PubMed Health⌕ Search

Biomedical subjects

Anindita Das

Publications and source records attributed to Anindita Das.

17 recordsLinked to original sources
PubMed2006

Cyclic GMP-dependent protein kinase Ialpha attenuates necrosis and apoptosis following ischemia/reoxygenation in adult cardiomyocyte.

Cyclic GMP-dependent protein kinases protein kinase G (PKG) Ialpha and PKGIbeta are major mediators of cGMP signaling in the cardiovascular system. PKGIalpha is present in the heart, although its role in protection against ischemia/reperfusion injury is not known. We investigated the direct effect of PKGIalpha against necrosis and apoptosis following simulated ischemia (SI) and reoxygenation (RO) in cardiomyocytes. Adult rat cardiomyocytes were infected with adenoviral vectors containing hPKGIalpha or catalytically inactive mutant hPKGIalphaK390A. After 24 h, the cells were subjected to 90 min of SI and 2 h RO for necrosis (trypan blue exclusion and lactate dehydrogenase release) or 18 h RO for apoptosis studies. To evaluate the role of K(ATP) channels, subgroups of cells were treated with 5-hydroxydecanoate (100 microm), HMR1098 (30 microm), or glibenclamide (50 microm), the respective blockers of mitochondrial, sarcolemmal, or both types of K(ATP) channels prior to SI. The necrosis observed in 33.7 +/- 1.6% of total myocytes in the SI-RO control group was reduced to 18.6 +/- 0.8% by PKGIalpha (mean +/- S.E., n = 7, p < 0.001). The apoptosis observed in 17.9 +/- 1.3% of total myocytes in the SI-RO control group was reduced to 6.0 +/- 0.6% by PKGIalpha (mean +/- S.E., n = 7, p < 0.001). In addition, PKGIalpha inhibited the activation of caspase-3 after SI-RO in myocytes. Myocytes infected with the inactive PKGIalphaK390A mutant showed no protection. PKGIalpha enhanced phosphorylation of Akt, ERK1/2, and JNK, increased Bcl-2, inducible nitric-oxide synthase, endothelial nitric-oxide synthase, and decreased Bax expression. 5-Hydroxydecanoate and glibenclamide abolished PKGIalpha-mediated protection against necrosis and apoptosis. However, HMR1098, had no effect. A scavenger of reactive oxygen species, as well as inhibitors of phosphatidylinositol 3-kinase, ERK, JNK1, and NOS, also blocked PKGIalpha-mediated protection against necrosis and apoptosis. These results show that opening of mitochondrial K(ATP) channels and generation of reactive oxygen species, in association with phosphorylation of Akt, ERK, and JNK, and increased expression of NOS and Bcl-2, play an essential role in the protective effect of PKGIalpha.

Animals↗
PubMed2006

Hypercholesterolemia enhances tolerance to lethal systemic hypoxia in middle-aged mice: possible role of VEGF downregulation in brain.

Hypercholesterolemia (HCL) is commonly associated with impaired vascular relaxation response and augmented vasoconstriction. Interestingly, it was shown that animals with HCL were less vulnerable to seizures and several clinical studies also revealed a better outcome after stroke in the patients with HCL. To this context, the present study was designed to test the hypothesis that HCL would enhance the animals' resistance to severe systemic hypoxia and in turn prolong their survival time under such noxious condition. Four groups of middle-aged (mean age: 51.1 +/- 2.8 weeks) male C57BL/6J wild-type mice (C57BL-WT) and low-density lipoprotein receptor knockout mice (LDLR-KO) were included in the study: two groups were exposed to severe normobaric hypoxia (5% F(I)O(2)) and other two groups were used for brain tissue sample collection and Western blot analysis. The survival time under the hypoxic condition was recorded for each animal. Individual blood samples were collected immedtately after the cessation of spontaneous breathing for measuring plasma total cholesterol (TCL) and triglycerides. The results show that the hypoxia survival time was longer in LDLR-KO than C57BL-WT (i.e. 3.7 +/- 0.5 versus 2.3 +/- 0.2 min; P < 0.05). A positive correlation was found between TCL and the survival time (r (2) = 0.43; P < 0.05). Furthermore, a significant downregulation of vascular endothelial growth factor (VEGF) was observed in the brain tissue of LDLR-KO, as compared with C57BL-WT (n, = 3/group; P < 0.05), whereas expression of heme oxygenase 1 was similar in these two groups. We conclude that HCL enhances resistance to lethal systemic hypoxia (i.e. 61% increase in survival time) in middle-aged mice. This paradoxical protective effect of HCL was associated with a concomitant downregulation of cerebral VEGF expression, which could potentially blunt the hypoxia-triggered and VEGF-mediated pathophysiological events leading to death.

Animals↗
PubMed2006

Rapamycin confers preconditioning-like protection against ischemia-reperfusion injury in isolated mouse heart and cardiomyocytes.

Rapamycin (sirolimus) is an antibiotic that inhibits protein synthesis through mammalian target of rapamycin (mTOR) signaling and is used as an immunosuppressant in the treatment of organ rejection in transplant recipients. Recently, the antigrowth properties of rapamycin have been utilized for cardiovascular benefit as stents impregnated with rapamycin effectively reduce coronary restenosis. We report here a novel role of this drug in protection against ischemia/reperfusion (I/R) injury. Adult male ICR mice were treated with rapamycin (0.25 mg/kg, IP) or volume-matched DMSO (solvent for rapamycin). The hearts were subjected to 20 min of global ischemia and 30 min of reperfusion in Langendorff mode. The blocker of mitochondrial KATP channel, 5-hydroxydecanoate (5-HD, 100 microM) was given 10 min before ischemia. Infarct size in the DMSO treated group was 28.2 +/- 1.3% and was reduced to 10.1 +/- 2.8% in the rapamycin-treated mice (64% decrease, P < 0.001). 5-HD blocked the protective effect (infarct area 32.2 +/- 1.8%, P < 0.001 vs. rapamycin). The infarct limiting effect of rapamycin was not associated with improved recovery of ventricular function. We further examined the effect of rapamycin in protection against necrosis and apoptosis in adult cardiomyocytes subjected to simulated ischemia and reoxygenation. Myocytes treated with rapamycin in doses from 25-100 nM demonstrated significantly lower trypan blue-positive necrotic cells and TUNEL-positive apoptotic nuclei, supporting the protective role of drug in the intact heart. These data suggest that rapamycin induces potent preconditioning-like effect against myocardial infarction through opening of mitochondrial KATP channels. We propose that rapamycin may be a novel therapeutic strategy to limit infarction, apoptosis, and remodeling following I/R injury in the heart.

Animals↗
PubMed2005

Phosphodiesterase-5 inhibition with sildenafil attenuates cardiomyocyte apoptosis and left ventricular dysfunction in a chronic model of doxorubicin cardiotoxicity.

BACKGROUND: Sildenafil, a phosphodiesterase-5 inhibitor, induces cardioprotection against ischemia/reperfusion injury via opening of mitochondrial K(ATP) channels. It is unclear whether sildenafil would provide similar protection from doxorubicin-induced cardiotoxicity. METHODS AND RESULTS: Male ICR mice were randomized to 1 of 4 treatments: saline, sildenafil, doxorubicin (5 mg/kg IP), and sildenafil (0.7 mg/kg IP) plus doxorubicin (n=6 per group). Apoptosis was assessed with the use of terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling and in situ oligo ligation methods. Desmin distribution was determined via immunofluorescence. Bcl-2 expression was analyzed by Western blot. Left ventricular function was assessed by measuring developed pressure and rate pressure product in Langendorff mode. ECG changes indicative of doxorubicin cardiotoxicity were also measured. For in vitro studies, adult ventricular cardiomyocytes were exposed to doxorubicin (1 micromol/L), sildenafil (1 micromol/L) with or without N(G)-nitro-L-arginine methyl ester (L-NAME) (100 micromol/L), or 5-hydroxydecanoate (100 micromol/L) 1 hour before doxorubicin and incubated for 18 hours. Doxorubicin-treated mice demonstrated increased apoptosis and desmin disruption, which was attenuated in the sildenafil+doxorubicin group. Bcl-2 was decreased in the doxorubicin group but was maintained at basal levels in the sildenafil+doxorubicin group. Left ventricular developed pressure and rate pressure product were significantly depressed in the doxorubicin group but were attenuated in the sildenafil+doxorubicin group. ST interval was significantly increased in the doxorubicin group over 8 weeks. In the sildenafil+doxorubicin group, ST interval remained unchanged from baseline. Doxorubicin caused a significant increase in apoptosis, caspase-3 activation, and disruption of mitochondrial membrane potential in vitro. In contrast, sildenafil significantly protected against doxorubicin cardiotoxicity; however, this protection was abolished by both L-NAME and 5-hydroxydecanoate. CONCLUSIONS: Prophylactic treatment with sildenafil prevented apoptosis and left ventricular dysfunction in a chronic model of doxorubicin-induced cardiomyopathy.

3',5'-Cyclic-GMP Phosphodiesterases↗
PubMed2005

Pharmacological preconditioning with sildenafil: Basic mechanisms and clinical implications.

The phosphodiesterase type-5 (PDE5) inhibitor, sildenafil, is the first drug developed for treatment of erectile dysfunction in patients. Experimental data in animals show that sildenafil has a preconditioning-like cardioprotective effect against ischemia/reperfusion injury in the intact heart. Mechanistic studies suggest that sildenafil exerts cardioprotection through NO generated from eNOS/iNOS, activation of protein kinase C/ERK signaling and opening of mitochondrial ATP-sensitive potassium channels. Additional studies show that the drug attenuates cell death resulting from necrosis and apoptosis, and increases the Bcl2/Bax ratio through NO signaling in adult cardiomyocytes. Emerging new data also suggest that sildenafil may be used clinically for treatment of pulmonary arterial hypertension and endothelial dysfunction. Future demonstration of the cardioprotective effect in patients with the relatively safe and effective FDA-approved PDE5 inhibitors such as sildenafil could have an enormous impact on bringing the long-studied phenomenon of ischemic and pharmacologic preconditioning to the clinical forefront.

3',5'-Cyclic-GMP Phosphodiesterases↗
PubMed2005

Phosphodiesterase-5 inhibitor sildenafil preconditions adult cardiac myocytes against necrosis and apoptosis. Essential role of nitric oxide signaling.

We investigated the effect of sildenafil in protection against necrosis or apoptosis in cardiomyocytes. Adult mouse ventricular myocytes were treated with sildenafil (1 or 10 microM) for 1 h before 40 min of simulated ischemia (SI). Necrosis was determined by trypan blue exclusion and lactate dehydrogenase release following SI alone or plus 1 or 18 h of reoxygenation (RO). Apoptosis was assessed by terminal deoxynucleotidyl transferase-mediated nick end labeling assay and mitochondrial membrane potential measured using a fluorescent probe 5,5',6,6'-tetrachloro-1,1',3,3'-tetraethylbenzimidazolyl-carbocyanine iodide (JC-1). Sildenafil reduced necrosis as indicated by decrease in trypan blue-positive myocytes and leakage of lactate dehydrogenase compared with untreated cells after either SI or SI-RO. The number of terminal deoxynucleotidyl transferase-mediated nick end labeling-positive myocytes or loss of JC-1 fluorescence following SI and 18 h of RO was attenuated in the sildenafil-treated group with concomitant inhibition of caspase 3 activity. An early increase in Bcl-2 to Bax ratio with sildenafil treatment was also observed in myocytes after SI-RO. The increase of Bcl-2 expression by sildenafil was inhibited by nitric-oxide synthase (NOS) inhibitor, L-nitro-amino-methyl-ester. The drug also enhanced mRNA and protein content of inducible NOS (iNOS) and endothelial NOS (eNOS) in the myocytes. Sildenafil-induced protection against necrosis and apoptosis was absent in the myocytes derived from iNOS knock-out mice and was attenuated in eNOS knock-out myocytes. The up-regulation of Bcl-2 expression by sildenafil was also absent in iNOS-deficient myocytes. Reverse transcription-PCR, Western blots, and immunohistochemical assay confirmed the expression of phosphodiesterase-5 in mouse cardiomyocytes. These data provide strong evidence for a direct protective effect of sildenafil against necrosis and apoptosis through NO signaling pathway. The results may have possible therapeutic potential in preventing myocyte cell death following ischemia/reperfusion.

3',5'-Cyclic-GMP Phosphodiesterases↗
PubMed2005

Neurosyphilis in a tertiary care hospital in north India.

BACKGROUND AND OBJECTIVES: The clinical diagnosis of neurosyphilis is very rarely encountered today in the developed world although syphilis remains a significant health problem in few areas of the industrialized countries and in most of the third world nations. This apparent decline may be due to increase in number of asymptomatic neurosyphilis and cases presenting as subtle, illdefined syndromes rather than classic presentation of tabes dorsalis and general paresis in the post penicillin era. This retrospective study was carried out to report the neurosyphilis cases diagnosed at a tertiary care hospital in North India, and to analyse the laboratory and clinical parameters of these cases. METHODS: Suspected cases of neurosyphilis presenting at Postgraduate Institute of Medical Education and Research (PGIMER), Chandigarh over a period of 13 yr (January 1990 to December 2002) were identified. Diagnosis of neurosyphilis was based on clinical presentation, prior history of syphilis, routine CSF biochemistry (protein and leukocytes) and serological evidence [serum and CSF venereal disease research laboratory (VDRL) and Treponema pallidum particle agglutination (TPPA) tests]. RESULTS: A total of 25 cases of neurosyphilis were identified, 18 (72%) with reactive CSF-VDRL, 22 (88%) with elevated CSF protein and 24 (96%) with CSF mononuclear leukocytosis. Serum VDRL was reactive in all 25 cases. Three patients were asymptomatic (2 primary syphilis; 1 early latent stage), 8 had secondary and 14 had tertiary syphilis. Two of the neurosyphilis cases were also seropositive for HIV. Radiology was abnormal in 7 (28%) patients. INTERPRETATION AND CONCLUSION: Neurosyphilis still remains a problem in a country like India and a high index of suspicion and clinical expertise are required for appropriate diagnosis and proper management especially in the era of AIDS pandemic.

Cerebrospinal Fluid↗
PubMed2004

Tumor-derived p53 mutants induce oncogenesis by transactivating growth-promoting genes.

We have studied the mechanism of mutant p53-mediated oncogenesis using several tumor-derived mutants. Using a colony formation assay, we found that the majority of the mutants increased the number of colonies formed compared to the vector. Expression of tumor-derived p53 mutants increases the rate of cell growth, suggesting that the p53 mutants have 'gain of function' properties. We have studied the gene expression profile of cells expressing tumor-derived p53-D281G to identify genes transactivated by mutant p53. We report the transactivation of two genes, asparagine synthetase and human telomerase reverse transcriptase. Quantitative real-time PCR confirms this upregulation. Transient transfection promoter assays verify that tumor-derived p53 mutants transactivate these promoters significantly. An electrophoretic mobility shift assay shows that tumor-derived p53-mutants cannot bind to the wild-type p53 consensus sequence. The results presented here provide some evidence of a possible mechanism for mutant p53-mediated transactivation.

Amino Acid Substitution↗
PubMed2004

Cardioprotection with phosphodiesterase-5 inhibition--a novel preconditioning strategy.

Phosphodiesterase type-5 (PDE-5) inhibitors including sildenafil, vardenafil and tadalafil are a new class of vasoactive drugs that have been developed for treatment of erectile dysfunction in patients. A growing number of studies in recent years suggest that sildenafil may be used clinically for treatment of pulmonary hypertension and endothelial dysfunction. In addition, recent studies primarily from our laboratory suggested that sildenafil has preconditioning-like powerful cardioprotective effect in the animal models of ischemia-reperfusion injury. Sildenafil has been found to exert cardioprotection through nitric oxide generated from endothelial and/or inducible nitric oxide synthases and opening of mitochondrial ATP-sensitive potassium channels. Future demonstration of the cardioprotective effect in patients with the relatively safe and effective FDA-approved PDE-5 inhibitors, such as sildenafil, could have an enormous impact on bringing the long-studied phenomena of ischemic and pharmacologic preconditioning to the clinical forefront.

3',5'-Cyclic-GMP Phosphodiesterases↗
PubMed2004

Protein kinase C plays an essential role in sildenafil-induced cardioprotection in rabbits.

Sildenafil citrate (Viagra) is the most widely used pharmacological drug for treating erectile dysfunction in men. It has potent cardioprotective effects against ischemia-reperfusion injury via nitric oxide and opening of mitochondrial ATP-sensitive K(+) channels. We further investigated the role of protein kinase C (PKC)-dependent signaling pathway in sildenafil-induced cardioprotection. Rabbits were treated (orally) with sildenafil citrate (1.4 mg/kg) 30 min before index ischemia for 30 min and reperfusion for 3 h. The PKC inhibitor chelerythrine (5 mg/kg i.v.) was given 5 min before sildenafil. Infarct size (% of risk area) reduced from 33.65 +/- 2.17 in the vehicle (saline) group to 15.07 +/- 0.63 in sildenafil-treated groups, a 45% reduction compared with vehicle (mean +/- SE, P < 0.05). Chelerythrine abolished sildenafil-induced protection, as demonstrated by increase in infarct size to 31.14 +/- 2.4 (P < 0.05). Chelerythrine alone had an infarct size of 33.5 +/- 2.5, which was not significantly different compared with DMSO-treated group (36.8 +/- 1.7, P > 0.05). Western blot analysis demonstrated translocation of PKC-alpha, -, and -delta isoforms from cytosol to membrane after treatment with sildenafil. However, no change in the PKC-beta and -epsilon isoforms was observed. These data provide direct evidence of an essential role of PKC, and potentially PKC-alpha, -, and -delta, in sildenafil-induced cardioprotection in the rabbit heart.

Alkaloids↗
PubMed2004

Central nervous system fungal infection--a report of three cases.

Fungal infections of the central nervous system in three apparently immunocompetent patients are being reported. Two cases of cerebral aspergillosis presenting as intracranial granulomas such as rhinocerebral and intracranial forms, and one of cryptococcal meningitis could be successfully diagnosed by newer diagnostic modality such as antigen detection techniques. The case with cryptococcal infection had typical neuroimaging feature which helped to suspect the underlying diagnosis. Aspergillus galactomannan detection in the cerebrospinal fluid helped in the early diagnosis and appropriate therapy of one patient.

Adult↗
PubMed2003

Suppression of the protein tyrosine phosphatase receptor type O gene (PTPRO) by methylation in hepatocellular carcinomas.

A diet lacking folic acid and choline and low in methionine (folate/methyl deficient diet, FMD diet) fed to rats is known to produce preneoplastic nodules (PNNs) after 36 weeks and hepatocellular carcinomas (tumors) after 54 weeks. FMD diet-induced tumors exhibit global hypomethylation and regional hypermethylation. Restriction landmark genome scanning analysis with methylation-sensitive enzyme NotI (RLGS-M) of genomic DNA isolated from control livers, PNNs and tumor tissues was performed to identify the genes that are differentially methylated or amplified during multistage hepatocarcinogenesis. Out of the 1250 genes analysed, 2 to 5 genes were methylated in the PNNs, whereas 5 to 45 genes were partially or completely methylated in the tumors. This analysis also showed amplification of 3 to 12 genes in the primary tumors. As a first step towards identifying the genes methylated in the PNNs and primary hepatomas, we generated a rat NotI-EcoRV genomic library in the pBluescriptKS vector. Here, we describe identification of one methylated and downregulated gene as the rat protein tyrosine phosphatase receptor type O (PTPRO) and one amplified gene as rat C-MYC. Methylation of PTPRO at the NotI site located immediate upstream of the trancription start site in the PNNs and tumors, and amplification of C-MYC gene in the tumors were confirmed by Southern blot analyses. Bisulfite genomic sequencing of the CpG island encompassing exon 1 of the PTPRO gene revealed dense methylation in the PNNs and tumors, whereas it was methylation free in the livers of animals on normal diet. Reverse transcription-polymerase chain reaction (RT-PCR) analysis showed significant decrease in the expression of PTPRO in the tumors and in a transplanted rat hepatoma. The expression of PTPRO mRNA in the transplanted hepatoma after demethylation with 5-azacytidine, a potent inhibitor of DNA methyltransferases, further confirmed the role of methylation in PTPRO gene expression. These results demonstrate alteration in methylation profile and expression of specific genes during tumor progression in the livers of rats in response to folate/methyl deficiency, and further implicate the potential role of PTPRO as a novel growth regulatory gene at least in the hepatocellular carcinomas.

Animals↗
PubMed2003

Rapid diagnosis of vaginal carriage of group B beta haemolytic streptococcus by an enrichment cum antigen detection test.

BACKGROUND & OBJECTIVES: Group B beta haemolytic streptococcus (GBS) is a frequent colonizer of the maternal genital tract causing peripartum fever, puerperal sepsis, neonatal sepsis and neonatal meningitis. The conventional methods for detection of maternal colonization take 24-48 h. We made an attempt to standardize a rapid enrichment cum antigen detection test to screen pregnant women for GBS colonization in less than 8 h, so as to enable early institution of measures to prevent neonatal sepsis. METHODS: Vaginal swabs of 100 women >36 wk of gestation were inoculated onto enrichment broth (Todd Hewitt broth with lysed horse blood and antibiotics). After incubation for 1,2,4,6, and 18 h, the broth was cultured on sheep blood agar. In culture positive cases, the enrichment broth was subjected to antigen detection by latex agglutination test (LAT). For further evaluation of the rapid test, another group of 100 pregnant women were screened for GBS carriage by 6 h enrichment broth culture followed by antigen detection test. RESULTS: Five of the first group yielded GBS on culture and all were positive for GBS antigen after 6 h enrichment. Thirteen of the second group were positive for the antigen, but GBS could be isolated in ten only. This enrichment cum antigen detection test showed sensitivity, specificity, and positive and negative predictive values of 100, 98.4, 83.3 and 100 per cent respectively and could detect as few as 10(3) cfu/ml organisms. Maternal vaginal carriage of GBS was 7.5 per cent (15/200). INTERPRETATION & CONCLUSION: Six hours of enrichment followed by antigen detection proved to be a rapid and reliable method for detection of GBS colonization. This test is easy to perform making it an ideal test for screening GBS vaginal colonization at labour and starting chemoprophylaxis, where indicated on the same day, before the woman is discharged.

Adult↗
PubMed2001

Restoration of transforming growth factor-beta signaling enhances radiosensitivity by altering the Bcl-2/Bax ratio in the p53 mutant pancreatic cancer cell line MIA PaCa-2.

In this study, we investigated whether lack of transforming growth factor beta (TGF-beta) type II receptor (RII) expression and loss of TGF-beta signaling played a role in radiation resistance of pancreatic cancer cells MIA PaCa-2 that possess a mutated p53 gene. Transfection of this cell line with a RII cDNA led to a stimulation of the transcriptional activity of p3TP-Lux, a TGF-beta-responsive reporter construct. The RII transfectants (MIA PaCa-2/RII) showed a significant increase in sensitivity to radiation when compared with MIA PaCa-2/vector cells. The increase in sensitivity to radiation was reversed by neutralizing antibodies to TGF-beta, indicating that these changes were dependent on TGF-beta signaling. Compared with MIA PaCa-2/vector cells, MIA PaCa-2/RII cells showed a greater than 3-fold increase in apoptosis after radiation. Enhanced radiation sensitivity of MIA PaCa-2/RII cells was associated with an induction of Bax mRNA and protein that was followed by a release of cytochrome c and activation of caspase-3 and poly(ADP-ribose) polymerase cleavage after radiation exposure. Overexpression of Bcl-x(L) or treatment with antisense oligodeoxynucleotides targeted against Bax significantly inhibited radiation-induced apoptosis in MIA PaCa-2/RII but not in MIA PaCa-2/Vector cells, suggesting that Bax induction is necessary for radiation-induced TGF-beta signaling-mediated apoptosis. Thus, restoration of TGF-beta signaling sensitized these cells to ionizing radiation, although these cells possess a mutated p53 gene. In addition, disruption of RII function by dominant negative mutant of RII inhibited the radiation-induced TGF-beta signaling and apoptosis in primary cultures of mouse embryonic fibroblasts. Together, these observations imply that RII is an important component of radiation-induced TGF-beta signaling, and loss of function of RII may enhance resistance to radiation-induced apoptosis.

Apoptosis↗
PubMedDate not supplied

Par-4, a pro-apoptotic gene, inhibits radiation-induced NF kappa B activity and Bcl-2 expression leading to induction of radiosensitivity in human prostate cancer cells PC-3.

Ionizing radiation caused induction NF kappa B activity and Bcl-2 protein expression in the radioresistant p53 null human prostate cancer cell line, PC-3. Exposure of PC-3 cells to Ad5-I kappa B super-repressor inhibited radiation-induced Bcl-2 expression indicating that radiation-induced NF kappa B activity is required for the induction of Bcl-2 protein. PAR-4, a novel pro-apoptotic protein is a potent down-modulator of NF kappa B activity and bcl-2 protein expression. This study was undertaken to investigate the impact of PAR-4 expression on radiation-induced NF kappa B activity and Bcl-2 expression and its resultant radiation response in PC-3 cells. Western blot analysis indicated that enforced expression of PAR-4 in PC-3 cells down regulated radiation-induced bcl-2 protein, whereas in vector transfected cells radiation caused an induction of bcl-2 protein. In both transfectant cell lines, the bax protein levels remained unaltered after radiation. When compared to PC-3/Vector cells, PC-3/PAR-4 cells showed significant sensitivity to radiation-induced clonogenic inhibition and apoptosis. Thus, the down-regulation of bcl-2 protein by ectopic PAR-4 expression altered bcl-2: bax ratio in PC-3/PAR-4 cells and this led enhanced radiosensitivity. PAR-4 was found to inhibit the radiation-induced NF kappa B activity and NF kappa B transcriptional activity is essential for bcl-2 upregulation. In PC-3/Vector cells, radiation caused an increase in NF kappa B activity leading to upregulation of bcl-2 protein. However, in PC-3/PAR-4 cells, the radiation-induced NF kappa B activity was inhibited resulting in the transrepression of bcl-2 promoter and down-modulation of bcl-2 protein. In addition, PAR-4 was found to directly inhibit the phosphorylation and degradation of I kappa B alpha, which led to the loss of NF kappa B activity causing repression of endogenous and radiation-induced Bcl-2 protein. Together, these mechanisms suggest that PAR-4 is functionally required to cause radiation-induced apoptosis by abrogating the survival and anti-apoptotic effects of NF kappa B activity and bcl-2 function respectively.

Apoptosis↗