Antitumor activity of Pachycereus marginatus ( DC . ) Britton & Rose extracts against murine lymphoma L 5178 YR and skin melanoma B 16 F 10 cells

Pachycereus marginatus (DC.) Britton & Rose is a species belonging to the family Cactaceae. In traditional medicine, it is recommended to treat diabetes and gastrointestinal infections; however, there are no studies related to its use in cancer treatment. The in vitro antitumor effect of P. marginatus hexane, chloroform, methanol, and methanol-aqueous partition stem extracts, against murine lymphoma L5178Y-R and skin melanoma B16F10 cells, was evaluated in liquid medium by the colorimetric 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) reduction assay. The extracts resulted in up to 84, 85, 84, and 82% cytotoxicity (p < 0.05) to L5178Y-R cells, respectively, and up to 39, 51, 48, and 42% cytotoxicity (p < 0.05) to B16F10 cells, respectively. Vehicle controls were not cytotoxic for tumor cells, and along with the extracts they did not affect viability of resident murine thymus and spleen lymphocytes. Taken together, the present results showed that P. marginatus extracts possess antitumor potential against L5178Y-R lymphoma and B16F10 skin melanoma cells.


INTRODUCTION
In the search for alternatives to treat diseases, researchers worldwide have taken the task of finding native plants with potential health benefit.Among them, numerous cacti species have been reported to possess *Corresponding author.E-mail: rgomez60@hotmail.com.Tel: (83) 29-41-10 x 6453.Fax: (83) 52-42-12.
Author(s) agree that this article remain permanently open access under the terms of the Creative Commons Attribution License 4.0 International License antitumor properties (Shetty et al., 2012;Harlev et al., 2013;Lema-Rumińska and Kulus, 2014).Pachycereus marginatus (DC.)Britton & Rose (Caryophyllales: Cactaceae), also known as Stenocereus marginatus,Cereus marginata, Central Mexico organ pipe, organo, Jarritos, Chilayo, and Mexican fencepost cactus, belongs to the Pachycereeae tribe.It has columnar trunks that can reach 20 m high and is commonly used as fodder, living fences, fuel wood, and as an alternative medicine.The species is endemic to Mexico, where it grows wild in states that have a dry and hot climate such as Nuevo León, Guanajuato, Aguascalientes, Oaxaca, Puebla, San Luis Potosí, Zacatecas, Ciudad de México, Tamaulipas, Guerrero, Michoacán, Hidalgo, Jalisco, Morelos, Veracruz, Tlaxcala, and Querétaro (Hernández et al., 2004), and in Texas, New Mexico, Arizona, and Southern California (Paredes-Flores et al., 2007;Arias and Terrazas, 2009).P. marginatus is traditionally used to treat gastrointestinal infections (Hernández et al., 2003); its antimicrobial potential in plants, animals, and humans has been reported, as well as its activity to improve wound healing, and promote plant growth (Jordan-Hernandez, 2012).However, the antitumor potential of P. marginatus has not yet been reported.
Cancer results from the interaction of internal (as genetic predisposition) and external factors that lead to cell degeneration, resulting in precancerous lesions and ultimately malignant tumors.If it is not promptly treated, cancer cells can spread to other organs (metastasis).
Cancer is one of the main causes of morbidity and mortality worldwide, with about 14 million new cases and 8.2 million deaths in 2012 and new cases are expected to increase 22 million in the next 20 years (Stewart and Wild, 2014).In men, most cancers affect prostate, lungs, and gastrointestinal tissues; in women, cancers in the breast, lung, cervix, and stomach are common (Stewart and Wild, 2014).In Mexico, according to the International Union Against Cancer, cancer is the third leading cause of death and estimated that each year 128,000 new cases are reported (Stewart and Wild, 2014).
The aim of the present study was to evaluate the in vitro cytotoxic activity of the hexane, chloroform, methanol, and aqueous methanol partition extracts of P. marginatus against murine lymphoma L5178Y-R and skin melanoma B16F10 cells.

Preparation of plant extracts
Pachycereus marginatus used in the present study was identified with voucher number 025588.Stems were rinsed with tap water to eliminate dust and other contaminating material, dried at 37°C for 36 h, and pulverized.Sixty grams of the powdered stems were sequentially extracted with 600 ml of hexane, chloroform, and methanol by Soxhlet system during 40 h each.The extracts were concentrated to dryness using a rotary evaporator Büchi (Brinkmann Instruments Inc., Switzerland) and stored at 6°C until use and dissolved in distilled water for biological activity testing.The methanol-aqueous partition of the extract was prepared by using 11 g of methanol extract and dissolving it in methanol, then in hexane, and shaking for 10 min.The resulting solution was separated with a funnel and washed with ethyl acetate.This solution was washed with water and then dried (referred as aqueous partition).Stock solutions were then prepared at 1 mg ml -1 in complete RPMI 1640 (for L5178Y-R cells), AIM-V medium (for lymphocyte cultures) or DMEM medium (for B16F10 cells), and sterilized by filtering through a 0.22-microns membrane (Millipore, Bedford, MA).P. marginatus hexane, chloroform, methanol, and methanol-aqueous partition stem extracts were tested at concentrations ranging from 0.03 to 500 µg ml -1 .

L5178Y-R and B16F10 cells preparation and culture
In order to determine the direct in vitro effect of the extracts on tumor cell growth, L5178Y-R cell and B16F10 cultures were collected and the cellular suspensions obtained were washed three times in RPMI 1640 (for L5178Y-R cells) or DMEM medium (for B16F10 cells), and suspended and adjusted to 5 × 10 4 cells ml -1 in those culture media.One hundred microliters of the cell suspensions were then added to flat-bottomed 96-well plates (Becton Dickinson, Cockeysville, MD), containing 100 µl triplicate cultures of complete RPMI 1640 or DMEM media (unstimulated controls), the extracts at various concentrations (extracts were dissolved in complete RPMI 1640 (for L5178Y-R cells) or DMEM medium (for B16F10 cells), extract-free vehicles (vehicles were similarly processed as with P. marginatus extracts, but without plant material), and vincristine as a positive control.After incubation for 44 h at 37°C with 5% CO2, MTT (0.5 mg ml -1 , final concentration) was added, and cultures were additionally incubated for 4 h.Next, cell cultures were incubated for 16 h with extraction buffer (100 µl well -1 ) and optical densities, resulting from dissolved formazan crystals, were then read in a microplate reader (DTX 880 Multimode detector, Becton Dickinson, Austria) at 570 nm (Gomez-Flores et al., 2009).The percentage of cytotoxicity was calculated as follows: % Cytotoxicity = 100-[(A570 in extract treated cells/ A570 in untreated cells) × (100)] % Cytotoxicity, as compared with untreated control (culture medium).Optical density for untreated control was 0.83 ± 0.006.Data represent mean ± SE of three replicate determinations from three independent experiments.Vehicle controls for hexane, methanol, chloroform, and methanol-aqueous partition were not cytotoxic for L5178Y-R cells; vincristine caused about 75% cytotoxicity to L5178Y-R cells at concentrations ranging from 0.24 to 125 µg•ml -1 .*p < 0.05, **p < 0.01 , compared with untreated control.

Animals
Six-week-old female BALB/c mice (22 to 28 g) were purchased from Harlan Sprague-Dawley Inc. (Indianapolis, IN, USA).They were kept in a pathogen-and stress-free environment at 24°C, under a light-dark cycle (light phase, 06:00 to 18:00 h), and given water and food ad libitum.

Cell preparation and culture
Thymus and spleen were removed immediately after mouse death, and a single cell-suspension was prepared by disrupting the organs in RPMI 1640 medium as previously reported (Gomez-Flores et al., 2009).The cell suspensions were washed three times in this medium, suspended, and adjusted to 1 × 10 7 cells ml -1 in complete RPMI 1640 medium.

T cell proliferation assay
T cell proliferation was determined by a colorimetric technique using MTT (Gomez-Flores et al., 2009).Thymus and spleen cell suspensions (100 µl of 1 × 10 7 cells ml -1 ) were added to flatbottomed 96-well plates (Becton Dickinson) containing triplicate cultures (100 µl) of RPMI 1640 medium supplemented with 5% fetal bovine serum (unstimulated control), Con A (2.5 µg ml -1 ), and plant extracts at various concentrations for 48 h at 37°C in 95% air-5% CO2 atmosphere.After incubation for 44 h, MTT (0.5 mg/ml, final concentration) was added, and cultures were additionally incubated for 4 h.Cell cultures were then incubated for 16 h with extraction buffer (100 µl), and optical densities, resulting from dissolved formazan crystals, were then read in a microplate reader (Becton Dickinson) at 570 nm.The lymphocyte proliferation index (LPI) was calculated as follows: LPI = A570 in extract-treated cells/ A570 in untreated cells

Statistical analysis
The results were expressed as mean ± SE of three replicate determinations from three independent experiments.Statistical significance was assessed by the analysis of variance (ANOVA), p < 0.05, and pos-hoc Tukey, using statistical package for social sciences (SPSS) 21.

In vitro cytotoxic activity of P. marginatus extracts
The hexane, chloroform, and methanol extracts, respectively caused significant (p < 0.05) 20 to 85%, 32 to 84%, and 32 to 72% cytotoxicity to L5178Y-R cells at concentrations ranging from 1.9 to 500 µg ml -1 , respectively, and the methanol-aqueous partition caused significant (p < 0.01) 65 to 78% cytotoxicity at concentrations ranging from 3.9 to 125 µg ml -1, respectively, and 72 and 59% cytotoxicity at 250 and 500 µg ml -1 , respectively, as compared with untreated control (Table 1).There was no statistical significance between extract treatment groups, as determined by ANOVA, p < , 43 to 51% cytotoxicity at concentrations of 25 to 100 µg ml -1 , respectively, 44 to 48% cytotoxicity at concentrations of 25 to 100 µg ml -1 respectively, and 40 to 42% cytotoxicity at concentrations of 25 to 100 µg ml -1 , respectively (Table 2), as compared with untreated control.The hexane extract treatment caused the lowest cytotoxicity, whereas the other extract treatments were not significantly different from each other (ANOVA, p < 0.05, and pos-hoc Tukey).Vehicle controls for the extracts were not cytotoxic for L5178Y-R or B16F10 cells (data not shown).In addition, extracts were not cytotoxic for murine resident thymus and spleen cells (Table 3), except for the methanol-aqueous partition that caused significant (p < 0.05) 40% cytotoxicity to thymic cells at the concentration of 125 µg ml -1 (Table 3).
An important goal in medicinal plant research is to avoid extensive and repetitive studies on known plants with limited anticancer potential, and focus on promising plants that have not been investigated; the present study was performed following these basic principles.We evaluated antitumor activity of P. marginatus crude extracts against murine lymphoma L5178Y-R and skin melanoma B16F10 cells, and demonstrated cytotoxicity with hexane, chloroform, methanol, and methanolaqueous partition extracts.Cacti with antitumor potential include Lophophora williamsii (Lem.ex Salm-Dyck) Coult., also known as peyote, which was found to be cytotoxic to murine L5178Y-R and fibroblastoma L929, and human myeloid U937 and mammary gland MCF7 tumor cells (Franco-Molina et al., 2003); Lophocereus schottii (Engelm.)Britton & Rose has antitumor activity against murine lymphoma (Orozco-Barocio et al., 2013), and Opuntia spp.has been reported to have anti-tumor activity (Supino et al., 1996;Veronesi et al., 1999;De Palo et al., 2002;Zou et al., 2005).
The increasing resistance of mammalian tumor cells to chemotherapy, which causes adverse side effects, reduces its clinical efficacy.Thus, it is critical to discover and to develop novel anticancer agents from natural sources, such as plants, to overcome such resistance and side-effects.Cacti-based chemotherapy is a potential alternative to current cancer treatment.After applying in vitro bioreactor systems, valuable cacti metabolites could be produced for a large scale at limited costs and time (Lema-Rumińska and Kulus, 2014).

Table 2 .
Effect of P. marginatus extracts on B16F10 cells toxicity[%].as compared with untreated control (culture medium).Optical density for untreated control was 0.29 ± 0.01.Data represent mean ± SE of three replicate determinations from three independent experiments.Vehicle controls for hexane, methanol, chloroform, and methanol-aqueous partition were not cytotoxic for B16F10 cells; vincristine caused about 75% cytotoxicity to B16F10 cells at concentrations ranging from 0.24 to 125 µg•ml -1 .*p < 0.05, **p < 0.01 , compared with untreated control.