br Breast adenocarcinoma cell lines
Breast adenocarcinoma cell lines (MCF-7) is one of the most fre-quent incident cancers among women, with an estimated 266,120 new cases, and 40,920 breast cancer (BC) deaths will occur in the United States' women in 2018 (Siegel et al., 2017). Therefore, BC is a challenge among research communities around the world; this is because of the BC incidence rate keep increasing by 0.4% annually worldwide ac-cording to the final report conducted by Jemal et al. (2017). Current protocols of treatment include radiation therapy, surgical intervention, and chemotherapy which induce numerous side eﬀects including nausea, fatigue, vomiting, weak of the immune system and hair loss (Griﬃn et al., 1996). Thus, the search for alternative treatment is ne-cessary.
Apoptosis also called programmed cell death, is a mechanism that allows self-destruction of the cells when stimulated by an appropriate trigger. Distinct morphological changes such as cell shrinkage, nuclear condensation, and pyknosis, complemented with biochemical phe-nomena (BP) such as the cleavage of DNA between the nucleosomes (Elmore, 2007; Peter, 2011). New insights into the process of apoptosis lead to diﬀerent parameters which can be used to detect and measure apoptosis. One of these parameters is the appearance of phosphati-dylserine (PS) on the surface of the cell membrane and loss of the asymmetry which is a specific signal for macrophages to recognise and remove the apoptotic cells (Fadok et al., 1992; Verhoven et al., 1995). Apoptosis is mainly created by either death receptors (extrinsic pathway) involving caspases 8 and 10 or the mitochondrial pathway (intrinsic pathway) via caspase 9 (Elmore, 2007). The alteration of the mitochondrial membrane potential translocates the pro-apoptotic pro-tein Bax which leads to the release of cytochrome C, into the cytoplasm, and subsequent activation of caspases and lead to apoptosis (Wyllie, 1997). The constant breast cell number is maintained and regulated via an equilibrium between cell proliferation and apoptosis. After that, any changes in this balance lead to the loss of homeostasis which can cause the growth of cancer cells (Hao et al., 1998). Thus, the arrestation of Hygromycin B machinery and induction of apoptosis are the key mechanism to prevent and suppress the breast cancer cells (BCs). Hence, there is a massive interest in the development of new apoptosis-inducing agents with eﬃcacy and more specificity with minimal side eﬀects. Recently, our group has successfully extracted the Graviola fruit using ionic liquid method resulted in high productivity. Thus, this study aimed to in-vestigate the possible inhibitions induced in breast cancer cell lines the MCF-7using the ionic liquid extract of Graviola fruit.
2. Materials and methods
2.1. Chemicals and reagents
All the chemicals and reagents used in this project are laboratory grade. 1-Butyl-3-Methylimidazolium Chloride 96% [C4MIM] [Cle] re-agent was purchased from (Alfa, USA), cell culture media, including Dulbecco's modified Eagle's medium (DMEM), penicillin/streptomycin and fetal bovine serum (FBS) were obtained from GIBCO® (Invitrogen, USA). The 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium Bromide (MTT) was purchased from Sigma-Aldrich, (USA). The accu-tase cell detachment solution purchased from (ICT, USA). RNase A/PI cell cycle assay kit and Annexin V/PI apoptosis assay kit was purchased from Beckman Coulter, (USA). These chemicals were used to investigate the anticancer eﬀect of IL-GFE on cancer cell lines.
2.2. Sample collection and preparation
identified from the Adikafirdaus Resources Farm in Kampong Bawong, Perak, Malaysia. After that, the fruits were washed with distilled water to remove all traces of dust and insects. The fresh pulp of each fruit separated from the pericarp and seeds, then the pulp was frozen at −20 °C and lyophilised using a Christ Alpha freeze drier for 72 h ac-cording to Hoeing (2001), with slight modification. After that, the powder was weighed and placed in an airtight bottle and stored until further extraction procedure (Gyamfi et al., 2011).
A plant sample deposited in the herbarium at the Kulliyyah of Architecture and Environmental Design (KAED), International Islamic University Malaysia with voucher number KAED/HBL/S1A047/2018/ 707. The name of the plant was checked on www.theplantlist.org.
2.3. Fruit extraction
One gram of the dried sample was mixed with 30 mL of 0.5 mol/L of 1-butyl-3-methylimidazolium chloride [C4MIM] [Cle] solution. Then the suspension was heated with microwave oven under irradiation power of 700 W for 3 min. After each irradiation, the obtained extracts (IL-GFE) were cooled down to 25 °C and then filtered through Whatman 3 mm filter paper and transferred into a test tube. The tubes were frozen at −20 °C then lyophilised using a Christ Alpha freeze drier for 72 h. Then, the powder was stored in 4 °C freezer until use (Bhan et al., 2017; Zhang et al., 2014).