Figure 2. Secretable TRAIL produced by fibroblasts induces apoptosis of neighboring UMUC3 tumor cells.

A. Schematic representation of plasmid encoding green fluorescence protein (GFP) fused with sTRAIL or TRAIL (as a less secretable control). B. Cell proliferation of cancer cells, UMUC3 and fibroblasts, NIH3T3 transfected with TRAIL, sTRAIL, GFP at different concentrations (MTT assay, n = 4). UMUC3 cells are sensitive to both TRAIL and sTRAIL, while NIH3T3 are resistant. C. The releasing of sTRAIL protein into the supernatant was determined using ELISA (against TRAIL). Both NIH3T3 and MRC-5 (lung fibroblasts) transfected with sTRAIL secreted TRAIL protein. However, minimal soluble TRAIL was detected in both cell line when transfecting with TRAIL (n = 3, ** P < 0.01, compared to sTRAIL LPD treatment). D. The neighboring effect of the sTRAIL protein was determined by a non-contact co-culture study. After co-cultured for 24 h, the UMUC3 cells in the bottom were counted. Cells were visualized by Calcein AM staining (shown in green). Numbers in white calculated the % of total cells in each treatment compared to the total tumor cells in the GFP LPD group. Apoptosis of bottom UMUC3 cells were assessed using annexin V/PI staining and flow cytometry (n = 3).