Figure 1.
TMI-1 induces dose-dependent reduction of cell viability.
A: Three different ADAM-17 inhibitors were used in the study. Chemical structures of TMI-1, TMI-005 and TMI-2 are represented. Interaction between hydroxamate moiety and enzymatic catalytic Zn-binding site is shown. LogD was calculated using the mean value obtained from three different prediction algorithms: ChemDraw, Mollinspi and AlogP. B: Cell viability was assessed on SUM149 cells at day 5. All compounds were diluted in DMSO at indicated concentrations. Cell viability assay was done with the Alamar blue staining kit. The “input” bar corresponds to the fluorescence level at Day 0. Experiments were done in triplicate for each concentration tested. Mean values +/− s.e.m is represented. These results are representative of three experiments.
Table 1.
TMI-1 inhibits cell viability and induces caspase-3/7 activity in different tumoral mammary cell lines.
Figure 2.
TMI-1 induces tumor cell death by apoptosis and cell cycle arrest.
A: TMI-1 induces cell cycle arrest in the G0/G1 phase. SUM149 cells were treated for 48 h with TMI-1 or DMSO. Cells were stained, after BrdU incorporation, with anti BrdU-FITC antibody and 7-AAD and analyzed with the BD Facscalibur flow cytometer. B: TMI-1 induces dose-dependent apoptosis. Cell apoptosis was assessed by annexin V/7-AAD double staining after 48 h of treatment with 2.5 to 20 µM TMI-1, TMI-2 or TMI-005. Only TMI-1 induces apoptosis in SUM149 cells. C: TMI-1, not TMI-2 and TMI-005, induces a dose-dependent increase of caspase-3/7 activity after 24 h. General caspase inhibitor Z-VAD was included as positive control. D: TMI-1 induces caspase-dependent apoptosis. SUM149 cell viability was measured using the Alamar blue staining kit after 5 days of treatment with increasing doses of TMI-1, in the presence or absence of 20 µM Z-VAD. E: TMI-1 induces caspase-dependent apoptosis. Annexin V/7-AAD staining is inhibited in the presence of 20 µM Z-VAD. ANOVA, P<0.0005,*** Bonferroni's Multiple Comparison test. F: Apoptosis quantification in the non tumoral breast cell line MCF-10A treated by increasing doses of TMI-1. Treatment does not enhance annexin V/7-AAD staining. These results are reproduced four times. G: Caspase-3/7 activity in MCF-10A. Increasing doses of TMI-1 do not activate caspase-3/7 activity. Mean values +/− s.e.m. are represented in C, D, E, F. Experiments were done at least three times.
Figure 3.
TMI-1-induced apoptosis requires mainly extrinsic death pathway.
A: TMI-1 induces a dose–dependent activation of caspase-8 and caspase-9. SUM149 cells were treated for 48 h with 2.5 to 20 µM TMI-1. Treatment of SUM149 cells in the presence of Z-VAD (20 µM) was used as positive control. Caspase-8 and caspase-9 activities were assessed using pro-fluorescence LETD and LEHD tetrapeptide sequences, respectively. Results are presented as percent positive cells. B: TMI-1-induced apoptosis is caspase-8 dependent. SUM149 cells were treated with TMI-1 (20 µM), in the presence or absence of the specific caspase-8 inhibitor Z-IETD (50 µM). This experiment was measured by annexin V test and results are presented as percent annexin V positive cells. ANOVA, P<0.0005,*** Bonferroni's Multiple Comparison test. C: SUM149 cells expressing the FADD-DN construct protein are resistant to TMI-1-induced apoptosis. SUM149 cells were transfected with pB-FADD-DN wild type (FADD-DN) or pB-FADD-DN-muted (FADD-DNm) followed by treatment for 72 h with TMI-1 (1.25–20 µM). Apoptosis was determined by percentage of positive annexin V cell. Results shown are representative of 3 independent experiments; bars represent the mean +/− s.e.m.. Transfection efficiency determined by the use of pMax-GFP construct was >65%. D: TMI-1 induces slight intracellular accumulation of ROS. SUM149 cells were treated with TMI-1 (2.5–20 µM) for 48 h or CCCP (30 µM), used as positive control. Results were presented as mean of fluorescence intensity of hydroethidine oxydation. ANOVA, P<0.0005,*** Bonferroni's Multiple Comparison test.
Figure 4.
Anti-tumoral effect of TMI-1 in vivo.
A: Effect of TMI-1 on the TgNeu27 cell line derived from a MMTV-ErbB2/neu tumor. ErbB2/neu expression by immunofluorescence (red staining) and western-blot analyzes showed expression and tyrosine phosphorylation of the transgene (top). This cell line is highly sensitive to TMI-1 as seen by cell growth inhibition and caspase-3/7 activation (bottom)B: MMTV-ErbB2/neu transgenic mice were injected daily IP with TMI-1 (n = 4) or with the vehicle of TMI-1 (n = 3). C: Tumor-cumulated volumes were assessed by adding the volumes of the primary tumors to the volumes of the other tumors developed in the same mouse. The number of tumors developed in the same mouse within the observation time (30 days) is indicated on the graph. D: TUNEL assay on tumors derived from MMTV-ErbB2/neu mice treated with vehicle (left) or TMI-1 (right) for 30 days (magnification: X100 (top), X400 (bottom)).
Figure 5.
Association of TMI-1 with therapeutic drugs in vitro.
SUM149 cells were incubated for 5 days with serial dilutions of TMI-1 (0.58–9.32 µM) and Docetaxel (14.87×10−6–238×10−6 µM) (A), doxorubicin (0.088–1.41 µM) (B), and lapatinib (0.17–2.72 µM) (C) individually or in combination at constant molar ratio. Determination between antagonism, additivity or synergism was evaluated using median-effect principle proposed by the Chou-Talalay method [11]. The CalcuSyn software was used to determine the CI.
Figure 6.
TMI-1 targeting of cancer stem cells.
A: TMI-1 inhibits primary tumorspheres formation. SUM149 cells were treated with TMI-1 (2 µM) for 3 days. Cells were then cultured for 5 days to allow tumorsphere formation (see material and methods). Results are presented as the mean of tumorspheres counted. Bars represent the mean +/− s.e.m.. **P<0.005 as calculated using Mann-Whitney test. B: TMI-1 inhibits secondary tumorspheres formation. Dissociated primary tumorspheres were cultured for 5 days. Results are presented as the mean of tumorspheres counted. Bars represent the mean +/− s.e.m.. **P<0.005 as calculated using Mann-Whitney test. C: TMI-1 affects the pool of the ALDH-1 positive population. SUM149 cells were treated with TMI-1 (2 µM), TMI-005 (2 µM) or doxorubicin (0.352 µM) for 3 days. Cells were stained with Aldefluor kit and analyzed by flow cytometry. DEAB inhibitor was used to block aldehyde dehydrogenase activity. D: Overall results are represented as ratio between percent ALDH+ cells in treated conditions vs percent ALDH+ cells in DMSO. These results are representative of four experiments.
Table 2.
TMI-1 effects on cell viability and caspase-3/7 activation in 46 different cell types.