The induction of apopto sis in Caco2 cells was further evident from the quantitative selleck Bosutinib APOPercentage apoptosis assay, which showed a clear tes tosterone HSA stimulated apoptotic response Inhibitors,Modulators,Libraries 12 and 24 h post treatment. Similar results were also obtained in HCT116 cells, while mAR deficient non transformed IEC06 intestinal cells did not responded to testosterone HSA treatment, as indi cated by the APOPercentage apoptosis assay. In line with these findings, testosterone HSA induced time dependent activation of caspase 3, indicating the participation of caspases as executors in mAR depend ent cell death. These effects were attributed to mAR activa tion and Inhibitors,Modulators,Libraries were independent of classical intracellular androgen receptors, since both the apoptotic response and the caspase 3 activation were not inhibited in the presence of the anti androgen flutamide.

In line with this, membrane bound iAR could not be detected in isolated Inhibitors,Modulators,Libraries membrane preparations of Caco2 cells by using specific iAR antibodies. In con trast, these membrane preparations were positive for the expression of Na K ATPase, a protein implicated in cellu lar ion homeostasis used as a positive membrane control in this experiment. To establish the functional role of actin reorganization in regulating the pro apoptotic responses induced by mAR, as previously reported for var ious cell systems, we assessed mAR dependent apoptosis and caspase 3 activation Inhibitors,Modulators,Libraries in the presence of anti actin drugs. As shown in Fig. 5A, B, in Caco2 cells pre treated with cytochalasin B, at a concentration which blocks actin redistribution without exerting toxic effects, the mAR induced apoptotic response and caspase 3 activation were abolished.

These results indicate that actin redistribution is a manda tory step for the apoptotic response of mAR stimulated colon cancer cells. We further evaluated the steroid Inhibitors,Modulators,Libraries hor mone specificity of the mAR induced apoptotic responses by using non conjugated testosterone and estradiol deriv atives. As shown in fig. 5A, free estradiol could not gener ate any apoptotic response, while free DHT clearly showed activity. Finally, considering the estimated KD of 2. 9 nM for mAR, we further performed titra tion experiments using a wide range of testosterone HSA and free DHT concentrations in the presence or absence of caspase inhibitor respectively.

These experiments indicate that even in the nM range the testosterone conjugate and DHT have very similar pro apoptotic effects. These effects were abolished in the presence of the caspase Ganetespib OSA inhibitor. mAR activation by testosterone HSA was followed by extensive reduction of tumor incidence in vivo The findings provided so far established that mAR activa tion results in colon cancer cell regression in vitro. Thus, we aimed to further evaluate the in vivo effects of albumin conjugated androgens in colon cancer animal models.