A285 Aims: CCI-779 (Wyeth-Ayerst, PA, USA) is an ester derivative of the macrolide rapamycin with improved pharmacokinetic properties. Similarly to its parental compound, CCI-779 is a potent immunosoppressant, but also a potential antitumor drug, proved to act against a broad range of human cancer cells in vitro and in tumor xenograft models. By inhibiting the serine/threonine mTOR kinase, that links mitogen stimulation to protein synthesis and cell cycle progression, CCI-779 potently suppresses tumor cell growth by arresting cells in G1 phase, and by potentially inducing apoptosis. Indeed, the response to rapamycins is different among cell lines, being only cytostatic or also cytotoxic, and several mechanisms for the intrinsic resistance have been documented. On the other hand, there are few data on the effector mechanisms responsible for induction of apoptosis in sensitive cells, and no data, in our knoledge, on apoptotic response of melanoma cells to CCI-779. In the present study, in a panel of four human metastatic melanoma cell lines we evaluated the cytostatic/cytotoxic response in vitro to CCI-779, the involvement of the mitochondrial pathway of apoptosis, and the constitutive differential expression of several key components of the apoptotic machinery, possibly explaining the variable cell sensitivity. Methods: SK-Mel-5, SK-Mel-28, HT-144, and Me665/2/21 melanoma cell lines were tested with increasing doses of CCI-779 (1 pM to 10 μM) for 6 days, and IC 50 for cell proliferation was determined by MTT assay. At the higher dose of 20 μM applied for 18 hr, we evaluated i) cell detachment, by counting separately floating and adhering cells, ii) caspase-3/-7 and caspase-9 enzymatic activity, as measured by the cleavage of specific synthetic fluorescent substrates, iii) the expression status of several positive and negative modulators of apoptosis, by SDS-PAGE followed by Western blot. Results: The melanoma cell lines showed a very different response to CCI-779, with IC 50 for cell growth ranging from 8.9 μM (for SK-Mel-5 cells) to 21 nM (for Me665/2/21 cells). The concentration of 20 μM proved to be apoptogenic only for HT-144 and Me665/2/21 cell lines showing the lower IC50 a significant cell detachment from the monolayer was associated with a remarkable increase over control cells of the downstream caspase-3/-7 activity and, to a lesser extent, of caspase-9 activity, both in still adhering cells and even more in detached cells. Concomitantly to the caspases activation, a significant loss of Apaf-1, the scaffold protein of the multimeric complex apoptosome formed along the mitochondrial pathway of apoptosis, was observed. No significant correlation was found among the varying sensitivity to CCI-779 and the constitutive expression status of caspase-3, caspase-9, Apaf-1, Bcl-2 and Bcl-XL. Conclusions: In those melanoma cell lines sensitive to CCI-779, the mitochondrial pathway of apoptosis is put into motion, along with the peculiar, but not yet clear, event of Apaf-1 loss. This preliminary study will be examined closely in order i) to clarify the role of mitochondria (in particular of Apaf-1) in such apoptotic machinery and ii) to demonstrate novel mechanisms of resistance to apoptosis, in the attempt to convert the general cytostatic response into a significant apoptotic death of melanoma cells.
Read full abstract