Rakhesh Madhusoodhanan

Curcumin inhibits NFκB mediated radioprotection and modulate apoptosis related genes in human neuroblastoma cells

Curcumin has been shown to exhibit growth inhibitory effects and induce apoptosis in a broad range of tumors. Accordingly, we investigated the radiosensitizing effects of curcumin in human neuroblastoma cells. SK-N-MC cells exposed to either 2Gy alone, or pretreated with curcumin (100nM) or NFκB inhibitor peptide SN50 (50nM) and exposed to 2Gy were harvested after 48h. Radioresistance was measured using clonogenic and MTT assay, NFκB DNA-binding activity using electrophoretic mobility shift assay, and apoptosis using Annexin V-FITC staining. Pathway (apoptosis) specific microarrays were used to measure gene expression and validated using QPCR. Radiation markedly enhanced the NFκB DNA-binding activity. Pre-treating the cells either with curcumin or SN50 significantly suppressed the radiation induced NFκB. Also, curcumin or SN50 pretreatment enhanced the radiation induced inhibition of cell survival. Microarray analysis revealed that curcumin enhanced the radiation induced activation of caspases, other pro-apoptotic and death effector molecules and, inhibit anti-apoptotic/survival molecules. In addition, curcumin markedly suppressed the radiation induced TNF super family genes. These results suggest that curcumin is a potent radiosensitizer and may act by overcoming the effects of radiation-induced NFκB mediated pro-survival gene expression in neuroblastoma.

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.

Curcumin Regulates Low-Linear Energy Transfer {gamma}-Radiation-Induced NF{kappa}B-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.

Surfactant protein-A and Toll-like receptor-4 Modulate Immune Functions of Preterm Baboon Lung Dendritic Cell Precursor Cells

Lung infections are important risk factors for an increased morbidity and mortality in prematurely-delivered babies. Immaturity of the innate immune components makes them extremely susceptible to infection. Recently, we isolated lung dendritic cell (DC)-precursor cells from preterm fetal baboons. The isolated cells were found to be defective in phagocytosing Escherichia coli under basal conditions. In this study, we investigated the effects of exogenously-added purified native lung surfactant protein (SP)-A and recombinant toll-like receptor (TLR)-4-MD2 proteins on phagocytic uptake and cytokine secreting ability of fetal baboon lung DC-precursor cells. The cells were pulsed with SP-A and/or TLR4-MD2 proteins and the phagocytic function was investigated by incubating the cells with fluorescent-labeled E. coli bioparticles and analyzed by spectrofluorometry. The amounts of TNF-α secreted in cell-free supernatants were measured by ELISA. Our results demonstrate that SP-A and TLR4-MD2 proteins, whether added alone or together, induce phagocytosis of E. coli (p<0.05). The SP-A does not affect TNF-α secretion, while the TLR4-MD2 protein induces TNF-α. However, simultaneous addition of SP-A with TLR4-MD2 protein reduces the TLR4-MD2-protein induced TNF-α to basal level. In conclusion, our results indicate that an exogenous administration of SP-A can potentially induce phagocytic activity and anti-inflammatory effect in preterm babies, and help control infection and inflammation.

A TLR4-interacting SPA4 peptide inhibits LPS-induced lung inflammation

The interaction between surfactant protein-A (SP-A) and TLR4 is important for host defense. We have recently identified an SPA4 peptide region from the interface of SP-A–TLR4 complex. Here, we studied the involvement of the SPA4 peptide region in SP-A–TLR4 interaction using a two-hybrid system, and biological effects of SPA4 peptide in cell systems and a mouse model. HEK293 cells were transfected with plasmid DNAs encoding SP-A or a SP-A-mutant lacking SPA4 peptide region and TLR4. Luciferase activity was measured as the end-point of SP-A–TLR4 interaction. NF-κB activity was also assessed simultaneously. Next, the dendritic cells or mice were challenged with Escherichia coli-derived LPS and treated with SPA4 peptide. Endotoxic shock-like symptoms and inflammatory parameters (TNF-α, NF-κB, leukocyte influx) were assessed. Our results reveal that the SPA4 peptide region contributes to the SP-A–TLR4 interaction and inhibits the LPS-induced NF-κB activity and TNF-α. We also observed that the SPA4 peptide inhibits LPS-induced expression of TNF-α, nuclear localization of NF-κB-p65 and cell influx, and alleviates the endotoxic shock-like symptoms in a mouse model. Our results suggest that the anti-inflammatory activity of the SPA4 peptide through its binding to TLR4 can be of therapeutic benefit.

Effect of Black Raspberry Extract in Inhibiting NFκ B Dependent Radioprotection in Human Breast Cancer Cells

Black raspberry extracts (RSE) have been shown to inhibit cancer cell growth and stimulate apoptosis. Also, studies have demonstrated that RSE inhibits transcriptional regulators including NF κ B. Accordingly, we investigated the effect of RSE in inhibiting radiation (IR) induced NF κ B mediated radioprotection in breast adenocarcinoma cells. MCF-7 cells were exposed to IR (2Gy), treated with RSE (0.5, 1.0, 2.0 μ g/ml) or treated with RSE (1.0 μ g/ml) followed by IR exposure, and harvested after 1, 3, 6, 24, 48, and 72 h. NF κ B DNA-binding activity was measured by EMSA and phosphorylated I κ B α by immunoblotting. Expression of IAP1, IAP2, XIAP and survivin were measured by QPCR and immunoblotting. Cell survival was measured using MTT assay and cell death using Caspase-3/7 activity. Effect of RSE on IR induced MnSOD, TNF α , IL-1 α and MnSOD activity was also determined. RSE inhibited NF κ B activity in a dose-dependent manner. Also, RSE inhibited IR-induced sustained activation of NF κ B, and NF κ B regulated IAP1, IAP2, XIAP, and survivin. In addition, RSE inhibited IR-induced TNF α , IL-1 α , and MnSOD levels and MnSOD activity. RSE suppressed cell survival and enhanced cell death. These results suggest that RSE may act as a potent radiosensitizer by overcoming the effects of NF κ B mediated radioprotection in human breast cancer cells.

Abstract 486: Abscopal effect of low-LET ionizing radiation on Rel protein signal transduction

Ascertaining the ionizing radiation (IR)-induced bystander response and identifying its mechanistic regulation would raise a paradigm shift in our understanding of the radiobiological effects. Recent evidence from our lab and others clearly prompted that the transcriptional regulator NFκB would play a key role in induced responses in non-targeted bystander cells. In this study, for the first time, we investigated the orchestration of NFκB signaling after IR in an organ (heart) distant from the exposure field. The C57/BL6 mice either mock irradiated, exposed to single dose IR (SDR: 2 or 10Gy) or fractionated IR (FIR, 2 Gy/day for 5 days) were sacrificed 24h post-irradiation. All IR exposures were limited to the lower abdomen area (1cm diameter), while the rest of the animal was protected by a specially designed cerrobend shield. EMSA analysis was used to examine the changes in NFκB DNA binding activity in the heart. Transcriptional activation of 88 mediators associated with NFκB signaling pathway was assessed using QPCR profiling. Phosphorylation of ERK1/2 and p38 that play a key role in NFκB signal transduction were examined using immunoblotting. Induced DNA fragmentation was measured using TdT nick end labeling. IR significantly induced NFκB DNA binding activity in the heart. Further more, real-time QPCR profiling showed a significant and differential modulation in the transcriptional responses of NFκB signaling pathway molecules. To that end, of the 88 genes analyzed, 28, 51 and 41 genes were upregulated after 2Gy, 10Gy and FIR, respectively. More importantly, at these exposures13 out of 28, 27 out of 51 and 25 out of 41 induced mediators of NFkB pathway were significantly upregulated. Conversely, lower abdominal IR exposure markedly suppressed 44, 20 and 30 genes after 2 Gy, 10Gy and FIR, respectively in the heart of the exposed animals. Compared to 2Gy, FIR significantly induced 26 genes, while 10Gy upregulated 27 genes. In addition, 6 and 15 genes were significantly upregulated after acute dose of 2 and 10Gy as opposed to fractionated dose. Consistently, immunoblotting analysis revealed a profound phosphorylation of both ERK1/2 and p38 in the heart. Interestingly, distant IR exposure significantly enhanced DNA fragmentation in the heart tissue. Taken together, these data clearly indicated an induced abscopal response in mice heart after clinically relevant doses of IR exposure. More importantly, these data implies that orchestration of NFκB signal transduction may play a key role in induced abscopal responses. Funding Support: Presbyterian Health Foundation, OKC. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 486.

Radiation induced molecular modulations in human breast adenocarcinoma cells after single (10Gy) and fractionated (2Gy x 5) dose irradiation.

CTRC-AACR San Antonio Breast Cancer Symposium: 2008 Abstracts Abstract #5133 Background : Radiotherapy a recognized treatment modality for breast cancer patients is now considered mandatory for most patients undergoing conservative surgery and is considered appropriate for women at high risk of recurrence after mastectomy. However, relapse due to radio-resistance of cancer cells significantly affects the success of radiotherapy and cancer patient survival. Ascertaining the fractionated irradiation (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 6 functional pathways including Cell cycle and DNA damage repair, Apoptosis and cell senescence, Signal transduction and transcription, Adhesion, Invasion and metastasis and Angiogenesis; (ii) nuclear translocation and DNA-binding activity of NFκB and; (iii) expression of radio-responsive TNFα, IL-1α, pAKT, cIAP1, cIAP2 and survivin molecules after single dose (SDR) radiation and FIR protocols in human breast adenocarcinoma cells. Methods : MCF-7 cells exposed to either SDR (10Gy) or FIR (2Gy x 5 days) and harvested after 24h were analyzed for alterations in gene expression using real-time Q-PCR-profiling. NFκB-DNA binding activity was analyzed using electrophoretic mobility shift assay and pIκB levels was measured using immunoblotting. Relative changes in the expression patterns of TNFα, IL-1α, pAKT, cIAP1, cIAP2 and survivin were determined using Q-PCR analysis and immunoblotting. Results : Compared to the SDR, FIR treated cells showed an induced expression 61 (Cell cycle & DNA damage repair - 12, Apoptosis & cell senescence - 10, Signal transduction & transcription - 7, Adhesion - 8, Invasion & metastasis – 11 and Angiogenesis – 13) genes. Furthermore, we observed a relatively induced DNA binding activity of NFκB and phosphorylation of IκBα after FIR. Like-wise, we observed an induced expression of TNF α , IL-1 α , pAKT, cIAP1, cIAP2 and survivin mRNA in cells exposed to FIR . The changes in these genes were confirmed at the protein level. Discussion : These results along with the differential gene expression pattern support our hypothesis that FIR can induce molecular targets. In this initial analysis, NFκB and known NFκB regulated radio-responsive genes were found to be modulated. Currently, we are in the process of identifying similar FIR-induced targets and evaluate their potential for selective drug-targeted manipulation. Citation Information: Cancer Res 2009;69(2 Suppl):Abstract nr 5133.

Abstract 4397: A TLR4-interacting peptide (SPA4) reduces the lipopolysaccharide-stimulated NF-kappaB signaling, cytokines and migration of colon cancer cells

Proceedings: AACR 103rd Annual Meeting 2012‐‐ Mar 31‐Apr 4, 2012; Chicago, IL Persistent inflammation in patients with colitis can lead to cancer. However, molecular mechanisms responsible for inflammation-induced cancer are not understood. Activation of TLR4-NF-kappaB signaling is associated with inflammation. Thus, we hypothesized that inhibition of TLR4-NF-kappaB signaling will help reduce inflammatory response and inflammation-induced cancer progression. In this work, we studied the effects of a TLR4-interacting surfactant protein-A-derived (SPA4) peptide on lipopolysaccharide (LPS)-induced TLR4-NF-kappaB signaling and cancer progression in colon adenocarcinoma SW480 cells. The SW480 cells were challenged with Escherichia coli-derived LPS. Cells were then treated with varying amounts of SPA4 peptide. Any changes in the expression of TLR4, intracellular-signaling molecules (IKBalpha, p65, phosphorylated IKBalpha, phosphorylated p65, RelB, Cox-2) and cytokines (IL-1beta, IL-6), and activity of NF-kappaB were studied by immunocytochemistry, immunoblotting and NF-kappaB reporter assay, respectively. Simultaneously, cell cycle progression, viability and migratory pattern of SW480 cells were followed. We found that the binding of SPA4 peptide to TLR4 inhibits the LPS-induced expression of TLR4, phosphorylated p65, Cox-2, IL-1beta and IL-6, activity of NF-kappaB and migration of SW480 cells. Although there was no significant change in cell cycle progression, the SPA4 peptide significantly inhibited the viability of SW480 cells. In conclusion, our preliminary results suggest that the inhibition of TLR4-NF-kappaB signaling by SPA4 peptide may be useful in prevention and treatment of inflammation and inflammation-induced cancer in patients with colitis. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4397. doi:1538-7445.AM2012-4397