Dimethyl suberimidate as a specific inductor of apoptosis in transformed cells

A modification of protein-protein interactions can be considered to be a way to regulate cell death. Chemical cross-linking agents have been traditionally used for protein complexing. This study has been undertaken to test a possibility to induce and(or) to modify cell death by a homobifunctional cross-linker dimethyl suberimidate (DMS). It was shown that the protein cross-linking by DMS resulted in a death of transformed cells by apoptosis. DMS-induced apoptosis was accompanied by cell cycle perturbations and down-regulation of p21/Waf1 mRNA expression. The RT-PCR analysis of bcl-2 family genes revealed the engagement of mitochondria in DMS-induced cytotoxicity. Then, the influence of DMS treatment on TNF-dependent and Fas-mediated apoptosis was investigated. Cell pre-incubation with DMS resulted in their increasing sensitivity for the TNF cytotoxic effect, though activities of anti-Fas cytotoxic antibodies were inhibited. The effects observed are probably due to cross-linking of TNF-receptors. Thus, this study first demonstrated that a chemical cross-linker DMS in capable of inducing apoptosis in transformed cells and modifying TNF-dependent and Fas-mediated apoptosis.     

Growth factors prevent changes in Bcl-2 and Bax expression and neuronal apoptosis induced by nitric oxide

Recent studies have shown that nitric oxide (NO) donors can trigger apoptosis of neurons, and growth factors such as insulin-like growth factor-1 (IGF-1) and basic fibroblast growth factor (bFGF) can protect against NO-induced neuronal cell death. The purpose of this study was to elucidate the possible mechanisms of NO-mediated neuronal apoptosis and the neuroprotective action of these growth factors. Both IGF-1 and bFGF prevented apoptosis induced by NO donors, sodium nitroprusside (SNP) or 3-morpholinosydnonimin (SIN-1) in hippocampal neuronal cultures. Incubation of neurons with SNP induced caspase-3-like activation following downregulation of Bcl-2 and upregulation of Bax protein levels in cultured neurons. Treatment of neurons with a bax antisense oligonucleotide inhibited the caspase-3-like activation and neuronal death induced by SNP. In addition, treatment of neurons with an inhibitor of caspase-3, Ac-DEVD-CHO, together with SNP did not affect the changes in the protein levels, although it inhibited NO-induced cell death. Pretreatment of cultures with either IGF-1 or bFGF prior to NO exposure inhibited caspase-3-like activation together with the changes in Bcl-2 and Bax protein levels. These results suggest that the changes in Bcl-2 and Bax protein levels followed by caspase-3-like activation are a component in the cascade of NO-induced neuronal apoptosis, and that the neuroprotective actions of IGF-1 and bFGF might be due to inhibition of the changes in the protein levels of the Bcl-2 family.     

Chelerythrine induces apoptosis through a Bax/Bak-independent mitochondrial mechanism

Although murine embryonic fibroblasts (MEFs) with Bax or Bak deleted displayed no defect in apoptosis signaling, MEFs with Bax and Bak double knock-out (DKO) showed dramatic resistance to diverse apoptotic stimuli, suggesting that Bax and Bak are redundant but essential regulators for apoptosis signaling. Chelerythrine has recently been identified as a Bcl-xL inhibitor that is capable of triggering apoptosis via direct action on mitochondria. Here we report that in contrast to classic apoptotic stimuli, chelerythrine is fully competent in inducing apoptosis in the DKO MEFs. Wild-type and DKO MEFs are equally sensitive to chelerythrine-induced morphological and biochemical changes associated with apoptosis phenotype. Interestingly, chelerythrine-mediated release of cytochrome c is rapid and precedes Bax translocation and integration. Although the BH3 peptide of Bim is totally inactive in releasing cytochrome c from isolated mitochondria of DKO MEFs, chelerythrine maintains its potency and efficacy in inducing direct release of cytochrome c from these mitochondria. Furthermore, chelerythrine-mediated mitochondrial swelling and loss in mitochondrial membrane potential (DeltaPsi(m)) are inhibited by cyclosporine A, suggesting that mitochondrial permeability transition pore is involved in chelerythrine-induced apoptosis. Although certain apoptotic stimuli have been shown to elicit cytotoxic effect in the DKO MEFs through alternate death mechanisms, chelerythrine does not appear to engage necrotic or autophagic death mechanism to trigger cell death in the DKO MEFs. These results, thus, argue for the existence of an alternative Bax/Bak-independent apoptotic mechanism that involves cyclosporine A-sensitive mitochondrial membrane permeability.     

Comparative studies on biological activity of certain microtubule-interacting taxanes

We have compared the nature of interaction of certain taxanes with microtubular protein, and the mechanism of action underlying cytotoxic activity. Taxanes induced tubulin assembly in vitro, but only taxanes bearing side chain were capable of inducing the formation of stable tubulin polymers. Electron microscopy detections showed that taxane-induced polymers are structurally similar to microtubules formed by paclitaxel, with differences in length. Otherwise, light microscopy views have shown that intracellular microtubule network is deeply reorganized by taxanes into short fibers, unlike paclitaxel-bundled microtubules. Taxanes inhibited the growth of various human tumor cell lines, but cell cycle analysis did not always indicate a block in the G2/M phase. These agents alter some apoptotic signal transduction pathways, probably by a mechanism distinct from microtubule interaction. Briefly, the effectiveness of taxanes is closely related to their chemical structure, and depends on their interaction with microtubular protein. By virtue of this mechanism, some of these taxanes may provide usefulness for therapeutic improvements.     

Peloruside A enhances apoptosis in H-ras-transformed cells and is cytotoxic to proliferating T cells

Peloruside A (peloruside), a compound isolated from the marine sponge Mycale hentscheli , inhibits growth of human (HL-60) and mouse (32D-ras) myeloid leukemic cells, as well as non-transformed 32D cells. Using the MTT cell proliferation assay and trypan blue dye exclusion tests, little difference was seen in growth inhibition between 32D and 32D- ras cells; however, peloruside was more cytotoxic to the oncogene-transformed cells. Peloruside also blocked 32D- ras cells more readily in G2/M of the cell cycle, leading to apoptosis. Annexin-V/propidium iodide staining of 32D and 32D- ras cells showed that 1.6 microM peloruside induced significant cell death by 36 hours in 32D cells (16% survival), but to comparable levels as early as 14 hours in 32D- ras cells (11% survival). There was no evidence for activation of either of the initiator caspases-8 or -9 by 0.1 microM peloruside following 12 hours of exposure. Peloruside inhibited T cell proliferation and IL-2 and IFN gamma production in both the mixed lymphocyte reaction and following CD3 cross-linking, and this effect was shown to be a non-specific cytotoxic effect. It is concluded that peloruside preferentially targets oncogene-transformed cells over non-transformed cells by inducing transformed cells to undergo apoptosis.     

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.     

4-Hydroxynonenal modulation of p53 family gene expression in the SK-N-BE neuroblastoma cell line

4-Hydroxynonenal (HNE), a product of lipid peroxidation, inhibits proliferation of several tumor cells. The p53 tumor suppressor protein plays a critical role in cell cycle control, by inducing p21 expression, and in apoptosis, by inducing bax expression. Recently, two other proteins with many p53-like properties, TAp73 (p73) and TAp63 (p63), have been discovered. SK-N-BE human neuroblastoma cells express the three p53 family proteins and can be used for the study of their induction. We investigated HNE action in the control of proliferation, differentiation, and apoptosis in SK-N-BE cells and the HNE effect on the expression of p53, p63, p73, p21, bax, and G1 cyclins. Retinoic acid (RA) was used as a positive control. HNE inhibited cell proliferation without inducing differentiation; it decreased S-phase cells and increased the number of apoptotic cells. RA reduced the proportion of S-phase cells and did not induce apoptosis. HNE increased p53, p73, p63, p21, and bax expression at different time points. HNE reduced cyclin D2 expression and the phosphorylation of pRb protein. Our results demonstrated that HNE inhibits SK-N-BE cell proliferation by increasing the expression of p53 family proteins and p53 target proteins which modulate cell cycle progression and apoptosis.     

Bax-dependent apoptosis induced by ceramide in HL-60 cells

Ceramide is an important lipid messenger involved in mediating a variety of cell functions including apoptosis. In this study, we show that antisense bax inhibits cytochrome c release, poly(ADP-ribose)polymerase cleavage and cell death induced by ceramide in HL-60 cells. In addition, ceramide induces translocation of Bax to mitochondria. The addition of the broad spectrum caspase inhibitor zVAD-fmk prevented ceramide-induced apoptotic cell death but did not inhibit translocation of Bax and mitochondrial cytochrome c release. Furthermore, ceramide inhibits the expression of the antiapoptotic protein Bcl-xL with an increase in the ratio of Bax to Bcl-xL. These data provide direct evidence that Bax plays an important role in regulating ceramide-induced apoptosis.     

CrossWork: software-assisted identification of cross-linked peptides

The increased interest in chemical cross-linking for probing protein structure and interaction has led to a large increase in literature describing new cross-linkers and search programs. However, this has not led to a corresponding increase in the analysis of large and complex proteins. A major obstacle is that the new cross-linkers are either not readily available and/or have a low reactivity. In combination with aging search programs that are slow and have low sensitivity, or new search programs that are described but not released, these efforts do little to advance the field of cross-linking. Here we present a method pipeline for chemical cross-linking, using two standard cross-linkers, BS3 and BS2G, combined with our freely available CrossWork search program. By this approach we generate cross-link data sufficient to derive structural information for large and complex proteins. CrossWork searches batches of tandem mass-spectrometric data, and identifies cross-linked and non-cross-linked peptides using a standard PC. We tested CrossWork by searching mass-spectrometric datasets of cross-linked complement factor C3 against small (1 protein) and large (1000 proteins) search spaces, and show that the resulting distance constraints agree with the established structures. We further investigated the structure of the multi-domain ERp72, and combined the individual domains of ERp72 into a single structure.     

Signalling pathways involved in the direct effects of IGFBP-5 on breast epithelial cell attachment and survival

We have demonstrated previously that IGFBP-5 can confer survival against apoptosis induced by ceramide, C2, or a small synthetic arginine-glycine-aspartic acid (RGD)-containing peptide in a direct manner. The endogenous ceramide-induced pathway is normally counter-balanced by survival signals mediated by sphingosine kinase (SK) and protein kinase C (PKC). In order to investigate whether these pathways are involved in the IGFBP-5 survival effect, we have used inhibitors of SK (N, N-di-methyl sphingosine, DMS) and PKC (chelerythrine chloride, CC). The effect of pre-incubating Hs578T breast cancer cells with IGFBP-5 on cell adhesion or on subsequent cell death induced by C2 or RGD was investigated with and without the presence of DMS or CC. Cell death was determined by trypan blue cell counts and apoptosis confirmed by morphological assessment and flow cytometry. Cell attachment was determined by a cell adhesion assay. The presence of IGFBP-5 significantly inhibited cell death induced by C2 or RGD, compared to the triggers of apoptosis alone (P<0.01 in both cases). In the presence of either IGFBP-5, CC or DMS, there was no significant effect on cell death compared to the control. IGFBP-5 in the presence of either inhibitor resulted in a significant increase in cell death; IGFBP-5 also lost its ability to confer survival on C2 and RGD-induced apoptosis and in contrast significantly increased cell death. In the cell adhesion assay, IGFBP-5 significantly increased cell attachment over basal levels. In the presence of either inhibitor the IGFBP-5 effect on cell adhesion was reversed and cell attachment was reduced to below basal levels. These data suggest that IGFBP-5 promotes the attachment and survival of Hs578T cells by modulating the balance between ceramide and opposing survival signals.     

bcl-2和bax及NF-kB在白藜芦醇诱导肝癌细胞凋亡中的作用

目的探讨白藜芦醇诱导肝癌细胞凋亡的途径。方法白藜芦醇体外处理HepG2肝癌细胞24h后,以免疫组化检测凋亡调控基因bc1-2和bax及NF-kB的表达。结果白藜芦醇处理组HepG2细胞bc1-2的阳性积分和NF-kB的阳性细胞密度均明显低于对照组(P<0.01);而bax阳性积分明显高于对照组(P<0.01)。结论白藜芦醇能下调HepG2细胞bc1-2基因的表达,上调bax的表达,同时抑制NF-kB的活化,这可能是其诱导HepG2细胞凋亡的途径之一。     

Persian shallot, <Emphasis Type="Italic">Allium hirtifolium</Emphasis> Boiss,induced apoptosis in human hepatocellular carcinoma cells

This study investigated the potential of Persian shallot extract as an anticancer agent in HepG2 tumor cell line, an in vitro human hepatoma cancer model system. The inhibitory effect of Persian shallot on the growth of HepG2 cells was measured by MTT assay. To explore the underlying mechanism of cell growth inhibition of Persian shallot, the activity of Persian shallot in inducing apoptosis was investigated through the detection of annexin V signal by flow cytometry and expression of some apoptosis related genes such p21, p53, puma, caspase-8 family-Bcl-2 proteins like bid, bim, bcl-2 and bax were measured by real-time PCR in HepG2 cells. Persian shallot extract inhibited the growth of HepG2 cells in a dose-dependent manner. The IC₅₀ value (inhibiting cell growth by 50%) was 149 μg/ml. The results of real-time PCR revealed a significant up-regulation of bid, bim, caspase-8, puma, p53, p21 and bax genes and a significant downregulation of bcl-2 gene in HepG2 cells treated with Persian shallot extract significantly. Therefore, this is the first report on an increased expression of bid, bim, caspase-8, puma, p53, p21 and bax genes and down regulation of bcl-2 gene indicating that the Persian shallot extract possibly induced the process of cell death through the intrinsic and extrinsic apoptosis pathways and triggers the programmed cell death in HepG2 tumor cell lines by modulating the expression of pro-/anti-apoptotic genes. Furthermore, we showed that Persian shallot extract increased annexin V signal and expression, resulting in apoptotic cell death of HepG2 cells after 24 h treatment. Therefore, according to the results of this study, the Persian shallot extract could be considered as a potential candidate for production of drug for the prevention or treatment of human hepatoma.     

Monascus-fermented red mold rice exhibits cytotoxic effect and induces apoptosis on human breast cancer cells

Red mold rice (RMR) is a traditional food and folk medicine to Asian people and has recently become a popular health supplement. RMR has been shown to have some anticancer activities, although the mechanism for inducing cell death of human breast cancer cells is still not fully understood. In this study, bioactive extracts of RMR fermented by Monascus purpureus NTU 803 were analyzed for effects on apoptosis induction in human breast cancer cells. The RMR ethanol extract and ethyl acetate extract contain monacolin K, total phenols, and flavonoids, the three components that have been reported to have anticancer activity. Red mold rice extracts (RMRE) exhibited selective cytotoxic effect on MCF-7 cells. RMRE treatment induced apoptosis and cell cycle arrest at G2/M phase. Apoptosis was confirmed by annexin V–fluorescein isothiocyanate (FITC)/propidium iodide staining, the observation of characteristic chromatin condensation, nuclear DNA fragmentation, and poly(ADP-ribose) polymerase cleavage. Furthermore, the RMRE-induced apoptosis in MCF-7 cells may occur through a mitochondria-dependent pathway while triggering an appropriate balance of bax/bcl-2 and activation of caspase-9 and caspase-3 in a time-dependent manner. To conclude, RMRE exhibits direct cytotoxic and proapoptotic effects on MCF-7 cells and could be considered as a potential functional food for breast cancer prevention.     

Lithium sensitizes tumor cells in an NF-kappa B-independent way to caspase activation and apoptosis induced by tumor necrosis factor (TNF). Evidence for a role of the TNF receptor-associated death domain protein

We have previously shown that lithium salts can considerably increase the direct cytotoxic effect of tumor necrosis factor (TNF) on various tumor cells in vitro and in vivo. However, the underlying mechanism has remained largely unknown. Here we show that the TNF-sensitizing effect of lithium chloride (LiCl) is independent of the type of cell death, either necrosis or apoptosis. In the case of apoptosis, TNF/lithium synergism is associated with an enhanced activation of caspases and mitochondrial cytochrome c release. Sensitization to apoptosis is specific for TNF-induced apoptosis, whereas Fas-mediated or etoposide-induced apoptosis remains unaffected. LiCl also potentiates cell death induced by artificial oligomerization of a fusion protein between FKBP and the TNF receptor-associated death domain protein. TNF-induced activation of NF-kappa B-dependent gene expression is not modulated by LiCl treatment. These results indicate that LiCl enhances TNF-induced cell death in an NF-kappa B-independent way, and suggest that the TNF receptor-associated death domain protein plays a crucial role in the TNF-sensitizing effect of LiCl.     

Ribosome inactivating proteins and apoptosis

Ribosome inactivating proteins (RIPs) are protein toxins that are of plant or microbial origin that inhibit protein synthesis by inactivating ribosomes. Recent studies suggest that RIPs are also capable of inducing cell death by apoptosis. Though many reports are available on cell death induced by RIPs, the mechanism involved is not well studied. Comparison of pathways of apoptosis and cellular events induced by various RIPs suggests a central role played by mitochondria, probably acting as an integrator of cellular stress and cell death. The purpose of this review is to compare the various apoptotic pathways that may be involved and propose a general pathway in RIP-induced cell death.     

Actin cytoskeleton is required for early apoptosis signaling induced by anti-Fas antibody but not Fas ligand in murine B lymphoma A20 cells.

Murine B lymphoma A20 cells are highly sensitive to Fas-mediated death signals induced by anti-Fas antibody Jo2 or cross-linked Fas ligand (FasL). We have found that the microfilament poison cytochalasin D blocks Fas-mediated apoptosis induced by Jo2 but not FasL in A20 cells. The induction of Fas-mediated apoptosis by Jo2 was antagonized by anti-Fcgamma RII/RIII receptor (FcgammaR) antibody, and defective in FcgammaR-negative A20 cells. Since the induction of Jo2-mediated apoptosis in FcgammaR-negative A20 cells was reversed by the addition of wild type A20 cells or the cross-linking agent protein A, Fas-expressing bystander A20 cells seem to be killed by other A20 cells that capture and cross-link monomeric Jo2 via FcgammaR. Although cytochalasin D affected FcgammaR-mediated cross-linking of Jo2 molecules, the drug markedly inhibited the intracellular signaling pathway induced by Jo2. The blockade of Jo2-induced apoptosis by cytochalasin D occurred upstream of caspase-8 activation. Thus, these observations suggest that actin cytoskeleton is required for early apoptosis signaling induced by Jo2, but not physiological FasL.     

Monocyte Derived Microvesicles Deliver a Cell Death Message via Encapsulated Caspase-1

Apoptosis depends upon the activation of intracellular caspases which are classically induced by either an intrinsic (mitochondrial based) or extrinsic (cytokine) pathway. However, in the process of explaining how endotoxin activated monocytes are able to induce apoptosis of vascular smooth muscle cells when co-cultured, we uncovered a transcellular apoptosis inducing pathway that utilizes caspase-1 containing microvesicles. Endotoxin stimulated monocytes induce the cell death of VSMCs but this activity is found in 100,000 g pellets of cell free supernatants of these monocytes. This activity is not a direct effect of endotoxin, and is inhibited by the caspase-1 inhibitor YVADcmk but not by inhibitors of Fas-L, IL-1β and IL-18. Importantly, the apoptosis inducing activity co-purifies with 100 nm sized microvesicles as determined by TEM of the pellets. These microvesicles contain caspase-1 and caspase-1 encapsulation is required since disruption of microvesicular integrity destroys the apoptotic activity but not the caspase-1 enzymatic activity. Thus, monocytes are capable of delivering a cell death message which depends upon the release of microvesicles containing functional caspase-1. This transcellular apoptosis induction pathway describes a novel pathway for inflammation induced programmed cell death.