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. 2015 Aug 19;10(8):e0135814.
doi: 10.1371/journal.pone.0135814. eCollection 2015.

Moringa oleifera as an Anti-Cancer Agent against Breast and Colorectal Cancer Cell Lines

Abdulrahman Khazim Al-Asmari  1 , Sulaiman Mansour Albalawi  1 , Md Tanwir Athar  1 , Abdul Quaiyoom Khan  1 , Hamoud Al-Shahrani  1 , Mozaffarul Islam  1
Affiliations

Moringa oleifera as an Anti-Cancer Agent against Breast and Colorectal Cancer Cell Lines

Abdulrahman Khazim Al-Asmari et al. PLoS One. .

Abstract

In this study we investigated the anti-cancer effect of Moringa oleifera leaves, bark and seed extracts. When tested against MDA-MB-231 and HCT-8 cancer cell lines, the extracts of leaves and bark showed remarkable anti-cancer properties while surprisingly, seed extracts exhibited hardly any such properties. Cell survival was significantly low in both cells lines when treated with leaves and bark extracts. Furthermore, a striking reduction (about 70-90%) in colony formation as well as cell motility was observed upon treatment with leaves and bark. Additionally, apoptosis assay performed on these treated breast and colorectal cancer lines showed a remarkable increase in the number of apoptotic cells; with a 7 fold increase in MD-MB-231 to an increase of several fold in colorectal cancer cell lines. However, no significant apoptotic cells were detected upon seeds extract treatment. Moreover, the cell cycle distribution showed a G2/M enrichment (about 2-3 fold) indicating that these extracts effectively arrest the cell progression at the G2/M phase. The GC-MS analyses of these extracts revealed numerous known anti-cancer compounds, namely eugenol, isopropyl isothiocynate, D-allose, and hexadeconoic acid ethyl ester, all of which possess long chain hydrocarbons, sugar moiety and an aromatic ring. This suggests that the anti-cancer properties of Moringa oleifera could be attributed to the bioactive compounds present in the extracts from this plant. This is a novel study because no report has yet been cited on the effectiveness of Moringa extracts obtained in the locally grown environment as an anti-cancer agent against breast and colorectal cancers. Our study is the first of its kind to evaluate the anti-malignant properties of Moringa not only in leaves but also in bark. These findings suggest that both the leaf and bark extracts of Moringa collected from the Saudi Arabian region possess anti-cancer activity that can be used to develop new drugs for treatment of breast and colorectal cancers.

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Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Fig 1
Fig 1. (A) GC-MS analyses of MO leaves.
Typical TIC-GC/MS chromatogram of ethanolic extract of Moringa leaf analyzed on GC system (Agilent 7890A series, USA) equipped with an apolar 5-MS (5% phenyl polymethyl siloxane) capillary column (Agilent 19091S-43; 30 m×0.25 mm i.d. and 0.25-μm film thickness) attached with Mass Detector. After comparing with the spectra in the NIST Library, It showed the presence of twelve compounds. To facilitate the interpretation, only the peaks showing bioactive anticancer compounds such as isopropyl thiocynate, D-allose, and hexadeconoic acid are marked. (B) Chemical structures of the active compounds as mentioned above are shown here.
Fig 2
Fig 2. GC-MS analyses of MO bark.
(A) Typical TIC-GC/MS chromatogram of Moringa bark analyzed on GC system equipped with an apolar 5-MS capillary column attached with Mass Detector. It showed the presence of seventeen compounds. The peaks showing bioactive anticancer compounds in bark such as eugenol and hexadeconoic acid are marked. (B) Chemical structures of the active compounds as mentioned above are shown here.
Fig 3
Fig 3. Cell survival assay.
(A) MDA-MB-231 (B) HCT-8 cell lines. Cells were treated with different concentrations of plant extracts; (L) leaves; (B) bark and (S) seed. Equal numbers of cells in 20 μl were taken in 380 μl of cell counting solution. Cell viability was analyzed on Muse cell analyzer. Column (B & D) representing the quantitative analysis of the viable cells. A significant number of dead cells were observed upon extract treatment as compared with control. * Statistically significant (P≤0.05).
Fig 4
Fig 4. Cell motility assays.
(A) MDA-MB-231 and (C) HCT-8 cell lines. Treatment patterns and annotation of the extracts were the same as descried in fig 3. A significant decrease in cell motility was observed upon extracts treatment. Columns of the bar graphs (B&D) showing 80–95% decrease in cell motility. * Statistically significant (P≤0.05).
Fig 5
Fig 5. Colony formation assay.
Anchorage dependent colony formation assay shows a significant reduction in colony formation in (A) MDA-MB-231 and (C) HCT-8 cell lines treated with different concentration of plant extracts. Quantitative analyses are given in the form of bar graphs for MDA-MB-231(B) and HCT-8 (D) respectively. A dramatic decrease in colony formation is evident from the figure. Statistically significant values were marked with asterisk. (P≤0.05).
Fig 6
Fig 6. Apoptosis assay.
Assessment of apoptosis in (A) MDA-MB-231 and (C) HCT-8 cell lines treated with the extracts of MO leaves (L), bark (B) and seed (S) for 24 h. The cells cultured either in DMEM or in RPMI media were used as control. The apoptotic rates were detected by annexin V-PI dual staining. Q1 quadrant (annexin V−, PI+) represented dead cells; Q2 quadrant (annexin V+, PI+) represented late apoptotic cells; Q4 quadrant (annexin V+, PI–) represented early apoptotic cells; Q3 quadrant (Annexin V−, PI−) represented live cells. The percentage of total apoptotic cells (Q2+Q4) was calculated and shown in the bar graphs MDA-MB-231(B) and HCT-8 (D) cell lines respectively. The extent of apoptosis was significantly high both in leaves and bark treated cell lines. However, few apoptotic cells were also observed only in HCT-8 when treated with the seed extract. * Statistically significant (P≤0.05).
Fig 7
Fig 7. Cell cycle assay.
Analysis of cell cycle arrest in plant extract treated (A) MDA-MB-231 and (C) HCT-8 cell lines. Cells were treated with 500μg/ml extracts of leaves (L) Bark (B) and seeds(S). Statistical analyses of the findings were shown in the form of bar graphs for MDA-MB-231(B) and HCT-8 (D) cell lines respectively. A significant G2/M enrichment was observed in leaves (L) and bark (B) treated cell lines. * Statistically significant (P≤0.05).
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Grants and funding

This work was funded by Prince Sultan Military Medical City. The funder had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.