Investigating the Vinblastine Induced-Chromosomal Abnormality in the Already Gamma Irradiated L929 Cell Line Using Micronucleus Assay in Cytokinesis Blocked Binucleated Cells

Objectives: Vast number of studies show the relationship between aneuploidy and cancer. Ionizing radiation in addition to induce all kinds of damages to the cells and structure of chromosomes, is also able to induce aneuploidy through direct damages to chromosome division apparatus. Also irradiation of the cells induces mutations in several genes which might be involved in cell division fidelity and play a role in reversing the effect of aneugens. Therefore, irradiation of cells and tissues might produce sensitivity to agents with aneugenic capability in irradiated cells. Methods: To investigate the persistent genomic effect of ionizing irradiation on chromosomal instability, L929 cells were gamma irradiated with the dose of 2 Gy. Cells were left to recover from the harmful effect of irradiation. They were treated with low dose of vinblastine (0.5 ng.ml-1) 72h post-gamma irradiation. Finally, the induced chromosomal abnormalities were scored using micronucleus assay in cytokinesis-blocked binucleated cells (MnBi). Results: Irradiation-recovered L929 cells treated with vinblastine showed a statistically higher frequency of MnBi compared to non-irradiated and vinblastine treated cells. Conclusion: The results indicate that gamma irradiation, in addition to direct induction of chromosomal damages, is also able to create persisting genomic sensitivity in the cells to chromosomal instability, which is detectable when exposed to the second stimulus.

Efficacy of hyperthermia in human colon adenocarcinoma cells is improved by auraptene.

Colon adenocarcinoma is one of the most common cancers worldwide, and resistance to current therapeutic modalities is a serious drawback in its treatment. Auraptene is a natural coumarin with considerable anticancer effects. The goal of this study was to introduce a novel combinatorial approach for treatment against colon adenocarcinoma cells. To do so, HT29 cells were pretreated with nontoxic auraptene and then hyperthermia was induced. Afterwards, the viability of the cells was assessed, changes induced in the cell cycle were analyzed, and the expression patterns of candidate genes were studied. Results from the MTT assay demonstrated significant (p < 0.01) decreases in cell viability when 20 µg/mL auraptene was used for 72 h, heat shock was induced, and cells were allowed to recover for 24 h. Flow cytometry analysis also indicated considerable changes in the distribution of cells between the sub-G1/G1 and G2/M phases of cell cycle after the combinatorial treatment. Real-time RT-PCR studies revealed significant (p < 0.01) up-regulation of P21 in the cells pretreated with auraptene after heat shock, whereas no significant change was observed in HSP27 expression. Our findings not only indicate, for the first time, that the efficacy of hyperthermia was improved by auraptene pretreatment, but also suggest that this coumarin could be used in the future to achieve more effective therapeutic outcomes.

Synergy between Auraptene, Ionizing Radiation, and Anticancer Drugs in Colon Adenocarcinoma Cells.

Colorectal cancer is a growing health concern with increasing mortality rates, and resistance to anticancer drugs and radiotherapy is a serious drawback in its treatment. Auraptene is a natural prenyloxycoumarin with valuable anticancer effects. The aim of current study was to determine the synergy between auraptene, ionizing radiation, and chemotherapeutic drugs in colon adenocarcinoma cells for the first time. To do so, HT29 cells were treated with combination of auraptene + cisplatin, + doxorubicin, or + vincristine. Furthermore, cells were pretreated with nontoxic auraptene and then exposed to various doses of X-radiation. Assessment of cell viability not only indicated significant (p < 0.05) synergic effects of auraptene and anticancer agents, also revealed more significant (p < 0.01) increase in the toxicity of applied radiations in auraptene pretreated cells. Interesting synergy between auraptene and radiotherapy was then confirmed by morphological alterations, DAPI staining, and flow cytometric analysis of the cell cycle. Moreover, real-time reverse transcription polymerase chain reaction analysis indicated significant (p < 0.01) overexpression of p21, but not GATA6, in auraptene pretreated cells after radiotherapy, and also significant (p < 0.01) down regulation of CD44 and ALDH1 by auraptene. According to present results, auraptene could be considered as an effective natural coumarin to improve the outcome of current chemoradiotherapy options. Copyright © 2017 John Wiley & Sons, Ltd.

Origanum vulgare leaf extract protects mice bone marrow cells against ionizing radiation.

OBJECTIVE: Ionizing radiation produces free radicals which induce DNA damage and cell death. Origanum vulgare leaf extract (OVLE) is a natural compound and its capability of scavenging free radicals and its antioxidant activity have been demonstrated by many researchers. In this study, using micronucleus assay, radioprotective effect of OVLE against clastogenic and cytotoxic effect of gamma irradiation has been investigated in mice bone marrow cells. MATERIALS AND METHODS: OVLE was injected intraperitoneally to the BALB/c mice 1hr prior to gamma irradiation (3Gy) at the doses of 100 and 200 mg/kg. Twenty four hours after irradiation or treatment, animals were killed and smears were prepared from the bone marrow cells. The slides were stained with May Grunwald-Giemsa method and analyzed microscopically. The frequency of micronucleated polychromatic erythrocytes (MnPCEs), micronucleated normochromatic erythrocyte (MnNCEs) and cell proliferation ratio PCE/PCE+NCE (polychromatic erythrocyte/polychromatic erythrocyte + normochromatic erythrocyte) were calculated. RESULTS: The results showed that gamma irradiation (3Gy) increased the frequency of MnPCEs, MnNCEs and reduced the PCE/PCE+NCE ratio in mice bone marrow compared to the non-irradiated control group (p<0.0001). Injection of OVLE significantly reduced the frequency of MnPCEs (p<0.0001) and MnNCEs (p<0.05) and increased the PCE/PCE+NCE ratio as compared to the irradiated control group (p<0.05). CONCLUSION: It seems that OVLE with its antioxidant properties and its capability of scavenging free radicals and reactive oxygen species can reduce the cytotoxic effects of gamma irradiation in mice bone marrow cells.

In vitro differentiation of adipose-tissue-derived mesenchymal stem cells into neural retinal cells through expression of human PAX6 (5a) gene.

The neural retina is subjected to various degenerative conditions. Regenerative stem-cell-based therapy holds great promise for treating severe retinal degeneration diseases, although many drawbacks remain to be overcome. One important problem is to gain authentically differentiated cells for replacement. Paired box 6 protein (5a) (PAX6 (5a)) is a highly conserved master control gene that has an essential role in the development of the vertebrate visual system. Human adipose-tissue-derived stem cell (hADSC) isolation was performed by using fat tissues and was confirmed by the differentiation potential of the cells into adipocytes and osteocytes and by their surface marker profile. The coding region of the human PAX6 (5a) gene isoform was cloned and lentiviral particles were propagated in HEK293T. The differentiation of hADSCs into retinal cells was characterized by morphological characteristics, quantitative real-time reverse transcription plus the polymerase chain reaction (qPCR) and immunocytochemistry (ICC) for some retinal cell-specific and retinal pigmented epithelial (RPE) cell-specific markers. hADSCs were successfully isolated. Flow cytometric analysis of surface markers indicated the high purity (~97 %) of isolated hADSCs. After 30 h of post-transduction, cells gradually showed the characteristic morphology of neuronal cells and small axon-like processes emerged. qPCR and ICC confirmed the differentiation of some neural retinal cells and RPE cells. Thus, PAX6 (5a) transcription factor expression, together with medium supplemented with fibronectin, is able to induce the differentiation of hADSCs into retinal progenitors, RPE cells and photoreceptors.

Developmental study of mercury effects on the fruit fly (Drosophila melanogaster).

Environmental pollution caused by heavy metals such as mercury is one of the most important human problems. It might have severe teratogenic effects on embryonic development. Some pharmacological and physiological aspects of fruit flies (Drosophila melanogaster) are similar to humans. So the stages of egg to adult fruit fly, as a developmental model, were employed in the study. Wild adult insects were maintained in glass dishes containing standard medium at 25 °C in complete darkness. Five pairs of 3-day old flies were then transferred to standard culture dishes containing different concentrations of mercury ion. They were removed after 8 hours. We considered the following: The rate of larvae becoming pupae and pupae to adults; the time required for the development; the hatching rate in the second generation without mercury in the culture; the morphometric changes during development in both length and width of the eggs through two generations; larvae, pupae and adult thorax length and width. The results showed that mercury in culture (20-100 mg/l) increase the duration of larvae (p<0.01) and pupae (p<0.01) development, the rate of larvae becoming pupae (p<0.001); pupae maturation (p<0.05), the hatching rate (p<0.01), the length (p<0.05) and width of larvae (p<0.01) and pupae (p<0.001) and the length in the adult thorax (p<0.01) decreased significantly. There was no effect upon the size of eggs. There were also no larvae hatching in concentrations of 200 mg/l of mercury. Negative effects of mercury as a heavy metal are possibly due to the interference of this metal in cellular signaling pathways, such as: Notch signaling and protein synthesis during the period of development. Since it bonds chemically with the sulfur hydride groups of proteins, it causes damage to the cell membrane and decreases the amount of RNA. This is the cause of failure of many enzyme mechanisms.

Study of Radioprotective Effect of Green Tea against Gamma Irradiation Using Micronucleus Assay on Binucleated Human Lymphocytes.

OBJECTIVES: The aim of this study was to investigate the radioprotective effect of green tea against genotoxicity induced by gamma irradiation in cultured blood lymphocytes from 5 human volunteers. MATERIALS AND METHODS: Peripheral blood samples were collected from volunteers before and 1, 3 and 5 hr after drinking a decoction 4 g green tea in 280 ml boiling water for 5 constitutive days with the same quantity. At each time point, the whole blood samples were exposed to 200 cGy of (60) Co gamma irradiation and then were cultured with mitogenic stimulation to determine the chromosomal aberration in micronucleus assay on cytokinesis-blocked binucleated cells. RESULTS: As expected, for each volunteer, the results showed a significant increase in the incidence of micronuclei after exposure to gamma irradiation as compared to non-irradiated control samples. Only lymphocytes blood sample collected 3 hr after drinking green tea exhibited a significant decrease in incidence of micronuclei compared to non-treated irradiated samples. CONCLUSION: These results suggest the radioprotective ability of green tea against ionizing radiation in human lymphocytes, at specified time after consumptior.

Acute restraint stress increases the frequency of vinblastine-induced micronuclei in mouse bone marrow cells.

Acute physiological stress induces remarkable effects on the nervous, endocrine, and immune systems and also on cellular metabolism and cell division processes. Stress-induced instability of cellular mechanisms might play an important role in increasing cell division disorders. In this study, a relationship between stress and micronucleus (MN) induction in mouse (balb/c) bone marrow cells following vinblastine treatment, or stress or stress and vinblastine treatment in comparison to a non-stressed control group was investigated. In order to test the effects of treatments on MN induction, an in vivo MN assay was performed on bone marrow cells. The results revealed a significantly greater increase in MNs in bone marrow cells (polychromatic erythrocytes) from the stressed/vinblastine treated mice. The data indicate the ability of exposure to an emotional stressor to enhance the damaging actions on bone marrow cells of an aneugenic agent.