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1.  Fractionated Radiation Exposure of Rat Spinal Cords Leads to Latent Neuro-Inflammation in Brain, Cognitive Deficits, and Alterations in Apurinic Endonuclease 1 
PLoS ONE  2015;10(7):e0133016.
Ionizing radiation causes degeneration of myelin, the insulating sheaths of neuronal axons, leading to neurological impairment. As radiation research on the central nervous system has predominantly focused on neurons, with few studies addressing the role of glial cells, we have focused our present research on identifying the latent effects of single/ fractionated -low dose of low/ high energy radiation on the role of base excision repair protein Apurinic Endonuclease-1, in the rat spinal cords oligodendrocyte progenitor cells’ differentiation. Apurinic endonuclease-1 is predominantly upregulated in response to oxidative stress by low- energy radiation, and previous studies show significant induction of Apurinic Endonuclease-1 in neurons and astrocytes. Our studies show for the first time, that fractionation of protons cause latent damage to spinal cord architecture while fractionation of HZE (28Si) induce increase in APE1 with single dose, which then decreased with fractionation. The oligodendrocyte progenitor cells differentiation was skewed with increase in immature oligodendrocytes and astrocytes, which likely cause the observed decrease in white matter, increased neuro-inflammation, together leading to the observed significant cognitive defects.
PMCID: PMC4514622  PMID: 26208353
2.  Acquired genetic alterations in tumor cells dictate the development of high-risk neuroblastoma and clinical outcomes 
BMC Cancer  2015;15:514.
Determining the driving factors and molecular flow-through that define the switch from favorable to aggressive high-risk disease is critical to the betterment of neuroblastoma cure.
In this study, we examined the cytogenetic and tumorigenic physiognomies of distinct population of metastatic site- derived aggressive cells (MSDACs) from high-risk tumors, and showed the influence of acquired genetic rearrangements on poor patient outcomes.
Karyotyping in SH-SY5Y and MSDACs revealed trisomy of 1q, with additional non-random chromosomal rearrangements on 1q32, 8p23, 9q34, 15q24, 22q13 (additions), and 7q32 (deletion). Array CGH analysis of individual clones of MSDACs revealed genetic alterations in chromosomes 1, 7, 8, and 22, corresponding to a gain in the copy numbers of LOC100288142, CD1C, CFHR3, FOXP2, MDFIC, RALYL, CSMD3, SAMD12-AS1, and MAL2, and a loss in ADAM5, LOC400927, APOBEC3B, RPL3, MGAT3, SLC25A17, EP300, L3MBTL2, SERHL, POLDIP3, A4GALT, and TTLL1. QPCR analysis and immunoblotting showed a definite association between DNA-copy number changes and matching transcriptional/translational expression in clones of MSDACs. Further, MSDACs exert a stem-like phenotype. Under serum-free conditions, MSDACs demonstrated profound tumorosphere formation ex vivo. Moreover, MSDACs exhibited high tumorigenic capacity in vivo and prompted aggressive metastatic disease. Tissue microarray analysis coupled with automated IHC revealed significant association of RALYL to the tumor grade in a cohort of 25 neuroblastoma patients. Clinical outcome association analysis showed a strong correlation between the expression of CFHR3, CSMD3, MDFIC, FOXP2, RALYL, POLDIP3, SLC25A17, SERHL, MGAT3, TTLL1, or LOC400927 and overall and relapse-free survival in patients with neuroblastoma.
Together, these data highlight the ongoing acquired genetic rearrangements in undifferentiated tumor-forming neural crest cells, and suggest that these alterations could switch favorable neuroblastoma to high-risk aggressive disease, promoting poor clinical outcomes.
Electronic supplementary material
The online version of this article (doi:10.1186/s12885-015-1463-y) contains supplementary material, which is available to authorized users.
PMCID: PMC4496850  PMID: 26159519
High-risk aggressive neuroblastoma; Genetic rearrangements; Karyotyping; Array CGH; Tumor progression; Clinical outcomes
3.  Acquired Tumor Cell Radiation Resistance at the Treatment Site Is Mediated Through Radiation-Orchestrated Intercellular Communication 
Radiation resistance induced in cancer cells that survive after radiation therapy (RT) could be associated with increased radiation protection, limiting the therapeutic benefit of radiation. Herein we investigated the sequential mechanistic molecular orchestration involved in radiation-induced radiation protection in tumor cells.
Radiation, both in the low-dose irradiation (LDIR) range (10, 50, or 100 cGy) or at a higher, challenge dose IR (CDIR), 4 Gy, induced dose-dependent and sustained NFκB-DNA binding activity. However, a robust and consistent increase was seen in CDIR-induced NFκB activity, decreased DNA fragmentation, apoptosis, and cytotoxicity and attenuation of CDIR-inhibited clonal expansion when the cells were primed with LDIR prior to challenge dose. Furthermore, NFκB manipulation studies with small interfering RNA (siRNA) silencing or p50/p65 overexpression unveiled the influence of LDIR-activated NFκB in regulating CDIR-induced DNA fragmentation and apoptosis. LDIR significantly increased the transactivation/translation of the radiation-responsive factors tumor necrosis factor-α (TNF- α), interleukin-1 α (IL-1α), cMYC, and SOD2. Coculture experiments exhibit LDIR-influenced radiation protection and increases in cellular expression, secretion, and activation of radiation-responsive molecules in bystander cells. Individual gene-silencing approach with siRNAs coupled with coculture studies showed the influence of LDIR-modulated TNF- α, IL-1α, cMYC, and SOD2 in induced radiation protection in bystander cells. NFκB inhibition/overexpression studies coupled with coculture experiments demonstrated that TNF- α, IL-1 α, cMYC, and SOD2 are selectively regulated by LDIR-induced NFκB.
Together, these data strongly suggest that scattered LDIR-induced NFκB-dependent TNF-α, IL-1α, cMYC, and SOD2 mediate radiation protection to the subsequent challenge dose in tumor cells.
PMCID: PMC4034458  PMID: 24411622
4.  Novel Synthetic Monoketone Transmute Radiation-Triggered NFκB-Dependent TNFα Cross-Signaling Feedback Maintained NFκB and Favors Neuroblastoma Regression 
PLoS ONE  2013;8(8):e72464.
Recently, we demonstrated that radiation (IR) instigates the occurrence of a NFκB-TNFα feedback cycle which sustains persistent NFκB activation in neuroblastoma (NB) cells and favors survival advantage and clonal expansion. Further, we reported that curcumin targets IR-induced survival signaling and NFκB dependent hTERT mediated clonal expansion in human NB cells. Herein, we investigated the efficacy of a novel synthetic monoketone, EF24, a curcumin analog in inhibiting persistent NFκB activation by disrupting the IR-induced NFκB-TNFα-NFκB feedback signaling in NB and subsequent mitigation of survival advantage and clonal expansion. EF24 profoundly suppressed the IR-induced NFκB-DNA binding activity/promoter activation and, maintained the NFκB repression by deterring NFκB-dependent TNFα transactivation/intercellular secretion in genetically varied human NB (SH-SY5Y, IMR-32, SK–PN–DW, MC-IXC and SK–N-MC) cell types. Further, EF24 completely suppressed IR-induced NFκB-TNFα cross-signaling dependent transactivation/translation of pro-survival IAP1, IAP2 and Survivin and subsequent cell survival. In corroboration, EF24 treatment maximally blocked IR-induced NFκB dependent hTERT transactivation/promoter activation, telomerase activation and consequent clonal expansion. EF24 displayed significant regulation of IR-induced feedback dependent NFκB and NFκB mediated survival signaling and complete regression of NB xenograft. Together, the results demonstrate for the first time that, novel synthetic monoketone EF24 potentiates radiotherapy and mitigates NB progression by selectively targeting IR-triggered NFκB-dependent TNFα-NFκB cross-signaling maintained NFκB mediated survival advantage and clonal expansion.
PMCID: PMC3743919  PMID: 23967300
5.  Molecular basis of ‘hypoxic’ breast cancer cell radio-sensitization: phytochemicals converge on radiation induced Rel signaling 
Heterogeneously distributed hypoxic areas are a characteristic property of locally advanced breast cancers (BCa) and generally associated with therapeutic resistance, metastases, and poor patient survival. About 50% of locally advanced BCa, where radiotherapy is less effective are suggested to be due to hypoxic regions. In this study, we investigated the potential of bioactive phytochemicals in radio-sensitizing hypoxic BCa cells.
Hypoxic (O2-2.5%; N2-92.5%; CO2-5%) MCF-7 cells were exposed to 4 Gy radiation (IR) alone or after pretreatment with Curcumin (CUR), curcumin analog EF24, neem leaf extract (NLE), Genistein (GEN), Resveratrol (RES) or raspberry extract (RSE). The cells were examined for inhibition of NFκB activity, transcriptional modulation of 88 NFκB signaling pathway genes, activation and cellular localization of radio-responsive NFκB related mediators, eNos, Erk1/2, SOD2, Akt1/2/3, p50, p65, pIκBα, TNFα, Birc-1, -2, -5 and associated induction of cell death.
EMSA revealed that cells exposed to phytochemicals showed complete suppression of IR-induced NFκB. Relatively, cells exposed EF24 revealed a robust inhibition of IR-induced NFκB. QPCR profiling showed induced expression of 53 NFκB signaling pathway genes after IR. Conversely, 53, 50, 53, 53, 53 and 53 of IR-induced genes were inhibited with EF24, NLE, CUR, GEN, RES and RSE respectively. In addition, 25, 29, 24, 16, 11 and 21 of 35 IR-suppressed genes were further inhibited with EF24, NLE, CUR, GEN, RES and RSE respectively. Immunoblotting revealed a significant attenuating effect of IR-modulated radio-responsive eNos, Erk1/2, SOD2, Akt1/2/3, p50, p65, pIκBα, TNFα, Birc-1, -2 and −5 with EF24, NLE, CUR, GEN, RES or RSE. Annexin V-FITC staining showed a consistent and significant induction of IR-induced cell death with these phytochemicals. Notably, EF24 robustly conferred IR-induced cell death.
Together, these data identifies the potential hypoxic cell radio-sensitizers and further implies that the induced radio-sensitization may be exerted by selectively targeting IR-induced NFκB signaling.
PMCID: PMC3599951  PMID: 23452621
Breast cancer; Hypoxia; Radio-sensitization; Phytochemicals; NFκB; Curcumin; EF24; Neem leaf extract; Genistein; Resveratrol; Raspberry extract
6.  The taccalonolides, novel microtubule stabilizers, and γ-radiation have additive effects on cellular viability 
Cancer letters  2011;307(1):104-111.
The taccalonolides are novel antimitotic microtubule stabilizers that have a unique mechanism of action independent of a direct interaction with tubulin. Cytotoxicity and clonogenic assays show that taccalonolide A and radiation act in an additive manner to cause cell death. The taxanes and epothilones have utility when combined with radiotherapy and these findings further suggest the additive effects of microtubule targeting agents with radiation on cellular proliferation are independent of direct tubulin binding and are instead a result of the downstream effects of these agents. These studies suggest that diverse antimitotic agents, including the taccalonolides, may have utility in chemoradiotherapy.
PMCID: PMC3104079  PMID: 21507571
taccalonolide; microtubule stabilizer; γ-radiation; antimitotic; radio-sensitizer
7.  Radiation-Triggered NF-κB Activation is Responsible for the Angiogenic Signaling Pathway and Neovascularization for Breast Cancer Cell Proliferation and Growth 
Tumors require blood supply to survive, grow, and metastasize. This involves the process of angiogenesis signaling for new blood vessel growth into a growing tumor mass. Understanding the mechanism of the angiogenic signaling pathway and neovascularization for breast cancer cell proliferation and growth would help to develop molecular interventions and achieve disease free survival. Our hypothesis is that the surviving cancer cell(s) after radiotherapy can initiate angiogenic signaling pathway in the neighboring endothelial cells resulting in neovascularization for breast cancer cell growth. The angiogenic signaling pathway is initiated by angiogenic factors, VEGF and FGF-2, through activation of a transcriptional regulator NF-κB, which in turn is triggered by therapeutic doses of radiation exposure Human breast adenocarcinoma cells (MCF-7 cells) were exposed to Cesium-137 (137Cs) γ rays to a total dose of 2 Gy at a dose rate of 1.03 Gy/min. The results of mobility shift assay showed that radiation at clinical doses (2 Gy) could induce NF-κB DNA-binding activity. Then, we examined the communication of angiogenic signals from irradiated MCF-7 cells to vascular endothelial cells. At the protein level, the western blot showed induction of angiogenic factors VEGF and FGF-2 in MCF-7 cells irradiated with 2 Gy. Inhibition of NF-κB activation attenuated VEGF and FGF-2 levels. These factors are secreted into the medium. The levels of VEGF and FGF-2 in the extra cellular medium were both increased, after 2 Gy exposures. We also observed corresponding expression of VEGFR2 and FGFR1 in non-irradiated endothelial cells that were co-cultured with irradiated MCF-7 cells. In support of this, in vitro tube formation assays provided evidence that irradiated MCF-7 cells transmit signals to potentiate cultured non-irradiated endothelial cells to form tube networks, which is the hallmark of neovascularization. Inhibition of NF-κB activation attenuated irradiated MCF-7-induced tube network formation. The data provide evidence that the radiation exposure is responsible for tumor growth and maintenance by inducing an angiogenic signaling pathway through activation of NF-κB.
PMCID: PMC3411495  PMID: 22872788
breast cancer; angiogenic factors; NF-κB activation; neovascularization
8.  Curcumin regulates low-LET γ-radiation induced NFκB dependent telomerase activity in human neuroblastoma cells 
We recently reported that curcumin attenuates radiation (IR) induced survival signaling and proliferation in human neuroblastoma (NB) cells. Also, in endothelial system, we 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 NB cells.
Methods and Materials
SK-N-MC or SH-SY5Y cells exposed to IR, treated with curcumin (10nM–100nM) with or without IR were harvested after 1h through 24h. NFκB dependent regulation was investigated either by luciferase reporter assays using pNFκB-, pGL3-354-, pGL3-347-, pUSE-IκBα-Luc, p50/p65 or RelA siRNA transfected cells. NFκB activity was analyzed using EMSA and hTERT expression using QPCR. TA was determined using TRAP assay and, cell survival using MTT and clonogenic assay.
Curcumin profoundly inhibited IR-induced NFκB. Consequently, curcumin significantly inhibited IR-induced TA and hTERT mRNA at all time points investigated. Furthermore, IR-induced TA is regulated at the transcriptional level by triggering 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 NFκB-recognition site on telomerase promoter or inhibition of NFκB by 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.
These results strongly suggest that curcumin inhibits IR-induced TA in an NFκB dependent manner in human NB cells.
PMCID: PMC3071590  PMID: 21236599
Telomerase activity; NFκB; Radiosensitization; Neuroblastoma; Curcumin
9.  Irreversible EGFR Inhibitor EKB-569 Targets Low-LET γ-Radiation-Triggered Rel Orchestration and Potentiates Cell Death in Squamous Cell Carcinoma 
PLoS ONE  2011;6(12):e29705.
EKB-569 (Pelitinib), an irreversible EGFR tyrosine kinase inhibitor has shown potential therapeutic efficiency in solid tumors. However, cell-killing potential in combination with radiotherapy and its underlying molecular orchestration remain to be explored. The objective of this study was to determine the effect of EKB-569 on ionizing radiation (IR)-associated NFκB-dependent cell death. SCC-4 and SCC-9 cells exposed to IR (2Gy) with and without EKB-569 treatment were analyzed for transactivation of 88 NFκB pathway molecules, NFκB DNA-binding activity, translation of the NFκB downstream mediators, Birc1, 2 and 5, cell viability, metabolic activity and apoptosis. Selective targeting of IR-induced NFκB by EKB-569 and its influence on cell-fate were assessed by overexpressing (p50/p65) and silencing (ΔIκBα) NFκB. QPCR profiling after IR exposure revealed a significant induction of 74 NFκB signal transduction molecules. Of those, 72 were suppressed with EKB-569. EMSA revealed a dose dependent inhibition of NFκB by EKB-569. More importantly, EKB-569 inhibited IR-induced NFκB in a dose-dependent manner, and this inhibition was sustained up to at least 72 h. Immunoblotting revealed a significant suppression of IR-induced Birc1, 2 and 5 by EKB-569. We observed a dose-dependent inhibition of cell viability, metabolic activity and apoptosis with EKB-569. EKB-569 significantly enhanced IR-induced cell death and apoptosis. Blocking NFκB improved IR-induced cell death. Conversely, NFκB overexpression negates EKB-569 -induced cell-killing. Together, these pre-clinical data suggest that EKB-569 is a radiosensitizer of squamous cell carcinoma and may mechanistically involve selective targeting of IR-induced NFκB-dependent survival signaling. Further pre-clinical in-vivo studies are warranted.
PMCID: PMC3248439  PMID: 22242139

Results 1-9 (9)