TMI-1 induces tumor cell death by apoptosis and cell cycle arrest.

<p><b>A:</b> 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>B:</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. <b>C:</b> 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. <b>D:</b> 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. <b>E:</b> TMI-1 induces caspase-dependent apoptosis. Annexin V/7-AAD staining is inhibited in the presence of 20 µM Z-VAD. ANOVA, <i>P</i><0.0005,*** Bonferroni's Multiple Comparison test. <b>F:</b> 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. <b>G:</b> 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.