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Objectives: Nuclear factor κB (NF-κB) plays an intrinsic role in promoting growth, angiogenesis, and metastasis in pancreatic cancer (PC) and serves as a mechanism underlying therapeutic resistance. Accordingly, we investigated the efficacy of bioactive phytochemicals in inhibiting radiotherapy (RT)-induced NF-κB activity, signaling, and NF-κB-dependent regulation of cell death., Methods: Panc-1, BxPC-3, and MIA PaCa-2 cells exposed to 10 Gy (single high dose [SDR]) or 2 Gy/d for 5 days (fractionated radiation [FIR]) with or without curcumin (CUR), neem leaf extract (NLE), or black raspberry extract (RSE) were analyzed., Results: Radiotherapy profoundly induced NF-κB-DNA-binding activity with relatively robust activation after FIR. Curcumin, NLE, and RSE significantly inhibited both constitutive and RT-induced NF-κB. Furthermore, quantitative polymerase chain reaction profiling of 88 NF-κB pathway molecules demonstrated that CUR, NLE, and RSE comprehensively, yet differentially inhibited FIR/SDR-induced genes. Functionally, CUR, NLE, and RSE markedly conferred RT-inhibited cell viability/survival, robustly activated caspase-3/7 activity, and subsequent cell death. More importantly, NF-κB overexpression and silencing studies demonstrate that these compounds potentiate RT-induced cell death by targeting RT-induced NF-κB., Conclusions: These data strongly imply that CUR, NLE, and RSE may serve as effective "deliverables" to potentiate RT in PC cure and further throw light that these phytochemicals-induced cell killing may involve selective regulation of RT-induced NF-κB.

Unlabelled: Induction of apoptosis is directly correlated with the biological effectiveness of ionizing radiation (IR). Accordingly, we investigated the efficacy of neem leaf extract (NLE) on IR-associated apoptotic transcriptional modulation and cell death in neuroblastoma (NB)., Materials and Methods: NB xenografts exposed to single dose (SDR, 10 Gy) or fractionated (FIR, 2 Gy/d×5d) with or without NLE were examined for transcriptional activation of 84 apoptotic pathway genes using quantitative polymerase chain reaction. Apoptosis was measured using TdT nick-end labeling., Results: FIR induced 55 and suppressed 10 genes, while SDR induced 49 and suppressed 5 genes. Of these, 46 and 4 genes were commonly up/down-regulated after FIR and SDR. NLE inhibited IR-induced NAIP, BIRC6, BIRC8, NOL3 and enhanced BAK1, BAX, BCL10, CASP1, CASP10 CARD8 and CRADD. Furthermore, NLE conferred FIR- and SDR-induced cell death., Conclusion: These data imply that NLE may exert radiosensitization by activating pro-apoptotic signaling and negating survival signaling and may thus potentiate radiotherapy in NB.

Conceptual approaches of heat-induced cytotoxic effects against tumor cells must address factors affecting therapeutic index, i.e., the relative toxicity for neoplastic versus normal tissues. Accordingly, we investigated the effect of hyperthermia treatment (HT) on the induction of DNA fragmentation, apoptosis, cell-cycle distribution, NFkappaB mRNA expression, DNA-binding activity, and phosphorylation of IkappaBalpha in the normal human Mono Mac 6 (MM6) cells. For HT, cells were exposed to 43 degrees C. FACS analysis showed a 48.5% increase in apoptosis, increased S-phase fraction, and reduced G2 phase fraction after 43 degrees C treatments. EMSA analysis showed a dose-dependent inhibition of NFkappaB DNA-binding activity after HT. This HT-mediated inhibition of NFkappaB was persistent even after 48 h. Immunoblotting analysis revealed dose-dependent inhibition of IkappaBalpha phosphorylation. Similarly, RPA analysis showed that HT persistently inhibits NFkappaB mRNA. These results demonstrate that apoptosis upon HT exposure of MM6 cells is regulated by IkappaBalpha phosphorylation mediated suppression of NFkappaB.

Ascertaining the upstream regulatory mechanisms of hyperthermia-induced apoptosis is important to understand the role of hyperthermia in combined modality cancer therapy. Accordingly, we investigated whether (i) hyperthermia-induced apoptosis is mediated through the nitric oxide (NO) signaling pathway and (ii) inhibition of post-translational modification of IkappaBalpha and down regulation of NFkappaB-DNA binding activity is an intermediate step in NO-dependent apoptosis in MCF-7 breast cancer cells. For hyperthermia treatment, the cells were exposed to 43 degrees C. Intracellular NO levels measured by the fluorescent intensity of DAF-2A and iNOS expression by immunobloting revealed an increased level of iNOS dependent NO production after 43 degrees C. Apoptosis measured by Annexin V expression and cell survival by clonogenic assay showed a 20% increase in apoptosis after 43 degrees C treatments. EMSA analysis showed a dose-dependent inhibition of NFkappaB-DNA binding activity. The hyperthermia-mediated inhibition of NFkappaB was persistent even after 48 h. Inhibition of NO by L-NAME rescued the NFkappaB-DNA binding activity and inhibits heat-induced apoptosis. Similarly, over-expression of NFkappaB by transient transfection inhibits heat-induced apoptosis. These results demonstrate that apoptosis upon hyperthermia exposure of MCF-7 cells is regulated by NO-mediated suppression of NFkappaB., (Copyright 2009 Wiley-Liss, Inc.)

Ascertaining the time-dependent regulation of induced apoptosis and radioresistance is important to understand the relationship between the level of spontaneous apoptosis in cells and their radiosensitivity. Accordingly, we investigated the time-dependent expression of apoptosis related genes and radioresistance in neuroblastoma cells. Serum-starved human SK-N-MC cells were exposed to low linear energy transfer (LET) radiation (2 Gy) and incubated for 15, 30, 45 min, and 48 h. Radioresistance was investigated by examining the NF kappa B DNA-binding activity, cellular toxicity, DNA fragmentation, and expression of apoptotic signal transduction molecules. NF kappa B DNA binding activity was analyzed using electrophoretic mobility shift assay (EMSA). Cellular toxicity was measured using MTT assay. DNA fragmentation was quantified by labeling with fluorescein-conjugated deoxynucleotides. Microarray analysis was performed using cDNA microarray and relative gene expression was measured as % GAPDH and, subsequently validated using Q-PCR. Induction of NF kappa B analyzed using EMSA showed an increased DNA-binding activity at all time points investigated. Induced DNA fragmentation was observed after 15, 30, and 45 min post-radiation. Relatively, induced fragmentation was reduced after 48 h. Compared to the untreated controls cellular toxicity was induced with low LET radiation after 15, 30, and 45 min. Conversely, cytotoxicity was relatively less at 48 h after low LET radiation. Microarray analysis after low LET radiation revealed time-dependent modulation of apoptosis-related genes that are involved in radio-adaptation, spontaneous apoptosis-related early-responsive genes and late response genes. These results suggest that the time-dependent regulation of apoptotic response may determine the relationship between the level of spontaneous apoptosis in cells and their radiosensitivity.

Critical illness myopathy (CIM) causes significant morbidity. In this study, we investigated the effect of repeated mild hypoxia on the skeletal muscle inflammation. Sprague-Dawley rats anesthetized with 2% inhaled isoflurane were divided into two groups (n = 6 each), normoxia and hypoxia (12.5% for 12 min followed by 35% for 12 min, at which point the cycle was repeated for three times). We measured the tissue oxygen tension and perfusion (simultaneously) in hind limb skeletal muscle. Inflammation in skeletal muscle was assessed by light microcopy (Hematoxylin-Eosin staining) and apoptosis (Fluorescein-FragEL DNA fragmentation detection) and expressed as percent normoxia. Compared to the control group, hypoxia significantly (P < 0.001) altered histomorphometrics. Similarly, DNA fragmentation analysis revealed that hypoxia significantly (P < 0.001) induced apoptosis. We conclude that after a mild but repeated hypoxic insult there is marked histological alterations and induced apoptosis in skeletal muscle. We postulate that variable periods of hypoxia in the critically ill may be playing a role in the etiology of CIM.

Recently, we demonstrated that furosemide improves renal hemodynamics and attenuates ischemia/reperfusion (I/R)-associated changes in angiogenesis-related gene expression. However, the effect of furosemide on I/R-induced apoptosis is not known. We utilized a rat model of acute ischemic nephropathy to test the hypothesis that furosemide attenuates I/R-induced apoptosis. Male Sprague-Dawley rats anesthetized with urethane (50 mg/kg) were randomly allocated into four groups (n = 6 each): sham operated saline infusion, sham operated with furosemide (30 microg/kg/hr) infusion, unilateral renal ischemia (1 hr) followed by six hours of reperfusion, and I/R with furosemide infusion. Apoptosis was measured in kidney samples and compared between groups using ANOVA with Bonferroni correction. Apoptosis-related gene expression was assessed using microarray analysis and validated with RT-PCR. Phosphorylation of Akt was analyzed using ELISA, and data were compared between groups using the Mann Whitney U test. Compared to the control group, I/R significantly (p < 0.001) induced apoptosis in both the cortex and medulla. Similarly, microarray analysis revealed that I/R induced (< or = two-fold increase compared to control group) 73 apoptosis-related genes. Phosphorylation of Akt was significantly (p < 0.05) downregulated after I/R. Treatment with furosemide significantly (p < 0.001) reduced I/R-induced apoptosis in both the cortex and medulla and attenuated the expression of 72 I/R-induced apoptosis-related genes. Compared to the I/R group, furosemide significantly (p < 0.01) upregulated the phosphorylation of Akt. These data suggest that a low dose furosemide infusion may attenuate I/R-induced apoptosis and associated gene transcription, and imply a possible novel molecular basis for the mechanism of action of furosemide in acute renal failure.

Background: Renal cell apoptosis contributes significantly to the pathogenesis of acute renal failure. Anesthetic agents have been shown to modulate apoptotic signal transduction in various tissues. We examined the effects of 6 h of different general anesthetic techniques on renal cell apoptosis in rat kidneys., Methods: Twenty-one male Sprague-Dawley rats were randomly allocated into four groups: (i) control, non-anesthetized rats (n= 3) and rats anesthetized with (ii) inhaled isoflurane (n= 6), (iii) intraperitoneal pentobarbital (n= 6), and (iv) intraperitoneal urethane (n= 6). Animals were sacrificed 6 h after the induction of anesthesia., Results: Apoptosis was assessed by terminal deoxynucleotidyl transferase-fluorescein end-labeling analysis. RNA was extracted from the left kidney to probe cDNA microarrays. Gene expression was measured as a percentage of glyceraldehyde 3-phosphate dehydrogenase (GAPDH) and subsequently confirmed using reverse transcriptase-polymerase chain reaction (RT-PCR). Compared with the control (no anesthesia), urethane significantly (P < 0.001) induced apoptosis in both the renal cortex and medulla. Isoflurane significantly (P < 0.001) inhibited apoptosis in the medulla. Microarray analysis revealed that urethane up-regulated more (74) genes than pentobarbital (16) and isoflurane (10). Isoflurane down-regulated more genes (85) than pentobarbital (74) and urethane (12). These anesthetic-induced modulations were significant (P < 0.05) for 60 isoflurane-, 30 pentobarbital- and 4 urethane-modulated genes., Conclusion: Our results suggest that general anesthetic drugs have an effect on renal cell apoptosis and apoptotic signal transduction, and thus may potentially affect the risk of subsequent acute renal failure.

Recently we demonstrated the effect of fenoldopam on ischemia/reperfusion (I/R) induced NFkappaB mediated pro-inflammatory signal transduction. However, the effect of fenoldopam on I/R-induced apoptosis is not known. We utilized a rat model of acute ischemic nephropathy to test the hypothesis that fenoldopam attenuates I/R-induced apoptosis. Sprague-Dawley rats were anesthetized by intraperitoneal administration of 50 mg/kg urethane and randomly allocated into 4 groups (n=6 each): (1) sham-operated, (2) sham operation with infusion of 0.1 microg/kg/min fenoldopam, (3) unilateral renal ischemia followed by 4 h of reperfusion, and (4) I/R with fenoldopam infusion. Kidney samples were fixed and paraffin-embedded to measure apoptosis. Data were compared between groups using ANOVA with Bonferroni correction. RNA was extracted from each left kidney to probe cDNA microarray and measure gene expression as percent of positive control. Compared to the control group, I/R significantly (P < 0.001) induced apoptosis in both the cortex and medulla. Similarly, microarray analysis revealed that IR induced 73 apoptosis-related genes. Treatment with fenoldopam significantly reduced (P < 0.001) I/R-induced apoptosis both in the cortex and medulla and attenuated all 73 I/R-induced apoptosis-related genes. Data from this rat model of ischemic nephropathy suggest that fenoldopam may attenuate I/R-induced apoptosis and apoptosis-related gene transcription.