Figure 6

Bortezomib (BTZ) inhibits ovarian cancer cell growth in mice. (a) Mouse ovarian surface epithelial cancer cells that constitutively expressed luciferase (MOSEC/LUC) were treated with BTZ at different concentrations (from 0.01 to 10 μM) for 48 h. Cell viability was measured with the MTT assay. (b) The effects of a 24-h exposure to BTZ (0.1 and 0.5 μM) were analyzed in MOSEC/LUC cells by immunoblotting; specifically, the levels of HSP70, phosphorylated STAT1, and cleaved-caspase-3 (c and d) were determined. The dose-dependent effects of JAKi I (AG490) on BTZ -induced cytotoxicity in MOSEC/LUC were measured by immunoblotting to determine the levels of cleaved-caspase-3. (e) Ten million MOSEC/LUC cells were intraperitoneally injected into C57BL/6 mice. Subsequently, the mice were intraperitoneally injected with 100 μl of HBSS (vehicle alone), 20 μg/ml of BTZ (for each mouse), 100 μl of JAKi (AG490; concentration: 350 μM), or both BTZ and AG490 in combination three times a week. At 10 min after the intraperitoneal injection of luciferin, the mice were imaged using the IVIS 200 in vivo imaging system (f). Data are expressed as mean±standard error of the mean. The results are representative of at least three independent experiments. (g) Immunohistochemistry for phosphorylated STAT1 and cleaved-caspase-3 (brown color) in tumor sections obtained using the HBSS control (left), BTZ alone (middle), or the combination of BTZ with AG490 (right). Cell nuclei were counterstained with hematoxylin. Scale bars represent 20 μm