CLS Formation Requires Activation of Apoptotic Pathways Mediated by Mitochondrial Cytochrome c Release and Caspase3 Activation

There are two major mechanisms mediating apoptosis: intrinsic and extrinsic. Cell surface Fas-receptor signaling upon FasL binding triggers caspase-8 activity and caspase cascade (Schulze-Osthoff et al. 1998). Involvement of this pathway in CLS formation by melanoma cells was ruled out in our studies, since antagonistic anti-Fas antibody did not block the in vitro tube-like organization process and CLS formation (Figure 3A). Fas and FasL expression on melanoma cells was also analyzed by flow cytometry and neither Fas nor FasL was seen during CLS formation (data not shown). These findings suggest that extrinsic apoptotic pathways did not participate in CLS formation.

Alternatively, under the stress conditions, mitochondrial cytochrome c is released into the cytosol. On release, cytochrome c interacts with Apaf-1 and pro-caspase-9 to form apoptosomes activating downstream effector caspase-3 responsible for

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Figure 3. Pathways of capillary-like structure (CLS) formation. (A) Fas-FasL croslinkage does not modify CLS formation. (A1) Cells treated with anti-Fas blocking antibody; (A2) Control, non-treated cells. (B) Flow cytometry analysis of melanoma cells stained with PI. (B1) Control: cells were detached from plastic 10 h after seeding; (B2) cells were detached from Matrigel 10 h after seeding; (B3) cells were incubated with 10 |M RT for 24 h, detached from plastic, seeded on Matrigel, then detached from Matrigel and analyzed. (C) CLS formation requires activation of apoptotic pathways mediated by mitochondrial cytochrome c release and caspase-3 activation. (Ca) Time-dependent cytochrome c release into cytosol. Lane 1, cells grown on plastic for 10 h, lanes 2-5, cells grown on Matrigel for 2, 4, 6, and 8 h. (Cb) Active caspase-3 in cytosol. Lane 1, cells grown on plastic; lanes 2-5, cells grown on Matrigel for 2, 4, 8, and 10 h . (Cc) Effect of antioxidants on active caspase-3. Lane 1, active caspase-3 in control cells; lane 2, active caspase-3 in cells pre-incubated with 10 | M RT for 24 h.

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Figure 3. Pathways of capillary-like structure (CLS) formation. (A) Fas-FasL croslinkage does not modify CLS formation. (A1) Cells treated with anti-Fas blocking antibody; (A2) Control, non-treated cells. (B) Flow cytometry analysis of melanoma cells stained with PI. (B1) Control: cells were detached from plastic 10 h after seeding; (B2) cells were detached from Matrigel 10 h after seeding; (B3) cells were incubated with 10 |M RT for 24 h, detached from plastic, seeded on Matrigel, then detached from Matrigel and analyzed. (C) CLS formation requires activation of apoptotic pathways mediated by mitochondrial cytochrome c release and caspase-3 activation. (Ca) Time-dependent cytochrome c release into cytosol. Lane 1, cells grown on plastic for 10 h, lanes 2-5, cells grown on Matrigel for 2, 4, 6, and 8 h. (Cb) Active caspase-3 in cytosol. Lane 1, cells grown on plastic; lanes 2-5, cells grown on Matrigel for 2, 4, 8, and 10 h . (Cc) Effect of antioxidants on active caspase-3. Lane 1, active caspase-3 in control cells; lane 2, active caspase-3 in cells pre-incubated with 10 | M RT for 24 h.

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