Tocotrienol-induced caspase-8 activation is unrelated to death receptor apoptotic signaling in neoplastic mammary epithelial cells

Tocotrienols, a subclass in the vitamin E family of compounds, have been shown to induce apoptosis by activating caspase-8 and caspase-3 in neoplastic mammary epithelial cells. Since caspase-8 activation is associated with death receptor apoptotic signaling, studies were conducted to determine the exact death receptor/ligand involved in tocotrienol-induced apoptosis. Highly malignant +SA mouse mammary epithelial cells were grown in culture and maintained in serum-free media. Treatment with 20 microM gamma-tocotrienol decreased+SA cell viability by inducing apoptosis, as determined by positive terminal dUTP nick end labeling (TUNEL) immunocytochemical staining. Western blot analysis showed that gamma-tocotrienol treatment increased the levels of cleaved (active) caspase-8 and caspase-3. Combined treatment with caspase inhibitors completely blocked tocotrienol-induced apoptosis. Additional studies showed that treatment with 100 ng/ml tumor necrosis factor-alpha (TNF-alpha), 100 ng/ml FasL, 100 ng/ml TNF-related apoptosis-inducing ligand (TRAIL), or 1 microg/ml apoptosis-inducing Fas antibody failed to induce death in +SA cells, indicating that this mammary tumor cell line is resistant to death receptor-induced apoptosis. Furthermore, treatment with 20 microM gamma-tocotrienol had no effect on total, membrane, or cytosolic levels of Fas, Fas ligand (FasL), or Fas-associated via death domain (FADD) and did not induce translocation of Fas, FasL, or FADD from the cytosolic to the membrane fraction, providing additional evidence that tocotrienol-induced caspase-8 activation is not associated with death receptor apoptotic signaling. Other studies showed that treatment with 20 microM gamma-tocotrienol induced a large decrease in the relative intracellular levels of phospho-phosphatidylinositol 3-kinase (PI3K)-dependent kinase 1 (phospho-PDK-1 active), phospho-Akt (active), and phospho-glycogen synthase kinase3, as well as decreasing intracellular levels of FLICE-inhibitory protein (FLIP), an antiapoptotic protein that inhibits caspase-8 activation, in these cells. Since stimulation of the PI3K/PDK/Akt mitogenic pathway is associated with increased FLIP expression, enhanced cellular proliferation, and survival, these results indicate that tocotrienol-induced caspase-8 activation and apoptosis in malignant +SA mammary epithelial cells is associated with a suppression in PI3K/PDK-1/Akt mitogenic signaling and subsequent reduction in intracellular FLIP levels.     

The design, synthesis, and anti-tumor mechanism study of N-phosphoryl amino acid modified resveratrol analogues

A novel series of trans-N-phosphoryl amino acid modified resveratrol analogues were synthesized and evaluated in vitro for their cytotoxic effects against CNE-1 and CNE-2 cell lines. These analogues showed good anti-proliferative activity, among which 8d, 8e, 8j, and 9d displayed much stronger inhibition effect than resveratrol and 8d showed the most potent activity with IC(50) value at 3.45+/-0.82microM. The anti-tumor effects of 8d, 8e, 8j, and 9d were due to the induction of apoptosis, confirmed by the DNA fragmentation and flow cytometry analysis using PI (propidium iodide) staining and Annexin-V-FITC/PI staining assay. The PI staining assay also showed that 8d, 8e, 8j, and 9d caused cell cycles arrest at G(0)-G(1) phase which finally led to cell apoptosis. Further mechanism study on compound 8d against CNE-2 cells has shown the PARP cleavage, which is a hallmark of caspase-3 activation, as well as the activation of caspase-9, and the intracellular ROS generation. These results all suggest that 8d induced a mitochondrial-dependent apoptosis pathway.     

Cotreatment with apicidin overcomes TRAIL resistance via inhibition of Bcr-Abl signaling pathway in K562 leukemia cells

TNF-related apoptosis-inducing ligand (TRAIL) is a pro-apoptotic cytokine that is capable of inducing apoptosis in a wide variety of cancer cells but not in normal cells. Although many cancer cells are sensitive to TRAIL-induced apoptosis, chronic myeloid leukemia (CML) develops resistance to TRAIL. In this study, we investigated whether apicidin, a novel histone deacetylase inhibitor, could overcome the TRAIL resistance in CML-derived K562 cells. Compared to treatment with apicidin or TRAIL alone, cotreatment with apicidin and TRAIL-induced apoptosis synergistically in K562 cells. This combination led to activation of caspase-8 and Bcl-2 interacting domain (Bid), resulting in the cytosolic accumulation of cytochrome c from mitochondria as well as an activation of caspase-3. Treatment with apicidin resulted in down-regulation of Bcr-Abl and inhibition of its downstream target, PI3K/AKT-NF-κB pathway. In addition, apicidin decreased the level of NF-κB-dependent Bcl-x_L, leading to caspase activation and Bid cleavage. These results suggest that apicidin may sensitize K562 cells to TRAIL-induced apoptosis through caspase-dependent mitochondrial pathway by regulating expression of Bcr-Abl and its related anti-apoptotic proteins. Therefore, the present study suggests that combination of apicidin and TRAIL may be an effective strategy for treating TRAIL-resistant Bcr-Abl expressing CML cells.     

Fluid shear stress inhibits TNF-alpha-induced apoptosis in osteoblasts: a role for fluid shear stress-induced activation of PI3-kinase and inhibition of caspase-3

In bone, a large proportion of osteoblasts, the cells responsible for deposition of new bone, normally undergo programmed cell death (apoptosis). Because mechanical loading of bone increases the rate of new bone formation, we hypothesized that mechanical stimulation of osteoblasts might increase their survival. To test this hypothesis, we investigated the effects of fluid shear stress (FSS) on osteoblast apoptosis using three osteoblast cell types: primary rat calvarial osteoblasts (RCOB), MC3T3-E1 osteoblastic cells, and UMR106 osteosarcoma cells. Cells were treated with TNF-alpha in the presence of cyclohexamide (CHX) to rapidly induce apoptosis. Osteoblasts showed significant signs of apoptosis within 4-6 h of exposure to TNF-alpha and CHX, and application of FSS (12 dyne/cm(2)) significantly attenuated this TNF-alpha-induced apoptosis. FSS activated PI3-kinase signaling, induced phosphorylation of Akt, and inhibited TNF-alpha-induced activation of caspase-3. Inhibition of PI3-kinase, using LY294002, blocked the ability of FSS to rescue osteoblasts from TNF-alpha-induced apoptosis and blocked FSS-induced inhibition of caspase-3 activation in osteoblasts treated with TNF-alpha. LY294002 did not, however, prevent FSS-induced phosphorylation of Akt suggesting that activation of Akt alone is not sufficient to rescue cells from apoptosis. This result also suggests that FSS can activate Akt via a PI3-kinase-independent pathway. These studies demonstrate for the first time that application of FSS to osteoblasts in vitro results in inhibition of TNF-alpha-induced apoptosis through a mechanism involving activation of PI3-kinase signaling and inhibition of caspases. FSS-induced activation of PI3-kinase may promote cell survival through a mechanism that is distinct from the Akt-mediated survival pathway.     

IGF-1 regulates cardiac fibroblast apoptosis induced by osmotic stress

In this study we have determined the ability of IGF-1 to protect cardiac fibroblasts against osmotic-induced apoptosis and investigated the potential mechanism(s) underlying this protection. Treatment with IGF-1 (1-100 ng/ml) promoted a dose dependent increase in cell survival against osmotic cell death. Both Akt and ERK1/2 were rapidly phosphorylated by IGF-1 and blocked by wortmannin and PD98059, inhibitors of their upstream activators respectively. However, IGF-1-induced protection was mediated via a wortmannin-dependent but PD98059-independent pathway as determined by cell survival assay suggesting a role of PI3-K/Akt. Furthermore, IGF-1 appeared to reduce the activation of a number of early components in the apoptotic pathway in a wortmannin dependent manner including the osmotic stress-induced perturbation in mitochondrial membrane potential, cleavage and activation of caspase-3 and DNA fragmentation. Thus, the results suggest that IGF-1 regulates osmotic stress-induced apoptosis via the activation of the PI3-K/Akt pathway at a point upstream of the mitochondria and caspase-3.     

Cell cycle specific induction of HL-60 cell differentiation and apoptosis by mycophenolic acid

HL-60 cells undergo terminal differentiation and apoptosis in response to different types of sub-toxic and toxic perturbations respectively. The mechanism by which cells sense different amounts of perturbation to activate pathways that lead to the engagement of a relevant biological response is not known. The response of HL-60 cells to treatment with the immunosuppressant mycophenolic acid (MPA), a specific inhibitor of dGTP/GTP-synthesis, allowed quantitation of a metabolic perturbation which triggered a cellular response. 1.5 microM MPA induced 38% terminal differentiation to CD14 positive, early monocyte-like cells and 22% cell death by apoptosis, whereas 3 microM MPA induced 70% apoptosis but no differentiation. Despite the difference in biological outcomes, 72 h exposure to both 1.5 microM and 3 microM MPA caused a similar ( approximately 75%) depletion of total GTP levels. Cells synchronized by centrifugal elutriation were treated with MPA. Elutriated cells were overall less sensitive to the effects of MPA but 3 microM MPA induced significantly less apoptosis and more differentiation in an elutriation-enriched G1-population than in a population normally distributed in the cell cycle, suggesting that the effects of MPA in S-phase may subsequently lead to cell death. However, analysis of apoptosis by using a terminal deoxynucleotidyltransferase assay and measurement of bromodeoxyuridine incorporation showed that apoptosis was engaged in G1. Analysis of the phosphorylation status of the retinoblastoma protein demonstrated that Rb was hypophosphorylated prior to apoptosis and that in apoptotic cells, separated by flow cytometry, Rb protein was absent, presumably due to proteolysis. The loss of Rb protein did not appear to permit transit to S-phase, and was not accompanied by an expression of c-Myc. Surprisingly, therefore, an antimetabolite inducing a loss of GTP brought about cell death by apoptosis in the G1 phase of the cell cycle.     

Characterization of the stimulatory effect of medroxyprogesterone acetate and chlormadinone acetate on growth factor treated normal human breast epithelial cells

OBJECTIVE: Evidence is increasing that adding progestogens to hormone replacement therapy may be more harmful than beneficial, however it is debatable whether all progestogens act equally on breast cells. Mitogenic growth factors from stromal breast tissue are important in growth-regulation of breast cells, and may modify responses to progestogens. We investigated the effect of two C-21 derivatives, medroxyprogesterone acetate (MPA) and chlormadinone acetate (CMA) on growth-factor treated normal breast epithelial cells and tried to explore the underlying mechanisms of proliferation. METHOD: MCF10A (human epithelial, estrogen- and progesterone-receptor negative normal breast cells) were incubated with MPA or CMA at 0.1 and 1 microM for 7 days with the growth factors (GFs) EGF, bFGF and IGF-I at 1pM. The same combinations, as well as growth factors alone, were also incubated with the proliferation inhibitors PD98059 and LY294002 at 1 microM for 4 days. Cell proliferation rate was measured by the ATP-assay. RESULTS: MPA 0.1 and 1 microM, and CMA 1 microM in combination with GFs both significantly increased cell proliferation rate, with MPA having the greatest effect. MPA- and CMA-induced proliferation of GF stimulated cells was blocked by both PD98059 (selective inhibitor of MAP kinases) and LY294002 (phosphatidylinositol 3-kinase inhibitor); GF stimulated cells could not be significantly reduced by any of the inhibitors used. CONCLUSION: MPA and CMA have a stimulatory effect on benign growth factor stimulated MCF10A cells, possibly via activation of MAP kinase and subsequent substrates and activation of PI3-kinase. GF induced proliferation appear to be mediated by pathways other than those investigated here. Growth factors and progestogens therefore have an additive, synergistic effect on cell proliferation, eliciting their effects via different pathways.     

Apoptosis induced by NO via phosphorylation of p38 MAPK that stimulates NF-kappaB, p53 and caspase-3 activation in rabbit articular chondrocytes

Nitric oxide (NO), reported as an important inducer of apoptosis, plays a considerable role in the pathogenetic mechanisms of articular diseases. This research aimed at investigating the role of p38 MAPK signal transduction pathway on apoptosis induced by NO in rabbit articular chondrocytes. In the present study, NO was produced by a novel NO donor NOC-18. Rabbit articular chondrocytes were cultured as monolayer, and the first passage cells were used for the experiments. We detected apoptosis induced by NO using Annexin V-FITC/PI flow cytometry and TUNEL assay. Measurement of caspase-3 has reflected its activity level. Western blotting was performed to show the protein expressions of p38, NF-kappaB, p53 and caspase-3. Furthermore, we examined the inhibitory effects in the NO pathway with p38-specific inhibitor SB203580. Treatment with NOC-18 caused accelerated apoptosis in a concentration dependent manner. This acceleration was able to be reduced when added to SB203580. Besides, the inhibitor could significantly decrease NO-induced p38, NF-kappaB, p53 and caspase-3 protein expressions, as well as caspase-3 intracellular activity (P<0.05). These results suggest that p38 MAPK signal transduction pathway is critical to NO-induced chondrocyte apoptosis, and p38 plays a role by way of stimulating NF-kappaB, p53 and caspase-3 activation.     

Induction of apoptosis by benzamide and its inhibition by aurin tricarboxylic acid (ATA) in Chinese hamster V79 cells

To investigate the effect of benzamide and nicotinamide, well known inhibitors of poly(ADP-ribose) polymerase, in Chinese hamster V79 cells at the physiological condition of cell growth, we have tested the ability of the inhibitors to induce apoptosis. Apoptosis was detected by nuclear fragmentation, nucleosomal ladder formation, cytochrome-c release from the mitochondria and caspase-3 activation. Benzamide treatment alone increased nuclear fragmentation in dose (2.5-10 mM) and time (4-48 h)-dependent manner. Such treatment also increased nucleosomal ladders. However, 5 mM benzamide pre-treatment inhibited the nucleosomal ladders induced by gamma-irradiation indicating the role of poly(ADP-ribose) polymerase was different in irradiated cells and in un-irradiated cells. Release of cytochrome-c from the mitochondria and caspase-3 activity were also increased by such treatment. Treatment with 200 microM of aurin tricarboxylic acid (ATA), an inhibitor of DNases, inhibited the nucleosomal ladders induced by benzamide or gamma-irradiation without changing the cytochrome-c release or caspase-3 activation. This result showed that ATA inhibited the nucleosomal ladders possibly by inhibiting DNase(s) involved in apoptosis.     

Myeloperoxidase-dependent caspase-3 activation and apoptosis in HL-60 cells: protection by the antioxidants ascorbate and (dihydro)lipoic acid

The heme enzyme myeloperoxidase (MPO) has recently been implicated in hydrogen peroxide H(2)O(2)-induced apoptosis of HL-60 human leukemia cells. The purpose of this study was to investigate the molecular mechanism(s) of MPO-mediated apoptosis, in particular caspase-3 activation, and to determine the effects of the antioxidants ascorbate and (dihydro)lipoic acid. Incubation of HL-60 cells (1 x 10(6) cells/ml media) with H(2)O(2) (0-200 microM) resulted in dose-dependent stimulation of caspase-3 activity, DNA fragmentation, and morphological changes associated with apoptosis. Caspase-3 activity, DNA fragmentation and apoptosis were maximal at approximately 50 microM H(2)O(2). Pre-incubation of the cells with the MPO-specific inhibitor 4-aminobenzoic acid hydrazide (ABAH) and the heme enzyme inhibitor 3-aminotriazole (100 microM each) resulted in complete and partial inhibition, respectively, of intracellular MPO, caspase-3 activity, and apoptosis following addition of 50 microM H(2)O(2). Enhancement of cellular antioxidant status by pre-incubation of the cells with dehydro-ascorbic acid and lipoic acid, which are reduced intracellularly to ascorbate and dihydrolipoic acid, respectively, afforded protection against caspase-3 activation and apoptosis following addition of H(2)O(2). Addition of high concentrations of H(2)O(2) (200 microM) to cells pre-incubated with lipoic acid, however, resulted in cytotoxicity. Overall, our data indicate that MPO-derived oxidants, rather than H(2)O(2) itself, are involved in caspase-3 activation and apoptosis in HL-60 cells, and the antioxidants ascorbate and (dihydro)lipoic acid inhibit caspase-3 activation and apoptosis in these cells, likely via scavenging the MPO-derived oxidants.     

Arsenic trioxide sensitizes promonocytic leukemia cells to TNFalpha-induced apoptosis via p38-MAPK-regulated activation of both receptor-mediated and mitochondrial pathways

Treatment with the anti-leukemic drug arsenic trioxide (As(2)O(3), 1-4 microM) sensitizes U937 promonocytes and other human myeloid leukemia cell lines (HL60, NB4) to apoptosis induction by TNFalpha. As(2)O(3) plus TNFalpha increases TNF receptor type 1 (TNF-R1) expression, decreases c-FLIP(L) expression, and causes caspase-8 and Bid activation, and apoptosis is reduced by anti-TNF-R1 neutralizing antibody and caspase-8 inhibitor. The treatment also causes Bax translocation to mitochondria, cytochrome c and Omi/HtrA2 release from mitochondria, XIAP down-regulation, and caspase-9 and caspase-3 activation. Bcl-2 over-expression inhibits cytochrome c release and apoptosis, and also prevents c-FLIP(L) down-regulation and caspase-8 activation, but not TNF-R1 over-expression. As(2)O(3) does not affect Akt phosphorylation/activation or intracellular GSH content, nor prevents the TNFalpha-provoked stimulation of p65-NF-kappaB translocation to the nucleus and the increase in NF-kappaB binding activity. Treatments with TNFalpha alone or with As(2)O(3) plus TNFalpha cause TNF-R1-mediated p38-MAPK phosphorylation/activation. P38-MAPK-specific inhibitors attenuate the As(2)O(3) plus TNFalpha-provoked activation of caspase-8/Bid, Bax translocation, cytochrome c release, and apoptosis induction. In conclusion, the sensitization by As(2)O(3) to TNFalpha-induced apoptosis in promonocytic leukemia cells is an Akt/NF-kappaB-independent, p38-MAPK-regulated process, which involves the interplay of both the receptor-mediated and mitochondrial executioner pathways.     

The antidepressants imipramine, clomipramine, and citalopram induce apoptosis in human acute myeloid leukemia HL-60 cells via caspase-3 activation.

Some widely used antidepressants such as imipramine, clomipramine, and citalopram have been found to possess antineoplastic effects. In the present study, these compounds were found to induce apoptotic cell death in human acute myeloid leukemia HL-60 cells. Apoptosis induced by the antidepressants was identified by electron microscopy and conventional agarose gel electrophoresis and was quantitated by propodium iodide staining and the terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) via flow cytometry. Treatment with apoptosis-inducing concentrations of the antidepressants (80 microM imipramine, 35 microM clomipramine, or 220 microM citalopram) caused induction of caspase-3/caspase-3-like activity, which was monitored by the cleavage of poly(ADP-ribose) polymerase (PARP), the loss of the 32 kD caspase-3 (CPP32) precursor, and the cleavage of the fluorescent CPP32-like substrate PhiPhiLux. Pretreatment with a potent caspase inhibitor benzyloxycarbonyl-Val-Ala-Asp-fluoromethyl-ketone (zVAD-fmk) inhibited antidepressant-induced CPP32/CPP32-like activity and apoptosis. Furthermore, activation of caspase induced by the antidepressants was preceded by the hypergeneration of intracellular reactive oxygen species (ROS). These results suggested that the antidepressants may induce apoptosis via a caspase-3-dependent pathway, and induction of apoptosis by the antidepressants may provide a clue for the mechanism of their antineoplastic effects.     

Zinc-mediated regulation of caspases activity: dose-dependent inhibition or activation of caspase-3 in the human Burkitt lymphoma B cells (Ramos)

Divalent cations, including Zinc and Manganese ions, are important modulators of cell activation. We investigated the ability of these two divalent cations to modulate apoptosis in human Burkitt lymphoma B cells line (Ramos). We found that Zinc (from 10 to 50 microM) inhibited Manganese-induced caspase-3 activation and apoptosis of Ramos cells. Higher concentration of Zinc (50 to 100 microM) did not prevent Manganese-mediated apoptosis but rather increased cell death among Ramos cells. This Zinc-mediated cell death was associated with apoptotic features such as cell shrinkage, the presence of phosphatidylserine residues on the outer leaflet of the cells, chromatin condensation, DNA fragmentation and decrease of mitochondrial transmembrane potential. Zinc-mediated apoptosis was associated with caspase-9 and caspase-3 activation as revealed by the appearance of active p35 fragment of caspase-9 and p19 and p17 of caspase-3 as well as in vivo cleavage of PARP and of a cell-permeable fluorogenic caspase-3 substrate (Phiphilux-G(1)D(2)). Both Zinc-mediated apoptosis and caspase-3 activation were prevented by the cell-permeable, broad-spectrum inhibitor of caspases (zVAD-fmk) or overexpression of bcl-2. In addition, we show that Zinc-induced loss of transmembrane mitochondrial potential is a caspase-independent event, since it is not modified by the presence of zVAD-fmk, which is inhibited by overexpression of bcl-2. These results indicate that depending on its concentration, Zinc can exert opposite effects on caspase-3 activation and apoptosis in human B lymphoma cells: concentrations below 50 microM inhibit caspase-3 activation and apoptosis whereas higher concentrations of Zinc activate a death pathway associated with apoptotic-like features and caspase-3 activation.     

Arisostatins A induces apoptosis through the activation of caspase-3 and reactive oxygen species generation in AMC-HN-4 cells

A microbial secondary metabolite, arisostatins A (As-A), was originally discovered as a substance carrying the antibiotic activity against Gram-positive bacteria and shown to possess potent anti-tumor properties. The mechanism by which arisostatins A initiates apoptosis remains poorly understood. In the present report we investigated the effect of arisostatins A on activation of the apoptotic pathway in HN-4 cells. Arisostatins A was shown to be responsible for the inhibition of HN-4 cell growth by inducing apoptosis. Treatment with 4 microM arisostatins A for 24h produced morphological features of apoptosis and DNA fragmentation in HN-4 cells. Arisostatins A caused dose-dependent apoptosis and DNA fragmentation of HN-4 cells used as a model. Treatment with caspase inhibitor significantly reduced the arisostatins A-induced caspase 3 activation. In addition, arisostatins A-induced apoptosis was associated with the generation of reactive oxygen species (ROS), which was prevented by an antioxidant NAC (N-acetyl-cysteine). These data indicate that cytotoxic effect of arisostatins A on HN-4 cells is attributable to the induced apoptosis and that arisostatins A-induced apoptosis is mediated by caspase-3 activation pathway, loss of mitochondrial transmembrane potential (DeltaPsi(m)), and release of cytochrome c into cytosol.     

Involvement of caspase-10 in advanced glycation end-product-induced apoptosis of bovine retinal pericytes in culture

Apoptosis appears to be the death mechanism of pericyte loss observed in diabetic retinopathy. We have previously shown that advanced glycation end-products (AGE-MGX) induce apoptosis of retinal pericytes in culture associated with diacylglycerol (DAG)/ceramide production. In the present study, we investigated possible caspase involvement in this process. Bovine retinal pericytes (BRP) were cultured with AGE-MGX and apoptosis examined after annexin V staining. Effects of peptidic inhibitors of caspases were determined on DAG/ceramide production and apoptosis. Pan-caspase inhibitor z-VAD-fmk (50 microM) was able to inhibit both DAG/ceramide production and apoptosis, whereas caspase-3-like inhibitor z-DEVD-fmk (50 microM) or caspase-9 inhibitor z-LEHD-fmk (50 microM) was only active on apoptosis. This differential effect strongly suggests involvement of initiator caspase(s) upstream and effector caspase(s) downstream DAG/ceramide production in AGE-mediated apoptosis. Pericyte treatment with caspase-8 inhibitor z-IETD-fmk (50 microM) did not protect cells against AGE-induced apoptosis and we failed to detect caspase-8 in pericytes by immunoblotting assay. Interestingly, one inhibitor of caspase-10 and related caspases z-AEVD-fmk (50 microM) inhibited both AGE-MGX-induced apoptosis and DAG/ceramide formation in pericytes. Cleavage of caspase-10 precursor into its active subunits was demonstrated by immunoblotting assay in pericytes incubated with AGE-MGX. These results strongly suggest that caspase-10, but not caspase-8, might be involved in the early phase of AGE-induced pericyte apoptosis, in contrast to caspase-9 and -3-like enzymes involved after DAG/ceramide production. This finding may provide new therapeutic perspectives for early treatment in diabetic retinopathy.     

Apigenin enhances the cytotoxic effects of tumor necrosis factor-related apoptosis-inducing ligand in human rheumatoid arthritis fibroblast-like synoviocytes

Activated rheumatoid arthritis (RA) fibroblast-like synoviocytes (RAFLSs) play a central role in both initiating and driving RA. Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) has been documented to induce apoptosis only in a small proportion of RAFLSs, which is followed by an induction of proliferation in surviving cells. Apigenin, a chemopreventive bioflavonoid, exhibits proapoptotic activity in many types of cells. In the present study, we sought to determine whether apigenin could enhance the cytotoxic effect of TRAIL on activated RAFLSs. Human RAFLSs isolated from patients with RA were treated with TRAIL (1 nM), apigenin (20 μM), or their combination, and subjected to apoptosis analysis after a 24-h incubation and proliferation analysis after a 72-h incubation. Apoptosis assay revealed that TRAIL or apigenin alone induced a marked apoptosis in RAFLS and their combination yielded a synergistic increase in RAFLS apoptosis. Immunoblotting analysis of apoptosis regulators demonstrated that combined treatment with apigenin increased caspase-3 expression and activity and decreased the Bcl-2/Bax ratio relative to treatment with TRAIL alone. The presence of apigenin significantly restrained TRAIL-induced RAFLS proliferation, coupled with restoration of the expression of two cell-cycle inhibitors p21 and p27. Moreover, the combination with apigenin blunted TRAIL-induced activation of the phosphatidylinositol 3-kinase (PI3-K)/Akt pathway. Our data collectively demonstrate that apigenin sensitizes RAFLS to TRAIL-induced apoptosis and counteracts TRAIL-dependent RAFLS proliferation, which is likely mediated through inactivation of PI3-K/Akt signaling pathway.     

Arsenic trioxide-induced apoptosis and its enhancement by buthionine sulfoximine in hepatocellular carcinoma cell lines

We treated four hepatocellular carcinoma cell lines, HLE, HLF, HuH7, and HepG2 with ATO and demonstrated that arsenic trioxide (ATO) at low doses (1--3 muM) induced a concentration-dependent suppression of cell growth in HLE, HLF, and HuH7. HLE cells underwent apoptosis at 2 microM ATO, which was executed by the activation of caspase-3 through the mitochondrial pathway mediated by caspase-8 activation and Bid truncation. When these cell lines were exposed to ATO in combination with l-S,R-buthionine sulfoximine (BSO) which inhibits GSH synthesis, a synergistic growth suppression was induced, even in HepG2 showing a lower sensitivity to ATO than other cell lines tested. The intracellular GSH levels after the treatment with ATO plus BSO were considerably decreased in HLE cells compared with those after the treatment with ATO or BSO alone. The production of reactive oxygen species (ROS) which was examined by 2' ,7' -dichlorodihydrofluorescein diacetate, increased significantly after the treatment with ATO plus BSO in HLE cells. These findings indicate that ATO at low concentrations induces growth inhibition and apoptosis, and furthermore that the ATO-BSO combination treatment enhances apoptosis through increased production of ROS in hepatocellular carcinoma cells.     

A novel sesquiterpenoid dimer parviflorene F induces apoptosis by up-regulating the expression of TRAIL-R2 and a caspase-dependent mechanism

Parviflorene F (1), a novel sesquiterpenoid dimer isolated from Curcuma parviflora Wall, is a cytotoxic compound. In this study, we examined the mechanism of its cytotoxic effect in HeLa cells. Treatment with 1 enhanced the mRNA and protein expression of TRAIL-R2 (tumor necrosis factor alpha-related apoptosis inducing ligand receptor 2). Apoptosis was induced by 1 as revealed by the distribution of DNA and Annexin V/PI staining using flow cytometry. In addition, 1-induced apoptosis was inhibited by human recombinant TRAIL-R2/Fc chimera protein, TRAIL-neutralizing fusion protein. Also, we found that 1 induced the activation of caspase-8, caspase-9, and caspase-3, indicating that the cytotoxic effect of 1 is correlated with apoptosis by a caspase-dependent mechanism through TRAIL-R2. In addition, 1 enhanced TRAIL-induced cell death against HeLa and TRAIL-resistant DLD1 cells. Taken together, up-regulation of TRAIL-R2 by 1 may contribute to sensitization of TRAIL-induced cell death.     

Histone deacetylase inhibitors potentiate TNF-related apoptosis-inducing ligand (TRAIL)-induced apoptosis in lymphoid malignancies

New therapies are required for chronic lymphocytic leukemia (CLL), an incurable disease characterized by failure of mature lymphocytes to undergo apoptosis. Activation of cell surface death receptors, such as via TRAIL receptor ligation, may provide a novel therapeutic target for various malignancies. However, CLL and other lymphoid malignancies are resistant to TRAIL. We report that low concentrations of histone deacetylase (HDAC) inhibitors, such as depsipeptide, which alone failed to induce apoptosis, markedly sensitize CLL cells and other primary lymphoid malignancies to TRAIL-induced apoptosis. These combinations caused little or no toxicity to normal lymphocytes. HDAC inhibitors sensitized resistant cells to TRAIL-induced apoptosis by facilitating formation of an active death-inducing signalling complex (DISC), leading to the rapid activation of caspase-8. The facilitated DISC formation also occurred in the absence of TRAIL-R2 upregulation. Thus, the combination of HDAC inhibitors and TRAIL may be valuable in the treatment of various hemopoietic malignancies.