Figure 1.
Cacospongionolide and Scalaradial: structures and original sponges.
Chemical structures of cacospongionolide and scalaradial isolated from marine sponges Fasciospongia cavernosa and Cacospongia scalaris, respectively.
Figure 2.
Effect of SC and CSP on cell viability and morphology.
T47D (A), A431(B), HCT116(C) and HeLa (D) cells were incubated with SC (1, 10 and 100 µM) and CSP (1, 10 and 100 µM) for 24 h. Thereafter cell viability was determined by MTT assay as described in Materials and Methods. Data are expressed as mean ± S.E.M. of six separate experiments (***p<0.001 vs. untreated cells). T47D cells (E) were treated with SC (10 µg/ml), CSP (10 µg/ml) or Dauno (100 µM) for 24 h, visualised by phase-contrast microscopy and photographed. The untreated cells (Control) exhibited normal morphologic aspects, whereas the cells treated with CSP, SC and Dauno were suggestive of apoptosis. Data illustrated in (C) are from a single experiment and are representative of three separate experiments.
Figure 3.
Effect of SC and CSP on DNA fragmentation.
The T47D cells were treated with SC (10 µg/ml), CSP (10 µg/ml) or Dauno (100 µM) for 24 h, thereafter DNA fragmentation was analyzed by comet assay (A), agarose gel electrophoresis (B) and diphenylamine assay (C). Basal DNA damage from untreated cells, DNA damage from SC or CSP cells (A). Data are from a single experiment and representative of 50 randomly selected cells stained with ethidium bromide (EtBr). Data illustrated in (B) are from a single experiment and representative of three separate experiments, while data in (C) are expressed as mean ± S.E.M. of three separate experiments. ***p<0.001 vs. control (untreated cells).
Figure 4.
Effect of SC and CSP on apoptosis induction.
Detection of dead and dying cells by FACS (A). Cells treated overnight with SC, CSP and cisPt were stained with ΔΨm-sensitive dye DiOC6(3) and the vital dye propidium iodide (PI). The white portions of the columns refer to the DiOC6(3)low/PI+ population (dead) and the remaining part of the column corresponds to the DiOC6(3)low/PI- (dying) population. Results are means ± S.E.M. of three independent experiments. Expression profile of apoptosis-related proteins (B). Cell lysates from HeLa cells, treated with SC (10 µM) and CSP (10 µM) overnight, were analyzed by human apoptosis profiler. Data are expressed as means ± S.D. of two independent experiments.
Figure 5.
Effect of SC and CSP on cell viability.
FACS analysis shows the effect of SC (10 µg/ml) or CSP (10 µg/ml) on HCT116 cell viability in the presence or absence of Z-VAD-fmk (50 µM) and pifthrin-alpha (30 µM). Data are the mean ± SD of three experiments in triplicate.
Figure 6.
Effect of SC and CSP on p50 and p65 nuclear translocation.
Representative Western blot of p50 and p65 as well as the densitometric analysis shows p50 and p65 nuclear translocation in T47D cells incubated with CSP (10 µg/ml) or SC (10 µg/ml) for 24 h. The results are from a single experiment and are representative of three separate experiments. Densitometric data are expressed as mean ± S.E.M. of three separate experiments. ***p<0.001 vs. untreated cells. The expression of GAPDH is shown as an equal loading control.