Figure 1.
Chemical structure of subditine (1) angustoline (2), angustidine (3), angustine (4), nauclefine (5) isolated from the bark of Nauclea subdita.
The structure of new compound, subditine (1) were elucidated using various spectroscopic method which were 1D-NMR (1H, 13C, DEPT), 2D-NMR (HSQC, HMBC, NOESY), UV, IR and LCMS while the structure of the other four known compounds were confirmed through the comparison of NMR data with literature values.
Table 1.
1H-NMR (400 MHz) and 13C-NMR (100 MHz) Spectral Data of Subditine (1) and Angustidine* (3) in CDCl3 and DMSO-d6 respectively.
Figure 2.
1H-1H COSY and HMBC correlations of subditine (1).
Table 2.
Subditine (1) angustoline (2), angustidine (3), angustine (4), nauclefine (5) and standard drug paclitaxel screening on LNCaP and PC-3 human prostate cancer and RWPE human normal prostate epithelial cell-lines using MTT assays.
Figure 3.
Subditine (1) induced cytoskeletal rearrangement at the peripheral.
LNCaP and PC-3 cells were treated with subditine (1) at various concentrations for 24 hours. Cells were fixed and stained with Hoechst (blue) and phalloidin (red) dye which stained nucleus and polymerized actin (F-actin), respectively. Bar chart showing average fluorescent intensity of phalloidin (mean ± S.D.; *p<0.05). Dose-dependent increased of phalloidin intensity in LNCaP cells were observed after subditine treatment.
Figure 4.
Subditine (1) treatment leads to nuclear DNA fragmentation.
LNCaP and PC-3 cells were treated with subditine (1) (12.5 µM and 25 µM) for 24 h. Cells were then fixed and stained with Hoescht 33342 (blue). Red circles indicate DNA shrinkage or fragmentation. Images were captured using Cellomic arrayscan system.
Figure 5.
Subditine (1) enhanced ROS production in LNCaP and PC-3.
LNCaP and PC-3 cells were treated with subditine (1) (12.5 µM, 25 µM, 50 µM) for 24 h. Cells were then fixed and stained with DHE dye. ROS levels were indirectly determined by measuring DHE dye incorporation in the nuclear using Cellomic HCS arrayscan. Increased DHE dye intensity in the nucleus was detected upon treatment of subditine. Hoechst (blue) and DHE dye (green). Bar chart showing the average fluorescent intensity of DHE stain (mean ± S.D.; *p<0.05).
Figure 6.
Subditine (1) induced glutathione reductase (GR) gene expression.
LNCaP and PC-3 cells were treated with subditine (1) (12.5 µM) for 18 h. (A) Human oxidative stress and antioxidant defence qPCR-array was used to identify genes significantly up- or down-regulated in subditine (1)-treated LNCaP or PC-3 cells. Gene profiling analyses were performed three times in independent experiments. (Arrow indicates location of GR in the scatter plots) (B) Transcriptional changes of GR were evaluated using quantitative real-time-PCR. Levels of GR mRNA were normalized using β-actin housekeeping gene and expressed as fold change in comparison to untreated control.
Figure 7.
Dose-dependent effect of subditine (1) on cell membrane permeability, MMP and cytochrome c release.
LNCaP and PC-3 cells were treated with subditine (1) for 24 h. Cells were then fixed and stained with membrane permeability dye, MMP, cytochrome c and Hoechst as described in Materials and Methods. (A) Stained cells were visualized using HSC arrayscan system to check nuclear morphology, membrane permeability, MMP integrity, cytochrome c release; Blue (nuclear), Green (Membrane permeability), Red (MMP), Cyan (cytochrome c release). (B) Bar chart showing the average fluorescent intensities of membrane permeability, MMP and cytochrome c (mean ± S.D.; *p<0.05).
Figure 8.
Subditine (1) induced caspase 9, 3/7 activation in LNCaP and PC-3 cells.
LNCaP and PC-3 cells were treated with subditine (12.5 µM) and caspase 8, 9, 3/7 activities were determined using bio-illuminescent assays at the indicated time point. Subditine (1) induced caspase 9, 3/7 activation in both LNCaP and PC-3 cells. No significant fold-change was detected in caspase 8 activity throughout 30 hours.
Figure 9.
Western blotting analyses of apoptosis-associated molecules after subditine (1) treatment.
LNCaP and PC-3 cells were treated with paclitaxel (positive control) or various concentrations of subditine (1) for 24 hours. Cells were lysed, subjected to SDS-PAGE and Western blotting. (A) Membranes were probed with Bcl-2, Bcl-xL and p53 antibodies. Protein loading was assessed with antibody to β-actin. Normalization for loading differences was done by dividing the densitometry values for individual bands with β-actin in the same lane (n.d.-not determined). (B) Bar charts showing densitometry quantification of Bcl-2, Bcl-xL and p53 expression in subditine (1)-treated cells relative to control (mean ± S.D.; *p<0.05).
Figure 10.
Schematic diagram of possible mechanism of subditine (1)-induced apoptosis in LNCaP and PC-3 cells.
Subditine (1) treatment leads to increase ROS production, which induced GR expression. Excessive ROS disrupts the MMP, promotes cytochrome c release from mitochondria and activates downstream caspase 9, 3/7. We also showed that subditine (1) modulates the expression of apoptotic regulatory proteins (Bcl-2, Bcl-xL, p53 (*in LNCaP)) involved in the complex intrinsic apoptosis signal cascades. Apoptosis is evident through increased cell permeability and DNA fragmentation. Overall, subditine (1) mediated cell death through ROS-induced signal-transduction cascades and activation of a set of caspases.