We therefore examined the effect of rPEDF on the proliferation of endocrine sensitive MCF 7 and endocrine resistant MCF 7,5C breast cancer cells. As shown in Figure 6a, rPEDF significantly reduced the growth of resistant MCF 7,5C cells but had no effect on parental MCF 7 cells. The add to favorites growth inhibitory effect of rPEDF was concentration dependent, with maxi mum inhibition observed Inhibitors,Modulators,Libraries at 100 nM, and this inhi bitory effect of rPEDF was completely blocked by the addition of antibodies specific to PEDF, thus confirming that the effect of PEDF was specific. To determine whether the anti proliferative effect of rPEDF on MCF 7,5C cells was due to apoptosis, we next performed a TUNEL assay. Figure 6b showed Inhibitors,Modulators,Libraries that rPEDF markedly increased apoptosis in MCF 7,5C cells, with 41.

8% of cells Inhibitors,Modulators,Libraries being TUNEL positive, compared with the untreated cells that showed very few TUNEL positive cells. Because rPEDF treatment caused endocrine resistant MCF 7,5C cells to undergo apoptosis, we also examined whether knockdown of PEDF expression in MCF 7 cells would cause them to undergo apoptosis. Inhibitors,Modulators,Libraries We found that PEDF knockdown in MCF 7 cells did not inhibit Inhibitors,Modulators,Libraries the growth of these cells or cause them to undergo apoptosis in the presence of rPEDF, thus confirm ing that the ability of rPEDF to induce apoptosis is specific for MCF 7,5C cells. Since rPEDF was shown to effectively inhibit the growth of endocrine resistant MCF 7,5C breast cancer cells in vitro, we next evaluated the effect of rPEDF on MCF 7,5C tumor growth in vivo. Endocrine resistant MCF 7,5C breast cancer cells were injected subcutaneously into the mammary fat pads of ovariectomized nude mice.

When palpable tumors were established, the animals were randomized into two groups and then treated with either rPEDF or PBS vehicle control that was administered every 2 days by intraperitoneal injection. We found that rPEDF reduced the growth Tofacitinib Citrate of MCF 7,5C tumors at all of the time points examined. The average tumor area was reduced from 0. 42 cm2 in the PBS treated group to 0. 12 cm2 in the rPEDF treated group. The differences between the two groups were statistically significant, as calculated by repeated measures analysis of variance. We next determined whether the anti tumor activity of rPEDF in vivo was due, in part, to its ability to inhibit angiogenesis. For this purpose, MCF 7,5C xenografts were excised at the end of the experiment and were sectioned and analyzed by immunohisto chemistry using antibody to CD34, a well known marker for newly formed blood vessels angiogenesis. As shown in Figure 6d, tumors from mice treated with PBS showed intense staining for CD34, indicating the presence of extensive angiogenesis in the tumors, whereas micro vessel density in tumors from mice treated with rPEDF was markedly lower.