Therefore, we verified the role of Fas-L/Fas in the process of PEDF-induced apoptosis. min at 37 C. After rinsing three times, the cells were fixed with 4% paraformaldehyde for 10 min at 37 C, followed by blocking with 5% BSA for 30 min at room temperature. The Fas protein was detected using a monoclonal anti-Fas antibody (1:100) and a secondary antibody conjugated to Alexa Fluor 488 (1:200). Cell nuclei were stained with DAPI (1:2000). All slides were viewed under a confocal laser-scanning microscope (LSM710, Zeiss, Jena, Germany). Western Blot Analysis Western blot analysis was performed as described elsewhere (25). Antibodies for caspase 8/9, PARP, Fas-L, Fas, phospho-p53, and p53 were used at 1:1000 dilution. Antibodies for -actin and GAPDH were used at 1:10,000 dilution. The bound antibody was visualized using HRP-conjugated secondary antibodies. Animal Studies The A549 heterotopic transplanted tumor model was established as described Mitoquinone mesylate previously, with some modifications (22). When tumors were visible, the mice were randomly assigned to two groups with six mice in each group. One group received peritoneal injections with 5 mg/kg PEDF per mouse, whereas the other group received the same volume of PBS as a control. Mice received five injections every other day until the overall dose reached 25 mg/kg. Nude mice were weighed, and the tumor length and width diameters were measured every 2 days. The tumor volume was determined according to the Mitoquinone mesylate following equation: volume = (length width2) 0.5. 24 days after Rabbit Polyclonal to IRF3 the first injection of A549 cells, tumors were dissected, weighed, and stored at ?80 C for Western blot and immunohistochemistry analyses. All animal studies were performed under an institutionally approved protocol according to the USPHS Guide for the Care and Use of Laboratory Animals. Microvessel Density Assay Frozen sections were treated with non-immune goat serum to block nonspecific binding (background). The sections were then incubated with 1:100 dilution of the rat monoclonal antibody against CD31 (BD Biosciences) at 4 C overnight. After rinsing with PBS, the sections were subjected to the cy3-labeled goat anti-rabbit antibody (1:200) at 37 C for 30 min. The cell nuclei were stained with Mitoquinone mesylate DAPI (1:2000) at room temperature for 10 min. All slides were viewed under a fluorescence microscope (Axio Observer Z1, Zeiss). The tumor vasculature was quantified according to the Weidner method (26). TUNEL Assay in A549 Xenografts Paraffin sections from each tumor were analyzed by TUNEL staining using an cell death detection kit (Merck Millipore). A brown coloration indicated apoptotic cells. The number of apoptotic cells was counted in five randomly selected fields using a conventional optical microscope. Statistical Analysis All data are expressed as mean S.D. SPSS 13.0 software was used for the one-way analysis of variance in all statistical analyses (SPSS, Chicago, IL). < 0.05 was considered statistically significant. RESULTS Inhibitory Effects of PEDF on Tumor Proliferation and Angiogenesis in the Heterotopic Transplanted Human Lung Cancer Nude Mice Model We first investigated the antitumor activity of PEDF = 6) or rPEDF (= 6). The rPEDF-treated group exhibited slower growth kinetics than the PBS-treated group, and a 72.6% reduction in tumor volumes was observed by day 24 (Fig. 1= 6, = 6, < 0.05; **, < 0.01. < 0.01 PBS control. < 0.01 PBS control. = 50 m. < 0.01 PBS control. = 50 m. Induction of Tumor Cell Apoptosis by PEDF in Vivo To evaluate whether PEDF influences tumor cell apoptosis and and < 0.05; **, < 0.01 controls. = 50 m. < 0.05; **, < 0.01 control. Open in a separate window FIGURE 3. Effects of PEDF overexpression on cell viability and apoptosis in A549 and Calu-3 cells. = 50 m. < 0.01 the vector-only group. Data are representative of three independent experiments. = 200 m. < 0.01 the vector-only group. PEDF-induced A549 and Calu-3 Cell Apoptosis Is Mediated Primarily by Caspase 8 Activation The activation of caspase 8 and caspase 9 has been shown to be involved in PEDF-induced human umbilical vein endothelial cell (HUVEC) apoptosis (17). To examine whether this process is also involved in PEDF-induced lung cancer cell apoptosis, the cleavage of caspase 8/9/PARP was detected by Western blot analysis. Both and studies demonstrated that PEDF significantly increased the levels of cleaved caspase 8/9/PARP (Fig. 4, and and and through through < 0.01 PEDF + dimethyl sulfoxide (< 0.05, PEDF +.