PEDF induces apoptosis in human endothelial cells by activating p38 MAP kinase dependent cleavage of multiple caspases

We examined how pigment epithelium derived factor (PEDF), an effective endogenous antiangiogenic protein, decreases survival of primary cultures of human umbilical vein endothelial cells (HUVECs) in a low serum environment supplemented with the endothelial cell growth factor (VEGF). We provide evidence that induction of apoptosis by PEDF is associated with activation of p38 followed by cleavage of caspases 3, 8, and 9 by treatment with PEDF, and PEDF's actions are caspase dependent. A key mediator in the executioner effects of PEDF is p38 since the inhibition of p38 activity blocked apoptosis and prevented cleavage of caspases 3, 8, and 9. Although PEDF-induced phosphorylation of JNK1, the inhibition of JNK1 had no effect on apoptosis, even though it prevented phosphorylation of JNK1 by PEDF. Based on these findings, we propose that the antiangiogenic action of PEDF is dependent on activation of p38 MAPkinase which regulates cleavage of multiple caspases cascades.     

Mitochondria and caspases in induced apoptosis in human luteinized granulosa cells

Apoptosis occurs as a physiologic process in the ovarian life cycle. Staurosporine, a protein kinase inhibitor, is reported to induce apoptosis. Here, we hypothesize that staurosporine will induce apoptosis in human luteinized granulosa cells and that mitochondria and the caspase cascade participate in this process. Luteinized granulosa cells isolated from in vitro fertilization patients were treated with staurosporine. Microscopy revealed that staurosporine treatment resulted in cells exhibiting evidence of apoptosis, including cell detachment, loss of cell processes, membrane shrinkage, and formation of apoptotic bodies. In the staurosporine-treated cells, flow cytometry and confocal microscopy showed a decrease in the mitochondrial cardiolipin levels. Western analysis showed cleavage of caspase-9, an initiator caspase, of caspase-3, an executioner caspase, and of a caspase substrate, poly-(ADP-ribose)-polymerase (PARP) in staurosporine-treated cells. These data support our hypothesis and that this is the first demonstration of the involvement of mitochondria and of cleavage of caspases in human luteinized granulosa cell apoptosis. This may serve as a useful model to delineate the mechanism of apoptosis in the ovary, such as corpus luteum regression.     

TRAIL causes cleavage of bid by caspase-8 and loss of mitochondrial membrane potential resulting in apoptosis in BJAB cells.

A new member of the TNF family, tumor necrosis factor-related apoptosis-inducing ligand (TRAIL), has been shown to induce apoptosis. However, the mechanism for TRAIL-induced apoptosis remains to be clarified. SDS-PAGE and Western blot analysis showed that cleavage of Bid was induced by a 1-h incubation of BJAB cells with TRAIL and was blocked by a caspase-8 inhibitor. Flow cytometry demonstrated that loss of mitochondrial membrane potential in BJAB cells began about 1.5 h after the treatment with TRAIL and was apparent at 2 h in comparison with the control. DNA ladder formation, which is characteristic for apoptosis, in the cells treated with TRAIL was detected at 2 h and observed most effectively at 3 h. The time course study suggests that TRAIL causes cleavage of Bid via activation of caspase-8, subsequently the loss of mitochondrial membrane potential, resulting in apoptosis in BJAB cells.     

Epstein-Barr virus BHRF1 functions downstream of Bid cleavage and upstream of mitochondrial dysfunction to inhibit TRAIL-induced apoptosis in BJAB cells

We have previously reported that TNF-related apoptosis inducing ligand (TRAIL) causes cleavage of Bid via activation of caspase-8 and the loss of mitochondrial membrane potential (DeltaPsim), resulting in apoptosis. Experiments with BJAB clones expressing Epstein-Barr virus (EBV) anti-apoptotic protein BHRF1 showed that BHRF1 drastically inhibited TRAIL-mediated apoptosis. Although Western blot analysis demonstrated that TRAIL-induced Bid cleavage was not inhibited by BHRF1, the decrease in DeltaPsim caused by TRAIL was effectively blocked by BHRF1. These findings suggest that in BJAB cells, BHRF1 acts downstream of Bid cleavage and upstream of mitochondrial damage, resulting in inhibition of TRAIL-induced apoptosis.     

Functional role of caspases in sphingosine-induced apoptosis in human hepatoma cells

We have previously shown that sphingosine increased caspase-3 activity and induced apoptosis in human hepatoma cells. Our data also suggest that other caspases may be involved in sphingosine-triggered apoptosis. In order to clarify this issue, we used different approaches to study the functional role of several initiator or executioner caspases in apoptosis induced by sphingosine. Activation of procaspases-2, -7, and -8, was clearly demonstrated during sphingosine-triggered apoptosis. Pretreatment with chemical inhibitors for caspase-7 and -8, attenuated apoptotic cell death induced by sphingosine. Conversely, pretreatment with specific caspase-2 inhibitor Z-VDVAD-FMK did not show any protective effect. In addition, enforced expression of constitutively activated AKT kinase which is known to inhibit apoptosis induced by sphingosine, potently suppressed activation of procaspases-7 and -8. In summary, these data suggest that in addition to caspases-3, caspase-7 and -8 are involved in the apoptosis induced by sphingosine.     

Involvement of caspases in 4-hydroxy-alkenal-induced apoptosis in human leukemic cells

4-Hydroxynonenal (HNE), a reactive and cytotoxic end-product of lipid peroxidation, has been suggested to be a key mediator of oxidative stress-induced cell death and in various cell types has been shown to induce apoptosis. We have demonstrated that HNE, at micromolar concentrations, induces dose- and time-dependent apoptosis in a leukemic cell line (CEM-C7). Interestingly, much higher concentrations of HNE (> 15-fold) were required to induce apoptosis in leukocytes obtained from normal individuals. We also demonstrate that HNE causes a decrease in clonogenicity of CEM-C7 cells. Furthermore, our data characterize the caspase cascade involved in HNE-induced apoptosis in CEM-C7 cells. Using specific fluorogenic substrates and irreversible peptide inhibitors, we demonstrate that caspase 2, caspase 3, and caspase 8 are involved in HNE-induced apoptosis, and that caspase 2 is the first initiator caspase that activates the executioner caspase 3, either directly or via activation of caspase 8. Our studies also suggest the involvement of another executioner caspase, which appears to be similar to caspase 8 but not caspases 2 and 3, in its specificity. The demonstration of decreased clonogenicity by HNE in the leukemic cells, and their higher susceptibility to HNE-induced apoptosis as compared to the normal cells, suggests that such compounds may have potential for leukemia chemotherapy.     

Activation of caspases and cleavage of Bid are required for tyrosine and phenylalanine deficiency-induced apoptosis of human A375 melanoma cells

Deprivation of tyrosine (Tyr) and phenylalanine (Phe) inhibits growth and induces programmed cell death (apoptosis) of human A375 melanoma cells. Herein, we found that activation of caspases and release of mitochondrial cytochrome c are required for this process. Culturing A375 cells in Tyr/Phe-free medium, containing 10% dialyzed fetal bovine serum, results in activation of caspase-3-like activity. This is accompanied by decreased cell viability and increased apoptosis. Tyr/Phe deprivation also stimulates proteolytic cleavage of the DNA repair enzyme, poly(ADP-ribose) polymerase (PARP). Western blot analysis showed that caspases 3, 7, 8, and 9 are activated by deprivation of Tyr/Phe. Tyr/Phe deprivation decreases mitochondrial membrane potential, induces cleavage of Bid, increases translocation of Bax from the cytosol to mitochondria, and results in release of cytochrome c from the mitochondria to the cytosol. Apoptosis due to Tyr/Phe deprivation is almost completely inhibited by the broad-spectrum cell-permeable caspase inhibitor, benzyloxycarbonyl-Val-Ala-Asp-fluoromethyl ketone (Z.VAD.fmk). This inhibitor suppresses the cleavage of Bid, the release of cytochrome c from the mitochondria to the cytosol, and the cleavage of PARP. Decylubiquinone, a mitochondrial permeability transition pore inhibitor, does not suppress the activation of caspase 8 but suppresses release of cytochrome c, activation of caspase 9, and induction of apoptosis. These results indicate that activation of caspases, cleavage of Bid, and mitochondrial release of cytochrome c are required for apoptosis induced by Tyr/Phe deprivation.     

Role of mitochondria and caspases in vitamin D-mediated apoptosis of MCF-7 breast cancer cells

Vitamin D(3) compounds are currently in clinical trials for human breast cancer and offer an alternative approach to anti-hormonal therapies for this disease. 1alpha,25-Dihydroxyvitamin D(3) (1alpha,25(OH)(2)D(3)), the active form of vitamin D(3), induces apoptosis in breast cancer cells and tumors, but the underlying mechanisms are poorly characterized. In these studies, we focused on the role of caspase activation and mitochondrial disruption in 1alpha,25(OH)(2)D(3)-mediated apoptosis in breast cancer cells (MCF-7) in vitro. The effect of 1alpha,25(OH)(2)D(3) on MCF-7 cells was compared with that of tumor necrosis factor alpha, which induces apoptosis via a caspase-dependent pathway. Our major findings are that 1alpha,25(OH)(2)D(3) induces apoptosis in MCF-7 cells by disruption of mitochondrial function, which is associated with Bax translocation to mitochondria, cytochrome c release, and production of reactive oxygen species. Moreover, we show that Bax translocation and mitochondrial disruption do not occur after 1alpha,25(OH)(2)D(3) treatment of a MCF-7 cell clone selected for resistance to 1alpha,25(OH)(2)D(3)-mediated apoptosis. These mitochondrial effects of 1alpha,25(OH)(2)D(3) do not require caspase activation, since they are not blocked by the cell-permeable caspase inhibitor z-Val-Ala-Asp-fluoromethylketone. Although caspase inhibition blocks 1alpha,25(OH)(2)D(3)-mediated events downstream of mitochondria such as poly(ADP-ribose) polymerase cleavage, external display of phosphatidylserine, and DNA fragmentation, MCF-7 cells still execute apoptosis in the presence of z-Val-Ala-Asp-fluoromethylketone, indicating that the commitment to 1alpha,25(OH)(2)D(3)-mediated cell death is caspase-independent.     

alpha-Tocopherol increases caspase-3 up-regulation and apoptosis by beta-carotene cleavage products in human neutrophils

It has been found that beta-carotene cleavage products (CarCP), besides having mutagenic and toxic effects on mitochondria due to their prooxidative properties, also initiate spontaneous apoptosis of human neutrophils. Therefore, it was expected that antioxidants such as alpha-tocopherol would inhibit the stimulation of apoptosis and caspase-3 activity by CarCP. However, we found that alpha-tocopherol increases caspase-3 up-regulation and stimulation of apoptosis of human neutrophils by CarCP. Ascorbic acid does not alter this caspase-3 up-regulating and proapoptotic effect exerted by alpha-tocopherol. Both alpha-tocopherol and ascorbic acid, in the absence of CarCP, decrease intracellular caspase-3 activity and spontaneous apoptosis of neutrophils. Uric acid alone or in combination with CarCP does not exert apparent effects on caspase-3 activity and apoptosis. Up-regulating effect of alpha-tocopherol is not observed in the presence of retinol that markedly stimulates apoptosis by itself, whereas increase of caspase-3 activity is induced by concomitant addition of alpha-tocopherol and beta-ionone, a cyclohexenyl degradation product of beta-carotene with shorter aliphatic chain.     

Berberine increases the expression of cytokines and proteins linked to apoptosis in human melanoma cells

Molecular Biology Reports - Melanoma is the most lethal form of skin cancer, and its incidence has increased considerably in the last decades. Melanoma presents difficult treatment with strong...     

Potent and selective nonpeptide inhibitors of caspases 3 and 7 inhibit apoptosis and maintain cell functionality

Caspases have been strongly implicated to play an essential role in apoptosis. A critical question regarding the role(s) of these proteases is whether selective inhibition of an effector caspase(s) will prevent cell death. We have identified potent and selective non-peptide inhibitors of the effector caspases 3 and 7. The inhibition of apoptosis and maintenance of cell functionality with a caspase 3/7-selective inhibitor is demonstrated for the first time, and suggests that targeting these two caspases alone is sufficient for blocking apoptosis. Furthermore, an x-ray co-crystal structure of the complex between recombinant human caspase 3 and an isatin sulfonamide inhibitor has been solved to 2.8-A resolution. In contrast to previously reported peptide-based caspase inhibitors, the isatin sulfonamides derive their selectivity for caspases 3 and 7 by interacting primarily with the S(2) subsite, and do not bind in the caspase primary aspartic acid binding pocket (S(1)). These inhibitors blocked apoptosis in murine bone marrow neutrophils and human chondrocytes. Furthermore, in camptothecin-induced chondrocyte apoptosis, cell functionality as measured by type II collagen promoter activity is maintained, an activity considered essential for cartilage homeostasis. These data suggest that inhibiting chondrocyte cell death with a caspase 3/7-selective inhibitor may provide a novel therapeutic approach for the prevention and treatment of osteoarthritis, or other disease states characterized by excessive apoptosis.     

Selective down-regulation of IP(3)receptor subtypes by caspases and calpain during TNF alpha -induced apoptosis of human T-lymphoma cells

There are at least three types of inositol 1,4,5-trisphosphate receptor (IP(3)R) [IP(3)-gated Ca(2+)channels], which are expressed in different cell types and mammalian tissues. In this study, we have identified three IP(3)R subtypes in human Jurkat T-lymphoma cells. All three subtypes have a molecular mass of about 260 kDa, and display Ca(2+)channel properties in an IP(3)-dependent manner. We have also demonstrated that TNFalpha promotes the activity of different proteases (e.g. caspase-8, caspase-3 and calpain), alters the TCR-mediated Ca(2+)response and subsequently induces apoptosis in Jurkat cells. During the first 6 h of incubation with TNFalpha, several IP(3)R subtype-related changes occur (e.g. proteolysis of IP(3)R subtypes, inhibition of IP(3)binding and impairment of IP(3)-mediated Ca(2+)flux) concomitantly with an elevation of protease (caspase-8, caspase-3 and calpain) activity. Furthermore, the caspase inhibitor, Z-VAD-fmk, significantly reduces TNFalpha-mediated perturbation of IP(3)R1 and IP(3)R2 (but not IP(3)R3) function; whereas the calpain inhibitor I, ALLN, is capable of blocking the inhibitory effect of TNFalpha on IP(3)R3 function. These findings suggest that IP(3)R1 and IP(3)R2 serve as cellular substrates for caspases, and IP(3)R3 is a substrate for calpain. We propose that the selective down-regulation of IP(3)R subtype-mediated Ca(2+)function by caspase-dependent and calpain-sensitive mechanisms may be responsible for the early onset of the apoptotic signal by TNFalpha in human T-cells.     

Ribozyme cleavage leads to decreased expression of fibroblast growth factor receptor 3 in human multiple myeloma cells, which is associated with apoptosis and downregulation of vascular endothelial growth factor

The aim of this study was to investigate the fibroblast growth factor receptor 3 (FGFR3) mRNA cleavage by ribozymes targeting FGFR3, effect of growth inhibition and associated with mechanism on multiple myeloma (MM). We designated two ribozyme-expressing plasmids that target the FGFR3 genes, Rz52 and Rz32. In vitro catalytic activity of Rz52 and Rz32 in KMS11 cells decreased FGFR3 mRNA expression to 45% (p < 0.05) and 80% (p < 0.5), respectively, of that of the control. In vivo examination of the Rz52-transfected KMS11 clone showed that FGFR3 mRNA expression decreased to 20% (p < 0.05) of the control. In the Rz52-transfected H929 clone, FGFR3 mRNA decreased to 50% of the control. Protein expression of FGFR3 decreased to 70% of the parental KMS11 and H929 clones. DNA synthesis in the Rz52-transfected KMS11 clone decreased to 20% of that of the control, whereas the viability of cells decreased to 2% (p < 0.01) of that of the control. Ribozyme cleavage-associated increase in apoptosis of Rz52 KMS11 transfectants was twice that of the control. The inhibition of FGFR3 expression by ribozymes was associated with decreased vascular endothelial growth factor (VEGF) expression and upregulation of Flt-1 but not of the KDR receptor. Our data indicate that FGFR3 is an important cell survival and antiapoptotic factor for MM cells and that ribozyme-targeted downregulation of FGFR3 might be useful as a novel therapeutic intervention in MM characterized by t(4;14).     

Equol induces apoptosis through cytochrome c-mediated caspases cascade in human breast cancer MDA-MB-453 cells

This study investigated the role of the caspase activation cascade in extrinsic and intrinsic apoptosis induced by equol in human breast cancer MDA-MB cells. First, the antiproliferative effect of equol was determined in cells treated with 1-100 μM equol for 24, 48, and 72 h. Equol significantly inhibited cell proliferation in a dose-dependent manner (p < 0.05). Exposure to 50 or 100 μM equol for 72 h strongly promoted apoptosis. Under the same conditions, remarkable cytochrome c release was observed. Subsequently, caspase-9, which acts in mitochondria-mediated apoptosis, was cleaved by equol at high concentrations, but caspase-8 activation of receptor-mediated apoptosis was not observed. At both equol concentrations, the caspase-8 and -9 activity assays showed similar patterns. In addition, equol treatment activated caspase-3, which is downstream from caspase-9, and this was accompanied by the cleavage of capase-6 and -7. Activation of these caspases leads to increased activation of PARP, lamin, and ICAD. This study suggests that equol induces the intrinsic pathway of apoptosis via caspase-9 and cytochrome c, independent of caspase-8, in human breast cancer MDA-MB-453 cells.     

Liver poly(ADP-ribose)polymerase is resistant to cleavage by caspases

In hepatocytes the DNA repair enzyme poly(ADP-ribose)polymerase (PARP) is not proteolytically cleaved during apoptosis. The reason for this was investigated using a cell-free system that consisted of isolated nuclei from hepatocytes or thymocytes and cytosolic extracts from hepatocytes or thymocytes undergoing apoptosis. It was found that liver PARP is resistant to proteolytic cleavage by the caspases present in the cytosolic extracts. Furthermore, liver PARP was not cleaved by recombinant human caspase-3. It is concluded that PARP proteolysis cannot be used as a marker for hepatocyte apoptosis.     

Rb-independent induction of apoptosis by bovine papillomavirus type 1 E7 in response to tumor necrosis factor alpha

Bovine papillomavirus type 1 (BPV-1) is a small DNA virus that causes fibropapillomas of the host. BPV-1 has served as the prototype for studies of the molecular biology of the papillomaviruses. BPV-1 efficiently induces anchorage-independent growth and focus formation in murine C127 cells. The transforming properties of BPV-1 primarily reside in two genes, E5 and E6. Each of these genes is sufficient to transform cells. Although no independent transformation activity has been detected for E7, it was shown to be required for full transformation of C127 by BPV-1. We investigated the biological activities of BPV-1 E7 in several assays. Our results indicate that expression of BPV-1 E7 sensitizes cells to tumor necrosis factor alpha (TNF)-induced apoptosis. The TNF-induced apoptosis in E7-expressing cells was accompanied by increased release of arachidonic acid, indicating that phospholipase A(2) was activated. Unlike the E7 proteins from the cancer-related human papillomaviruses, the BPV-1 E7 protein does not associate efficiently with the retinoblastoma protein (pRB) in vitro, nor does it significantly affect the pRB levels in cultured cells. Furthermore, BPV-1 E7 sensitizes Rb-null cells to TNF-induced apoptosis. These studies indicate that BPV-1 E7 can sensitize cells to apoptosis through mechanisms that are independent of pRB.