α-Santalol, a derivative of sandalwood oil, induces apoptosis in human prostate cancer cells by causing caspase-3 activation

The anticancer effects of α-santalol, a major component of sandalwood oil, have been reported against the development of certain cancers such as skin cancer both in vitro and in vivo. The primary objectives of the current study were to investigate the cancer preventive properties of α-santalol on human prostate cancer cells PC-3 (androgen independent and P-53 null) and LNCaP (androgen dependent and P-53 wild-type), and determine the possible mechanisms of its action. The effect of α-santalol on cell viability was determined by trypan blue dye exclusion assay. Apoptosis induction was confirmed by analysis of cytoplasmic histone-associated DNA fragmentation using both an apoptotic ELISA kit and a DAPI fluorescence assay. Caspase-3 activity was determined using caspase-3 (active) ELISA kit. PARP cleavage was analyzed using immunoblotting. α-Santalol at 25-75 μM decreased cell viability in both cell lines in a concentration and time dependent manner. Treatment of prostate cancer cells with α-santalol resulted in induction of apoptosis as evidenced by DNA fragmentation and nuclear staining of apoptotic cells by DAPI. α-Santalol treatment also resulted in activation of caspase-3 activity and PARP cleavage. The α-santalol-induced apoptotic cell death and activation of caspase-3 was significantly attenuated in the presence of pharmacological inhibitors of caspase-8 and caspase-9. In conclusion, the present study reveals the apoptotic effects of α-santalol in inhibiting the growth of human prostate cancer cells.     

Endoplasmic reticulum stress,autophagic and apoptotic cell death,and immune activation by a natural triterpenoid in human prostate cancer cells

Though the current therapies are effective at clearing an early stage prostate cancer, they often fail to treat late-stage metastatic disease. We aimed to investigate the molecular mechanisms underlying the anticancer effects of a natural triterpenoid, ganoderic acid DM (GA-DM), on two human prostate cancer cell lines: the androgen-independent prostate carcinoma (PC-3), and androgen-sensitive prostate adenocarcinoma (LNCaP). Cell viability assay showed that GA-DM was relatively more toxic to LNCaP cells than to PC-3 cells (IC₅₀s ranged 45-55 µM for PC-3, and 20-25 µM for LNCaP), which may have occurred due to differential expression of p53. Hoechst DNA staining confirmed detectable nuclear fragmentation in both cell lines irrespective of the p53 status. GA-DM treatment decreased Bcl-2 proteins while it upregulated apoptotic Bax and autophagic Beclin-1, Atg5, and LC-3 molecules, and caused an induction of both early and late events of apoptotic cell death. Biochemical analyses of GA-DM-treated prostate cancer cells demonstrated that caspase-3 cleavage was notable in GA-DM-treated PC-3 cells. Interestingly, GA-DM treatment altered cell cycle progression in the S phase with a significant growth arrest in the G2 checkpoint and enhanced CD4 ⁺ T cell recognition of prostate tumor cells. Mechanistic study of GA-DM-treated prostate cancer cells further demonstrated that calpain activation and endoplasmic reticulum stress contributed to cell death. These findings suggest that GA-DM is a candidate for future drug design for prostate cancer as it activates multiple pathways of cell death and immune recognition.     

Inflammatory Cytokines and Survival Factors from Serum Modulate Tweak-Induced Apoptosis in PC-3 Prostate Cancer Cells

Tumor necrosis factor-like weak inducer of apoptosis (TWEAK, TNFSF12) is a member of the tumor necrosis factor superfamily. TWEAK activates the Fn14 receptor, and may regulate cell death, survival and proliferation in tumor cells. However, there is little information on the function and regulation of this system in prostate cancer. Fn14 expression and TWEAK actions were studied in two human prostate cancer cell lines, the androgen-independent PC-3 cell line and androgen-sensitive LNCaP cells. Additionally, the expression of Fn14 was analyzed in human biopsies of prostate cancer. Fn14 expression is increased in histological sections of human prostate adenocarcinoma. Both prostate cancer cell lines express constitutively Fn14, but, the androgen-independent cell line PC-3 showed higher levels of Fn14 that the LNCaP cells. Fn14 expression was up-regulated in PC-3 human prostate cancer cells in presence of inflammatory cytokines (TNFα/IFNγ) as well as in presence of bovine fetal serum. TWEAK induced apoptotic cell death in PC-3 cells, but not in LNCaP cells. Moreover, in PC-3 cells, co-stimulation with TNFα/IFNγ/TWEAK induced a higher rate of apoptosis. However, TWEAK or TWEAK/TNFα/IFNγ did not induce apoptosis in presence of bovine fetal serum. TWEAK induced cell death through activation of the Fn14 receptor. Apoptosis was associated with activation of caspase-3, release of mitochondrial cytochrome C and an increased Bax/BclxL ratio. TWEAK/Fn14 pathway activation promotes apoptosis in androgen-independent PC-3 cells under certain culture conditions. Further characterization of the therapeutic target potential of TWEAK/Fn14 for human prostate cancer is warranted.     

Cell death induced by MPPa-PDT in prostate carcinoma in vitro and in vivo

Lack of effective photosensitizers has become a major limit for extensive application of photodynamic therapy. In this study, the photocytotoxicity and mode of death induced by a newly developed photosensitizer MPPa, a derivative of chlorophyll a, were investigated in PC-3M cell line, a highly metastatic variant of poorly differentiated androgen-independent proctanec adenocarcinoma PC-3. MTT reduction assay was used to measure cytotoxicity in both PC-3M and HUVEC, after which a flow cytometer was used to measure apoptotic rate and cell cycle, and then Caspase-3, -8, -9 were investigated. Finally, an animal model was set up to embody the curative effect and for histopathological examinations. The photocytotoxicity of MPPa showed both light- and drug-dose dependent characteristics and no significant dark cytotoxicity was observed in PC-3M cells. In HUVEC, MPPa exhibited an obviously low cytotoxicity. By other in vitro studies, we found MPPa-PDT induced apoptotic mainly via the mitochondrial/Caspase-9/Caspase-3 pathway and could restrain the cell cycle progression from the more sensitive G0/G1-phases. In vivo, the tumour growth was significantly inhibited after PDT, and many apoptotic cells could be seen by histopathological examinations. These results indicate the death way of cells induced by MPPa is mainly via mild apoptotic and the cure effect is obvious, suggesting that MPPa is a potential photosensitizer of photodynamic therapy for prostate cancer.     

Pipernonaline from Piper longum Linn. induces ROS-mediated apoptosis in human prostate cancer PC-3 cells

The antiproliferation effects of pipernonaline, a piperine derivative, were investigated on human prostate cancer PC-3 cells. It inhibited growth of androgen independent PC-3 and androgen dependent LNCaP prostate cells in a dose-dependent (30–90 μM) and time-dependent (24–48 h) manner. The growth inhibition of PC-3 cells was associated with sub-G₁ and G₀/G₁ accumulation, confirmed by the down-regulation of CDK2, CDK4, cyclin D1 and cyclin E, which are correlated with G₁ phase of cell cycle. Pipernonaline up-regulated cleavage of procaspase-3/PARP, but did not change expression of proapoptotic bax and antiapoptotic bcl-2 proteins. Its caspase-3 activation was confirmed by the caspase-3 assay kit. In addition, pipernonaline caused the production of reactive oxygen species (ROS), increase of intracellular Ca²⁺, and mitochondrial membrane depolarization, which these phenomena were reversed by N-acetylcysteine, a ROS scavenger. The results suggest that pipernonaline exhibits apoptotic properties through ROS production, which causes disruption of mitochondrial function and Ca²⁺ homeostasis and leads to its downstream events including activation of caspase-3 and cleavage of PARP in PC-3 cells. This is the first report of pipernonaline toward the anticancer activity of prostate cancer cells, which provides a role for candidate agent as well as the molecular basis for human prostate cancer.     

Effects of dihydroxy gymnemic triacetate (DGT) on expression of apoptosis associated proteins in human prostate cancer cell lines (PC3)

Abstract

Prostate cancer is the most common malignancies among men. The present study is aimed at the investigation of dihydroxy gymnemic triacetate (DGT) from Gymnema sylvestre on mitochondrial apoptotic pathway and cell cycle arrest. Treatment of DGT resulted in a dose-dependent inhibition of growth of PC-3 cells. The cell cycle arrest was observed at the G2/M phase and accumulation of apoptotic cells was observed in DGT-treated prostate cancer cell lines. The occurrence of apoptosis in these cells was observed by DNA fragmentation. These events were associated with increased levels of pro-apoptotic proteins Bax, Bad and reduced levels of the antiapoptotic proteins Bcl-2, Bcl-xL and Mcl-1. DGT also induces the activation of caspase-9 and caspase-3. The above results, clearly, suggest that DGT induces apoptosis by the intrinsic pathways which could be very useful for the treatment of prostate cancer.     

Thiosulfinates from Allium tuberosum L. induce apoptosis via caspase-dependent and -independent pathways in PC-3 human prostate cancer cells

This study was aimed to evaluate the apoptotic effects of thiosulfinates purified from Allium tuberosum L. on PC-3 human prostate cancer cells, and to elucidate detailed apoptosis mechanisms. Thiosulfinates significantly decrease viable cell numbers in dose- and time-dependent manners by apoptotic cell death via DNA fragmentation, chromatin condensation, and an increased sub-G1 phase. Apoptosis induced by thiosulfinates is associated with the activation of initiator caspase-8 and -9, and the effector caspase-3. In this study, thiosulfinates stimulated Bid cleavage, indicating that the apoptotic action of caspase-8-mediated Bid cleavage leads to the activation of caspase-9. Thiosulfinates decreased the expression of the anti-apoptotic protein Bcl-2 and increased the expression of the pro-apoptotic protein Bax. Thiosulfinates also increased the expression of AIF, a caspase-independent mitochondrial apoptosis factor, in PC-3 cells. These results indicate that thiosulfinates from A. tuberosum L. inhibit cell proliferation and induce apoptosis in PC-3 cells, which may be mediated via both caspase-dependent and -independent pathways.     

Modulation of the tumor cell death pathway by androgen receptor in response to cytotoxic stimuli

Despite an initial response from androgen deprivation therapy, most prostate cancer patients relapse to a hormone-refractory state where tumors still remain dependent on androgen receptor (AR) function. We have previously shown that AR breakdown correlates with the induction of cancer cell apoptosis by proteasome inhibition. However, the involvement of AR in modulating the cell death pathway has remained elusive. To investigate this, we used an experimental model consisting of parental PC-3 prostate cancer cells that lack AR expression and PC-3 cells stably overexpressing wild type AR gene. Here, we report that both chemotherapeutic drugs (cisplatin) and proteasome inhibitors induced caspase-3-associated cell death in parental PC-3 cells whereas non-caspase-3 associated cell death in PC3-AR cells. The involvement of AR in modulating tumor cell death was further confirmed in PC-3 cells transiently expressing AR. Consistently, treatment with the clinically used proteasome inhibitor Bortezomib (Velcade/PS-341) of (AR+) LNCaP prostate cancer cells caused AR cleavage and cell death with low levels of caspase activation. However, co-treatment with Bortezomib and the AR antagonist Bicalutamide (Casodex) caused significant decrease in AR expression associated with an increase in caspase-3 activity in both LNCaP and PC3-AR cells. Thus our results provide compelling evidence for involvement of AR in deciding types of tumor cell death upon cytotoxic stimuli, and specifically, blockade of AR activities could change necrosis to apoptosis in tumor cells. Our findings may help guide clinicians based on AR status in the design of favorable treatment strategies for prostate cancer patients.     

A novel synthetic protoapigenone analogue, WYC02-9, induces DNA damage and apoptosis in DU145 prostate cancer cells through generation of reactive oxygen species

The protoapigenone analogue WYC02-9, a novel synthetic flavonoid, has been shown to act against a variety of experimental tumors. However, its effects on prostate cancer and its mechanism of action are unknown. Thus, WYC02-9 was investigated for its cytotoxicity against DU145 prostate cancer cells, as was the underlying mechanisms by which WYC02-9 might induce DNA damage and apoptotic cell death through reactive oxygen species (ROS). WYC02-9 inhibited the cell growth of three prostate cancer cell lines, especially DU145 cells. In DU145 cells, WYC02-9 increased the generation of intracellular ROS, followed by induction of DNA damage and activation of the ATM-p53-H2A.X pathway and checkpoint-related signals Chk1/Chk2, which led to increased numbers of cells in the S and G2/M phases of the cell cycle. Furthermore, WYC02-9 induced apoptotic cell death through mitochondrial membrane potential decrease and activation of caspase-9, caspase-3, and PARP. The above effects were all prevented by the ROS scavenger N-acetylcysteine. Administration of WYC02-9 in a nude mouse DU145 xenograft model further identified the anti-cancer activity of WYC02-9. These findings therefore suggest that WYC02-9-induced DNA damage and mitochondria-dependent cell apoptosis in DU145 cells are mediated via ROS generation.     

The p75(NTR) tumor suppressor induces cell cycle arrest facilitating caspase mediated apoptosis in prostate tumor cells

The p75 neurotrophin receptor (p75(NTR)) is a death receptor which belongs to the tumor necrosis factor receptor super-family of membrane proteins. This study shows that p75(NTR) retarded cell cycle progression by induced accumulation of cells in G0/G1 and a reduction in the S phase of the cell cycle. The rescue of tumor cells from cell cycle progression by a death domain deleted (DeltaDD) dominant-negative antagonist of p75(NTR) showed that the death domain transduced anti-proliferative activity in a ligand-independent manner. Conversely, addition of NGF ligand rescued retardation of cell cycle progression with commensurate changes in components of the cyclin/cdk holoenzyme complex. In the absence of ligand, p75(NTR)-dependent cell cycle arrest facilitated an increase in apoptotic nuclear fragmentation of the prostate cancer cells. Apoptosis of p75(NTR) expressing cells occurred via the intrinsic mitochondrial pathway leading to a sequential caspase-9 and -7 cascade. Since the death domain deleted dominant-negative antagonist of p75(NTR) rescued intrinsic caspase associated apoptosis in PC-3 cells, this shows p75(NTR) was integral to ligand independent induction of apoptosis. Moreover, the ability of ligand to ameliorate the p75(NTR)-dependent intrinsic apoptotic cascade indicates that NGF functioned as a survival factor for p75(NTR) expressing prostate cancer cells.     

Dictyopteris undulata extract induces apoptosis in human colon cancer cells

The present study investigated the cytotoxic and apoptotic effects of an ethanol extract derived from the marine brown alga Dictyopteris undulata against human colon adenocarcinoma cells. The Dictyopteris undulata extract (DUE) showed cytotoxic activity against SW480 cells in a dose-dependent manner, with 50% inhibition of cell viability at a concentration of 40 μg/mL. DUE also induced programmed cell death in SW480 cells, as evidenced by apoptotic body formation, DNA fragmentation, an increase in the population of apoptotic sub-G1 phase cells, and mitochondrial membrane depolarization. Moreover, DUE significantly modulated the expression of apoptosisassociated proteins, resulting in a decrease in B cell lymphoma-2 expression and an increase in Bcl-2-associated X protein expression, as well as the activation of caspase-9 and caspase-3. Furthermore, DUE showed apoptotic cell death in two other colon cancer cell lines, SNU407 and HT29. These observations suggest that DUE may prove useful as a therapeutic agent for the attenuation of colon cancer.     

Bcl-xL mediates a survival mechanism independent of the phosphoinositide 3-kinase/Akt pathway in prostate cancer cells

Among various molecular strategies by which prostate cancer cells evade apoptosis, phosphoinositide 3-kinase (PI3K)/Akt signaling represents a dominant survival pathway. However, different prostate cancer cell lines such as LNCaP and PC-3 display differential sensitivity to the apoptotic effect of PI3K inhibition in serum-free media, reflecting the heterogeneous nature of prostate cancer in apoptosis regulation. Whereas both cell lines are equally susceptible to LY294002-mediated Akt dephosphorylation, only LNCaP cells default to apoptosis, as evidenced by DNA fragmentation and cytochrome c release. In PC-3 cells, Akt deactivation does not lead to cytochrome c release, suggesting that the intermediary signaling pathway is short-circuited by an antiapoptotic factor. This study presents evidence that Bcl-xL overexpression provides a distinct survival mechanism that protects PC-3 cells from apoptotic signals emanating from PI3K inhibition. First, the Bcl-xL/BAD ratio in PC-3 cells is at least an order of magnitude greater than that of LNCaP cells. Second, ectopic expression of Bcl-xL protects LNCaP cells against LY294002-induced apoptosis. Third, antisense down-regulation of Bcl-xL sensitizes PC-3 cells to the apoptotic effect of LY294002. The physiological relevance of this Bcl-xL-mediated survival mechanism is further underscored by the protective effect of serum on LY294002-induced cell death in LNCaP cells, which is correlated with a multifold increase in Bcl-xL expression. In contrast to Bcl-xL, Bcl-2 expression levels are similar in both cells lines, and do not respond to serum stimulation, suggesting that Bcl-2 may not play a physiological role in antagonizing apoptosis signals pertinent to BAD activation in prostate cancer cells.     

NKX3.1 potentiates TNF-α/CHX-induced apoptosis of prostate cancer cells through increasing caspase-3 expression and its activity

NKX3.1, a prostate-specific homeobox gene, plays an important role in prostate cancer and usually functions as tumor suppressor gene. Previously we have demonstrated that forced expression of NKX3.1 reduced cell growth and invasion in prostate cancer cell line PC-3. Presently, we investigated the effect of NKX3.1 on the sensitivity of the prostate cancer cells to apoptosis inducer tumor necrosis factor-α (TNF-α) and cycloheximide (CHX). PC-3 cells were transfected with NKX3.1 expression plasmid (pcDNA3.1-NKX3.1) and LNCaP cells were transfected with siRNA expression plasmid (pRNAT-RNAi1) targeting NKX3.1. The cell morphology and apoptotic rate were analyzed by Hoechst 33342 staining and Flow Cytometry in absence or presence of TNF-α and CHX. The activity of caspase-3 was determined using DEVD-pNA as substrate. Simultaneously, the effect of NKX3.1 on caspase-3 expression was detected using RT-PCR and Western blot. The results showed that ectopic expression of NKX3.1 promoted TNF-α/CHX-induced apoptosis in PC-3 cells, whereas knockdown of NKX3.1 protected LNCaP cells from apoptosis induced by TNF-α/CHX. The pro-apoptosis activity of NKX3.1 might partially contribute to its elevation of caspase-3 expression and activity. Manipulating NKX3.1 expression should be a promising therapeutic strategy for treating both androgen-dependent and androgen-independent prostate cancer.     

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.     

Apoptosis-like cell death of human breast cancer cell line MCF-7 induced by buprenorphine hydrochloride

The analgesic buprenorphine hydrochloride (Bph) induced apoptosis-like cell death in the caspase-3-deficient human breast cancer cell line, MCF-7. This apoptosis-like cell death activated key molecules in the mitochondrial apoptotic pathway: cytochrome c, caspase-9, caspase-7, and caspase-6. Bph caused the release of fluorescent protein from the mitochondria of MCF-7 cells transfected with the pDsRed2-Mito-vector in a time-dependent manner, suggesting disruption of the mitochondrial membrane. Zn(2+) as high as 2 mM did not inhibit the DNase that took part in this apoptosis. Thus, this unidentified DNase might resemble other DNases involved in apoptosis-like cell death whose activity is not inhibited by zinc ion.     

Mechanisms of abemaciclib,a CDK4/6 inhibitor,induced apoptotic cell death in prostate cancer cells in vitro

The therapeutic effects of abemaciclib (ABE), an inhibitor of cyclin- dependent kinases (CDK) 4/6, on the proliferation of two types of prostate cancer (PC) cells were revealed. In this study, in vitro cytotoxic and apoptotic effects of ABE on metastatic castration-resistant prostate cancer (mCRPC) androgen receptor (AR) negative PC-3 and AR mutant LNCaP PC cells were analyzed with WST-1, Annexin V, cell cycle, reactive oxygen species (ROS), mitochondrial membrane potential, RT-PCR, western blot, and apoptosis protein array. ABE considerably inhibited the growth of PC cells in a dose-dependent manner (p<0.01) and caused significant apoptotic cell death through the suppression of CDK4/6-Cyclin D complex, ROS generation and depolarization of mitochondria membrane potential. However, PC-3 cells were more sensitive to ABE than LNCaP cells. Furthermore, the expression levels of several pro-apoptotic and cell cycle regulatory proteins were upregulated by ABE in especially PC-3 cells with the downregulation of apoptotic inhibitor proteins. Our results suggest that ABE inhibits PC cell growth and promotes apoptosis and thus ABE treatment may be a promising treatment strategy in especially mCRPC. Further preclinical and clinical studies should be performed to clarify the clinical use of ABE for the treatment of PC.     

Induction of apoptosis in prostate tumor PC-3 cells and inhibition of xenograft prostate tumor growth by the vanilloid capsaicin

Capsaicin, the pungent ingredient of hot chilli pepper, has been recently shown to induce apoptosis in several cell lines through a not well known mechanism. Here, we investigated the role of the vanilloid capsaicin in the death regulation of the human cancer androgen-resistant cell line PC-3. Capsaicin inhibited the growth of PC-3 with an IC₅₀ of 20 μM cells and induced cell apoptosis, as assessed by flow cytometry and nuclei staining with DAPI. Capsaicin induced apoptosis in prostate cells by a mechanism involving reactive oxygen species generation, dissipation of the mitochondrial inner transmembrane potential (ΔΨ_m) and activation of caspase 3. Capsaicin-induced apoptosis was not reduced by the antagonist capsazepine in a dose range from 0.1 μM to 20 μM, suggesting a receptor-independent mechanism. To study the in vivo effects of capsaicinoids, PC-3 cells were grown as xenografts in nude mice. Subcutaneous injection of either capsaicin or capsazepine (5 mg/kg body weight) in nude mice suppressed PC-3 tumor growth in all tumors investigated and induced apoptosis of tumor cells. Our data show a role for capsaicin against androgen-independent prostate cancer cells in vitro and in vivo and suggest that capsaicin is a promising anti-tumor agent in hormone-refractory prostate cancer, which shows resistance to many chemotherapeutic agents.     

B cell receptor cross-linking triggers a caspase-8-dependent apoptotic pathway that is independent of the death effector domain of Fas-associated death domain protein.

We have previously reported that B cell receptors, depending on the degree to which they are cross-linked, can promote apoptosis in various human B cell types. In this study, we show that B cell receptors can trigger two apoptotic pathways according to cross-linking and that these pathways control mitochondrial activation in human Burkitt's lymphoma cells. Whereas soluble anti-mu Ab triggers caspase-independent mitochondrial activation, cross-linked anti-mu Ab induces an apoptotic response associated with a caspase-dependent loss of mitochondrial transmembrane potential. This B cell receptor-mediated caspase-dependent mitochondrial activation is associated with caspase-8 activation. We show here that caspase-8 inhibitors strongly decrease cross-linking-dependent B cell receptor-mediated apoptosis in Burkitt's lymphoma BL41 cells. These inhibitors act upstream from the mitochondria as they prevented the loss of mitochondrial membrane potential observed in B cell receptor-treated BL41 cells. Caspase-8 activation in these cells was also evident from the detection of cleaved fragments of caspase-8 and the cleavage of specific substrates, including Bid. Our data show that cross-linked B cell receptors induced an apoptotic pathway involving sequential caspase-8 activation, loss of mitochondrial membrane potential, and the activation of caspase-9 and caspase-3. Cells expressing a dominant negative mutant of Fas-associated death domain protein were sensitive to cross-linked B cell receptor-induced caspase-8 activation and apoptosis; therefore, this caspase-8 activation was independent of the death effector domain of Fas-associated death domain protein.