The effect of elemene on lung adenocarcinoma A549 cell radiosensitivity and elucidation of its mechanism

OBJECTIVE: To investigate the effect of elemene on the radiosensitivity of A549 cells and its possible molecular mechanism. METHODS: Apoptosis of A549 cells was detected by flow cytometry and fluorescence microscopy. The effect of double-strand break (DSB) damage repair in A549 cells was evaluated using the neutral comet assay. Protein expression levels were detected using western blotting, and the correlation between protein levels was analyzed. RESULTS: Elemene exhibited a radiosensitizing effect on A549 cells. The level of apoptosis induced by elemene combined with radiation was significantly greater (p<0.01) than that elicited by either radiation or elemene alone. Following radiation and subsequent repair for 24 h, the tail intensity of A549 cells treated with a combination of elemene and radiation was greater than that of cells treated with either elemene or radiation alone (p<0.01). This result indicates that elemene inhibits cellular DSB repair. Both elemene combined with radiation and radiation alone decreased the protein expression of DNA-PKcs and Bcl-2 compared to elemene alone (p<0.01), while p53 protein expression was increased (p<0.01). A negative correlation was observed between DNA-PKcs and p53 expression (r=−0.569, p=0.040), while a positive correlation was found between DNA-PKcs and Bcl-2 expression (r=0.755, p=0.012). CONCLUSIONS: Elemene exhibits a radiosensitizing effect on A549 cells, and its underlying molecular mechanism of action may be related to the downregulation of DNA-PKcs gene expression. .

Enhancement of radiation induced cell death in chicken B lymphocytes by withaferin A.

Withaferin A (WA), a plant withanolide, has shown significant radiosensitizing effect in vitro and in vivo. Inhibition of DNA repair has been suggested as a mechanism of radiosensitization by WA. To test this, the effect of withaferin A on survival of DT40 chicken B-lymphocyte cell line and its repair deficient single gene mutants Rad54-/-, Ku70-/- and double mutant Ku70-/- /Rad54-/- after irradiation was studied. Exponentially growing cells were treated for 1 hr with 5 microM WA and then exposed to different doses of X-rays. Cell survival was studied by clonogenic assay. WA significantly reduced survival of DT40, Ku70-/- and Ku70-/- /Rad54-/-, but not Rad54-/- cells, suggesting that WA enhances radiosensitivity by interfering with homologous repair, the major pathway of DSB repair in these cells. Inhibition of DNA repair is further indicated in a significant decrease in surviving fraction of DT40 cells by post-irradiation incubation with WA. This could have relevance to cancer radiotherapy.

Ginseng extract exhibits antimutagenic activity against induced mutagenesis in various strains of Salmonella typhimurium.

Ginseng has been reported to exhibit antioxidant and antimutagenic activity. The present study was undertaken with a view to confirm whether the antioxidant activity of Ginseng is responsible for its antimutagenic action. The concentrated root extract of Panax ginseng (Ginseng extract I) and its lyophilized powder (Ginseng extract II) obtained from two different manufacturing houses, were tested against mutagenesis using the well-standardized Ames microsomal test system. The extracts exhibited antimutagenic effect against hydrogen peroxide induced mutagenesis in TA100 strain, and against mutagenesis produced by 4-nitroquinoline-N-oxide in both TA98 and TA100 strains of Salmonella typhimurium. Both the extracts failed to show any antimutagenic potential against tert-butyl hydroperoxide (an oxidative mutagen) in TA102 strain, a strain highly sensitive to active oxygen species. The extracts also indicated a weak antioxidant activity in a series of in vitro test systems viz., 1,1-diphenyl picryl hydrazyl (DPPH) assay, hydrogen peroxide scavenging and superoxide anion scavenging. The results indicate that the protective effects shown by ginseng extract(s) against 4-nitroquinoline-n-oxide and hydrogen peroxide induced mutagenesis in TA98 and TA100 could mainly be due to its property to initiate and promote DNA repair rather than free radical scavenging action.

Radiosensitization by targeting radioresistance-related genes with protein kinase A inhibitor in radioresistant cancer cells

Here we determined which radiation-responsive genes were altered in radioresistant CEM/IR and FM3A/IR variants, which showed higher resistance to irradiation than parental human leukemia CEM and mouse mammary carcinoma FM3A cells, respectively and studied if radioresistance observed after radiotherapy could be restored by inhibition of protein kinase A. The expressions of DNA-PKcs, Ku70/80, Rad51 and Rad54 genes that related to DNA damage repair, and Bcl-2 and NF-kappaB genes that related to antiapoptosis, were up-regulated, but the expression of proapototic Bax gene was down-regulated in the radioresistant cells as compared to each parental counterpart. We also revealed that the combined treatment of radiation and the inhibitor of protein kinase A (PKA) to these radioresistant cells resulted in synergistic inhibition of DNA-PK, Rad51 and Bcl-2 expressions of the cells, and consequently restored radiosensitivity of the cells. Our results propose that combined treatment with radiotherapy and PKA inhibitor can be a novel therapeutic strategy to radioresistant cancers.

Further phenotypic characterization of pso mutants of Saccharomyces cerevisiae with respect to DNA repair and response to oxidative stress

The sensitivity responses of seven pso mutants of Saccharomyces cerevisiae towards the mutagens N-nitrosodiethylamine (NDEA), 1,2:7,8-diepoxyoctane (DEO), and 8-hydroxyquinoline (8HQ) further substantiated their allocation into two distinct groups: genes PSO1 (allelic to REV3), PSO2 (SNM1), PSO4 (PRP19), and PSO5 (RAD16) constitute one group in that they are involved in repair of damaged DNA or in RNA processing whereas genes PSO6 (ERG3) and PSO7 (COX11) are related to metabolic steps protecting from oxidative stress and thus form a second group, not responsible for DNA repair. PSO3 has not yet been molecularly characterized but its pleiotropic phenotype would allow its integration into either group. The first three PSO genes of the DNA repair group and PSO3, apart from being sensitive to photo-activated psoralens, have another common phenotype: they are also involved in error-prone DNA repair. While all mutants of the DNA repair group and pso3 were sensitive to DEO and NDEA the pso6 mutant revealed WT or near WT resistance to these mutagens. As expected, the repair-proficient pso7-1 and cox11-Delta mutant alleles conferred high sensitivity to NDEA, a chemical known to be metabolized via redox cycling that yields hydroxylamine radicals and reactive oxygen species. All pso mutants exhibited some sensitivity to 8HQ and again pso7-1 and cox11-Delta conferred the highest sensitivity to this drug. Double mutant snm1-Delta cox11-Delta exhibited additivity of 8HQ and NDEA sensitivities of the single mutants, indicating that two different repair/recovery systems are involved in survival. DEO sensitivity of the double mutant was equal or less than that of the single snm1-Delta mutant. In order to determine if there was oxidative damage to nucleotide bases by these drugs we employed an established bacterial test with and without metabolic activation. After S9-mix biotransformation, NDEA and to a lesser extent 8HQ, lead to significantly higher mutagenesis in an Escherichia coli tester strain WP2-IC203 as compared to WP2, whereas DEO-induced mutagenicity remained unchanged

G2 repair and chromosomal damage in lymphocytes from workers occupationally exposed to low-level ionizing radiation

The effect of the G2 repair of chromosomal damage in lymphocytes from workers exposed to low levels of X- or g-rays was evaluated. Samples of peripheral blood were collected from 15 radiation workers, 20 subjects working in radiodiagnostics, and 30 healthy control donors. Chromosomal aberrations (CA) were evaluated by scoring the presence of chromatid and isochromatid breaks, dicentric and ring chromosomes in lymphocytes with/without 5mM caffeine plus 3mM-aminobenzamide (3-AB) treatment during G2. Our results showed that the mean value of basal aberrations in lymphocytes from exposed workers was higher than in control cells (p< 0.001). The chromosomal damage in G2, detected with caffeine plus 3-AB treatment was higher than the basal damage (untreated conditions), both in control and exposed populations (p< 0.05). In the exposed workers group, the mean value of chromosomal abnormalities in G2 was higher than in the control (p< 0.0001). No correlation was found between the frequency of chromosome type of aberrations (basal or in G2), and the absorbed dose. Nevertheless, significant correlation coefficients (p< 0.05) between absorbed dose and basal aberrations yield (r = 0.430) or in G2 (r = 0.448) were detected when chromatid breaks were included in the total aberrations yield. Under this latter condition no significant effect of age, years of employment or smoking habit on the chromosomal aberrations yield was detected. However, analysis of the relationship between basal aberrations yield and the efficiency of G2 repair mechanisms, defined as the percentage of chromosomal lesions repaired in G2, showed a significant correlation coefficient (r = -0.802; p< 0.001). These results suggest that in addition to the absorbed dose, the individual G2 repair efficiency may be another important factor affecting the chromosomal aberrations yield detected in workers exposed to low-level ionizing radiation

2-Deoxy-D-glucose induced modulation of DNA damage repair, survival, mutagenesis and recombinogenesis in 8-MOP+UVA treated yeast.

Cellular and genomic effects of post-treatment repair modulation by 2-deoxy-D-glucose (2-DG) and yeast extract were studied in 8-MOP + UVA treated cells of Saccharomyces cerevisiae. The type of lesions and their repair in phosphate buffer glucose (PBG) differed with UVA dose. At low UVA dose (1.4 kJ/m2), lesions were sublethal and mutagenic and did not repair by recombinogensis. The fraction of potentially lethal lesions and lesions repaired by recombinogenesis increased with UVA dose. Cellular repair in PBG was largely error-free and was inhibited by 2-DG. Yeast extract enhanced cellular repair and also recombinogensis; 2-DG in presence of yeast extract promoted error-prone repair. Pulsed-field gel electrophoresed chromosomal DNA bands did not show observable alterations immediately after 8-MOP + UVA treatment. On post-treatment incubation in PBG, the intensity ratio (rho n), of each band altered in a biphasic manner showing decrease first, followed by either increase or no change upto 24 hr depending upon UVA exposure dose. Presence of 2-DG in PBG inhibited decrease in rho n in a concentration dependent manner. Yeast extract reduced the time of first phase of DNA repair. 2-DG and yeast extract together reduced the time of first phase of repair and also inhibited the subsequent increase in rho n, which was observed in the case of yeast extract in PBG. It is proposed that (i) 2-DG in PBG inhibits excision of DNA damage and error-free repair; (ii) yeast extract stimulates the error-prone repair associated with cell cycle and recombinogenesis; (iii) 2-DG in presence of yeast extract allows excision of damage but inhibits build up through recombinogenesis inducing instead, cell cycle associated error-prone repair. A simple schematic model has been proposed to explain these events.

Effect of nalidixic acid on recombination and DNA repair of Escherichia coli K-12 strains.

Bacterial DNA gyrase enzyme plays a role in replication, transcription, recombination and repair. Escherichia coli gyrase enzyme is composed of two types of subunit, the gyr A and gyr B gene products called subunit A and B. Each type of subunit can be inhibited with temperature-sensitive mutations or by treating cells with specific antibiotic. Quinolones class of drugs such as nalidixic acid, oxonilic acid and norfloxacin affect gyr A protein. This effect due to inactivation of gyrase arises from the change in DNA superhelical tension. Mutant bacteria resistant to nalidixic acid show structural alterations in subunit A. Nalidixic acid resistant mutants of E. coli strains showed lower recombination efficiency, linkage, DNA repair, and mutation frequency than their wild types.

Presence of RNA from yeast inhibits the photoreactivation of UV-irradiated DNA by Phr A photolyase from Escherichia coli.

Photoreactivation of UV-irradiated DSNA with phr A photolyase from Escherichia coli was studied in the presence of yeast RNA. Mixing of RNA with UV-irradiated DNA before its treatment with photolyase inhibited the photoreactivation of DNA. Denatured (by sonication) RNA was found to be more effective in blocking photolyase action. Agarose gel electrophoresis experiments suggest that this inhibition of photoreactivation is due to interference in the binding of photolyase with UV-irradiated DNA by yeast RNA.

In vitro radiation responses of human intracranial meningiomas & their modifications by 2-deoxy-D-glucose.

Radiation responses of 16 human intracranial meningiomas [hemangiopericyte, 4; fibrous, 4; transitional, 4; vascular, 3; and meningiothelial, 1] and their modifications by 2-deoxy-D-glucose (2-DG) have been studied in vitro using organ cultures derived from post-operative tumour specimens. Treatment induced cytogenetic damage viz., micronuclei formation and DNA content dispersion, were analysed as indices of the radiation damage. Results showed that the 60Co gamma ray induced micronuclei frequencies in meningiomas varied over a large range (0.7-6.2%). Presence of 2-DG (at equimolar concentration with glucose) for 4 h after irradiation, increased the radiation damage by a factor of up to 2.8 in nearly 70 per cent of the cases, although a decrease was observed in 20 per cent cases. These observations can be explained on the basis of the energy linked modulations of the competitive processes of repair and fixation of radiation induced DNA lesions. The results of the study suggest that the therapy of meningiomas could be improved by combining radiotherapy with administration of 2-DG.