Ethanolic extract of Caryocar brasiliense fruit peel promotes death and cell cycle control in canine osteosarcoma cells




Apoptosis; Autophagy; Cycle block; Cellular metabolism; Cytoprotection.


The Cerrado biome is a source of chemical molecules with great medicinal potential. The substances extracted from pequi, the fruit of Caryocar brasiliense, have antiparasitic, antioxidant, and antineoplastic properties. Canine osteosarcoma is a highly aggressive and metastatic bone tumor, not very responsive to current chemotherapy modalities. This study aimed to show the effects of the ethanol extract of pequi peel (EEPP) on canine osteosarcoma cells, in addition to suggesting a metabolic route that explains the action of the extract. D-17 cells were seeded and exposed to EEPP at concentrations of 0, 10, and 100 µg/mL. After treatment, the cell nuclei were marked with DAPI and quantified using fluorescence microscopy. The expression of proteins p53, Ki-67, Bcl-2, Akt, AMPK, and mTOR was analyzed using immunocytochemistry. By using DAPI, we found a reduction in the number of quantified nuclei, time, and dose-dependent. The labeling of p53, Ki-67, and Bcl-2, antibodies decreased in groups exposed to EEPP. The opposite was observed with Akt, AMPK, and mTOR antibodies, where the protein was not found in the control group but was expressed in groups exposed to EEPP. We suggest a possible metabolic route where EEPP promotes cell death and cell cycle control in D-17 cells.


Arnhold E. (2013). Package in the R environment for analysis of variance and complementary analyses. Brazilian Journal of Veterinary Research and Animal Science. 50(6):488-92.

Banerji V., & Gibson S. B. (2012). Targeting metabolism and autophagy in the context of haematologic malignancies. International journal of cell biology. 2012.

Bouaoun, L., Sonkin, D., Ardin, M., Hollstein, M., Byrnes, G., Zavadil, J., & Olivier, M. (2016). TP53 variations in human cancers: new lessons from the IARC TP53 database and genomics data. Human mutation. 37(9):865-876.

Chakrabarty S., Roy M., Hazra B., & Bhattacharya R. (2002). Induction of apoptosis in human cancer cell lines by diospyrin, a plant-derived bisnaphthoquinonoid, and its synthetic derivatives. Cancer letters. 188(1-2):85-93.

Ching C. B., & Hansel D. E. (2010). Expanding therapeutic targets in bladder cancer: the PI3K/Akt/mTOR pathway. Laboratory investigation. 90(10):1406-14.

Dowsett, M., Nielsen, T. O., A’Hern, R., Bartlett, J., Coombes, R. C., Cuzick, J., & McShane, L. (2011). Assessment of Ki67 in breast cancer: recommendations from the International Ki67 in Breast Cancer working group. Journal of the National cancer Institute. 103(22):1656-1664.

Espindola-Darvenne L. S. (2007). Cerrado: fonte de descoberta de novos medicamentos. Brasília méd. 193-8.

Fedchenko N., & Reifenrath J. (2014). Different approaches for interpretation and reporting of immunohistochemistry analysis results in the bone tissue–a review. Diagnostic pathology. 9(1):1-12.

Ferreira C. M. (2019). Análise química de extratos de Caryocar brasiliense com potencial antioxidante. Repository UFG. 2019:1-133.

Gan R. Y., & Li H. B. (2014). Recent progress on liver kinase B1 (LKB1): expression, regulation, downstream signaling and cancer suppressive function. International journal of molecular sciences. 15(9):16698-718.

García-Martínez J. M., & Alessi D. R. (2008). mTOR complex 2 (mTORC2) controls hydrophobic motif phosphorylation and activation of serum-and glucocorticoid-induced protein kinase 1 (SGK1). Biochemical Journal. 416(3):375-85.

Gerdes J., Lemke H., Baisch H., Wacker H. H., Schwab U., & Stein H. (1984). Cell cycle analysis of a cell proliferation-associated human nuclear antigen defined by the monoclonal antibody Ki-67. The journal of immunology. 133(4):1710-5.

Gerdes J., Schwab U., Lemke H., & Stein H. (1983). Production of a mouse monoclonal antibody reactive with a human nuclear antigen associated with cell proliferation. International journal of cancer. 31(1):13-20.

Gerdes, J., Stein, H., Pileri, S., Rivano, M. T., Gobbi, M., Ralfkiaer, E., & Delsol, G. (1987). Prognostic relevance of tumour-cell growth fraction in malignant non-Hodgkin's lymphomas. The Lancet. 330(8556): 448-449.

Happo, L., Cragg, M. S., Phipson, B., Haga, J. M., Jansen, E. S., Herold, M. J., & Strasser, A. (2010). Maximal killing of lymphoma cells by DNA damage–inducing therapy requires not only the p53 targets Puma and Noxa, but also Bim. Blood, The Journal of the American Society of Hematology. 116(24):5256-5267.

Jung C. H., Ro S. H., Cao J., Otto N. M., & Kim D. H. (2010). mTOR regulation of autophagy. FEBS letters. 584(7):1287-95.

Kan, J. Y., Yen, M. C., Wang, J. Y., Wu, D. C., Chiu, Y. J., Ho, Y. W., & Kuo, P. L. (2016). Nesfatin-1/Nucleobindin-2 enhances cell migration, invasion, and epithelial-mesenchymal transition via LKB1/AMPK/TORC1/ZEB1 pathways in colon cancer. Oncotarget. 7(21):31336.

Laplante M., & Sabatini D. M. (2009). mTOR signaling at a glance. Journal of cell science. 122(20):3589-94.

Leung H. W. C., Wu C. H., Lin C. H., & Lee H. Z. (2005). Luteolin induced DNA damage leading to human lung squamous carcinoma CH27 cell apoptosis. European journal of pharmacology. 508(1-3):77-83.

Luo Y., Xu W., Li G., & Cui W. (2018). Weighing in on mTOR complex 2 signaling: the expanding role in cell metabolism. Oxidative medicine and cellular longevity. 2018.

Miranda-Vilela, A. L., Grisolia, C. K., Longo, J. P. F., Peixoto, R. C., de Almeida, M. C., Barbosa, L. C. P., & Báo, S. N. (2014). Oil rich in carotenoids instead of vitamins C and E as a better option to reduce doxorubicin-induced damage to normal cells of Ehrlich tumor-bearing mice: hematological, toxicological and histopathological evaluations. The Journal of nutritional biochemistry. 25(11):1161-1176.

Miranda-Vilela A. L., Resck I. S., & Grisolia C. K. (2008). Antigenotoxic activity and antioxidant properties of organic and aqueous extracts of pequi fruit (Caryocar brasiliense Camb.) pulp. Genetics and Molecular Biology. 31(4):956-63.

Moya, A., Sakamaki, K., Mason, B. M., Huisman, L., Forêt, S., Weiss, Y., & Ball, E. E. (2016). Functional conservation of the apoptotic machinery from coral to man: the diverse and complex Bcl-2 and caspase repertoires of Acropora millepora. BMC genomics. 17(1): 62.

Netten, H., Young, I. T., Prins, M., van Vliet, L. J., Tanke, H., Vrolijk, H., & Sloos, W. (1994). Automation of fluorescent dot counting in cell nuclei. In Proceedings of 12th International Conference on Pattern Recognition. IEEE. 1:84-87.

Peixoto, P. M., Teijido, O., Mirzalieva, O., Dejean, L. M., Pavlov, E. V., Antonsson, B., & Kinnally, K. W. (2017). MAC inhibitors antagonize the pro-apoptotic effects of tBid and disassemble Bax/Bak oligomers. Journal of bioenergetics and biomembranes. 49(1):65-74.

Pereira, A. S., Shitsuka, D. M., Parreira, F. J., & Shitsuka, R. (2018). Metodologia da pesquisa científica.[e-book]. Santa Maria. Ed. UAB/NTE/UFSM. Disponível em: https://repositorio. ufsm. br/bitstream/handle/1/15824/Lic_Computacao_Metodologia-Pesquisa-Cientifica. pdf.

Santidrián, A. F., González-Gironès, D. M., Iglesias-Serret, D., Coll-Mulet, L., Cosialls, A. M., de Frias, M., & Viollet, B. (2010). AICAR induces apoptosis independently of AMPK and p53 through up-regulation of the BH3-only proteins BIM and NOXA in chronic lymphocytic leukemia cells. Blood. 116(16):3023-3032.

Sobecki, M., Mrouj, K., Colinge, J., Gerbe, F., Jay, P., Krasinska, L., & Fisher, D. (2017). Cell-cycle regulation accounts for variability in Ki-67 expression levels. Cancer research. 77(10):2722-2734.

Souza, M. R., de Carvalho, R. K., de Carvalho, L. S., de Sá, S., Andersen, M. L., de Araújo, E. G., & Mazaro-Costa, R. (2019). Effects of subchronic exposure to Caryocar brasiliense peel ethanolic extract on male reproductive functions in Swiss mice. Reproductive Toxicology. 87:118-124.

Sun X., & Kaufman P. D. (2018). Ki-67: more than a proliferation marker. Chromosoma. 127(2):175-86.

Szewczyk M., Lechowski R., & Zabielska K. (2015). What do we know about canine osteosarcoma treatment?–review. Veterinary research communications. 39(1):61-7.

Team RC. R: (2013). A language and environment for statistical computing.

Zhao, G. S., Gao, Z. R., Zhang, Q., Tang, X. F., Lv, Y. F., Zhang, Z. S., & Guo, Q. N. (2018). TSSC3 promotes autophagy via inactivating the Src-mediated PI3K/Akt/mTOR pathway to suppress tumorigenesis and metastasis in osteosarcoma, and predicts a favorable prognosis. Journal of Experimental & Clinical Cancer Research. 37(1):188.




How to Cite

NEPOMUCENO, L. L. .; SOARES, N. P. .; BORGES, J. C. de A. .; VIEIRA, V. de S.; PEREIRA, D. K. S.; PEREIRA, K. F.; CRUZ, V. de S. .; FERREIRA, J. L. .; ARNHOLD, E. .; ARAÚJO, E. G. de . Ethanolic extract of Caryocar brasiliense fruit peel promotes death and cell cycle control in canine osteosarcoma cells . Research, Society and Development, [S. l.], v. 9, n. 10, p. e7299109194, 2020. DOI: 10.33448/rsd-v9i10.9194. Disponível em: Acesso em: 28 jun. 2022.



Agrarian and Biological Sciences