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S Pervaiz

Publications and source records attributed to S Pervaiz.

At least 19 recordsLinked to original sources
PubMed2003

Decrease in intracellular superoxide sensitizes Bcl-2-overexpressing tumor cells to receptor and drug-induced apoptosis independent of the mitochondria.

At least two mechanisms of early cytosolic acidification during apoptotic signaling have been described, one that involves caspase 8 activation downstream of receptor ligation and another dependent on mitochondria-derived hydrogen peroxide during merocil-induced apoptosis. Here, we show that Bcl-2 inhibits both mechanisms of acidification. Moreover, Bcl-2 overexpression resulted in a slightly elevated constitutive level of superoxide anion and pH in CEM leukemia cells. Interestingly, decreasing intracellular superoxide concentration with an inhibitor of the beta-nicotinamide adenine dinucleotide phosphate oxidase or by transient transfection with a dominant-negative form of the guanosine triphosphate-binding protein Rac1 resulted in a significant increase in the sensitivity of CEM/Bcl-2 cells to CD95- or merocil-induced apoptosis. This increase in sensitivity was a direct result of a significant increase in caspase 8 activation and caspase 8-dependent acidification in the absence of caspase 9 activity or cytochrome c release. These findings suggest a mechanism of switching from mitochondria-dependent to mitochondria-independent death signaling in the same cell, provided the intracellular milieu is permissive for upstream caspase 8 activation, and could have implications for favorably tailoring tumor cells for drug treatment even when the mitochondrial pathway is compromised by Bcl-2.

Apoptosis↗
PubMed2001

Activation of the RacGTPase inhibits apoptosis in human tumor cells.

The small GTP-binding protein Rac is a downstream effector of the oncogene product p21-ras. Rac is involved in actin polymerization, Jun kinase activation, and intracellular superoxide anion production, through distinct pathways in tumor cells. Here we investigated the role of activated Rac in the response of tumor cells to apoptosis triggered by anti-cancer drugs or the cell surface death receptor CD95. Using M14 melanoma cells stably transfected with a constitutively active form of Rac1, we show that activated Rac inhibits tumor cell response to apoptosis. The inhibitory effect of activated Rac on apoptotic signaling is mediated by the interaction of Rac with intracellular oxidase and the subsequent production of superoxide, which is supported by experiments performed with M14 and NIH3T3 cells transiently transfected with the loss-of-function mutants of Rac in an activated RacV12 background. Consistent with these findings, we also demonstrate that inhibition of the Rac pathway in the HaRas-expressing T24 bladder carcinoma cell line induces a decrease in superoxide anion concentration, and results in a significant increase in tumor cell sensitivity to apoptosis. These findings demonstrate the existence of a novel Rac-dependent survival pathway mediated by intracellular superoxide in tumor cells.

3T3 Cells↗
PubMed2001

Intracellular acidification triggered by mitochondrial-derived hydrogen peroxide is an effector mechanism for drug-induced apoptosis in tumor cells.

We recently showed that two photoproducts of merocyanine 540, C2 and C5, triggered cytochrome C release; however, C5 was inefficient in inducing caspase activity and apoptosis in leukemia cells, unlike C2. Here we show that HL60 cells acidified upon exposure to C2 but not C5. The intracellular drop in pH and caspase activation were dependent upon hydrogen peroxide production, and were inhibited by scavengers of hydrogen peroxide. On the contrary, caspase inhibitors did not block hydrogen peroxide production. In turn, increased intracellular hydrogen peroxide concentration was downstream of superoxide anion produced within 2 h of exposure to C2. Inhibitor of NADPH oxidase diphenyleneiodonium neither inhibited superoxide production nor caspase activation triggered by C2. However, exposure of purified mitochondria to C2 resulted in significantly increased superoxide production. Furthermore, cytochrome C release from isolated mitochondria induced by C2 was completely inhibited in the presence of scavengers of hydrogen peroxide. Contrarily, scavenging hydrogen peroxide had no effect on the cyclosporin A-sensitive mitochondrial permeability transition induced by C5. Our data suggest a scenario where drug-induced hydrogen peroxide production induces intracellular acidification and release of cytochrome C, independent of the inner membrane pore, thereby creating an intracellular environment permissive for caspase activation.

Apoptosis↗
PubMed2001

Reactive oxygen-dependent production of novel photochemotherapeutic agents.

The reactive nature of species derived from oxygen, such as singlet oxygen and hydrogen peroxide, has been exploited in the clinical setting for targeting bacteria, viruses, and tumor cells by photodynamic excitation of a variety of chromophores. This modality, termed photodynamic therapy (PDT), is currently being used to treat some forms of cancer. However, the applicability of conventional PDT is limited due to the absolute dependence on simultaneous exposure of the target to the photoactive compound and light. In 1990, we demonstrated that the need for simultaneous exposure of the biological target to light and photosensitizer could be circumvented by prior exposure (activation) of the sensitizer molecule to light and its subsequent use as any other anti-cancer or anti-viral drug. By dint of the nature of the protocol, this process was termed preactivation. Since then, the generation of biologically active molecules in vitro by preactivation has been validated using a variety of chromophores, such as merocyanine 540, Photofrin II, and naphthalimide. Here we briefly review the role of reactive oxygen species in the photodynamic effect, and provide an explanation for the mechanism of preactivation. We propose that photo-oxidation not only provides a novel means for the generation of biologically active molecules, but could also explain, at least in part the mechanism of conventional PDT. It is likely that the light-dependent breakdown of the chromophore to generate novel active compounds, in addition to reactive oxygen species, also contributes to the photodynamic damage observed on simultaneous exposure of the chromophore and target tissue to light during PDT.-Pervaiz, S. Reactive oxygen-dependent production of novel photochemotherapeutic agents.

Animals↗
PubMed2001

Resveratrol--from the bottle to the bedside?

Resveratrol, a naturally occurring plant antibiotic has been the focus of a number of studies investigating its biological attributes, which include anti-oxidant activity, anti-platelet aggregation effect, anti-atherogenic property, estrogen-like growth promoting effect, growth inhibiting activity, immunomodulation, and chemoprevention. More recently, since the first report on the apoptosis inducing activity of resveratrol in human cancer cells, the interest in this molecule as a potential chemotherapy agent has significantly intensified. Not only has its role as an anti-cancer agent been corroborated, but the precise mechanism(s) of the anti-cancer activity of resveratrol is/are being elucidated. Our group has been active in studying the cross talk between the caspase family of proteases and mitochondria, in drug-induced apoptosis. In this regard, we have shown that the cancer preventive activity of resveratrol could be attributed to its ability to trigger apoptosis in human leukemia and breast carcinoma cells. The cytotoxicity of resveratrol is restricted against these transformed cell types due to its ability to selectively upregulate CD95-CD95L interaction on the tumor cell surface, unlike normal peripheral blood cells. Despite the involvement of the CD95 signaling pathway, apoptosis induced by resveratrol is not accompanied by robust caspase 8 activation, but involves mitochondrial release of cytochrome C and downstream activation of caspases 9 and 3. We also extrapolate these in vitro findings in a murine model of carcinogensis, and demonstrate in vivo induction of apoptosis in mouse skin papillomas. These findings highlight the chemotherapeutic potential of this polyphenolic compound.

Antineoplastic Agents, Phytogenic↗
PubMed2000

Induction of mitochondrial permeability transition and cytochrome C release in the absence of caspase activation is insufficient for effective apoptosis in human leukemia cells.

Induction of mitochondrial permeability transition (MPT) and cytosolic translocation of cytochrome C are considered essential components of the apoptotic pathway. Hence, there is the realization that mitochondrial-specific drugs could have potential for use as chemotherapeutic agents to trigger apoptosis in tumor cells. Recently, we showed that photoproducts of merocyanine 540 (pMC540) induced tumor cell apoptosis. In this study, we focused on identifying mitochondrial-specific compounds from pMC540 and studied their apoptotic potential. One purified fraction, C5, induced a drop in mitochondrial transmembrane potential and cytosolic translocation of cytochrome C in HL60 human leukemia cells. Moreover, the addition of C5 to purified rat liver mitochondria induced MPT as indicated by mitochondrial matrix swelling, which was completely inhibited by cyclosporin A, an inhibitor of the inner-membrane pore. Supernatant of C5-treated mitochondria showed a dose-dependent increase in cytochrome C, which was also inhibited in the presence of cyclosporin A, strongly indicating a direct effect on the inner-membrane pore. Despite the strong mitochondrial reactivity, C5 elicited minimal cytotoxicity (less than 25%) against HL60 leukemia and M14 melanoma cells because of inefficient caspase activation. However, prior exposure to C5 significantly enhanced the apoptotic response to etoposide or the CD95 receptor. Thus, we demonstrate that MPT induction and cytochrome C release by the novel compound C5, in the absence of effective caspase activation, is insufficient for triggering efficient apoptosis in tumor cells. However, when used in combination with known apoptosis inducers, such compounds could enhance the sensitivity of tumor cells to apoptosis. (Blood. 2000;95:1773-1780)

Animals↗
PubMed2000

Melanoma antigen recognition by tumour-infiltrating T lymphocytes (TIL): effect of differential expression of melan-A/MART-1.

We have isolated, from an individual patient with metastatic melanoma, a series of eight TIL clones capable of lysing autologous melanoma cell targets. Six of the eight clones expressed TCRAV2S1 and lysed targets expressing HLA-A2 and the Melan-A/MART-1 peptide: AAGIGILTV. Polymerase chain reaction-single stranded conformational polymorphism (PCR-SSCP) analysis showed that the Melan-A/MART-1-specific clones were predominant in the bulk culture prior to cloning. However, the tumour progressed in vivo even in the presence of these tumour cell-lytic clones. Using the anti-Melan-A/MART-1 MoAb (A-103), we noted that Melan-A/MART-1 expression on three melanoma cell lines varied considerably during in vitro culture, in the absence of T cell immunoselection, relative to cell density. Tumour cells which spontaneously decreased Melan-A/MART-1 expression were less susceptible to specific TIL lysis. Melan-A/MART-1 expression and susceptibility to lysis increased in cells cultured at lower density. These data suggest that modulation of tumour antigen may account for tumour progression in the presence of tumour cell-lytic T lymphocytes. The observations suggest a possible explanation for the common finding of Melan-A/MART-1-specific lytic TIL in clinically progressing melanomas, as well as a possible pathway for therapeutic intervention.

Amino Acid Sequence↗
PubMed1999

Superoxide anion inhibits drug-induced tumor cell death.

Intracellular superoxide (O(2)*- was manipulated in M14 melanoma cells by overexpression or repression of Cu/Zn SOD using a tetracycline-inducible expression system. Scavenging intracellular O(2)*- increased tumor cell sensitivity to daunorubicin, etoposide, and pMC540, whereas expression of the antisense SOD mRNA significantly decreased cell sensitivity to drug treatment. Whereas Cu/Zn SOD overexpressing cells exhibited higher activation of the executioner caspase 3 upon drug exposure, caspase 3 activation was significantly lower when Cu/Zn SOD was repressed by antisense expression. These data show that intracellular O(2)*- regulates tumor cell response to drug-induced cell death via a direct or indirect effect on the caspase activation pathway.

Apoptosis↗
PubMed1999

Purified photoproducts of merocyanine 540 trigger cytochrome C release and caspase 8-dependent apoptosis in human leukemia and melanoma cells.

If the interplay between caspase proteases and mitochondria decide the fate of the cell during apoptosis, they may constitute useful molecular targets for novel drug design. We have shown that photoactivated merocyanine 540 (pMC540) triggers caspase-mediated apoptosis in HL60 leukemia and M14 melanoma cells. Because pMC540 is a mixture of photoproducts, we set out to purify the biologically active component(s) from this mixture and to investigate their ability to directly activate intracellular caspases and/or trigger mitochondrial events associated with apoptosis. Two photoproducts, namely C1 and C2, purified and characterized by mass spectroscopy and nuclear magnetic resonance (NMR) analysis, effectively induced apoptosis in HL60 and M14 cells. Interestingly, both C1 and C2 induced non-receptor-dependent activation of caspase 8, which was responsible for the downstream activation of caspase 3 and cell death. Both compounds induced the release of cytochrome C from mitochondria of tumor cells and from purified rat liver mitochondria; however, different mechanisms were operative in cytochrome C translocation in response to C1 or C2. C1-induced cytochrome C release was mediated by the mitochondrial permeability transition (MPT) pore and accompanied by a decrease in mitochondrial transmembrane potential (triangle uppsim), whereas cytochrome C release in response to C2 was independent of MPT pore opening. These findings do not exclude the possibility that changes in mitochondrial triangle uppsim are critical for apoptosis in some instances, but support the notion that this may not be a universal step in the apoptotic process. Thus, identification of two novel anticancer agents that directly activate effector components of the apoptotic pathway could have potential implications for the development of newer chemotherapeutic drugs.

Animals↗
PubMed1999

Reactive oxygen intermediates regulate cellular response to apoptotic stimuli: an hypothesis.

Production of reactive oxygen intermediates (ROI) has been thought for a long time to adversely affect the physiology and survival of a cell. There is now a growing body of evidence to suggest that ROI such as superoxide anion (O2*-) and hydrogen peroxide (H2O2) can influence the growth, as well as death, of animal cells in vitro. The observation that cells release O2*- or its dismutation product H2O2, either constitutively in the case of tumor cells or following cytokine stimulation, has led to the speculation that they might possibly serve as intercellular messengers to stimulate proliferation via mechanisms common to natural growth factors. However, as the balance between cell populations in an organism is tightly controlled by the rate of proliferation and death of constituent cells, an increase in cell numbers could reciprocally be viewed as deregulation of cell death. Hence, it is equally important to decipher how ROI influence the response of cells to signals that activate cell death pathway(s). We propose that ROI not only regulate proliferation but also affect cell sensitivity to triggers which activate the cellular suicide program (apoptosis) versus those that cause accidental (necrotic) cell death.

Animals↗
PubMed1998

Apoptosis induced by hydrogen peroxide is mediated by decreased superoxide anion concentration and reduction of intracellular milieu.

Hydrogen peroxide (H2O2) is considered to be a mediator of apoptotic cell death but the mechanism by which it induces apoptosis is unclear. Here, we show that cells undergoing apoptosis from exposure to H2O2 display a significant decrease in intracellular concentration of superoxide (O2-) which is associated with a reduction of the intracellular milieu, as measured by an increase in the GSH/GSSG ratio and a decrease in intracellular pH. The notion that a decrease in intracellular O2- concentration triggers apoptosis is supported by the observation that H2O2-mediated apoptosis could be retarded in cells in which the intracellular O2- concentration is maintained at or above the cellular baseline level by inhibition of the major O2- scavenger superoxide dismutase (Cu/Zn SOD). Taken together, our observations indicate that a decrease in the intracellular O2- concentration, reduction and acidification of the intracellular milieu constitute a signal for H2O2-mediated apoptosis, thereby inducing a reductive as opposed to an oxidative stress.

Apoptosis↗
PubMed1998

Chemopreventive agent resveratrol, a natural product derived from grapes, triggers CD95 signaling-dependent apoptosis in human tumor cells.

Resveratrol, a constituent of grapes and other food products, has been shown to prevent carcinogenesis in murine models. We report here that resveratrol induces apoptotic cell death in HL60 human leukemia cell line. Resveratrol-treated tumor cells exhibit a dose-dependent increase in externalization of inner membrane phosphatidylserine and in cellular content of subdiploid DNA, indicating loss of membrane phospholipid asymmetry and DNA fragmentation. Resveratrol-induced cell death is mediated by intracellular caspases as observed by the dose-dependent increase in proteolytic cleavage of caspase substrate poly (ADP-ribose) polymerase (PARP) and the ability of caspase inhibitors to block resveratrol cytotoxicity. We also show that resveratrol treatment enhances CD95L expression on HL60 cells, as well as T47D breast carcinoma cells, and that resveratrol-mediated cell death is specifically CD95-signaling dependent. On the contrary, resveratrol treatment of normal human peripheral blood lymphocytes (PBLs) does not affect cell survival for up to 72 hours, which correlates with the absence of a significant change in either CD95 or CD95L expression on treated PBLs. These data show specific involvement of the CD95-CD95L system in the anti-cancer activity of resveratrol and highlight the chemotherapeutic potential of this natural product, in addition to its recently reported chemopreventive activity.

Antibodies, Monoclonal↗
PubMed1998

Caspase proteases mediate apoptosis induced by anticancer agent preactivated MC540 in human tumor cell lines.

The molecular events involved in tumor cell death induced by novel photoproducts of merocyanine 540 (pMC540) are poorly understood. Using HL60 leukemia and M14 melanoma cell lines we investigated the role of the apoptotic pathway in pMC540-mediated cell death. Tumor cells exposed to pMC540 showed cell size shrinkage and an increase in the sub-diploid DNA content. A loss of membrane phospholipid asymmetry associated with apoptosis was induced by pMC540 in both tumor cell lines as evidenced by the externalization of phosphatidylserine. A dose-dependent increase in caspase-3 protease activity suppressed by the tetrapeptide inhibitor DEVD-CHO was observed in both cell lines. Western blot analysis of poly (ADP-ribose) polymerase, a caspase substrate, showed the classical cleavage pattern (116 to 89 kDa) associated with apoptosis in pMC540-treated cell lysates. Furthermore, caspase inhibition blocked the externalization of membrane PS, indicating that the loss of membrane phospholipid asymmetry is a downstream event of caspase activation. These findings demonstrate that tumor cell death induced by pMC540 is mediated by caspase proteases.

Apoptosis↗
PubMed1997

Comparative analysis of cardiac troponin I and creatine kinase-MB as markers of acute myocardial infarction.

BACKGROUND: The World Health Organization (WHO) criteria for the diagnosis of acute myocardial infarction (AMI) includes presentation of chest pain over 20 min, evolutionary changes on the electrocardiogram (ECG), and abnormal levels of cardiac enzymes. HYPOTHESIS: A multicenter study was conducted to evaluate the efficacy of cardiac troponin I (cTnI) in detecting and ruling out AMI. METHODS: The normal range for cTnI in 149 apparently healthy subjects without known history of cardiac or other diseases was 0 to 0.5 ng/ml. Cutoffs of 2.5 ng/ml for c TnI and 5.0 ng/ml for creatine kinase-MB (CK-MB) were used. RESULTS: The diagnostic sensitivity of blood collected from 291 consecutive patients with suspicion of AMI was 95.0 and 96.4%, respectively, for samples obtained at 4-48 h after AMI onset. CK-MB was more sensitive during the early 4-8 h interval (84 vs. 74%); both had 100% sensitivity from 12-36 h. CTnI remained at 100% for 72 h, while CK-MB declined to 57%. The clinical specificity was 97.4 vs. 85.8%, respectively, on non-AMI patients with cardiac and noncardiac diseases, and those with renal disease. CONCLUSION: cTnI is an excellent marker for detecting and ruling out AMI, because it has better specificity and a wider diagnostic window than the accepted standard, CK-MB.

Biomarkers↗