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Cytotoxic Activity of Stem of Pycnarrhena cauliflora through Apoptosis Induction on Human Breast Cancer Cell Line T47D

Cytotoxic activities of extracts of Pycnarrhena cauliflora leaves, stems and roots on some human cancer cells have been known. However, the cytotoxicity of stem of Pycnarrhena cauliflora on human breast cancer cell line T47D through an apoptosis induction has not been proven yet. This study was an effort in understanding the cytotoxic activity of stem of Pycnarrhena cauliflora through apoptosis induction. The crude ethanol extract was partitioned between n-hexane and dichloromethane at pH 3, 7 and 9, which was subsequently tested for their cytotoxic activity against human breast cancer cell line T47D by using 3-(4,5-dimethylthiazol-2-yl)2,5-diphenyltetrazolium bromide (MTT). Flow cytometric analysis with Annexin V-FLUOS/ propidium iodide (PI) double staining was used for apoptosis analysis. The dichloromethane fraction at pH 7 was considered the most potent with an IC50 value of 59.30 μg/mL and induced apoptosis on the T47D cell line. The result indicated the potential of dichloromethane fraction of the stem of P.cauliflora as a source of bioactive compounds and provided a basis of further studies to isolate a new anticancer compound.


INTRODUCTION
Cancer is one of the major causes of death worldwide. According to GLOBOCAN, the number of new cases and death caused by cancer in 2018 was 8.1 million and 9.6 million (Bray et al., 2018). One in 8 men and 11 women of deaths in the world is caused by cancer. Breast cancer is the second most common malignancies (11.6%) and the second cause of cancer deaths worldwide (6.6% of total cases) .
Various strategies for cancer treatment have been carried out, but the morbidity and mortality rates are still high. Cancer treatment strategies can be carried out either single or in combination through surgery, chemotherapy and radiation methods (Taraphdar et al., 2001;Vijayalaxmi et al., 2002;Rogers, 2003). Chemotherapy is the most important method in treating cancer, especially for metastatic cancers. The main problem in treating cancer with chemotherapy is the emergence of serious side effects (Sausville and Longo, 2005) and multidrug resistance (Tsuruo, 2003;Li et al., 2005). This led to the discovery of new anticancer drugs.
Natural resources have played an important role in the field of cancer drug discovery and development. During 1981 to 2006, natural resources represented about 47.1% of 155 of drugs in clinical application (Newman and Cragg, 2007). World Health Organization (WHO) predicted about 80% of the developing world's population relies on traditional herbal medicines for their primary care (Alves and Rosa, 2005).
Pycnarrhena cauliflora (Menispermaceae) is one of indigenous plants of West Kalimantan which is locally well known as sengkubak or sanksang. The Dayak and Malay ethnic groups used the leaves of the plant as a natural taste substance (Afrianti, 2007). The ethanolic extract from leaves of P. cauliflora showed a weak antioxidant activity by scavenging of DPPH free radical with IC 50 value of 565 µg/mL (Masriani et al., 2011). In addition, the ethanolic extract from leaves, stems and roots of P. cauliflora exhibited cytotoxic activity on human cervical cancer HeLa cell line. The roots of ethanolic extract induced apoptosis and caused cell cycle arrest at the G0/G1 phase (Masriani et al., 2013). The crude alkaloid of P. cauliflora root showed potent cytotoxic activity and high selectivity on T47D cancer cells. The cytotoxic effect was triggered by inducing of apoptosis and arresting of cell cycle at G2/M phase .
Anticancer activity against breast cancer T47D cell lines of P. cauliflora stem has not been well studied. Accordingly, we evaluated cytotoxicity of ethanol extract of P. cauliflora stem in the breast cancer T47D cell lines, furthermore we confirmed it activities in the apoptosis induction process. It was expected that the results of this study can provide a basis of further studies in a new anticancer drug development.

Extraction of crude extract and solvent partitioning
The air-dried powder of P.cauliflora (4.6 Kg) stem was macerated in ethanol (24 L) for 3 x 5 days. The resulted residue (245 g, SE) was dissolved in distilled water and partitioned with successive solvent of n-hexane and dichloromethane at pH 3, 7 and 10 to obtain n-hexane fraction (8 g, SH) and dichloromethane fractions at pH 3 (10 g, SD3), pH 7 (15 g, SD7) and pH 10 (5 g, SD10). The extract and fractions were dissolved in dimethyl sulphoxide (DMSO) to give a stock solution of 100 mg/ mL and stored (-20 °C) until used.

Cell line and culture
Human breast cancer cell line T47D was used for in vitro cytotoxicity assays. The cell line used in this study was obtained from Parasitology Laboratory, Faculty of Medicine, Gadjah Mada University, Yogyakarta, Indonesia. RPMI-1640 medium, supplemented with 10% FBS, 2% penicillin/streptomycin and 5% amphotericin was used as the culture medium for T47D cell. The cell line was grown and maintained in a humidified incubator at 37 °C with 5% CO 2 atmosphere.

Cell cytotoxicity assay (MTT assay)
The cytotoxic effect of n-hexane fraction (SH) and dichloromethane fractions at pH 3 (SD3), pH 7 (SD7) and pH 10 (SD10) on T47D cell line was determined by MTT colorimetric assay (Mosmann, 1983). Briefly, cells (1 × 10 4 cells/well) were seeded into 96-well flat bottom culture plates and allowed for overnight to adhere in incubator at 37 °C with 5% CO 2 humidified atmosphere. Afterwards, cells were treated with culture medium (cell control) and various concentrations of fractions (final concentration from 31.25 to 500.00 μg/mL) and incubated for 24 h. Three replicate wells were used per concentration. At each end time point, the media was discarded and cells were washed with PBS once. Each well was added with 110 μL of mixture of MTT solution (5 mg/mL) and complete medium (1:9).The cells were again incubated for 3-4 h. At the end of incubation, the reaction was stopped by 100 μL sodium dodecyl sulphate in HCl 0.1 N to each well and permitted for overnight in dark at room temperature for complete dissolution of formazan crystals. The absorbance of each well was read at 595 nm using an enzyme-linked immunosorbent assay (ELISA) reader. The results were expressed as the percentage of viability cells in comparison to the control cells, using the following formula: The concentration required to inhibit 50% the cancer cells growth population or IC 50 was calculated by using regression analysis of viable cells percentage graph against concentration.

Flow cytometric analysis of cell apoptosis
The T47D cells (5 × 10 5 cells/well) were seeded onto 6-well plates (Iwaki, Japan) in a final volume 2 mL/well. The cells were incubated with 2 mL/well of the three different concentrations of SD7 and incubated at 37 °C with 5% CO 2 for 24 h. The cells were treated with SD7 with three different concentrations (60.0, 120.0 and 180.0 µg/mL). After 24 h, the cells were harvested, collected and re-suspended in 100 μL of 1 × binding buffer containing 2 μL of Annexin V-FLUOS and 2 μL of PI and incubated for 15 min in the dark at room temperature, according to the manufacturer's recommendations (Roche, Germany). The results were then analyzed using flow cytometry. The analysis of the cell was executed by flow cytometry (FACScan; BD Biosciences) equipped with a Cell Quest software (BD Biosciences). Cells were divided to four quadrants namely living, early apoptotic, late apoptotic and necrotic cell.

Data analysis
Data were analyzed by using Microsoft Excel 2007 to obtain graph and linear regression for counting viability cell percentage and IC 50 value . The extract with smallest IC 50 indicates the highest cytotoxic activity.

RESULT AND DISCUSSION
The results of cytotoxic activity of P. cauliflora stem fractions on T47D cancer cell lines are presented in  Whereas, with the same concentration, the percentage of cancer cell viability for dichloromethane fraction at pH 3 was 93.33%, dichloromethane fraction at pH 10 was 79.30% and n-hexane fraction was 98.17%. For some different fractions, dichloromethane fraction at pH 7 (SD7) showed the highest cytotoxicity, with IC 50 value of 59.29 µg/mL. On another hand, the dichloromethane fraction at pH 10 (SD10) had the lowest cytotoxicity, with an IC 50 value of 130.32 µg/mL (Table 1). According to the fractions of cytotoxicity levels, the order of cytotoxicity was SD7 > SD3 > SH > SD9.
The cytotoxic effects of plant extracts are usually caused by secondary metabolites in them. We assumed that alkaloids were compounds that were responsible for cytotoxic activity of P. cauliflora stem. Bisbenzylisoquinoline alkaloid has been reported to have various interesting biological activities including cytotoxic activities (Atta-ur-Rahman, et al., 2004;Wang et al., 2010;Lv et al., 2013), anticholinesterase (Cometa et al., 2012) and antimicrobial activities (Lohombo-Ekomba et al., 2004). The potent cytotoxic activity of dichloromethane fraction at pH 7 might be related to the high concentration of bisbenzylisoquinoline. Studies of the species of genus Pycnarrhena has been reported the presence of secondary metabolite compounds  (Sioumis and Vashist, 1972;Loder and Nearn, 1972;Siwon et al. 1981;van Beek et al., 1982;Abouchacra et al., 1987). Bisbenzylisoquinoline alkaloids from P. ozantha have been showed antitumor activity (Loder and Nearn, 1972). Masriani (2014) isolated bisbenzylisoquinoline alkaloid from P. cauliflora root. The compound also demonstrated potent toxicity activity against cancer cells Hela, MCF-7, T47D, WiDr and Raji with IC 50 < 4 µg/ mL.
One of important properties possessed by anticancer drug candidate is an ability to induce apoptosis (Frankfurt and Krishan, 2003). To confirm whether the cytotoxic activity observed was due to apoptosis, the cell was stained by Annexin-V FLUOS/PI. Necrosis and apoptosis are two well-described pathways. As the most active substance, the dichloromethane fraction at pH 7 was selected for analysis of apoptosis on breast cancer cells T47D. The results of flow cytometer analysis (Table 2 and    dose, however the increase of total number of apoptosis was in line with increasing dose (Figure 3). The number of cells undergoing necrosis due to treatment of dichloromethane fraction at pH 7 is not increased when compared to cells undergoing apoptosis. These data clearly suggest that SD7 induced apoptosis in T47D cell line.

CONCLUSION
Dichloromethane fraction at pH 7 of P. cauliflora stem showed the highest cytotoxic activity on human breast cancer cell line T47D through induction of apoptosis. The result indicated the potential of dichloromethane fraction of P.cauliflora stem as a source of bioactive compounds and provided a basis of further studies to isolate a new anticancer compounds.