B. Kolund zˇ ija et al. / Bioorg. Med. Chem. Lett. 24 (2014) 65–71
69
not show significant accumulation in sub-G1 phase of the cell cycle
on HeLa cells.
The activities of caspase-3 and -8, in 5h, 5m, 5n, and 5q-treated
HeLa cells were detected by specific caspase inhibitors using flow
cytometry (Fig. 5). Bearing in mind the absence of DNA fragmenta-
tion and the fact that these compounds do not lead to significant
accumulation of sub-G1 phase of the cell cycle, one could conclude
that the cell death induced by these compounds should be inde-
pendent of caspase-3. Unexpectedly, the analysis revealed the
presence of active caspase-3 in HeLa cells treated with 5h, 5n,
and 5q while 5m showed that inhibitors of caspase-3, had almost
no effect on the activity of this compound. Thus, only specific cas-
pase-8 inhibitor significantly suppressed the caspase activity and
we can conclude that 5m shows activity mainly via mitochondrial
pathway through activation of caspase-8 in HeLa cells. In general,
these caspase activities, coupled with the absence of DNA fragmen-
tation, may indicate a possible inhibition of the endonuclease
2
6–29
activity DFF40/CAD by these compounds.
Further studies are
needed for more detailed understanding of the relationship be-
tween DFF40/CAD endonuclease and caspase-3 activity.
Figure 4. DNA fragmentation detection by 2% agarose gel electrophoresis. C1 and
C2 represent control samples while 5h, 5m, 5n and 5q lanes represent treatment
with IC50 of respective compounds.
Blocking angiogenesis could be a strategy to arrest tumor
growth and anti-angiogenic drug development has attracted a lot
of research interest. Without blood vessels, tumors cannot grow
beyond a critical size or metastasize to another organ.30 The tube
formation activity of EA.hy926s is important in vitro endpoint for
angiogenesis. Examined compounds 5h, 5m, 5n, and 5q inhibited
tubulogenesis as it is shown in Figure 6A where untreated EA.-
hy926s formed elongated tube-like structures. The treatment with
IC20 sub-toxic dose of investigated compounds resulted in a signif-
icant decrease in capillary tube formation and this effect was very
strong for 5h. The IC20 values of these compounds on tube forma-
pounds on cell cycle distribution of malignant cells is shown in
Table S1 (Supplementary material). There were no significant
changes of sub-G1 populations on all tested cell lines after 24 h
treatment with IC50 dose of investigated compounds. The percent-
age change in sub-G1 in A549 cells treated with 5q for 24 h was
marked with increase to 15.29%. In addition, any relevant change
in the basal level in G1, S and G2-M population was not observed
in the investigated cells after 24 h of treatment.
Moreover, the A549 and LS174 cells showed significant changes
in G2/M arrest (29.31% and 68.89%, respectively) after 24 h of
treatment with 5h (see Fig. 2 and Table S1). The G2/M arrest is
associated with evident reduction in S phase.
Figure 3 shows the results of fluorescence microscopy of HeLa
cells coupled with acridine orange/ethidium bromide (AO/EB) dou-
ble staining for the occurrence of morphological changes and DNA
condensation after treatment with 5h, 5m, 5n and 5q. HeLa cells
treated with IC50 and 2IC50 concentrations of these compounds
exhibited nuclear shrinkage and chromatin condensation,
compared to the untreated control cells.
tion inhibition of EA.hy926 ranged from 4.9 to 5.5 lM (sub-toxic
effect of these compounds is illustrated in Figure 6B. Our observa-
tions were consistent with previously cited reports that many nat-
ural and synthetic chalcones possess anti-angiogenic activity.
Next, we examined the effect of the investigated compounds on
the activity of matrix metalloproteinases. HeLa cells were treated
with sub-cytotoxic concentrations (IC20) of 5h, 5m, 5n and 5q for
2
4 h in serum-free RPMI-1640. The supernatant was collected
and tested for matrix metalloproteinase activity by gelatin zymog-
raphy. As it is shown in Figure 7, activity of MMP-s was signifi-
cantly reduced compared to the controls C1 and C2. Our results
showed that the investigated compounds inhibited the activity of
these enzymes. MMP-2 has been strongly implicated in angiogen-
esis and promotion of the tumor metastatic potential playing cru-
cial role not only in invasion, but also in determination of cancer
The presence of a ladder DNA fragmentation pattern typical for
apoptotic cells was evaluated after 24 h of exposure of HeLa cells to
IC50 of 5h, 5m, 5n, and 5q. However, alterations in nuclear mor-
2
5
phology may not necessarily involve DNA fragmentation. Despite
the significant cytotoxicity of compounds observed towards HeLa
cells, no DNA ladder formation was detected upon their treatment
3
1–
cell transformation, growth, apoptosis and signal transduction.
3
3
The inhibitory effects confirmed by 5h, 5m, 5n and 5q against
MMP-2 secretion could be a starting point for further development
(Fig. 4). In this regard, it should be noted that 5h, 5m, 5n, and 5q do
Figure 5. Effects of 5h, 5m, 5n and 5q on caspase-3 and -8 inhibitors. Data are shown as mean ± SD. HeLa cells were exposed to IC90 concentrations of investigated
compounds for 24 h in the presence of specific caspase inhibitors (final concentration—40 M) as described.
l