Fig 1.
Decreased cell viability in cancer cells treated with TM5275 and TM5441.
A. The structures and chemical names of TM5275 and TM5441 are shown. B. The indicated cell lines were treated with increasing concentrations of TM5275 (black) or TM5441 (red). Viability was measured by relative luminescence units for increasing concentrations of TM5275 and TM5441 and were graphed as % survival (± SD) in comparison to DMSO control treated cells. Data was plotted and a best fit line was drawn, n = 3.
Fig 2.
Decreased proliferation in cancer cells treated with TM5275 and TM5441.
Representative fluorescence-activated cell sorting (FACS) plots are shown in the upper panels with BrdU positive cells (purple) shown within the upper gate. The mean percentage (± SD) of HT1080 and HCT116 BrdU positive cells after treatment with DMSO (white bar), TM5275 (black bar) or TM5441 (red bar) was graphed, n = 3. * indicates P values compared to DMSO controls < 0.05, ** indicates P < 0.01, and *** indicates P < 0.001.
Fig 3.
Increased apoptosis in cancer cells treated with TM5275 and TM5441.
A. Caspase 3/7 activity of HT1080 and HCT116 cells treated with increasing concentrations of TM5275 (black) or TM5441 (red) after 48 hours was plotted as the average fold change (± SD) compared to DMSO-treated cells, n = 3. B. Representative FACS plots of HT1080 and HCT116 cells treated with DMSO, TM5275 (50 μM) and TM5441 (50 μM) are shown in the upper panels with annexin V-FITC and propidium iodide staining. The average percentage of cells in early (Annexin V positive, PI negative) and late (Annexin V and PI positive) apoptosis (± SD) was graphed for the indicated concentrations of TM5275 (black bars) or TM5441 (red bars) in each cell line, n = 3. C. Average plasmin activity (black bars, ± SD) and average caspase 3/7 activity (grey bars, ± SD) was plotted as a fold change for 50 μM TM5275-treated cells compared to control DMSO-treated cells for each time point, n = 2. * indicates P values compared to DMSO controls < 0.05, *** indicates P < 0.001, and **** indicates P < 0.0001.
Fig 4.
Treatment with TM5275 or TM5441 increases intrinsic apoptosis.
A. Representative immunoblots for HT1080 and HCT116 cells treated with 25 μM or 50 μM TM5275 or TM5441 as indicated for caspase 8, 9, 3, PARP, and their cleavage products, n = 3. The numbers over the cleaved protein bands indicate the ratio of the cleaved protein pixel density to the corresponding tubulin control band pixel density. B. Representative JC-1 FACS plots of cells treated for 48 hours with 50 μM TM5275 or TM5441. The graphs show the average % (± SD) of JC-1 membrane depolarization in control DMSO-treated and TM inhibitor-treated cells n = 3. * indicates P values compared to DMSO controls < 0.05, ** indicates P < 0.01, *** indicates P < 0.001, and **** indicates P < 0.0001.
Fig 5.
Pre-clinical activity of TM5441 in vivo.
A. Individual tumor volume was plotted over time (days) for vehicle and TM5441 (20 mg/kg/day) treated mice (n = 5 per group). B. Average time (±SD) to reach maximal tumor growth for vehicle and TM5441-treated mice was plotted over time (days). C. Average tumor relative luciferase units (RLUs) (±SD) for mice treated with vehicle or TM5441 was plotted over time (days). D. Mouse survival (%) was plotted for mice treated with vehicle (black line) or TM5441 (red line) over time (days). E. Representative histology sections of tumors stained with H&E. Yellow arrows indicate the area selected for the inset panels. Blue arrows indicate hemorrhagic areas with red blood cells. Green arrows indicate EC lining the vessels. Scale bar = 50 μm. F. Representative histological sections of tumors stained for CD31 (red) as described in Materials and Methods and counterstained with nuclear 4’6’-diamidino-2-phenylindole (DAPI) (blue) staining. Yellow arrows indicate the area selected for the inset panels. White arrows indicate CD31 positive cells lining the vessel wall. Clear arrows with a white outline indicate the absence of distinct CD31 positive cells. The graph shows the percentage of CD31 positive staining (± SD, n = 5 tumors per group with 5 sections per tumor, n = 25 total per group). Scale bar = 50 μm. G. Representative histology sections of tumors stained with TUNEL as described in Materials and Methods. TUNEL staining (brown) counterstained with methyl green (green). Yellow arrows indicate the area selected for the inset panels. Red arrows indicate TUNEL positive cells. The graph shows the average TUNEL optical density (± SD, n = 5 tumors per group with 5 sections per tumor, n = 25 total per group). Scale bar = 50 μm. * indicates P values compared to vehicle control < 0.05, and *** indicates P < 0.001.
Fig 6.
TM5441 inhibits EC branching morphogenesis.
A. Representative photographs of HUVEC cultured in Matrigel with the indicated concentrations of TM5441. The graph represents the average number of HUVEC branch points (± SD) at the indicated TM5441 concentrations, n = 3. B. The graph represents the average % survival (± SD) in HUVEC at the indicated concentrations of TM5441, n = 3. C. The graph represents the average fold change of caspase 3/7 activity (± SD) compared to DMSO controls for the indicated concentrations of TM5441, n = 3. * indicates P values compared to DMSO control wells < 0.05.