Differential response induced by exposure to low-dose ionizing radiation in SHSY-5Y and normal human fibroblast cells

Radiation therapy has been used in the treatment of a wide variety of cancers for nearly a century and is one of the most effective ways to treat cancer. Low-dose ionizing radiation (IR) can interfere with cell division of cancer and normal cells by introducing oxidative stress and injury to DNA. The differences in the response to IR-induced DNA damage and increased reactive oxygen species between normal human fibroblasts (NHFs) and cancerous SHSY-5Y cells were considered. H2AX staining and comet assays revealed that NHF cells responded by initiating a DNA repair sequence whereas SHSY-5Y cells did not. In addition, NHF cells appeared to quench the oxidative stress induced by IR, and after 24 h no DNA damage was present. SHSY-5Y cells, however, did not repair their DNA, did not quench the oxidative stress, and showed characteristic signs that they were beginning to undergo apoptosis. These results indicate that there is a differential response between this cancerous and normal cell line in their ability to respond to low-dose IR, and these differences need to be exploited in order to treat cancer effectively. Further study is needed in order to elucidate the mechanism by which SHSY-5Y cells undergo apoptosis following radiation and why these normal cells are better equipped to deal with IR-induced double-strand breaks and oxidative stress.     

Differential response of excision proficient and deficient L5178Y cells to UVC irradiation and benzamide

L5178Y-R and L5178Y-S cells differ in sensitivity to UVC radiation (D0 values: 2.8 and 9.0 J m-2 respectively, exposure in Fischer's medium). The UVC sensitivity is related to the excision repair ability. Benzamide (Bz), an inhibitor of adenosine diphosphoribosyl transferase (ADPRT), does not modify the lethal effect of UVC radiation in L5178Y-R cells, whereas it sensitizes L5178Y-S cells. The content of NAD+ after irradiation decreases only in the latter cells and this decrease can be prevented by 2 mM Bz treatment. In agreement with the survival data, in L5178Y-R cells neither the proportion of abnormal cells nor the frequency of chromatid aberration are affected by 2 mM Bz treatment, in contrast with L5178Y-S cells. Bz slightly reverses inhibition of 3H-thymidine incorporation only in L5178Y-S cells, but it does not affect the proportions of cells in the different phases of the cell cycle in either cell strain after UVC exposure. These data could be taken as an indirect indication of the involvement of ADPRT in DNA repair in UVC-irradiated L5178Y-S cells. However, the increase in the number of DNA strand breaks in UVC-exposed, Bz-treated cells compared with UVC-exposed untreated cells is the same in both L5178Y strains.     

Interspecies variation in survival and growth of filamentous heterotrophic bacteria in response to UVC radiation

Ultraviolet radiation is an important environmental constraint on the evolution of life. In addition to its harmful effects, ultraviolet radiation plays an important role in generating genetic polymorphisms and acting as a selective agent. Understanding how prokaryotes cope with high radiation can give insights on the evolution of life on Earth. Four representative filamentous bacteria from the family Cytophagaceae with different pigmentation were selected and exposed to different doses of UVC radiation (15-32,400Jm(-2)). The effect of UVC radiation on bacterial survival, growth and morphology were investigated. Results showed high survival in response to UVC for Rudanella lutea and Fibrisoma limi, whereas low survival was observed for Fibrella aestuarina and Spirosoma linguale. S. linguale showed slow growth recovery after ultraviolet exposure, R. lutea and F. limi showed intermediate growth recovery, while F. aestuarina had the fastest recovery among the four tested bacteria. In terms of survival, S. linguale was the most sensitive bacterium whereas R. lutea and F. limi were better at coping with UVC stress. The latter two resumed growth even after 2h exposure (～10,800Jm(-2)). Additionally, the ability to form multicellular filaments after exposure was tested using two bacteria: one representative of the high (R. lutea) and one of the low (F. aestuarina) survival rates. The ability to elongate filaments due to cell division was preserved but modified. In R. lutea 10min exposure reduced the average filament length. The opposite was observed in F. aestuarina, where the 5 and 10min exposures increased the average filament length. R. lutea and F. limi are potential candidates for further research into survival and resistance to ultraviolet radiation stress.     

DNA interstrand crosslinks are induced in cells prelabelled with 5-bromo-2'-deoxyuridine and exposed to UVC radiation

It has been observed previously that 5-bromo-2'-deoxyuridine (BrdU) potentiates the effect of UVC radiation on the level of sister chromatid exchanges. It is not known which type of DNA damage is responsible for this enhancing effect and we have proposed this to be the DNA interstrand crosslink (ICL) which, theoretically, may arise in cells that are labelled with BrdU for one round of replication and exposed to UVC radiation. The aim of the present investigation was to verify if ICLs are indeed formed during this irradiation scenario. CHO-K1 cells were prelabelled with BrdU and exposed to UVC. ICLs were detected by a modified version of the comet assay that relies on the reduction of induced DNA migration in the agarose gel. Carboplatin was used as a positive control. We found that BrdU+UVC treatment indeed results in a reduction of the damage induced by gamma-radiation. Furthermore, we observed that CL-V4B cells exposed to BrdU+UVC, but not to UVC alone, showed a very high level of chromosomal damage. These cells have a deficient Rad51C paralog that renders them extremely sensitive towards ICLs. Interestingly, the cytogenetic results did not correlate with cell survival, where it was found that the CL-V4B cells tolerate BrdU+UVC better than the wild type cells. The possible reasons are discussed. Taken together our results indicate that ICLs are formed in DNA that was prelabelled with BrdU and exposed to UVC radiation.     

Neutrophil infiltration in normal human skin after exposure to different ultraviolet radiation sources

Exposure of the skin to UV radiation can lead to a local infiltration of neutrophils. Not much is known on whether the infiltration of neutrophils in the irradiated skin is UV source dependent. In this study we compared different UV sources (solar-simulated radiation [SSR], narrowband [NB]-UVB, broadband [BB]-UVB and UVA1) in their potency to induce neutrophil infiltration in normal human skin after exposure to two times the minimal erythema dose of UV radiation. Biopsies were collected from irradiated buttock skin 6 and 24 h after irradiation and from nonirradiated skin. The presence, distribution and amount of skin-infiltrated neutrophils were determined using immunohistochemical staining. Analysis revealed that SSR was most effective in inducing neutrophil infiltration. NB-UVB gave a neutrophil influx pattern similar to that seen with SSR but in smaller numbers. BB-UVB and UVA1 were far less potent in inducing neutrophil infiltration compared with SSR or NB-UVB. Our findings indicate that neutrophil infiltration in the UV-irradiated skin is UV source dependent. When the spectra emitted by the different UV sources were compared UVB seemed to be more effective than UVA in inducing neutrophil infiltration. Furthermore, our results suggest that longer wavelengths within the UVB range are mostly responsible for the infiltration of neutrophils in the UV-irradiated skin.     

p38 MAPK and ERK activation by 9-cis-retinoic acid induces chemokine receptors CCR1 and CCR2 expression in human monocytic THP-1 cells

9-cis-Retinoic acid (9CRA) plays an important role in the immune response; this includes cytokine production and cell migration. We have previously demonstrated that 9CRA increases expression of chemokine receptors CCR1 and CCR2 in human monocytes. To better understand how 9CRA induces CCR1 and CCR2 expression, we examined the contribution of signaling proteins in human monocytic THP-1 cells. The mRNA and surface protein up-regulation of CCR1 and CCR2 in 9CRA-stimulated cells were weakly blocked by the pretreatment of SB202190, a p38 MAPK inhibitor, and PD98059, an upstream ERK inhibitor. Activation of p38 MAPK and ERK1/2 was induced in both a time and dose-dependent manner after 9CRA stimulation. Both p38 MAPK and ERK1/2 phosphorylation peaked at 2 h after a 100 nM 9CRA treatment. 9CRA increased calcium influx and chemotactic activity in response to CCR1-dependent chemokines, Lkn-1/CCL15, MIP-1alpha/CCL3, and RANTES/CCL5, and the CCR2-specific chemokine, MCP-1/CCL2. Both SB202190 and PD98059 pretreatment diminished the increased calcium mobilization and chemotactic ability due to 9CRA. SB202190 inhibited the expression and functional activities of CCR1 and CCR2 more effectively than did PD98059. Therefore, our results demonstrate that 9CRA transduces the signal through p38 MAPK and ERK1/2 for CCR1 and CCR2 up-regulation, and may regulate the pro-inflammatory process through the p38 MAPK and ERK-dependent signaling pathways.     

Mild addition of carbon nucleophiles to pyridine and quinoline N-oxides under different activation conditions

In the course of our syntheses of functionalised pyridine and quinoline derivatives, we examined the reactivities of pyridine and quinoline N-oxides towards the nucleophilic addition of acidic carbon derivatives. Different activating reagents were used, such as PyBroP, triflic anhydride and a combination of pyrrolidine phosphoramide and triflic anhydride.     

p21Cip/WAF1 activation is an important factor for the ERK pathway dependent anti-proliferation of colorectal cancer cells

p21Cip/WAF1, an important regulator of cell proliferation, is induced by both p53- and extracellular signal regulated kinase (ERK) pathways. The induction of p21Cip/WAF1 occurs by prolonged activation of the ERKs caused by extracellular stimuli, such as zinc. However, not all the cells appeared to respond to ERK pathway dependent p21Cip/WAF1 induction. Here we investigated the cause of such difference using colorectal cancer cells. p21Cip/WAF1 induction and concomitant reduction of bromodeoxyuridine (BrdU) incorporation were observed by zinc treatment within HT-29 and DLD-1. However, HCT-116 cells with high endogenous p21Cip/WAF1 levels did not show any additional increment of p21Cip/WAF1 levels by zinc treatment and did maintain high BrdU incorporation level. The p21Cip/WAF1 induction by zinc depended upon prolonged activation of extracellular signal regulated kinase (ERK) was not observed in HCT-116 cells. The percentage of BrdU positive cells was 50% higher in p21Cip/WAF1 -/- HCT-116 cells compared to p21Cip/WAF1 +/+ HCT- 116 cells, and no cells induced p21Cip/WAF1 incorporated BrdU in its nucleus, yet confirming the importance of p21Cip/WAF1 induction in anti- proliferation. These results again support that p21Cip/WAF1 induction is a determinant in the regulation of colonic proliferation by the ERK pathway.     

Production of reactive oxygen species in endothelial cells under different pulsatile shear stresses and glucose concentrations

A hemodynamic Lab-on-a-chip system was developed in this study. This system has two unique features: (1) it consists of a microfluidic network with an array of endothelial cell seeding sites for testing them under multiple conditions, and (2) the flow rate and the frequency of the culture medium in the microchannel are controlled by a pulsation free pump to mimic the flow profile of the blood in the blood vessel under different physiological conditions. The investigated physiological conditions were: (1) the resting condition in a normal shear stress of 15 dyne cm(-2) with a normal heart rate of 70 bpm, (2) an exhaustive exercise condition with a high shear stress of 30 dyne cm(-2) and a fast heart rate of 140 bpm, and (3) a constant high shear stress of 30 dyne cm(-2). Two chemical conditions were investigated (10 mM and 20 mM glucose) to mimic hyperglycemic conditions in diabetes patients. The effects of various shear stresses either alone or in combination with different glucose concentrations on endothelial cells were examined using the developed hemodynamic Lab-on-a-chip system by assessing two parameters. One is the intracellular level of reactive oxygen species (ROS) determined by a fluorescent probe, H(2)DCFDA. Another is the mitochondrial morphology revealed with a fluorescent dye, MitoTracker Green FM. The results showed that ROS level was elevated nearly 4-fold after 60 min of exhaustive exercise. We found that the pulsatile nature of the fluid was the determination factor for causing ROS generation in the cells as almost no increase of ROS was detected in the constant shear stress condition. Similarly, much higher level of ROS was detected when 10 mM glucose was applied to the cells under normal or high pulsatile shear stresses compared with under a static condition. These results suggest that it is necessary to use pulsatile shear stress to represent the physiological conditions of the blood flow, and demonstrate the advantage of utilizing this newly developed hemodynamic Lab-on-a-chip system over the conventional non-pulsatile system in the future shear stress related studies.     

Linifanib induces apoptosis in human ovarian cancer cells via activation of FOXO3 and reactive oxygen species

Linifanib is known as an inhibitor of receptor tyrosine kinase. Even though it has been widely recognized as efficient inhibitor of receptor tyrosine kinases, anti-carcinogenic effect has not been investigated enough in ovarian cancer. In this study, we investigated the anti-cancer effect of linifanib on human ovary cancer SKOV3 cells. WST-1, cell counting assay, and observation of morphological changes were performed to evaluate the cytotoxic effect of linifanib in SKOV3 cells. We analyzed SKOV3 cells treated with linifanib using Muse cell analyzer. We focused on investigating the effect of linifanib on DNA damage in nucleus. Additionally, intracellular reactive oxygen species (ROS) level was measured through Muse cell analyzer. Western blotting was performed to evaluate the protein expression level related to apoptosis. We found that linifanib inhibited proliferation of SKOV3 cells. Our results showed that linifanib induced apoptosis in SKOV3 cells. Additionally, linifanib induced DNA damage in SKOV3 cells. We found that intracellular ROS level increased after treatment of linifanib in SKOV3 cells. Interestingly, FOXO3 was transferred from cytosol into nucleus after linifanib treatment. Taken together, our results supported that linifanib inhibited the proliferation of human ovary cancer SKOV3 cells, which suggested that linifanib might have the potential to be developed as drugs for ovarian cancer treatment.     

Distinct patterns of activation-dependent changes in conformational mobility between ERK1 and ERK2

Hydrogen/deuterium exchange measurements by mass spectrometry (HX-MS) can be used to report localized conformational mobility within folded proteins, where exchange predominantly occurs through low energy fluctuations in structure, allowing transient solvent exposure. Changes in conformational mobility may impact protein function, even in cases where structural changes are unobservable. Previous studies of the MAP kinase, ERK2, revealed increases in HX upon activation occured at the hinge between conserved N- and C-terminal domains, which could be ascribed to enhanced backbone flexibility. This implied that kinase activation modulates interdomain closure, and was supported by evidence for two modes of nucleotide binding that were consistent with closed vs open conformations in active vs inactive forms of ERK2, respectively. Thus, phosphorylation of ERK2 releases constraints to interdomain closure, by modulating hinge flexibility. In this study, we examined ERK1, which shares 90% sequence identity with ERK2. HX-MS measurements of ERK1 showed similarities with ERK2 in overall deuteration, consistent with their similar tertiary structures. However, the patterns of HX that were altered upon activation of ERK1 differed from those in ERK2. In particular, alterations in HX at the hinge region upon activation of ERK2 did not occur in ERK1, suggesting that the two enzymes differ with respect to their regulation of hinge mobility and interdomain closure. In agreement, HX-MS measurements of nucleotide binding suggested revealed domain closure in both inactive and active forms of ERK1. We conclude that although ERK1 and ERK2 are closely related with respect to primary sequence and tertiary structure, they utilize distinct mechanisms for controlling enzyme function through interdomain interactions.     

Involvement of ROS and JNK1 in selenite-induced apoptosis in Chang liver cells

Selenium is a dietary essential trace nutrient with important biological roles. Selenocompounds were reported to induce apoptosis in many types of tumor cells. In this study, we investigated the signaling pathway involved in the selenite-induced apoptosis using Chang liver cells as a non-malignant cell model. The Chang liver cell apoptosis induced by selenite (10 microM) was confirmed by DNA fragmentation and typical apoptotic nuclear changes. Treatment of selenite increased intracellular reactive oxygen species (ROS) level and c-Jun N-terminal kinase1 (JNK1) phosphorylation. The selenite-induced cell death was attenuated by SP600125, a specific inhibitor of JNK, and by dominant negative JNK1 (DN-JNK1). Antioxidants such as glutathione (GSH), N-acetyl cysteine (NAC), curcumin, epigallocatechin gallate (EGCG) and epicatechin (EC) inhibited selenite-induced intracellular ROS elevation and JNK1 phosphorylation. Our results suggest that selenite-induced apoptosis in Chang liver cells was preceded by the ROS generation and JNK1 activation.     

Non-stoichiometric oxygen in Cr2O3 catalysts of different dispersities

The content of non-stoichiometric oxygen in Cr₂O₃ catalysts of different dispersity has been determined by a direct thermodesorption method. A sharp decrease in the content of non-stoichiometric oxygen after calcination of the samples above 500°C was observed.

---

Cr₂O₃ . 500°C.

     

Regulation of hepatocyte growth factor-mediated urokinase plasminogen activator secretion by MEK/ERK activation in human stomach cancer cell lines

The regulatory mechanisms for the proliferation and the particular invasive phenotypes of stomach cancers are not still fully understood. Up-regulations of hepatocytes growth factor (HGF), its receptor (c-Met), and urokinase-type plasminogen activator (uPA) are correlated with the development and metastasis of cancers. In order to investigate roles of HGF/c-Met signaling in tumor progression and metastasis in stomach cancers, we determined effects of a specific MEK1 inhibitor (PD098059) and a p38 kinase inhibitor (SB203580) on HGF-mediated cell proliferation and uPA expression in stomach cancer cell lines (NUGC-3 and MKN-28). HGF treatment induced the phosphorylations of ERK and p38 kinase in time- and dose- dependent manners. Pre-treatment with PD098059 reduced HGF-mediated cell proliferation and uPA secretion. In contrast, SB203580 pre-treatment enhanced cell proliferation and uPA secretion due to induction of ERK phosphorylation. Stable expression of dominant negative-MEK1 in NUGC-3 cells showed a decrease in HGF-mediated uPA secretion. These results suggest that interaction of a MEK/ERK and a p38 kinase might play an important role in proliferation and invasiveness of stomach cancer cells.     

COX-2 inhibits anoikis by activation of the PI-3K/Akt pathway in human bladder cancer cells

Cyclooxygenase-2 (COX-2) has been reported to be associated with tumor development and progression as well as to protect cells from apoptosis induced by various cellular stresses. Through a tetracycline-regulated COX-2 overexpression system, we found that COX-2 inhibits detachment-induced apoptosis (anoikis) in a human bladder cancer cell line, EJ. We also found that the inhibition of anoikis by COX-2 results from activation of the PI-3K/Akt pathway as evidenced by suppression of the COX-2 effect on anoikis by a PI-3K inhibitor, LY294002. Furthermore, COX-2 enhanced Mcl-1 expression in the anoikis process, implying that Mcl-1 also may be involved in mediating the survival function of COX-2. Together, these results suggest that COX-2 inhibits anoikis by activation of the PI-3K/Akt pathway and probably by enhancement of Mcl-1 expression in human bladder cancer cells. This anti- anoikis effect of COX-2 may be a part of mechanisms to promote tumor development and progression.     

Resveratrol imparts photoprotection of normal cells and enhances the efficacy of radiation therapy in cancer cells

Solar radiation spans a whole range of electromagnetic spectrum including UV radiation, which are potentially harmful to normal cells as well as ionizing radiations which are therapeutically beneficial towards the killing of cancer cells. UV radiation is an established cause of a majority of skin cancers as well as precancerous conditions such as actinic keratosis. However, despite efforts to educate people about the use of sunscreens and protective clothing as preventive strategies, the incidence of skin cancer and other skin-related disorders are on the rise. This has generated an enormous interest towards finding alternative approaches for management of UV-mediated damages. Chemoprevention via nontoxic agents, especially botanical antioxidants, is one such approach that is being considered as a plausible strategy for prevention of photodamages including photocarcinogenesis. In this review, we have discussed the photoprotective effects of resveratrol, an antioxidant found in grapes and red wine, against UVB exposure-mediated damages in vitro and in vivo. In addition, we have also discussed studies showing that resveratrol can act as a sensitizer to enhance the therapeutic effects of ionizing radiation against cancer cells. Based on available literature, we suggest that resveratrol may be useful for (1) prevention of UVB-mediated damages including skin cancer and (2) enhancing the response of radiation therapies against hyperproliferative, precancerous and neoplastic conditions.     

Genetic variability in the response of normal murine hematopoietic progenitor cells to extracorporeal photochemotherapy

Normal hematopoietic progenitor cells from 129S6/SvEv mice are substantially less sensitive to Merocyanine 540 (MC540)-mediated photodynamic therapy (PDT) than hematopoietic progenitors from sex- and age-matched C57BL/6 mice. When exposed to a combination of MC540 and light commonly used for the extracorporeal purging of hematopoietic stem cells, granulocyte/macrophage progenitors (CFU-GM) from C57BL/6 mice are depleted 7.9-fold whereas CFU-GM from 129S6/SvEv and (C57BL/6 x 129S6/SvEv) F1 mice are depleted 1.4- and 2-fold, respectively. The same rank order of sensitivity is also found with regard to unipotent progenitors of granulocytes and macrophages and with regard to early and late erythroid progenitors. The resistance of hematopoietic progenitors from 129S6/SvEv mice to MC540-PDT appears to be the result of reduced dye binding rather than the result of high levels of intracellular glutathione. These findings have practical implications for the design of preclinical tests of PDT in animal models. They may also provide a useful tool for future investigations into the molecular determinants of sensitivity to MC540-PDT.     

The nature of molecular oxygen binding and activation in Mn-O2 complex

Non-empirical calculations of CASSCF energies, electric dipole moments, Einstein coefficients, matrix elements of the operator of spin-orbital interaction between states of different multiplicity in a model complex ^6,8[Mn-O₂] of C _2v symmetry have been made in 3-21G, 6-31G, 6-31G** basis sets. The crosssections of the potential energy surface (PES) of the ground and excited states were built. It is found that oxygen bonding to manganese is possible when excited atoms of manganese collide with molecular oxygen, singlet oxygen with Mn[⁶ S _5/2] atoms, or in a close contact O₂[X³Σ _g ^? ] + Mn[⁶ S _5/2] and is determined by charge transfer states ^6,8CTS(Mn⁺O ₂ ^? ). Mechanisms of singlet oxygen activation/deactivation are determined by a considerably increased probability of electric dipole transitions b ¹Σ _g ⁺ ?a ¹Δ_g, a ¹Δ_g?X³Σ _g ^? , b ¹Σ _g ⁺ ?X³Σ _g ^? induced in oxygen in the collision process.