Abstract

Curcuma longa L., also known as turmeric, has been widely studied for its various therapeutic properties, including antineoplastic action. The ethanolic extract of the plant contains several phenolic compounds, especially curcumin. Osteosarcoma is a predominant bone tumor in dogs and humans, characterized by high metastatic potential and an unfavorable prognosis. The aim of this study was to investigate the effects of turmeric ethanol extract on canine osteosarcoma cells from established culture. The cells were cultured and treated with different curcumin concentrations (0, 10 μM, 20 μM, 50 μM, 100 μM, and 1000 μM) and exposure times (24h, 48h, and 72h). We first performed tetrazolium reduction technique (MTT) assay and calculated IC₅₀. An immunocytochemistry assay was performed after extract treatment to verify the expression of mutated p53 and therefore study the proliferative potential of malignant cells; Bcl-2 and Ki-67 were used to assess apoptosis and the degree of malignancy, respectively. The extract enhanced the proliferation of canine osteosarcoma cells, reaching 3,819.74% at 50 μM of curcumin. The extract also significantly altered the expression of mutated p53 and Ki-67 proteins but not that of Bcl-2, suggesting that it did not induce this antiapoptotic pathway. Overall, these results are prerequisite to better understanding how natural compounds such as turmeric ethanolic extract affect cell proliferation and could be used to treat various diseases.

Keywords:
bone neoplasms; D-17; natural compounds; therapeutic safety

Resumo

A Curcuma longa L., planta conhecida popularmente como açafrão, tem sido amplamente estudada por suas diversas propriedades terapêuticas, incluindo a ação antineoplásica. O extrato etanólico da planta contém diversos compostos fenólicos, com destaque para a curcumina. O osteossarcoma é um tumor ósseo predominante em cães e humanos, caracterizado por apresentar alto potencial metastático e prognóstico desfavorável. Procurou-se investigar os efeitos de diferentes concentrações de curcumina do extrato etanólico de açafrão sobre células de osteossarcoma canino de cultura estabelecida. As células foram cultivadas e submetidas ao tratamento com extrato com diferentes concentrações de curcumina (0, 10 μM, 20 μM, 50 μM, 100 μM e 1000 μM) e tempos de exposição (24h, 48h e 72h) pelo EEA. Inicialmente, foram realizados: técnica de redução do tetrazólio (MTT) e cálculo da IC₅₀. Posteriormente, após o tratamento com o extrato, realizou-se o ensaio de imunocitoquímica para verificar a expressão de p53 mutada e estudar o potencial proliferativo das células malignas; Bcl-2, com intuito de averiguar o estímulo de via antiapoptótica; e o marcador Ki-67, que sinaliza aumento no grau de malignidade. O extrato promoveu proliferação de células de osteossarcoma canino, com incremento de até 3819,74% na concentração de 50μM de curcumina. O composto também alterou a expressão das proteínas p53 mutante e Ki-67 significativamente, mas não alterou a expressão de Bcl-2, mostrando que não induziu a via antiapoptótica mediada por esta. Estes resultados demonstram que o extrato etanólico do açafrão apresenta potencial proliferativo sobre células de osteossarcoma canino, sugerindo a necessidade de conscientização e conhecimento dos reais efeitos de determinados compostos naturais, considerados seguros ao serem utilizados como tratamento de diversas enfermidades.

Palavras-chave
compostos naturais; D-17; neoplasias ósseas; segurança terapêutica

Introduction

Plant-derived products are among the most promising sources for medicine production (¹1 Salehi B, Ata A, Kumar NVA, Sharopov F, Ramírez-Alarcón K, Ruiz-Ortega A, Ayatollahi SA, Fokou PVT, Kobarfard F, Zakaria ZA, Iriti M, Taheri Y, Martorell M, Sureda A, Setzer WN, et al. Antidiabetic potential of medicinal plants and their active components. Vol. 9, Biomolecules. 2019.,²2 Sharifi-Rad J, Sureda A, Tenore GC, Daglia M, Sharifi-Rad M, Valussi M, Tundis R, Sharifi-Rad M, Loizzo MR, Oluwaseun Ademiluyi A, Sharifi-Rad R, Ayatollahi SA, Iriti M. Biological activities of essential oils: From plant chemoecology to traditional healing systems. Vol. 22, Molecules. 2017.). Indeed, 25% of medications prescribed worldwide are plant-derived; among the 252 medicines that make up the essential drugs list of the World Health Organization (WHO), 11% are entirely of plant origin (³3 Nakaziba R, Anyolitho MK, Amanya SB, Sesaazi CD, Byarugaba F, Ogwal-Okeng J, Alele PE. Traditional medicinal vegetables in northern uganda: An ethnobotanical survey. Int J Food Sci. 2021;2021.,⁴4 Veeresham C. Natural products derived from plants as a source of drugs. Vol. 3, Journal of Advanced Pharmaceutical Technology and Research. 2012. p. 200-1.). Cerrado, the second largest biome in Brazil, offers many plants featured in popular medicines despite habitat destruction (⁵5 Cortelo PC, Demarque DP, Dusi RG, Albernaz LC, Braz-Filho R, Goncharova EI, Bokesch HR, Gustafson KR, Beutler JA, Espindola LS. A molecular networking strategy: High-throughput screening and chemical analysis of brazilian cerrado plant extracts against cancer cells. Cells. 2021;10(3):1-13. Available from: https://10.3390/cells10030691
https://10.3390/cells10030691... ). Its widespread biodiversity includes natural products that are often used as therapeutics (⁶6 Teklehaymanot T. An ethnobotanical survey of medicinal and edible plants of Yalo Woreda in Afar regional state, Ethiopia. J Ethnobiol Ethnomed. 2017;13(1).).

Although secondary plant compounds harbor therapeutic potential, some of their substances can harm human health. More studies should explore the pharmacological mechanisms and their possible side effects (⁷7 Fatima I, Kanwal S, Mahmood T. Evaluation of biological potential of selected species of family Poaceae from Bahawalpur, Pakistan. BMC Complement Altern Med. 2018;18(1):1-13.) in addition to identifying which compound is responsible for each therapeutic action (⁸8 Seepe HA, Nxumalo W, Amoo SO. Natural products from medicinal plants against phytopathogenic fusarium species: Current research endeavours, challenges and prospects. Molecules. 2021;26(21).,⁹9 Clemen-Pascual LM, Macahig RAS, Rojas NRL. Comparative toxicity, phytochemistry, and use of 53 Philippine medicinal plants. Toxicol Reports. 2022;9:22-35.). The final product may have undergone acquisition, preparation, or conservation that do not render its phytochemical composition identical to that of the native plant (¹⁰10 Morlock GE, Heil J, Bardot V, Lenoir L, Cotte C, Dubourdeaux M. Effect-directed profiling of 17 different fortified plant extracts by high-performance thin-layer chromatography combined with six planar assays and high-resolution mass spectrometry. Molecules. 2021;26(5).).

Curcuma longa L., popularly known as turmeric, belongs to the family Zingiberaceae of genus Curcuma; it is a food spice appreciated worldwide to color and flavor foods. Although the plant originates from the tropical forests of India, the biggest supplier worldwide (¹¹11 Borges J, Saturnino K, Cruz V, Araújo E. Ação Antioxidante Da Curcumina (Curcuma Longa L.) Na Injúria De Isquemia E Reperfusão Tecidual. Enciclopédia Biosf. 2019;16(29):48-62.,¹²12 Girme A, Saste G, Pawar S, Ghule C, Mirgal A, Patel S, Tiwari A, Ghoshal S, Bharate SB, Bharate SS, Reddy DS, Vishwakarma RA, Hingorani L. Quantitative Determination and Characterization of a Kashmir Saffron (Crocus sativus L.)-Based Botanical Supplement Using Single-Laboratory Validation Study by HPLC-PDA with LC-MS/MS and HPTLC Investigations. ACS Omega. 2021;6(36):23460-74.), it is also widely consumed and produced in Mara Rosa, a central municipality in Brazil. Turmeric contains a large fraction (6.8%) of curcumin, a highly therapeutic molecule (¹³13 Marchi JP, Tedesco L, Melo ADC, Frasson AC, França VF, Wietzikoski Sato S, Wietzikoski EC. Curcuma longa l., o açafrão da terra, e seus benefícios medicinais. Arq Ciências da Saúde da UNIPAR. 2016;20(3).). Curcumin reduces lipogenesis and exerts antipruritic, anti-inflammatory, antineoplastic, analgesic, antimicrobial, antifungal, antiviral, and antioxidant effects (¹⁴14 Lee SY, Cho SS, Li YC, Bae CS, Park KM, Park DH. Anti-inflammatory Effect of Curcuma longa and Allium hookeri Co-treatment via NF-κB and COX-2 Pathways. Sci Rep. 2020;10(1):1-11.

15 Nascimento PRS do, Silva Júnior EL da, Branco AC da SC. Aplicações farmacológicas da Cúrcuma longa L. como planta medicinal: Uma revisão. Res Soc Dev. 2020;9(10):e2629108430.

16 Czernicka L, Grzegorczyk A, Marzec Z, Antosiewicz B, Malm A, Kukula-Koch W. Antimicrobial potential of single metabolites of Curcuma longa assessed in the total extract by thin-layer chromatography-based bioautography and image analysis. Int J Mol Sci. 2019;20(4).

17 Chen C, Long L, Zhang F, Chen Q, Chen C, Yu X, Liu Q, Bao J, Long Z. Antifungal activity, main active components and mechanism of Curcuma longa extract against Fusarium graminearum. PLoS One. 2018;13(3):1-19.-¹⁸18 Soares NP, Nepomuceno LL, Cruz V de S, Arnhold E, Vieira V de S, Borges JC de A, Pereira DKS, Pereira KF, Araújo EG de. Curcumin promotes extrinsic apoptosis in canine osteosarcoma cells. Res Soc Dev. 2020;9(10):e7289109231. Available from: https://10.33448/rsd-v9i10.9231
https://10.33448/rsd-v9i10.9231... ). In practice curcumin has been widely sold as a phytotherapeutic agent to treat inflammatory bone diseases in canine (¹⁹19 Comblain F, Serisier S, Barthelemy N, Balligand M, Henrotin Y. Review of dietary supplements for the management of osteoarthritis in dogs in studies from 2004 to 2014. J Vet Pharmacol Ther. 2016;39(1):1-15.) and human patients (²⁰20 Mathieu S, Soubrier M, Peirs C, Monfoulet LE, Boirie Y, Tournadre A. A Meta-Analysis of the Impact of Nutritional Supplementation on Osteoarthritis Symptoms. Nutrients. 2022;14(8):1-21.). However, its mechanisms and related cell signaling pathways remain poorly characterized.

A previous study showed that pure curcumin inhibits tumor development in canine osteosarcoma (CO) cells by activating extrinsic apoptosis through the JNK kinase and the caspase-3 pathways, which are mediated by the cAMP/AMPK signal transduction pathway. Furthermore, curcumin can reduce the expression of the p53 mutant protein without interfering in the autophagic pathway of AKT/mTOR (¹⁸18 Soares NP, Nepomuceno LL, Cruz V de S, Arnhold E, Vieira V de S, Borges JC de A, Pereira DKS, Pereira KF, Araújo EG de. Curcumin promotes extrinsic apoptosis in canine osteosarcoma cells. Res Soc Dev. 2020;9(10):e7289109231. Available from: https://10.33448/rsd-v9i10.9231
https://10.33448/rsd-v9i10.9231... ). However, other compounds found in turmeric extract such as phenols are antioxidants that neutralize free radicals (²¹21 Sabir SM, Zeb A, Mahmood M, Abbas SR, Ahmad Z, Iqbal N. Phytochemical analysis and biological activities of ethanolic extract of curcuma longa rhizome. Brazilian J Biol. 2021;81(3):737-40.) and could interfere with the antiapoptotic capabilities of curcumin.

CO is an aggressive neoplasm that represents up to 85% of all bone tumors in dogs. Most tumors originate in the metaphyseal region of the bones in the appendicular skeleton. The risk factors for CO include sex, breed, history of trauma, and whether the dog is neutered. Female dogs have a lower risk, while larger dogs (>25 kg) are at greater risk. Affected animals usually exhibit lameness and swelling and may experience pathological fracture, especially in the femur. CO prognosis is generally poor due to the high global rate of metastasis. Common treatment includes amputation and follow-up chemotherapy (adjuvant); the disease-free interval and the global survival time are typically 291 and 284 days, respectively. Few therapeutic alternatives have emerged in recent decades, warranting new advances to treat CO (²²22 Poon AC, Matsuyama A, Mutsaers AJ. Recent and current clinical trials in canine appendicular osteosarcoma. Can Vet J. 2020;61(3):301-8.). Mutated tumor suppressor genes are potentially related to the proliferation and migration of neoplastic cells, leading to metastasis (²³23 Bieging KT, Mello SS, Attardi LD. Unravelling mechanisms of p53-mediated tumour suppression. Vol. 14, Nature Reviews Cancer. 2014. p. 359-70.). Ideal therapeutic agents would reduce such proliferation and induce apoptosis in tumor cells to improve chances of survival (¹⁸18 Soares NP, Nepomuceno LL, Cruz V de S, Arnhold E, Vieira V de S, Borges JC de A, Pereira DKS, Pereira KF, Araújo EG de. Curcumin promotes extrinsic apoptosis in canine osteosarcoma cells. Res Soc Dev. 2020;9(10):e7289109231. Available from: https://10.33448/rsd-v9i10.9231
https://10.33448/rsd-v9i10.9231... ).

The present study investigated how the ethanolic extract of Curcuma longa L. (TEE) from the region of Mara Rosa affected canine osteosarcoma cells and analyzed the expression of biomarkers relevant to the progression of this neoplasm.

Material and methods

The experiment was developed at the Multiuser Laboratory for the Evaluation of Molecules, Cells, and Tissues in the Veterinary and Zootechny School at the Federal University of Goiás.

Preparation of the turmeric ethanolic extract (Curcuma longa L.)

A turmeric ethanolic extract was obtained and prepared as previously described (²⁴24 Teles JC de AB, Soares; NP, Graziani; D, Júnior; ER de O, Lima; EM, Nepomuceno; LL, Vieira; V de S, Cruz; V de S, Saturnino; KC, Arnhold; E, Eugênio Gonçalves de Araújo. Curcumin rich extract from central brazil Curcuma longa protects human umbilical vein endothelial cells under oxidative stress. ABCS Heal Sci. 2022;In Press(MS 2021-005):1-10.). The rhizomes of Curcuma longa L. were acquired from the Turmeric Products Cooperative of Mara Rosa (Cooperaçafrão), located in the town of Mara Rosa, state of Goiás, Brazil. The rhizomes were first removed from the soil, then washed and dried on a kiln to be processed. Afterwards, 100 g of the dry and ground material were added to 1000 ml of absolute ethanol, which underwent cold percolation. The supernatant was filtered out, and the solvent was evaporated on a rotary evaporator (IKA® RV 05 Basic) (²⁵25 Péret-Almeida L, da Cunha Naghetini C, de Aguiar Nunan E, Junqueira RG, Beatriz Abreu Glória M. In vitro antimicrobial activity of the ground rhizome, curcuminoid pigments and essential oil of Curcuma longa L. Cienc e Agrotecnologia. 2008;32(3):875-81.).

The composition of the extract was previously characterized (²⁴24 Teles JC de AB, Soares; NP, Graziani; D, Júnior; ER de O, Lima; EM, Nepomuceno; LL, Vieira; V de S, Cruz; V de S, Saturnino; KC, Arnhold; E, Eugênio Gonçalves de Araújo. Curcumin rich extract from central brazil Curcuma longa protects human umbilical vein endothelial cells under oxidative stress. ABCS Heal Sci. 2022;In Press(MS 2021-005):1-10.). According to TEE, the extract contains on average 16.7% curcumin, as measured by fluorescent high pressure liquid chromatography. The total content of phenolic compounds was determined from the standard curve of gallic acid (gallic acid equivalent: GAE) as 691.49 GAE/100 g. The extract's ability to scavenge free radicals was assessed from its discoloration of DPPH (2,2 diphenyl-1-picrylhydrazyl) and a standard reagent (Trolox) equivalent to vitamin E. The TEE has an antioxidant activity of approximately 18.45 μM Trolox/g of sample; the dislocation percentage of DPPH is 55% at IC₅₀ of 18.1 μg/mL. The crude extract was stored in a refrigerator and protected from photodegradation at every step.

Cell culture

Canine metastatic osteosarcoma cells (D-17, BCRJ 0276, batch 000573, pass 239, species Canis familiaris) originated from ATCC (American Type Culture Collection - Manassas, VA, USA) were acquired from the Cell Bank of Rio de Janeiro (UFRJ-Rio de Janeiro, Brazil). The cells were cultured in Dulbecco's Modified Eagle's Medium (DMEM Thermo Fisher, Waltham, MA, USA) enriched with 10% fetal bovine serum, penicillin, and streptomycin (10,000 I.U./mL), amphotericin B, and L-glutamine (all reagents from Cultilab, Campinas, Brazil) and maintained in a humidified incubator at 37°C and 5% CO₂.

Cell viability and cytotoxicity assay

After culture, the cells were quantified on a Neubauer chamber, and 1 × 10⁴ cells in 200 μL of DMEM per well were seeded in 96-well plates. The plates were maintained in a humidified incubator at 37°C with 5% CO₂ for 24 hours. Then the medium was discarded, and the wells were treated with TEE diluted in 1% of DMSO (dimethyl sulfoxide) at various concentrations of curcumin calculated from its molecular weight (368.38 g/mol) and its concentration in TEE (16.7%): 0 μM, 10 μM, 20 μM, 50 μM, 100 μM, and 1000 μM (Table 1). The cells were exposed to TEE at 24, 48, or 72 hours. The control group was treated only with DMSO. The wells were treated randomly, and each treatment was performed in quintuplicate for three independent experiments.

A cell viability assay - the reduction of tetrazolium salt (MTT assay) - was performed after the treatment period. The culture medium was discarded, and 10 μL of tetrazolium (MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium) were added to each well. The plates were incubated for three hours. Fifty μL of sodium dodecyl sulfate (SDS-Vivantis Biochemical) were added to each well to terminate the reaction, and the plates remained incubated for 24 hours at room temperature. Optical density was measured with a spectrophotometer (Awareness Technology Stat Fax 2100, 425-540 nm, Palm City, FL, USA). Three wavelengths were used to discern the absorbances measured by the spectrophotometer because the size of the molecules, density, dilution, and other factors can influence the physical properties of the samples and thus interfere with the ideal wavelength (²⁶26 Nepomuceno LL, Carvalho LS de, Soares NP, Cruz V de S, Arnhold E, Ferreira JL, Araújo EG de. Padronização Metodológica in Vitro Do Ensaio Colorimétrico Mtt Para Avaliação Da Atividade De Formazan E Dosagem Do Fármaco: Padronização Do Teste Colorimétrico Mtt. In: Samylla MCS, editor. Farmacologia Aplicada à Enfermagem: Aspectos Teóricos e Práticos. 1st ed. 2021. p. 149-61. Available from: https://10.37885/201202541
https://10.37885/201202541... ).

Cell viability (CV) was determined by the equation

where A indicates absorbance. Data were collected from three independent studies. One-way analysis of variance (ANOVA) and Tukey's post-hoc test were performed in R using the package easyanova^® to compare groups as previously described (¹⁸18 Soares NP, Nepomuceno LL, Cruz V de S, Arnhold E, Vieira V de S, Borges JC de A, Pereira DKS, Pereira KF, Araújo EG de. Curcumin promotes extrinsic apoptosis in canine osteosarcoma cells. Res Soc Dev. 2020;9(10):e7289109231. Available from: https://10.33448/rsd-v9i10.9231
https://10.33448/rsd-v9i10.9231... ). A p-value <0.05 was considered statistically significant.

Immunocytochemistry

The D-17 cells were seeded on culture slides with a Falcon™ (Corning, Glendale, EUA) chamber at a concentration of 1 × 10⁴; they were cultured and exposed to TEE for 48 hours at the studied concentrations (0, 10, 20, 50, 100, or 1000 μM). After this period, the cells were fixed with 4% paraformaldehyde for 30 minutes.

All steps were executed using the automatic processor BOND-MAX (Leica Biosystems, Nussloch, Germany) with the following reagents: BOND Polymer Refine Detection, BOND Wash Solution, BOND Epitope Retrieval 1, BOND Dewax Solution, BOND Epitope Retrieval 2, BOND DAB (3,3’-diaminobenzidine chromogen), and hematoxylin. The antibodies used were anti-Bcl-2, anti-p53 (SC71785 monoclonal of mouse), and anti-Ki-67, which were diluted 1:500 in 1.5% bovine serum albumin. A group that was not treated with primary antibody served as a control.

Immunostaining was evaluated by counting the total number of cells in five distinct fields and determining the percentage of antibody-stained cells, a procedure adapted from Fedchenko & Reifenrath (²⁷27 Fedchenko N, Reifenrath J. Different approaches for interpretation and reporting of immunohistochemistry analysis results in the bone tissue - a review. Vol. 9, Diagnostic pathology. 2014. p. 221.). To establish the expression intensity score (EIS), a blind semi-quantitative analysis was performed to establish an expression intensity score (EIS) whose values ranged from 0 to 3 according to the shades on a colorimetric scale (¹⁸18 Soares NP, Nepomuceno LL, Cruz V de S, Arnhold E, Vieira V de S, Borges JC de A, Pereira DKS, Pereira KF, Araújo EG de. Curcumin promotes extrinsic apoptosis in canine osteosarcoma cells. Res Soc Dev. 2020;9(10):e7289109231. Available from: https://10.33448/rsd-v9i10.9231
https://10.33448/rsd-v9i10.9231... ) (Figure 1). The positively stained cells ratio (PSCR) was calculated as the ratio of the number of stained cells to the total number of cells multiplied by 100. From this value several categories were established: 0 for 0%, 1 for <1%, 2 for 1%-10%, 3 for 11%-33%, 4 for 34%-66%, and 5 for >67%. The PSCR was added to the EIS to define a semi-quantitative combined scoring system (SCSS) whose average and standard deviation were calculated and outliers excluded. ANOVA and the Kruskal-Wallis test were performed with R software and package easyanova^®, where p < 0.05 indicated statistical significance (¹⁸18 Soares NP, Nepomuceno LL, Cruz V de S, Arnhold E, Vieira V de S, Borges JC de A, Pereira DKS, Pereira KF, Araújo EG de. Curcumin promotes extrinsic apoptosis in canine osteosarcoma cells. Res Soc Dev. 2020;9(10):e7289109231. Available from: https://10.33448/rsd-v9i10.9231
https://10.33448/rsd-v9i10.9231... ).

Results

Cell viability and cytotoxicity assay

The experiment was performed with different concentrations and exposure times to determine if the TEE affected viability of the D-17 cells. The averages of the treatments and the exposure times were compared. The compound exponentially and significantly affected proliferation until the concentration of TEE approached 50 µM. Cell proliferation decreased at higher concentrations. Extract exposure times did not significantly affect cell proliferation (Table 2).

This effect can be better understood when an average of the cell proliferation values was taken on the three experimental times in relation to the used concentrations, as demonstrated on Table 3.

Automated immunocytochemistry was performed to better understand the mechanisms that determined CO cell viability. The intensity of Bcl-2, an antiapoptotic marker, did not significantly differ across extract concentrations (Table 3 and Figure 2). However, that of the mutant p53 protein, which does independently affect cellular proliferation, increased with TEE concentration, and peaked at 1000 µM of curcumin (Table 4 and Figure 2). This behavior matched that of Ki-67, a marker for tumor malignancy (Table 3 and Figure 2).

Discussion

This work explored how turmeric (Curcuma longa L.) affects proliferation of canine CO cells. To date no studies have examined the effect of this extract on proliferation of cancer cell lineages let alone that of canine CO cells: reports focus solely on the benefits of C. longa L. This is unsurprising given the low toxicity of the plant and its isolated compounds; a retrospective study on the toxicity of turmeric and its main component, curcumin, found that doses as high as 3.6 g of daily curcumin for six months or 4 g of fermented turmeric extract for 12 weeks was safe in humans (²⁸28 Soleimani V, Sahebkar A, Hosseinzadeh H. Turmeric (Curcuma longa) and its major constituent (curcumin) as nontoxic and safe substances: Review. Phyther Res. 2018;32(6):985-95.).

Contrary to this study's findings, many have concluded that the constituents of turmeric, especially curcumin, are highly effective against cancer. Their anti-proliferative and anti-cancer activities have proven promising in pre-clinical studies, though rare side effects have been described in normal cells (²⁸28 Soleimani V, Sahebkar A, Hosseinzadeh H. Turmeric (Curcuma longa) and its major constituent (curcumin) as nontoxic and safe substances: Review. Phyther Res. 2018;32(6):985-95.

29 Giannoulaki P, Kotzakioulafi E, Chourdakis M, Hatzitolios A, Didangelos T. Impact of Crocus sativus L. on metabolic profile in patients with diabetes mellitus or metabolic syndrome: A systematic review. Nutrients. 2020;12(5):1-17.-³⁰30 Vahdati Hassani F, Naseri V, Razavi BM, Mehri S, Abnous K, Hosseinzadeh H. Antidepressant effects of crocin and its effects on transcript and protein levels of CREB, BDNF, and VGF in rat hippocampus. DARU, J Pharm Sci. 2014;22(1):1-9.).Yet we have found that TEE boosts cell proliferation of D-17 cells according to MTT assay (Figure 2). The cells exposed to 50 μM of curcumin showed the highest percentage of cell viability, which declined at higher TEE concentrations. The exposure time to the extract did not significantly influence cell proliferation.

We expected that curcumin would cause extrinsic apoptosis of D-17 cells given previous findings in cells of the same lineage (¹⁸18 Soares NP, Nepomuceno LL, Cruz V de S, Arnhold E, Vieira V de S, Borges JC de A, Pereira DKS, Pereira KF, Araújo EG de. Curcumin promotes extrinsic apoptosis in canine osteosarcoma cells. Res Soc Dev. 2020;9(10):e7289109231. Available from: https://10.33448/rsd-v9i10.9231
https://10.33448/rsd-v9i10.9231... ). Curcumin can induce intrinsic and extrinsic apoptosis in different tumor cell lineages in addition to inducing the production of reactive oxygen species, which positively regulate apoptosis receptors on the tumor cell membrane. The compound also regulates the expression and activity of native p53 to inhibit the proliferation of tumor cells and increase apoptosis while inhibiting NF-κB and COX-2, which are involved in the superexpression of antiapoptotic genes such as Bcl-2 (³¹31 Mortezaee K, Salehi E, Mirtavoos-mahyari H, Motevaseli E, Najafi M, Farhood B, Rosengren RJ, Sahebkar A. Mechanisms of apoptosis modulation by curcumin: Implications for cancer therapy. J Cell Physiol. 2019;234(8):12537-50.). Curcumin previously inhibited the growth of MG-63 CO cells, inducing apoptosis by activating caspase-3 and reducing expression levels of Bcl-2 (³²32 Zhang Y, Chen P, Hong H, Wang L, Zhou Y, Lang Y. JNK pathway mediates curcumin-induced apoptosis and autophagy in osteosarcoma MG63 cells. Exp Ther Med. 2017;14(1):593-9.). Here, on the contrary, TEE did not kill canine CO cells.

Importantly, this study used turmeric ethanolic extract, which comprises various compounds. Although the mechanisms of its major compounds are defined, its remaining phytochemical products are poorly characterized and therefore may exert unknown side effects or exponential toxicity. The metabolites of the extract may also interact with metabolic pathways to activate genes that cultivate neoplasms (³³33 Akhtar MF, Saleem A, Sharif A, Akhtar B, Nasim M Bin, Peerzada S, Raza M, Ijaz H, Ahmed S, Shabbir M, Ali S, Akbar Z, Ul Hassan SS. Genotoxic and cytotoxic action potential of Terminalia citrina, a medicinal plant of ethnopharmacological significance. EXCLI J. 2016;15:589-98.,³⁴34 Amarasiri SS, Attanayake AP, Arawwawala LDAM, Jayatilaka KAPW, Mudduwa LKB. Acute and 28-Day Repeated-Dose Oral Toxicity Assessment of Abelmoschus moschatus Medik. In Healthy Wistar Rats. Evidence-based Complement Altern Med. 2020;2020.) or protect tumor cells.

The pharmacological effects and mechanisms of the isolated compounds of turmeric are predominantly deemed beneficial, but this does not indicate that the extract studied here is safe under the same conditions. The cancer cells studied here increasingly proliferated when exposed to greater concentrations of TE and thus curcumin, which in isolation has proven efficacious against cancer cell lineages through the STAT3 apoptotic pathway. Curcumin also inhibited proliferation of thyroid papillary adenocarcinoma cells in a concentration-dependent manner (³⁵35 Khan AQ, Ahmed EI, Elareer N, Fathima H, Prabhu KS, Siveen KS, Kulinski M, Azizi F, Dermime S, Ahmad A, Steinhoff M, Uddin S. Curcumin-mediated apoptotic cell death in papillary thyroid cancer and cancer stem-like cells through targeting of the JAK/STAT3 signaling pathway. Int J Mol Sci. 2020;21(2).).

The total amount of phenols in TEE is reportedly 691.49 mg of EAG/100 g. Recently, the ethanolic extract of the pequi shell (Caryocar brasiliense, Camb.), whose total phenol content (696.91 mg of EAG/100 g) is similar to that of the TEE used in this study, exerted protective effects in human coronary endothelial cells (³⁶36 Braga KMS, Araujo EG, Sellke FW, Abid MR. Pequi fruit extract increases antioxidant enzymes and reduces oxidants in human coronary artery endothelial cells. Antioxidants. 2022;11(3):474. Available from: https://10.3390/ANTIOX11030474
https://10.3390/ANTIOX11030474... ). Most phenols in turmeric are curcuminoids, but other phenolic compounds such as caffeic acid, coumaric acid, and quercetin (²¹21 Sabir SM, Zeb A, Mahmood M, Abbas SR, Ahmad Z, Iqbal N. Phytochemical analysis and biological activities of ethanolic extract of curcuma longa rhizome. Brazilian J Biol. 2021;81(3):737-40.) are antioxidants found in the pequi extract.

We qualitatively and semi-quantitatively evaluated the expression levels of mutated p53 protein, Ki-67, and Bcl-2 using automated immunocytochemistry (²⁷27 Fedchenko N, Reifenrath J. Different approaches for interpretation and reporting of immunohistochemistry analysis results in the bone tissue - a review. Vol. 9, Diagnostic pathology. 2014. p. 221.,³⁷37 Nepomuceno LL, Soares NP, Borges JC de A, Vieira V de S, Pereira DKS, Pereira KF, Cruz V de S, Ferreira JL, Arnhold E, Araújo EG de. Extrato etanólico da casca de fruta de Caryocar brasiliense promove a morte e o controle do ciclo celular em células osteossarcoma caninas. Res Soc Dev. 2020;9(10):e7299109194.). The data revealed relatively strong expression levels of mutated p53 protein and Ki-67, especially in the group treated with 1000 μM of the extract. The mutated p53 protein was found in and thus affected the cellular proliferation of every experimental group; its expression is a reliable marker of proliferation and tumor progression because the mutation in the TP53 gene changes the structure of p53 protein, which loses its ability to repair DNA or induce tumor cell death (³⁸38 Kandoth C, McLellan MD, Vandin F, Ye K, Niu B, Lu C, Xie M, Zhang Q, McMichael JF, Wyczalkowski MA, Leiserson MDM, Miller CA, Welch JS, Walter MJ, Wendl MC, et al. Mutational landscape and significance across 12 major cancer types. Nature. 2013;502(7471):333-9.)(³⁹39 Sabapathy K, Lane DP. Understanding p53 functions through p53 antibodies. Vol. 11, Journal of Molecular Cell Biology. 2019. p. 317-29.). Ki-67 also indicates potential malignancy potential in various types of neoplasms, including canine OS cells (³⁷37 Nepomuceno LL, Soares NP, Borges JC de A, Vieira V de S, Pereira DKS, Pereira KF, Cruz V de S, Ferreira JL, Arnhold E, Araújo EG de. Extrato etanólico da casca de fruta de Caryocar brasiliense promove a morte e o controle do ciclo celular em células osteossarcoma caninas. Res Soc Dev. 2020;9(10):e7299109194.,⁴⁰40 Vieira VS, Cruz VS, Nepomuceno LL, Soares NP, Arnhold E, Teixeira WFP, Vieira DS, Borges JCA, Paixão FM, Araújo EG, Vieira VS. Crude extract of Jatobá leaves promotes canine osteosarcoma cell D17 proliferation. Vet World. 2022;15(5):1283-9.

41 Wang Y xing, Wang Y yuan, Yang C gang, Bu H, Yang W tao, Wang L, Xu W mang, Zhao X long, Zhao W xing, Li L, Song S ling, Yang J lun. An interobserver reproducibility analysis of size-set semiautomatic counting for Ki67 assessment in breast cancer. Breast. 2020;49:225-32.-⁴²42 Robertson S, Acs B, Lippert M, Hartman J. Prognostic potential of automated Ki67 evaluation in breast cancer: different hot spot definitions versus true global score. Breast Cancer Res Treat. 2020;183(1):161-75.).

The expression level of Bcl-2 in canine CO cells did not significantly change here (Table 4), unlike previous studies that have found higher expression levels of Bcl-2 protein in cells treated with 50 μM. Although the mechanisms here remain unknown, its unchanging expression here could be related to other constituents in the extract that have antiapoptotic effects in our cells. Bcl-2 regulates cell apoptosis, as it encodes a protein in the mitochondrial membrane that inhibits cell death by apoptosis and activating cell proliferation (⁴³43 Jong Kyong KIM, Alan Diehl J. Nuclear cyclin D1: An oncogenic driver in human cancer. Vol. 220, Journal of Cellular Physiology. 2009. p. 292-6.,⁴⁴44 Adams JM, Cory S. The BCL-2 arbiters of apoptosis and their growing role as cancer targets. Cell Death Differ. 2018;25(1):27-36.).

The outcome of this study disagrees with similar studies that evaluated the cytotoxicity of other plant extracts on cancer cells by measuring cell proliferation and expression of oncogenes (³⁷37 Nepomuceno LL, Soares NP, Borges JC de A, Vieira V de S, Pereira DKS, Pereira KF, Cruz V de S, Ferreira JL, Arnhold E, Araújo EG de. Extrato etanólico da casca de fruta de Caryocar brasiliense promove a morte e o controle do ciclo celular em células osteossarcoma caninas. Res Soc Dev. 2020;9(10):e7299109194.,⁴⁰40 Vieira VS, Cruz VS, Nepomuceno LL, Soares NP, Arnhold E, Teixeira WFP, Vieira DS, Borges JCA, Paixão FM, Araújo EG, Vieira VS. Crude extract of Jatobá leaves promotes canine osteosarcoma cell D17 proliferation. Vet World. 2022;15(5):1283-9.,⁴⁵45 Kowalczyk T, Sitarek P, Skała E, Toma M, Wielanek M, Pytel D, Wieczfińska J, Szemraj J, Śliwiński T. Induction of apoptosis by in vitro and in vivo plant extracts derived from Menyanthes trifoliata L. in human cancer cells. Cytotechnology. 2019;71(1):165-80.,⁴⁶46 Mbaveng AT, Kuete V, Efferth T. Potential of central, Eastern and Western Africa medicinal plants for cancer therapy: Spotlight on resistant cells and molecular targets. Vol. 8, Frontiers in Pharmacology. 2017.). This warrants precaution when administering plants and their derivatives as phytotherapeutics, whose efficacy and safety are assured almost instantaneously because they are native and organic. However, plant metabolites can cause unknown and potentially deleterious effects such as subacute chronic toxicity, carcinogenesis (³⁴34 Amarasiri SS, Attanayake AP, Arawwawala LDAM, Jayatilaka KAPW, Mudduwa LKB. Acute and 28-Day Repeated-Dose Oral Toxicity Assessment of Abelmoschus moschatus Medik. In Healthy Wistar Rats. Evidence-based Complement Altern Med. 2020;2020.,⁴⁷47 Chahardehi AM, Arsad H, Lim V. Zebrafish as a successful animal model for screening toxicity of medicinal plants. Vol. 9, Plants. 2020. p. 1-35.), or the proliferation of pre-existent atypical cell clones, such as reported here.

This study is the first to show the proliferative properties of turmeric extract on canine OS cells. Our findings highlight the importance of validating every potentially therapeutic compound even if it is naturally derived; the mechanisms of action and related signaling pahways should be revealed to confirm safety and efficacy of prescribed doses.

Conclusion

Turmeric ethanolic extract increased the in vitro proliferation of D-17 canine OS cells and the expression of proteins related to oncogenesis and tumor malignancy. More studies should explore the apoptotic mechanisms of turmeric before using it to treat CO in dogs or humans.

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