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Figure 3 | Molecular Cancer

Figure 3

From: Molecular mechanism implicated in Pemetrexed-induced apoptosis in human melanoma cells

Figure 3

MTA induced caspase-dependent apoptosis. (A) Kinetics of caspase cascade activation. Induction of apoptosis in melanoma cells by MTA was accompanied by activation of caspases-2, -3, -8, -9 and −10. Hs294T cells were treated with 0.84 μM MTA for the indicated time points. Total cell lysates were obtained according to the manufacturer’s instructions. Assays were performed in triplicate in 96-well plates. Caspase activity results are represented as a fold change of the control, comparing obtained results (ORmta) with the activity obtained for MTA-untreated cells (ORcontrol) by computing ORmta/ORcontrol. Caspase-2 and −10 activities were enhanced after only 24 h, pointing to their role as inititor caspases. The activity of caspase-3 and −9 also increased significantly after 24 h MTA exposure, confirming their executioner role in this process. After 48 h MTA treatment, caspase-8 activity had also significantly increased, in addition to further increases in caspase-2 and −3 activities. Data represent mean ± SD of three determinations from three separate experiments. (B) Hs294T cells were pretreated with or without caspase inhibitors for 1 h and then challenged with 0.84 μM MTA for 72 h. Cell viability was assessed using the XTT assay. Blockade with the pancaspase inhibitor (z-VAD-FMK) inhibited significantly but not totally the MTA effect. The most effective caspase inhibitors were those which inhibited caspase-2 (z-VDVAD-FMK), caspase-3 (z-DEVD-FMK), caspase-9 (z-LEHD-FMK) and caspase-10 (z-AEVD-FMK) which confirmed the role of these caspases in mediating the effects due to MTA. The caspase-8 inhibitor (z-IETD-FMK) did not significantly reverse the MTA effect. These results are representative of three independent experiments. Similar results were obtained for all melanoma cell lines.

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