Antiproliferative activities of several plant extracts from Turkey on rat brain tumor and human cervix carcinoma cell lines

Abstract

Turkey has a wide range of flora and fauna due to its climatic diversity. Medicinal plants from Turkey have been used since ancient times for their primary health care. In this study, we examined antiproliferative activities of the extracts from Crataegus monogyna, Vitis vinifera, Glycrrhiza glabra, Alnus glutinosa L. gaertn, and Alcea rosea against rat brain tumor (C6) and human cervical cancer (HeLa) cell lines. The results were compared with the standard anticancer drugs 5-Flurouracil (5-FU) and Cisplatin. C. monogyna, V. vinifera and A. rosea exhibited better antiproliferative activity than 5-FU and cisplatin at 100-75 µg/mL concentrations, against C6 cell lines. On the other hand, C. monogyna and V. vinifera extracts showed considerable antiproliferative activity against HeLa cells compared with 5-FU and cisplatin at 100-75 µg/mL. It can be suggested that, C. monogyna, A. glutinosa L. gaertn, V. vinifera and A. rosea extracts could be developed as an anticancer drug.

GRAPHICAL ABSTRACT

Keywords:

• HeLa
• C6
• antiproliferative activity
• plant extract

Introduction

Cancer is one of the leading causes of death and rapidly becoming global pandemic (Jemal et al. 2009). Many factors play an important role in the development of cancer, such as lifestyle, environment, and nutrition. Cervical cancer is the second most common type of cancer among women worldwide. There were more than half a million new cases of cervical cancer diagnosed in 2010 (De Sanjose et al. 2010; Forouzanfar et al. 2011). Over the years, the cancer treatment methods have undergone revolutionary changes. On the other hand, chemotherapy of solid tumors is still limited by the lack of anticancer drugs; therefore, new medicines have yet to be developed to cure cancer (Ferguson et al. 2004).

Natural products have attracted considerable attention from synthetic community for reasons of wide range of plant diversity along with thousands of natural compounds. Previous studies have demonstrated the role and importance of herbs and spices in the prevention of cardiovascular diseases, carcinogenesis, inflammation, atherosclerosis, and so on. (Hossain et al. 2008). It is known that many medicinal plants contain effective chemopreventive and antitumor substances (Borrelli et al. 2004). But less than 1% of known 250,000 plant species are investigated for their secondary metabolites and biological activity (Farnsworth 1988).

In this present work, five medicinal plants from Turkey, namely Crataegus monogyna (Rosaceae), Vitis vinifera L. (Vitaceae), Glycrrhiza glabra (Fabaceae), Alnus glutinosa L. gaertn (Betulaceae), and Alcea rosea (Malvaceae), were investigated for their antiproliferative activities against C6 and HeLa cancer cell lines. The inhibition potential of plant extracts is aimed to be determined in terms of cell proliferation.

Materials and methods

Plant materials

A. rosea, C. monogyna, V. vinifera L., G. glabra, and A. glutinosa L. gaertn were collected from Tokat and Amasya at their flowering seasons and authenticated by Dr Bedrettin Selvi (Departmant of Biology, Gaziosmanpasa University, Tokat, Turkey). All species were dried in a dark place, at room temperature, until obtaining a stabile weight.

Preparation of extracts

Plant extract have been prepared according to literature method (Chon et al. 2009). 10 g of dried and grounded plant material was soaked in 200 mL methanol for three days at room temperature. The mixture was filtered and the solvent was evaporated under vacuum. Stock solution of the samples, 5-florouracil (5-FU) and cisplatin were prepared in sterile dimethyl sulfoxide (DMSO) and were diluted with Dulbecco's modified eagle's medium (DMEM; 1:20). The final concentration of DMSO was kept below 1% in all tests. The stock solutions were stored at ±4°C until usage.

Cell culture and cell proliferation assay

Antiproliferative effects of the plants were investigated against HeLa and C6 cell lines using proliferation BrdU enzyme-linked immuno sorbent assay (ELISA) (Demirtas et al. 2009; Demirtas & Sahin 2013). HeLa and C6 cells were cultured in DMEM, Sigma, supplemented with 10% (v/v) fetal bovine serum (Sigma, Germany) and PenStrep solution (Sigma, Germany). Cultured cells were detached from the flasks with trypsin-EDTA (Sigma, Germany). After centrifugation of the cells, pellets were resuspended to 3 × 10⁵ cells/mL in DMEM. Cells were plated in 96-well plates (COSTAR, Corning, USA) at a density of 3 × 10³ cells/well and incubated at 37°C with 5% CO₂ overnight for attachment. All the materials used in experiment were dissolved in sterile DMSO. Tests were carried out in triplicate for each experiment. Cells were treated with crude extracts at final concentrations of 5, 10, 20, 30, 40, 50, 75 and 100 µg/mL. Controls, negative and positive control wells were treated with culture medium, sterile DMSO, 5-FU and cisplatin, respectively. Treated cells were incubated at 37°C with 5% CO₂ for 24 h. Cell proliferation was measured by using BrdU Cell Proliferation ELISA (Roche, Germany), a colorimetric immunoassay based on BrdU incorporation into the cellular DNA according to manufacturer's procedure. Briefly, cells were pulsed with BrdU labeling reagent for 4 h followed by fixation in FixDenat solution for 30 min at room temperature. Thereafter, cells were incubated with 1:100 dilution of anti-BrdU-POD for 1.30 h at room temperature. Finally, the immune reaction was detected by adding the substrate solution and the color developed was read at 450 nm with a microplate reader.

Statistical analysis

The results of investigation in vitro are presented as means ± SD of three independent measurements. Statistical comparisons were tested with one-way ANOVA. The P values of <.01 were considered statistically significant. All statistical calculations were performed using SPSS (Version 13.5) software.

Results and discussion

Herbal medicine plays an important role in the prevention and treatment of cancer (Xiong et al. 2015). Traditional medicinal herbs such as C. monogyna (Ozyurek et al. 2012; Rodrigues et al. 2012), A. glutinosa L. gaertn, G. glabra, A. rosea and V. vinifera L. (Karaman & Kocabas 2001) have been used in the treatment of different diseases in Turkey. In this study, different parts of six plant species (Table 1) were tested for their antiproliferative activity against C6 and HeLa cancer cell lines. The antiproliferative potential of the plant extracts was compared with positive control drugs, 5-FU and Cisplatin. In terms of antiproliferative activity against C6 cell lines, C. monogyna extract exhibited the highest performance with its lowest IC₅₀ value (29.8 µg/mL) among other extracts (Table 2). C. monogyna (common hawthorn) is widely used in traditional medicine for its beneficial effects (Carvalho 2010). Recent studies revealed that, secondary metabolites are responsible from the bioactivity of the plants and the biological effect is mostly the result of synergy or additive effect of the other types of natural compounds (Ramful et al. 2011). Therefore, the higher antiproliferative activity of C. monogyna extract (87.33% at 100 µg/mL) can be attributed to its secondary metabolite content, mainly phenolic compounds. Main phenolics of C. monogyna were determined as quercetin derivatives and phenolic acids (Rodrigues et al. 2012). It is known that quercetin has inhibitory effect on various tumor cells (Kandaswami et al. 2005). In addition, phenolic acids of C. monogyna extract, such as gallic and caffeic acid derivatives, had shown antiproliferative effect against several cancer lines including HeLa cells (Gomes et al. 2003). Another study reported a systematic comparison of four different hawthorn parts relating their human inhibitory activity on human tumor cell lines. It was found that the flower buds extract of C. monogyna represents GI₅₀ (cell growth inhibition) value of 63.55 µg/mL for HeLa cells (Rodrigues et al. 2012).

Table 1. Selected plants used in the study for screening of antiproliferative activity.

Plants	Family	Parts used	Usage of the plants in folk medicine
A. glutinosa	Betulaceae	Leaves	Treatment of several types of cancer (Hartwell 1967), some of its constituents exhibit various biological properties, including anti-inflammatory and cytotoxic activities (Acero & Muñoz-Mingarro 2012).
C. monogyna	Rosaceae	Flowers	Treatment of ailments including high-blood pressure, heart and digestive disorders (Tadic et al. 2008)
V. vinifera	Vitaceae	Leaves	The leaves of the plant, which have astringent and hemostatic properties, are used in the treatment of diarrhea, hemorrhage, varicose veins, hemorrhoids, inflammatory disorder, pain, hepatitis, and free radical-related diseases and externally for centuries in Anatolia to heal wounds and drain furuncles (Bombardelli & Morazzonni 1995; Baytop 1999; Lardos & Kreuter 2000)
G. glabra	Fabaceae	Roots	Treatment of upper respiratory tract ailments including coughs, hoarseness, sore throat, and bronchitis (Saxena 2005)
A. rosea	Malvaceae	Flowers	A. rosea possesses anti-inflammatory, antibacterial, and analgesic effects (Wang et al. 1989; Mert et al. 2010; Seyyednejad et al. 2010). The roots of A. rosea have been used in Iranian traditional medicine for a wide range of ailments, including bronchitis, diuretic, diarrhea, constipation, inflammation, severe coughs, and angina (Aghili Khosarani & Makhzan-Al-Adviah 1992; Zargari 1992)

Table 2. Antiproliferative activities (IC₅₀ = µgmL⁻¹) of the plant extracts against C6 and HeLa cell lines.

	Antiproliferative activity IC50 (µg/mL)
Plant species	C6 cell line HeLa cell line	HeLa cell line
A. glutinosa L. gaertn	45.7	53.8
C. monogyna	29.8	34.0
V. vinifera L.	40.1	48.8
G. glabra	30.6	61.1
A. rosea	37.63	14.48

Note: Data were presented as mean ± SD (n = 3). IC₅₀: 50% inhibition of cell growth.

C. monogyna, V. vinifera, A. glutinosa L. gaertn, and A. rosea possessed considerable antiproliferative activity against C6 cell lines (P < .01, Figure 1). Inhibition of A. glutinosa and V. vinifera plant extracts against C6 cell lines was found to be 84.33% and 84.00%, at 100 µg/mL concentration, respectively. Not only V. vinifera, but also C. monogyna and A. glutinosa extract exhibited higher antiproliferative activity than standard drugs against HeLa cells at 75 µg/mL concentration (P < .01, Figure 2). Previous study showed that A. glutinosa (stem bark) exhibits inhibitory activity against the human LoVo colon cancer, PC3 prostate cancer, and U373 glioblastoma cell lines (Frederich et al. 2009). Main chemical constituents of A. glutinosa were reported as hirsutanonol, oregonin, genkwanin, rhododendron, and glutinic acid (Guz et al. 2002; O'Rourke et al. 2005) which can be attributed its biological activity. Genkwanin, rhododendrin, and glutinic acid are the main constituents of A. glutinosa L. gaertn (O'Rourke et al. 2005). It can be suggested that the main constituents of the plant extracts including anthocyanins and polyphenols play an important role in the inhibition of C6 and HeLa cancer cell lines.

Figure 1. The antiproliferative activities of the plant extracts against C6 cell lines. The results were expressed as percentage of cell proliferation inhibition compared with standard drugs. Data were presented as mean ± SD (n = 3).

Figure 2. The antiproliferative activities of the plant extracts against HeLa cell lines. The results were expressed as percentage of cell proliferation inhibition compared with standard drugs. Data were presented as mean ± SD (n = 3).

Considering the antiproliferative activity described by the popular use of C. monogyna, the results of antiproliferative activity tests against HeLa and C6 cell lines supported the ethnomedicinal use of this plant. C. monogyna had higher antiproliferative activity against C6 cells than positive controls, 5-FU and Cisplatin at 50–100 µg/mL concentrations (P < .01, Figure 1).

For HeLa cells, the highest inhibition value (89.33%) was obtained with V. vinifera extract at 75 µg/mL concentration (P < .01, Figure 2). On the other hand, V. vinifera extract effect decreases in lower concentrations (5–50 µg/mL). Different parts of V. vinifera have been widely investigated against different types of cancer (Kaliora et al. 2008; Amico et al. 2009; Lazze et al. 2009; Sung & Lee 2010; Nechita et al. 2012; Apostolou et al. 2013; Esfahanian et al. 2013; Espino et al. 2013; Kountouri et al. 2013; Giovannelli et al. 2014; Liang et al. 2014; Sahpazidou et al. 2014) and it has been reported that V. vinifera has an antiproliferative effect against many cancer cell lines. Naturally occurring bioactive component of V. vinifera that is responsible from the cardioprotective and cancer chemopreventive activities (Jang et al. 1997) is identified as resveratrol (3,4′,5-trihydroxystilbene) (Frankel et al. 1993).

Finally, we investigated the antiproliferative activity of G. glabra extract, which exhibited considerable antiproliferative activity against HeLa cell lines (Figure 2) at the highest concentration (100 µg/mL). It is reported that G. glabra showed biological activity similar to that of anti-microtubule drugs which are widely used for the treatment of malignancy (DiPaola et al. 1999; Rafi et al. 2002). The plant-derived phytochemicals are able to inhibit the proliferation and apoptosis in tumor cells and therefore there is an increasing interest in plant secondary metabolites and their antiproliferative activities (Alesiani et al. 2010).

Previous studies on A. rosea plant were focused on its biological activities such as hepatotoxicity (Hussain et al. 2014), tyrosinase inhibitory activity (Namjooyan et al. 2011), antibacterial activity (Seyyednejad et al. 2010). Studies on antiproliferative activity of A. rosea plant are not sufficient.

The finding from our study showed that C. monogyna, V. vinifera, and A. glutinosa L. gaertn exhibited high antiproliferative activity against C6 and HeLa cancer cell lines at the concentration of 75 and100 µg/mL. This may indicate that extracts of the plants may have potential antiproliferative activity for different cancer cell lines.

Conclusions

In this study, we report antiproliferative activities of several plant extracts, which are used in Turkish folk medicine, against HeLa and C6 cell lines. According to the results obtained, it is feasible to speculate that C. monogyna, V. vinifera, A. glutinosa L. gaertn, and A. rosea plant extracts could be a promising alternative natural source of chemotherapy agents.

Acknowledgements

The authors thank Dr Ali Karagoz and Prof. Nazlı Arda from Istanbul University, Department of Molecular Biology, for help in providing C6 (Rat Brain tumor cells) and HeLa (human cervix carcinoma) cells. The authors are also grateful to Dr Nihal Deligonul for the grammatical revision of the manuscript. This work is funded by Gaziosmanpasa University Scientific Research Projects (BAP-File No.2010/39), Tokat, Turkey.

Disclosure statement

No potential conflict of interest was reported by the authors.