Biomedical Research

Abstract

Breast cancer is the type of cancer that kills more women in the world and the conventional cancer treatment has high costs and many side effects. The bioactive compounds from diet alone or in combination with others substances may be a potential source for the prevention and treatment of breast cancer. The aim was to investigate the effect of curcumin, piperine and resveratrol alone and associated in MCF-7 and MDA-MB-231 breast cancer cells culture. Curcumin, piperine and resveratrol showed cytotoxicity for MCF-7 and MDA-MB-231 cells with IC50 values of 24.50 μM, 94.50 μM and 131.00 μM, and 23.30 μM, 276.00 μM and 306.00 μM, respectively. The combination of curcumin+piperine, curcumin+resveratrol, piperine+resveratrol showed synergy in MCF-7 cells, and piperine+resveratrol in MDA-MB-231 cells. We conclude that the combination of bioactive compounds presents in diet as curcumin, piperine and resveratrol is better than used in isolated forms, and these results may be useful in future clinical trials with breast cancer.

Keywords

Breast cancer cells, Resveratrol, Piperine, Curcumin, Synergism.

Introduction

Cancer is one of the leading causes of death worldwide. According to WHO in 2015, there were 8.8 million deaths and breast cancer contributed 571.000 deaths. Breast cancer is the type of cancer that kills more women in the world, particularly in low- and middle-income countries [1]. Current treatments for cancer include cytotoxic chemotherapy. However, some limitations are observed as high toxicity, in which it implies many adverse effects, but contributes to the survival of the patient [2].

Previous studies have demonstrated that bioactive compounds are a promising source to search for novel drugs for the treatment of various diseases. Curcumin (1,7-bis(4-hydroxy-3- methoxyphenyl)-1,6-hepta-diene-3,5-dione) is a lipophilic phenolic compound extracted from the rhizome of turmeric and possess various biological effects such antioxidant, antiinflammatory, antimicrobial and anticancer [3-5]. Piperine {1- [5-(1,3-benzodioxol-5-yl)-1-oxo-2,4-pentadienyl]piperidine} is an alkaloid found in the fruits of Piper nigrum L. and possess various biological effects such as antioxidant, antiinflammatory and anticancer [6-9]. Resveratrol (3,4 ’ ,5- trihydroxy-trans-stilbene) is a naturally occurring polyphenol found in several edible natural products, including grapes, berries, peanuts and in a number of herbal medicines [10]. Besides has been shown to possess antioxidant effect, anti-inflammatory, anti-Leishmaniaamazonensis and as well chemoprevention of cancer [11-14]. The structures of these molecules are shown in the Figure 1.

Figure 1: Chemical structure of resveratrol (A), curcumin (B) and piperine (C).

The bioactive compounds from diet alone or in combination with other substances may be a potential source for the prevention and treatment of breast cancer [15]. Rai et al. [16] demonstrated that resveratrol synergistically induced the chemotherapeutic potential of doxorubicin by in vitro and in vivo model of breast cancer. The combination of resveratrol, quercetin and catechin was able to reduce the primary tumor in the in vivo model of breast cancer [17]. Here, we report the synergistic activity of curcumin+piperine, curcumin +resveratrol and piperine+resveratrol on estrogen receptorpositive breast cancer cells (MCF-7) and triple negative breast cancer cells (MDA-MB-231).

Material and Methods

Reagents

Unless otherwise stated, all reagents were of analytical grade from Sigma-Aldrich (St. Louis, MO, EUA).

Cell culture

Monolayer cultures of human breast carcinoma MCF-7 and MDA-MB-231 cells were obtained from American Type Culture Collection (ATCC) and were grown in DMEM supplemented with 10% FBS, 100 units/mL of penicillin, 100 μg/mL of streptomycin and 5 μg/mL of insulin; the cells were kept at 37°C in a humidified atmosphere of % CO₂ in air. When the cultures reached 70%-80% confluence, the cells were treated with various concentrations of resveratrol, piperine and curcumin or both compounds in associations for 24 hours.

Cell viability assay

The cell viability assay was performed using MTT assay [13]. After treatment, the cells were washed with phosphate buffered saline (PBS) and then incubated for 3 h in 0.5 mL of MTT solution (0.5 mg/mL of PBS) at 37°C in 5% CO₂ in an incubator, and then 500 μL isopropanol was added to each well to dissolve the resulting formazan crystals. The absorbance was measured at a wavelength of 570 nm. The results are expressed in percentage of viable cells compared to untreated control.

Combination index (CI)

After cell viability analysis, determination of IC₅₀ was determined by the method described by Chou using the CalcSyn (Biosoft) software 2.0. For the analysis of the compounds used in association the combination index equation was used, where: (D)1 and (D)2 are the doses of two compounds used in combination and (Dx)1 and (Dx)2 are the doses of two compounds used alone. For the analysis of the combination, CI<1, CI=1 and CI>1 indicated, respectively, synergic, additive and antagonistic effects.

CI=((D)1/(Dx)1)+((D)2/(Dx)2)

Statistical analysis

IC₅₀ were calculated according to a nonlinear regression using a second-order polynomial equation, with 95% confidence intervals, using Graph Pad Prism 5.0 software. The results were expressed as the IC₅₀ standard error of the mean (SEM). The data were analysed by Student’s t test when comparing two groups or by one-way analysis of variance (ANOVA) for more than two groups. p values of 0.05 were considered significant.

Results

Curcumin, piperine or resveratrol treatments on cellular viability of MCF-7 and MDA-MB-231 cells

In the present study, we investigated the cytotoxicity in the presence of different concentrations of compounds (curcumin, piperine or resveratrol) on MCF-7 cells evaluated by MTT method. As shown in Figure 2A, resveratrol, curcumin and piperine presented a dose-dependent cytotoxic effect on MCF-7 cells. In order to evaluate the effect of the compounds on other breast cancer cell line, we performed the MTT viability assay on MDA-MB-231 cells. As shown in Figure 2B, resveratrol, curcumin and piperine also presented a dosedependent cytotoxic effect on MDA-MB-231 cells; however curcumin showed greater inhibition (56.1%) from the concentration of 25 μM than resveratrol and piperine.Our results showed that curcumin, piperine or resveratrol reduced MCF-7 and MDA-MB-231 cells viability after 24 hours of treatment, with IC₅₀ values of 24.50 μM, 94.50 μM and 131.00 μM; and 23.30 μM, 276.00 μM and 306.00 μM, respectively (Table 1).

Figure 2: The effects of resveratrol, curcumin or piperine on the cell viability of MCF-7 and MDA-MB-231 cells. MCF-7 (A) and MDAMB- 231 (B) cells were treated for 24 hours with different concentrations of resveratrol, curcumin or piperine, and the cell viability was then determined by the MTT assay. The results are representative of three experiments performed in triplicate ± SEM. *p<0.05, **p<0.001, ***p<0.0001 in relation of control.

Table 1: Analysis of IC₅₀ values obtained after treatment with various concentrations of curcumin, piperine or resveratrol for 24 hours.

Effect of curcumin, piperine and resveratrol in association on cellular viability of MCF-7 and MDAMB- 231 cells

After observing the effect of isolated compounds and calculating their IC₅₀ values, the compounds were associated in pairs. For this, the concentrations used for curcumin in MCF-7 cells were 3.06, 6.12, 12.25, 18.37, 24.50 and 30.62 μM, with association of 5.91, 11.81, 23.62, 35.44, 47.25 and 59.06 μM of piperine or 8.19, 16.37, 32.75, 49.12, 65.50 and 81.87 μM of resveratrol. The association between the compounds reduced the cell viability in a dose-dependent manner (Figures 3A and 3B). In the same way, the association of 11.81, 23.62, 47.25, 70.87, 94.50 and 118.12 μM of piperine with 8.19, 16.37, 32.75, 49.12, 65.50 and 81.87 μM of resveratrol also reduced the cell viability in a dose-dependent manner (Figure 3C). The association of 5.8, 11.6, 17.5, 23.3 and 29.1 μM of curcumin with 69.0, 138.0, 207.0, 276.0 and 345.0 μM of piperine or 76.5, 153.0, 229.5, 306.0 and 382.5 μM of resveratrol reduced the cell viability of MDA-MB-231 cells in a dose-dependent manner (Figures 3D and 3E). Besides, the association of 69.0, 138.0, 207.0, 276.0 and 345.0 μM of piperine with 76.5, 153.0, 229.5, 306.0 and 382.5 μM of resveratrol reduced the cell viability in a dose-dependent manner (Figure 3F).

Figure 3: The effects of the association of bioactive compounds in breast cancer cells MCF-7 and MDA-MB-231. The cells were treated with different concentrations of curcumin plus piperine (A and D), curcumin plus resveratrol (B and E) and piperine plus resveratrol (C and F) for 24 hours, and the cell viability was determined by MTT assay. The percentage of viable cells was calculated as the ratio of treated cells to control cells. Data represent the mean ± SEM of three independent experiments.* p<0.05, ** p<0.01, *** p<0.0001, in relation of control.

Combination index of curcumin, piperine and resveratrol

An important strategy for the treatment of many types of cancer is a combination therapy. Therefore, we assessed whether compounds could be having an additive, synergistic or antagonic effect one with other, using the CalcSyn (Biosoft) software. Thus, in MCF-7 cells the combination index values for curcumin+piperine, curcumin+resveratrol, piperine +resveratrol were 0.6666, 0.5862 and 0.4759, respectively, that indicates synergism (Table 2). On the other hand, in the MDA-MB- 231 cells the combination index values for curcumin +piperine and curcumin+resveratrol were 4.2564 and 2.5907, respectively, which indicates antagonism. The combination index values for piperine+resveratrol were 0.2126 and indicate synergism; this result was 2.24-times lower than that of MCF-7 cells (Table 2).

Table 2: Combination index analysis of the associations of curcumin, piperine and resveratrol in MCF-7 and MDA-MB-231 cells.

Discussion

Natural products play an important role in the treatment of several pathologies, among them cancer [18]. The use of combination of natural products and chemotherapeutic agents, in reduced concentrations, is associated with a decrease in toxic effects and an improvement in treatment efficacy [19,20]. The inhibitory effect of curcumin, piperine or resveratrol treatments has been described on several types of tumour cells. Previously, we have reported that resveratrol in association with melphalan enhanced the cytotoxic effects on MCF-7 and MDA-MB-231 cells in vitro, with 1.67-time more pronounced effect on MCF-7 cells [13]. Patel and colleagues [21] demonstrated that the combination of curcumin and citral, a lemon essential oil, induced death by apoptosis with activation of the p53 protein in MCF-7 and MDA-MB-231 cells. Bayet- Robert and colleagues [22] performed a clinical phase I dose escalation trial of combination docetaxel chemotherapy with curcumin in advanced and metastatic breast cancer patients, and showed that combination of curcumin plus docetaxel has antitumor activity and a superior response rate in comparison to monotherapy with docetaxel. The combination of piperine and paclitaxel produces synergistic effects in ovarian adenocarcinomas SKOV-3 cells, through loss of membrane integrity of mitochondria leading the cells to apoptosis [23].

We determine the IC₅₀ of curcumin, piperine and resveratrol for MCF-7 and MDA-MB-231 cells, which were 24.50 μM, 94.50 μM and 131.00 μM, and 23.3 μM, 276.00 μM and 306.00 μM, respectively. In the literature, there is IC₅₀s values of curcumin, established for SHI-1 monocyticleukemia cells and HT-29 colon cancer cells, which was of 14.13 μM and 50.0 μM, respectively [24,25]. Besides, for piperine the IC₅₀s values in melanoma cells were: 221.0 μM in SK-MEL-28 cells, 200.0 μM in B16-F0 cells, 225.0 μM in A375 cells and 250.0 μM in ASPC-1 cells [26]. Likewise for resveratrol, the IC₅₀s values were 120.0 μM and 370.0 μM for MCF-7 and MDA-MB-231 breast cancer cells, respectively, and 127.0 μM in MGC803 gastric cancer cells [13,27].

Drug association therapy has several important advantages as reduced dosages and treatment duration, lower treatment costs and decrease drug resistance development, besides the associations of natural products in cancer are fundamental, because they act in inhibition of cell growth, viability, invasion, migration and autophagy, among others [19]. Our data showed for the first time that the combination of curcumin +piperine, curcumin resveratrol and piperine+resveratrol has a synergistic effect against MCF-7 cells, and piperine +resveratrol has a synergistic effect against MDA-MB-231 breast cancer cells. Shoba and colleagues [28], described that piperine increases the serum concentration and the oral bioavailability of curcumin in rats and humans. The curcumin has also been shown to act synergistically with piperine in the suppression of hepatocellular carcinoma in rats, and this effect might be owing to increased bioavailability of curcumin in the presence of piperine [29]. Recently, the combination of resveratrol and curcumin has been shown to induce apoptosis in MCF-10A-Tr cells (cigarette smoke condensate-transformed breast epithelial cell line) through p21Waf1/Cip1 mediated inhibition of the Hedgehog-Gli cascade [30,31].

Conclusion

In conclusion, we can affirm that the combination of bioactive compounds present in diet as curcumin, piperine and resveratrol, may be useful as possible application in future clinical trials with breast cancer.

Acknowledgements

This work was supported by ConselhoNacional de DesenvolvimentoCientífico e Tecnológico, http:// www.cnpq.br; Coordenação de Aperfeiçoamento de Pessoal de Nível Superior, http://www.capes.gov.br; Fundação do Câncer, HTTP://www.cancer.org.br, and Fundação de Amparo à Pesquisa do Estado do Rio de Janeiro, http://www.faperj.br. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Author Contributions

B.S., C.L.A.P., C.F., J.L.S. and E.F. designed and analyzed all experiments, B.S., C.L.A.P., and C.F. performed experiments. All authors reviewed the manuscript.

References

1. DeSantis CE, Bray F, Ferlay J, Lortet-Tieulent J, Anderson BO, Jemal A. International Variation in Female Breast Cancer Incidence and Mortality Rates. Cancer Epidemiol Biomarkers Prev 2015;10:1495-506.
2. Palumbo MO, Kavan P, Miller WH Jr, Panasci L, Assouline S, Johnson N, Cohen V, Patenaude F, Pollak M, Jagoe RT, Batist G. Systemic cancer therapy: achievements and challenges that lie ahead. Front Pharmacol 2013;7:4-57.
3. Priyadarsini KI. The chemistry of curcumin: from extraction to therapeutic agent. Molecules 2014;12:20091-112.
4. Hussain Z, Thu HE, Amjad MW, Hussain F, Ahmed TA, Khan S. Exploring recent developments to improve antioxidant, anti-inflammatory and antimicrobial efficacy of curcumin: A review of new trends and future perspectives. Mater SciEng C Mater Biol Appl 2017;77:1316-1326.
5. Shi J, Zhang X, Shi T, Li H. Antitumor effects of curcumin in human bladder cancer in vitro. Oncol Lett 2017;1:1157-1161.
6. Meghwal M, Goswami TK. Piper nigrum and piperine: an update. Phytother Res 2013;8:1121-30.
7. Srinivasan K. Antioxidant potential of spices and their active constituents. Crit Rev Food Sci Nutr 2014;3:352-72.
8. Ying X, Yu K, Chen X, Chen H, Hong J, Cheng S, Peng L. Piperine inhibits LPS induced expression of inflammatory mediators in RAW 264.7 cells. Cell Immunol 2013;1-2:49-54.
9. Greenshields AL, Doucette CD, Sutton KM, Madera L, Annan H, Yaffe PB, Knickle AF, Dong Z, Hoskin DW. Piperine inhibits the growth and motility of triple-negative breast cancer cells. Cancer Lett 2015;1:129-40
10. Signorelli P, Ghidoni R. Resveratrol as an anticancer nutrient: molecular basis, open questions and promises. J Nutr Biochem 2005;8:449-66.
11. Singh AK, Vinayak M. Resveratrol alleviates inflammatory hyperalgesia by modulation of reactive oxygen species (ROS), antioxidant enzymes and ERK activation. Inflamm Res 2017;24.
12. Ferreira C, Soares DC, Nascimento MT, Pinto-da-Silva LH, Sarzedas CG, Tinoco LW, Saraiva EM. Resveratrol is active against Leishmaniaamazonensis: in vitro effect of its association with Amphotericin B. Antimicrob Agents Chemother 2014;10:6197-208.
13. Casanova F, Quarti J, da Costa DC, Ramos CA, da Silva JL, Fialho E. Resveratrol chemosensitizes breast cancer cells to melphalan by cell cycle arrest. J Cell Biochem 2012;8:2586-96.
14. Ferraz da Costa DC, Fialho E, Silva JL. Cancer Chemoprevention by Resveratrol: The p53 Tumor Suppressor Protein as a Promising Molecular Target. Molecules 2017;22:E1014.
15. Li Y, Li S, Meng X, Gan RY, Zhang JJ, Li HB. Dietary natural products for prevention and treatment of breast cancer. Nutrients 2017;7:E728.
16. Rai G, Mishra S, Suman S, Shukla Y. Resveratrol improves the anticancer effects of doxorubicin in vitro and in vivo models: A mechanistic insight. Phytomedicine 2016;3:233-42.
17. Schlachterman A, Valle F, Wall KM, Azios NG, Castillo L, Morell L, Washington AV, Cubano LA, Dharmawardhane SF. Combined resveratrol, quercetin, and catechin treatment reduces breast tumor growth in a nude mouse model. TranslOncol 2008;1:19-27.
18. Efferth T. Cancer combination therapies with artemisinin-type drugs. BiochemPharmacol 2017;139:56-70.
19. Yang J, Liu RH. Synergistic effect of apple extracts and quercetin 3-beta-d-glucoside combination on antiproliferative activity in MCF-7 human breast cancer cells in vitro. J Agric Food Chem 2009;18:8581-6.
20. Kapinova A, Stefanicka P,  Kubatka P, PavolZubor P, Uramova S, Kello M, Mojzis J, Blahutova D, Qaradakhi T, Zulli A, Caprnda M, Danko J,  Lasabova Z, Busselberg D, Kruzlia P. Are plant-based functional foods better choices against cancer than single phytochemicals? A critical review of current breast cancer research. Biomed Pharmacother 2017;95:1465-77.
21. Patel PB, Thakkar VR, Patel JS. Cellular Effect of Curcumin and Citral Combination on Breast Cancer Cells: Induction of Apoptosis and Cell Cycle Arrest. J Breast Cancer 2015;3:225-34.
22. Bayet-Robert M, Kwiatkowski F, Leheurteur M, Gachon F, Planchat E, Abrial C, Mouret-Reynier MA, Durando X, Barthomeuf C, Chollet P. Phase I dose escalation trial of docetaxel plus curcumin in patients with advanced and metastatic breast cancer. Cancer Biol Ther 2010;9:8-14.
23. Pal MK, Jaiswar SP, Srivastav AK, Goyal S, Dwivedi A, Verma A. Synergistic effect of piperine and paclitaxel on cell fate via cyt-c, Bax/Bcl-2-caspase-3 pathway in ovarian adenocarcinomas SKOV-3 cells. Eur J Pharmacol 2016;791:751-762.
24. Zhu GH, Dai HP, Shen Q, Ji O, Zhang Q, Zhai YL. Curcumin induces apoptosis and suppresses invasion through MAPK and MMP signaling in human monocyticleukemia SHI-1 cells. Pharm Biol 2016;8:1303-11.
25. Singh N, Shrivastav A, Sharma RK. Curcumin induces caspase and calpain-dependent apoptosis in HT29 human colon cancer cells. Mol Med Rep 2009;4:627-31.
26. Fofaria NM, Kim SH, Srivastava SK. Piperine causes G1 phase cell cycle arrest and apoptosis in melanoma cells through checkpoint kinase-1 activation. PLoS One 2014;5:e94298.
27. Jing X, Cheng W, Wang S, Li P, He L. Resveratrol induces cell cycle arrest in human gastric cancer MGC803 cells via the PTEN-regulated PI3K/Aktsignaling pathway. Oncol Rep 2016;1:472-8.
28. Shoba G, Joy D, Joseph T, Majeed M, Rajendran R, Srinivas PS. Influence of piperine on the pharmacokinetics of curcumin in animals and human volunteers. Planta Med 1998;4:353-6.
29. Patial VSM, Sharma S, Pratap K, Singh D, Padwad YS. Synergistic effect of curcumin and piperine in suppression of DENA-induced hepatocellular carcinoma in rats. Environ Toxicol Pharmacol 2015;2:445-52.
30. Mohapatra P, Satapathy SR, Siddharth S, Das D, Nayak A, Kundu CN. Resveratrol and curcumin synergistically induces apoptosis in cigarette smoke condensate transformed breast epithelial cells through a p21(Waf1/Cip1) mediated inhibition of Hh-Glisignaling. Int J Biochem Cell Biol 2015;66:75-84.
31. Chou TC. Theoretical basis, experimental design, and computerized simulation of synergism and antagonism in drug combination studies. 2006; 58(3):621-81. Erratum in: Pharmacol Rev 2007;59:124.