Mohan Natarajan

High glucose induced nuclear factor kappa B mediated inhibition of endothelial cell migration.

Delayed wound healing and accelerated atherosclerosis are common vascular complications of diabetes mellitus. Although elevated blood glucose level is the major contributing factor, mechanisms that mediate these complications are not clearly understood. In the present study, we have demonstrated that elevated glucose inhibits endothelial cell migration, thereby delaying wound healing. Our results clearly indicated that high glucose (10 or 30 mM) induced activation of nuclear factor kappa B (NF-kappaB) inhibited endothelial cell migration (P<0.05). High glucose induced NF-kappaB DNA binding activity may mediate this inhibition of migration by regulating intracellular nitric oxide. In vitro wound healing model in human aortic endothelial cells (HAEC) were used to evaluate cell migration under the influence of high glucose. The migration inhibited by high glucose was restored by NF-kappaB inhibitors (including E3-4-methylphenyl sulfonyl-2-propenenitrile, N-tosyl-Lys-chloromethylketone (TLCK), or over-expression of inhibitor subunit of kappaB) and endothelial nitric oxide synthase inhibitors (N-methyl-L-arginine (L-NMMA); and Nomega-nitro-L-arginine methyl ester (L-NAME)). Furthermore, NF-kappaB inhibitors attenuated high glucose induced eNOS expression and intracellular nitric oxide (NO) production. Cytoskeletal immunofluorescence staining confirmed differences in actin distribution in HAEC incubated in high glucose in the presence or absence of NF-kappaB and NO inhibitors, explaining the differences observed in migration. In summary, our results for the first time suggest therapeutic strategies involving inhibition of NF-kappaB activation induced by high glucose, which may improve wound healing and help avoid some of the vascular complications of diabetes.

Diabetic eNOS knockout mice develop distinct macro- and microvascular complications

Functional consequences of impaired endothelial nitric oxide synthase (eNOS) activity causing organ-specific abnormalities on a diabetic setting are not completely understood. In this study, we extensively characterized a diabetic mouse model (leprdb/db) in which eNOS expression is genetically disrupted (eNOS−/−). The eNOS−/−/ leprdb/db double-knockout (DKO) mice developed obesity, hyperglycemia, hyperinsulinemia and hypertension. Analysis of tissues from DKO mice showed large islets in the pancreas and fat droplets in hepatocytes. Interestingly, the aorta was normal and atherogenic lesions were not observed. Abnormalities in the aorta including poor re-endothelialization and increased medial wall thickness were evident only in response to deliberate injury. In contrast, significant glomerular capillary damage in the kidney was identified, with DKO mice demonstrating a robust diabetic nephropathy similar to human disease. The vascular and renal impairments in DKO mice were pronounced despite lower fasting plasma glucose levels compared to leprdb/db mice, indicating that eNOS is a critical determinant of hyperglycemia-induced organ-specific complications and their severity in diabetes. Results provide the first evidence that absence of eNOS in diabetes has a greater deleterious effect on the renal microvasculature than on the larger aortic vessel. The DKO model may suggest novel therapeutic strategies to prevent both vascular and renal complications of diabetes.

Antioxidant activity of melatonin in Chinese hamster ovarian cells: changes in cellular proliferation and differentiation

Melatonin is an endogenously generated molecule with free radical scavenging and antioxidant properties. Here, we studied the antiproliferative role of melatonin and other antioxidants on transformed Chinese hamster ovarian cells. Melatonin reduces cell proliferation in a dose- and time-dependent manner. Natural antioxidants which appear in edible plants including resveratrol and vitamin E mimicked the effect of melatonin. Flow cytometer analysis revealed that melatonin treatment reduces the number of cells in S-phase and increases cells in both G0/G1 and G2/M gaps. In addition, melatonin, as well as trolox, caused a clear morphological change by inducing the cells to become spindle shaped and fibroblast-like. Its effect is a reversible phenomenon that disappeared when melatonin was withdrawn from the culture medium. GSH levels are increased after melatonin treatment but pharmacologically blockade of GSH synthesis did not abolish melatonins antiproliferative effect. Reduction of cell proliferation and the apparent induction of cell differentiation overlapped with melatonins ability to change the intracellular redox state of CHO cells. We conclude that the cellular redox state may be involved in cellular transformation caused by antioxidants such as melatonin and trolox.

Critical role of glutathione in melatonin enhancement of tumor necrosis factor and ionizing radiation-induced apoptosis in prostate cancer cells in vitro

Abstract:  The role of antioxidants in reducing cancer initiation and progression has been highlighted in recent years. Not only antioxidants limit cancer cell growth but also, in some situations, they promote the effectiveness of conventional treatments. Melatonin, an endogenously synthesized antioxidant, reduces cell growth of several tumor types both in vivo and in vitro. Additionally, the indole limits the collateral damage induced by many chemotherapeutic agents. By using a cellular model of human prostate cancer, we studied the ability of melatonin to enhance apoptosis induced by tumor necrosis factor or gamma radiation. It has been reported that melatonin reduces prostate cancer cell growth and, more recently, it promotes cell differentiation. In this work, we also show that melatonin elevates p21 protein levels and increases antioxidant capacity of prostate cancer cells. In addition, melatonin significantly enhances hrTNFα induced cell death by decreasing NFκB activation. Bcl‐2 and survivin down‐regulation appears to be associated to apoptosis stimulation under NFκB inhibition. On the contrary, melatonin does not promote irradiation‐induced cell death due to an increment in intracellular glutathione content. In conclusion, prevention of NFκB activation by melatonin enhances the effectiveness of cytokine treatment in prostate cancer cells but it is not sufficient to enhance cell death triggered by other therapies which generate free radicals. A crucial role of glutathione in survival mechanisms of prostate cancer cells should be carefully considered.

Radiation-triggered Tumor Necrosis Factor (TNF) α-NFκB Cross-signaling Favors Survival Advantage in Human Neuroblastoma Cells

Induced radioresistance in the surviving cancer cells after radiotherapy could be associated with clonal selection leading to tumor regrowth at the treatment site. Previously we reported that post-translational modification of IκBα activates NFκB in response to ionizing radiation (IR) and plays a key role in regulating apoptotic signaling. Herein, we investigated the orchestration of NFκB after IR in human neuroblastoma. Both in vitro (SH-SY5Y, SK-N-MC, and IMR-32) and in vivo (xenograft) studies showed that IR persistently induced NFκB DNA binding activity and NFκB-dependent TNFα transactivation and secretion. Approaches including silencing NFκB transcription, blocking post-translational NFκB nuclear import, muting TNF receptor, overexpression, and physiological induction of either NFκB or TNFα precisely demonstrated the initiation and occurrence of NFκB → TNFα → NFκB positive feedback cycle after IR that leads to and sustains NFκB activation. Selective TNF-dependent NFκB regulation was confirmed with futile inhibition of AP-1 and SP-1 in TNF receptor muted cells. Moreover, IR increased both transactivation and translation of Birc1, Birc2, and Birc5 and induced metabolic activity and clonal expansion. This pathway was further defined to show that IR-induced functional p65 transcription (not NFκB1, NFκB2, or c-Rel) is necessary for activation of these survival molecules and associated survival advantage. Together, these results demonstrate for the first time the functional orchestration of NFκB in response to IR and further imply that p65-dependent survival advantage and initiation of clonal expansion may correlate with an unfavorable prognosis of human neuroblastoma.

High glucose-induced IKK-Hsp-90 interaction contributes to endothelial dysfunction.

A decline in the bioavailability of nitric oxide (NO) that causes endothelial dysfunction is a hallmark of diabetes. The availability of NO to the vasculature is regulated by endothelial nitric oxide synthase (eNOS) activity and the involvement of heat shock protein-90 (Hsp-90) in the regulation of eNOS activity has been demonstrated. Hsp-90 has been shown to interact with upstream kinases [inhibitor kappaB kinases (IKK)alpha, beta, and gamma] in nonvascular cells. In this study, we have investigated the interaction of Hsp-90-IKKbeta in endothelial cells under conditions of high glucose (HG) as a possible mechanism that diminishes Hsp-90-eNOS interaction, which could contribute to reduced bioavailability of NO. We report for the first time that IKKbeta interacts with Hsp-90, and this interaction is augmented by HG in vascular endothelial cells. HG also augments transcriptional (3.5 +/- 0.65-fold) and translational (1.97 +/- 0.17-fold) expression as well as the catalytic activity of IKKbeta (2.45 +/- 0.4-fold). Both IKKbeta and eNOS could be coimmunoprecipitated with Hsp-90. Inhibition of Hsp-90 with geldanamycin (2 microM) or Radicicol (20 microM) mitigated (0.45 +/- 0.04-fold and 0.93 +/- 0.16-fold, respectively) HG induced-IKKbeta activity (2.5 +/- 0.42-fold). Blocking of IKKbeta expression by IKK inhibitor II (15 microM wedelolactone) or small interferring RNA (siRNA) improved Hsp-90-eNOS interaction and NO production under conditions of HG. These results illuminate a possible mechanism for the declining eNOS activity reported under conditions of HG.

Efficacy of Mycobacterium indicus pranii Immunotherapy as an Adjunct to Chemotherapy for Tuberculosis and Underlying Immune Responses in the Lung

Background The 9-month-long chemotherapy of tuberculosis often results in poor compliance and emergence of drug-resistant strains. So, improved therapeutic strategy is urgently needed. Immunotherapy could be beneficial for the effective management of the disease. Previously we showed the protective efficacy of Mycobacterium indicus pranii (MIP) when given as prophylactic vaccine in animal models of tuberculosis. Methods We sought to investigate whether MIP can be used as an adjunct to the chemotherapy in guinea pig models of tuberculosis. Efficacy of MIP was evaluated when given subcutaneously or by aerosol. Results MIP-therapy as an adjunct to the chemotherapy was found to be effective in accelerating bacterial killing and improving organ pathology. MIP-immunotherapy resulted in higher numbers of activated antigen-presenting cells and lymphocytes in the infected lungs and also modulated the granulomatous response. Early increase in protective Th1 immune response was observed in the immunotherapy group. Following subsequent doses of MIP, decrease in the inflammatory response and increase in the immunosuppressive response was observed, which resulted in the improvement of lung pathology. Conclusion MIP immunotherapy is a valuable adjunct to chemotherapy for tuberculosis. Aerosol route of immunotherapy can play a crucial role for inducing immediate local immune response in the lung.

Setup and characterization of a human head and neck squamous cell carcinoma xenograft model in nude rats

OBJECTIVE: To develop and characterize a new head and neck cancer animal model. STUDY DESIGN: A human head and neck squamous cell carcinoma (HNSCC) xenograft model in nude rats was established via subcutaneous inoculation of a human-origin HNSCC cell line, SCC-4. The tumor was evaluated for growth characteristics, pathologic features by hematoxylin-eosin (HE) staining, and immunohistochemistry of epidermal growth factor receptor (EGFR). 2-[18F] fluoro-2-deoxy-D-glucose (18F-FDG) positron emission tomography (PET) imaging characteristics were studied too. RESULTS: A new HNSCC animal model was successfully established. Tumor sizes reached about 1 cm3 on day 15 after tumor cell inoculation. HE staining pathology has confirmed that this tumor is a typical SCC. EGFR immunohistochemistry demonstrated this tumor model to be strongly EGFR positive. 18F-FDG PET study has shown that 18F-FDG accumulated in tumors. CONCLUSIONS: This study has demonstrated that this tumor model is an appropriate HNSCC tumor model for animal studies on HNSCC.

NFκB activity and transcriptional responses in human breast adenocarcinoma cells after single and fractionated irradiation

Radiotherapy is considered mandatory for breast cancer patients undergoing conservative surgery and for women at high risk of recurrence. However, relapse due to radio-resistance affects the success of radiotherapy. Ascertaining the fractionated radiation (FIR) modulated molecular targets is important to make tumors more susceptible to molecular targeted therapy. Accordingly, we investigated the (i) expression of 84 genes representing six functional pathways; (ii) NFκB DNA binding activity and; (iii) expression of radio-responsive molecules after single dose (10Gy) radiation (SDR) and FIR (2Gyx5). MCF-7 cells exposed to SDR or FIR were analyzed for alterations in gene expression using QPCR-profiling. NFκB DNA binding activity was analyzed using EMSA and pIκB using immunoblotting. Expression of TNFα, IL-1α, pAKT, IAP1, IAP2, XIAP, survivin, MnSOD, BID and Bak were determined using QPCR and/or immunoblotting. Compared to SDR, FIR significantly induced 60 genes and completely suppressed 14 genes. Furthermore, FIR induced NFκB-DNA binding activity and IκBα phosphorylation. Like-wise, FIR induced the expression of IAP1, IAP2, XIAP Survivin, MnSOD, TNFα, pAKT and IL-1α. The results of the study clearly show distinct differences in the molecular response of cells between SDR and FIR exposures. We identified several potential targets that may affect radio-resistance following FIR.

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.