Natarajan Aravindan

Spinal glial glutamate transporters downregulate in rats with taxol-induced hyperalgesia

Changes in the expression of glial glutamate transporters (GLAST and GLT-1) were examined in the spinal cord of rats with chemotherapy (taxol)-induced mechanical hyperalgesia. Immunohistochemical studies show that the expression of both GLAST and GLT-1 in the L4-L5 spinal dorsal horn is decreased by 24% (P<0.001) and 23% (P<0.001), respectively, in rats with taxol-induced hyperalgesia as compared with those in control rats. These changes were further confirmed using an enzyme-linked immunosorbent assay that confirmed downregulation of GLAST by 36% (P<0.05) and GLT-1 by 18% (P<0.05) in the L4-L5 spinal cord of taxol-treated rats. These data indicate that downregulation of glutamate transporters may contribute to the development of hyperalgesia induced by taxol and suggest that glutamate transporters may be a new target for treatment of pain.

Venous thromboembolism in the intensive care unit

Most ICU patients have a significant number of risk factors for VTE. The high incidence of DVT in the ICU population and the recognition of a high incidence of PE at autopsy confirm this. We have alluded to the difficulty of clinical diagnosis of VTE and the need for diagnostic investigations. We have reviewed currently available diagnostic investigations with regard to their sensitivity and specificity and their practicability in ICU patients, and have formulated recommended diagnostic algorithms (Figs. 4 and 5). The most important factor in the management of VTE is prevention. In the ICU, all patients are at high risk for VTE, and therefore, at a minimum should receive subcutaneous prophylactic heparin unless it is contraindicated. Alternative methods of prophylaxis are available, and should be considered for patients who have contraindications to heparin.

Furosemide Prevents Apoptosis and Associated Gene Expression in a Rat Model of Surgical Ischemic Acute Renal Failure

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 μg/kg/hr) infusion, unilateral renal ischemia (1hr) 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 (≤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.

Curcumin regulates low-linear energy transfer γ-radiation-induced NFκB-dependent telomerase activity in human neuroblastoma cells

PURPOSE We recently reported that curcumin attenuates ionizing radiation (IR)-induced survival signaling and proliferation in human neuroblastoma cells. Also, in the endothelial system, we have demonstrated that NFκB regulates IR-induced telomerase activity (TA). Accordingly, we investigated the effect of curcumin in inhibiting IR-induced NFκB-dependent hTERT transcription, TA, and cell survival in neuroblastoma cells. METHODS AND MATERIALS SK-N-MC or SH-SY5Y cells exposed to IR and treated with curcumin (10-100 nM) with or without IR were harvested after 1 h through 24 h. NFκB-dependent regulation was investigated either by luciferase reporter assays using pNFκB-, pGL3-354-, pGL3-347-, or pUSE-IκBα-Luc, p50/p65, or RelA siRNA-transfected cells. NFκB activity was analyzed using an electrophoretic mobility shift assay and hTERT expression using the quantitative polymerase chain reaction. TA was determined using the telomerase repeat amplification protocol assay and cell survival using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltertrazolium bromide and clonogenic assay. RESULTS Curcumin profoundly inhibited IR-induced NFκB. Consequently, curcumin significantly inhibited IR-induced TA and hTERT mRNA at all points investigated. Furthermore, IR-induced TA is regulated at the transcriptional level by triggering telomerase reverse transcriptase (TERT) promoter activation. Moreover, NFκB becomes functionally activated after IR and mediates TA upregulation by binding to the κB-binding region in the promoter region of the TERT gene. Consistently, elimination of the NFκB-recognition site on the telomerase promoter or inhibition of NFκB by the IκBα mutant compromises IR-induced telomerase promoter activation. Significantly, curcumin inhibited IR-induced TERT transcription. Consequently, curcumin inhibited hTERT mRNA and TA in NFκB overexpressed cells. Furthermore, curcumin enhanced the IR-induced inhibition of cell survival. CONCLUSIONS These results strongly suggest that curcumin inhibits IR-induced TA in an NFκB dependent manner in human neuroblastoma cells.

Effect of furosemide infusion on renal hemodynamics and angiogenesis gene expression in acute renal ischemia/reperfusion

Loop diuretics are known to affect renal hemodynamics and possibly gene transcription, but the specific effect of furosemide on renal angiogenesis gene expression after acute ischemia is not known. We utilized an acute renal failure model in rats to test the hypothesis that furosemide improves renal hemodynamics and alters the transcriptional signature of acute ischemic nephropathy. Twenty-four male Sprague-Dawley rats were anesthetized by the intraperitoneal administration of 50 mg/kg urethane. Animals were divided into four groups (n = 6 each): (1) sham-operated group infused with saline; (2) sham-operated group infused with 30 μg/kg/hr furosemide (equivalent to a human dosage of 2 mg/hr); (3) unilateral renal ischemia (1 hr, left renal artery cross-clamping) followed by 6 hr of reperfusion; and (4) renal ischemia/reperfusion (I/R) with furosemide. Renal artery blood flow (RBF), renal cortical perfusion (RCP), and renal corticomedullary tissue oxygen tension (PO2) were recorded throughout. Following 6 hr of reperfusion, left kidney RNA was used to probe microarrays. Gene expression was measured as percent positive control and confirmed using reverse transcriptase polymerase chain reaction. Physiologic data were analyzed by calculating area under the curve, and gene expression data were compared by using multiple analysis of variance with Tukeys post-hoc tests. Furosemide significantly increased RBF (P < 0.05) and PO2 (P < 0.05) in postischemic kidneys. Furosemide attenuated nine of the 13 ischemia-induced and 41 of 78 ischemia-suppressed angiogenesis-related genes. This attenuation was statistically significant (P < 0.05) for 17 I/R injury-suppressed genes. Data from this rat model of ischemic nephropathy suggest that furosemide improves renal hemodynamics and attenuates ischemia-related changes in gene expression.

Anti-pancreatic cancer deliverables from sea: first-hand evidence on the efficacy, molecular targets and mode of action for multifarious polyphenols from five different brown-algae.

Pancreatic cancer (PC) remains the fourth leading cause of cancer death with an unacceptable survival that has remained relatively unchanged over the past 25 years. The presence of occult or clinical metastases at the time of diagnosis together with the lack of effective chemotherapies pose a dire need for designing new and targeted therapeutic deliverables that favors the clinical outcome. Herein, we investigated the anti-tumorigenic potential of polyphenols from five different brown-algae in human PC cells (MiaPaCa-2, Panc-1, BXPC-3 and Panc-3.27). Total anti-oxidant capacity (TAC) analysis on stepwise polyphenol separations with increasing polarity (Hexane-DCM-EA-methanol) identified high levels of TAC in DCM and EA extractions across all seaweeds assessed. All DCM and EA separated polyphenols induced a dose-dependent and sustained (time-independent) inhibition of cell proliferation and viability. Further, these polyphenols profoundly enhanced DNA damage (acridine orange/Ethidium bromide staining and DNA fragmentation) in all the cell lines investigated. More importantly, luciferase reporter assay revealed a significant inhibition of NFκB transcription in cells treated with polyphenols. Interestingly, QPCR analysis identified a differential yet definite regulation of pro-tumorigenic EGFR, VEGFA, AKT, hTERT, kRas, Bcl2, FGFα and PDGFα transcription in cells treated with DCM and EA polyphenols. Immunoblotting validates the inhibitory potential of seaweed polyphenols in EGFR phosphorylation, kRas, AurKβ and Stat3. Together, these data suggest that intermediate polarity based fractions of seaweed polyphenols may significantly potentiate tumor cell killing and may serve as potential drug deliverable for PC cure. More Studies dissecting out the active constituents in potent fractions, mechanisms of action and synergism, if any, are warranted and are currently in process.

Effect of Fenoldopam on Ischemia/Reperfusion-Induced Apoptosis

Recently we demonstrated the effect of fenoldopam on ischemia/reperfusion (I/R) induced NFκB 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 μg/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 I/R 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.

Metastatic neuroblastoma cancer stem cells exhibit flexible plasticity and adaptive stemness signaling

High-risk neuroblastoma (HR-NB) presenting with hematogenous metastasis is one of the most difficult cancers to cure. Patient survival is poor. Aggressive tumors contain populations of rapidly proliferating clonogens that exhibit stem cell properties, cancer stem cells (CSCs). Conceptually, CSCs that evade intensive multimodal therapy dictate tumor progression, relapse/recurrence, and poor clinical outcomes. Herein, we investigated the plasticity and stem-cell related molecular response of aggressive metastatic neuroblastoma cells that fit the CSC model. Well-characterized clones of metastatic site-derived aggressive cells (MSDACs) from a manifold of metastatic tumors of clinically translatable HR-NB were characterized for their CSC fit by examining epithelial-to-mesenchymal transition (EMT) (E-cadherin, N-Cadherin), survival (NFκB P65, p50, IκB and pIκB) and drug resistance (ABCG2) by immunoblotting; pluripotency maintenance (Nanog, SOX2) by immunofluorescence; and EMT and stemness related transcription of 93 genes by QPCR profiling. Plasticity of MSDACs under sequential alternation of culture conditions with serum and serum-free stem-cell conditions was assessed by clonal expansion (BrdU incorporation), tumorosphere formation (anchorage independent growth), EMT and stemness related transcriptome (QPCR profiling) and validated with MYC, SOX2, EGFR, NOTCH1 and CXCL2 immunoblotting. HR-NB MSDACs maintained in alternated culture conditions, serum-free stem cell medium to growth medium with serum and vice versa identified its flexible revocable plasticity characteristics. We observed signatures of stem cell-related molecular responses consistent with phenotypic conversions. Successive reintroduction to the favorable niche not only regained identical EMT, self-renewal capacity, pluripotency maintenance, and other stem cell-related signaling events, but also instigated additional events depicting aggressive adaptive plasticity. Together, these results demonstrated the flexible plasticity of HR-NB MSDACs that typically fit the CSC model, and further identified the intrinsic adaptiveness of the successive phenotype switching that clarifies the heterogeneity of HR-NB. Moreover, the continuous ongoing acquisition of stem cell-related molecular rearrangements may hold the key to the switch from favorable disease to HR-NB.

Abscopal effect of low-LET γ-radiation mediated through Rel protein signal transduction in a mouse model of nontargeted radiation response

Ascertaining the ionizing radiation (IR)-induced bystander response and its preceding molecular regulation would increase our understanding of the mechanism of acute and delayed radiobiological effects. Recent evidence clearly prompted that radiation-induced nuclear factor kappa B (NF-κB) would play a key role in bystander responses in nontargeted cells. Accordingly, we investigated the orchestration of NF-κB signaling after IR in a nontargeted distant organ. Heart tissues from C57/BL6 mice either mock irradiated or exposed (limited to lower abdomen 1 cm diameter) to single-dose IR (SDR: 2 or 10 Gy) or fractionated IR (FIR, 2 Gy per day for 5 days) were examined for onset of abscopal NF-κB signal transduction, translated activity, downstream functional signaling and associated DNA damage. Radiation significantly induced NF-κB DNA binding activity in nontargeted heart. Transcriptional profiling showed that 51, 46 and 26 of 88 genes were significantly upregulated after 2 Gy, 10 Gy and FIR. Of these genes, 22 showed dose- and fractionation-independent upregulation. Immunohistochemistry revealed a robust increase in p65 and cMyc expression in distant heart after SDR and FIR. Immunoblotting revealed increased phosphorylation of p38 after 2 Gy and extracellular signal-regulated kinases 1/2 after 10 Gy in nontargeted heart. In addition, IR exposure significantly enhanced DNA fragmentation in nontargeted heart. Together, these data clearly indicated an induced abscopal response in distant organ after clinically relevant IR doses. More importantly, the results imply that orchestration of NF-κB signal transduction in nontargeted tissues may serve as an effector and could play a key role in induced abscopal responses.

Fenoldopam Improves Corticomedullary Oxygen Delivery and Attenuates Angiogenesis Gene Expression in Acute Ischemic Renal Injury

Background/Aims: Vasoactive compounds are known to affect intrarenal hemodynamics and gene transcription, but specific effects of fenoldopam in the setting of acute renal ischemia are not known. We utilized a rat model of acute ischemic nephropathy to test the hypothesis that fenoldopam improves corticomedullary tissue oxygen tension (PtO2) and attenuates angiogenesis gene expression in acute renal ischemia. Methods: Rats anesthetized with 50 mg/kg urethane were divided into 4 groups (n = 6 each): (1) sham with infusion of 0.9% saline; (2) sham with infusion of 0.1 µg·kg–1·min–1 fenoldopam; (3) unilateral renal ischemia followed by 6 h of reperfusion with saline, and (4) ischemia/reperfusion with fenoldopam. Renal artery blood flow (RBF), renal cortical perfusion (RCP), and PtO2 were recorded throughout. Total RNA from left kidneys was used to probe microarrays. Gene expression was measured as percent positive control (GAPDH) and confirmed using RT-PCR. Results: Fenoldopam significantly increased RBF (p < 0.05), RCP (p < 0.01) and PtO2 (p <0.01) in both non-ischemic and post-ischemic kidneys. Fenoldopam attenuated 11 of the 13 ischemia-induced genes and 44 of 78 ischemia-suppressed genes. This attenuation was statistically significant (p <0.05) for five genes. Conclusion: Data from this rat model of ischemic nephropathy suggest that fenoldopam improves intrarenal hemodynamics and attenuates ischemia-related changes in angiogenesis gene expression.