single intraperitoneal injection) and Phenobarbital promoted (0.05% through drinking water for 14 successive weeks) cancer-bearing rats. Immunohistochemistry was employed to detect cell proliferating markers proliferating cell nuclear antigen (PCNA), DNA fragmentation was determined by agarose gel electrophoresis and terminal deoxynucleatide transferase dUTP nick labeling (TUNEL) staining and caspase by enzyme-linked immunosorbent assay. We found inhibition of cell proliferation, induction of apoptosis and activation of caspase-3 in genistein treated animals. From these results, we conclude that genistein inhibit cell proliferation, induced apoptosis. This activation of caspsase-3 in genistein treated liver cancer bearing animals correlated well with its apoptosis inducing effect.     

Ghrelin modulates testicular germ cells apoptosis and proliferation in adult normal rats

Under normal condition in the most mammals, spermatogenesis is closely associated with the balance between germ cells proliferation and apoptosis. The present study was designed to determine the effects of ghrelin treatment on in vivo quality and quantity expression of apoptosis and proliferation specific indices in rat testicular germ cells. Twenty eight adult normal rats were subdivided into equal control and treatment groups. Treatment group received 3 nmol of ghrelin as subcutaneous injection for 30 consecutive days or vehicle to the control animals. The rats from each group (n=7) were killed on days 10 and 30 and their testes were taken for immunocytochemical evaluation and caspase-3 assay. Immunohistochemical analysis indicated that the accumulations of Bax and PCNA peptides are generally more prominent in spermatocytes and spermatogonia of both groups. Likewise, the mean percentage of immunoreactive spermatocytes against Bax increased (P<0.01) in the ghrelin-treated group on day 10, while despite of 30% increment in the Bax level of spermatocytes in the treated rats on day 30, however, it was not statistically significant. During the experimental period, only a few spermatogonia represented Bax expression and the changes of Bax immunolabling cells were negligible upon ghrelin treatment. Likewise, there were immunostaining cells against Bcl-2 in each germ cell neither in the control nor in the treated animals. In fact, ghrelin balanced Bax/Bcl-2 ratio toward at increase of Bax level in the spermatocytes and therefore may stimulate apoptosis in these germ cells. In contrast, ghrelin administration significantly suppressed proliferation-associated peptide PCNA in the spermatocytes as well as spermatogonia (P<0.05). Whereas, caspase-3 activity did not show any marked alteration during the experiment in both groups (P>0.05). Upstream of Bax substance parallel to down-regulation of PCNA demonstrate that ghrelin may prevent massive accumulation of germ cells during normal spermatogenesis. These observations also indicate that ghrelin may be considered as a modulator of spermatogenesis in normal adult rats and could be potentially implicated for abnormal spermatogenesis in some testicular germ cell tumors.     

Effects of differential overexpression of Bcl-2 on apoptosis, proliferation, and telomerase activity in Jurkat T cells.

The effects of Bcl-2 overexpression on several of its multifunctional characteristics, which include anti-apoptotic properties, impeding of cell proliferation, and telomerase activity, were examined in four Jurkat T cell clones overexpressing different levels of Bcl-2. When treated with anti-Fas or staurosporine, only three of the four clones showed resistance to apoptosis that correlated with the level of Bcl-2 expression. Surprisingly, the clone having no anti-apoptotic characteristic expressed the highest level of Bcl-2. When all the clones were treated with anti-Fas the processing of caspase-2, -3, and -7 but not -8 was inhibited in the resistant clones to a similar extent by the differential overexpression of Bcl-2. However, with staurosporine treatment the processing of all the caspases examined was inhibited to a similar degree by the different levels of Bcl-2 expression in the resistant clones. These results suggest that Bcl-2 blocked Fas-mediated cell death by acting downstream of caspase-8, which is in contrast to staurosporine-induced apoptosis where Bcl-2 is acting upstream of caspase-8. When the anti-proliferative effect of Bcl-2 was examined, a direct correlation between a decrease in cell proliferation and the level of Bcl-2 overexpressed in the clones was observed. The clone overexpressing the greatest amount of Bcl-2 protein, which had no resistance to apoptosis, had the slowest proliferative rate. This suggests that the anti-apoptotic effect of Bcl-2 can be separated from its anti-proliferative effect. The possible effect of overexpression of Bcl-2 on telomerase activity, which is known to control the proliferative capacity of normal cells and cellular senescence, was also determined. Our results suggest that Bcl-2 had no effect on telomerase activity or telomere length in the clones. In summary, our results further suggest that some properties of Bcl-2, such as anti-apoptotic and inhibition of cell proliferation, are individual features of a multifaceted protein.     

Cadmium Induces PC12 Cells Apoptosis via an Extracellular Signal-Regulated Kinase and c-Jun N-Terminal Kinase-Mediated Mitochondrial Apoptotic Pathway

To investigate the role of mitogen-activated protein kinase (MAPK) and downstream events in cadmium (Cd)-induced neuronal apoptosis executed via the mitochondrial apoptotic pathway, this study used the PC-12 cell line as a neuronal model. The result showed that Cd significantly decreased cell viability and the Bcl-2?/?Bax ratio and increased the percentage of apoptotic cells, release of cytochrome c, caspase-3, and poly(ADP-ribose) polymerase cleavage, and nuclear translocation of apoptosis-inducing factor (AIF) and endonuclease G. In addition, exposure to Cd-induced phosphorylation of extracellular signal-regulated kinase (ERK), c-Jun N-terminal kinase (JNK), and p38 MAPK. Inhibition of ERK and JNK, but not p38 MAPK, partially protected the cells from Cd-induced apoptosis. ERK and JNK inhibition also blocked alteration of the Bcl-2?/?Bax ratio and cytochrome c release and suppressed caspase-3 and poly(ADP-ribose) polymerase cleavage and AIF and endonuclease G nuclear translocation. Taken together, these data suggest that the ERK- and JNK-mediated mitochondrial apoptotic pathway played an important role in Cd-induced PC12 cells apoptosis.     

Chk1 suppresses a caspase-2 apoptotic response to DNA damage that bypasses p53, Bcl-2, and caspase-3

Evasion of DNA damage-induced cell death, via mutation of the p53 tumor suppressor or overexpression of prosurvival Bcl-2 family proteins, is a key step toward malignant transformation and therapeutic resistance. We report that depletion or acute inhibition of checkpoint kinase 1 (Chk1) is sufficient to restore gamma-radiation-induced apoptosis in p53 mutant zebrafish embryos. Surprisingly, caspase-3 is not activated prior to DNA fragmentation, in contrast to classical intrinsic or extrinsic apoptosis. Rather, an alternative apoptotic program is engaged that cell autonomously requires atm (ataxia telangiectasia mutated), atr (ATM and Rad3-related) and caspase-2, and is not affected by p53 loss or overexpression of bcl-2/xl. Similarly, Chk1 inhibitor-treated human tumor cells hyperactivate ATM, ATR, and caspase-2 after gamma-radiation and trigger a caspase-2-dependent apoptotic program that bypasses p53 deficiency and excess Bcl-2. The evolutionarily conserved "Chk1-suppressed" pathway defines a novel apoptotic process, whose responsiveness to Chk1 inhibitors and insensitivity to p53 and BCL2 alterations have important implications for cancer therapy.     

Role of caspases,Bid, and p53 in the apoptotic response triggered by histone deacetylase inhibitors trichostatin-A (TSA) and suberoylanilide hydroxamic acid (SAHA)

Histone deacetylase activity is potently inhibited by hydroaximc acid derivatives such as suberoylanilide hydroxamic acid (SAHA) and trichostatin-A (TSA). These inhibitors specifically induce differentiation/apoptosis of transformed cells in vitro and suppress tumor growth in vivo. Because of its low toxicity, SAHA is currently evaluated in clinical trials for the treatment of cancer. SAHA and TSA induce apoptosis, which is characterized by mitochondrial stress, but so far, the critical elements of this apoptotic program remain poorly defined. To characterize in more detail this apoptotic program, we used human cell lines containing alterations in important elements of apoptotic response such as: p53, Bcl-2, caspase-9, and caspase-3. We demonstrate that caspase-9 is critical for apoptosis induced by SAHA and TSA and that efficient proteolytic activation of caspase-2, caspase-8, and caspase-7 strictly depends on caspase-9. Bcl-2 efficiently antagonizes cytochrome c release and apoptosis in response to both histone deacetylase inhibitors. We provide evidences that translocation into the mitochondria of the Bcl-2 family member Bid depends on caspase-9 and that this translocation is a late event during TSA-induced apoptosis. We also demonstrate that the susceptibility to TSA- and SAHA-induced cell death is regulated by p53.     

Interaction of cadmium and zinc during prenatal development in the rat

Cadmium chloride, zinc chloride, or a mixture of the two, labeled with 115m-Cd or 65-Zn was administered intraperitoneally to Wistar rats on day 9 of gestation. On day 20 fetuses of Cd-treated rats exhibited malformations, but those of rats given zinc or zinc plus cadmium did not. No radioactive cadmium was recovered in the fetuses or fetal membranes, although some was found in the placentas. Simultaneous administration of zinc did not alter the distribution of cadmium, but cadmium significantly increased the amount of zinc in the fetus and placenta. In a second experiment, cadmium or cadmium plus zinc was administered on day 9 of gestation and embryonic units were removed on days 10, 11, and 12. On day 10 cadmium was found in the embryonic unit and maternal uterus, and cadmium in both was significantly reduced by simultaneous administration of zinc. The cadmium concentration in uterus and embryonic units decreased sharply on day 11 and 12 and by day 12 did not differ in animals treated with cadmium or with cadmium plus zinc. It is concluded that cadmium reaches the placenta or embryo at an organogenetically sensitive time, and that zinc may protect the embryo by decreasing the exposure to cadmium this time.     

Verrucarin A, a protein synthesis inhibitor, induces growth inhibition and apoptosis in breast cancer cell lines MDA-MB-231 and T47D

Verrucarin A (VA), a protein synthesis inhibitor, derived from the pathogen fungus Myrothecium verrucaria, inhibits growth of leukemia cell lines and activates caspases and apoptosis and inflammatory signaling in macrophages. We have investigated VA-induced growth inhibition in breast cancer cells MDA-MB-231 and T47D and, particularly, the mechanism of VA-induced apoptosis. VA treatment brought about apoptotic cell death in a dose- and time-dependent manner which was associated with chromatin condensation, cell shrinkage, nuclear fragmentation and intracellular ROS production. Mitochondrial membrane depolarization, activation of caspase-3, down-regulation of Bcl-2 expression and up-regulation of Bax and p53 expression were observed. VA thus affects the viability of both the breast cancer cells by triggering ROS-mediated intrinsic mechanism of apoptosis.     

Quercetin-induced apoptosis of HL-60 cells by reducing PI3K/Akt

To explore the effect and mechanism of quercetin on proliferation and apoptosis of leukemia cells, and provide a theoretical basis for its clinical application. HL-60 leukemia cell lines was treated with different dose quercetin, the proliferation activity of leukemia cells was assessed by MTT method; the morphological changes of apoptosis of HL-60 cells, including nuclear condensation and DNA fragmentation, were observed by Hoechst 33258 fluorescence staining, the apoptosis rate and caspase 2,3 activation were assessed by flow cytometry, and the cell signal pathway including phosphatidylinositol 3-kinase (PI3K), phosphorylated protein kinase B (pAkt), Bcl-2, Bax were detected by western blotting. Quercetin could significantly decrease the proliferation activity of HL-60 cells through the blockade of G(0)/G(1) phase, and induce the apoptosis of HL-60 cells in a time- and dose-dependent manner. Quercetin caused leukemia cells apoptosis by decreasing the protein expression of PI3K and Bax, the inhibitory phosphorylation of Akt, the decreased levels of Bcl-2 protein and increased activations of caspase-2 and -3, and increased poly(ADP-ribose) polymerase cleavage. Our results indicate that the apoptotic processes caused by quercetin are mediated by the decrease of pAkt and Bcl-2 levels, the increase of Bax level, and the activation of caspase families in HL-60 cells.     

Necrosis predominates in the cell death of human colon adenocarcinoma HT-29 cells treated under variable conditions of photodynamic therapy with hypericin.

Photodynamic therapy (PDT) represents a new rapidly-developing anticancer approach based on administration of a non- or weakly-toxic photosensitizer and its activation with light of appropriate wavelength. Hypericin, one of the promising photosensitizers, is known to induce apoptosis with high efficiency in various cell line models. However, here we report the prevalence of necrosis accompanied by suppression of caspase-3 activation in colon adenocarcinoma HT-29 cells exposed to an extensive range of PDT doses evoked by variations in two variables -- hypericin concentration and light dose. Necrosis was the principal mode of cell death despite different PDT doses and the absence of anti-apoptotic Bcl-2 expression, even if the same condition induced caspase-3 activity at similar toxicity in HeLa cells. Introduction of Bcl-2 into HT-29 cells invoked caspase-3 activation, changed the Bcl-X(L) expression pattern, increased the apoptosis ratio with no effect on overall toxicity, and supported arrest in the G(2)/M-phase of cell cycle. Since it is known that Bcl-2 suppression in HT-29 is reversible and linked to the over-expression of mutated p53 and also considering our data, we suggest that the mutation in p53 and events linked to this feature may play a role in cell death signalling in HT-29 colon cancer cells.     

Kinetics of Apoptosis and Expression of Apoptosis-Related Proteins in Rat CA3 Hippocampus Cells After Experimental Diffuse Brain Injury

The present study examined kinetics of apoptosis and expression of apoptosis-related proteins Bcl-2, Bax, and caspase-3 in the CA3 hippocampus cells after diffuse brain injury (DBI) induced experimentally in rats. Percentage of apoptotic cells and expressions of above proteins were examined by flow cytometry and immunohistochemistry. Substantial neuronal apoptosis was documented in the CA3 hippocampus cells after DBI (22.26 ± 2.97 % at 72 h after DBI vs. 2.92 ± 0.88 % in sham-operated animals). Expression of Bc1-2 decreased, while expression of Bax and caspase-3 increased after DBI, with caspase-3 expression peaking after that of Bax (72 vs. 48 h, respectively). Further, the Bc1-2/Bax expression ratio decreased prior to increase of caspase-3 expression. In conclusion, cell apoptosis and altered expressions of Bcl-2, Bax, and caspase-3 are present in the CA3 region of hippocampus after experimental DBI. Changes in the Bc1-2/Bax expression ratio may facilitate activation of caspase-3 and aggravate neuronal apoptosis after brain injury.     

Ivermectin inhibits the growth of glioma cells by inducing cell cycle arrest and apoptosis in vitro and in vivo

Glioma, the most predominant primary malignant brain tumor, remains uncured due to the absence of effective treatments. Hence, it is imperative to develop successful therapeutic agents. This study aimed to explore the antitumor effects and mechanisms of ivermectin (IVM) in glioma cells in vitro and in vivo. The effects of IVM on cell viability, cell cycle arrest, apoptosis rate, and morphological characteristics were determined respectively by MTT assay/colony formation assay, flow cytometry, and transmission electron microscope. In addition, the expression levels of cycle-related and apoptosis-associated proteins were individually examined by Western blot analysis. Moreover, cell proliferation and apoptosis analyses were carried out by TUNEL, Ki-67, cleaved caspase-3, and cleaved caspase-9 immunostaining assay. Our results demonstrated that IVM has a potential dosage-dependent inhibition effect on the apoptosis rate of glioma cells. Meanwhile, the results also revealed that IVM induced apoptosis by increasing caspase-3 and caspase-9 activity, upregulating the expressions of p53 and Bax, downregulating Bcl-2, activating cleaved caspase-3 and cleaved caspase-9, and blocking cell cycle in G0/G1 phase by downregulating levels of CDK2, CDK4, CDK6, cyclin D1, and cyclin E. These findings suggest that IVM has an inhibition effect on the proliferation of glioma cells by triggering cell cycle arrest and inducing cell apoptosis in vitro and in vivo, and probably represents promising agent for treating glioma.     

镉对小鼠精子和生精细胞超微结构及生精细胞bcl-2、bax基因表达的影响

研究镉暴露对小鼠附睾精子和睾丸生精细胞超微结构的变化以及镉对生精细胞凋亡相关基因bcl-2、bax表达水平的影响。采用24只雄性ICR小鼠随机分为4组,每组6只,分别以0.183、0.915、1.83mg/kg氯化镉腹腔注射,每天1次,连续5次,设阴性对照生理盐水组。于第6天透射电镜观察附睾精子超微结构、睾丸生精细胞核和线粒体超微结构的变化,免疫组化方法检测生精细胞Bcl-2、Bax表达水平。透射电镜观察显示,0.183mg/kg组精子超微结构无显著性变化,0.915mg/kg组精子头部两侧膜与头部胞质间隙轻微扩大,线粒体嵴间腔扩大且轻度空泡化,但与对照组相比无统计学意义(P>0.05)。1.83mg/kg组头部两侧膜与胞质间隙扩大,与对照组相比有显著性差异(P<0.05),尾部线粒体嵴间腔扩大且轻度空泡化,与对照组相比有显著性差异(P<0.05)。3种剂量处理组睾丸生精细胞核超微结构异常发生率显著高于对照组(P<0.05),且随着处理浓度的升高异常发生率升高;1.83mg/kg组线粒体肿胀空泡化发生率显著高于对照组(P<0.05)。3种剂量实验组生精细胞Bcl-2表达水平(吸光度)显著低于对照组(P<0.01),0.915mg/kg组Bax表达水平显著高于对照组和0.183、1.83mg/kg组(P<0.01)。3种剂量实验组Bcl-2/Bax吸光度比值显著低于对照组(P<0.01);0.915mg/kg组Bcl-2/Bax比值显著低于1.83mg/kg组(P<0.01)。上述结果提示:高浓度镉诱导附睾精子超微结构改变,高中低浓度镉致睾丸生精细胞超微结构的改变,生精细胞超微结构发生凋亡现象。镉对Bcl-2、Bax表达水平的改变可能是生精细胞凋亡的分子机制之一。     

Cadmium induces mitochondria-dependent apoptosis of normal human hepatocytes

The heavy metal cadmium, an environmental pollutant, has been widely demonstrated to be toxic, in particular for liver. In murines, cadmium induces apoptosis of hepatocytes and hepatomas. In human cells, apoptosis induced by cadmium has been exclusively demonstrated in tumoral cell lines. Nothing was known in normal liver, in vitro or in vivo. In the present study, we examined the effects of cadmium in nonmalignant human hepatocytes. For that purpose, we investigated whether cadmium was able to induce apoptosis of normal human hepatocytes (NHH) in primary culture and of a SV40-immortalized human hepatocyte (IHH) cell line. Treatment of IHH and NHH with cadmium induced the presence of a sub-G₁ population at 10 and 100 μmol/L, respectively. DAPI staining of both cell types treated with cadmium 100 μmol/L revealed the induction of nuclear apoptotic bodies, supporting the hypothesis of apoptosis. In IHH and NHH, cadmium 100 μmol/L induced PARP cleavage into a 85 kDa fragment. In order to investigate the involvement of mitochondria in cadmium-induced apoptosis, we measured the mitochondrial membrane potential (Δ_Ψm). We observed that in IHH and NHH, cadmium 100 μmol/L induced a decrease of Δ_Ψm. As expected, cadmium under the same conditions enhanced caspase-9 and caspase-3 activities. In addition, cadmium from 1 to 100 μmol/L induced the expression of p53 and phosphorylation of its Ser15 in IHH and NHH. In conclusion, we showed in this study that human hepatocytes were sensitive to cadmium and apoptosis induced at concentrations suggested in the literature to inhibit p53 DNA-binding and DNA repair.     

Analysis of p53 and NF-κB signaling in modulating the cardiomyocyte fate during hypertrophy

Cardiac hypertrophy leading to eventual heart failure is the most common cause of mortality throughout the world. The triggering mechanisms for cardiac hypertrophy are not clear but both apoptosis and cell proliferation have been reported in sections of failing hearts. In this study, we utilized both angiotensin II (AngII) treatment of cardiomyocytes and aortic ligation in rats (Rattus norvegicus, Wistar strain) for induction of hypertrophy to understand the cellular factors responsible for activation of apoptotic or anti-apoptotic pathway. Hypertrophy markers (ANF, β-MHC), apoptotic proteins (Bax, Bad, Fas, p53, caspase-3, PARP), and anti-apoptotic or cell proliferation marker proteins (Bcl2, NF-κB, Ki-67) were induced significantly during hypertrophy, both in vitro as well as in vivo. Co-localization of both active caspase-3 and Ki-67 was observed in hypertrophied myocytes. p53 and NF-κBp65 binding to co-activator p300 was also increased in AngII treated myocytes. Inhibition of p53 resulted in downregulation of apoptosis, NF-κB activation, and NF-κB-p300 binding; however, NF-κB inhibition did not inhibit apoptosis or p53-p300 binding. Blocking of either p53 or NF-κB by specific inhibitors resulted in decrease in cell proliferation and hypertrophy markers, suggesting that p53 initially binds to p300 and then this complex recruits NF-κB. Thus, these results indicate the crucial role of p53 in regulating both apoptotic and cell proliferation during hypertrophy.     

Immunohistochemical expression of Bcl-2, p53 and caspase-9 in hypothalamus magnocellular centers of nNOS knockout mice following water deprivation

Abstract

We studied the interactions between apoptosis regulator proteins (Bcl-2, p53 and caspase-9) and neuronal nitric oxide in vasopressinergic magnocellular centers of the hypothalamus using neuronal nitric oxide synthase (nNOS) gene knockout mice. nNOS gene deletion resulted in accumulation of Bcl-2, p53 and caspase-9 in the paraventricular (PVN) and supraoptic (SON) nuclei in controls. Dehydration increased the levels of all three apoptosis regulator proteins studied in nuclei of wild type mice. In the hypothalamus magnocellular centers of nNOS knockout mice, however, expression of Bcl-2, p53 and caspase-9 was unchanged after dehydration. The number of magnocellular neurons did not change in the SON and PVN of nNOS deficient mice compared to wild type, and after dehydration, cell death was not observed in either nucleus of wild type or knockout mice despite activation of apoptosis regulator protein expression. Thus, we demonstrated that gene disruption of nNOS prevents activation of Bcl-2, p53 and caspase-9 expression during water deprivation, and that nNOS deficiency did not affect survival of magnocellular neurons of the hypothalamus.     

Bcl-2 rescues ceramide- and etoposide-induced mitochondrial apoptosis through blockage of caspase-2 activation

Recent studies indicate that caspase-2 is involved in the early stage of apoptosis before mitochondrial damage. Although the activation of caspase-2 has been shown to occur in a large protein complex, the mechanisms of caspase-2 activation remain unclear. Here we report a regulatory role of Bcl-2 on caspase-2 upstream of mitochondria. Stress stimuli, including ceramide and etoposide, caused caspase-2 activation, mitochondrial damage followed by downstream caspase-9 and -3 activation, and cell apoptosis in human lung epithelial cell line A549. When A549 cells were pretreated with the caspase-2 inhibitor benzyloxycarbonyl-Val-Asp(-OMe)-Val-Ala-Asp(-OMe)-fluoromethyl ketone or transfected with caspase-2 short interfering RNA, both ceramide- and etoposide-induced mitochondrial damage and apoptosis were blocked. Overexpression of Bcl-2 prevented ceramide- and etoposide-induced caspase-2 activation and mitochondrial apoptosis. Furthermore, caspase-2 was activated when A549 cells were introduced with Bcl-2 short interfering RNA or were treated with Bcl-2 inhibitor, which provided direct evidence of a negative regulatory effect of Bcl-2 on caspase-2. Cell survival was observed when caspase-2 was inhibited in Bcl-2-silencing cells. Blockage of the mitochondrial permeability transition pore and caspase-9 demonstrated that Bcl-2-modulated caspase-2 activity occurred upstream of mitochondria. Further studies showed that Bcl-2 was dephosphorylated at serine 70 after ceramide and etoposide treatment. A protein phosphatase inhibitor, okadaic acid, rescued Bcl-2 dephosphorylation and blocked caspase-2 activation, mitochondrial damage, and cell death. Taken together, ceramide and etoposide induced mitochondria-mediated apoptosis by initiating caspase-2 activation, which was, at least in part, regulated by Bcl-2.