The avian ChB6 alloantigen triggers apoptosis in a mammalian cell line

Many developing B lymphocytes are deleted by apoptosis. However, the mechanism signaling their demise remains poorly understood. Like mammals, chicken B cells are selected during their development; >95% of the cells in the bursa of Fabricius die without entering the secondary immune system. The molecule chB6 (Bu-1) has been used as a marker to identify B cells in the chicken. ChB6 is a type I transmembrane glycoprotein whose function is enigmatic. We have provided evidence that chB6 can induce a rapid form of cell death exhibiting characteristics of apoptosis. Here we further examine cell death induced by chB6 in a transfected mouse cell line. ChB6 is shown to cause apoptosis in this cell line as detected by a TUNEL assay for DNA fragmentation. This apoptosis is subject to regulation by signals from growth factor or by Bcl-x(L). Furthermore, we show that Ab binding to chB6 leads to cleavage of caspase 8, caspase 3, and poly(ADP ribose) polymerase. Overall, these data support the hypothesis that chB6 is a novel death receptor on avian B cells.     

LIGHT/IFN‐γ triggers β cells apoptosis via NF‐κB/Bcl2‐dependent mitochondrial pathway

LIGHT recruits and activates naive T cells in the islets at the onset of diabetes. IFN‐γ secreted by activated T lymphocytes is involved in beta cell apoptosis. However, whether LIGHT sensitizes IFNγ‐induced beta cells destruction remains unclear. In this study, we used the murine beta cell line MIN6 and primary islet cells as models for investigating the underlying cellular mechanisms involved in LIGHT/IFNγ – induced pancreatic beta cell destruction. LIGHT and IFN‐γ synergistically reduced MIN6 and primary islet cells viability; decreased cell viability was due to apoptosis, as demonstrated by a significant increase in Annexin V⁺ cell percentage, detected by flow cytometry. In addition to marked increases in cytochrome c release and NF‐κB activation, the combination of LIGHT and IFN‐γ caused an obvious decrease in expression of the anti‐apoptotic proteins Bcl‐2 and Bcl‐xL, but an increase in expression of the pro‐apoptotic proteins Bak and Bax in MIN6 cells. Accordingly, LIGHT deficiency led to a decrease in NF‐κB activation and Bak expression, and peri‐insulitis in non‐obese diabetes mice. Inhibition of NF‐κB activation with the specific NF‐κB inhibitor, PDTC (pyrrolidine dithiocarbamate), reversed Bcl‐xL down‐regulation and Bax up‐regulation, and led to a significant increase in LIGHT‐ and IFN‐γ‐treated cell viability. Moreover, cleaved caspase‐9, ‐3, and PARP (poly (ADP‐ribose) polymerase) were observed after LIGHT and IFN‐γ treatment. Pretreatment with caspase inhibitors remarkably attenuated LIGHT‐ and IFNγ‐induced cell apoptosis. Taken together, our results indicate that LIGHT signalling pathway combined with IFN‐γ induces beta cells apoptosis via an NF‐κB/Bcl2‐dependent mitochondrial pathway.     

Direct stimulation of poly(ADP ribose) polymerase in permeabilized cells by double-stranded DNA oligomers

Poly(ADP ribosyl)ation, a post-translational modification of nuclear proteins catalyzed by poly (ADP ribose) polymerase, is an immediate response of most eukaryotic cells to DNA strand breaks and has been implicated in DNA repair and other cellular phenomena associated with DNA strand breakage. Poly(ADP ribose) polymerase activity levels have been frequently assayed by incubating permeabilized cells with radioactively labeled NAD+ as substrate. In such assays enzyme activation has routinely been achieved indirectly by prior exposure of living cells to carcinogens or by adding DNase I to permeabilized cells, thereby introducing strand breaks in chromosomal DNA. Here we show that, as an alternative method, the direct activation of purified poly(ADP ribose) polymerase by double-stranded oligonucleotides (N. A. Berger and S. I. Petzold, 1985, Biochemistry 24, 4352-4355) can be adopted for permeabilized cell systems. The inclusion of a palindromic decameric deoxynucleotide in the reaction buffer stimulated the enzyme activity in permeabilized Molt-3 human lymphoma cells up to 30-fold (at 50 micrograms/ml [corrected] oligonucleotide concentration) in a concentration-dependent manner. The activating effect of oligonucleotides was also evident when ethanol-fixed HeLa cells were postincubated with NAD+ to allow poly(ADP ribose) synthesis to occur in situ, which was detected as specific anti-poly (ADP ribose) immunofluorescence. We conclude that double-stranded oligonucleotides can be conveniently used as chemically and stoichiometrically well-defined poly (ADP ribose) polymerase activators in permeabilized or ethanol-fixed mammalian cells.     

Nitric oxide induces apoptosis in cutaneous T cell lymphoma (HuT-78) by downregulating constitutive NF-κB

Constitutive active NF-κB have been shown to protect cutaneous T cell lymphoma (CTCL) cells from apoptosis. In the present study, we have studied the cytotoxic potential of nitric oxide generating compound, sodium nitroprusside (SNP) on CTCL cell line, HuT-78. Treatment of cells with SNP resulted in decrease in mitochondrial membrane potential, cytochrome c release, activation of caspase-3 and poly (ADP ribose) polymerase cleavage. SNP treatment inhibited activation of NF-κB in a concentration dependent manner. SNP increased the expression of IκBα without affecting the phosphorylation of IκBα. Downregulation of NF-κB by SNP decreased p65 nuclear translocation as evident by confocal fluorescence microscopy. Further it was found that SNP treatment caused downregulation of Bcl-2 family member (Bcl-xl) in HuT-78 cells. Thus, we have provided evidence that SNP induces apoptosis in CTCL cell line, HuT-78 by downregulating constitutive NF-κB and thereby Bcl-xl expression.     

Swainsonine Induces Caprine Luteal Cells Apoptosis via Mitochondrial‐Mediated Caspase‐Dependent Pathway

Swainsonine (SW) is an indolizidine alkaloid isolated from a number of poisonous plants. We have previously reported that SW inhibited luteal cell progesterone production by inducing caprine luteal cell apoptosis in vitro; however, the molecular mechanism of this phenomenon remains unclear. In this study, SW‐treated luteal cells showed apoptosis characteristics, including nuclear fragmentation, DNA ladder formation, and phosphatidylserine externalization. Further studies showed that SW activated caspase‐9 and caspase‐3, which subsequently cleaved poly(ADP‐ribose) polymerase. SW also increased in Bax/BcL‐2 ratios, promoted Bax translocation from the cytosol to mitochondria, and triggered the release of cytochrome c from mitochondria into the cytoplasm. However, Fas and Fas ligand induction or caspase‐8 activity did not appear any significant changes. Additional analysis also showed that pan‐caspase inhibitor, caspase‐9 inhibitor, or caspase‐3 inhibitor almost completely protected the cells from SW‐induced apoptosis, but not caspase‐8 inhibitor. Overall, these data demonstrated that SW induced luteal cells apoptosis through a mitochondrial‐mediated caspase‐dependent pathway.     

Imiquimod‐induced apoptosis of melanoma cells is mediated by ER stress‐dependent Noxa induction and enhanced by NF‐κB inhibition

Melanoma is characterized by dysregulated intracellular signalling pathways including an impairment of the cell death machinery, ultimately resulting in melanoma resistance, survival and progression. This explains the tumour's extraordinary resistance to the standard treatment. Imiquimod is a topical immune response modifier (imidazoquinoline) with both antiviral and antitumour activities. The mechanism by which imiquimod triggers the apoptosis of melanoma cells has now been carefully elucidated. Imiquimod‐induced apoptosis is associated with the activation of apoptosis signalling regulating kinase1/c‐Jun‐N‐terminal kinase/p38 pathways and the induction of endoplasmic stress characterized by the activation of the protein kinase RNA‐like endoplasmic reticulum kinase signalling pathway, increase in intracellular Ca²⁺ release, degradation of calpain and subsequent cleavage of caspase‐4. Moreover, imiquimod triggers the activation of NF‐κB and the expression of the inhibitor of apoptosis proteins (IAPs) such as, X‐linked IAP (XIAP) together with the accumulation of reactive oxygen species (ROS). Also, imiquimod triggers mitochondrial dysregulation characterized by the loss of mitochondrial membrane potential (Δψm), the increase in cytochrome c release, and cleavage of caspase‐9, caspase‐3 and poly(ADP‐ribose) polymerase (PARP). Inhibitors of specific pathways, permit the elucidation of possible mechanisms of imiquimod‐induced apoptosis. They demonstrate that inhibition of NF‐kB by the inhibitor of nuclear factor kappa‐B kinase (IKK) inhibitor Bay 11‐782 or knockdown of XIAP induces melanoma apoptosis in cells exposed to imiquimod. These findings support the use of either IKK inhibitors or IAP antagonists as adjuvant therapies to improve the effectiveness topical imiquimod in the treatment of melanoma.     

Poly (ADP‐ribose) polymerase inhibition protects against myocardial ischaemia/reperfusion injury via suppressing mitophagy

Myocardial ischaemia/reperfusion (I/R) injury attenuates the beneficial effects of reperfusion therapy. Poly(ADP‐ribose) polymerase (PARP) is overactivated during myocardial I/R injury. Mitophagy plays a critical role in the development of myocardial I/R injury. However, the effect of PARP activation on mitophagy in cardiomyocytes is unknown. In this study, we found that I/R induced PARP activation and mitophagy in mouse hearts. Poly(ADP‐ribose) polymerase inhibition reduced the infarct size and suppressed mitophagy after myocardial I/R injury. In vitro, hypoxia/reoxygenation (H/R) activated PARP, promoted mitophagy and induced cell apoptosis in cardiomyocytes. Poly(ADP‐ribose) polymerase inhibition suppressed H/R‐induced mitophagy and cell apoptosis. Parkin knockdown with lentivirus vectors inhibited mitophagy and prevented cell apoptosis in H/R‐treated cells. Poly(ADP‐ribose) polymerase inhibition prevented the loss of the mitochondrial membrane potential (ΔΨm). Cyclosporin A maintained ΔΨm and suppressed mitophagy but FCCP reduced the effect of PARP inhibition on ΔΨm and promoted mitophagy, indicating the critical role of ΔΨm in H/R‐induced mitophagy. Furthermore, reactive oxygen species (ROS) and poly(ADP‐ribosylation) of CypD and TSPO might contribute to the regulation of ΔΨm by PARP. Our findings thus suggest that PARP inhibition protects against I/R‐induced cell apoptosis by suppressing excessive mitophagy via the ΔΨm/Parkin pathway.     

Theileria parva-transformed T cells show enhanced resistance to Fas/Fas ligand-induced apoptosis

Lymphocyte homeostasis is regulated by mechanisms that control lymphocyte proliferation and apoptosis. Activation-induced cell death is mediated by the expression of death ligands and receptors, which, when triggered, activate an apoptotic cascade. Bovine T cells transformed by the intracellular parasite Theileria parva proliferate in an uncontrolled manner and undergo clonal expansion. They constitutively express the death receptor Fas and its ligand, FasL but do not undergo apoptosis. Upon elimination of the parasite from the host cell by treatment with a theilericidal drug, cells become increasingly sensitive to Fas/FasL-induced apoptosis. In normal T cells, the sensitivity to death receptor killing is regulated by specific inhibitor proteins. We found that anti-apoptotic proteins such as cellular (c)-FLIP, which functions as a catalytically inactive form of caspase-8, and X-chromosome-linked inhibitor of apoptosis protein (IAP) as well as c-IAP, which can block downstream executioner caspases, are constitutively expressed in T. parva-transformed T cells. Expression of these proteins is rapidly down-regulated upon parasite elimination. Antiapoptotic proteins of the Bcl-2 family such as Bcl-2 and Bcl-x(L) are also expressed but, in contrast to c-FLIP, c-IAP, and X-chromosome-linked IAP, do not appear to be tightly regulated by the presence of the parasite. Finally, we show that, in contrast to the situation in tumor cells, the phosphoinositide 3-kinase/Akt pathway is not essential for c-FLIP expression. Our findings indicate that by inducing the expression of antiapoptotic proteins, T. parva allows the host cell to escape destruction by homeostatic mechanisms that would normally be activated to limit the continuous expansion of a T cell population.     

In vivo analysis reveals different apoptotic pathways in pre‐ and postmigratory cerebellar granule cells of rabbit

Naturally occurring neuronal death (NOND) has been described in the postnatal cerebellum of several species, mainly affecting the cerebellar granule cells (CGCs) by an apoptotic mechanism. However, little is known about the cellular pathway(s) of CGC apoptosis in vivo. By immunocytochemistry, in situ detection of fragmented DNA, electron microscopy, and Western blotting, we demonstrate here the existence of two different molecular mechanisms of apoptosis in the rabbit postnatal cerebellum. These two mechanisms affect CGCs at different stages of their maturation and migration. In the external granular layer, premigratory CGCs undergo apoptosis upon phosphorylation of checkpoint kinase 1 (Chk1), and hyperphosphorylation of retinoblastoma protein. In postmigratory CGCs within the internal granular layer, caspase 3 and to a lesser extent 7 and 9 are activated, eventually leading to poly‐ADP‐ribose polymerase‐1 (PARP‐1) cleavage and programmed cell death. We conclude that NOND of premigratory CGCs is linked to activation of DNA checkpoint and alteration of normal cell cycle, whereas in postmigratory CGCs apoptosis is, more classically, dependent upon caspase 3 activation. © 2004 Wiley Periodicals, Inc. J Neurobiol 60: 437–452, 2004     

Comparative cellular and molecular analysis of cytotoxicity and apoptosis induction by doxorubicin and <Emphasis Type="Italic">Baneh</Emphasis> in human breast cancer T47D cells

It is now widely accepted that dietary phytochemicals inhibit cancer progression and enhance the effects of conventional chemotherapy. In this report, we comparatively studied the cellular and molecular aspects of apoptosis induction by the methanolic extract of Baneh fruit skin in comparison to Doxorubicin (Dox), a well-known anticancer drug, in human breast cancer T47D cells. The MTT assay was used to determine the antiproliferative effects. The flow cytometric and microscopic analyses were done to evaluate the apoptosis induction. Furthermore, western blot analyses have been done to study the role of key molecular players of apoptosis including caspase 3 and PARP. The Baneh extract showed strong antiproliferative activity against T47D cells in a dose- and time-dependent manner that was comparable to and even stronger than Dox in certain concentrations. Analysis of Baneh-treated cells by flow cytometry and fluorescence microscopy indicated strong apoptosis induction and nuclear morphological alterations similar to or greater than Dox. Finally, molecular analysis of apoptosis by western blotting proved activation of caspase 3 followed by poly ADP ribose polymerase (PARP) cleavage more efficiently in Baneh than in Dox treated cancer cells. These findings indicate that Baneh extract contains phytochemicals which act as inhibitor of cell proliferation and inducer of apoptosis in human breast cancer T47D cells that makes it a potentially good candidate for new anticancer drug development.     

Light‐emitting diode irradiation induces AKT/mTOR‐mediated apoptosis in human pancreatic cancer cells and xenograft mouse model

The beneficial effects of light‐emitting diode (LED) irradiation have been reported in various pathologies, including cancer. However, its effect in pancreatic cancer cells remains unclear. Herein, we demonstrated that blue LED of 460 nm regulated pancreatic cancer cell proliferation and apoptosis by suppressing the expression of apoptosis‐related factors, such as mutant p53 and B‐cell lymphoma 2 (Bcl‐2), and decreasing the expression of RAC‐β serine/threonine kinase 2 (AKT2), the phosphorylation of protein kinase B (AKT), and mammalian target of rapamycin (mTOR). Blue LED irradiation also increased the levels of cleaved poly‐(ADP‐ribose) polymerase (PARP) and caspase‐3 in pancreatic cancer cells, while it suppressed AKT2 expression and inhibited tumor growth in xenograft tumor tissues. In conclusion, blue LED irradiation suppressed pancreatic cancer cell and tumor growth by regulating AKT/mTOR signaling. Our findings indicated that blue LEDs could be used as a nonpharmacological treatment for pancreatic cancer.     

Nitric oxide induces apoptosis in cutaneous T cell lymphoma (HuT-78) by downregulating constitutive NF-kappaB

Constitutive active NF-kappaB have been shown to protect cutaneous T cell lymphoma (CTCL) cells from apoptosis. In the present study, we have studied the cytotoxic potential of nitric oxide generating compound, sodium nitroprusside (SNP) on CTCL cell line, HuT-78. Treatment of cells with SNP resulted in decrease in mitochondrial membrane potential, cytochrome c release, activation of caspase-3 and poly (ADP ribose) polymerase cleavage. SNP treatment inhibited activation of NF-kappaB in a concentration dependent manner. SNP increased the expression of IkappaBalpha without affecting the phosphorylation of IkappaBalpha. Downregulation of NF-kappaB by SNP decreased p65 nuclear translocation as evident by confocal fluorescence microscopy. Further it was found that SNP treatment caused downregulation of Bcl-2 family member (Bcl-xl) in HuT-78 cells. Thus, we have provided evidence that SNP induces apoptosis in CTCL cell line, HuT-78 by downregulating constitutive NF-kappaB and thereby Bcl-xl expression.     

Inhibition of taurolithocholate 3-sulfate-induced apoptosis by cyclic AMP in rat hepatocytes involves protein kinase A-dependent and -independent mechanisms

The mechanisms underlying the inhibition of bile acid-induced apoptosis by cyclic AMP (cAMP) were studied in 24-h-cultured rat hepatocytes. Taurolithocholate 3-sulfate (TLCS, 100 micromol/l) led to a sustained activation of mitogen activated protein (MAP) kinases (JNK, p38(MAPK), and ERKs), dephosphorylation of protein kinase B (PKB), activation of caspases 3 and 8, and hepatocyte apoptosis. cAMP prevented TLCS-induced apoptosis, shifted the persistent TLCS-induced MAP kinase response to a transient pattern, and prevented PKB dephosphorylation. TLCS-induced CD95 and TRAIL receptor-2 trafficking to the plasma membrane were significantly inhibited. Blockade of protein kinase A (PKA) abolished the inhibitory effect of cAMP on TLCS-induced CD95 membrane targeting, but not TRAIL receptor-2 membrane targeting, PKB and MAP kinase responses. H89, an inhibitor of PKA, had no effect on cAMP-induced inhibition of TLCS-triggered poly(ADP) ribose polymerase (PARP) cleavage and caspase activation, but abolished the cAMP-induced inhibition of TLCS-triggered TUNEL- and Annexin V staining. It is concluded that cAMP inhibits bile acid-induced apoptosis via PKA-dependent and -independent mechanisms.     

PSAP induces a unique Apaf-1 and Smac-dependent mitochondrial apoptotic pathway independent of Bcl-2 family proteins

Presenilin-associated protein (PSAP) has been identified as a mitochondrial proapoptotic protein. However, the mechanism by which PSAP induces apoptosis remains unknown. To this end, we have established an inducible expression system. Using this system, we have examined the roles of B-cell lymphoma 2 (Bcl-2) family proteins, cytochrome c, Smac (Smac/Diablo, second mitochondria-derived activator of caspases/direct IAP binding protein with low PI), and Apaf-1 (apoptotic protease-activating factor) in PSAP-induced apoptosis. Our results demonstrate that knockdown of Apaf-1 abolished PSAP-induced caspase activation and poly(ADP ribose) polymerase (PARP) cleavage, indicating that the apoptosome formation triggered by cytochrome c is crucial for PSAP-induced apoptosis. Our data also demonstrate that knockdown of Smac abolished PSAP-induced caspase activation and PARP cleavage, indicating that, in addition to Apaf-1 or apoptosome formation, Smac is also essential for PSAP-induced apoptosis. However, interestingly, our data demonstrate that overexpression of Bcl-2 and Bcl-xL did not protect cells from PSAP-induced apoptosis, and that knockdown of Bid, Bax, and Bak had no effect on PSAP-induced cytochrome c and Smac release, indicating that PSAP-induced apoptosis is not regulated by Bcl-2 family proteins. These results strongly suggest that PSAP evokes mitochondrial apoptotic cascades via a novel mechanism that is not regulated by Bcl-2 family proteins, but that both the formation of cytochrome c-Apaf-1 apoptosome and the presence of Smac are absolutely required for PSAP-induced apoptosis.     

Rhein induces apoptosis in HL-60 cells via reactive oxygen species-independent mitochondrial death pathway

Rhein is an anthraquinone compound enriched in the rhizome of rhubarb, a traditional Chinese medicine herb showing anti-tumor promotion function. In this study, we first reported that rhein could induce apoptosis in human promyelocytic leukemia cells (HL-60), characterized by caspase activation, poly(ADP)ribose polymerase (PARP) cleavage, and DNA fragmentation. The efficacious induction of apoptosis was observed at 100 microM for 6h. Mechanistic analysis demonstrated that rhein induced the loss of mitochondrial membrane potential (DeltaPsi(m)), cytochrome c release from mitochondrion to cytosol, and cleavage of Bid protein. Rhein also induced generation of reactive oxygen species (ROS) and the phosphorylation of c-Jun N-terminal kinase (JNK) and p38 kinase. However, these actions seem not to be associated with the apoptosis induction because antioxidants including N-acetyl cysteine (NAC), Tiron, and catalase did not block rhein-induced apoptosis, although they could block the generation of ROS and the phosphorylation of JNK and p38 kinase. Our data demonstrate that rhein induces apoptosis in HL-60 cells via a ROS-independent mitochondrial death pathway.     

Spatiotemporal activation of caspase‐dependent and ‐independent pathways in staurosporine‐induced apoptosis of p53wt and p53mt human cervical carcinoma cells

Background information. Caspase‐dependent and ‐independent death mechanisms are involved in apoptosis in a variety of human carcinoma cells treated with antineoplastic compounds. Our laboratory has reported that p53 is a key contributor of mitochondrial apoptosis in cervical carcinoma cells after staurosporine exposure. However, higher mitochondrial membrane potential dissipation and greater DNA fragmentation were observed in p53^wt (wild‐type p53) HeLa cells compared with p53^mt (mutated p53) C‐33A cells. Here, we have studied events linked to the mitochondrial apoptotic pathway. Results. Staurosporine can induce death of HeLa cells via a cytochrome c/caspase‐9/caspase‐3 mitochondrial‐dependent apoptotic pathway and via a delayed caspase‐independent pathway. In contrast with p53^wt cells, p53^mt C‐33A cells exhibit firstly caspase‐8 activation leading to caspase‐3 activation and Bid cleavage followed by cytochrome c release. Attenuation of PARP‐1 [poly(ADP‐ribose) polymerase‐1] cleavage as well as oligonucleosomal DNA fragmentation in the presence of z‐VAD‐fmk points toward a major involvement of a caspase‐dependent pathway in staurosporine‐induced apoptosis in p53^wt HeLa cells, which is not the case in p53^mt C‐33A cells. Meanwhile, the use of 3‐aminobenzamide, a PARP‐1 inhibitor known to prevent AIF (apoptosis‐inducing factor) release, significantly decreases staurosporine‐induced death in these p53^mt carcinoma cells, suggesting a preferential implication of caspase‐independent apoptosis. On the other hand, we show that p53, whose activity is modulated by pifithrin‐α, isolated as a suppressor of p53‐mediated transactivation, or by PRIMA‐1 (p53 reactivation and induction of massive apoptosis), that reactivates mutant p53, causes cytochrome c release as well as mitochondrio—nuclear AIF translocation in staurosporine‐induced apoptosis of cervical carcinoma cells. Conclusions. The present paper highlights that staurosporine engages the intrinsic mitochondrial apoptotic pathway via caspase‐8 or caspase‐9 signalling cascades and via caspase‐independent cell death, as well as through p53 activity.     

Tocotrienols promote apoptosis in human breast cancer cells by inducing poly(ADP‐ribose) polymerase cleavage and inhibiting nuclear factor kappa‐B activity

Objectives

Tocotrienols and tocopherols are members of the vitamin E family, with similar structures; however, only tocotrienols have been reported to achieve potent anti‐cancer effects. The study described here has evaluated anti‐cancer activity of vitamin E to elucidate mechanisms of cell death, using human breast cancer cells.

Materials and methods

Anti‐cancer activity of a tocotrienol‐rich fraction (TRF) and a tocotrienol‐enriched fraction (TEF) isolated from palm oil, as well as pure vitamin E analogues (α‐tocopherol, α?, δ? and γ?tocotrienols) were studied using highly aggressive triple negative MDA‐MB‐231 cells and oestrogen‐dependent MCF‐7 cells, both of human breast cancer cell lines. Cell population growth was evaluated using a Coulter particle counter. Cell death mechanism, poly(ADP‐ribose) polymerase cleavage and levels of NF‐κB were determined using commercial ELISA kits.

Results

Tocotrienols exerted potent anti‐proliferative effects on both types of cell by inducing apoptosis, the underlying mechanism of cell death being ascertained using respective IC₅₀ concentrations of all test compounds. There was marked induction of apoptosis in both cell lines by tocotrienols compared to treatment with Paclitaxel, which was used as positive control. This activity was found to be associated with cleavage of poly(ADP‐ribose) polymerase (a DNA repair protein), demonstrating involvement of the apoptotic cell death signalling pathway. Tocotrienols also inhibited expression of nuclear factor kappa‐B (NF‐κB), which in turn can increase sensitivity of cancer cells to apoptosis.

Conclusion

Tocotrienols induced anti‐proliferative and apoptotic effects in association with DNA fragmentation, poly(ADP‐ribose) polymerase cleavage and NF‐κB inhibition in the two human breast cancer cell lines.

     

Peroxynitrite-induced apoptosis involves activation of multiple caspases in HL-60 cells

In this study, we show that caspases2, 3, 6, and 7 were activated during peroxynitrite-induced apoptosis inhuman leukemia HL-60 cells and that processing of these caspases wasaccompanied by cleavage of poly(ADP-ribose) polymerase and lamin B. Treatment of cells with DEVD-fluoromethyl ketone (FMK), a selectiveinhibitor for caspase 3-like proteases, resulted in a marked diminution of apoptotic cells. VAVAD-FMK, an inhibitor of caspase 2, partially inhibited the apoptotic response to peroxynitrite. However, selective inactivation of caspase 6 by VEID-FMK did not affect apoptosis rates.These data suggest that caspase 3-like proteases and caspase 2, but notcaspase 6, are required for peroxynitrite-induced apoptosis in thiscell type. Moreover, we demonstrate that peroxynitrite treatmentstimulated activation of caspases 8 and 9, two initial caspases in theapoptotic signaling pathway, and preincubation of cells with theirinhibitor, IETD-FMK, inhibited activation of caspase 3-like proteasesand caspase 2 at the concentration that prevents the apoptosis. Theseobservations, together, suggest that caspase 8 and/or caspase 9 mediates activation of caspase 3-like proteases and caspase 2 duringthe apoptosis induced by peroxynitrite in HL-60 cells.