INTRODUCTION:

Nowadays, cancer is a complex disease which is non-curable in most cases¹. Cancer is difficult to conquer in all living systems. Cancer cells exhibit characteristic features, such as avoidance of apoptosis, impaired cell cycle control, and self-sufficiency in growth signaling^2,3. Colorectal cancer is still the leading cause of cancer related deaths in both men and women. Furthermore, its annual incidence and mortality rates have both risen over the past 25 years⁴. Today, chemotherapy is one of the most widely used therapeutic strategies against cancers. However, it has some limitations, such as the toxicity of normal cells and the gradual increment of cancer cells resistance. The discovery of new drugs as alternative strategies in cancer treatment is therefore highly desirable⁵. Plants are regarded as very promising from this perspective since they represent substantial sources of substances with various therapeutic uses⁶. Most anticancer drugs today are produced from plants^5,7.

Allium cepa L., commonly known as brown or yellow onion, is a worldwide culinary spice belonging to the family Liliaceae. Allium cepa L. is an essential ingredient in many African sauces and mostly produced locally⁸. Allium cepa L. has some active compounds, such as phenolic acids, thiosulfinates, saponins, and flavonoids. The plant has a variety of pharmacological activities including anticancer, antidiabetic, antimicrobial, cardiovascular, and antioxidant effects⁹. The flavonoids of Allium cepa L. act as antioxidants that can prevent oxidation and stabilize free radicals which are formed by carcinogenic compounds and inhibit the production of heat shock proteins that cause resistance to cancer drug therapy. Saponins function as immunomodulators and prevent the proliferation of various cancer cells. Previous studies showed that different natural compounds can improve the efficiency of chemotherapeutic agents, decrease the resistance of cancer cells, and alleviate the adverse side effects of chemotherapy¹⁰. Moreover, anticancer drugs from plant extracts have been used as alternative therapies to prevent negative reactions and have a role as cell growth inhibitors at the safe doses¹¹.

Various studies investigated the cytotoxic effects of different Allium cepa L. species. Onion oil inhibits the growth of the HL-60 human promyelocytic leukemia cell line by inducing the differentiation of HL-60 into the mature cells of granulocytic lineage12. Moreover, sodium n-propyl thiosulfate (NPTS) and alk(en)yl thiosulfates present in Allium cepa L suppress the growth of the HL-60 cells through the induction of apoptosis initiated by oxidative stress, indicating that the alk(en)yl thiosulfates ylthiosulfates are accounted partly for the anti-carcinogenic properties of onion and have the potential to prevent tumors¹³. Allium cepa L. is also known for having the cytotoxic effect on K562 cells and Lucena cells¹⁴.

Cytotoxicity tests are recommended for all medical devices as they allow a rapid evaluation, standard protocols, and produce quantitative as well as comparable data due to their sensitivity which allow toxic materials to be discarded prior to animal study¹⁵. Generally, three types of cytotoxicity tests are employed: the extract dilution method, the direct contact method, and the indirect contact (agar diffusion) method. The direct contact method enables weak cytotoxicity to be detected because of its high sensitivity¹⁶. Whereas, the extract dilution method is more commonly adopted for the in vitro cytotoxicity evaluation of materials and devices used in the body since it can be applied to a wide variety of raw materials and finished the products that may release toxins from the exposed surfaces. Extraction conditions, like time and temperature, are dependent on the physicochemical characteristics of the materials being tested and the extraction device¹⁷. Thus, the aim of this study was to evaluate the cytotoxic effects of Allium cepa L. extract on WiDr cells.

MATERIAL AND METHODS:

Ethical Clearance:

All treatment procedures have been tested through The Medical and Health Research Ethics Committee (MHREC), Faculty of Medicine, Gadjah Mada University, Yogyakarta, Indonesia (Approval Reference Number: KE/FK/0106/EC/2018).

Chemicals and Reagents:

This research used materials, such as Dulbecco’s modified Eagle medium (DMEM) (Gibco, USA), fetal bovine serum (FBS) (Rocky Mountain Biologicals, Inc., USA), Hepes (Sigma Aldrich, USA), penicillin-streptomycin (Gibco, USA), fungizone, phosphate buffer saline (PBS), trypsin-EDTA solution (Sigma Aldrich, USA), dimethyl sulfoxide (DMSO) (Sigma Aldrich, USA), 3-(4,5-dimethyltiazole-2-il)-2,5-diphenyltetrazolium bromide (MTT), SDS, HCl 0.1 N, RNAse (BD Bioscience, San Diego, CA). All chemicals and reagents used were of the analytical grade.

Preparation of Allium cepa L. extract:

Several Allium cepa L. were bought from the local market in Jombang, East Java, Indonesia. They were cleaned and chopped into small pieces and shade-dried. They were mashed to powdery form using a mechanical blender and passed through the coarse sieve (0.2 mm). The Allium cepa L. powder was macerated with ethanol 96% for 72 h at 37 °C. The extract was evaporated in waterbath at 60°C. The residue was stored in a refrigerator at -4°C.

Cell culture of WiDr cells:

WiDr cells were collected from Parasitology Laboratory, Faculty of Medicine, Gadjah Mada University, Yogyakarta, Indonesia. Cells were cultured in DMEM media and supplemented with 10% (v/v) fetal bovine serum, 3% streptomycin-penicillin and 1% fungizone in 5% CO₂ incubator at 37 °C. Cells were maintained in 25 cm² flask with 7 mL media and harvested using 0.25 trypsine-EDTA after reaching 80% on confluency.

Determination of IC₅₀ value:

The IC₅₀ value of Allium cepa L. extract against WiDr cells was determined by the 3-(4,5-Dimethylthiazol-2-yl) 2,5-diphenyltetrazolium bromide (MTT) assay. Cells were cultured in 96-well plates at a density of 1x10⁴ cells/well with 100 µL of volume and incubated at 37 ºC, 5% CO₂ for overnight. Cells were added with various concentration of Allium cepa L. extract (50, 100, 200, 400 µg/mL) for 24 h. Then, the media were removed and 100 µL of DMEM and 10 µL MTT (5 mg MTT/mL solution) were added to every single well. The plates were incubated for 4 h. On the other hand. The control cells received only media without Allium cepa L. extract sample. The crystal of formazan which was formed in viable cells were solubilized with 100 µL of SDS-stopper HCl 0.1 N. The absorbance of 595 nm was measured by microplate enzyme-linked immunosorbent assay (ELISA) reader by Benchmark Microplate Reader (Bio-Rad, USA). The different absorbance in both treated and the untreated groups were calculated to determine the IC₅₀value with linear regression analysis using Microsoft Excel 2016 (Microsoft Inc., USA)

RESULTS AND DISCUSSION:

The anticancer activity of Allium cepa L. extract was tested against WiDr cells by MTT assay. The MTT assay results revealed that the cytotoxicity of treated cells decreased gradually with the increase of the sample concentration. The highest reduction of WiDr cells was found at the concentration of 400 µg/mL where the cytotoxic of WiDr cells was 15.7 µg/mL. Meanwhile, the lowest reduction was at the concentration of 50 µg/mL with the cytotoxicity of WiDr cells at 2.78 µg/mL. The reductions of cell growth from two other concentration (100 µg/mL, 200 µg/mL) were at 6.12 µg/mL and 9.36 µg/mL. The Allium cepa L. extract showed IC₅₀ value of 1363.29 µg/mL at 24 h on WiDr cells. This study proved that the treatment of Allium cepa L. extract slowly reduced the cell growth of WiDr cells by changing the concentration of the sample and it did not reach 50% of IC₅₀ value. The MTT assay against WiDr cells and cytotoxicity graph of Allium cepa L. extract effect on WiDr cells are shown in Figure 1A and B.

Figure 1A. The MTT assay against WiDr cells: A1) untreated cells; A2-A5) represent the different concentration of Allium cepa L. extract, 50 µg/mL, 100 µg/mL, 200 µg/mL, and 400 µg/mL,

Figure 1B. Cytotoxic activity of Allium cepa L. extract against WiDr cells.

Based on the potential properties of Allium cepa L., the analysis of this study began with the previous study result of Allium cepa L. extract in their anticancer activity and the cytotoxic effect on K562 cells as well as Lucena cells. It showed that the capacity of Allium cepa L. to induce the cell death was verified because several compounds of Allium cepa L., like flavonol quarcetin and quarcetin derivates, the major flavonol; were present in the Allium cepa L. species. The study showed that the bioavailability of quercetin could be better when ingested through the onion and the different components present in onion, which probably have an additive action. Allium cepa L. extract was only matched for K562 cells which resulted in the apoptosis inducement, however, a necrosis occurred in Lucena cells^14,18,19.

In this study, Allium cepa L. extracts shows a high number of IC₅₀ value of 1363.29 µg/mL which means that it is no longer active as a potent anticancer drug on WiDr cells, but it may be more potent if the researcher uses quarcetin, oil, sodium n-prophyl thiosulfate, and alk(en)yl thiosulfate and more compounds of Allium cepa L. species. It is related to the previous study that showed that sodium n-prophyl thiosulfate suppress the growth of HL-60 cells through the induction of apoptosis initiated by oxidative stress¹³.

To date, sodium n-prophyl thiosulfate has shown to have an inhibitory effect on tumor cell growth as well as the immune-enhancing and antithrombotic effects²⁰. It can be considered reporting that organosulfur compounds in the Allium plants can induce phase II detoxification enzymes including quinone reductase (QR), glutathione S-transferase (GST), epoxide hydrolase (EH), and uridine diphosphate glucuronosyl transferase (UGT) in cultured cells because the sodium n-prophyl thiosulfate is a bioactive organosulfur compound²¹.

On other hand, the use of selective extract compounds may be one of the important things that can be considered. Ethyl acetate of extract of Allium cepa L. has potent inhibitory effects on animal fatty acid synthase (FAS) and can induce an apoptosis in FAS over-expressing human breast cancer MDA-MB-231 cells²². Methanolic extract of Allium cepa L. induces apoptosis in the heart-derived H9C2 cells²³. In addition, apoptosis can be initiated by the oxidative stress, which, in turn, was mediated by the generation of reactive oxygen intermediates²⁴. Oxidative stress activates caspases, a family of cysteine proteases that are involved in the induction of cell death by the apoptosis mechanism²⁵.

From this study, we propose that the cell death of WiDr cells may come from the oxidative damage or also to that of cytotoxicity caused by the compounds. In fact, it seems difficult to explain the cellular sensitivity to antitumor agents by one single related key factor because multiple factors may be involved in the cellular sensitivity. However, to date, no report has been found on Allium cepa L. extract for potential anticancer agent against human colon cancer, WiDr cells. Hence, the findings of this study proved that the crude extract of Allium cepa L. species still need to be developed in terms of the doses required as an anticancer drug.

CONCLUSION:

Thus, it is possible to suggest that there are others ways to utilize Allium cepa L. species for observation as an anticancer drug. However, Allium cepa L. extract showed cytotoxicity with IC₅₀ value of 1363.29 µg/mL on WiDr cells. The result of the study showed that Allium cepa L. is no longer active as an anticancer drug, but it will be more active using its direct-bioactive compound. Allium cepa L. requires some intense observations to maximize its potential as one of chemotherapeutic agents in the future. Further studies are needed to obtain the right dose of Allium cepa L. that can inhibit the cell growth well and determine the accuracy of its mechanism on WiDr cells as a therapeutic model of human colon cancer.

ACKNOWLEDGEMENT:

This study was supported by PMDSU Grant Funds with reference number 1341/UN3.14/LT/2018 from The Ministry of Research, Technology, and Higher Education (KEMENRISTEKDIKTI) of The Republic of Indonesia.

CONFLICT OF INTEREST:

The authors declare no conflict of interest.

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Received on 22.02.2019 Modified on 16.03.2019

Research J. Pharm. and Tech. 2019; 12(7):3483-3486.

DOI: 10.5958/0974-360X.2019.00591.2