A Novel Chenodeoxycholic Derivative HS-1200 Enhances Radiation-induced Apoptosis in Human MCF-7 Breast Cancer Cells / 대한방사선종양학회지

PURPOSE: To examine whether a synthetic bile acid derivatives (HS-1200) sensitizes the radiation-induced apoptosis in human breast cancer cells (MCF-7) and to investigate the underlying mechanism. MATERIALS AND MEHTODS: Human breast cancer cells (MCF-7) in exponential growth phase were treated with HS-1200 for 24 hours at 37degrees C with 5% CO2 in air atmosphere. After removal of HS-1200, cells were irradiated with 2~8 Gy X-ray, and then cultured in drug-free media for 24-96 hours. The effect of radiation on the clonogenicity of MCF-7 cells was determined with clonogenic cell survival assay with 16muM of HS-1200. The induction of apoptosis was determined using agarose gel electrophoresis and Hoechst staining. The expression level of apoptosis-related molecules, such as PARP, Bax, Bcl-2, Bak and AIF, were assayed by Western blotting analysis with 40muM of HS-1200 combined with 8 Gy irradiation. To examine the cellular location of cytochrome c, bax and AIF immunofluorescent stainings were undertaken RESULTS: Treatment of MCF-7 cells with 40muM of HS-1200 combined with 8 Gy irradiation showed several changes associated with enhanced apoptosis by agarose gel electrophoresis and Hoechst staining. HS-1200 combined with 8 Gy irradiation treatment also enhanced production of PARP cleavage products and increased Bax/Bcl-2 ratio by Western blotting. Loss of mitochondrial membrane potential (delta psi m) and increased cytochrome c staining indicated that cytochrome c had been released from the mitochondria in HS-1200 treated cells. CONCLUSION: We demonstrated that combination treatment with a synthetic chenodeoxycholic acid derivative HS-1200 and irradiation enhanced radiation-induced apoptosis of human breast cancer cells (MCF-7). We suggest that the increased Bax/Bcl-2 ratio in HS-1200 co-treatment group underlies the increased radiosensitivity of MCF-7 cells. Further futures studies are remained elusive.

Signal Transduction Factors on the Modulation of Radiosusceptibility in K562 Cells / 대한방사선종양학회지

PURPOSE: The human chronic myelogenous leukemia cell line, K562, expresses the chimeric bcr-abl oncoprotein, whose deregulated protein tyrosine kinase activity antagonizes the induction of apoptosis via DNA damaging agents. Previous experiments have shown that nanomolar concentrations of herbimycin A (HMA) coupled with X-irradiation have a synergistic effect in inducing apoptosis in the Ph-positive K562 leukemia cell line, but genistein, a PTK inhibitor, is non selective for the radiation-induced apoptosis of p210bcr/abl protected K562 cells. In these experiments, the cytoplasmic signal transduction pathways, the induction of a number of transcription factors and the differential gene expression in this model were investigated. MATERIALS AND METHODS: K562 cells in the exponential growth phase were used in this study. The cells were irradiated with 0.5-12 Gy, using a 6 MeV Linac (Clinac 1800, Varian, USA). Immediately after irradiation, the cells were treated with 0.25 microM of HMA and 25 microM of genistein, and the expressions and the activities of abl kinase, MAPK family, NF-kB, c-fos, c-myc, and thymidine kinase1 (TK1) were examined. The differential gene expressions induced by PTK inhibitors were also investigated. RESULTS: The modulating effects of herbimycin A and genistein on the radiosensitivity of K562 cells were not related to the bcr-abl kinase activity. The signaling responses through the MAPK family of proteins, were not involved either. In association with the radiation-induced apoptosis, which is accelerated by HMA, the expression of c-myc was increased. The combined treatment of genistein, with irradiation, enhanced NF-kB activity and the TK1 expression and activity. CONCLUSION: The effects of HMA and genistein on the radiosensitivity of the K562 cells were not related to the bcr-abl kinase activity. In this study, another signaling pathway, besides the MAPK family responses to radiation to K562 cells, was found. Further evaluation using this model will provide valuable information for the optional radiosensitization or radioprotection.

Smad6 Gene and Suppression of Radiation-Induced Apoptosis by Genistein in K562 cells / 대한방사선종양학회지

PURPOSE: The genes involved on the suppression of radiation-induced apoptosis by genistein in K562 leukemia cell line was investigated. MATERIALS AND METHODS: K562 cells in exponential growth phase were irradiated with a linear accelerator at room temperature. Forx-ray irradiation and drug treatment, cultures were prepared at 2x105 cells/mL. The cells were irradiated with 10 Gy (Clinac 1800C, Varian, USA). Stock solutions of herbimycin A (HMA, Calbiochem, UK) and genistein (Calbiochem, UK) were prepared in dimethylsulfoxide (DMSO, Sigma, UK). After incubation at 37degreesC for 24 h, PCR-select cDNA subtractive hybridization, dot hybridization, DNA sequencing and Northern hybridization were examined. RESULTS: Smad6 gene was identified from the differentially expressed genes in K562 cells incubated with genistein which had been selected by PCR-select cDNA subtractive hybridization. The mRNA expression of Smad6 in K562 cells incubated with genistein was also higher than control group by Northern hybridization analysis. CONCLUSION: We have shown that Smad6 involved on the suppression of radiation-induced apoptosis by genistein in K562 leukemia cell line. It is plausible that the relationship between Smad6 and the suppression of radiation-induced apoptosis is essential for treatment development based on molecular targeting designed to modify radiation-induced apoptosis.

Studies on the Radiation Induced Apoptosis by Morphological and Biochemical Analysis in A431 Cells / 대한핵의학회잡지

PURPOSE: We performed this study to evaluate the process of radiation induced apoptosis in A431 skin epithelial cancer cell line. MATERIALS AND METHODS: Low to high dose radiation (0, 2, 5, 10, 25 Gy) was given to A431 cells by Cs-137 cell irradiator. Apoptosis was evaluated by cell morphology, dye exclusion test, and DNA laddering. RESULTS: Cell viability decreased as the radiation dose increased. Number of apoptotic bodies increased as radiation dose increased. It increased most significantly at 12 hours after irradiation. Lactate dehydrogenase activity in culture medium increased according to radiation dose and time after irradiation. CONCLUSION:: Radiation-induced apoptosis which was the main course of cell death in A431 cells could be analyzed quantitatively by counting apoptotic bodies under microscope. Apoptosis increased as radiation dose increased.

The Expression of Oncogenes on the Radiation-induced Apoptosis in SCK Mammary Adenocarcinoma Cell Line / 대한방사선종양학회지

PURPOSE:The expression of p53, p21/WAF/CIP, Bcl-2, and Bax underlying the radiation-induced apoptosis in different pH environments using SCK mammary adenocarcinoma cell line was investigated. MATERIALS AND METHODS: Mammary adenocarcinoma cells of A/J mice (SCK cells) in exponential growth phase were irradiated with a linear accelerator at room temperature. The cells were irradiated with 12 Gy and one hour later, the media was replaced with fresh media at a different pHs. After incubation at 37 degrees C for 0-48 h, the extent of apoptosis was determined using agarose gel electrophoresis and flow cytometry. The progression of cells through the cell cycle after irradiation in different pHs was also determined with flow cytometry. Western blot analysis was used to monitor p53, p21/WAF/CIP, Bcl-2, and Bax protein levels. RESULTS:The induction of apoptosis by irradiation in pH 6.6 medium was markedly less than that in pH 7.5 medium. The radiation-induced G2/M arrest in pH 6.6 medium lasted markedly longer than that in pH 7.5 medium. Considerable amounts of p53 and p21 proteins already existed at pH 7.5 and increased the level of p53 and p21 significantly after 12 Gy X-irradiation. An incubation at pH 6.6 after 12 Gy X-irradiation did not change the level of p53 and p21 protein levels significantly. Bcl-2 proteins were not significantly affected by radiation and showed no correlation with cell susceptibility to radiation-induced apoptosis in different pHs. An exposure to 12 Gy of X-rays increased the level of Bax protein at pH 7.5 but at pH 6.6, it was slight. CONCLUSION: The molecular mechanism underlying radiation-induced apoptosis in different pH environments using SCK mammary adenocarcinoma cell line was dependent of the expression p53 and p21/WAF/CIP proteins. We may propose following hypothesis that an acidic stress augments the radiation-induced G2/M arrest, which inhibiting the irradiated cells undergo post-mitotic apoptosis. The effects of environmental acidity on anti-apoptotic and pro-apoptotic function of Bcl-2 family was unclear in SCK mammary adenocarcinoma cell line.

The Cell Cycle Dependence and Radiation-induced Apoptosis in SCK Mammary Adenocarcinoma Cell Line / 대한방사선종양학회지

PURPOSE: The relationship between environmental pH on the radiation induced-apoptosis in SCK mammary adenocarcinoma cells and cell cycle dependence was investigated. MATERIAL AND METHODS: Mammary adenocarcinoma cells of A/J mice (SCK cells) in exponential growth phase were irradiated with a 137Cs irradiator at room temperature. The cells were irradiated 1 hour after the media was replaced with fresh media at a different pHs. After incubation at 37degrees C for 0-48 h, the extent of apoptosis was determined using agarose gel electrophoresis and flow cytometry. The progression of cells through the cell cycle after irradiation in different pHs was also determined with flow cytometry. RESULTS: The induction of apoptosis by irradiation in pH 6.6 medium was markedly less than that in pH 7.5 medium. When the cells were irradiated and maintained in pH 7.5 medium, the percentage of cells in G2/M phase rapidly increased to about 70% at 12 h after an exposure to 12Gy and returned to control level by 36 h. The percentage of cells in G1 phase decreased as the percentage of cells in G2/M increased. On the other hand, in pH 6.6 medium the percentage of cells in G2/M phases gradually increased to about 45% at 24 h after 12Gy irradiation and then slowly recessed and consequently, as much as 30-35% of the cells were still in the G2/M phase 48 h after irradiation. The percentage of cells in G1 phase then increased as the G2/M arrest began to recede. The radiation-induced G2/M arrest in pH 6.6 medium lasted markedly longer than that in pH 7.5 medium. CONCLUSION: Radiation-induced apoptosis in SCK tumor cells are reversely suppressed in an acidic environment. Radiation-induced G2/M arrest is prolonged in an acidic environment indicating that the suppression of radiation- induced apoptosis and prolongation of radiation-induced G2/M arrest in an acidic environment are related.

The Cell Cycle Dependence and Radiation-induced Apoptosis in SCK Mammary Adenocarcinoma Cell Line / 대한방사선종양학회지

PURPOSE: The relationship between environmental pH on the radiation induced-apoptosis in SCK mammary adenocarcinoma cells and cell cycle dependence was investigated. MATERIAL AND METHODS: Mammary adenocarcinoma cells of A/J mice (SCK cells) in exponential growth phase were irradiated with a 137Cs irradiator at room temperature. The cells were irradiated 1 hour after the media was replaced with fresh media at a different pHs. After incubation at 37degrees C for 0-48 h, the extent of apoptosis was determined using agarose gel electrophoresis and flow cytometry. The progression of cells through the cell cycle after irradiation in different pHs was also determined with flow cytometry. RESULTS: The induction of apoptosis by irradiation in pH 6.6 medium was markedly less than that in pH 7.5 medium. When the cells were irradiated and maintained in pH 7.5 medium, the percentage of cells in G2/M phase rapidly increased to about 70% at 12 h after an exposure to 12Gy and returned to control level by 36 h. The percentage of cells in G1 phase decreased as the percentage of cells in G2/M increased. On the other hand, in pH 6.6 medium the percentage of cells in G2/M phases gradually increased to about 45% at 24 h after 12Gy irradiation and then slowly recessed and consequently, as much as 30-35% of the cells were still in the G2/M phase 48 h after irradiation. The percentage of cells in G1 phase then increased as the G2/M arrest began to recede. The radiation-induced G2/M arrest in pH 6.6 medium lasted markedly longer than that in pH 7.5 medium. CONCLUSION: Radiation-induced apoptosis in SCK tumor cells are reversely suppressed in an acidic environment. Radiation-induced G2/M arrest is prolonged in an acidic environment indicating that the suppression of radiation- induced apoptosis and prolongation of radiation-induced G2/M arrest in an acidic environment are related.