Acetylmelodorinol isolated from Sphaerocoryne affinis seeds inhibits cell proliferation and activates apoptosis on HeLa cells

Abstract

Background: Cervical cancer is a major global health concern with a high prevalence in low- and middle-income countries. Natural products, particularly plant-derived compounds, have shown immense potential for developing anticancer drugs. In this study, we aimed to investigate the anticancer properties of the pericarp and seeds of Sphaerocoryne affinis fruit on human cervical carcinoma cells (HeLa) and isolate the bioactive compound from the active fraction.

Methods: We prepared solvent fractions from the ethanol extracts of the pericarp and the seed portion by partitioning and assessing their cytotoxicity on HeLa cells. Subsequently, we collected acetylmelodorinol (AM), an anticancer compound, from the ethyl acetate fraction of seeds and determined its structure using nuclear magnetic resonance. We employed cytotoxicity assay, western blotting, Annexin V apoptosis assay, measurement of intracellular reactive oxygen species (ROS) levels, 4',6-diamidino-2-phenylindole (DAPI) staining, and a terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay, to evaluate the anticancer properties of AM on HeLa.

Results: The solvent fractions from the seed displayed considerably higher cytotoxic activity against HeLa cells than those of the pericarp. We isolated and identified acetylmelodorinol as an anticancer compound from the ethyl acetate fraction from S. affinis seed extract. Treatment with acetylmelodorinol inhibited HeLa cell proliferation with an IC₅₀ value of 2.62 ± 0.57 µg/mL. Furthermore, this study demonstrated that acetylmelodorinol treatment disrupted cell cycle progression by reducing the expression of cyclin E, CDK1/2, and AKT/mTOR pathways, increasing the intracellular ROS levels, reducing BCL-2/BCL-XL expression, causing DNA fragmentation and nuclear shrinkage, and triggering apoptosis through caspase 3 and 9 activation in a dose-and time-dependent manner.

Conclusion: In contrast to previous reports, this study focuses on the inhibitory effects of AM on the AKT/mTOR pathway, leading to a reduction in cell proliferation in cervical cancer cells. Our findings highlight the promising potential of acetylmelodorinol as an effective treatment for cervical cancer. Additionally, this study establishes a foundation for investigating the molecular mechanisms underlying AM's properties, fostering further exploration into plant-based cancer therapies.

Keywords: Acetylmelodorinol; Anti-proliferation; Anticancer; Cervical cancer; HeLa cells; Sphaerocoryne affinis.

Fig. 1

Cytotoxicity of solvent fractions from SAP and SAS extracts. HeLa cell viability after 48-h treatment with solvent fractions of SAP (a) and SAS (b) extracts were measured and expressed as percent inhibition of cell viability of treatment groups compared with that of the non-treatment control group. Significant cytotoxic effects were observed exclusively in the solvent fractions of SAS. Solvent fractions: circle, EA; square, Hex; triangle, water (Wat). X axis, the logarithm of the concentration (µg/mL) to the base 10. Data are represented as mean ± SD; n = 3

Fig. 2

Isolation and identification of AM from SAS-EA. a Viability of Hela cells cultured with fractions E2 to E8 at concentrations of 50, 25, and 12.5 µg/mL for 48 h. E4 exhibited the strongest inhibitory effect on HeLa cell proliferation. AM was isolated from E4. b Chemical structure of AM was identified using ESI-TOF–MS. Data are represented as mean ± SD; n = 3

Fig. 3

Cytotoxicity assessment of AM in HeLa cells. a The dose-dependent cytotoxicity of AM was assayed at concentrations ranging from 0.39 to 100 µg/mL against HeLa and OUMS-36 cells and expressed as percent inhibition of cell viability of treatment groups with AM compared with that of the non-treatment control group. X axis, the logarithm of the AM concentration (µg/mL) to the base 10. n = 3. Error bar, mean ± SD. b The time-dependent cytotoxicity on HeLa cells of AM at IC₅₀ (square, 2.6 µg/mL) and IC₉₀ (triangle, 6.25 µg/mL) concentrations for various times (0, 4, 8, 12, 24, 36, 48, and 60 h). Non-treatment group was used as a control (circle). Data are expressed as relative cell viability at each time to that at time 0. c Representative images of the clonogenic assay. HeLa cells were treated with AM at IC₅₀ (2.6 µg/mL) and IC₉₀ (6.25 µg/mL) concentrations for 36 h and then cultured in a compound-free medium for 10 d. Subsequently, the cells were stained with crystal violet and observed under a microscope. d Numbers of colonies containing over 50 cells formed by the 10-d culture in the clonogenic forming assay in panel (c). Data are represented as mean ± SD and statistically analyzed by one-way analysis of variance (ANOVA); Statistically significant differences were observed between the treated groups and the non-treated group; ‡, p < 0.0001; n = 3

Fig. 4

Effects of AM on cell proliferation in HeLa cells. a Western blot analysis displaying the reduced expression of cyclin E and CDK1/2 and the complete inhibition of expression of total-AKT, phospho-AKT, and phospho-mTOR upon treatment with IC₅₀ (2.6 µg/mL) and IC₉₀ (6.25 µg/mL) concentrations of AM for 12 h. The representative images are cropped from larger membranes. Original images of blots are shown in Fig. S7. b Relative band intensities of cyclin E and CDK1/2 in cells treated with AM to those of non-treated cells support the decrease in their expression upon AM treatment. Data are represented as mean ± SD and statistically analyzed by one-way ANOVA. Statistically significant differences were observed between the treated groups and the non-treated group; *, p < 0.05; #, p < 0.01; n = 3

Fig. 5

Analysis of pro-apoptotic effects of AM on HeLa cells. a Representative plots obtained by flow cytometry using Annexin A5 Apoptosis Detection kit were used to quantify cell viability and apoptosis following treatment with AM at IC₅₀ and IC₉₀ concentrations for 12 h. b Proportion of viable cells, early and late apoptotic cells, and dead cells analyzed using flow cytometry. c Representative TUNEL assay images captured at a magnification of 20 × , where brown cells indicate positive DNA fragmentation. d Fluorescent images of DAPI staining observed at 60 × magnification indicate nuclear fragmentation. e Flow cytometry using the DCFH-DA probe indicated the distribution of cell groups treated with AM at IC₅₀ and IC₉₀ concentrations compared with the non-treated cells, reflecting intracellular ROS levels in HeLa cells. f The average intensity of DCFH-DA supported the observed trend of increased ROS levels with AM treatment. g Western blot images of caspase 3, caspase 9, and pro-apoptotic (BAX, BAK) and anti-apoptotic markers (BCL2, BCL-XL) in HeLa cells treated with IC₅₀ and IC₉₀ doses of AM for 12 h. The representative images are cropped from larger membranes. Original images of blots are shown in Fig. S9. h Relative expression levels of pro-apoptotic (BAX, BAK) and anti-apoptotic markers (BCL2, BCL-XL) of AM-treated group to that of the non-treated group, calculated from the intensities of bands obtained by western blotting. i Ratio of expression levels of pro-apoptotic to those of anti-apoptotic proteins (BAX/BCL2 and BAK/BCL-XL). IC₅₀, 2.6 µg/mL; IC₉₀, 6.25 µg/mL. Data are represented as mean ± SD and statistically analyzed by one-way ANOVA. Statistically significant differences were observed between the treated and the non-treated groups; *, p < 0.05; #, p < 0.01; †, p < 0.001; n = 3. Scale bars indicate 50 μm