Figure 5.

Paracrine-acting WNT16B promotes the resistance of prostate carcinoma to cytotoxic chemotherapy. (a) Viability of prostate cancer cells 3 d after treatment with a half-maximal inhibitory concentration (IC₅₀) of MIT and medium conditioned by fibroblasts with (PSC27^WNT16B) or without (PSC27^C) WNT16B. (b) Bright field microscopic view of PC3 cells cultured with control or PSC27^WNT16B-conditioned medium photographed 24 h after exposure to vehicle or the IC₅₀ of MIT. Arrowheads denote apoptotic cell bodies. Scale bars, 50 µm. (c) Acute tumor cell responses to chemotherapy in vitro. Quantification of apoptosis by assays reflecting combined caspase 3 and 7 activity measured 24 h after the exposure of PC3 cells to vehicle or the IC₅₀ of MIT. Data in a and c are mean ± s.e.m. of triplicate experiments, and P values were determined by ANOVA followed by t test. RLU, relative luciferase unit. (d) In vivo responses of PC3 tumors to MIT chemotherapy. Grafts were comprised of PC3 cells alone or PC3 cells combined with either PSC27 prostate fibroblasts expressing a control vector (PC3+PSC27^C) or PSC27 prostate fibroblasts expressing WNT16B (PC3+PSC27^WNT16B). MIT was administered every 2 weeks for three cycles, and grafts were harvested and tumor volumes determined 1 week after the final MIT treatment. Each data point represents an individual xenograft. Horizontal lines are group means of ten tumors, with P values determined by ANOVA followed by t test. (e) Acute tumor cell responses to chemotherapy in vivo. Quantification of apoptosis by cleaved caspase 3 (C-caspase 3) IHC and of DNA damage by γ-H2AX immunofluorescence in PC3 and fibroblast xenografts measured 24 h after in vivo treatment with vehicle (C) or MIT. Values represent a minimum of 100 cells counted from each of 3–5 tumors per group. Data are mean ± s.e.m., and P values were determined by ANOVA followed by t test.