Cefepime br Table br Computed ApoC values
Computed ApoC50 values for 1 and CDDP according results of Annexin V/PI double staining method after 24 h incubation.
Compound ApoC50 [μM]
a n.d. - not determined as no activity was observed in the concentration range 1–100 μM.
cell cycle distribution of MCF-7 and AsPC-1 cells due to the treatment with 1 indicate that both cell lines experienced distress during DNA duplication, while their phenotypic particularities most probably were accountable for initiation different check points, thus leading to diverse distribution of cells during mitosis. However, if present results indicate that 1 induces Cefepime shifts different than for CDDP, thus determi-nation whether 1 has the ability to establish a strong interaction with DNA or is involved in a mechanism involved in the regulation of the DNA replication phase remains to be elucidated.
3.4.3. Compared to CDDP, apoptosis induced by 1 reveals as lower caspase-dependence with possible interruption in activation of extrinsic pathway To determine the role of caspases activation in process of apoptotic death due to 1, impact of Z-VAD-fmk pan-caspase inhibitor on initiation and evolution of cell death was monitored, as was the activation of
either caspase-8 or -9 after 6 h incubation of 1 at ApoC50 concentration. As represented in Fig. 5A, apoptotic death shows low caspase-de-pendency in both cell lines (17 ± 2% and 25 ± 1% for MCF-7 and AsPC-1 cells, respectively). While co-incubation with Z-VAD-fmk has almost equivalent consequence on initiation of apoptotic death (28 ± 7% and 22 ± 3% in MCF-7 and AsPC-1 cells, respectively), evolution of apoptosis in MCF-7 samples was nearly irrespectively due to caspases' activity (11 ± 1%) contrary to AsPC-1 cells (32 ± 11%). According to those data, it is obvious that apoptosis induced by 1 is partially caspase-dependent in both treated cell lines . Ad-ditionally, pan-caspase inhibition yielded significant augmentation of necrotic events (41 ± 8% and 67 ± 2% in MCF-7 and AsPC-1 cells, respectively), which is an expected consequence of interrupted apop-totic cascades .
Moreover, when treated with 1, both cell lines display strikingly suppressed activation of caspase-8 (Fig. 5B) and not for caspase-9. While for MCF-7 percentages of cells with single activated caspase-9 or both activated caspases were at the level of non-treated controls, modestly stimulated activation of caspase-9, accompanied by sig-nificant decrease in cross-talk activation of extrinsic pathway, was the prominent finding for AsPC-1 cells. Such noticeable decline in active form of caspase-8, even below basal levels in both cell lines, connotes a possibility that 1 may directly interfere with the process of caspase-8 activation.
Considering that the cell cycle changes in both cell lines treated with 1 indicate a disturbed DNA replication process (Fig. 4), possible role of caspase-2 remains to be investigated during further studies .
Fig. 4. Distribution of cells within phases of mitotic division afterwards 24 h incubation with 1 and CDDP. Results are represented as the mean ± SD percentages of two replicates from independent experiments.
Fig. 5. Caspase-dependency of cell death determined over percentages of cell death inhibition after co-incubation with pan-caspase inhibitor with investigated compounds applied at their ApoC50 concentration (A). Analyses are done after 6 h treatment by means of Annexin V/PI double staining method. Results are expressed as the mean ± SD of two replicates from independent experiments. Percentages of positive for activated caspase-8, caspase-9 or both caspases determined after 6 h incubation with investigated compounds applied at their ApoC50 concentrations (B). Results are represented as the mean ± SD of two replicates from two in-dependent experiments.
S. Bjelogrlić et al.