Caspase-8/FLICE functions as an executioner caspase in anticancer drug-induced apoptosis

Caspase-8 plays an essential role in apoptosis triggered by death receptors. Through the cleavage of Bid, a proapoptotic Bcl-2 member, it further activates the mitochondrial cytochrome c/Apaf-1 pathway. Because caspase-8 can be processed also by anticancer drugs independently of death receptors, we investigated its exact role and order in the caspase cascade. We show that in Jurkat cells either deficient for caspase-8 or overexpressing its inhibitor c-FLIP apoptosis mediated by CD95, but not by anticancer drugs was inhibited. In the absence of active caspase-8, anticancer drugs still induced the processing of caspase-9, -3 and Bid, indicating that Bid cleavage does not require caspase-8. Overexpression of Bcl-x(L) prevented the processing of caspase-8 as well as caspase-9, -6 and Bid in response to drugs, but was less effective in CD95-induced apoptosis. Similar responses were observed by overexpression of a dominant-negative caspase-9 mutant. To further determine the order of caspase-8 activation, we employed MCF7 cells lacking caspase-3. In contrast to caspase-9 that was cleaved in these cells, anticancer drugs induced caspase-8 activation only in caspase-3 transfected MCF7 cells. Thus, our data indicate that, unlike its proximal role in receptor signaling, in the mitochondrial pathway caspase-8 rather functions as an amplifying executioner caspase.     

Celecoxib derivatives induce apoptosis via the disruption of mitochondrial membrane potential and activation of caspase 9

Celecoxib is a potent nonsteroid antiinflammatory drug (NSAID) that has shown great promise in cancer chemoprevention and treatment. The tumor suppression activity of celecoxib and other NSAIDs have been related to the induction of apoptosis in many cancer cell lines and animal models. While celecoxib is a specific inhibitor of cyclooxygenase (COX)-2, recent data indicate that its apoptotic properties may also be mediated through COX-independent pathways. In our study, we evaluated second generation celecoxib derivatives, lacking COX-2 inhibitory activity, in a premalignant and malignant human oral cell culture model to determine their potential anticancer effect and mechanisms responsible for the COX-independent apoptotic activity. Celecoxib and its derivatives delayed the progression of cells through the G(2)/M phase and induced apoptosis. The derivatives with apolar substituents at the terminal phenyl moiety of celecoxib greatly enhanced apoptosis and cell cycle delay. Apoptosis and cell cycle arrest appeared to be independent of derivative induced inhibition of PDK1 and phosphorylation of Akt and Erk1/2. Derivatives induced apoptosis was mediated by the cleavage and activation of caspase-9 and caspase-3, but not caspase 8, implicating the mitochondrial pathway for apoptosis induction. Inhibitors of caspase-3 and caspase-9 and cyclosporin A, a mitochondrial membrane potential stabilizer, attenuated derivative induced apoptosis. Inhibition of caspase-3 prevented the activation of caspase 8, while the inhibition of caspase-9 inhibitor blocked activation of both caspase 3 and 8 by the derivatives. Apoptosis was independent of Bcl-2. These results indicate that the second generation celecoxib derivatives induce apoptosis in human oral cancer lines by the disruption of mitochondrial membrane potential activating caspase 9 and downstream caspase 3 and 8. This suggests that the modification of the celecoxib structure can lead to highly effective COX-independent growth inhibitory and apoptotic agents in chemoprevention and therapy.     

Methadone, commonly used as maintenance medication for outpatient treatment of opioid dependence, kills leukemia cells and overcomes chemoresistance

The therapeutic opioid drug methadone (d,l-methadone hydrochloride) is the most commonly used maintenance medication for outpatient treatment of opioid dependence. In our study, we found that methadone is also a potent inducer of cell death in leukemia cells and we clarified the unknown mechanism of methadone-induced cell killing in leukemia cells. Methadone inhibited proliferation in leukemia cells and induced cell death through apoptosis induction and activated apoptosis pathways through the activation of caspase-9 and caspase-3, down-regulation of Bcl-x(L) and X chromosome-linked inhibitor of apoptosis, and cleavage of poly(ADP-ribose) polymerase. In addition, methadone induced cell death not only in anticancer drug-sensitive and apoptosis-sensitive leukemia cells but also in doxorubicin-resistant, multidrug-resistant, and apoptosis-resistant leukemia cells, which anticancer drugs commonly used in conventional therapies of leukemias failed to kill. Depending on caspase activation, methadone overcomes doxorubicin resistance, multidrug resistance, and apoptosis resistance in leukemia cells through activation of mitochondria. In contrast to leukemia cells, nonleukemic peripheral blood lymphocytes survived after methadone treatment. These findings show that methadone kills leukemia cells and breaks chemoresistance and apoptosis resistance. Our results suggest that methadone is a promising therapeutic approach not only for patients with opioid dependence but also for patients with leukemias and provide the foundation for new strategies using methadone as an additional anticancer drug in leukemia therapy, especially when conventional therapies are less effective.     

Arsenic trioxide triggers a regulated form of caspase-independent necrotic cell death via the mitochondrial death pathway

Cell death is generally believed to occur either by accidental, lytic necrosis or by programmed cell death, that is, apoptosis. The initiation and execution of cell death, however, is far more complex and includes pathways like caspase-independent apoptosis or actively triggered necrosis. In this study, we investigated the mechanisms of cell death induced by arsenic trioxide (arsenite, As2O3), a clinically efficient agent in anticancer therapy. As2O3-induced cell death coincides with cytochrome c release, facilitates mitochondrial permeability transition and is sensitive to inhibition by Bcl-x(L), indicating that cell demise is regulated through the mitochondrial apoptosis pathway. Nevertheless, only little caspase-3 activation was observed and As2O3-induced cell death was only weakly obstructed by the broad spectrum caspase inhibitor z-VAD-fmk. Moreover, disruption of caspase-9 or -2 failed to decrease the amount of As2O3-mediated cell death. Interestingly, As2O3-induced cell death had a predominantly necrosis-like phenotype as assessed by Annexin-V/propidium iodide staining and LDH release. Finally, blocking glutathione synthetase by buthionine sulfoximine enhanced the As2O3-mediated necrosis-like cell death without increasing caspase-3 cleavage. As2O3 does, however, not directly inhibit caspases, but appears to interfere with caspase activation. Altogether, our data clearly delineate a mode of As2O3-triggered cell death that differs considerably from that induced by conventional anticancer drugs. These findings may explain the capability of As2O3 to efficiently kill even chemoresistant tumor cells with disturbed apoptosis signaling and caspase activation, a frequent finding in malignancy.     

Cisplatin (CDDP) specifically induces apoptosis via sequential activation of caspase-8, -3 and -6 in osteosarcoma

Purpose: Osteosarcoma is a common malignant tumor. The first choice of treatment plan for osteosarcoma is chemotherapy. In particular, preoperative chemotherapy is most important in clinical treatment in orthopedics. In these chemotherapies, multiple anticancer drugs such as Adriamycin (ADM), CDDP, cyclophosphamide (CPM), methotrexate (MTX) and vincristine (VCR) are commonly used in combination. Recently, anticancer drugs have been shown to trigger apoptosis in various cancer cells. However, many studies on this topic have been examined using leukemia cell lines, and many kinds of cancer cells established from solid tumor are resistant to the induction of apoptosis by anticancer drugs. So in this study, we examined the effects of the anticancer drugs ADM, CDDP, CPM, MTX and VCR on osteosarcoma cells in vitro. We also examined the signaling pathways of each anticancer drug by studying the induction of apoptosis and activation of caspases in the osteosarcoma cells. Methods: We examined the effects of the anticancer drugs ADM, CDDP, CPM, MTX and VCR, which are used clinically for the treatment of osteosarcoma, on cells of the human osteosarcoma (HOS) cell line. The cytotoxic effects of the anticancer drugs were evaluated using the MTT assay. We used both flow cytometry and activation of caspases to confirm the induction of apoptosis in the HOS cells. To dissect the pathway of the caspase cascade in apoptosis in HOS cells, we used the tetrapeptides YVAD-CHO, DMQD-CHO, VEID-CHO and IETD-CHO, which selectively inhibit caspase-1, -3, -6 and -8, respectively. Results: ADM, CDDP, CPM and VCR, but not MTX, induced death of HOS cells in a dose-dependent manner. CDDP at 10 μM, CPM at 7.5 μM, ADM at 20 μM and VCR at 150 μM caused 80% cell death of HOS cells after 12 h. However, the percentages of apoptotic cells were 5.6% (medium alone), 75.9% (CDDP), 20.0% (CPM), 22.2% (ADM), 20.5% (VCR) and 13.1% (MTX). In addition, direct measurement of caspase-3 activity revealed that CDDP but not the other drugs activated caspase-3 in HOS cells. These analyses revealed that only CDDP induced apoptosis of HOS cells via activation of caspases. Furthermore, DMQD-CHO, VEID-CHO and IETD-CHO inhibited CDDP-induced apoptosis of HOS cells, suggesting that caspase-3, -6 and -8 are involved in the signaling pathway of CDDP-induced apoptosis. In contrast, none of the caspase inhibitors inhibited cell death induced by the other anticancer drugs. Conclusions: This study demonstrates that CDDP specifically induces apoptosis via activation of caspases and the other anticancer drugs induce death of HOS cells via different signaling pathways. It also demonstrates that caspase-8 is a key molecule in the earliest stage of the signaling pathway of CDDP-induced apoptosis of HOS cells, and caspase-3 works downstream of caspase-8. Received: 29 March 1999 / Accepted: 20 July 1999     

Chemotherapy triggers apoptosis in a caspase-8-dependent and mitochondria-controlled manner in the non-small cell lung cancer cell line NCI-H460

Chemotherapy-induced apoptosis is generally thought to be dependent on a pathway headed by caspase-9. This model is primarily based on studies performed in leukemia cells; however, little is known about caspase cascades in relatively resistant solid tumor cells, including non-small cell lung cancer (NSCLC) cells. Using the NSCLC cell line NCI-H460 (H460), here, we studied the effect of stable expression of various caspase inhibitors on apoptosis induced by the anticancer drugs cisplatin, topotecan, and gemcitabine. Interestingly, overexpression of caspase-9S and X-linked inhibitor of apoptosis (XIAP), both able to inhibit caspase-9 activity, failed to block apoptosis. In contrast, stable expression of caspase-8 inhibitors, such as cytokine response modifier A (CrmA) and dominant-negative caspase-8, almost completely abrogated apoptosis and also enhanced clonogenic survival. Caspase-8 activation in H460 cells was not mediated by death receptors, inasmuch as overexpression of dominant-negative Fas-associated death domain (FADD-DN) did not prevent procaspase-8 cleavage and subsequent apoptosis. However, stable expression of Bcl-2 and Bcl-xL did suppress these apoptotic events, including the release of cytochrome c from mitochondria, which was observed in drug-treated H460 cells. In the NSCLC cell line H460, we, thus, provide evidence for the existence of a novel drug-inducible apoptotic pathway in which activation of caspase-8, and not of caspase-9, forms the apical and mitochondria-dependent step that subsequently activates the downstream caspases.     

Bax deficiency affects caspase-2 activation during ultraviolet radiation-induced apoptosis

There has been a considerable debate as to whether caspase-2 is an initiator or effector caspase. Recently, a new model of intrinsic pathway of apoptosis has been proposed, which suggests caspase-2 to be an initiator caspase. For example, ultraviolet radiation (UV) and other DNA damage-inducing agents were shown to first activate caspase-2 and then regulate the mitochondrial and postmitochondrial events. Active caspase-2 was found to engage mitochondria by promoting Bax translocation to the mitochondria. Consequently, Bax was proposed to play a central role in bridging the active caspase-2 with mitochondria by affecting mitochondrial permeability, cytochrome c release into the cytosol and caspase-9 activation. In the present study, we investigated the role of Bax in UV-induced apoptosis and caspase-2 activation. Our results indicate that UV-induced apoptosis and caspase-2 activation were diminished in Bax-deficient cells, suggesting that Bax appears to play an important role in UV-induced apoptosis as well as caspase-2 activation, and that it also appears to reside upstream of caspase-2. Bax deficiency also affected the activation of caspase-3 and -8 and abolished caspase-9 activation during UV-induced apoptosis, suggesting that the absence of caspase-9 activation may affect caspase-2, -3 and -8 activation in Bax-deficient cells. Based on our results, we propose that activation of caspases is not a linear cascade of events, but is rather connected via complex feedback loops.     

c-Jun NH2-terminal kinase-mediated up-regulation of death receptor 5 contributes to induction of apoptosis by the novel synthetic triterpenoid methyl-2-cyano-3,12-dioxooleana-1, 9-dien-28-oate in human lung cancer cells

Death receptor (DR) 4 or 5, on binding to its ligand, tumor necrosis factor-related apoptosis-inducing ligand (TRAIL), triggers apoptosis via activating the caspase-8-mediated caspase cascade. Certain anticancer drugs up-regulate the expression of these receptors and thereby induce apoptosis or enhance TRAIL-induced apoptosis. In this study, we explored the ability of methyl-2-cyano-3,12-dioxooleana-1,9-dien-28-oate (CDDO-Me) to activate the extrinsic DR-mediated apoptotic pathway in human lung cancer cells. We found that CDDO-Me not only activated caspase-8 but also induced expression of DRs, particularly DR5, in a p53-independent mechanism. Correspondingly, CDDO-Me augmented TRAIL-induced apoptosis in these cells regardless of p53 status as evidenced by enhanced DNA fragmentation and activation of caspase cascades, suggesting that CDDO-Me-induced DRs are functionally active. Moreover, silencing of DR5 expression using small interfering RNA suppressed apoptosis induced by CDDO-Me alone or by combination of CDDO-Me and TRAIL, indicating that DR5 up-regulation is required for induction of apoptosis by CDDO-Me and for enhancement of TRAIL-induced apoptosis by CDDO-Me. CDDO-Me rapidly activated c-Jun NH(2)-terminal kinase (JNK) before DR up-regulation and caspase-8 activation. Moreover, application of the JNK-specific inhibitor SP600125 blocked CDDO-Me-induced increases in JNK activation, DR up-regulation, caspase-8 activation, and DNA fragmentation. These results show that activation of JNK pathway results in CDDO-Me-induced DR up-regulation, caspase-8 activation, and apoptosis. Collectively, we conclude that CDDO-Me induces apoptosis via the JNK-mediated DR up-regulation in human lung cancer cells.     

Caspases as key executors of methyl selenium-induced apoptosis (anoikis) of DU-145 prostate cancer cells

Apoptosis induction may be a mechanism mediating the anticancer activity of selenium. Our earlier work indicated that distinct cell death pathways are likely involved in apoptosis induced by the CH3SeH and the hydrogen selenide pools of selenium metabolites. To explore the role of caspases in cancer cell apoptosis induced by selenium, we examined the involvement of these molecules in the death of the DU-145 human prostate carcinoma cells induced by methylseleninic acid (MSeA), a novel penultimate precursor of the putative critical anticancer metabolite CH3SeH. Sodium selenite, a representative of the genotoxic selenium pool, was used as a reference for comparison. The results show that MSeA-induced apoptosis was accompanied by the activation of multiple caspases (caspase-3, -7, -8, and -9), mitochondrial release of cytochrome c (CC), poly(ADP-ribose) polymerase (PARP) cleavage, and DNA fragmentation. In contrast, selenite-induced apoptotic DNA fragmentation was observed in the absence of these changes, but was associated with the phosphorylation of c-Jun-NH2-terminal kinase 1/2 and p38 mitogen-activated protein kinase/stress-activated protein kinase 2. A general caspase inhibitor, benzyloxycarbonyl-Val-Ala-Asp-(OMe) fluoromethyl ketone, blocked MSeA-induced cleavage of procaspases and PARP, CC release, and DNA nucleosomal fragmentation, but did not prevent cell detachment. Furthermore, PARP cleavage and caspase activation were confined exclusively to detached cells, indicating that MSeA induction of cell detachment was a prerequisite for caspase activation and apoptosis execution. This process therefore resembled "anoikis," a special mode of apoptosis induction in which adherent cells lose contact with the extracellular matrix. Additional experiments with irreversible caspase inhibitors show that MSeA-induced anoikis involved caspase-3- and -7-mediated PARP cleavage that was initiated by caspase-8 and probably amplified through CC-caspase-9 activation and a feedback activation loop from caspase-3. Taken together, the data support a methyl selenium-specific induction of DU-145 cell apoptosis that involves cell detachment as a prerequisite (anoikis) and is executed principally through caspase-8 activation and its cross-talk with multiple caspases.     

Smac induces cytochrome c release and apoptosis independently from Bax/Bcl-x(L) in a strictly caspase-3-dependent manner in human carcinoma cells

The mitochondrial apoptosis pathway mediates cell death through the release of various pro-apoptotic factors including cytochrome c and Smac, the second mitochondrial activator of caspases, into the cytosol. Smac was shown previously to inhibit IAP proteins and to facilitate initiation of the caspase cascade upon cytochrome c release. To investigate Smac function during apoptosis and to explore Smac as an experimental cancer therapeutic, we constructed an expression system based on a single adenoviral vector containing Smac under control of the Tet-off system supplied in cis. Conditional expression of Smac induced apoptosis in human HCT116 and DU145 carcinoma cells regardless of the loss of Bax or overexpression of Bcl-x(L). Nevertheless, apoptosis induced by Smac was associated with cytochrome c release and breakdown of the mitochondrial membrane potential. This indicates that Smac acts independently of Bax and Bcl-x(L) during initiation of apoptosis and triggers a positive feedback loop that results in Bax/Bcl-x(L)-independent activation of mitochondria. In caspase-proficient cells, Smac-induced apoptosis could be inhibited partially by cell-permeable LEHD (caspase-9 inhibitor) and DEVD (caspase-3 inhibitor) peptides. Furthermore, loss of caspase-3 expression in MCF-7 cells carrying a caspase-3 null mutation completely abrogated the sensitivity for Smac-induced apoptotic or nonapoptotic, necrosis-like cell death, while re-expression of caspase-3 conferred sensitivity. Altogether, caspase-3 but not caspase-9 activation was necessary for execution of Smac-induced cell death. Notably, Smac did not induce caspase-9 processing in the absence of caspase-3. Thus, caspase-9 processing occurs secondary to caspase-3 activation during Smac-induced apoptosis. Altogether, Smac is capable of circumventing defects in mitochondrial apoptosis signaling such as loss of Bax or overexpression of Bcl-x(L) that are frequently observed in tumor cells resistant to anticancer therapy. Consequently, Smac appears to be a promising therapeutic target in anticancer treatment.     

Mistletoe lectin activates caspase-8/FLICE independently of death receptor signaling and enhances anticancer drug-induced apoptosis

Mistletoe lectin I (ML-I) is a major active component in plant extracts of Viscum album that is increasingly used in adjuvant cancer therapy. ML-I exerts potent immunomodulating and cytotoxic effects, although its mechanism of action is largely unknown. We show that treatment of leukemic T- and B-cell lines with ML-I induced apoptosis, which required the prior activation of proteases of the caspase family. The involvement of caspases is demonstrated because (a) a peptide caspase inhibitor almost completely prevented ML-I-induced cell death and (b) proteolytic activation of caspase-8, caspase-9, and caspase-3 was observed. Because caspase-8 has been implicated as a regulator of apoptosis mediated by death receptors, we further investigated a potential receptor involvement in ML-I-induced effects. Cell death triggered by ML-I was neither attenuated in cell clones resistant to CD95 nor in cells that were rendered refractory to other death receptors by overexpressing a dominant-negative FADD mutant. In contrast, ML-I triggered a receptor-independent mitochondria-controlled apoptotic pathway because it rapidly induced the release of cytochrome c into the cytosol. Because ML-I was also observed to enhance the cytotoxic effect of chemotherapeutic drugs, these data may provide a molecular basis for clinical trials using MLs in anticancer therapy.     

Caspase 9 is required for p53-dependent apoptosis and chemosensitivity in a human ovarian cancer cell line.

The p53 gene suppresses tumor cell growth by inducing cell cycle arrest or apoptosis. Loss of its apoptosis activity has been implicated not only in tumor development but also in chemoresistance. We previously reported that targeting p53 for degradation by the human HPV E6 gene in the ovarian cancer cell line PA1 leads to an increase in the chemoresistant phenotype. Here we investigate the relationship between loss of p53-dependent caspase activation and chemosensitivity. In PA1-neo cells with wild-type p53, the activation of caspases including caspases 9, 8, 7 and 3 and cleavage of PARP were detected following adriamycin or etoposide treatment, whereas no such changes were observed in PA1-E6 cells whose p53 is degraded, suggesting that loss of p53 impairs caspase activation. Importantly, we showed that loss of caspase activation in PA1-E6 cells correlates with increased cell survival. Moreover, PA1 cells overexpressing a dominant negative caspase 9 were found to have decreased caspase-dependent apoptosis, as compared with vector control cells. Furthermore, these dominant negative caspase 9 expressing cells were resistant to chemotherapeutic agent-induced killing. Our results suggest that caspase 9 may be an important target for anticancer drug development. Thus, identifying novel compounds that can activate caspase 9 may be a strategy for overcoming a defect in the p53 apoptosis pathway.     

Current status of the molecular mechanisms of anticancer drug-induced apoptosis. The contribution of molecular-level analysis to cancer chemotherapy

Apoptosis is an important phenomenon in cytotoxicity induced by anticancer drugs. Here, we review the current status of the molecular mechanisms of anticancer drug-induced apoptosis in order to assess the contribution of molecular-level analysis to cancer chemotherapy. It is apparent that the molecular mechanisms by which anticancer drugs induce apoptosis are mediated by death receptor-dependent and -independent pathways, which are related to the release of cytochrome c through voltage-dependent anion channels in the mitochondrial inner membrane. The release of cytochrome c is the central gate in turning on/off apoptosis, and is regulated by the interaction of proapoptotic proteins, including Bid, Bax and Bak, and antiapoptotic proteins including Bcl-2 and Bcl-X(L), and a specific class of inhibitors of apoptosis proteins (IAPs) including Akt, survivin, and heat-shock proteins. The caspase cascade is activated by the release of cytochrome c, which is initiated by the formation of apoptosomes consisting of procaspase-9, Apaf-1 and cytochrome c in the presence of dATP, and results in the activation of caspase-9 and caspase-3, thereby leading to apoptosis. Drug sensitivity can be enhanced by the introduction of proapoptotic genes and the inhibition of antiapoptotic proteins. The latter process is mediated by antisense oligonucleotides and is associated with apoptosis. The signal transduction pathways that are triggered by the central gate in mitochondria play a critical role in anticancer drug-induced apoptosis. The modulation of signal transduction pathways targeting the proteins involved in these signal transduction pathways using antisense IAPs, and growth factor antibodies may be a good strategy for enhancing therapeutic efficacy of anticancer drugs in cancer chemotherapy.     

Staurosporine induces apoptosis through both caspase-dependent and caspase-independent mechanisms.

Sensitivity of tumor cells to anticancer therapy depends on the ability of the drug to induce apoptosis. However, multiple signaling pathways control this induction and thus determine this sensitivity. We report here that staurosporine, a well known inducer of apoptosis in a wide range of cell lines, displays distinct ability to trigger apoptosis in two different L1210 sublines (termed L1210/S and L1210/0). Staurosporine treatment resulted in an early cell death (within 3 h) in L1210/S cells, while in L1210/0 cells, death occurred only after 12 h. In both instances, death occurred by apoptosis. A broad spectrum caspase inhibitor, Z-VAD-fmk, blocked early apoptosis in L1210/S cells but did not confer any protection on late apoptosis in L1210/0 cells. Protection by Z-VAD-fmk observed in L1210/S cells was not lasting and unmasked a secondary process of cell death that also exhibited characteristics of apoptosis. Thus, staurosporine induces apoptotic cell death through at least two redundant parallel pathways. These two pathways normally coexist in L1210/S cells. However, the early cell death mechanism depending on caspase activation disguises the late caspase-independent apoptotic process. Staurosporine-induced apoptosis in L1210/0 cells develops only by the caspase-independent mechanism due to a general defect in caspase activation.     

Apo2L/TRAIL differentially modulates the apoptotic effects of sulindac and a COX-2 selective non-steroidal anti-inflammatory agent in Bax-deficient cells

The nonsteroidal anti-inflammatory drugs (NSAIDs) are believed to mediate their anticancer effects by inducing apoptosis but the molecular mechanisms of their apoptotic effects remain largely unknown. Here we report that two different NSAIDs, sulindac sulfide and SC-'236 engage the death receptor 5 (DR5) and mitochondrial pathways to mediate apoptosis in human colon cancer cells. We show that sulindac sulfide and SC-'236-induced apoptosis is coupled with upregulation of DR5, caspase 8 activation and Bid cleavage. Thus, a cross talk appears to exist between the DR5 and mitochondrial pathways during apoptosis induced by these NSAIDs. We further show that sulindac sulfide and SC-'236-induced DR5 upregulation occurs independent of the COX inhibitory effects of these NSAIDs. Using Bax-proficient (Bax+/-) and Bax-deficient (Bax-/-) HCT116 human colon cancer cells, we further demonstrate that Apo2L/TRAIL differentially modulates the apoptotic effects of sulindac sulfide and SC-'236. For example, sulindac sulfide upregulates DR5 in both Bax-deficient and proficient cells, but Apo2L/TRAIL efficiently potentiates sulindac sulfide-induced apoptosis as well as activation of caspase-8, -9 and -3 only in Bax-proficient cells. SC-'236 also upregulates DR5 in both Bax-proficient and Bax-deficient cells but Apo2L/TRAIL potentiates SC-'236-mediated apoptosis and caspases-8 and -3 activation in both Bax-proficient and Bax-deficient cells. Further, in Bax-deficient cells, neither sulindac sulfide nor SC-'236 in combination with Apo2L/TRAIL effectively promotes the release of cytochrome c from mitochondria into cytosol and caspase-9 activation. Collectively, our results suggest that unlike sulindac sulfide, SC-'236 in combination with Apo2L/TRAIL can overcome Bax deficiency to induce apoptosis. These results have important clinical implications in that the tumors harboring Bax mutations are likely to develop resistance to sulindac but not to SC-'236-like NSAIDs. In conclusion, the data presented herein form the basis of future in-depth studies to further explore the utility of Apo2L/TRAIL and NSAIDs, in combination, as a novel cancer preventive/therapeutic strategy.     

Molecular mechanisms of apoptosis induced by magnolol in colon and liver cancer cells.

Magnolol has been reported to have anticancer activity. In this study we found that treatment with 100 microm magnolol induced apoptosis in cultured human hepatoma (Hep G2) and colon cancer (COLO 205) cell lines but not in human untransformed gingival fibroblasts and human umbilical vein endothelial cells. Our investigation of apoptosis in Hep G2 cells showed a sequence of associated intracellular events that included (a) increased cytosolic free Ca(2+); (b) increased translocation of cytochrome c (Cyto c) from mitochondria to cytosol; (c) activation of caspase 3, caspase 8, and caspase 9; and (d) downregulation of bcl-2 protein. Pretreatment of the cells with the phospholipase C inhibitor 1-[6-[[(17 beta)-3-methoxyestra-1,3,5(10)-trien-17-yl]amino]hexyl]-1 H-pyrrole-2,5-dione (U73122) or the intracellular chelator of Ca(2+) 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid acetoxymethyl ester (BAPTA/AM) inhibited the subsequent magnolol augmentation of [Ca(2+)](i) and also the activation of caspase-8 and caspase-9, so that the occurrence of apoptosis in those cells was greatly reduced. Pretreatment of the cells with ZB4 (which disrupts the Fas response mechanism) also decreased the subsequent magnolol-induced caspase-8 activation and reduced the occurrence of apoptosis. We interpreted these findings to indicate that the above-listed sequence of intracellular events led to the apoptosis seen in Hep G2 cells and that [Ca(2+)](i), Cyto c, and Fas function as intracellular signals to coordinate those events.     

Drug-induced Myc-mediated apoptosis of cancer cells is inhibited by stress protein Hsp70

The Myc oncoprotein serves a dual function by stimulating cells both towards growth and apoptosis. The latter functions are often abrogated during tumor development. The Hsp70 stress protein is a potent anti-apoptotic molecule, but its potential role in protecting cells from Myc-mediated apoptosis has not been investigated. Our results show that activated Myc potentiated apoptosis induced by the cancer drugs etoposide (ETO) and camptothecin (CAMP) in v-Myc-expressing human U-937 monoblastic cells and in Rat1 cells containing a conditionally active Myc/estrogen receptor (MycER) fusion protein. However, both heat shock and ectopic Hsp70 expression protected the cells from Myc-mediated apoptosis after drug treatment in both systems. The increased susceptibility to the anti-tumor drugs by activated Myc was enhanced by siRNA-mediated knockdown of Hsp70 expression in U-937 cells. Addressing the mechanisms by which Myc and Hsp70 promotes and inhibits drug-induced apoptosis, respectively, we found that v-Myc stimulated cytochrome c release and activation of effector caspase-9, -3 and -7, but not of initiator caspase-8. Inhibition of caspase-9 specifically reduced v-Myc-stimulated apoptosis, whereas inhibition of caspase-8 and -3/7 reduced apoptosis both in v-myc-expressing and parental ETO-treated U-937 cells. Interestingly, Myc-stimulated activation of effector caspases was inhibited, but cytochrome c release was not affected by Hsp70 expression, suggesting that Hsp70 interferes with the proapoptotic function of Myc downstream of mitochondria, at the level of caspase-9 and downstream caspases. In conclusion, Hsp70 seems to have key function in inhibition of apoptosis mediated by Myc and may therefore play an important role in Myc-driven oncogenesis.     

PTEN sensitizes prostate cancer cells to death receptor-mediated and drug-induced apoptosis through a FADD-dependent pathway.

The PTEN tumor suppressor is frequently mutated in human tumors. Loss of PTEN function is associated with constitutive survival signaling through the phosphatidylinositol-3 kinase/Akt pathway. Therefore, we asked if reconstitution of PTEN function would lead to the reversal of resistance to apoptosis in prostate cancer cells. Adenovirus-mediated expression of PTEN completely suppressed constitutive Akt activation in LNCaP prostate cancer cells and enhanced apoptosis induced by a broad range of apoptotic stimuli. PTEN expression sensitized cells to death receptor-mediated apoptosis induced by tumor necrosis factor, anti-Fas antibody, and TRAIL. PTEN also sensitized cells to non-receptor mediated apoptosis induced by a kinase inhibitor staurosporine and chemotherapeutic agents mitoxantrone and etoposide. PTEN-mediated apoptosis was accompanied by caspase-3 and caspase-8 activation and was inhibited by a broad specificity caspase inhibitor Z-VAD-fmk. Bcl-2 overexpression also blocked PTEN-mediated apoptosis. Lipid phosphatase activity of PTEN is required for apoptosis as the PTEN G129E mutant selectively deficient in lipid phosphatase activity was unable to sensitize cells to apoptosis. PTEN-mediated apoptosis involves a FADD-dependent pathway for both death receptor-mediated and drug-induced apoptosis as coexpression of a dominant negative FADD mutant blocked PTEN-mediated apoptosis. Since in death receptor signaling, FADD mediates activation of caspase-8, which in turn cleaves BID, and since caspase-8 is activated in PTEN-mediated apoptosis, we examined BID cleavage in PTEN-mediated apoptosis. PTEN facilitated BID cleavage after treatment with low doses of staurosporine and mitoxantrone. BID cleavage was inhibited by dominant negative FADD. Taken together, these data are consistent with the hypothesis that PTEN promotes drug-induced apoptosis by facilitating caspase-8 activation and BID cleavage through a FADD-dependent pathway.     

Ganciclovir-induced apoptosis in HSV-1 thymidine kinase expressing cells: critical role of DNA breaks, Bcl-2 decline and caspase-9 activation

Although ganciclovir (GCV) is most often used in suicide anticancer gene therapy, the mechanism of GCV-induced cell killing and apoptosis is not fully understood. We analysed the mechanism of apoptosis triggered by GCV using a model system of CHO cells stably transfected with HSV-1 thymidine kinase (HSVtk). GCV-induced apoptosis is due to incorporation of the drug into DNA resulting in replication-dependent formation of DNA double-strand breaks and, at later stages, S and G2/M arrest. GCV-provoked DNA instability was likely to be responsible for the observed initial decline in Bcl-2 level and caspase-9/-3 activation. Further decline in the Bcl-2 level was due to cleavage of the protein by caspase-9, as demonstrated by use of caspase inhibitors and transfection with trans-dominant negative caspase expression vectors. Bcl-2 cleavage resulted in the appearance of a pro-apoptotic 23 kDa Bcl-2 fragment and in excessive cytochrome c release, dephosphorylation of BAD, cleavage of PARP and finally DNA degradation. Since Fas/CD95 and caspase-8 were only slightly activated we conclude GCV-induced apoptosis to occur in this cell system mainly by activating the mitochondrial damage pathway. This process is independent of p53 for which the cells are mutated. Caspase-9 mediated cleavage of Bcl-2 accelerates the apoptotic process and may explain the high potential of GCV to induce apoptosis. Data are also discussed as to implications for HSVtk gene therapy utilizing GCV.     

Defective caspase-3 relocalization in non-small cell lung carcinoma.

Many anticancer drugs exert their cytotoxicity through DNA damage and induction of apoptosis. Small cell lung carcinoma (SCLC) and non-small cell lung carcinoma (NSCLC) have different sensitivity to treatment with radiation and chemotherapeutic agents with SCLC being more sensitive than NSCLC both in vitro and in vivo. This difference might be related to the different susceptibility of small and non-small cell lung carcinoma to undergo apoptosis. The aim of this study was to investigate if deficiencies in the apoptotic pathways can explain the intrinsic resistance of NSCLC to anti-cancer treatment. Three different triggers were used to induce apoptosis. Etoposide and gamma-radiation, which are important parts of clinical lung cancer treatment, induce DNA-damage, whereas Fas ligation induces receptor-mediated apoptotic pathways. NSCLC cells were cross-resistant to all treatments, whereas SCLC cells, which do not express pro-caspase-8, were resistant to alphaFas-, but not to DNA-damage-induced apoptosis. Cytochrome c release, activation of caspase-9 and the executioner caspase-3 were observed in both types of lung cancer cells. However, cleavage of known nuclear substrates for caspase-3, such as PARP and DFF45/ICAD, was documented only in the sensitive SCLC cells but not in the resistant NSCLC cells. Moreover, relocalization of active caspase-3 from the cytosol into the nucleus upon treatment was observed only in the SCLC cell line. These results indicate that the inhibition of apoptosis in NSCLC occurs downstream of mitochondrial changes and caspase activation, and upstream of nuclear events.