Cytotoxic activity of crude saponins from Gaultheria trichophylla against human breast cancer cells MCF-7 and MDA-MB-468

Fiaz Alam¹, Qazi Najam us Saqib¹ and Abdul Waheed²

¹Department of Pharmacy, COMSATS Institute of Information Technology, Abbottabad, Pakistan; ²Wolfson Cell Science Centre, Faculty of Health & Medical Sciences, University of Surrey, Guildford, UK.

Abstract

This study was conducted to evaluate Gaultheria trichophylla crude extract and respective saponins fraction against human breast cancer cell lines. In MTT assay, cell viability was inhibited by G. trichophylla crude extract (500 µg/mL) and saponins (200 µg/mL) in a dose dependent manner with maximum inhibition of (82% and 85%) and (71% and 42%) against MCF-7 and MDA MB-468, respectively. In neutral red uptake assay, the cell viability was inhibited by crude extract and saponins (100 µg/mL) in a similar manner with maximum inhibitions of (96% and 93%) and (87% and 61%) against MCF-7 and MDA MB-468, respectively, with respect to 91% and 93% inhibition by actinomycin-D (4 µM). The DAPI (4',6-diamidino-2-phenylindole) (10 µg/mL) staining of MCF-7 cells treated with crude saponins showed shrunken nuclei with apparent nuclear fragmentation indicating apoptosis and in contrast, MDA MB-468 showed necrosis mode of cell death. The study exhibited that the G. trichophylla provides new evidences to further explore this plant for the novel targets in anticancer drug development.

Introduction

With more than 10 million new cases every year, cancer has become one of the most devastating diseases worldwide. Natural products have been the source of most of the active ingredients of medicines. According to a report half of the drugs approved since 1994 are based on natural products. Thirteen natural-product-related drugs were approved from 2005 to 2007. They cover a range of therapeutic indications, mainly the anti-cancer (Farnsworth and Soejarto, 1991). Therefore, as always, there is need to find alternative drugs with low toxicity, more effective and accessible to common man. Therefore, the present study was carried out to investigate the anticancer activity of extract and crude saponins fraction from Gaultheria trichophylla in cultured MCF-7 and MDA MB-468 human breast cancer cells.

G. trichophylla Royle (Family Ericaceae) is native to the Himalayas and therefore common name is Himalayan Snowberry. Plants belonging to genus Gaultheria like G. yunnanensis, G. fragrantissima and G. Procumbens are reported to have anti-inflammatory activity, antibacterial activity, and is used to treat arthritis. Gaultherin a natural salicylate isolated from G. yunnanensis possesses analgesic and anti-inflammatory activities. The phytochemical investigation of the species investigated reported to contain methyl salicylate, diterpenoids, acids, dilactone, alkaloids and other glycosides. Cytotoxic activities against the selected cancer cell lines are also reported in plants like G. itoana and G. yunnanensis

Materials and Methods

Chemicals: Neutral red solution, fetal bovine serum (FBS), actinomycin-D, Dulbecco’s modified Eagle medi-um (DMEM) and 4,6-diamidineo-2-phenylinldole (DAPI) were obtained from Sigma Chemical Co. (St Lois, MO, USA). MCF-7 and MDA-MB-468 cells were purchased from ATCC (American Type Culture Collection, USA) through an authorized distributor, LGC Standards, Teddington, UK.

Plant material: G. trichophylla plant (5 kg) was collected by the author from Kaghan valley, District Mansehra, KPK, Pakistan, in November, 2013. After authentication of the plant, its voucher specimen (CTPHM-GT01, 13) was deposited in the herbarium of the Department of Pharmacy, the COMSATS Institute of Information Technology, Abbottabad. The whole plant was washed under running water and dried in shade at room temperature and was ground to a coarse powder. The powder drug was stored in air tight and light resistant container before extraction.

Preparation of extract and crude saponins: The dried, powdered plant material (200 g) was extracted with methanol using soxhlet extractor for 20 hours. It was filtered through a Whatman Grade-I filter paper. The filtrate was evaporated on a vacuum rotary evaporator under reduced pressure at 40°C. Extracted percent yield of the methanol fraction (G. Me) was 21.9%. For the extraction of saponins from powdered materials (200 g) of whole plant of G. trichophylla, was extracted first with petroleum ether, and followed by extraction with methanol in soxhlet apparatus. The solvent was reduced on a rotary evaporator under vacuum to obtain dry semisolid extract. The methanol extract of the plant was further fractionated with n-butanol and water, in equal proportion. The n-butanol fraction was separated. The crude saponins were precipitated with petroleum ether; yield was approximately 4.5 g of crude saponins (G. Sa) (Borenfreund et al., 1988; Qadir et al., 2014) growth of cancer cells was quantified. Following a 24 hours exposure test period of drugs, cells were washed twice with phosphate buffer saline (PBS), and a 10 μL of MTT reagent (5 mg/mL in PBS) was added to each well including the blanks, which contained medium only. The plates were returned to the incubator for 4 hours at 37°C. Subsequently, cells were washed twice with PBS, and 100 µL/well DMSO was added in each well as solvent to dissolve the insoluble crystalline formazan products. The effect of plant extracts on cancer cells was quantified as the percentage of control absorbance of reduced dye at 550 nm on a microplate reader (LabtechLT-4000MS, Labtechm International Ltd., East Sussex, and UK). For each treatment, five replicates wells were examined, and each experiment was repeated three times. Standard error of mean was calculated between three experiments. The results were calculated as percentage growth inhibition, untreated (control) cells versus treated cells according to the following formula:

Absorbance of the media was subtracted, both from control and treated cells.

NRU assay: The neutral red uptake assay was performed according to the method of (Saha et al. , 2013).

Data presentation and statistical analysis: All data were compiled from a minimum of three experiments. Data for statistical analysis were expressed as the mean ± SEM. One-way ANOVA with Dunnett’s test, as specified, was performed using GraphPad Prism version 6.00 for Windows, GraphPad Software, San Diego California, USA.

Results

The results from the MTT and NRU assays of cellular viability in various concentrations of methanolic crude extract (G.Me) of G. trichophylla are listed in Table I. MCF-7 and MDA MB-468 cells were inhibited by crude extract with a rate increasing with increasing concentration. The inhibition rate of G. Me (500 μg/mL) against MCF-7 and MDA MB-468 cells was about 82% and 85% respectively in MTT assay. The inhibition rate of G. Me (500 μg/mL) against MCF-7 and MDA MB-468 cells was about 96% and 93% respectively in the NRU assay (Table I).

Table I

The inhibition of crude saponins of G. trichophylla (G. Sa) against MCF-7 and MDA MB-468 cancer cell lines were tested in dose of 200 μg/mL for MTT assays and in dose of 100 μg/mL for NRU assay. In MTT assay the saponins inhibited the growth of MCF-7 and MDA MB-468 by about 71% and 42%, respectively, with respect to actinomycin-D (4 µM) which showed the growth inhibition of about 62%. The response to G. Sa was more pronounced in NRU assay, and a growth inhibition of about 87% and 61% was observed against MCF-7 and MDA MB-468, respectively, with respect to actinomycin-D (4 µM) which showed the growth inhibition of 91% and 93%, respectively (Table II and III).

Table II

Table III

Both the untreated (control) and G. Sa (10 µg/mL) treated cells were stained with DAPI and visualized by confocal microscope (Figure 1). The control cells of MCF-7 were found with normal morphology and intact nuclei, while the treated cells were exhibiting the apoptotic characteristics, with condensed and fragmented nuclei, along with marked shrinkage in the cell membranes. This was a clear indication of apoptosis induced in MCF-7 cancer cells. DAPI staining of G. Sa treated MDA MB-468 cells showed necrotic cells characterized by swelling and increasing in size before the disruption.

Figure 1: Morphological assessment of control (A and B) and Gaultheria trichophylla saponins (G. Sa) (10 µg/mL, final concentration) treated MCF-7 cells (A2) and MDA MB-468 (B2) for 24 hours. The nuclei were stained with DAPI and observed under confocal microscope

Discussion

In the recent past, there has been unforeseen interest in the clinical utilization of saponins as chemotherapeutic agents and over 400 studies have been reported regarding the saponins and their ability to treat cancer or induce apoptosis. It is reported that saponins exert its cytotoxic activity through apoptosis through signaling pathways to prevent the tumor. Numerous steroid and triterpenoid saponins exhibit cytotoxic properties (Li et al., 2010). Our previous study also showed that the crude saponins caused the inhibition of colon cancer cells lines through mechanism involving apoptosis (Lau et al., 2004). MTT assay results showed that G. Me and G. Sa showed dose dependent antiproliferative activity and is effective in inhibiting the growth of MCF-7 and MDA MB-468 cells. The neutral red (NR) metabolic impairment assay also showed that G. Me and G. Sa inhibited the growth of MCF-7 and MDA MB-468 cells.

Apoptosis is a programmed cell death process that occurs in physiological and pathological conditions and a defect in apoptotic pathways has an important role in carcinogenesis. It is characterized by cell shrinkage, chromatin condensation, DNA fragmentation and formation of apoptotic bodies. The induction of apoptosis for the prevention of cancer is therefore desirable. Necrotic cell death, in contrast, is characterized by the loss of metabolic functions, and of the integrity of the cell membrane. Structurally, the cells’ organelles swell and become nonfunctional during the initial stages of necrosis (Saha et al., 2013). In this study G. Sa induced morphological changes in apoptotic cells, which were observed in DAPI staining of MCF-7 cells.

Conclusion

This study demonstrates that the treatment with G. Sa resulted in the apoptotic body formation, chromatin condensation and nuclear fragmentation. It clearly shows the potential of G. Sa to induce apoptosis against MCF-7 cancer cell lines. While, on the other hand DAPI staining of MDA MB-468 cells indicated necrosis mode of cell death.

References

Alam F, Saqib QN, Waheed A. Effect of crude saponins from Gaultheria trichophylla extract on growth inhibition in human colorectal cancer cells. Bangladesh J Pharmacol. 2015; 10: 160-65.

Borenfreund E, Babich H, Martin-Alguacil N. Comparisons of two in vitro cytotoxicity assays: The neutral red (NR) and tetrazolium MTT tests. Toxicol In Vitro. 1988; 2: 1-6.

Borenfreund E, Puerner JA. Toxicity determined in vitro by morphological alterations and neutral red absorption. Toxicol Lett. 1985; 24: 119-24.

Cassileth BR, Chapman CC. Alternative and complementary cancer therapies. Cancer. 1996; 77: 1026-34.

Cragg GM, Newman DJ, Snader KM. Natural products in drug discovery and development. J Nat Prod. 1997; 60: 52-60.

Dande PR, Talekar VS, Chakraborthy G. Evaluation of crude saponins extract from leaves of Sesbania sesban (L.) Merr. for topical anti-inflammatory activity. Int J Res Pharm Sci. 2010; 1: 296-99.

Farnsworth NR, Soejarto D. Global importance of medicinal plants. The conservation of medicinal plants. 1991, pp 25-51.

Green DR, Kroemer G. The pathophysiology of mitochondrial cell death. Science 2004; 305: 626-29.

Lau C, Ho C, Kim C, Leung K, Fung K, Tse T, et al. Cytotoxic activities of Coriolus versicolor (Yunzhi) extract on human leukemia and lymphoma cells by induction of apoptosis. Life Sci. 2004; 75: 797-808.

Li J, Li F, Lu Y-Y, Su X-J, Huang C-P, Lu X-W. A new dilactone from the seeds of Gaultheria yunnanensis. Fitoterapia 2010; 81: 35-37.

Liu W-R, Qiao W-L, Liu Z-Z, Wang X-H, Jiang R, Li S-Y, et al. Gaultheria: Phytochemical and pharmacological characteristics. Molecules 2013; 18: 12071-108.

Nanji AA, Hiller-Sturmhöfel S. Apoptosis and necrosis. Alcohol Health Res World. 1997; 21: 325-30.

Qadir MI, Ali M, Ibrahim Z. Anticancer activity of Morus nigra leaves extract. Bangladesh J Pharmacol. 2014; 9: 496-97.

Saha SK, Sikdar S, Mukherjee A, Bhadra K, Boujedaini N, Khuda-Bukhsh AR. Ethanolic extract of the Goldenseal, Hydrastis canadensis, has demonstrable chemopreventive effects on HeLa cells in vitro: Drug–DNA interaction with calf thymus DNA as target. Env Toxicol Pharmacol. 2013; 36: 202-14.

Thakur M, Melzig MF, Fuchs H, Weng A. Chemistry and pharmacology of saponins: Special focus on cytotoxic properties. Botanics Targets Therapy. 2011; 1: 19-29.