Fig 1.
Chemical structure of F8 (methyl 5-[(dimethylamino)carbonyl]-4-methyl-2-[(3-phenyl-2-propynoyl)amino]-3-thiophenecarboxylate).
Table 1.
Cytotoxicity values of F8.
Fig 2.
F8 compound-induced apoptosis in CEM cells. F8 ability to induce apoptosis was assessed in a phosphatidylserine externalization assay and measured through flow cytometry. Analysis was performed following a 24 h incubation with 24 h CC50 and 2X CC50 (2.89 μM and 5.78 μM). Controls included DMSO as the vehicle, H2O2 as the positive, and untreated. Significant phosphatidylserine externalization was evident for the CC50 and 2X CC50, given the p-value p<0.00001 (***).
Fig 3.
Depolarization of the mitochondria.
Significant mitochondrial depolarization was induced by compound F8 on CEM cells following a 4 h incubation treated with the CC50 and 2X CC50 (2.89 μM and 5.78 μM). The cells were stained with JC-1 reagent and analyzed by flow cytometry. Statistical analyses were obtained through the two-tailed Students paired t-test. The controls that were included are the same as previously stated.
Fig 4.
Significant ROS was induced by F8 CC50 and 2X CC50 (2.89 μM and 5.78 μM) in CCRF-CEM cells compared to the vehicle control, DMSO, following an 18 h incubation period. Statistical analyses were acquired using a two-tailed t-test, p = 0.0005 and p = 0.003.
Fig 5.
F8 induces caspase activation.
Compound F8 CC50 and 2X CC50 (2.89 μM and 5.78 μM) induced significant caspase 3/7 activation in CEM cells. The cells were incubated for 8 h and stained with NucView 488 caspase-3/7 substrate. Statistical analyses were obtained using the two-tailed Student’s paired t-test compared to the vehicle control (1% DMSO).
Fig 6.
F8 induced DNA fragmentation on CEM cells, a hallmark of apoptosis.
F8 cytotoxicity was analyzed via flow cytometer after a 72 h exposure on CEM cells utilizing CC25 and CC10 (1.44 μM and 0.57 μM). NIM-DAPI was used to stain each cell’s DNA amount and quantified in the flow. F8 displays DNA fragmentation evident in the Sub G0-G1 phase. Controls were included, DMSO as the vehicle and H2O2 as the positive control, and untreated cells, respectively.
Fig 7.
F8 causes hypophosphorylation in JAK/STAT pathway.
Human Phosphorylation Kinase Array C55 was conducted on the CCRF-CEM cell line and treated with 2X CC50 (5.78 μM). This assay was used to determine the phosphorylation status of MAPK and JAK-STAT membranes to gauge phosphorylation. Fold changes were determined using densitometry. Hyperphosphorylation was observed in the MAPK membrane versus the JAK-STAT. Hypophosphorylation was displayed in the JAK 1–2 and STAT 2–5.