Breast cancer is a major cancer commonly in women, were over one million women worldwide are diagnosed with this disease.1 Although there are many therapeutic strategies including chemoteraphy to treat cancer, high systemic toxicity and drug resistence limit the successful outcames in most cases.2 The other strategies are being developed to control and treat cancer, one such approach could be used the medicinal plants agents, would enhance efficacy while reducing toxicity to normal tissue.3 Several Sumatran plants have been studied for their anticancer properties.4-6 In this paper, cytotoxicity activity of of ginger (Zingiber officinale Rosc.) induced with arbuscular mycorrhizal fungi (AMF) against T47D cells line breast cancer was reported.
Ginger (Zingiber officinale Rosc.) family of zingiberaceae, has been widely used as a condiment though out the world for centuries. Ginger has been used as herbal medicine to tread a wide range of disorder such as anti inflamation, analgesic, dispepsia, nausea, vomiting, antibacteri, antioxidant and anticancer.7-8 Ginger contains active phenolic components such as gingerol and shogaol which have antioxidant and anticancer effects.9-10 Component carrier spicy ginger flavor that is gingerol, paradol, shogaol and zingerone have anti-inflammatory activity and chemopreventive effects of that shows prevention of cancer in experimental carcinogenesis.11
The present study uses ginger rhizome that has been induced by arbuscular mycorrhizal fungi (AMF), as one type of biological agent. The results of previous studies AMF may increase the resistance of plants to bacterial wilt disease, increase rhizome production and secondary metabolites from the rhizome of the ginger plant.12 The aim of the study was to evaluate the cytotoxic effect of the ethanolic extract of AMF induced ginger on breast cancer T47D cell lines.
MATERIALS AND METHODS
The rhizome of ninth months AMF induced ginger were cultivated and collected from screen house of Herbal Medicinal Study Centre Andalas University Padang Indonesia.
Human breast cancer T47D cell lines was obtain from Tissue Culture Laboratory of Medicine Gajah Mada University, Yogyakarta, Indonesia).
The plant meterial was sliced and air dried in green house for 3 days, follow by oven drying at temperature 40°C for 24 h. The dry rhizomes were grinded to powder using laboratory grinder. About 2 kg of powdered the rhizome of ginger was macerated in 7 L of 70% ethanol for 3 days. This process repeated 3 times. The ethanol extract was evaporated and concentrated with rotary evaporator at 40°C.13 the resulting extract was kept in the refrigerator. The extract is dried under reduced pressure by using rotary evaporator. Dried extract was dissolved in DMSO (Sigma) at stock solution of 100 mg/mL.
Human breast cancer cell line T47D were cultured in RPMI with 10 % complete mudium (Gibco). The medium was supplemented with 10 % heat inactivated fetal bovine serum, penecillin G and streptomycin 100 μg/ml. The cell lines were maintained at 37º C in 5% CO2 incubator.
Cytotoxic Assay and Cell Viability
Cells were seeded into 96-well plate (Nunc, Denmark) and precultured for 24 h, treated with ethanol extract of AMF induced ginger for 48 h. Cell Cytotoxity was determined by MTT assay, were 20 μL extract at concentration 0.1 μg/mL; 1.0 μg/mL; 10 μg/mL; 100 μg/mL disolved in DMSO completely were added into 180 μL cell suspension in RPMI media. After 24 h incubation, added 20 μL MTT (Merc, Germany) reagent in phosphate buffer saline (PBS) to each well. The plate were incubated at 37ºC, the medium was discharged and the purple percipitate wich had been formed in the cells were dissolved with 100 μL DMSO. The absorbance was measured at 550 nm wave length by Autoated Microplated Reader (Bio-Teck) and the cell death was calculated.12 Cell viability was estimated by trypan blue dye exclusion. After 24 h incubation, the culture was observed under inverted microscope and morphological change of cells were identified.
The relationship between the concentration of the test solution with cell viability shown in graphical form and the determined IC50 (concentration that inhibits 50% living cells) of the test solution. All experiments were repeated three times and the data were presented as the mean ± SD unless noted otherwise. Differences between data groups were evaluated for significance using one ways analysis of variance (ANOVA) followed by Duncan’s multiple range test, using the software SPSS 14.0 for windows. P values less than 0.05 indicate statistical significance.
Effect of Ethanol extract of AMF Induced ginger rhizome on T47D breast cancer cell line were determined by MTT Assay. The examined of the cytotoxicity effect of Ethanol extract of AMF Induced ginger in multiple concentration on human breast cancer T47D. The effective concentration was calculated from concentration-responce curve. The precentage of viability of each plate was shown in Table 1. Based on the MTT assay, it was found that Ethanol extract of AMF Induced ginger had IC50 12.5 ± 3.73 μL / mL (Table 2), dose-response relationship curve of the rhizomes of Ginger against breast cancer T47D cells (Figure 1).
|0.1 µg/mL||1.0 µg/mL||10 µg/mL||100 µg/mL|
|1||54.02 %||57.93 %||52.48 %||13.16 %|
|2||70.80 %||65.61 %||48.70 %||12.44 %|
|3||60.22 %||55.70 %||51.57 %||18.0 %|
|Average ± SD||61.68 % ± 12.1||59.74 % ± 7.9||50.91 % ± 4.9||14.53 % ± 4.2|
Morphological changes evaluation upon treatment with extract
Figure 2 showed the difference picture when viewed with an inverted microscope, were difference concentration gives different morphological profile of cells when compared to control. At concentration 0.1 μg/mL and 1.0 μg/mL, many cells grow and attached at the base of the flask, cells grow very dense and very little distance between one cell to another cell, it almost no difference compared to control. In the other hand at concentration of 10 μg/mL, the cells look less than concentration of 0.1 and 1.0 μg/mL, cells are visible only in small groups and the distance a group of cells with other cells look far. In a concentartion of 100 μg/mL, the cells look less, many cells was dead, no cell nucleus be visible, it is also evident from the low absorbance value.
Ginger has been widely used as a condiment thoughout the world for centuries. It has been used as herbal medicine to tread a wide range of disorder such as anti-inflammation, antioxidant and anticancer.3-4 Ginger root and its main phenolic compounds such as gingerols have anticarcinogenic activity, antioxidant and anti-inflammatory activity. Plant Materials used in the form of fresh samples of ginger plants induced by AMF, it has an activity increase of growth hormone ginger and chemical compound when compared with uninoculation plant.12 T47D cancer cell cultures commonly used in cancer research in vitro because it has the ability in each cycle of cell replication and have a fast replication capability, high homogenity, that is suited for cytotoxic test.12 T47D cancer cells are cells that have the function of the p53 gene is mutated, so it can not bind to p53 response elements on DNA. This results in reduced even loss of the ability of p53 gene to cell cycle regulation.
MTT assay is a colorimetric cytotoxic test method to determine the number of living cells based on changes in a solution of 3-(4,5-dimetilthiazol-2-il)-2,5-difeniltetrazolium bromide were colored yellow to purple formazan crystals by active mitochondria in living cells.12 Purple color intensity is directly propotional to the amount of the active cell metabolism. Formed the darker color, the higher the absorbance value and the more living cells.
The result of one Way ANOVA followed by Duncan Multiple Range Test (α=0.05), suggesting that the extract of ginger rhizome gave significantly different barriers to the growth of T47D breast cancer cells. The ethanol extract of ginger induced by AMF at a concentration of 100 μg / mL, had the highest activity inhibiting the growth of cancer cells T47D significantly different from the concentration of 10 μg / mL, 1.0 μg/mL. But the concentration of 1 μg / mL and 0.1 μg / mL was not significantly different (Table 1). The difference can be seen from the color produced by each concentration on the test plate. The color produced at a concentration of 100 μg / mL is slightly closer to purple or clear. When compared with a concentration of 0.1 μg / mL of concentrated purple and almost the same as the control. Purple formazan crystals produced as a result. Formazon crystals can penetrate the cell membrane and accumulates in the cells that are still alive. The data obtained that at a concentration of 100 μg / mL, significanly different from the concentration 10 μg / mL which when viewed on an inverted microscope many cells die, and many are the remains or carcasses from these cells, compared with a concentration of 1 μg / mL and 0.1 μg / mL were not different.
The inhibitory concentrations (IC50) of ethanol ginger extract on T47D breast cancer cells was 12.5 ± 3.73 μg / mL. These results suggest that ginger has a cytotoxic activity that is characterized by a decrease in the percentage of T47D breast cancer cell viability in a highly significant (p <0.05).