|Year : 2013 | Volume
| Issue : 4 | Page : 601-606
Cytotoxic effect of Pouteria torta leaf extracts on human oral and breast carcinomas cell lines
Silvia T Elias1, Paloma M Salles2, Josť E de Paula3, Luiz A Simeoni2, D‚maris Silveira2, Eliete N.S Guerra1, Andrea B Motoyama2
1 Department of Odontology, Institute of Biological Sciences, University of Brasilia, Brasilia, Brazil
2 Department of Pharmacy, Health Sciences Faculty, Institute of Biological Sciences, University of Brasilia, Brasilia, Brazil
3 Department of Botany, Institute of Biological Sciences, University of Brasilia, Brasilia, Brazil
|Date of Web Publication||11-Feb-2014|
Andrea B Motoyama
University of Brasilia, Department of Pharmacy Faculty of Health Sciences, Campus Universistario Darcy Ribeiro, 70910-900, Brasilia-DF
Source of Support: None, Conflict of Interest: None
Aims: 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
|How to cite this article:|
Elias ST, Salles PM, de Paula JE, Simeoni LA, Silveira D, Guerra EN, Motoyama AB. Cytotoxic effect of Pouteria torta leaf extracts on human oral and breast carcinomas cell lines. J Can Res Ther 2013;9:601-6
|How to cite this URL:|
Elias ST, Salles PM, de Paula JE, Simeoni LA, Silveira D, Guerra EN, Motoyama AB. Cytotoxic effect of Pouteria torta leaf extracts on human oral and breast carcinomas cell lines. J Can Res Ther [serial online] 2013 [cited 2019 Dec 15];9:601-6. Available from: http://www.cancerjournal.net/text.asp?2013/9/4/601/126454
| > Introduction|| |
The International 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.  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. 
Oral cancer ranks the eighth type in prevalence rates worldwide.  For the last decades, survival rates have not improved, remaining at around 60%,  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. 
On the other hand, breast cancer, which is the type most prevalent among women, presents an overall survival rate around 80% worldwide.  This is mainly, due to the availability of targeted-directed therapies, as well as early diagnostics methods. , Nonetheless, cases of resistance to primary and/or adjuvant therapy, as well as secondary tumor growth and metastasis still prevent its full eradication. , In this sense, total death numbers can sum up to 400,000.00 worldwide. 
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. ,, Currently, approximately 60% of new anti-cancer drugs come from natural sources  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. 
Pouteria (Sapotaceae) is a genus commonly found in the Cerrado, a savannah-like formation that accounts for 23% of the Brazilian area.  Several of its species have been used in folk medicine in Brazil  to treat fever, inflammation, skin eruptions and rashes, ulcers, diabetes, , diarrhea,  nausea, vomiting, back pain, and to promote lactation in breast-feeding mothers.  Pouteria torta (Mart.) Radlk, specifically, , 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. ,,, For instance, from flower and fruit extracts, the following compounds have been reported to be found: α- and β-amyrin. lupeol, taraxasterol, pseudo taraxasterol, cycloartenol, lanosterol, lanosta-7, 24-dien and erythrodiol palmitate, fatty acids, triglycerides and normal and branched hydrocarbons.  On the other hand, Pouteria torta (P. Torta) stems were shown to contain α-amyrin acetate, β-amyrin acetate, betulinic acid and ursolic acid, isolated from methanolic extract.  Lupeol acetate was described in the hexanic extract from leaves,  and a mixture of α- and β-friedelinol was isolated from ethanolic extract. 
In spite of 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 extracts
Leaves 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 Brasνlia 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 treatments
Two 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 Poηas (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).
For the cytotoxicity experiments, OSCC-3 and MCF-7 cells were seeded at the density of 1.5 x 10  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).
Cells 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 assay
DNA fragmentation assay was performed as described previously.  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 data
All data were analyzed using the PRISM 5.0 GraphPad software, according to statistical parameters described in figure legends.
| > 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 1]a. 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.
|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|
Click here to view
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 1]b. 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.
In these experiments, cells were treated with decreasing concentrations (1000, 750, 500, 250, 125 ΅g/mL) of extracts. [Figure 2]a 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).
|Figure 2: Dose-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 μg/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|
Click here to view
Mechanism of cell death
To investigate the mechanism 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 3]a.
Treatment of both PTE and PTA in MCF-7 cells resulted in a clear apoptotic pattern, similar to the positive control [Figure 3]b. 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.
| > Discussion|| |
This study aimed at investigating 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 cytotoxic 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,  and to have growth-inhibitory effects on hepatocellular carcinoma SMMC7721 cells.  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.  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 3]a. 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 μg/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.  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.
Recently, pouterin, a protein with lectin-like activity, was isolated from seeds of P. torta.  Pouterin was able to induce agglutination of human, rabbits and rats erythrocytes.  In addition, it also induced apoptosis in some mammalian cancer cells.  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. 
Additionally, upon cell metabolism, 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, , 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.  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.  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.
| > References|| |
|1.||World Cancer Report 2008 [internet]. In: Boyle P, Levin B, editors. World Health Organization/ International Agency for Research on Cancer. c2008. Available from: http://www.iarc.fr/en/publications/pdfs-online/wcr/2008/index.php[Lastnone accessed on 2012 Jul 31]. |
|2.||Estimativa 2012 [internet]. In: da Silva AP, Noronha CP, Silva JL, Ferreira JM, Oliveira JF, Santos MO, et al. Ministério da Saúde/ Instituto Nacional do Câncer. c2011, Available from: http://www.inca.gov.br/estimativa/2012/none [Last accessed on 2012 Jul 31]. |
|3.||Pedruzzi PA, Kowalski LP, Nishimoto IN, Oliveira BV, Tironi F, Ramos GH. Analysis of prognostic factors in patients with oropharyngeal squamous cell carcinoma treated with radiotherapy alone or in combination with systemic chemotherapy. Arch Otolaryngol Head Neck Surg 2008;134:1196-204. |
|4.||Shah JP, Gil Z. Current concepts in management of oral cancer-Surgery. Oral Oncol 2009;45:394-401. |
|5.||SEER Stat Fact Sheets: Breast [internet]. In: Howlader N, Noone AM, Krapcho M, Neyman N, Aminou R, Altekruse SF, editors. Surveillance Epidemiology and End Results. c2012. Available from: http://seer.cancer.gov/statfacts/html/breast.htmlnone [Last accessed on 2012 Jul 31]. |
|6.||Musgrove EA, Sutherland RL. Biological determinants of endocrine resistance in breast cancer. Nat Rev Cancer 2009;9:631-43. |
|7.||Cook KL, Shajahan AN, Clarke R. Autophagy and endocrine resistance in breast cancer. Expert Rev Anticancer Ther 2011;11:1283-94. |
|8.||Levêque D, Jehl F. Molecular pharmacokinetics of catharanthus (vinca) alkaloids. J Clin Pharmacol 2007;47:579-88. |
|9.||Magnotta M, Murata J, Chen J, De Luca V. Expression of deacetylvindoline-4-O-acetyltransferase in Catharanthus roseus hairy roots. Phytochemistry 2007;68:1922-31. |
|10.||Kingston DG, Newman DJ. Taxoids: Cancer-fighting compounds from nature. Curr Opin Drug Discov Devel 2007;10:130-44. |
|11.||Newman DJ, Cragg GM. Natural Products As Sources of New Drugs over the 30 Years from1981 to 2010. J Nat Prod 2012;75:311-35. |
|12.||Patwardhan B, Mashelkar RA. Traditional medicine-inspired approaches to drug discovery: Can Ayurveda show the way forward?. Drug Discovery Today 2009;14:804-11. |
|13.||Ratter JA, Ribeiro JF, Bridgewater S. The Brazilian cerrado vegetation and threats to its biodiversity. Ann Bot 1997;80:223-30. |
|14.||Brandão MG, Zanetti NN, Oliveira P, Grael CF, Santos AC, Monte-Mór RL. Brazilian medicinal plants described by 19 th century European naturalists and in the Official Pharmacopoeia. J Ethnopharmacol 2008;120:141-8. |
|15.||Ma J, Yang H, Basile MJ, Kennelly EJ. Analysis of polyphenolic antioxidants from the fruits of three pouteria species by selected ion monitoring liquid chromatography-mass spectrometry. J Agric Food Chem 2004;52:5873-8. |
|16.||Montenegro LH, Oliveira PE, Conserva LM, Rocham EM, Brito AC, Araújo RM, et al. Triterpenóides e avaliação do potencial antimalárico, larvicida, anti-radicalar e anticolinesterástico de Pouteria venosa I (Sapotaceae). Braz J Pharmacogn 2006;16:611-7. |
|17.||Manosroi A, Saraphanchotiwitthaya A, Manosroi J. In vitro immunomodulatory effect of Pouteria cambodiana (Pierre ex Dubard) Baehni extract. J Ethnopharmacol 2005;101:90-4. |
|18.||Perfeito JP, Santos ML, Lopez KS, Paula JE, Silveira D. Characterization and biological properties of Pouteria torta extracts: A preliminary study. Braz J Pharmacogn 2005;15:183-6. |
|19.||Silva CA, Simeoni LA, Silveira D. Genus Pouteria: Chemistry and biological activity. Revista Brasileira de Farmacognosia 2009;19:501-9. |
|20.||Elias ST, Diniz J, Almeida RS, Alvarenga N, Simeoni LA, Silveira D, et al. Cytotoxic effect of tobacco extracts on human oral squamous cell carcinoma cell-line. Oral Oncol 2010;46:869-73. |
|21.||Saleem M, Maddodi N, Abu Zaid M, Khan N, bin Hafeez B, Asim M, et al. Lupeol inhibits growth of highly aggressive human metastatic melanoma cells in vitro and in vivo by inducing apoptosis. Clin Cancer Res 2008;14:2119-27. |
|22.||Zhang L, Zhang Y, Yang X, Lv Z. Lupeol, a dietary triterpene, inhibited growth, and induced apoptosis through down-regulation of DR3 in SMMC7721 cells. Cancer invest 2009;27:163-70. |
|23.||Lee TK, Poon RT, Wo JY, Ma S, Guan XY, Myers JN, et al. Lupeol Suppresses Cisplatin-Induced Nuclear Factor- B Activation in Head and Neck Squamous Cell Carcinoma and Inhibits Local Invasion and Nodal Metastasis in an Orthotopic Nude Mouse Model. Cancer Res 2007;67:8800-9. |
|24.||Hernandez P, Rodriguez, PC, Delgado R, Walczak H. Protective effect of Mangifera indica L. polyphenols on human T lymphocytes against activation-induced cell death. Pharmacol Res 2007;55:167-73. |
|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. |
|26.||Boleti AP, Ventura CA, Justo GZ, Silva RA, de Sousa AC, Ferreira CV, et al. Pouterin, a novel potential cytotoxic lectin-like protein with apoptosis-inducing activity in tumorigenic mammalian cells. Toxicon 2008;51:1321-30. |
|27.||Rice-Evans CA, Miller NJ, Paganga G. Structure-antioxidant activity relationships of flavonoids and phenolic acids. Free Radic Biol Med 1996;20:933-56. |
|28.||Asano N, Kuno T, Hirose Y, Yamada Y, Yoshida K, Tomita H, et al. Preventive effects of a flavonoid myricitrin on the formation of azoxymethane-induced premalignant lesions in colons of rats. Asian Pac J Cancer Prev 2007;8:73-6. |
|29.||Ko CH, Shen SC, Lee TJ, Chen YC. Myricetin inhibits matrix metalloproteinase 2 protein expression and enzyme activity in colorectal carcinoma cells. Mol Cancer Ther 2005;4:281-90. |
[Figure 1], [Figure 2], [Figure 3]