Activation of the atypical NF-κB pathway induced by ionizing radiation is not affected by the p53 status.

DNA double-strand breaks induced by ionizing radiation can activate the atypical NF-κB pathway via ATM-mediated phosphorylation of NEMO/IKKγ. We aimed to determine whether the status of p53 influenced the activation of this particular NF-κB pathway. The NF-κB signaling was activated either by irradiation with a single 8 Gy dose or by TNFα cytokine in p53-proficient and p53-deficient variants of HCT116, RKO, and U2-OS human cancer cell lines. To assess pathway activation the kinetics of phosphorylation (Ser32) and proteolytic degradation of IκBα inhibitor and phosphorylation (Ser536) of RelA(p65) NF-κB subunit were analyzed. Though activation of the radiation-induced atypical pathway was delayed and weakened when compared to the cytokine-induced canonical pathway, no significant differences were noted between p53-proficient and p53-deficient variants, which indicated that activation of both NF-κB pathways was not affected by the p53 status. In marked contrast, the presence of p53 significantly affected downstream effects of NF-κB activation, i.e. transcription of NF-κB-dependent genes. However, different patterns of such interference were observed, which indicated gene-specific and cell-specific mechanisms of interactions between NF-κB and p53 at the transcription regulation level.

Plasma Rich in Growth Factors Promotes Autophagy in ARPE19 Cells in Response to Oxidative Stress Induced by Blue Light.

Age-related macular degeneration (AMD) causes the degeneration of photoreceptors and retinal cells leading to vision loss in older subjects. Among possible exogenous risk factors, it has been recently proposed that long-term exposure to blue light could aggravate the course of AMD. In the search for therapeutic options, plasma rich in growth factors (PRGF) has been shown to enhance cell antioxidant pathways and protect photoreceptors against the harm produced by blue light, although its mechanism of action remains unknown. One possible mechanism, autophagy, is one of the most conservative cell renewal systems used in eukaryotes to destroy cellular components that have been damaged by some kind of insult. The oxidative stress of exposure to blue light is known to induce cell autophagy. In this study, we examined the combined effects on autophagy of blue light and PRGF in a retinal cell line, ARPE19. In response to treatment with both PRGF and blue light, we detected the modulated expression of autophagy markers such as NF-kB, p62/sqstm1, Atg5, LC3 and Beclin1, and inflammatory markers such as IL1B and IL18. Our findings suggest that PRGF promotes cell autophagy in response to exposure to blue light.

Alpha-lipoic acid effectively attenuates ionizing radiation-mediated testicular dysfunction in rats: Crosstalk of NF-ĸB, TGF-ß, and PPAR-ϒ pathways.

Radiotherapy is one of the principal approaches employed in the treatment of pelvic cancers. Nevertheless, testicular dysfunction and infertility are among the most common adverse effects in young adult cancer survivors. Clinically, alpha-lipoic acid (LA) has been applied to improve the quality of sperm with a satisfactory effect. Therefore, the present study investigated the underlying mechanisms of the radioprotective effects of LA against testicular damage. Male Sprague-Dawley rats were exposed to 10 Gy of whole-body ϒ-radiation and LA (50 mg/kg, P.O.) was administered one week before and three days post-irradiation. LA showed remarkable capacity in preserving testicular tissue against radiation damage by improving histological and ultrastructural changes of disorganized seminiferous tubules, besides enhancing its diameter, germinal epithelial thickness, and Johnsen's score. Radiation instigated a significant decrease in sperm quality and quantity associated with depletion of serum testosterone levels, while the LA administration maintained spermatogenesis. Strikingly, LA exhibited antioxidant properties by restoring reduced glutathione levels and antioxidant enzyme activities such as catalase and glutathione-s-transferase, besides diminishing malondialdehyde levels in the testis of irradiated group. Furthermore, LA alleviated testicular inflammation through downregulation of nuclear factor-ĸB (NF-ĸB) expression with a subsequent reduction in interleukin (IL)-6 and cyclooxygenase-2 expression, accompanied by the augmented expression of the anti-inflammatory cytokine IL-10. Additionally, testicular fibrosis markers including Masson's trichrome and transforming growth factor (TGF)-ß expression were noticeably declined in LA-treated irradiated rats, together with the upregulation of peroxisome proliferator-activated receptor-ϒ expression. Collectively, LA ameliorates radiation-mediated spermatogenesis-defects and testicular-damage via suppression of oxidative stress/NF-ĸB/TGF-ß signaling.

Robust combination of liver stereotactic body radiotherapy modulates pharmacokinetics of sorafenib toward preferable parameters.

To evaluate the effect and mechanism of radiotherapy (RT)-sorafenib pharmacokinetics (PK) in different regimens with conventional or high dose irradiation. Between February 2012 and December 2018, 43 patients with portal vein tumor thrombosis treated with sorafenib plus conventional RT (58%) or stereotactic body radiation therapy (SBRT, 42%) were retrospectively reviewed. In vivo and in vitro studies of concurrent and sequential RT with sorafenib were designed. SBRT resulted in a 3-fold increase in complete recanalization compared to conventional RT group (28% vs. 8%, p = 0.014). Compared to the control group, the area under the concentration vs. time curve (AUC) of sorafenib was increased in the concurrent RT2Gy and RT9Gy groups and the sequential RT9Gy group by 132% (p = 0.046), 163% (p = 0.038) and 102% (p = 0.018), respectively; and was decreased by 59% in the sequential RT2Gy group (p = 0.036). Sequential RT2Gy and RT9Gy increased CYP3A4 activity by 82% (p = 0.028) and 203% (p = 0.0004), respectively, compared to that with the corresponding concurrent regimen. SBRT produced better recanalization than conventional RT with sorafenib. The AUC of sorafenib was modulated by RT. P-gp expression was not influenced by RT. The sequential RT regimen increased CYP3A4 activity that may increase the RT-sorafenib synergy effect and overall sorafenib activity. The biodistribution of sorafenib was modulated by local RT with the different regimens.

Ultrasound Stimulation Suppresses LPS-Induced Proinflammatory Responses by Regulating NF-κB and CREB Activation in Microglial Cells.

The purpose of this study was to investigate the effects and underlying mechanisms of low-intensity pulsed ultrasound (LIPUS) against lipopolysaccharide (LPS)-induced neuroinflammation. BV-2 microglia subjected to LPS administration (1 µg/mL) were treated with LIPUS stimulation. The levels of inflammatory mediators and brain-derived neurotrophic factor (BDNF) were quantified using the western blot. The results showed that LIPUS stimulation promoted the associated cAMP response element-binding protein (CREB)/BDNF expression in the LPS-treated microglia. Meanwhile, LIPUS treatment effectively suppressed the LPS-induced production of tumor necrosis factor-α, interleukin-1ß, interleukin-6, inducible nitric oxide synthase, and cyclooxygenase-2 in the microglial cells, in addition to inhibiting the LPS-induced expressions of toll-like receptor 4 and myeloid differentiation factor 88, as well as the LPS-induced activation of c-Jun N-terminal kinase and nuclear factor kappa B. Furthermore, LIPUS significantly decreased the Bax/Bcl-2 ratio in the microglia following LPS treatment. Our data indicated that LIPUS attenuated the proinflammatory responses as well as the decline in BDNF in LPS-treated microglia. This study provides a better understanding of how LIPUS stimulation regulates anti-inflammatory actions in microglia, providing further evidence suggesting that such stimulation may be regarded as a novel strategy for the treatment of neuroinflammation.

Role of NF-κB activation in mouse bone marrow stromal cells exposed to 900 MHz radiofrequency fields (RF).

Nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) is a primary transcription factor which plays a key role in several cellular processes including proliferation and survival. It is well-known that exposure to non-ionizing radiofrequency fields (RF), which are ubiquitous, interact with cellular components. The aim of the study was thus to examine whether exposure of mouse bone marrow stromal cells (BMSC) to RF also resulted in cellular interactions. BMSC were exposed to 900 MHz RF at 120 µW/cm2 power intensity for 4 hr/day for 5 consecutive days. The relative protein expression levels of NF-κB in the cytoplasm and nucleus of RF-exposed cells were compared to non-RF-exposed controls. At 30 min post-RF exposure a significant decrease in protein expression of NF-κB in the cytoplasm was accompanied by a concomitant increase in nuclear NF-κB protein expression levels. Similar responses were noted in the cytoplasm and nuclear NF-κB levels at 2 hr with a return to control concentrations in primary transcription factor at 24 hr post-RF treatment. Daily incubation of BAY 11-7082 an inhibitor of NF-κB for 90 min for 5 days followed by RF each day prevented the fall in cytoplasmic NF-κB and rise in nuclear primary transcription factor at 30 min and 2 hr. There were no marked alterations at 24 hr. Data showed that the effects of RF treatment on BMSC involved transient activation of NF-κB which may be attributed to RF-mediated cellular perturbation as evidenced by consequences of BAY 11-7082 inhibition.

Klotho Protein Protects Human Keratinocytes from UVB-Induced Damage Possibly by Reducing Expression and Nuclear Translocation of NF-κB.

BACKGROUND UV-related skin disease such as actinic keratosis is a major concern in public health. In view of the cell injury induced by UVB, Klotho protein it is an ideal therapy to eliminate UVB-induced cell damages and the associated signaling pathways. MATERIAL AND METHODS To gain insights into the potential role of Klotho and the underlying molecular mechanism, we constructed a Klotho-overexpress HaCaT cell line and assessed the protection against UVB insults. The effects of exposure to UVB radiation on the human keratinocyte HaCaT cells, including cell growth, apoptosis, and changes of selected biomarkers, were measured by CCK-8, flow cytometry, Quantitative real-time PCR, and Western blot analysis. RESULTS We found that enhanced NF-κB activity was accompanied by decreased expression of the anti-aging protein Klotho upon UVB stimulation, which was further confirmed with in vivo experiments. Overexpression of Klotho was able to considerably alleviate the UVB-induced damages to cells and reversed the UVB-caused biomarker changes to a great extent, which was comparable to the effects of administration of NF-κB inhibitor PDTC, suggesting the inhibition of nuclear translocation and DNA-binding activity of NF-κB. Furthermore, Klotho overexpression was proved to decrease the nuclear expression of NF-κB as much as the treatment with PDTC, which provides support for the direct regulation of NF-κB by Klotho. CONCLUSIONS Collectively, our work provides new insight into the potential role of Klotho in the context of UVB-induced injuries in human keratinocytes, as well as providing the basis for future study of new therapies against UV-related skin disease.

Ultraviolet B Radiation Stimulates the Interaction between Nuclear Factor of Activated T Cells 5 (NFAT5) and Nuclear Factor-Kappa B (NF-κB) in Human Lens Epithelial Cells.

PURPOSE: Nuclear factor-kappa B (NF-κB) has been proposed as a therapeutic target for the treatment of cataracts. The authors investigated the relationship between nuclear factor of activated T cells 5 (NFAT5) and NF-κB in ultraviolet B (UVB)-irradiated human lens epithelial (HLE) cells. METHODS: Human lens epithelial B-3 (HLE-B3) cells were exposed to UVB light at a dose of 10 mJ/cm2 and then incubated for 24 h. Cell viability was assessed by using the Cell Counting Kit-8 (CCK-8) assay. Gene expression level of NFAT5 was determined using real-time quantitative polymerase chain reaction (qPCR). Protein expression levels of NFAT5, NF-κB p65, and α-smooth muscle actin (α-SMA) and the association of NFAT5 with the NF-κB p65 subunit were measured by Western blot analysis and a co-immunoprecipitation assay, respectively. The cellular distribution of NFAT5 and NF-κB p65 was examined by triple immunofluorescence staining. RESULTS: At 24 h after UVB exposure, cell viability significantly decreased in a dose-dependent manner, and UVB light (15 and 20 mJ/cm2) significantly increased the ROS generation. UVB irradiation increased NFAT5 mRNA and protein levels and increased phosphorylation of NF-κB in HLE-B3 cells. α-SMA protein levels were increased in the irradiated cells. In addition, NFAT5 and NF-κB translocated from the cytoplasm to the nucleus, and binding between the p65 subunit and NFAT5 was increased. CONCLUSIONS: Exposure to UVB radiation induces nuclear translocation and stimulates binding between NFAT5 and NF-κB proteins in HLE-B3 cells. These interactions may form part of the biochemical mechanism of cataractogenesis in UVB-irradiated HLECs.

Cell fate regulated by nuclear factor-κB- and activator protein-1-dependent signalling in human melanocytes exposed to ultraviolet A and ultraviolet B.

BACKGROUND: Ultraviolet (UV) radiation constitutes an important risk factor for malignant melanoma, but the wavelength responsible for the initiation of this disease is not fully elucidated. Solar UV induces multiple signalling pathways that are critical for initiation of apoptotic cell death as a cellular defence against malignant transformation. OBJECTIVES: To evaluate the involvement of the transcription factors nuclear factor (NF)-κB and activator protein (AP)-1 in the signalling pathways induced by UVA or UVB irradiation in human melanocytes. METHODS: Primary cultures of normal human melanocytes were irradiated with UVA or UVB, and the concomitant DNA damage and redox alterations were monitored. The resulting activation of the NF-κB and AP-1 signalling pathways and subsequent apoptosis were studied. RESULTS: UVB irradiation causes DNA damage detected as formation of cyclobutane pyrimidine dimers, while UVA induces increased levels of 8-hydroxydeoxyguanosine and lipid peroxidation. UVA and UVB initiate phosphorylation of c-Jun N-terminal protein kinase and extracellular signal-regulated kinase, and the apoptosis signalling pathways converge into a common mechanism. Downregulation of c-Jun suppresses AP-1-mediated signalling and prevents apoptosis upstream of lysosomal and mitochondrial membrane permeabilization, whereas inhibition of NF-κB by SN50 increases apoptosis. CONCLUSIONS: We conclude that AP-1 induces proapoptotic signalling, whereas NF-κB is a key antiapoptotic/prosurvival factor in both UVA- and UVB-induced cellular damage in human melanocytes, which might in turn impact melanoma development and progression.

Systemic response to ultraviolet radiation involves induction of leukocytic IL-1ß and inflammation in zebrafish.

Ultraviolet radiation is a pervasive stimulus with wide-ranging effects on all living forms. The effects of UV vary from physiological to pathological, depending on levels of exposure, but the immune response at the organismal level is not well understood. We use the zebrafish embryo and larva to study immune responses to UV stress in vivo. UV exposure causes inflammation characterized by systemic induction of proinflammatory cytokines. Leukocytes are an important component of this systemic response and upregulate IL-1ß expression proportional to the dose of UV exposure. Increased levels of this proinflammatory cytokine counteract the lethal effect of high doses of UV.

A comparative study of UV-induced cell signalling pathways in human keratinocyte-derived cell lines.

Ultraviolet (UV) radiation can activate the p38 mitogen-activated protein kinase (MAPK), Jun N-terminal kinase (JNK) and nuclear factor-κB (NFκB) pathways in skin cells. HaCaT cells are widely used as a primary keratinocyte substitute to study these pathways. However, like most squamous cell carcinomas (SCCs), it contains a dysfunctional p53. It is unclear if HaCaT cells activate these signalling pathways similarly to SCC cells (Colo16) or to primary human epidermal keratinocytes (HEK). In this study, the UV activation (UVA, UVB, UVA+B, UVB+A) of p38 MAPK, JNK and NFκB pathways, and TNFα secretion by HEK, HaCaT and Colo16 cells were investigated. The signalling pathway activation was UV-type and dose-dependent with UVB+A radiation inducing a high p38 and JNK activation. HaCaT cells exhibited 2- to 4-fold higher activity of the p38 (771% at 60 min) and JNK (794% at 30 min) pathways following UVB+A radiation than did HEK cells (p38: 367% at 15 min and JNK: 184% at 30 min). While both HaCaT and Colo16 cells did not activate the NFκB pathway, Colo16 cells had a lower p38 and higher JNK activity than HaCaT cells. Irradiated HaCaT cells produced less TNFα (UVB: 3.5 pg/ml), while HEK cells produced the most (UVB: 1,296 pg/ml). When co-exposed to IL1α, irradiated HaCaT had the greatest fold of TNFα release (UVB: 16.2-fold, UVA+B: 8.9-fold and UVB+A: 6.1-fold). The pattern of activation and TNFα secretion of HaCaT cells mirrored that of Colo16 cells. It is likely that the presence of molecular alterations in HaCaT cells may be responsible for its different responses to that seen for HEK cells. The results of this study suggest caution in using HaCaT cells as a substitute for normal keratinocytes in investigating UV-induced cells signalling pathways.

Bortezomib-enhanced radiosensitization through the suppression of radiation-induced nuclear factor-κB activity in human oral cancer cells.

Oral cancer cells have a significantly augmented nuclear factor-κB (NF-κB) activity and the inhibition of this activity suppresses tumor growth. Bortezomib is a proteasome inhibitor and a drug used for molecular-targeted therapy (targets NF-κB). In this study, we investigated whether bortezomib would be effective as an inhibitor of proliferation and a radiosensitizer for the treatment of oral cancer. We demonstrate that bortezomib inhibits NF-κB activity and cell proliferation. The combined treatment with bortezomib and radiation (RT) suppressed NF-κB activity and cell growth in vitro and in vivo compared with RT treatment alone. To investigate the mechanisms by which bortezomib suppresses tumor growth, the expression of signaling molecules downstream of NF-κB were examined by ELISA. The combined treatment significantly inhibited the radiation-induced production of angiogenic factors and decreased the number of blood vessels in the tumor tissues. Although the expression of anti-apoptotic proteins was upregulated by RT, bortezomib downregulated the RT-induced expression of these proteins. Moreover, the expression of cleaved poly(ADP-ribose) polymerase in vitro and in vivo was enhanced by bortezomib, indicating that bortezomib inhibits tumor growth by inducing apoptosis. This study clearly demonstrates that bortezomib significantly inhibits tumor growth and that the combined treatment with bortezomib and RT results in a significant inhibition of tumor growth. The mechanisms underlying the inhibition of tumor growth by bortezomib include the suppression of angiogenesis and the induction of apoptosis. A novel molecular targeting therapy including bortezomib may be effective in the treatment of oral cancer by suppressing NF-κB activity.

Acceleration of astrocytic differentiation in neural stem cells surviving X-irradiation.

Neural stem cells (NSCs) are highly susceptible to DNA double-strand breaks; however, little is known about the effects of radiation in cells surviving radiation. Although the nestin-positive NSCs predominantly became glial fibrillary acidic protein (GFAP)-positive in differentiation-permissive medium, little or no cells were GFAP positive in proliferation-permissive medium. We found that more than half of the cells surviving X-rays became GFAP positive in proliferation-permissive medium. Moreover, localized irradiation stimulated differentiation of cells outside the irradiated area. These results indicate for the first time that ionizing radiation is able to stimulate astrocyte-specific differentiation of surviving NSCs, whose process is mediated both by the direct activation of nuclear factor-κB and by the indirect bystander effect induced by X-irradiation.

Leaf extract of Moringa oleifera prevents ionizing radiation-induced oxidative stress in mice.

The present study evaluated the hepatoprotective effect of aqueous ethanolic Moringa oleifera leaf extract (MoLE) against radiation-induced oxidative stress, which is assessed in terms of inflammation and lipid peroxidation. Swiss albino mice were administered MoLE (300 mg/kg of body weight) for 15 consecutive days before exposing them to a single dose of 5 Gy of 6°Co γ-irradiation. Mice were sacrificed at 4 hours after irradiation. Liver was collected for immunoblotting and biochemical tests for the detection of markers of hepatic oxidative stress. Nuclear translocation of nuclear factor kappa B (NF-κB) and lipid peroxidation were augmented, whereas the superoxide dismutase (SOD), catalase (CAT), reduced glutathione (GSH), and ferric reducing antioxidant power (FRAP) values were decreased by radiation exposure. Translocation of NF-κB from cytoplasm to nucleus and lipid peroxidation were found to be inhibited, whereas increases in SOD, CAT, GSH, and FRAP were observed in the mice treated with MoLE prior to irradiation. Therefore pretreatment with MoLE protected against γ-radiation-induced liver damage. The protection may be attributed to the free radical scavenging activity of MoLE, through which it can ameliorate radiation-induced oxidative stress.

Selenium-containing thioredoxin reductase inhibitor ethaselen sensitizes non-small cell lung cancer to radiotherapy.

It has been proposed that thioredoxin reductase (TR) is a mediator that allows non-small cell lung cancer (NSCLC) to develop resistance to irradiation; however, little is known regarding the detailed mechanisms of action. Thus, ethaselen {1, 2-[bis (1,2-benzisoselenazolone-3 (2H)-ketone)] ethane, BBSKE}, a novel organoselenium TR inhibitor, is currently being investigated in a phase I clinical trial in China. However, its radiosensitizing effect remains unexplored. In this study, we found that the activity of TR increased dramatically in both A549 and H1299 cells after radiation, and moreover, could be inhibited by pretreatment with BBSKE (5 µmol/l). As a TR inhibitor, BBSKE enhanced the efficacy of radiation therapy both in vivo and in vitro without observable toxicity. BBSKE was found to suppress irradiation-induced NF-κB activation dramatically when using A549 cells stably transfected with NF-κB luciferase reporter. These results show the critical role of TR in the radioresistance of NSCLC and suggest that BBSKE is a potentially promising agent for the treatment of patients with NSCLC clinically.

Induction of MET by ionizing radiation and its role in radioresistance and invasive growth of cancer.

BACKGROUND: Ionizing radiation (IR) is effectively used in cancer therapy. However, in subsets of patients, a few radioresistant cancer cells survive and cause disease relapse with metastatic progression. The MET oncogene encodes the hepatocyte growth factor (HGF) receptor and is known to drive "invasive growth", a regenerative and prosurvival program unduly activated in metastasis. METHODS: Human tumor cell lines (MDA-MB-231, MDA-MB-435S, U251) were subjected to therapeutic doses of IR. MET mRNA, and protein expression and signal transduction were compared in treated and untreated cells, and the involvement of the DNA-damage sensor ataxia telangiectasia mutated (ATM) and the transcription factor nuclear factor kappa B (NF-κB) in activating MET transcription were analyzed by immunoblotting, chromatin immunoprecipitation, and use of NF-κB silencing RNA (siRNA). Cell invasiveness was measured in wound healing and transwell assays, and cell survival was measured in viability and clonogenic assays. MET was inhibited by siRNA or small-molecule kinase inhibitors (PHA665752 or JNJ-38877605). Combinations of MET-targeted therapy and radiotherapy were assessed in MDA-MB-231 and U251 xenografts (n = 5-6 mice per group). All P values were from two-sided tests. RESULTS: After irradiation, MET expression in cell lines was increased up to fivefold via activation of ATM and NF-κB. MET overexpression increased ligand-independent MET phosphorylation and signal transduction, and rendered cells more sensitive to HGF. Irradiated cells became more invasive via a MET-dependent mechanism that was further enhanced in the presence of HGF. MET silencing by siRNA or inhibition of its kinase activity by treatment with PHA665752 or JNJ-38877605 counteracted radiation-induced invasiveness, promoted apoptosis, and prevented cells from resuming proliferation after irradiation in vitro. Treatment with MET inhibitors enhanced the efficacy of IR to stop the growth of or to induce the regression of xenografts (eg, at day 13, U251 xenografts, mean volume increase relative to mean tumor volume at day 0: vehicle = 438%, 5 Gy IR = 151%, 5 Gy IR + JNJ-38877605 = 76%; difference, IR vs JNJ-38877604 + IR = 75%, 95% CI = 59% to 91%, P = .01). CONCLUSION: IR induces overexpression and activity of the MET oncogene through the ATM-NF-κB signaling pathway; MET, in turn, promotes cell invasion and protects cells from apoptosis, thus supporting radioresistance. Drugs targeting MET increase tumor cell radiosensitivity and prevent radiation-induced invasiveness.

Modulation of NF-κB and FOXOs by baicalein attenuates the radiation-induced inflammatory process in mouse kidney.

The bioactive flavonoid baicalein has been shown to have radioprotective activity, although the molecular mechanism is poorly understood in vivo. C57BL/6 mice were irradiated with X-rays (15 Gy) with and without baicalein treatment (5 mg/kg/day). Irradiation groups showed an increase of NF-κB-mediated inflammatory factors with oxidative damage and showed inactivation of FOXO and its target genes, catalase and SOD. However, baicalein suppressed radiation-induced inflammatory response by negatively regulating NF-κB and up-regulating FOXO activation and catalase and SOD activities. Furthermore, baicalein inhibited radiation-induced phosphorylation of MAPKs and Akt, which are the upstream kinases of NF-κB and FOXOs. Based on these findings, it is concluded that baicalein has a radioprotective effect against NF-κB-mediated inflammatory response through MAPKs and the Akt pathway, which is accompanied by the protective effects on FOXO and its target genes, catalase and SOD. Thus, these findings provide new insights into the molecular mechanism underlying the radioprotective role of baicalein in mice.

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.

Beta-lapachone suppresses radiation-induced activation of nuclear factor-kappaB.

Anticancer effects of beta-lapachone (beta-lap) are due to generation of ROS and metabolic catastrophes as a result of NAD(P)H:quinone oxidoreductase (NQO1)-mediated futile cycling between the oxidized and reduced forms of beta-lap. It has been shown that NQO1 is also essential for the TNF-induced activation of NF-kappaB and that beta-lap suppresses the TNF-induced NF-kappaB activation. We investigated whether or not NQO1 is involved and beta-lap suppresses the radiation-induced NF-kappaB activation using A549 human lung cancer cells and NQO1-knock down A549 cells (shNQO1 A549 cells). Irradiation with 4 Gy markedly increased the DNA binding activity of NF-kappaB in A549 cells, but not in the shNQO1 A549 cells, thus demonstrating that NQO1 plays a pivotal role in irradiation-induced NF-kappaB activation. Treatment with 10 micronM beta-lap for 4 h almost completely abrogated the radiation-induced increase in NF-kappaB activation and the transcription of NF-kappaB target genes such as bcl2, gadd45beta and cyclinD1. Moreover, beta-lap markedly suppressed the activation of IkappaB kinase gamma (IKKgamma) and the subsequent phosphorylation of IkappaBalpha, thereby inhibiting NF-kappaB activation. It is concluded that beta-lap suppresses the radiation-induced activation of NF-kappaB by interrupting the involvement of NQO1 in the activation of NF-kappaB, thereby inhibiting the transcription of survival signals. The radiosensitization caused by beta-lap may, in part, be attributed to beta-lap-induced suppression of NF-kappaB activation.