Reprinted from NCBI, the National Center for Biotechnology Information. Advancing science and health by providing access to biomedical and genomic information. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4545797/
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.
Moringa oleifera L (MO) (Family: Moringaceae) is a perennial angiosperm plants, which includes several other species. It is a native of the Himalayan region that is widely cultivated throughout tropical and sub-tropical countries of the world including Saudi Arabia. The plant has numerous medicinal applications and is used as a traditional medicine for the treatment of various illnesses such as skin diseases, respiratory distress, ear and dental infections, hypertension, diabetes, anemia, and cancer. Additionally, the pharmacological importance of the leaves extract containing bio-active compounds are well described by Leone et al (2015).
In this study we focused upon the effect of Moringa oleifera extracts from leaves (MOL), bark (MOB), and seeds (MOS) to observe its efficacy as an anti-cancer agent on breast and colorectal cancers. To elucidate the effectiveness of these extracts we analyzed cell motility and clonogenic survival assays to assess the phenotypic changes in MDA-MB-231(breast) and HCT-8 (colorectal) cancer cell lines. To elaborate our study further, we also analyzed the cell survival, apoptosis, and cell cycle progression of these two lines after challenging them with the extracts of MO as stated above. The rationale behind selecting these lines are; easy access to availability and more importantly, prevalence of these cancers in a major population of the Kingdom of Saudi Arabia.
Colorectal cancer is the third most lethal cancer worldwide. Both genders, male and female are equally affected by this deadly disease. In the past year about 140,000 people were diagnosed with colon cancer in the United States and the estimated survival is 50% or less. Furthermore, the Saudi Cancer Registry has reported a sharp increase in colorectal cancer in the kingdom. Similarly, breast cancer among women is also a deadly cancer worldwide. A study conducted between 2001–2008 reports a significant rise in breast cancer among young women in Saudi Arabia. Noticeably, the incidence is more prominent in the eastern province of the kingdom consequently the women in these areas are more vulnerable to this disease.
The important characteristic features of cancer cells include the ability to proliferate, invade through the extra cellular matrix and migrate to other body parts to form secondary tumors. The migration of cancerous cells is dependent on the tumor micro environment from where they get nourishment and support by forming new-vasculature (a process called angiogenesis) and allowing them to spread. It is a challenging task for Oncologists and Medical Scientists to devise the best treatment regimen that kills the maximum number of cancer cells with minimal side effect rendering maximum benefits to the cancer patients.
As reported earlier, about 74% of the known anti-cancer medicines are derived from various plant species. Indeed, there are many household dietary products exhibiting anti-cancer potential with minimal side effect that are currently under clinical trials for cancer treatment. Among these, two important household dietary products that are very common among South Asian communities are Curcumin and Lycopene. Curcumin is a polyphenolic compound isolated from turmeric and this product exhibits anti-microbial, immunomodulatory, and potential cancer chemo preventive efficacy.
Lycopene is a carotenoid compound abundant in tomatoes and also present in tomato products. As reported earlier lycopene and its derivative exhibit anti-cancer properties. In addition to this the product can also be used in the treatment of cardiovascular diseases. The anti-cancer properties of curcumin are well studied. The molecular mechanism, by which curcumin acts is by inhibiting MAP kinase activity, negatively interfering with JAK/STAT signaling pathways, and inhibiting the expression of several transcription factors including NF-Kβ and STAT. The expression of apoptotic proteins such as caspases and the anti-apoptotic protein Bcl-2 were also reported to be modulated by curcumin. On the other hand, the anti-cancer mechanism of lycopene acts by inhibiting the PI3K/AKT signaling pathways as well as inducing apoptosis in the cancerous cells.
In this study we have shown the remarkable effects of Moringa leaves and bark on MDA-MB-231 and HCT-8 cancer cell lines. Moringa is a common vegetable used by inhabitants of tropical and sub-tropical countries. The extracts of leaves and bark tested in our current study induced a significant level of apoptosis as well as G2/M enrichment in breast and colorectal cancer cell lines. In addition, a remarkable change in the normal phenotypic properties of the cells was also observed. However, surprisingly, we did not observe any significant changes in cell lines exposed to Moringa seed’s extract. Furthermore, when GC-MS analyses of these extracts were performed, we observed considerable amounts of bioactive components present in the extracts of leaves and bark, which we have describe elaborately in the results section. The effective role of these compounds present in the extracts as an anti-cancer agent is well documented. Therefore, the aim of this study was to evaluate the anti-cancer potential of Moringa oleifera grown in the kingdom of Saudi Arabia, against breast and colorectal cancers. The importance of this study is that this locally grown plant has not been previously tested as an anti-cancer agent. Hence, the novelty of our research is that we have tested not only leaf extracts, but also the bark and the seed extracts against two cancer cell lines, which has not been studied in the past.
Materials and Methods
Collection of plant materials
Leaves (L), bark (B), and seeds (S) of Moringa oleifera were collected from the city of Tabuk, Kingdom of Saudi Arabia. Geographically the latitude of the city is 28° 22`60N and longitude is 36° 34`60E on degree minutes second (DMS) unit. Due to the ease of cultivation and abundance, it does not fall under the endangered plant species and hence safe to use for research purpose. (http://faculty.ksu.edu.sa/74413/Pages/Endangeredplants.aspx). Additionally, the parts of the plant used in this study are the gift of one of the co-authors (SMA) of this manuscript obtained from his garden plants. The study was performed in the research center, Prince Sultan Military Medical City, Riyadh.
Preparation of the extracts
The extractions were performed using Soxhlet apparatus. About 60g of dried leaves, bark, and seeds were grinded separately into coarse powder. Each of the coarsely powdered specimens was taken in round bottom flask and 600 ml of ethanol was added. The extraction was continued for 6–8 h until all the soluble constituents dissolved in the solvent. The soluble extracts were filtered and evaporated in rotary evaporator (Buchi, Switzerland; temp: 50°C; pressure 175 mbar) to yield semi solid masses. Extracts thus obtained, were collected and stored at 4°C until further use. For this study 250mg of extracts were dissolved in 1.0 ml of ethanol and filtered through a 0.22μM filter. The sterile extracts were always used in a cell culture hood under aseptic conditions.
Gas chromatography- mass spectrometry (GC-MS) analyses GC-MS analysis of the extracts were carried out in a GC system (Agilent 7890A series, USA) equipped with split/splitless injector and auto-sampler attached to 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) and fitted to Mass Detector (Agilent 5975C series, USA). The flow rate of the carrier gas, helium (He) was set to be at 1 ml.min−1 in split less mode. The injector temperature was adjusted at 250°C, while the detector temperature was fixed to 280°C. The column temperature was kept at 70°C for 1 min followed by linear programming to raise the temperature from 70° to 200°C (at 8°C min−1 with 2 min hold time), and 200°C to 250°C (at 10°C min−1 with 2 min hold time). The transfer line was heated at 280°C. Total run time was 27.2 min. Mass spectra were acquired in scan mode (70 eV); in the range of 50 to 550 m/z. Twenty microliter each of the extracts (250 mg/ml stock) were further diluted in 2 ml of methanol. One micro liter of this diluted sample was injected for GC-MS analyses.
Identification of compounds
Interpretation of mass spectra was conducted using the database of the National Institute of Standards and Technology (NIST, USA). The database caters for more than 62,000 patterns of known compounds. The spectrum of the extracts was matched with the spectrum of the known components stored in the NIST library.
Cancer cell lines namely HCT-8 (derived from the ileocecal adenocarcinoma of a 67 year old male; and MDA-MB-231 (obtained from breast mammary glands were kindly supplied by the cancer research facilities, King Saud Bin Abdulaziz Medical City, Riyadh, Kingdom of Saudi Arabia and had been originally procured from the American Type Culture Collection (ATCC), USA. These cell lines were cultured either in RPMI-1640 for HCT cell line or DMEM (Life technologies, USA) for MDA-MB-231 cell line, supplemented with 10% heat inactivated fetal bovine serum (PAA Laboratories, Germany), 2mM L-glutamine, 50μg/mL of penicillin-G, and 50μg/mL of streptomycin sulfate. The culture was maintained as described earlier.
Assays to determine the phenotypic changes
Motility assay: cell motility for MDA-MB-231 and HCT-8 lines was performed in 6 well cell culture plates as described earlier. Cells were allowed to grow either in absence (control) or in presence of MOL, MOB, and MOS (250μg/ml and 500 μg/ml). The extent of the gap filled by the cells was monitored microscopically after 24 hours of treatment.
In vitro Clonogenic survival assay: Anchorage dependent colony formation assay was performed according to the standard procedure. The results of the assay were depicted in the form of photographs and the quantitative analysis was shown in the form of bar graphs.
Cell viability, apoptosis and cell cycle arrest assays
All three cellular parameters were examined on the Muse Cell Analyzer (Millipore, USA).
Cell viability assay: the cell viability assay was performed in 24-h pre-treated MDA-MB-231 and HCT-8 cell lines by different concentrations of plant extracts. The assay was performed as per the manufacturer’s protocol. Briefly, 12×104 cells from control and extract treated cells were taken in 200 μl of PBS and mix with 380μl of cell counting solution (Millipore, USA cat # MCH 100102). Contents were gently mixed for a few seconds and immediately read on the machine using specific programming. A histogram and numeric values displayed the number of dead and live cells after treatment.
Apoptosis assay: in order to evaluate the extent of early and late apoptosis induced by the plant extracts, 12×104 cells from MDAMB-231 and HCT-8 cell lines were treated for 24 h with different concentrations of leaves, bark and seeds extracts. Thereafter the extent of apoptosis was examined on the Muse cell analyzer. The assay was performed by utilizing the Annexin V and Dead Cell Kit (Millipore, USA cat # MCH 100105). Using designated programming on the cell analyzer, the numbers of live, dead, early and late apoptotic cells were determined. Total apoptosis was calculated by combining the number of cells from late and early apoptosis quadrants of the histograms and is presented in the form of bar graphs.
Cell cycle assay: to determine the effect of plant extracts on cell cycle arrest the assay was performed using the cell cycle kit (Millipore, USA cat # MCH100106). At the completion of the plant extracts treatment which was done in the same way as described above, cells were trypsinized, and counted. About 12x 104 cells from control and treated groups were fixed in chilled 75% ethyl alcohol for 3h at -20°C. Next cells were washed once with PBS and incubated with 200μl of assay solution for 30 min in the dark at room temperature. After completion of the incubation period the cells were vortexed gently and read on the cell analyzer. The number of cells at each event namely, Go/G1, S and G2/M phases were determined in control and extracts treated cells.
Statistical calculations (Student’s t-test) were performed using Graph Pad Prism 4.0 software. The mean was reported with standard deviation (± SD). Differences were considered to be statistically significant when p values were ≤ 0.05.
Optimization of GC-MS Method
GC method was optimized by varying the oven temperature. In current gradient oven temperature programming, a good resolution of all the extracts has been seen in a relatively short duration of time. The fragmented ions were separated by the analyzer, according to their mass-to-charge ratio.
GC-MS analyses of Moringa oleifera’s (MO) leaves, barks and seeds show several carbohydrates/ sugar and long chain fatty acid moieties
The ethanol extracts of the leaves and bark of Moringa showed twelve and seventeen peaks on chromatogram respectively (Figs (Figs1A1A and and2A).2A). The extracts of leaves mainly comprises of thiocynates, hydrocarbons and fatty acids, while the bark extract chiefly consists of hydrocarbons, phenolics, phthalates, carboxylic acids, and long chain fatty acids. Analyses of seeds revealed either hydrocarbons or long chain fatty acids and its derivative (S1 Fig). Chemically, the composition of leaves, bark and seeds revealed on GC-MS, were isopropyl isothiocyanate, D-allose and cetene present exclusively in the leaves extract; eugenol, dibutyl phthalate, 2- chloropropionic acid and 5-eicosene, present only in the bark extract. The seeds extracts showed the presence of 1-butanamine, 1-dodecene, 2-decenal, 3-tetradecene, 2-tetradecene, 1-octadecene, hexadecanoic acid, 10-octadecenoic acid, and heptadecanoic acid. Some of the compounds and derivatives like octadecene (C18:1 in leaf & C18:1 in bark), tetradecene (C14:5 in leaf & C14:2 in bark), octadeconoic acid (C18:11 in leaf & C18:9 and C18:9 in bark) and palmitic acid has been found in both leaf and bark extracts. Interestingly, we did not detect phenolic compounds and flavonoids using GC-MS techniques. Our findings are in accordance with the previously published work. The molecular structures of some of the bioactive molecules are given in Figs Figs1B1B and and2B2B.