(D) Cell viability of T24 cells transfected with control shRNA or shRNA and treated with a combination of TRAIL (2 ng/mL) and Andro (8 M) for 24-h. Therefore, bioinformatics analysis suggested that relatively high expression might represent an effective therapeutic TRAIL-related target in bladder cancer cells. However, MTS assays revealed that this 50% inhibitory concentration (IC50) value of TRAIL was 38.35 ng/mL, indicating that low concentrations of TRAIL would be ineffective in T24 cells (Determine ?(Physique1C).1C). This suggested the necessity to identify appropriate TRAIL-specific sensitizers capable of overcoming TRAIL resistance in bladder cancer cells. Moreover, Andro represents a potential agonist for TRAIL therapy, with MTS assays revealing an IC50 value for Andro of 101.5 M in T24 cells (Determine ?(Figure11E). Open in a separate window Physique 1 Potential TRAIL-receptor mRNA expression in bladder cancer patients and the antitumor effects of TRAIL and Andro in T24 cells. (A) Log2-converted mRNA expression levels from the Oncomine database. (B) GSEA results showing that high expression was positively correlated with apoptosis-gene signatures. (C) T24 cells were treated with various concentrations of TRAIL for 24-h. (D) Two- and three-dimensional chemical representation of Andro derived from the PubChem Compound Database (https://pubchem.ncbi.nlm.nih.gov/). Red, grey, and light-blue nodes represent oxygen atoms, carbon LF3 atoms, and hydrogen atoms, respectively. (E) T24 cells were treated with various concentrations of Andro for 24-h. The p-value and IC50 values were calculated using GraphPad Prism software. Data represent the mean SD. *P < 0.05; **P < 0.01; ***P < 0.001 (= 3). Andro synergistically enhances TRAIL-induced inhibition of proliferation, colony formation and migration in T24 bladder cancer cells Both cell-counting and MTS assays suggested that single treatment with either TRAIL or Andro inhibited cell-proliferation rates. Interestingly, we found that combination treatment with TRAIL and Andro substantially enhanced this inhibitory effect on cell proliferation (Physique ?(Physique2A2A and B). Additionally, morphological changes in TRAIL and/or Andro-treated cells confirmed the inhibition of T24-cell proliferation associated with combined treatment versus single treatment (Physique ?(Figure2C).2C). Moreover, colony formation dramatically decreased following combined treatment relative to that observed following treatment with Andro or TRAIL alone (Physique ?(Figure22D). Open in a separate window Physique 2 TRAIL combined with Andro further inhibits T24-cell proliferation, migration, and colony formation. (A, B) Effects of TRAIL and/or Andro LF3 treatment around the T24 growth curve. Verification by cell-counting and MTS assays. (C) Images (200) show T24 cells following treatment with TRAIL or/and Andro for 72-h. (D) Effects of TRAIL and Andro treatment around the colony formation of BLCA cell lines. T24 cells were treated with DMSO (control), TRAIL (2 ng/mL), or Andro (8 M) alone or both TRAIL (2 ng/mL) and Andro (8 M) and incubated for 12 days. Cell colonies (>50 cells) were counted using an inverted microscope (100). (E) Effects of TRAIL and Andro treatment on T24-cell migration. T24 cells were treated with DMSO, TRAIL (2 ng/mL), and/or Andro (5 M) for 18 h. Images (100) show T24-cell migration after treatment. (F) Left panel: the protein levels of CD147. Right panel: MMP-9 in T24 cells treated with different concentrations of TRAIL (2 ng/ml) and/or Andro [4uM (+) or 8 uM (++)] for 18-h and measured by western blot. Data Mouse monoclonal to RBP4 represent the mean SD. *P < 0.05; **P < 0.01; ***P < 0.001 (= 3). Given that cancer cells exhibit potent migratory features, we conducted wound-healing assays as functional readings. The results indicated that treatment with TRAIL or Andro alone modestly decreased the ratio of migrating bladder cancer cells. In the TRAIL-treated group, the cell-migration ratio was 65.37 2.47%, whereas that in the Andro-treated group was 79.65 1.82%. However, combined treatment resulted in a migration ratio of 32.16 1.59% (Figure ?(Figure2E).2E). Evidence shows that matrix metalloproteinases (MMPs) play important functions in tumor progression, invasion, and metastasis 18. Therefore, we evaluated protein levels of CD147 LF3 and MMP-9 by immunoblot, revealing that CD147 and MMP-9 were downregulated after a 24-h incubation with both TRAIL and Andro relative to levels observed following single treatment with TRAIL or Andro alone (Physique ?(Figure2F).2F). These findings exhibited that combination treatment with TRAIL and Andro potently suppressed T24-cell growth and migration. Andro enhances TRAIL-induced apoptosis by initiating caspase activation in BLCA cells The canonical pathway associated with TRAIL-induced cell death involves binding to specific death receptors (DR4 or DR5) to initiate activation of extrinsic apoptosis 6, 7. MTS assays suggested that in the combination-treatment groups, cell viability was.