Fig 1.
Structures of the active nucleoside analogues (A), their respective IC50 values compared to camptothecin, in the Caco-2 and HT-29 cell lines (B), and (C) the leucocyte survival bargraph. (B) The bars represent the mean ± SEM of the IC50 values from four MTT assays carried out for each test nucleoside. The HT-29 and Caco-2 cells were exposed to the nucleoside analogues at concentrations ranging from 1 μM to 120 μM. Normal peripheral leukocytes (C) were exposed to 100 μM of nucleoside 1, 2, 5 and camptothecin for 24 hrs.
Fig 2.
Active nucleosides induce caspase 3 and caspase 8 activity in both HT-29 (A) and in Caco-2 (B) cells after various exposure periods to 100 μM camptothecin (a), nucleoside 1 (b), nucleoside 2 (c), and nucleoside 5 (d) Data points on the graphs represent the mean of the triplicate values of caspase activity after cells were exposed to the nucleoside analogues for the indicated time periods.
Table 1.
Summary of the mitochondrial membrane potential change induced by the nucleoside analogues and camptothecin in the HT-29 and Caco-2 colon cancer cell lines.
Fig 3.
Nucleosides 1, 2 and 5 cause the redistribution of cytochrome c from mitochondria to the cytosol in HT-29 (A) and Caco-2 (B).
The cells were exposed to 100 μM of nucleoside 1, 2, 5 and camptothecin, respectively, for 24 hours. The bars represent the mean ± SEM from three Elisa assays carried out for each nucleoside.
Table 2.
Percentage of HT-29 and Caco-2 cells in early and late apoptosis as determined by the annexin-V assay.
Fig 4.
In HT-29 cells, nucleoside 1 and 2 induce nuclear fragmentation, while nucleoside 5 causes a loss of normal nuclear structure and cytoplasmic vacuolisation.
Cells were exposed to 50 μM of nucleoside 1, 2 and 5 for 24 hours and stained with Hoechst 33342 stain. Scalebar: 20 μm.
Fig 5.
In Caco-2 cells, nucleoside 1 and 2 cause perinuclear vacuole formation, while nucleoside 5 causes a loss of normal nuclear structure, nuclear budding and cytoplasmic vacuolisation.
Cells were exposed to 50 μM of nucleoside 1, 2 and 5 for 24 hours and stained with Hoechst 33342 stain. Scalebar: 20 μm.
Fig 6.
Nucleoside-induced morphological changes in HT-29 cells include rapid perinuclear vacuole formation (nucleoside 1 and 2) and a loss of cellular adherence (nucleoside 5).
Cells were exposed to 50 μM of the nucleoside for the indicated time periods. Experiments were repeated three times. Scale bar: 20 μm.
Fig 7.
Nucleoside-induced morphological changes in Caco-2 cells include rapid perinuclear vacuole formation (nucleoside 1 and 2) and a loss of cellular adherence (nucleoside 5).
Cells were exposed to 50 μM of each nucleoside for the indicated times and the experiments were repeated three times. Scalebar: 20 μm.
Fig 8.
Nucleoside 2 treated vacuolated HT-29 and Caco-2 cells stained with acridine orange shows an absence of lysosomal staining (A) and a nucleoside 2 induced HT-29 multi-lobular structure (B).
Acridine orange stained HT-29 and Caco-2 cells, following exposure to 50 μM of nucleoside 2 (A). HT-29 cells were exposed to 50 μM of nucleoside 1 and the supernatant harvested and centrifuged at 500 x g for 5 mins. The multi-lobular cell was stained with Hoechst 33342 and acridine orange (B). Scalebar: 20 μm.
Fig 9.
Nucleoside 5 causes aggregation of actin in HT-29 cells, but not in Caco-2 cells.
Cells were treated with 50 μM of nucleoside 5 for 6 hours. Scalebar: 20μm.
Fig 10.
Vacuoles observed under phase contrast microscopy do not co-localize with MDC stained fluorescent granules.
HT-29 cells were exposed to 50 μM of nucleoside 2 for 3 hours before MDC staining.
Fig 11.
Nucleoside 2 and chloroquine cause the formation of large, highly fluorescent autophagic vacuoles in Caco-2 cells.
Cells were exposed to nucleoside 1, 2 and 5 at a concentration of 50 μM for three hours. Chloroquine (50 μM) was used as a positive control and camptothecin (20 μM) was included as a vacuole negative control. Arrows indicate autophagic vacuoles. Scalebars: 20 μm.
Fig 12.
Nucleoside 2 and chloroquine cause the formation of large highly fluorescent autophagic vacuoles in HT-29 cells.
Cells were exposed to nucleoside 1, 2 and 5 at a concentration of 50 μM for three hours. Chloroquine (50 μM) was used as a positive control at and camptothecin (20 μM) was included as a negative control. Arrows indicate autophagic vacuoles. Scalebars: 20 μm.