Anticancer Potential of Cratoxylum formosum Subsp . Pruniflorum ( Kurz . ) Gogel Extracts Against Cervical Cancer Cell Lines

Cervical cancer is common in the world with an estimated 527,624 new cases and 275,008 deaths of the total cancer among females in 2012 (Ferlay et al., 2013). In Thailand, it is the second diagnosed in patients compared with other cancers (Wilailak, 2009). Human papillomavirus (HPV), especially the high risk HPV type 16 and 18, is associated with cervical carcinogenesis (zur Hausen, 1999). Cisplatin and 5-fluorouracil (5-FU) are used to treat cervical cancer but there are reports about their drug resistance (Shen et al., 2012) and side effects throughout the treatment (Sun et al., 2014). Low expression of Raf kinase inhibitory protein (RKIP) and high expression of ERCC1 mRNA resistance to cisplatin treatment in cervical cells were related with tumor progression, metastasis and drug resistance (Britten et al., 2000; Martinho et al., 2013). Therefore, many studies are


Introduction
Cervical cancer is common in the world with an estimated 527,624 new cases and 275,008 deaths of the total cancer among females in 2012 (Ferlay et al., 2013).In Thailand, it is the second diagnosed in patients compared with other cancers (Wilailak, 2009).Human papillomavirus (HPV), especially the high risk HPV type 16 and 18, is associated with cervical carcinogenesis (zur Hausen, 1999).Cisplatin and 5-fluorouracil (5-FU) are used to treat cervical cancer but there are reports about their drug resistance (Shen et al., 2012) and side effects throughout the treatment (Sun et al., 2014).Low expression of Raf kinase inhibitory protein (RKIP) and high expression of ERCC1 mRNA resistance to cisplatin treatment in cervical cells were related with tumor progression, metastasis and drug resistance (Britten et al., 2000;Martinho et al., 2013).Therefore, many studies are RESEARCH ARTICLE Anticancer Potential of Cratoxylum formosum Subsp.Pruniflorum (Kurz.)Gogel Extracts Against Cervical Cancer Cell Lines Bundit Promraksa 1 , Jureerut Daduang 2 , Tueanjit Khampitak 1 , Ratree Tavichakorntrakool 2,3 , Amonrat Koraneekit 2 , Adisak Palasap 2 , Roongpet Tangrassameeprasert 1 , Patcharee Boonsiri 1 * carried out to search for new potential anticancer agents from natural sources (Sak, 2012).It was reported that phytochemicals in vegetables and fruits such as flavonoids can reduce risk of cancer (Romagnolo and Selmin, 2012).Quercetin can induce cell-cycle arrest in HeLa cell line (Vidya et al., 2010).Gallic acid have potential to inhibit cancer cell progression via PI3K/Akt pathway (Zhao and Hu, 2013) and to induce apoptosis (Palasap et al, 2014).

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isolated from 95% Teaw leaves ethanolic extract play role as anti-neuroinflammatory agents (Xiong et al., 2014).The anticancer potential of Teaw extracts was reported on HepG2 cell line by increasing caspase 3/7, 8, and 9 related apoptosis pathway (Nonpunya et al., 2014) and decreased the level of circular DNA of hepatitis B virus (HBV).Cratoxylum formosum subsp.pruniflorum (Kurz.)Gogel., Thai name "Teawdang" (Figure 1) is a subspecie of Cratoxylum.As there is a limited information about Teawdang and its effect on cervical cancer, therefore, this study aims to investigate the cytotoxic effect of Teawdang extracts on cervical cancer cell lines.Phenolic contents and antioxidant activity of Teawdang were also determined.

Preparation and extraction of vegetable materials
Teawdang was purchased from local markets in Khon Kaen province, Thailand during June to October 2013.The edible parts (stems and leaves) of Teawdang (2,870 g) were washed with distilled water and dried at 50˚C in a hot air oven.Dried vegetable was grinded to fine powder (557 g) and extracted by soaking in one liter of hexane for 5 days at room temperature.Next it was filtered through Whatman® No. 4 paper and hexane was evaporated by using a rotary evaporator (Buchi, Switzerland).The residue obtained was "crude hexane extract (CHE)".Then, the residue was further extracted with ethyl acetate followed by methanol respectively by the same procedure."Crude ethyl acetate (CEE)" and "crude methanol extract (CME)" were obtained respectively.The residue left was macerated in deionized water for 5 days at room temperature.Afterthat "crude water extract (CWE)" was obtained by removing water using a lyophilizer.All the extracts were stored at 4 ˚C for further analysis.The yield in percentage was calculated by using an equation below : % yield = (weight of extract after solvent evaporation/ weight of the vegetable powder) x 100

High performance liquid chromatographic (HPLC) profile of crude extracts
The HPLC analysis of crude extracts was performed according to a method of Palasap et al (2014).Standard compounds including gallic acid, catechin, caffeine, caffeic acid, ferulic acid, resveratrol and quercetin and each crude extract were dissolved in methanol to make final concentration 1 mg/mL.Then 100 µL of each sample was passed through a 0.22 µm pore size filter before injection to a column (C18 reversed phase, 5 µm particle size, 150x4.6 mm, Phenomenex).Mobile phase used was a step-gradient mixture of methanol and 0.5% phosphoric acid (methanol : 5%phosphoric acid ratio 5 : 95%, 70 : 30%, 90 : 10% and 5 : 95% at 0-17, 17-18, 18-20.5 and 20.5-25 min, respectively) with a flow rate of 1 mL/min.The compounds eluted were detected by using a spectrophotometer (Model 2489, Waters) at wavelength 270 nm.The retention time was recorded and peak areas of these compounds were calculated by using Clarity software.

Cell culture
The cell lines used in this study were Vero (Cercopithecus aethiops kidney normal), HeLa (adenocarcinoma with HPV 18 positive), SiHa (squamous cell carcinoma grade II with HPV 16 positive) and C-33A (carcinoma with non HPV infection) cells.All cell lines were cultured in DMEM with 10% BSA and 1% penicillin-streptomycin.

Cytotoxicity test
The cytotoxicity test of crude extracts on cervical cancer cell lines were performed in a 96-well plate.One hundred and sixty microliters of each suspension cell (8,000 cells/mL) was seeded into each well.Each crude extract (40 µL), diluted with 5%DMSO to make final concentration 50-400 µg/mL and filtered through a 0.22 µm pore size filter (Corning, USA), was added.After that, they were incubated at 37ºC for 24 hrs under 5% carbondioxide atmosphere.Negative control was 5%DMSO.
After incubation for 24 hrs, the existing media was removed and cell viability was detected by neutral red (NR) uptake assay.The pre-filtrate NR media (0.066 mg/mL) was passed through a 0.22 µm pore size filter and 200 µL of this solution was pipetted to each well in the plate.They were incubated for 3 hrs and then media was removed.Phosphate buffer saline (PBS) pH 7.4 was used for washing the cells for 2 times and 200 µL of lysis buffer (50% methanol and acetic acid at a ratio of 99:1) was added to break cell membrane.The absorbance at 540 nm was recorded by using a plate reader (Rayto, China).
The half maximal inhibitory concentration of crude extract (IC50) against cervical cancer cell lines were calculated by plotting the percent of cell inhibition (as in an equation below) versus the crude extract concentrations (Machana et al., 2011).

Statistic analysis
The results were expressed in mean±SD.Statistical significance was analyzed by one-way ANOVA using the SPSS software (version 17).P-values less than 0.05 (p<0.05) was considered statistically significant.

High performance liquid chromatographic profile of crude extracts
The yield of CHE, CEE, CME and CWE were 5.49, 2.95, 5.33 and 3.37% respectively.Their HPLC retention time and percentage of peak area of standard phenolic compounds and crude extracts were shown in Table 1.The total peak area of CHE, CEE, CME and CWE were 14675.76, 36396.23, 39307.13 and 59008.64mV•s respectively.Area under curve (AUC) was analyzed for phenolic concentration.The results demonstrate that all crude extracts contain caffeine, ferulic acid and resveratrol.Gallic acid was found in CEE, CME and CWE (14.48,10.02,8.78µg/mL respectively) whereas catechin was found only in CWE (50.07 µg/mL).CEE and CME showed peaks that matched with the retention time of gallic acid, caffeine, ferulic acid, resveratrol and quercetin.

Cytotoxicity of crude extracts on cervical cancer cell lines
In Table 2, CHE was not toxic to all the studied cell lines whereas CEE had cytotoxic effect on HeLa and SiHa.CME was toxic to all tested cell lines and its IC50 for C-33A was lower than the other extracts.CWE exhibited cytotoxic only for C-33A.All extracts, except CME, were not toxic to Vero.Phytochemical compound including gallic acid and quercetin were cytotoxic to all cell lines with lower IC50 for SiHa and HeLa than CEE and CME.The inhibitory efficacy of each crude extract (200 µg/mL) on cancer cells is shown in Figure 2. Compared to Vero cell, CEE can inhibit HeLa and SiHa cell lines significantly (p=0.032 and 0.008 respectively).For C-33A cell line, CWE significantly decreased the cell viability with slightly effect on Vero cell (p=0.001).

Discussion
In Thailand, the average incidence of cervical cancer    was about 16.7 per 100,000.There were 21.7, 16.4, 14.9 and 14.4 per 100,000 in the north, central, northeast and south of Thailand respectively.In the northeast area, Ubon ratchathani, Nakon Phanom and Khon Kaen were only 13.4, 13.8 and 15.1 per 100,000 respectively (Khuhaprema et al., 2013).As there is lower incidence rate of cervical cancer in northeast Thailand where Teawdang is normally consumed as side dish, therefore, phytochemicals in Teawdang and the other northeast Thai vegetables may involve in preventing and/or cytotoxic to cervical cancer.
In the present study, Teawdang was extracted by increasing polarity of solvents, from nonpolar (hexane) to high polar solvent (water).According to a previous study, gallic acid was soluble in many organic solvents used including methanol, ethyl acetate and water (Daneshfar et al., 2008).Hence, gallic acid was detected in CEE, CME and CWE in HPLC profile analysis (Table 1).Only catechin was found in CWE.This was similar to a previous report that catechin was found in aqueous extracts of Cladogynos orientalis (Machana et al., 2011).The high concentration of caffeine and caffeic acid were found in CWE followed by CME and CEE because of increasing solvent polarity.Caffeic acid can form ester with quinic acid to give a compound called "chlorogenic acid", which has antioxidant activity (Xu et al., 2012).This result agreed with a report that Teaw ethnolic extract contained chlorogenic acid, ferulic acid and dicaffeoylquinic acid (Maisuthisakul et al., 2007).The highest resveratrol found in CME was similar to the Gouania leptostachya methanolic extract (Dung et al., 2014).As the extracted phenolic compounds are related to polarity of the solvent used, this implies that the edible part of Teawdang contains more polar phenolic compounds than non-polar compounds.On the other hand, Teaw root extracted with hexane had anthraquinones such as formoxanthone, xanthone, gerontoxanthone I and madagascin which had antibacterial activity (Boonsri et al., 2006;Lee et al., 2014).
Different phenolic content in each crude extracts may cause different effects on cervical cancer cells (Di Domenico et al., 2012).Both CME and CEE contained gallic acid.Gallic acid was also found in Caesalpinia mimosoides Lamk (Kaya) extracts which had cytotoxic effect to cervical cancer cell lines (Palasap et al., 2014).In the present study, CEE was cytotoxic to SiHa and HeLa, which are HPV-infected cell lines, respectively.The study of Waiyaput et al. (2012) that Teaw can decrease hepatitis B virus and it was cytotoxic to liver cancer cell lines (HepG2 cell) supported our results that Teawdang extracts were cytotoxic to HPV infected cell lines.
Among Teawdang extracts, CME showed the lowest IC50 on C-33A cells, which is HPV-non infected cell lines, but not Vero cells.For standard treatment of cervical cancer, it was reported that C-33A cells were the most response to combination of chemotherapy and radiation (Saxena et al., 2005) which may cause side effects to the patients.In conclusion, Teawdang extracts had cytotoxic effect on both HPV-infected and HPV-non infected cervical cancer cell lines.Purification and identification of active compounds from CME and CEE should be further investigated.

Figure 2 .
Figure 2. Cell Viability of Various Cervical Cancer Cell Lines, After Treated with Crude Teawdang Extracts (200 µg/mL), Compared to Vero Cell Line.Data are expressed as mean±SD; * = statistically significant (p<0.05)