Comparison of radiosensitizing effect of Resveratrol on monolayer and spheroid culture of DU145 prostatic cell line

Radiotherapy is an established therapeutic modality for prostate cancer. Resveratrol, a natural antioxidant, has been shown to inhibit carcinogenesis and to block the process of tumor initiation and progression. No data is available on the response of cellular spheroid to Reseveratol. In this study we have examined the effect of Resveratol on the radiation response of human prostate cell line DU145 in monolayer and spheroid cultures. Radiosensitivity was assessed using viability and colony formation assay. Apoptosis and necrosis were assessed using acridine orange/ ethidium bromide double staining. The colony formation assay did not show any significant radio-sensitizing effect, but apoptosis assay showed significant radio-sensitizing effect of Resveratol on DU145 cells grown as monolayer. In the spheroid cells the results of apoptosis test were not significant and corresponded closely to the result of survival curve. While Resveratol could sensitize DU145 cells in monolayer to ionizing radiation, it did not have any effect on sensitivity of cells cultured in spheroid cultures

role of Rad51 protein in radioresistance of spheroid model of DU145 prostate carcinoma cell line

Rad51 is a protein with critical role in double strand break repair. Down-regulation of this protein has a significant effect in radiosensitivity of some cell lines like prostate carcinoma. Compared to monolayer cell culture model, the spheroids are more resistant to radiation. The aim of the current study was to determine the Rad51 protein level in DU145 spheroids, and monolayer cells before and after exposure to gamma irradiation. In the present study, western blot was used to determine the level of Rad51 protein in DU145 cell line grown as monolayer and spheroid. Western blot analysis showed that in the spheroid cells, Rad51 had an elevated level before and after radiation in comparison with monolayer cells. Higher doses of radiation induced elevated expression of Rad51 protein in both culture models. The level of at protein after exposure to gamma rays had been time dependent. Rad51 might act as a mediator of radiation resistance in tumor cells. Repression of Rad51 activity could be a prominent strategy to overcome radiation resistance of tumors

Reduced DNA damage in tumor spheroids compared to monolayer cultures exposed to ionizing radiation

Several cell lines when cultured under proper condition can form three dimensional structures called multicellular tumor spheroids. Tumor spheroids are valuable in vitro models for studying physical and biological behavior of real tumors. A number of previous studies using a variety of techniques have shown no relationship between radiosensitivity and DNA strand breaks in monolayer and spheroid model of cell culture. In the present study, the radiosensitivity of cells grown as monolayer and spheroid were measured with colony assay and the role of DNA strand breaks in this sensitivity was examined using single cell gel electrophoresis assay also known as Comet assay. In the present experiment, spheroids showed more radioresistance than monolayers as judged by the number of colonies which they produced after radiation. Under the same experimental conditions, less level of DNA damage was detected in spheroids using "comet assay" technique. It was concluded that the loss of radioresistance which was observed in monolayer cultures might have been attributed to the higher level of DNA damage occurred in the cells

Antisense RNA to the type I insulin-like growth factor receptor reversed the transformed phenotype of PC-3 human prostate cancer cell line in vitro

The insulin-like growth factor I receptor [IGF-IR] plays an essential role in the establishment and maintenance of transformed phenotype. Interference with the IGF- IR pathway by antisense causes reversal of the transformed phenotype in many rodent and human tumor cell lines. We stably transfected the PC-3 human prostate cancer cell line with an IGF-IR antisense RNA expression plasmid. The number of lGF-I receptors on the antisense-transfected PC-3 cells was reduced by 40.2% relative to the control-transfected cells. The transfected cells maintained their high expression of IGF-IR antisense RNA for up to one year in selective medium. The reduction in the expression of IGF-IR had no effect on the cell growth in monolayer. The clonogenicity of antisense-transfected cells was 24.7% of the clonogenicity of control-transfected cells in soft agar. There was a good correlation between IGF-IR level and inhibition of transformation in soft agar. These results indicate that reduction of IGF-IR by antisense RNA can reverse the transformed phenotype of human prostate cancer cells

study on the hemopoietic activity of human placenta in vitro, in the presence and absence of stimulators and inhibitors of transcription and translation

The hemopoietic activity of the human placenta was studied in vitro under various conditions in order to assess the optimae conditions of protein synthesis and colony stimulating factor [CSF] production by this tissue. The hemopoietic activity of placental tissue was assayed by the semi-solid agar medium technique. In order to obtain maximum hemopoietic activity, various stimulators were examined and it was determined that lipopolysaccharide was the most potent stimulator. Inhibitors of transcription and translation were also examined alone and in the presence of stimulators. They potently suppressed hemopoietic activity. Pulse-chase studies with the use of 3H-leucine were used to determine the kinetics of protein synthesis of placental tissue in vitro

Feedback regulation of colony stimulating factor production

The production of colony-stimulating factors [CSF] is delicately controlled through a complex network of humoral and environmental factors. We have studied some of the mechanisms which regulate the production of CSF as compared to general protein synthesis in the lung tissue in vitro. When lung tissue from mice was cultured for various times in serum free medium, the first detectable level of CSF activity in the lung conditioned medium [LCM] appeared 6 hr after initiation of the culture, continued to rise until 24 hr, and then levelled off for several days. Under similar conditions protein synthesis did not level off, but continuously rose after 24 hr. When the lung tissue which had been cultured previously for 6,24, or 48 hr was recultured in fresh tissue culture medium, de novo synthesis of CSF occurred as judged by CSF synthesis inhibition and stimulation studies. The amount of new CSF synthesized by these tissues decreased as the initial culturing period increased from 6 to 48 hr. There was also a decrease in the amount of total protein synthesis and release in the secondary lung cultures as a function of the initial culturing period. Endotoxin stimulation of 24 or 48 hr cultured lung tissues [plateau phase tissue] resulted in de novo synthesis of CSF by these tissues. However, when fresh lung tissue was cultured in 24or 48 hr LCM, no new CSF was produced by the fresh tissues, while under similar conditions, protein synthesis by these tissues was significant as judged by double-labelling experiments. On the other hand, 6 hr LCM was able to support both CSF production and protein synthesis by fresh lung tissues. The results suggest at least two distinct regulatory systems controlling CSF production by the lung in vitro: 1- Aging which is responsible for general and nonspecific decrease in the rate of protein synthesis and CSF production in this system, and 2-Feedback regulation of CSF production by the level of CSF which is formed in the LCM

Heterogeneity of macrophage populations: antibodies detecting various populations of macrophages

Two groups of antibodies against mouse resident alveolar and peritoneal macrophages have been raised individually in rabbits by immunization with whole cell suspensions of lavaged alveolar and peritoneal macrophages. The whole antisera reacted positively with neutrophils and lymphocytes. However, upon extensive absorptions with these cells the activity became restricted to macrophages. The immunoglobulins were purified up to 8 fold by repeated precipitations with saturated ammonium sulphate. The antibodies were labeled with fluorescein isothiocyanate and further used. The specificity of antibodies was confirmed by direct and indirect immunofluorescence and complement-dependent cytotoxicity. In the cytolysis of target cells in the presence of complement, the antibodies could lyse more than 80% of cells at dilutions as low as 1:512

role of alveolar macrophages in the production of colony-stimulating factor by the lung

The role of alveolar macrophages in the production of granulocyte/ macrophage colony-stimulating factor[s] by the rat lung was investigated. Lavaged lungs, when incubated at proper weight per volume of culture medium, produced the same amount of colony-stimulating factor as unlavaged ones. Both lavaged and unlavaged lungs produced similar types of colony-stimulating factor [s]. Prolonged incubation of lavaged and unlavaged lung tissues did not result in higher levels of activity beyond that produced by 48 hrs of incubation. Alveolar macrophages recovered from the lung did not produce colony-stimulating factors when they were cultured unstimulated and under similar conditions as lung conditioned medium. The results indicated that alveolar macrophages did not play a significant role in colony stimulating factor [s] production by the lungs and when unstimulated, they did not produce colony-stimulating factors directly