Effect of quercetin on secretion and gene expression of leptin in breast cancer

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J Tradit Chin Med 2017 June 15; 37(3): 321-325 ISSN 0255-2922 © 2017 JTCM. This is an open access article under the CC BY-NC-ND license.

RESEARCH ARTICLE TOPIC

Effect of quercetin on secretion and gene expression of leptin in breast cancer

Rahimifard Maryam, Sadeghi Faegheh, Asadi-Samani Majid, Nejati-Koshki Kazem aa Rahimifard Maryam, Cellular and Molecular Biology Department, Faculty of Science, Ahar Islamic Azad University, Ahar 5451116714, Iran Sadeghi Faegheh, Cellular and Molecular Biology Department, Faculty of Science, Ahar Islamic Azad University, Ahar 5451116714, Iran Asadi-Samani Majid, Student Research Committee, Shahrekord University of Medical Sciences, Shahrekord 8815713471, Iran Nejati-Koshki Kazem, Student Research Committee, Zanjan University of Medical Sciences, Zanjan 4515786349, Iran Correspondence to: Dr. Asadi-Samani Majid, Medical Plants Research Center, Shahrekord University of Medical Sciences, Shahrekord 8815713471, Iran. [email protected] Telephone: +98-3833330061 Accepted: April 8, 2016

analysis of real-time PCR showed that with increases in quercetin concentration, a decreasing trend was seen in mRNA levels of leptin of treated cells compared with the control cells (P < 0.05). Also, measurement of secreted leptin in the culture media showed a similar decreasing trend in the amount of leptin protein in the treated cells compared with the control cells (P < 0.05). CONCLUSION: Quercetin significantly inhibits the growth of T47D cells through inhibition of leptin secretion and gene expression in T47D breast cancer cells. Therefore, it might be an alternative approach to breast cancer therapy through leptin targeting. © 2017 JTCM. This is an open access article under the CC BY-NC-ND license.

Keywords: Quercetin; Cytotoxicity; Real-time polymerase chain reaction; Breast neoplasms

Abstract OBJECTIVE: To investigate the possible inhibitory action of pure quercetin on secretion and gene expression of leptin in the T47D breast cancer cell line.

INTRODUCTION Breast cancer is one of the most commonly diagnosed cancers worldwide. The incidence of breast cancer is approximately 10.4%, and it is the most common cancer in women, showing an annual 1%-2% increase.1,2 Because of the high morbidity and mortality of breast cancer, the development of an effective drug for its treatment is obviously required. There are many known risk factors for breast cancer including age, sex, hormonal factors, diet, radiation exposure, breast density, family history, obesity, and mutations in susceptibility genes (BRCA1, BRCA2, TP53, PTEN and leptin). Leptin is expressed in normal mammary tissue, breast cancer cell lines, and solid tumors.1-3 Leptin acts as a mitogen and induces growth and transformation of T47D breast cancer cells.4 Also, it has been shown that increased levels of leptin and de-

METHODS: In this experimental study, T47D cells were cultured in monolayers in RPMI 1640. IC50 was determined by MTT (3-[4,5-dimethylthiazol-2-yl]-2, 5-diphenyltetrazolium bromide) assay after 24 h treatment at different concentrations of quercetin. The levels of leptin gene expression were measured by reverse-transcription real-time polymerase chain reaction (PCR). Secreted leptin was measured in the supernatant of cells by an enzyme-linked immuno sorbent assay. RESULTS: Analysis of MTT assay data showed that quercetin has a cytotoxic effect on T47D breast cancer cells with 40 µM IC50 after 24 h exposure. Data JTCM | www. journaltcm. com

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creased adiponectin secretion are directly associated with breast cancer development.2 With increasing attention paid to the significant role of leptin as a therapeutic target in breast cancer, natural compounds capable of reducing the expression and secretion of leptin could contribute greatly to preventing and treating breast cancer. Medicinal plants and their compounds are rich sources for discovering drug therapies and they can be used to prevent and treat diseases.5-13 Quercetin is a plant flavonoid that is generally found in citrus fruits, onions, tea, and red wine. It exhibits a wide range of properties such as anticarcinogenic, anti-inflammatory, and anti-viral actions. Many studies have shown that quercetin is able to induce cytotoxic effects, including inhibition of cell proliferation and apoptosis in a variety of cancer cells.14-17 In this investigation, we aimed to study the effect of quercetin on secretion of and leptin gene expression in the epithelial-like cell line T47D.

vestigated by MTT assay after 24 h of treatment. In short, 2000 cells/well were cultivated in a 96-well culture plate. After 24 h incubation at 37 ℃ , cells were treated with different concentrations of quercetin (0-120 μM). Then, the medium of all wells was removed carefully and 50 μL MTT (2 mg/mL) was added to each well and incubated for 4.5 h in the dark, followed by the addition of 200 μL DMSO. Thereafter, Sorensen's glycine buffer (0.1 M glycine, 0.1 M NaCl, pH 10.5) was added and the absorbance of each well was read at 570 nm within 15-30 min. For data analysis, the mean OD of each well was calculated. Total RNA extraction and cDNA synthesis Total RNA was extracted by the TRIZOL Reagent according to the manufacturer's instructions. The concentration of prepared RNA was measured by a NanoDrop spectrophotometer (Termoscientific, Wilmington, DE, USA) and its integrity was confirmed by electrophoresis on a 1.2% agarose gel containing 1% formaldehyde. After the RNA was prepared, cDNA was synthesized by the First-Strand Synthesis kit according to the manufacturer's instructions. The synthesized cDNA was immediately used in real time PCR or stored at － 70 ℃ for later use.

MATERIALS AND METHODS Chemicals and reagents Quercetin (2-[3,4-dihydroxyphenyl]-3,5,7-trihydroxy-4H-chromen-4-one, "Sigma-Aldrich provides this product to early discovery researchers as part of a collection of unique chemicals"), MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide), glycine, and NaCl were obtained from Sigma (Deisenhofen, Germany). The leptin ELISA kit was purchased from Labor Diagnostika Nord GmbH and Co., KG (Nordhorn, Germany); fetal bovine serum (FBS) and phenol-red free Roswell Park Memorial Institute (RPMI) 1640 with L-glutamine were purchased from Gibco BRL (Life Technologies, Grand Island, NY, USA); and the T47D cell line was obtained from the National Cell Bank of Iran (Tehran, Iran). Sodium bicarbonate, streptomycin, and amphotericin B were purchased from Merck (Darmstadt, Germany); penicillin G was purchased from SERVA Co., (Heidelberg, Germany); Total RNA Isolation (TRIzol) reagent was purchased from Invitrogen (Eugene, OR, USA); the First-Strand cDNA Synthesis kit was purchased from Fermentas (Hanover, MD, USA); and the Syber Green-I reagent was purchased from Takara Bio (Otsu, Japan).

Real-time PCR The real-time PCR method was used for analysis of leptin gene expression levels in the control and treated T47D cells. The real-time PCR reaction was done in triplicate using the Syber Green Ⅰ reagent in the Rotor Gene ™ 6000 system (Corbett Research, Sydney, Australia) according to the manufacturer's instructions. The amplification conditions were as follows: leptin (2 min at 95 ℃ and a two-step cycle of 95 ℃ for 15 s and 60 ℃ for 40 s for 40 cycles). Changes in leptin gene expression levels between the control and treated T47D cells were calculated by the 2－ ΔΔCT method.18 Measurement of the secreted leptin To measure the possible effect of quercetin on the amount of secreted leptin in the treated cells compared with the control cells, leptin concentrations were measured in the supernatant from the cells by a human leptin ELISA kit (Labor Diagnostika Nord GmbH and Co., KG; Nordhorn, Germany) according to the manufacturer's instructions.

Cell culture The T47D cell line was provided by the National Cell Bank of Iran (Tehran, Iran). The T47D cells were cultured in RPMI 1640 (+ L-glutamine) supplemented with 10% FBS, streptomycin (100 mg/L), penicillin G (100 IU/mL), and amphotericin B (2.5 μg/mL) and incubated at 37 ℃ in a humid atmosphere containing 5% CO2.

Statistical analysis Data were expressed as mean ± standard deviation ( xˉ ± s). Data were processed with SPSS 18.0 (SPSS Inc., Released 2009. SPSS Statistics for Windows, version 18.0. Chicago, IL, USA). The differences in expression levels of leptin between the control and treated cells were analyzed by one-way analysis of variance and Dunett's multiple comparison tests. P < 0.05 was defined as significant.

Cell treatment and MTT assay The cytotoxic effect of quercetin on T47D cells was inJTCM | www. journaltcm. com

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RESULTS

Measurement of secreted leptin in the culture media showed a similar decreasing trend in the amount of leptin protein in the treated cells compared to the control cells. The differences between these concentrations were statistically significant (P < 0.05). This finding is in agreement with and confirms the real-time PCR results.

Analysis of the MTT assay data showed that quercetin had a cytotoxic effect on T47D breast cancer cells with 4.0 μM IC50 after 24 h exposure (Figure 1). The obtained IC50 was timedependent (P < 0.05). 1.2 Fold change

1.0 0.8

DISCUSSION

0.6

This study was conducted to investigate the possible inhibitory action of pure quercetin on secretion and leptin gene expression in the T47D breast cancer cell line. Our study demonstrated that quercetin, the flavonoid from quercetum (oak forest), could inhibit leptin gene expression and secretion in T47D breast cancer cells. Leptin, a circulating hormone secreted by adipocytes, is one of the main adipose cytokines acting as an important signalling molecule in energy regulation and food intake, and also controls body weight homeostasis.19,20 Leptin has been shown to stimulate DNA synthesis and cell growth, acting through multiple signalling cascades such as the Janus-activated kinase 2/signal transducers and activators of transcription 3, extracellular signal-regulated kinase 1/2, protein kinase Ca, and Akt/GSK3 pathways.21 Leptin-induced cell cycle progression is accompanied by up-regulation of cyclin-dependent kinase 2 and cyclin D1 levels and hyperphosphorylation/inactivation of the cell cycle inhibitor pRb.22 After recognition of the critical role of leptin in breast carcinogenesis, there have been many attempts to inhibit leptin function and secretion. For example, Rene Gonzalez et al 23 inhibited growth of murine mammary cancer cells and growth in a xenograft tumor model of human breast cancer cell lines by using a leptin peptide antagonist. In contrast, the present study aimed to directly inhibit leptin expression in a human cancer cell line by used quercetin to directly inhibit leptin expression. The use of a natural compound, quercetin, is expected to have fewer potential sideeffects and its lower cost is an advantage of the present study over previous studies.

0.4 0.2 0.0

Control

10 20 30 40 50 Drug concentration (μM) Figure 1 Cytotoxic effect of quercetin on T47D cells after 24 h of treatment

Quantitative amplification of diluted concentrations of cDNA (1/5, 1/10, 1/20) showed that the 1/10 dilution was the best template for the real-time reactions (Figure 2). Table 1 shows the data for the real-time PCR. Analysis of the real-time PCR showed that with an increase in the quercetin concentration, a decreasing trend was seen in the mRNA levels of leptin in treated cells compared with the control cells (Figure 2). There was no significant difference between DMSOtreated cells (as a solvent control) and untreated cells with regards to leptin gene expression (P > 0.05). The difference between the control and treated cells was statistically significant (P < 0.05). 120 Cell viability (%)

100 80 60 40 20 0

Control

20 40 60 80 100 120 Drug concentration (μM) Figure 2 Effect of quercetin on leptin expression levels in T47D breast cancer cells Table 1 Analysis of real-time polymerase chain reaction Item

－ ΔΔCT

Ct1

Ct2

Ct3

Average of Ct

Ct Reference

ΔCT

ΔΔCT

2

Untreated

12.680

12.270

12.960

12.636

15.68

3.044

0.000

1.000

DMSO treated

17.760

17.310

18.370

17.813

14.78

3.033

0.021

0.950

Quercetin (10 μM)

17.760

15.340

15.780

15.747

18.86

3.113

0.069

0.880

Quercetin (20 μM)

15.013

14.899

14.433

14.781

18.14

3.359

0.315

0.810

Quercetin (30 μM)

21.320

20.133

19.800

20.417

17.00

3.417

0.373

0.630

Quercetin (40 μM)

16.030

16.130

16.400

16.186

20.63

4.444

1.400

0.370

Quercetin (50 μM)

12.550

12.391

12.011

12.317

16.70

4.383

1.339

0.300

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ACKNOWLEDGEMENTS

Reduced cancer risk and no observed toxicity associated with intake of high amounts of natural products indicate that certain concentrations of phytochemicals derived from plants suggests that they may have cancer chemopreventative effects without leading to significant levels of toxicity. Natural agents can suppress the inflammatory process that results in neoplastic transformation, promotion, and progression of carcinogenesis and angiogenesis.24-31 The anticancer effects of quercetin on various cancer cells, including glioma, colon, breast, melanoma, and endometrial cancer cells, has been demonstrated.32-35 A phase Ⅰ clinical study showed that quercetin inhibited tumor growth and in fl ammation.36 In addition, studies have shown that the potential mechanism of quercetin-mediated inhibition of tumor progression involves the induction of cell cycle arrest and the promotion of cell apoptosis.15,21,37 Quercetin can induce apoptosis through induction of BAX with concomitant inhibition of BCL-2 in human breast cancer cells,37 and mitochondrial- and caspase-3-dependent pathways in human breast cancer MDA-MB-231 cells.15 It alters the AKT/mTOR pathway, COX-2 expression, NFkB signalling, TNF-a expression, and VEGF expression.38,39 In our study, quercetin inhibited leptin secretion and gene expression in T47D breast cancer cells. In another study by Nejati-Koshki et al, silibinin inhibited the expression and secretion of leptin in T47D breast cancer cells. However, a cytotoxicity assay showed that the IC50 of silibinin was 110 μM at 24 h 40 whereas quercetin in our study had a cytotoxic effect on T47D breast cancer cells at 40 μM IC50 at 24 h exposure. Nejati-Koshki et al 40 noted a probable role of estrogen receptor β in the inhibition of leptin expression and secretion. Nejati-Koshki et al 41 also have shown that curcumin alone and the combination of silibinin and curcumin can inhibit leptin gene expression and secretion in T47D breast cancer cells better than silibinin alone. A decrease in leptin gene expression and secretion by curcumin has been linked to OB-Ra, OBRb, and ERα gene expression. Furthermore, in light of the fact that leptin and its receptors directly increase each other's gene expression through an autocrine cycle,42 a decrease in leptin and/or leptin receptors can inhibit this cycle, and downregulate the expression of both leptin and its receptors. This may be another mechanism for decreasing leptin expression. Most likely quercetin, as with silibinin and curcumin, affects leptin receptors. However, this needs to be investigated further in future studies. In conclusion, our results indicate that quercetin has the potential to interact with the expression of the leptin gene, which has significant roles in carcinogenesis and proliferation of breast cancer cells. Therefore, it may be a novel strategy for prevention and treatment of breast cancer in the future. JTCM | www. journaltcm. com

The authors thank the Tabriz University of Medical Sciences and Ahar Islamic Azad University for all of the support they provided.

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