Figure 1.
The anticancer activity-guided purification and identification of a C-20 oxygenated ent-kaurane from R.serra.
(A), R. serra extracts and their subfractions inhibited NPC cell proliferation. Leaf ethanolic extract (LEE), stem ethanolic extract (SEE), leaf petroleum ether layer (LPEL), stem petroleum ether layer (SPEL), leaf ethyl acetate layer (LEAL), stem ethyl acetate layer (SEAL), leaf butanol layer (LBL), stem butanol layer (SBL), leaf water layer (LWL), stem water layer (SWL). After 72 hr treatment, cell viability was determined by the MTT assay. NPC cells treated with the vehicle control (DMSO) were used as the reference group with cell viability set at 100%. (B), S3 was analyzed by HPLC at 254 nm. F3 possessed the most effectively inhibitory effects on NPC cells. After 72 hr treatment, cell viability was determined by the MTT assay. NPC cells treated with the vehicle control (DMSO) were used as the reference group. (C), Compound 1 was analyzed by HPLC at 254 nm. (D), The structure of compound 1 was shown. The figures are representative of three experiments. The data are presented as mean ± S.D. of three separate experiments. * P<0.05, the significant differences between treatment and control groups.
Table 1.
1H and 13C NMR data of lasiodin in CD3OD (δ in ppm).
Figure 2.
Lasiodin inhibited cell proliferation and migration.
(A), Lasiodin inhibited NPC cell proliferation. The cells treated with the vehicle control (DMSO) were used as the reference group with cell viability set at 100%. (B), The cells were treated with lasiodin at the indicated does for 24 hr. The cellular morphology was observed by the phase contrast microscopy. NPC cells treated with the vehicle control (DMSO) were used as the reference group. (C), The colony formation efficiency was detected by the plate clone formation assay. After incubation for two weeks, the cells were stained with crystal violet staining solution. NPC cells treated with the vehicle control (DMSO) were used as the reference group. (D), Lasiodin mediated the NPC cell migration inhibition. Cell migration was analyzed by the scratch assay. CNE1 and CNE2 were grown to the full confluency. The cell monolayers were wounded with a sterile pipette tip, and washed with the medium to remove the detached cells from the plates. NPC cells were left either untreated or treated with the indicated doses of lasiodin. After 24 hr, the wound gap was observed and photographed. NPC cells treated with the vehicle control (DMSO) were used as the reference group. The figures are representative of three experiments. The data are presented as mean ± S.D. of three separate experiments. * P<0.05, the significant differences between treatment and control groups.
Figure 3.
Activation of the caspase-dependent apoptotic pathway by lasiodin.
(A), After treatment with lasiodin for 24 hr, both the adherent and floating cells were collected and stained with Annexin V-FITC and propidium iodide (PI). The apoptotic ratio was calculated in terms of the FITC-positive values of the cells. The apoptotic ratio was represented by the relative percentages of the FITC-positive cells versus the DMSO-treated cells (vehicle control). (B), CNE1 and CNE2 cells were treated with lasiodin at the doses of 0.8, 3.1, and 6.3 µM. After 24 hr treatment, the expressions of caspase-3, caspase-9, PARP, Bax, and Bcl-2 were determined, and the cleavage products of caspase-9, caspase-3, and PARP were detected by Western blotting. NPC cells treated with the vehicle control (DMSO) were used as the reference group. GAPDH was used as the control for sample loading. (C), CNE1 and CNE2 cells were treated with lasiodin at 6.3 µM. After 24 hr treatment, the releases of Cyto-C and Apaf-1 were determined by immunofluorescence imaging analysis to monitor the Cyto-C and Apaf-1 releases from inter-mitochondrial space into cytosol. NPC cells treated with the vehicle control (DMSO) were used as the reference group. The figures are representative of three experiments. The data are presented as mean ± S.D. of three separate experiments. * P<0.05, the significant differences between treatment and control groups.
Figure 4.
Inhibition of PI3K/AKT signaling by lasiodin.
(A), CNE1 and CNE2 cells were treated with lasiodin at the indicated doses. After 24 hr treatment, the expressions of the phosphorylated or total protein of AKT and PI3K were detected by Western blotting. NPC cells treated with the vehicle control (DMSO) were used as the reference group. GAPDH was used as the control for sample loading. (B), CNE1 and CNE2 cells were treated with the AKT-selective inhibitor (LY294002, 5 µM) for 4 hr, and then treated with lasiodin at the indicated doses. After 48 hr treatment, cell viability was determined by the MTT assay. The figures are representative of three experiments. The data are presented as mean ± S.D. of three separate experiments. * P<0.05, the significant differences between treatment groups without the inhibitor and control groups without the inhibitor. # P<0.05, the significant differences between treatment groups with the inhibitor and control groups with the inhibitor.
Figure 5.
Supression of MAPK signaling by lasiodin.
(A), CNE1 and CNE2 cells were treated with lasiodin at the indicated doses. After 24 hr treatment, the expressions of the phosphorylated or total protein of ERK1/2, JNK and p38 were detected by Western blotting. NPC cells treated with the vehicle control (DMSO) were used as the reference group. GAPDH was used as the control for sample loading. (B), CNE1 and CNE2 cells were treated with the ERK-selective inhibitor (U0126, 20 µM), JNK inhibitor (SB203580, 600 nM) and p38 inhibitor (SP600125, 2.5 µM) for 4 hr, respectively, and then treated with lasiodin at the indicated doses. After 48 hr treatment, cell viability was determined by the MTT assay. The figures are representative of three experiments. The data are presented as mean ± S.D. of three separate experiments. * P<0.05, the significant differences between treatment groups without the inhibitor and control groups without the inhibitor. # P<0.05, the significant differences between treatment groups with the inhibitor and control groups with the inhibitor.
Figure 6.
Suppression of COX-2 expression and NF-κB binding by lasiodin.
(A), CNE1 and CNE2 cells were treated with lasiodin at the indicated doses. After 24 hr treatment, the expressions of COX-2, IKK and NF-κB were detected by Western blotting. NPC cells treated with the vehicle control (DMSO) were used as the reference group. GAPDH was used as the control for sample loading. (B), CNE1 and CNE2 cells were treated with the COX-2-selective inhibitor (celecoxib, 20 µM) for 4 hr, and then treated with lasiodin at the indicated doses. After 48 hr treatment, cell viability was determined by the MTT assay. (C), CNE1 and CNE2 cells were treated with lasiodin at 6.3 µM for 24 hr. The binding of the transactivators to the COX-2 promoter was analyzed by the streptavidin-agrose pulldown assay. NPC cells treated with the vehicle control (DMSO) were used as the reference group. (D), CNE1 and CNE2 cells were treated with lasiodin at the dose of 6.3 µM for 24 hr. The nuclear extracts were prepared, and NF-κB was detected by Western blotting. NPC cells treated with the vehicle control (DMSO) were used as the reference group. (E), CNE1 and CNE2 cells were treated with lasiodin at 6.3 µM for 24 hr. The NF-κB nuclear translocations in CNE1 and CNE2 cells were determined by immunofluorescence imaging analysis. NPC cells treated with the vehicle control (DMSO) were used as the reference group. (F), The proposed mechanisms were shown. The figures are representative of three experiments. The data are presented as mean ± S.D. of three separate experiments. * P<0.05, the significant differences between treatment groups without the inhibitor and control groups without the inhibitor. # P<0.05, the significant differences between treatment groups with the inhibitor and control groups with the inhibitor.