Cancer is a frightful disease and represents one of the biggest health-care issues for the human race and demands a proactive strategy for cure. Exploration of natural product containing anticancer agent provide a promising line for research on cancer. Moringa plant (Moringa oleifera) is one of the medicinal plants used in traditional medicine for the treatment of cancer. Several studies have reported that water and alcoholic leaves extracts of M. oleifera have anticancer activity in some cancer cell line, including HepG2 liver cancer cells, A549 lung cancer cells, Caco-2 colon cancer cells and MDA-MB-231 breast cancer cells. This study was carried out to investigate the mechanism on selular and molecular basis of ethyl acetate fraction of M. oleifera leaves (EMO) against T47D breast cancer cell line by observing cell cycle progression. Cell cycle analysis was performed using flow cytometry and cyclin D1 expression was analyzed using immunocytochemistry method. Flow cytometry assay indicated that EMO induced cell cycle arrest on G0/G1 phases. Immunocytochemistry assay showed that the EMO decreased expression of cyclin D1.
Breast cancer has been widely known as the most common malignancies among women. It will afflict an estimated 9.1 million women in poorer countries over the next decade. If the 5 million women expected to die from breast cancer in the next decade, 70 percent will live in lowand middle-income countries1. There is evidence for a genetic contribution to the risk of developing breast cancer, as well as an association with modern affluence (diet and alcohol consumption). In addition, the influence of reproductive factors supports a hormonal role in the etiology of the disease2.
Moringa oleifera is an edible plant native to Northern Indian subcontinents, but recently the plants are widely cultivated and become naturalized in many countries throughout Asia and Africa3,4. M. oleifera is also called with various names such as horseradish tree, drumstick tree and locally named ‘kelor’. It belongs to the family of Moringaceaeand has been used in traditional medicine for centuries. The traditional uses of M. oleifera including the treatment of bacterial, fungal, viral and parasitic issues, along with asthma, circulatory, digestive and inflammatory disorders, malaria, typhoid fever, arthritis, hypertension, and diabetes5,6. Almost every part of the M. oleifera plant from the leaves to the fruit, bark and seeds can be used to treat a diverse array of ailments, but the leaves are the most widely cultivated due to its phytochemical composition and their associated medicinal properties3. It contains a rich source of rhamnose, glucosinolates and isothiocyanates. A study conducted by Manguro and Lemmen7 into the phenolics of MOE had characterised five flavonol glycosides using spectroscopic methods. The anticancer property can be attributed to specific components of MOE such as 4-(α-L-rhamnopyranosyloxy) benzyl glucosinolate, 4-(α-L-rhamnopyranosyloxy) benzyl isothiocyanate, benzyl isothiocyanate and niazimicin. The leaves contain quercetin-3-O-glucoside and kaempferol-3-O-glucoside which plays a role in antioxidant defence as it scavengers for free radicals thus reducing oxidative stress8. In addition thiocarbamates such as niazimicin found in the leaves, can be used as a chemopreventive agent9,10. Studies have suggested that the anticancer and chemopreventive property of M. oleifera extract can be attributed to niazimicin11,12.
A number of M. oleifera leaves extract cancer studies have been published. In previous studies M. oleifera aqueous crude leaf extracts has expressed anticancer effects in both A549 lung cancer cells and SNO oesophageal cancer cells13 as well as KB tumour cells14 in a ROS-dependent manner. Apriani et al.15 have reported that ethyl acetate fraction of M. oleifera leaves had potent anticancer activity and apoptosis inducing against breast cancer cell line T47D. This study is further research of Apriani et al.15, aims to investigate the mechanism on selular and molecular basis of ethyl acetate fraction of M. oleifera leaves (EMO) against T47D breast cancer cell line through observing cell cycle progression. Expression of cyclin D1 protein level was also investigated.
Material and Methods
Plant material: M. oleifera L. leaves were collected from Mangunreja, Tasikmalaya, Indonesia on Februari 2018, during rainy season.
Extraction: M. oleifera leaves were dried with an oven at 30oC. Dry powder of M. oleifera leaves were extracted with ethanol 96% for 3×24 hours. Filtrate was concentrated using rotary evaporator at 50oC. M. oleifera ethanol extract was then partitioned with n-hexane and ethyl acetate by liquid-liquid extraction. Ethyl acetate fraction was evaporated by using rotary evaporator to get ethyl acetate fraction of M. oleifera leaves (EMO).
Cell culture: Human breast cancer T47D culture cells culture were a collection of Paracytology Laboratorium, Universitas Gadjah Mada, Yogyakarta. Cells were cultured in RPMI (Gibco, USA) supplemented with 10% Fetal Bovine Serum (Gibco, USA).
Data analysis: Cell viability resulted by EMO treatment was analyzed statistically by probit analysis using SPSS 24.
Cell cycle analysis: FACS analysis was carried out to investigate cell cycle distribution. T47D cells (106 cells/well) were grown in 6-well plate and treated with EMO. After 24 hours treatment, cells were trypsinized and centrifuged at 2000 rpm for 3 minutes. Trypsinized adherent cells were collected and detected by adding 25 µL propidium iodide, 2.5 µL RNAse, 0.5 µL Triton-X, then incubated at room temperature for 10 minutes. The cell suspension was transferred into a flow cytometer (BD FACS-Calibur, USA).
Immunocytochemistry: T47D cells were grown with the density of 5×104 cells/cover slip in 24-well plate and incubated for 24 hours. The medium in each well was then replaced by the fresh medium containing various concentrations of EMO and then placed in a humidified incubator at 37oC for 24 hours. The cells were then harvested and were washed with PBS and fixed with cold methanol for 10 minutes at 4°C. After that, the cells in coverslips were placed each on a respective slide. The cells were washed with PBS and distilled water, then were blocked in a hydrogen peroxide (Millipore Sigma, Burlington, USA) blocking solution for 10 minutes at room temperature. Then, washed again with PBS, and incubated with pre-diluted blocking serum for 10 minutes at room temperature. Next, the cells were stained with primary Cyclin D1 antibody (Biocare Medical, Cali-fornia, USA) for 1 hour at room temperature. After three time-washing with PBS, the secondary antibody was applied for 15-20 min, and then washed with PBS three times. The slides were incubated with streptavidin-biotin complex (Biocare Medical, Cali-fornia, USA) for 10 minutes, and then washed with PBS three times. The slides were incubated in DAB (3, 3 diamino benzidine) (Alfa Aesar, Ward Hill, USA) solution for 3-5 minutes and washed with distilled water. Cells were counterstained with Mayer-Haematoxilin reagent for 3-4 minutes. After incubation, the coverslips were washed with distilled water and then immersed in absolute ethanol and in xylol. The protein expression was assessed under a light microscope (Olympus Life Science, Shinjuku, Tokyo, Japan).
Results and Discussion
Cytotoxic Activity of EMO on T47D Cells: Cytotoxic activity was used to evaluate the potential of EMO cytotoxicity on T47D cells. Furthermore, IC50 value was acquired as a parameter of EMO concentration to inhibit 50% T47D cell’s growth. According to Apriani et al.15, EMO giving IC50 value of 135.321 µg/mL. Based on the IC50 value, EMO was considered medium active as an anticancer because according to Kamuhabwa et al.16, an extract is considered active if it has an IC50 value less than 100 μg/mL, but it can be still developed as an anticancer because an extract is considered inactive if the IC50 value more than 500 μg/mL17.
EMO Induce Cell Cycle Arrest: According to the cytotoxic activity mentioned above, cell cycle distribution was determined by flow cytometry using. Figure 1 shows the distribution of T47D cells treated with variation concentration of EMO for 24 h incubation. T47D cells treated with 150, 200, 250 and 300 μg/mL of EMO were accumulated in G0/G1 phase, that is from 47.64% in untreated cells to 70.24%, 58.27%, 48.36%, and 55.77%, respectively, in treated cells. In addition, doxorubicin have been reported to induce cell accumulation in G0/G1 phase in T47D cells, which is 50.64%18. This study have proven that treatment of T47D cells with EMO resulted in significant G0/G1 phase arrest of cell cycle progression which indicates that one of the mechanism by which EMO may act to inhibit the proliferation of cancer cell is inhibition of cell cycle progression.
Generally, proliferation of cells is regulated by a variety of extracellular growth factor that control the progression of cells through the restriction point in late G1. In the absence of growth factors, cells unable to pass the restriction point and become quiescent, frequently entering the resting state known as G0. They can reenter the cell cycle in response to growth factors implies that the extracellular signalling pathways stimulated downstream of growth factor receptors ultimately act to regulate components of the cell cycle19. In this study, cells accumulation in G0/G1 phase indicates that T47D cells did not get any stimulus from extracellular growth factor signalling, so that the cells could not synthesize DNA.
Expression of Cyclin D1 was caused by EMO on T47D Cell: The cell cycle progression in G0/G1 phase might be caused by some proteins that play a role in cell cycle checkpoints. Cyclin D1 is an important regulator protein of G1 to S phase progression. Therefore, the expression of cyclin D1 was also investigated using immunocytochemistry method. As shown in Figure 2, immunocytochemistry evaluation indicated that cyclin D1 level decreased significantly confirmed by an intensive brown color in cytoplasm after being treated with 150, 200, 250 and 300 µg/mL EMO.
Cyclin D1 is synthesized in a response to growth factor stimulation through Ras/Raf/ERK signalling pathway18. Together with its binding partners cyclin dependent kinase 4 and 6 (CDK4 and CDK6), cyclin D1 forms active complexes that promote cell cycle progression by phosphorylating and inactivating the retinoblastoma protein (RB)20 and then cells will be able to enter the restriction point on the cell cycle. On the other hand, if appropriate growth factors are not available in G1, cyclin D1 level will decrease and could not associated with CDK 4 and CDK 6. In this study, the cyclin D1 level was decreased, cells cannot pass the restriction point and then enter a quiscent stage of the cell cycle called G0 in which they can remain for long periods of time without proliferating.
In conclusion, EMO exhibits potential ability as an anticancer through cell cycle arrest on T47D cells with cyclin D1 stabilization. Observation on its selectivity as part of safety aspect is also needed. Further, EMO has a potential compound to be explored and developed as a chemo preventive agent for breast cancer.
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