DNA damage response and repair in ovarian cancer: Potential targets for therapeutic strategies

Ovarian cancer is among the most lethal gynecologic malignancies with a poor survival prognosis. The current therapeutic strategies involve surgery and chemotherapy. Research is now focused on novel agents especially those targeting DNA damage response (DDR) pathways. Understanding the DDR process in ovarian cancer necessitates having a detailed knowledge on a series of signaling mediators at the cellular and molecular levels. The complexity of the DDR process in ovarian cancer and how this process works in metastatic conditions is comprehensively reviewed. For evaluating the efficacy of therapeutic agents targeting DNA damage in ovarian cancer, we will discuss the components of this system including DDR sensors, DDR transducers, DDR mediators, and DDR effectors. The constituent pathways include DNA repair machinery, cell cycle checkpoints, and apoptotic pathways. We also will assess the potential of active mediators involved in the DDR process such as therapeutic and prognostic candidates that may facilitate future studies.     

All three J-domain proteins of the Escherichia coli DnaK chaperone machinery are DNA binding proteins

DnaJ, DjlA and CbpA are the J-domain proteins of DnaK, the major Hsp70 of Escherichia coli. CbpA was originally discovered as a DNA binding protein. Here, we show that DNA binding is a property of DnaJ and DjlA as well. Of special interest in this respect is DjlA, as this cytoplasmic protein is membrane bound and, as shown here, its affinity for DNA is extremely high. The finding that all the three J-proteins of DnaK are DNA binding proteins sheds new light on the cellular activity of these proteins.     

A microarray analysis of the effects of moderate hypothermia and rewarming on gene expression by human hepatocytes (HepG2)

The gene expression changes produced by moderate hypothermia are not fully known, but appear to differ in important ways from those produced by heat shock. We examined the gene expression changes produced by moderate hypothermia and tested the hypothesis that rewarming after hypothermia approximates a heat-shock response. Six sets of human HepG2 hepatocytes were subjected to moderate hypothermia (31°C for 16 h), a conventional in vitro heat shock (43°C for 30 min) or control conditions (37°C), then harvested immediately or allowed to recover for 3 h at 37°C. Expression analysis was performed with Affymetrix U133A gene chips, using analysis of variance-based techniques. Moderate hypothermia led to distinct time-dependent expression changes, as did heat shock. Hypothermia initially caused statistically significant, greater than or equal to twofold changes in expression (relative to controls) of 409 sequences (143 increased and 266 decreased), whereas heat shock affected 71 (35 increased and 36 decreased). After 3 h of recovery, 192 sequences (83 increased, 109 decreased) were affected by hypothermia and 231 (146 increased, 85 decreased) by heat shock. Expression of many heat shock proteins was decreased by hypothermia but significantly increased after rewarming. A comparison of sequences affected by thermal stress without regard to the magnitude of change revealed that the overlap between heat and cold stress was greater after 3 h of recovery than immediately following thermal stress. Thus, while some overlap occurs (particularly after rewarming), moderate hypothermia produces extensive, time-dependent gene expression changes in HepG2 cells that differ in important ways from those induced by heat shock.     

Antifungal and antitumor activity of heterocyclic thiosemicarbazones and their metal complexes: current status

More than 75 substituted thiosemicarbazones and a number of metal complexes of each have been assayed for their antifungal activity. Their activity is significantly affected by the substituted groups attached at both¹ N and⁴ N of the thiosemicarbazone moiety. Greatest activity occurs for 2-substituted pyridine thiosemicarbazones with differences observed for 2-formylpyridine, 2-acetylpyridine and 2-benzoylpyridine derivatives and their metal complexes. Further, there are activity differences for⁴ N-alkyl-,⁴ N-aryl-,⁴ N-dialkyl- and 3-azacyclothiosemicarbazones and their metal complexes as well as changes in the substituent size among each of these subgroups. Cu(II) complexes are often more active than the uncomplexed thiosemicarbazones, with the latter showing similar activity to Ni(II) complexes in many instances. The reduction potential of the thiosemicarbazone ligand in a Cu(II) complex, the strength of the ligand field and various spectral properties can be correlated to the inhibitory activity.     

Synthesis and antiproliferative activity of some steroidal thiosemicarbazones,semicarbazones and hydrozones

Steroidal thiosemicarbazones, semicarbazones and hydrazones have received extensive attention of scientists recently because they exhibit some biological activities such as antibacterial, antiviral and anticancer. Using different steroids as starting materials, through different chemical methods, 24 steroidal compounds with thiosemicarbazone, semicarbazone or hydrazone groups in their structures, were synthesized, characterized by IR, NMR and MS. The antiproliferative activity of the compounds was evaluated against human gastric cancer (SGC-7901) and human liver cancer (Bel-7404) cells. The structure–activity relationship of these compounds was discussed. The results showed that compound 3 and 12a–12c exhibited significant inhibitory activity to Bel-7404 cells, and IC₅₀ values of them were 4.2, 11.0, 7.4 and 15.0 μM respectively (Cisplatin, IC₅₀: 11.6 μM).     

Effects of 1.8 GHz radiofrequency field on DNA damage and expression of heat shock protein 70 in human lens epithelial cells

To investigate the DNA damage, expression of heat shock protein 70 (Hsp70) and cell proliferation of human lens epithelial cells (hLEC) after exposure to the 1.8 GHz radiofrequency field (RF) of a global system for mobile communications (GSM). An Xc-1800 RF exposure system was used to employ a GSM signal at 1.8 GHz (217 Hz amplitude-modulated) with the output power in the specific absorption rate (SAR) of 1, 2 and 3 W/kg. After 2 h exposure to RF, the DNA damage of hLEC was accessed by comet assay at five different incubation times: 0, 30, 60, 120 and 240 min, respectively. Western blot and RT-PCR were used to determine the expression of Hsp70 in hLECs after RF exposure. The proliferation rate of cells was evaluated by bromodeoxyuridine incorporation on days 0, 1 and 4 after exposure. The results show that the difference of DNA-breaks between the exposed and sham-exposed (control) groups induced by 1 and 2 W/kg irradiation were not significant at any incubation time point (P > 0.05). The DNA damage caused by 3 W/kg irradiation was significantly increased at the times of 0 and 30 min after exposure (P < 0.05), a phenomenon that could not be seen at the time points of 60, 120 or 240 min (P > 0.05). Detectable mRNA as well as protein expression of Hsp70 was found in all groups. Exposure at SARs of 2 and 3 W/kg for 2 h exhibited significantly increased Hsp70 protein expression (P < 0.05), while no change in Hsp70 mRNA expression could be found in any of the groups (P > 0.05). No difference of the cell proliferation rate between the sham-exposed and exposed cells was found at any exposure dose tested (P > 0.05). The results indicate that exposure to non-thermal dosages of RF for wireless communications can induce no or repairable DNA damage and the increased Hsp70 protein expression in hLECs occurred without change in the cell proliferation rate. The non-thermal stress response of Hsp70 protein increase to RF exposure might be involved in protecting hLEC from DNA damage and maintaining the cellular capacity for proliferation.     

Anhydrobiosis in tardigrades--the last decade

The current state of knowledge about anhydrobiosis in tardigrades is presented. In response to adverse environmental conditions tardigrades arrest their metabolic activity and after complete dehydration enter the so-called “tun” state. In this ametabolic state they are able to tolerate exposure to various chemical and physical extremes. These micrometazoans have evolved various kinds of morphological, physiological and molecular adaptations to reduce the effects of desiccation. In this review we address behavioral adaptation, morphological features and molecules which determine the anhydrobiotic survival. The influence of the time spent in anhydrobiotic state on the lifespan and DNA and the role of the antioxidant defense system are also considered. Finally we summarize recent input from the “omics” sciences.     

Development of the embryonic heat shock response and the impact of repeated thermal stress in early stage lake whitefish (Coregonus clupeaformis) embryos

Lake whitefish (Coregonus clupeaformis) embryos were exposed to thermal stress (TS) at different developmental stages to determine when the heat shock response (HSR) can be initiated and if it is altered by exposure to repeated TS. First, embryos were subject to one of three different TS temperatures (6, 9, or 12 °C above control) at 4 points in development (21, 38, 60 and 70 days post-fertilisation (dpf)) for 2 h followed by a 2 h recovery to understand the ontogeny of the HSR. A second experiment explored the effects of repeated TS on the HSR in embryos from 15 to 75 dpf. Embryos were subjected to one of two TS regimes; +6 °C TS for 1 h every 6 days or +9 °C TS for 1 h every 6 days. Following a 2 h recovery, a subset of embryos was sampled. Our results show that embryos could initiate a HSR via upregulation of heat shock protein 70 (hsp70) mRNA at all developmental ages studied, but that this response varied with age and was only observed with a TS of +9 or +12 °C. In comparison, when embryos received multiple TS treatments, hsp70 was not induced in response to the 1 h TS and 2 h recovery, and a downregulation was observed at 39 dpf. Downregulation of hsp47 and hsp90α mRNA was also observed in early age embryos. Collectively, these data suggest that embryos are capable of initiating a HSR at early age and throughout embryogenesis, but that repeated TS can alter the HSR, and may result in either reduced responsiveness or a downregulation of inducible hsps. Our findings warrant further investigation into both the short- and long-term effects of repeated TS on lake whitefish development.     

Synthesis,biological evaluation and molecular docking of N-phenyl thiosemicarbazones as urease inhibitors

Urease is an important enzyme which breaks urea into ammonia and carbon dioxide during metabolic processes. However, an elevated activity of urease causes various complications of clinical importance. The inhibition of urease activity with small molecules as inhibitors is an effective strategy for therapeutic intervention. Herein, we have synthesized a series of 19 benzofurane linked N-phenyl semithiocarbazones (3a–3s). All the compounds were screened for enzyme inhibitor activity against Jack bean urease. The synthesized N-phenyl thiosemicarbazones had varying activity levels with IC₅₀ values between 0.077 ± 0.001 and 24.04 ± 0.14 μM compared to standard inhibitor, thiourea (IC₅₀ = 21 ± 0.11 μM). The activities of these compounds may be due to their close resemblance of thiourea. A docking study with Jack bean urease (PDB ID: 4H9M) revealed possible binding modes of N-phenyl thiosemicarbazones.     

Transcriptional regulation of the Trichoderma longibrachiatum egl1 gene

Abstract Detection of stress proteins in Porphyromonas gingivalis was investigated by SDS-PAGE and Western immunoblotting procedure using a polyclonal antibody (anti hsp60) and a monoclonal antibody (anti-Dnak). Results indicate that P. gingivalis can elicit a hsp60-like stress protein when submitted to different environmental stresses such as a heat shift from 35°C to 43°C, a pH drop from 7.2 to 6.0 or an increase in oxygen concentration. Virulent and non-virulent strains of P. gingivalis responded the same way. The other bacterial species tested also showed an increased synthesis of a GroEL-like protein after heat shock, as detected by the anti hsp60 antibody. However, the monoclonal anti-Dnak recognized an hsp70-like protein only in two of the tested species.     

Testosterone amplifies HSP70-2a,HSP90 and PCNA expression in experimental varicocele condition: Implication for DNA fragmentation

The DNA fragmentation and failure in post-meiotic maturation of the spermatozoa because of testosterone withdrawal can affect the fertilization potential in varicocele (VCL) patients. To find out the exact mechanism of VCL-induced failure in histone-protamine replacement process and DNA fragmentation, the correlations between the levels of expression of HSP70-2a, HSP90, PCNA, TP1/2 and PCNA genes and the patterns of DNA methylation were investigated before and after testosterone administration in rats. In total, 40 mature male Wistar rats (10 in each group) were assigned between control (with no intervention), control-sham (undergone a simple laparotomy), VCL-induced (VCL-sole), and testosterone-treated VCL-induced (VCLT) groups. The HSP70-2a, HSP90, PCNA, TP1, and TP2 genes expressions and the patterns of global DNA methylation were determined in all groups. A statistically significant (p < 0.05) reduction were found in the HSP70-2a, HSP90, PCNA, TP1 and TP2 genes expressions in VCL-sole group. In VCLT group, testosterone was shown to significantly (p < 0.05) up-regulate the HSP70-2a, HSP90, PCNA, and TP2expression levels, but TP1 expression has not been changed. Furthermore, the VCLT group exhibited higher DNA methylation rates compared to VCL-sole animals. In conclusion, testosterone, by up-regulating the HSP70-2a and HSP90 expressions and maintaining the pre-existing HSP70-2a and HSP90 proteins levels, may be the reason for the significant increment in TP2 expression during post-meiotic stage and can boost the global methylation rates of DNA via up-regulating the PCNA expression, suggesting that administration of testosterone can mitigate the VCL-impaired histone-protamine replacement and DNA methylation rates and protect the cellular DNA content from VCL-induced oxidative stress.     

The interaction of HspA1A with TLR2 and TLR4 in the response of neutrophils induced by ovarian cancer cells in vitro

Inducible heat shock protein (HspA1A) promotes tumor cell growth and survival. It also interacts with effector cells of the innate immune system and affects their activity. Recently, we showed that the direct contact of ovarian cancer cells, isolated from tumor specimens, with neutrophils intensified their biological functions. Our current experiments demonstrate that the activation of neutrophils, followed by an increased production of reactive oxygen species, by cancer cells involves the interaction of HspA1A from cancer cells with Toll-like receptors 2 and 4 expressed on the neutrophils’ surface. Our data may have a practical implication for targeted anticancer therapies based, among other factors, on the inhibition of HspA1A expression in the cancer cells.     

Genetic analysis of heat shock proteins in maize

A genetic analysis of heat shock protein (HSP) synthesis was performed in seedling leaf tissue of two maize inbred lines, their F₁ hybrid and F₂ progeny. Protein synthesis following a high temperature treatment was visualized by [³⁵S]-methionine in vivo labelling and two-dimensional gel electrophoresis. The parental lines' HSP synthesis patterns revealed both qualitative and quantitative polymorphisms implicative of differences in HSP structural genes and regulatory factors. The F₁ hybrid HSP profile indicated that synthesis of all parental HSPs conformed to dominant inheritance patterns, including complete dominance, over-dominance and co-dominance. Alleles for six low-molecularweight HSPs in F₂ progeny assorted according to typical 31 Mendelian ratios for dominant gene expression. There is evidence for unlinked gene loci of four different HSP gene pairs, but data for three other HSP gene pairs were inconclusive, perhaps reflecting linkage for one pair and complex regulatory factor interactions for the other two pairs of genes. These results clearly indicate the existence of genetic variability in HSP synthesis and emphasize the potential of partitioning their roles in thermal tolerance using genetic and molecular analyses.     

Induction of Antioxidant and Heat Shock Protein Responses During Torpor in the Gray Mouse Lemur,Microcebus murinus

A natural tolerance of various environmental stresses is typically supported by various cytoprotective mechanisms that protect macromolecules and promote extended viability. Among these are antioxidant defenses that help to limit damage from reactive oxygen species and chaperones that help to minimize protein misfolding or unfolding under stress conditions. To understand the molecular mechanisms that act to protect cells during primate torpor, the present study characterizes antioxidant and heat shock protein(HSP) responses in various organs of control(aroused)and torpid gray mouse lemurs, Microcebus murinus. Protein expression of HSP70 and HSP90 a was elevated to 1.26 and 1.49 fold, respectively, in brown adipose tissue during torpor as compared with control animals, whereas HSP60 in liver of torpid animals was 1.15 fold of that in control(P 0.05). Among antioxidant enzymes, protein levels of thioredoxin 1 were elevated to 2.19 fold in white adipose tissue during torpor, whereas Cu–Zn superoxide dismutase 1 levels rose to 1.1 fold in skeletal muscle(P 0.05). Additionally, total antioxidant capacity was increased to 1.6 fold in liver during torpor(P 0.05), while remaining unchanged in the five other tissues. Overall, our data suggest that antioxidant and HSP responses are modified in a tissue-specific manner during daily torpor in gray mouse lemurs. Furthermore, our data also show that cytoprotective strategies employed during primate torpor are distinct from the strategies in rodent hibernation as reported in previous studies.     

Effects of broad band electromagnetic fields on HSP70 expression and ischemia-reperfusion in rat hearts

Although exposure to broad band (0.2-20 MHz) electromagnetic fields (EMF) is part of the treatment of several diseases, little is known as to their effects on myocardial protein expression and resistance to ischemia-reperfusion (I/R). We exposed Sprague-Dawley rats to either high (H, 10 min/day at 200 V/m, 36.1 microT) or low (L, 2 min/day at 30 V/m, 11.4 microT) intensity broad band EMF for 15 days. At the end of the treatment, myocardial HSP70 was 32 +/- 8% (mean +/- SEM) higher in L (P = 0.01) than in control (C), whereas in H it remained the same as in C. Electron microscopy revealed sporadic ruptures of mitochondrial cristae in H hearts, with no differences in other parameters. Malondialdehyde was increased in treated hearts (P < 0.05), but especially in H (P = 0.008). To assess the protective role of HSP70 during I/R, hearts were Langendorff-perfused with Krebs-Henseleit. After I/R, C hearts displayed depressed rate. pressure (-13 +/- 7%) and increased end-diastolic (+9.2 +/- 2.8 mmHg) and perfusion pressures (+30 +/- 10 mmHg). In H and L, rate. pressure recovery was similar to C (-2 +/- 21% and -12 +/- 16%, respectively, P = NS). In contrast, both end-diastolic and perfusion pressures were higher in L than in H (30.8 +/- 5.4 vs 18.2 +/- 3.5, P = 0.01, and 54 +/- 8 vs 21 +/- 8 mmHg, P = 0.01, respectively) indicating diastolic derangement in L. In conclusion, the effects of broad band EMF on HSP70 appear to be biphasic, and HSP70 overexpression might not be directly related to improved protection against I/R.     

Tetrahydroindolocarbazoles (THICZs) as new class of urokinase (uPA) inhibitors: Synthesis,anticancer evaluation,DNA-damage determination,and molecular modelling study

Tetrahydroindolocarbazoles (THICZs) with versatile substituents, have been designed, synthesized, structure characterized, then investigated for their in-vitro anticancer screening, urokinase inhibition (uPA) evaluated, DNA-damage determination was further explored. Compounds 5, 8, 10 and 17 displayed the most promising antitumor activities against the breast cancer cell line as compared to the standard drug, doxorubicin with IC₅₀ = 5.24 ± 0.37, 4.00 ± 0.52, 7.20 ± 0.90 and 9.60 ± 1.10 µg/ml (versus 3.30 ± 0.48 µg/ml for doxorubicin). Compounds 5, 8, 10 and 17 represents the most significant uPA inhibitors of our study with IC₅₀ of 3.80, 2.70. 4.75, 10.80 (ng/ml) respectively. The expression levels of CDKN2A gene were decreased in 8, 10 and 17 cell lines as compared to those in positive control samples. Cell lines treated with 5, 8, 10 and 17 clearly observed a high score of damaged DNA cells. A deeper examination revealed that our hetroaromatics showed an extensive hydrogen bonding interactions that is required in the S pocket which is important for activity Arg 217, Gly 219, Gly 216, Lys 143 and Ser 190. So we present THICZs as promising uPA inhibitors expected as significant promise for further development as anti-invasiveness drugs.     

Genistein induces G2/M cell cycle arrest and apoptosis of human ovarian cancer cells via activation of DNA damage checkpoint pathways

Genistein is a major isoflavonoid in dietary soybean, commonly consumed in Asia. Genistein exerts inhibitory effects on the proliferation of various cancer cells and plays an important role in cancer prevention. However, the molecular and cellular mechanisms of genistein on human ovarian cancer cells are still little known. We show that exposure of human ovarian cancer HO-8910 cells to genistein induces DNA damage, and triggers G2/M phase arrest and apoptosis. Furthermore, we also found that checkpoint proteins ATM and ATR are phosphorylated and activated in the cells treated with genistein. It is also shown that genistein increases the phosphorylation and activation of Chk1 and Chk2, which results in the phosphorylation and inactivation of phosphatases Cdc25C and Cdc25A, and thereby the phosphorylation and inactivation of Cdc2 which arrests cells in G2/M phase. Moreover, genistein enhances the phosphorylation and activation of p53, while decreases the ratio of Bcl-2/Bax and Bcl-xL/Bax and the level of phosphorylated Akt, which result in cells undergoing apoptosis. These results demonstrate that genistein-activated ATM-Chk2-Cdc25 and ATR-Chk1-Cdc25 DNA damage checkpoint pathways can arrest ovarian cancer cells in G2/M phase, and induce apoptosis while the cellular DNA damage is too serious to be repaired. Thus, the antiproliferative, DNA damage-inducing and pro-apoptotic activities of genistein are probably responsible for its genotoxic effects on human ovarian cancer HO-8910 cells.