601Journal of Cancer Research and Therapeutics - October-December 2013 - Volume 9 - Issue 4

Silvia T. Elias1, Paloma M. Salles2,José E. de Paula3, Luiz A. Simeoni2,Dâmaris Silveira2, Eliete N.S. Guerra1,Andrea B. Motoyama2

1Department ofOdontology,2Department ofPharmacy, HealthSciences Faculty,3Department of Botany, Institute of Biological Sciences, University of Brasilia, Brasilia, Brazil

For correspondence: Dr. Andrea Barretto Motoyama, University of Brasilia, Department of Pharmacy Faculty of Health Sciences, Campus Universis-tario Darcy Ribeiro, 70910-900, Brasilia--DF, Brazil. E-mail: andreabm@unb.br

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Original Article

Cytotoxic effect of Pouteria torta leaf extracts on human oral and breast carcinomas cell lines

ABSTRACTAims: This study aimed at investigating the cytotoxic activity and the type of cell death induced by Pouteria torta (P. torta) leaf extracts on human oral squamous cell carcinoma and breast carcinoma cell lines.

Material and Methods: The effects of P. torta leaf hexanic (PTH), ethanolic (PTE) and aqueous (PTA) extracts at the concentration of 500 mg/mL were evaluated on OSCC-3 and MCF-7 cell lines, using crystal violet staining after 24 and 48 h of treatment. To obtain the dose-response curve, cells were treated with decreasing concentrations of the extracts (1000, 750, 500, 250, 125 mg/mL) for 24 h. To investigate the mechanism of cell death (apoptosis vs. necrosis), DNA fragmentation assay was performed.

Results: All extracts were cytotoxic to both OSCC-3 and MCF-7, albeit at differing levels. PTH and PTE were effective at the concentration of 500 �g/mL, resulting in nearly 50% of cell death in both cancer cell lines. PTA was more effective at lower concentrations, with more significant cell death at 125 �g/mL. Treatment with PTA and PTE caused apoptosis in MCF-7, whereas in OSCC-3 cells, the same effect could only be caused by PTH. On the other hand, PTA was able to induce necrosis in OSCC-3.

Conclusions: These findings demonstrated that P. torta leaf extracts may contain useful compounds to combat oral and breast cancer, and this study highlights the potential biological relevance of the Brazilian Cerrado Biome in cancer therapy.

KEYWORDS: Cancer therapy, Cerrado Biome, Cytotoxic assay, MCF-7, OSCC-3, Pouteria torta

INTRODUCTION

The I nternational Agency for Research on Cancer (IARC) estimates that approximately 12.5 million new cancer cases are diagnosed each year worldwide and 7.6 million deaths occur due to cancer.[1] In Brazil, more than 518,500 new cases of cancer are expected to be diagnosed in 2012, of which approximately 14,000 are of the oral cavity and 52,600 of the female breast.[2]

Oral cancer ranks the eighth type in prevalence rates worldwide.[1] For the last decades, survival rates have not improved, remaining at around 60%,[3] mainly because of low efficacy of current therapeutic options (which include surgery, radiotherapy, chemotherapy). This is further complicated by the occurrence of adverse effects and/or permanent scars, which considerably lowers the quality of life for survivors.[4]

On the other hand, breast cancer, which is the type most prevalent among women, presents an overall survival rate around 80% worldwide.[1] This is mainly, due to the availability of targeted-directed therapies, as well as early diagnostics methods.[1,5]

Nonetheless, cases of resistance to primary and/or adjuvant therapy, as well as secondary tumor growth and metastasis still prevent its full eradication.[6,7] In this sense, total death numbers can sum up to 400,000.00 worldwide.[1]

In the above scenario, it is crucial to develop alternative, more effective therapeutics for both oral and breast cancer. Historically, a number of natural products have been isolated and described as anti-cancer agents; vincristine, vinblastine and taxol are well-known and clinically used examples.[8-10] Currently, approximately 60% of new anti-cancer drugs come from natural sources[11] and it is predicted that still many more successful molecules will be developed upon modification of natural lead compounds. In fact, it is argued that natural compounds derived from traditional medicine may improve and speed the process of drug discovery, in the sense that they have already been used for many years. Thus, it is a validated strategy to investigate plants extracts, especially those used in folk and traditional medicine, for their anti-cancer potential.[12]

Pou teria (Sapotaceae) is a genus commonly found in the Cerrado, a savannah-like formation that accounts

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602 Journal of Cancer Research and Therapeutics - October-December 2013 - Volume 9 - Issue 4

for 23% of the Brazilian area.[13] Several of its species have been used in folk medicine in Brazil [14] to treat fever, inflammation, skin eruptions and rashes, ulcers, diabetes,[15,16] diarrhea,[17] nausea, vomiting, back pain, and to promote lactation in breast-feeding mothers.[18] Pouteria torta (Mart.) Radlk, specifically,[18,19] has been used as building material, remedies in folk medicine, as well as food source, due to its edible fruits.

The isolation and identification of several different compounds from Pouteria torta have been reported.[15,16,18,19] For instance, from flower and fruit extracts, the following compounds have been reported to be found: D- and E-amyrin. lupeol, taraxasterol, pseudo taraxasterol, cycloartenol, lanosterol, lanosta-7, 24-dien and erythrodiol palmitate, fatty acids, triglycerides and normal and branched hydrocarbons.[19] On the other hand, Pouteria torta (P. Torta) stems were shown to contain D-amyrin acetate, E-amyrin acetate, betulinic acid and ursolic acid, isolated from methanolic extract.[19] Lupeol acetate was described in the hexanic extract from leaves,[17] and a mixture of D- and E-friedelinol was isolated from ethanolic extract. [19]

In spite o f its diverse molecular composition, P torta leaf extracts have not yet been tested for its cytotoxic activity against human cancer cells. Therefore, the aim of the present study was to evaluate the cytotoxic and anti-proliferative activity of ethanolic, hexanic and aqueous P. torta leaf extracts on OSCC-3, a human oral squamous cell carcinoma cell line and on MCF-7 cells, a human breast cancer cell line.

MATERIALS AND METHODS

Preparation of crude extractsLeaves of Pouteria torta (Mart.) Radlk were collected on the campus of the University of Brasilia (UnB), in July, 2003. The species is identical to the exsiccate deposited by J. E. de Paula, Ph.D., in the Herbarium of the University of Brasília under the number JElias de Paula 3674. The plant material was dried at room temperature and powdered in a knife mill. The plant material (918 g) was macerated at room temperature first with hexane, followed by ethanol, for 7 days, and this process was repeated thrice. After filtration, the solvents were removed under reduced pressure, at temperatures below 40˚C to yield 46 g (5%) and 101 g (11%), respectively, of crude extracts. The aqueous extract was obtained by infusion of 400 g of the powdered material in 3 L distilled water. After filtration, the water was removed by lyophilization to yield 44 g (11%) of crude aqueous extract. For cell treatment, the aqueous extract was diluted in saline solution, and the hexanic and ethanolic extracts, in a solution of DMSO: ethanol (2:3). The P. torta leaf hexanic, ethanolic and aqueous extracts are herein referred to as PTH, PTE and PTA, respectively.

Cell culture and treatmentsTwo cancer cell lines were used in this study: OSCC-3, an oral squamous cell carcinoma cell line derived from tongue

and MCF-7 cell line isolated from adenocarcinoma of the mammary gland. The former was a generous gift from Dr. Marcio Poças (Biological Sciences Institute, UnB), and the latter was acquired from ATCC (American Type Culture Collection); Cells were maintained at 37 ˚C and 5% of CO

2 in Dulbecco’s

Modified Eagle Medium (DMEM) supplemented with 10% fetal bovine serum (FBS), penicillin and streptomycin. For all experiments, OSCC-3 cells were detached with trypsin (0.25%)/ EDTA (1mM) solution; and MCF-7 cells with trypsin (0.05%)/ EDTA (1mM) solution. Trypsin was inactivated by addition of bovine serum-containing-medium and plated as indicated. All cell culture reagents were purchased from Invitrogen (Carlsbad, USA).

Cell cytotoxicity For the cytotoxi city experiments, OSCC-3 and MCF-7 cells were seeded at the density of 1.5 x 10[4] cells/well on 24-well plates and then treated with PTA, PTE or PTH extracts at 500 mg/mL for indicated periods of time. Staurosporine (200nM) was used as positive control. Two negative controls were included: saline solution (for comparison with PTA treatment) and DMSO: EtOH (2:3) solution (for comparison with PTH and PTE treatments). After 24 and 48 h treatment, dead (floating) cells were washed out with PBS. Remaining adherent live cells were fixed with methanol 100% for 20 min and stained with crystal violet solution (0.2% in methanol) for 5 min at room temperature. After extensive washing, dye-uptake by remaining cells was assessed by recovery with citrate-based alcoholic solution (sodium citrate 0.1M in 50% ethanol) and absorbance at 570 nm was measured at spectrophotometer (Shimadzu, Japan).

Dose-response curveCells were seeded as described above and treated with increasing concentrations of the extracts (125, 250, 500, 750, 1000 mg/mL). After treatment for 24 h, cell death was assessed with crystal violet staining as described.

DNA fragmentation assayDNA fragmentation assay was performed as described previously. [20] Briefly, OSCC-3 and MCF-7 cells were treated with either P. torta extracts at 500 mg/mL, staurosporine (positive control) or vehicle (negative control) for 24 and 48 h, respectively. At the end of treatment, floating and adherent cells were collected, lysed in SDS buffer solution (0.2M EDTA, 50mM Tris pH 8.0, 5% w/v Triton X-100, 2% w/v SDS) and total DNA was purified using the phenol/chloroform method, followed by alcoholic precipitation. Three micrograms of the obtained material were subjected to electrophoresis on 1.5 % agarose gels, containing ethidium bromide (1 ng/mL). Bands were visualized under UV light and images were acquired on an Eagle eye Acquisition System.

Statistical analyses of dataAll data were an alyzed using the PRISM 5.0 GraphPad software, according to statistical parameters described in figure legends.

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603Journal of Cancer Research and Therapeutics - October-December 2013 - Volume 9 - Issue 4

RESULTS

Pouteria torta extracts reduce cell viability of OSCC-3 and MCF-7.The effect of P. torta leaf extracts was evaluated in the two cell lines. Treatment with PTH, PTE, PTA extracts, resulted in cell death in both cell lines, in comparison to vehicles, after 24 and 48 h of treatment. However, the extent and the timing of cell death differed among cell lines depending upon the extract used. In the OSCC-3 cell line, PTH was the most effective in inducing cell toxicity, resulting in 40% and 33% of live cells after 24 and 48 h of treatment, respectively. PTE and PTA also caused cell death in OSCC-3 cells, but the results seemed to be less pronounced: after 48 h of treatment with PTE or PTA, there were still 52% and 40% of live cells, respectively, when compared with the vehicle alone [Figure 1a]. Of note, the effects of PTE and PTA on OSCC-3 cells did not increase with time, as noticed by the percentage of cell death after 24 h vs. 48 h treatment.

The results obtained with MCF-7 cells were similar to those obtained in the cytotoxicity test in OSCC-3 cells, in the sense, that all extracts were able to induce cell death. However, on MCF-7 cells PTH and PTA were almost equally effective in inducing cell death after 48 h of treatment, resulting in approximately 70% of reduction of viable cells. On the other hand, PTE resulted in death in 60% of the treated cells after 48 h treatment [Figure 1b]. Interestingly, the results indicate that effects of the extracts on MCF-7 cells could be time-dependent as in all cases there was a reduction in viable cells from 24 h to 48 h treatment. Taken together, these results show that, albeit at different levels, all the evaluated extracts present toxicity to both cancer cell lines tested.

Dose–response assay In these experiments, cells were treated with decreasing concentrations (1000, 750, 500, 250, 125 �g/mL) of extracts. Figures 2a and b show that PTH induced a dose-dependent response in both cell lines. In OSSC-3, the lowest percentage of live cells (approximately 15%) was reached at the concentration

of 750 �g/mL PTH and in MCF-7 cells (approximately 10% of live cells), with 1000 �g/mL PTH. The dose-dependent response presented a dual pattern when the cells were treated with PTE or PTA. PTE treatment induced cell death in both oral and breast cancer cells in a dose-dependent manner when concentrations varied from 250 to 500 �g/mL (ranging from 80-90% to 50-60% viable cells). However, upon treatment with higher concentrations PTE was unable to further induce cell death. In fact, breast cancer cells treated with higher doses of PTE showed a higher rate of survival. PTA treatment resulted in a similar pattern, in the sense that at the concentration of 500 �g/mL (or lower, in the case of OSCC-3), cell death was induced to an extent of around 40% in both cell lines. However, an increase in the concentrations used resulted in a recovery of cell viability, a phenomenon that more markedly occurred in the OSCC-3 cell line. Curiously, PTA extract was the most effective when used at lower concentrations, with the most significant response at 125 �g/mL (around 30% and 50% of viability for OSCC-3 and MCF-7 cell lines, respectively).

Mechanism of cell deathTo investigate the me chanism of cell death (apoptosis vs. necrosis) induced by P. torta extracts in OSCC-3 and MCF-7 cells, DNA was extracted and subjected to electrophoresis on agarose gels. OSCC-3 treatment with PTH resulted in a “ladder pattern” consistent with an apoptotic response, derived from the DNA nicks between the nucleosomes. Additionally, the pattern is similar to the one observed upon treatment with positive control staurosporine, a well-established apoptosis inducer. Treatment of OSCC-3 cells with PTE resulted in a single, upper band, corresponding to intact DNA, whereas PTA treatment resulted in a smear in the gel, especially in the higher molecular weight range [Figure 3a].

Treatment of both PTE and PTA in MCF-7 cells resulted in a clear apoptotic pattern, similar to the positive control [Figure 3b]. PTH treatment, on the other hand, resulted in a single, intact band in the upper part of the gel without any smearing; indicating that no cell death could be detected upon this treatment.

Figure 1: Cytotoxicity of Pouteria torta extracts on OSCC-3 (a) and MCF-7 (b) cells. Depicted is average of three independent experiments, each performed in quadruplicates. Ctrl and Stp: negative and positive control, respectively. PTH, PTE and PTA: respectively: P. torta leaf hexanic, ethanolic and aqueous extracts. Negative controls were normalized to 100%. P < 0.05 compared to negative control.

Figure 2: Do se-response curve of OSCC-3 cells (a) and MCF-7 cells (b) treated with Pouteria torta extracts. Cells were treated with increasing concentrations (0, 125, 250, 500, 750 and 1000 zg/mL) of PTH, PTE or PTA for 24 h. Depicted is the average of 3 independent experiments, performed in triplicates, and represented as percentage of the negative control.

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DISCUSSION

This study aimed at i nvestigating the cytotoxic activity of three leaf extracts from P. torta on OSCC-3 cells from human oral squamous cell carcinoma, on MCF-7 breast cancer cell cultures. Cytotoxicity and cell death type (necrosis vs. apoptosis) induced by treatment with hexanic (PTH), ethanolic (PTE) and aqueous (PTA) extracts were assessed. Our data showed that all tested P. torta leaf extracts were cytotoxic, to different degrees, to both OSCC-3 and MCF-7. Apoptosis was induced by PTA and PTE in MCF-7 cells, and by PTH in OSCC-3 cells. Additionally, taken together our results suggest that PTA was able to cause necrosis in OSCC-3 cell line.

The observed cytotoxi c responses were not dose-dependent in the evaluated cell lines, except for PTH. Treatment with this extract resulted in a steady decrease in viable cells as the extract concentration was increased from 125 �g/mL to 1000 �g/mL. These results are in agreement with those reported by previous studies using different cancer cell lines. Lupeol, a dietary triterpene that can also be isolated from PTH, was described to be able to induce apoptosis in 451Lu and WM35 melanoma cells,[21] and to have growth-inhibitory effects on hepatocellular carcinoma SMMC7721 cells.[22] More importantly, it was also shown to selectively induce substantial cell death in head and neck squamous cell carcinoma but to exhibit only minimal effect on a normal tongue fibroblast cell line in vitro.[23] At this point, we are not able to determine how much lupeol is present in our extract. Nonetheless, the results highlight the potential of this extract as an anti-cancer drug or lead molecule to future drug.

PTA treatment resulted in an increase in cell death rates in concentrations varying from 125 �g/mL to 500 �g/mL in both cell lines. However, at higher concentrations no increase

in cell death percentage was observed in MCF-7 cells, and a slight increase in cell survival was noticed in OSCC-3 cells. These results suggest that PTA may inefficiently (through a mechanism yet unknown) induce cell death, which only becomes more noticeable with longer times of exposure to the extract. In fact, after 48 h-treatment, PTA induced necrosis in OSCC-3, as demonstrated by the DNA fragmentation assay [Figure 3a]. Further analyses are necessary to determine and isolate the components of PTA, as well as their respective activities and possible pharmacological cellular targets.

PTE treatment resulted in different patterns of cell survival in OSCC-3 and MCF-7 cells. The former seemed to be inhibited at the concentration of 500 �g/mL, and increasing extract concentrations did not significantly alter cell survival from that point. MCF-7 cells, on the other hand, showed a 50% decrease in cell survival when the concentration of 500 Pg/mL was used. However, at higher concentrations cell survival rate was higher, reaching almost 100% when a concentration of 1000 �g/mL was employed.

It should be noted that extracts are composed by a number of substances which may have not been isolated and fully characterized yet. So it is possible that molecules with opposing, as well as with synergistic activities can be found at differing relative concentrations within the same extract. Both PTA and PTE extracts contain polyphenol compounds, such as flavonols (rutin and myricitrin) and catechin derivatives (LAS, DS, unpublished observations), which exhibit anti-oxidant activity. The anti-oxidative, protective effect of these compounds against cell death has already been reported elsewhere.[24] It is speculated that by increasing the dosis, the concentration of polyphenols in the sample reach a level high enough to scavenge free radical and thus protect cells from toxic agents. Such hypothesis still awaits testing.

Figure 3a: DNA fragmentation induced by Pouteria torta extracts in OSCC-3 cells. Cells were treated with 500 zg/mL of extracts and positive (staurosporine) and negative (only vehicle) controls. 1: 1kb plus DNA molecular marker, 2 and 2’: negative controls; 3: positive control (staurosporine); 4: PTH; 5: PTE; 6: PTA.

Figure 3b: DNA fragmentation induced by Pouteria torta extracts in MCF-7 cells. Cells were treated with 500 zg/mL of extracts and positive (staurosporine) and negative (only vehicle) controls. 1: 1kb plus DNA molecular marker, 2: negative control; 3: positive control (staurosporine); 4: PTH; 5: PTE; 6: PTA.

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Recently, pouterin, a prote in with lectin-like activity, was isolated from seeds of P. torta. [25] Pouterin was able to induce agglutination of human, rabbits and rats erythrocytes.[25] In addition, it also induced apoptosis in some mammalian cancer cells.[26] It is believed that due to its lectin-like nature pouterin is mainly found in seeds, and may be absent from other plant parts. On the other hand, anti-oxidant and radical scavenging activities, which could favor cell proliferation, as well as prevent the initiation of the tumorigenic process, have also been reported in the more polar extracts and fractions.[27]

Additionally, upon cell metab olism, compounds may give rise to new ones, with new or opposing activities than that of the parental compound. For instance, myricitrin (myricetin-3-rhamnoside), a flavonoid found in several Pouteria species, including P. torta,[17,19] did not cause apoptosis in human leukemia HL-60 cells in in vitro experiments, while the aglycone myricetin exhibited dose- and time-dependent cytotoxicity in HL-60 cells.[28] Also, myricetin, but not myricitrin, inhibited MMP-2 enzyme activity in the human colorectal carcinoma cell lines COLO 320HSR, COLO 320DM, HT 29, COLO 205, and COLO 205-X.[29] These findings suggest that myricitrin may be a pro-drug, which is converted to the active form, myricetin, through intracellular metabolism.

At this point, it remains to be investigated which compounds are present in each extract, as well as their possible cell metabolites. Once such knowledge is available, it will be possible to uncover their exact roles in inducing cell death, as well as uncovering their pharmacological cellular target. Although such investigation is underway, we believe it to be out of the scope of the present work.

In summary, our results showed that, ethanolic, hexanic and aqueous P. torta leaf extracts possess cytotoxic activity against oral squamous and breast tumor cell lines. Their active components may later be isolated, characterized and used to develop new anti-cancer drugs. In light of the limited effectiveness of the present therapeutic modalities, and the high prevalence of these two types of cancer, it is especially important to search for more potent and/or diverse therapeutic options. In this sense, this study opens up new pharmacological avenues. Furthermore, it highlights the biological relevance of the Brazilian Cerrado plants in the development of new and alternative anti-cancer therapeutics.

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25. Boleti AP, Freire MG, Coelho MB, Silva W, Baldasso PA, Gomes VM, et al. Insecticidal and antifungal activity of a protein from Pouteria torta seeds with lectin-like properties. J Agric Food Chem 2007;55:2653-8.

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Cite this article as: Elias ST, Salles PM, de Paula JE, Simeoni LA, Silveira D, Guerra EN, et al. Cytotoxic effect of Pouteria torta leaf extracts on human oral and breast carcinomas cell lines. J Can Res Ther 2013;9:601-6.

Source of Support: Nil, Confl ict of Interest: None declared.

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