Influence of Estrogen and Polyamines on Mifepristone-induced Apoptosis in Prostate Cancer Cells / Journal of the Korean Cancer Association, 대한암학회지

PURPOSE: Although androgens are the main steroids controlling the growth of prostate glands, estrogens are also important in the regulation of its growth. Prostate cancer cells, like other cancer cells, maintain high levels of polyamines. In LNCaP cells, apoptosis is induced by mifepristone. During the process of cell death, the regulation of ROS production, caspase-3 activation and poly (ADP-ribose) polymerase cleavage were investigated in the presence of estrogen and polyamines to identify their possible roles. MATERIALS AND METHODS: The cell growth was assessed using the MTT assay, and the intracellular ROS production by the DCFH-DA assay. The p53 protein expression, activation of caspase-3 and PARP cleavage were checked by Western blotting, with specific antibodies to each. RESULTS: The growth and viability of the cells were significantly inhibited, in a dose- and time-dependent manners, by mifepristone (MIF) treatment. The production of ROS were dependent on the MIF dosage. The activation of caspase-3 and cleavage of PARP also increased with the duration of MIF treatment. The expression of p53 protein also increased with increases in the MIF incubation time. E2 severely inhibited the ROS production, caspase-3 activation and PARP cleavage. However, polyamines only inhibited the ROS production, without influencing the caspase-3 activation or PARP cleavage. CONCLUSION: In LNCaP cells, MIF induces apoptosis through ROS production. The expression of p53 protein, caspase-3 activation and PARP cleavage accompanied the process of apoptosis. The apoptotic processes were inhibited by E2, but polyamines only inhibited the ROS production, implying the multifunctional role of E2, in addition to its role as a free radical scavenger.

Polyamines Regulate Growth Factor-Induced Protein Phosphorylation in MCF-7 Human Breast Cancer Cells / Journal of the Korean Cancer Association, 대한암학회지

PURPOSE: Growth factors stimulate protein phosphorylation resulting in transmission of mitogenic signals. In breast cancer, protein kinases and their substrate proteins are importnat in cell proliferation and phathogenesis. Polymine is known as a mediator of stimuli-induced proliferation in many cell systems. In the present study, we report the importance of polyamines in protein phosphorylation in MCF-7 human breast cancer cells. MATERIALS AND METGODS: Protein phosphorylation study was done by incubating cells in the DMEM containing [gamma-(32)P]-ATP. Quantitation of phosphorylation was analysed by fluorescene image analyzer. Tyrosine phosphorylation was detected by anti-phosphotyrosine antibody. Shc was detected by radioimmunoprecipitation and Western blotting. RESULTS: E2, TGF-alpha, and EGF enhanced the protein phosphorylation in very similar pattern. Among those proteins, 67 kDa protein was most strongly phosphorylated. But the most prominent tyrosine phosphoprotein was 52 kDa protein. DFMO at 5 mM strongly inhibited the phosphorylation of the most proteins. Externally added polyamine could recover the inhibitory effect of DFMO in protein phosphorylation. Among the 5 major tyrosine phosphoproteins, 52 and 46 kDa proteins appeared to be Shc proteins. CONCLUSION: Polyamines modulate signal transduction in relation with estrogen receptor and EGF receptor through multiple steps of protein phosphorylations. Tyrosine phosphorylation of Shc proteins were most significantly influenced by polyamines in growth factor-stimulate breast cancer cell proliferation.

Effects of Polyamines on TNFalpha- or Tamoxifen-induced Apoptosis in Human Breast Cancer Cells / Journal of the Korean Cancer Association, 대한암학회지

PURPOSE: To investigate the effects of polyamines on tumor necrosis factor alpha (TNFalpha)-or tamoxifen (TAM)-induced apoptosis in estrogen receptor (ER)-positive MCF- 7 and ER-negative MDA-MB-231 human breast cancer cells. MATERIALS AND METHODS: Cell viability was assessed by using MTT assay. Reactive oxygen species (ROS) generation was measured using 2', 7'-dichlorofluorescin diacetste (DCFDA) by fluorescence plate reader. DNA fragmentation was assessed by 1.5% agarose gel electrophoresis. RESULTS: TNFalpah and TAM showed significant dose- and time- dependent inhibitory effects on the growth of MCF-7 human cells. However, the growth of MDA-MB-231 cells were not inhibited by TNFalpha or TAM treatment. The generation of ROS was increased in dose-and time-dependent manner by TNFalpha treatment in MCF-7 cells. Polyamines, especially spermine suppressed TNFalpha-induced ROS generation in MCF-7 cells. Antioxidant effects of polyamines were also demonstrated by DNA fragmentation, cell morphology as well as ROS generation assay. Polyamines also blocked TAM-induced cell death in MCF-7 cell. However, MDA-MB-231 cells showed resistance to the cytotoxic effects of TNFalpha or TAM. CONCLUSION: These results suggest that polyamines may prevent TNFalpha or TAM-induced apoptosis in MCF-7 human breast cancer cells.

KR 31378, a potent antioxidant, inhibits apoptotic death of A7r5 cells

This work describes the pharmacological inhibition by KR 31378 and its acetyl metabolite, KR 31612, of the apoptotic cell death induced by H2O2 in the A7r5 cells. Exposure of A7r5 cells to H2O2 (0.5 mM) induced a concentration-dependent cytotoxicity in association with oligonucleosomal DNA fragmentation. H2O2-induced cell death was potently suppressed by KR 31378, KR 31612, alpha-tocopherol or trolox. Additionally, the apoptotic death of A7r5 cells (DNA ladders on electrophoresis) was also strongly suppressed by KR 31378 and KR 31612, but to a less degree by alpha-tocopherol and trolox. As a mechanistic study, incubation with H2O2 markedly showed a decreased Bcl-2 level and, in contrast, increased Bax protein and cytochrome C release, which were significantly and concentration-dependently reversed by KR 31378 and KR 31612 as well as by alpha-tocopherol and trolox. KR 31378 and alpha-tocopherol significantly reduced lipid peroxidation in accordance with reduced intracellular ROS and peroxyl radical. These results suggest that KR 31378 has a therapeutic potential against the apoptotic injury via mediation of antioxidative stress.

Comparative Studies on the Polyarnine Involvement in MCF - 7 and MDA - MB - 231 Breast Cancer Cell Proliferation / 대한암학회지

No abstract available.