https://doi.org/10.17795/zjrms962

Effect of Quercetin on RAC1 Gene Expression as a Marker of Metastasis in Cervical Cancer Cells

authors:

avatar Arezu Chakerzehi 1 , avatar Neda Eivazi Arvanagh 1 , avatar Samaneh Saedi 2 , avatar Mahdiyeh Hematti 1 , avatar Javad Mohiti Ardakani 1 , avatar Ali Moradi 1 , * , avatar Abolfazl Shokouhi 1

Department of Medical Biochemistry, Shahid Sadughi University of Medical Sciences, Yazd, IR Iran
Department of Medical Bacteriology, Pasteur Institute, Tehran, IR Iran
Zahedan Journal of Research in Medical Sciences: Vol.17, issue 5
published online: May 31, 2015
article type: Research Article
received: January 28, 2014
revised: March 30, 2014
accepted: May 14, 2014

How To Cite Chakerzehi A, Eivazi Arvanagh N, Saedi S, Hematti M, Mohiti Ardakani J, et al. Effect of Quercetin on RAC1 Gene Expression as a Marker of Metastasis in Cervical Cancer Cells. Zahedan J Res Med Sci. 2015;17(5): -. https://doi.org/10.17795/zjrms962.

Abstract

Background:

Cancer metastasis is the most important cause of cancer death and different treatment strategies have targeted on preventing the occurrence of metastasis. Quercetin is a flavonoid and widely used as an antioxidant and recent studies have discovered its anticancer and antiproliferative capabilities. Rac1 is protein known to involve in tumor invasion and metastases.

Objectives:

In this study, we investigated the effect of quercetin on expression of Rac1 protein.

Materials and Methods:

In this experimental study, HeLa cells were treated with quercetin at 2 concentrations (20 and 40 μM) for 24 hours and then cell lysate was assessed by Western blotting analysis to investigate the impact of quercetin on expression of Rac1 protein.

Results:

Quercetin significantly decreased Rac1 expression of HeLa cells assessed by western blotting, in dose-dependent manners (P < 0.05).

Conclusions:

This study showed that quercetin decreased Rac1 expression as a marker of metastasis in cervical cancer cells. Therefore, the quercetin may provide an effective new strategy to reduce of tumor metastasis.

1. Background

Cervical cancer is one of the leading causes of death in women worldwide and its global incidence increased at an annual rate of 0.6% between 1980 and 2010 [1]. Although cervical cancer mortality rates have been decreasing, the recurrence and metastasis of cervical cancer to other parts such as the lymph nodes [2, 3], lungs [4, 5], bones [6, 7], liver [8] and bowels [9] are main factors contributing to mortality in patients with cervical carcinoma. Thus, apart from surgery and the destruction of cervical cancer cells by medications, inhibiting metastasis is an auxiliary strategy for treating patients with cancers. Cancer metastasis leads to poor clinical outcomes and mortality in patients with cancers. Metastasis process involves cell adhesion, migration, invasion and proteolytic degradation of extracellular matrix (ECM) [10]. During the invasion and metastasis, cancer cells move within tissues, thus the control of migration could be critical key in treatment of metastatic cancer [11]. Reorganization of the actin cytoskeleton is the primary mechanism of cell motility and it is essential for most types of cell migration. In the metastatic process, the actin cytoskeleton and its regulatory proteins are necessary for cancer cell migration. According to results, the ability of Rho GTPase family members has been proved in the regulation of cell movement, cell adhesion and migration that this could be an important role in the invasion and metastasis of cancer cells [12]. Rac1, small protein of Rho GTPase family, performed multiple cellular activities on actin. This protein plays an important role in degradation of adhesive joints cell- cell, resistance to apoptosis and cell proliferation. Thus, Rac1 accelerates the invasion of cancer cells [13, 14]. Therefore, study on this protein is important for investigating molecular mechanisms of cancer cells metastasis. Herbal medicines have been used for treatment a variety of cancers including leukemia, cervical, ovarian, testicular, lung, liver, esophageal, stomach, colon, and rectum cancer [15]. Flavonoids possess anticancer and chemopreventive properties through their antioxidant activity and evidences indicate that some flavonoids are potent chemopreventive agents with low cytotoxicity [16-18]. Flavonoids or phenolic acids are plant pigments and soluble in water. This group is responsible for the antioxidant capacity of fruits and vegetables [19]. Over 4000 different flavonoids have been identified in the major groups of flavonoids [20]. Quercetin (3, 3, 4, 5, 7-pantahydroxyflavone), a flavone-3ol-class of flavonoid, is ubiquitously present in apples, onions and other vegetables [21]. Quercetin is without carbohydrate [22, 23] and has been the most studied flavonoids to determine the biological effects of this component [20].

This compound has a wide range of medical, pharmaceutical and biological applications [24]. Quercetin exhibits a variety of biological functions, including anti-oxidative, anti-inflammatory, anti-cancer and anti-metastatic activities. Studies have shown that quercetin can inhibit the proliferation of a wide range of cancers, including colon, cervix, lung, breast and prostate [25, 26]. This polyphenolic compound has been reported to modulate signal transduction pathways associated with cell proliferation and differentiation, apoptosis, angiogenesis and metastasis [27, 28].

2. Objectives

According to the role of Rac1 in the migration and invasion of cancer cells and also the importance of cervical cancer, therefore, in present study we investigated anticancer effects of quercetin through its effects on metastasis based on the amount of Rac1 expression in cervical cancer cells.

3. Materials and Methods

In this experimental study, quercetin, penicillin, streptomycin and DMEM culture medium were purchased from GIBCO BRL (Grand Island NY, USA). Trypsin and fetal bovine serum (FBS) were obtained from GIBCO. Primary antibody anti-Rac1 from cell signaling (cut number: 2465s), Goat-anti rabbit IgG-HRP secondary antibody from Sigma and ECL kit were obtained from Isfahan CMG. Other solutions and reagents were from the Merck Company in this research.

3.1. Cell Culture

This study was performed in Yazd University of Medical Sciences. HeLa cells were obtained from the Pasteur Institute of Tehran, Iran. Cells were grown in Dulbeccos modified Eagles medium (DMEM) containing 10% fetal bovine serum, 0.30% sodium bicarbonate and antibiotics (streptomycin 100 µg/mL and penicillin 100 IU/mg) cells were maintained in a humidified atmosphere of 5% CO2 at 37°C and subcultured every 3 or 4 days with 0.05% trypsin (Figure 1). Then cells were treated with 20 and 40 µM concentration of stock solution of quercetin for 24 hours. These concentrations were according to IC50 = 80 µM of quercetin on cervical cancer cells [29], the cells are maximum viability in the concentrations lower than 80 µM. Moreover, we have control sample that all conditions were similar to test groups but without quercetin.

3.2. Preparation of Stock Solution Quercetin

Stock solution of quercetin (100 mM) prepared by dissolving 0.0151 grams quercetin powder in 0.5% DMSO was diluted with Dulbecco’s modified Eagles medium (DMEM) prior to use to obtain the desired concentration (20 and 40 µM). The final concentration of DMSO used for treatment was 0.1% (v/v).

3.3. Electrophoresis and Westernblotting

After 24 hours of treatment, cells washed with ice phosphate-buffered saline (PBS) and lysed in a lysis buffer [50 mM Tris-HCl (pH = 7.4), 1% Nonidet P-40, 40 mM NaF, 10 mM NaCl, 10 mM Na3VO4, 1 mM phenylmethanesulfony, l fL uoride (PMSF) and 10 mM dithiothreitol (DTT)]. The cell lysates were centrifuged at 12,000 rpm for 5 minutes. Total protein of supernatant was determined by the method of Bradford [30]. A total of 60 µg of this suspension were applied to 10% SDS-polyacrylamide electrophoresis gels (PAGEs) at 100 V for 1 hour. The separated proteins were then electrophoretically transferred to nitrocellulose overnight at 30 V at 4°C. The membranes were incubated in blocking solution containing 5% non-fat dry milk and Tris-buffered saline/0.1% Tween-20 for 2 hours to block non-specific binding sites. After 2 hours blocking, blots washed in Tris-buffered saline/0.1% Tween-20 (TBST) four times, then the blots were incubated overnight at 4°C with 1:1000 dilutions of primary antibody. After 24 hours, washed in TBST buffer (4 times) and probed for 1 hour with 1:5000 dilutions HRP-conjugated secondary antibody at room temperature. After washing with TBST buffer (4 times), the immunoreactive proteins were visualized using enhanced chemiluminescence (ECL) detection reagents. Protein expression levels were calculated by the program Gene Tools Gel Document sets and were reported as relative percentage. For western blotting analysis, acting used as internal standard. Also for quantifying of target proteins, resulting film scanning densitometry were determined relative to acting levels. The experiment was replicated three different times.

3.4. Statistical Analysis

The results analyzed using SPSS-16. All values were expressed as the mean ± standard error of mean (SEM). Data were analyzed by repeated measures analysis of variance (ANOVA) followed by post-hoc test for determine the significance of differences between treatment groups. Statistical significance was accepted for P < 0.05.

4. Results

In order to evaluate effect of quercetin on Rac1 expression levels, 20 and 40 µM concentrations of quercetin were used for HeLa cells since these concentrations and time are the best sources to evaluate effect of quercetin on these cells [29].

Analysis of Rac1 by western blotting revealed a significant reduction in Rac1 expression (21 kD) in cells treated with quercetin compared to untreated control (Figure 2). Figure 2 A showed that the expression levels of Rac1 decreased in a dose-dependent manner. Rac1 expression was obtained 100 ± 0, 10.4 ± 3.14, and 3.59 ± 2.4 in control group, 20 µM and 40 µM quercetin, respectively.

Effect of Quercetin on Expression of Rac1 Protein in HeLa Cells
A, Representative immunoblots protein samples (60 μg/lane) resolved on SDS-PAGE were probed with antibodies. β-actin was used as loading control; B, Densitometric analysis (* Statically significant (P < 0.05)).

A, Representative immunoblots protein samples (60 μg/lane) resolved on SDS-PAGE were probed with antibodies. β-actin was used as loading control; B, Densitometric analysis (* Statically significant (P < 0.05)).

Figure 2.

Effect of Quercetin on Expression of Rac1 Protein in HeLa Cells

5. Discussion

In the present study, we evaluated the mechanism of antimetastatic activity quercetin. The majority cancer deaths occur as an outcome of metastasis rather than the original tumor; therefore, inhibiting cancer-cell metastasis is an important aspect of cancer prevention. With increasing application of plant derived cancer chemotherapeutic agents probing the antiproliferative and antimetastatic effects of phytochemicals have gained enhancing momentum for anticancer drug design [29]. This study showed that the quercetin inhibited Rac1 expression as a marker of metastasis in cervical cancer cells that it can be shown that quercetin may have potential antimetastatic effects on cancer cells. Amount of the inhibitory becomes more by increasing concentrations of quercetin. A study was carried out on HeLa cells showed that quercetin could inhibit adhesion and migration and invasion this cells. Quercetin could enhance the inhibitory effect of cis-platin on HeLa cell adhesion, migration and invasion but the exact mechanism was not understood [31]. The polyphenols in green tea, including catechin derivatives, suppressed the invasive behavior of MDA-MB231 cells and the antiinvasive effect occurred. Other study were performed on breast cancer cells MCF-7, shown that EGCG blocked the adhesion of MCF-7 cells to ECM, fibronection and vitronectin and so suppressed migration and invasion of these cells. These effects consequenced from the inhibition of NFkB, VEGF, MMP-2 integrin receptor-α, β, VASP, FAK and Rac1 by EGCG [32]. Liu et al. investigated the Rac1 signaling pathway on liver cancer (HCC) in vivo and in vitro conditions. In this study it was revealed the anti-metastatic effect of melitin on the cancer cells via rac1 suppression. Expression of Rac1 was measured by using Western blotting technique in a variety of liver cancer cell lines and it was concluded that Rac1 expression in HCC cells would increase invasive and it directly would relate to metastasis of this cells [33]. In 2004, the protein expression of Rho, Rac1 and cdc42 in tumor samples from surgical specimens of 57 patients with tumors of the testis (Testicular cancer) and non-tumoric were measured by using Western blotting techniques. The results indicated that the expression of these proteins in tumor samples was significantly higher than non-tumoric samples, and it was also reported that expression of Rho, Rac1 and cdc42 proteins in advanced stages of cancer is higher than in the early stages of cancer [34]. Expression of Rac1 and PAK proteins, a potent effector of Rac1, was evaluated on four groups including tumor and non-tumoric tissue, metastatic and normal lymph nodes in the upper urinary tract cancer and the results showed that expression levels of active Rac1 and PAK in tumor and metastatic lymph nodes were significantly higher than their levels in samples non-tumoric [35]. Considering that active Rac1 can mediate looseness of adhesion connections and subsequently accelerate migratory phenotype [13, 14].

Based on this study, we speculated that quercetin may affect the Rac1 pathway, which has been reported to be critical for inhibiting migration and invasion in tumor cells. Quercetin is a nontoxic and non-allergic dietary flavonoid that has been shown to possess antimetastatic properties. Consequently, consumption of natural compounds is very important to increase efficiency of cancer treatment. Results from the present study and previous studies beneficial effects of the quercetin flavonoid on migration and cancer progression was showed, therefore, the use of flavonoids as treatment with other pharmacological agents was suggested but the exact further studies on cellular and animal models were recommended. In summary, we have demonstrated that treatment with quercetin inhibits expression of Rac1 protein in HeLa cells. This is the first study to demonstrate that quercetin might be a novel anticancer agent for the treatment of cervical cancer through inhibiting migration and invasion. These results displayed that Rac1 inhibition may apply a profound influence on quercetin-mediated inhibition of tumor metastasis. Future studies will focus on elucidating the role of Rac1 in quercetin-mediated signaling.

Acknowledgements

References

  • 1.

    Forouzanfar MH, Foreman KJ, Delossantos AM, Lozano R, Lopez AD, Murray CJ, et al. Breast and cervical cancer in 187 countries between 1980 and 2010: a systematic analysis. Lancet. 2011;378(9801):1461-84. [PubMed ID: 21924486].

  • 2.

    Takeuchi H, Kitajima M, Kitagawa Y. Sentinel lymph node as a target of molecular diagnosis of lymphatic micrometastasis and local immunoresponse to malignant cells. Cancer Sci. 2008;99(3):441-50. [PubMed ID: 18070155].

  • 3.

    Yuan SH, Liang XF, Jia WH, Huang JL, Wei M, Deng L, et al. Molecular diagnosis of sentinel lymph node metastases in cervical cancer using squamous cell carcinoma antigen. Clin Cancer Res. 2008;14(17):5571-8. [PubMed ID: 18765550]. https://doi.org/10.1158/1078-0432.CCR-08-0346.

  • 4.

    Yamamoto K, Yoshikawa H, Shiromizu K, Saito T, Kuzuya K, Tsunematsu R, et al. Pulmonary metastasectomy for uterine cervical cancer: a multivariate analysis. Ann Thorac Surg. 2004;77(4):1179-82. [PubMed ID: 15063230].

  • 5.

    Tangjitgamol S, Levenback CF, Beller U, Kavanagh JJ. Role of surgical resection for lung, liver, and central nervous system metastases in patients with gynecological cancer: a literature review. Int J Gynecol Cancer. 2004;14(3):399-422. [PubMed ID: 15228413].

  • 6.

    Ratanatharathorn V, Powers WE, Steverson N, Han I, Ahmad K, Grimm J. Bone metastasis from cervical cancer. Cancer. 1994;73(9):2372-9. [PubMed ID: 7513250].

  • 7.

    Thanapprapasr D, Nartthanarung A, Likittanasombut P, Na Ayudhya NI, Charakorn C, Udomsubpayakul U, et al. Bone metastasis in cervical cancer patients over a 10-year period. Int J Gynecol Cancer. 2010;20(3):373-8. [PubMed ID: 20375800].

  • 8.

    Park JY, Lim MC, Lim SY, Bae JM, Yoo CW, Seo SS, et al. Port-site and liver metastases after laparoscopic pelvic and para-aortic lymph node dissection for surgical staging of locally advanced cervical cancer. Int J Gynecol Cancer. 2008;18(1):176-80. [PubMed ID: 17506848].

  • 9.

    Kanthan R, Senger JL, Diudea D, Kanthan S. A review of duodenal metastases from squamous cell carcinoma of the cervix presenting as an upper gastrointestinal bleed. World J Surg Oncol. 2011;9:113. [PubMed ID: 21958048]. https://doi.org/10.1186/1477-7819-9-113.

  • 10.

    Chaffer CL, Weinberg RA. A perspective on cancer cell metastasis. Science. 2011;331(6024):1559-64. [PubMed ID: 21436443]. https://doi.org/10.1126/science.1203543.

  • 11.

    Ridley AJ, Schwartz MA, Burridge K, Firtel RA, Ginsberg MH, Borisy G, et al. Cell migration: integrating signals from front to back. Science. 2003;302(5651):1704-9. [PubMed ID: 14657486]. https://doi.org/10.1126/science.1092053.

  • 12.

    Jaffe AB, Hall A. Rho GTPases: biochemistry and biology. Annu Rev Cell Dev Biol. 2005;21:247-69. [PubMed ID: 16212495].

  • 13.

    Engers R, Springer E, Michiels F, Collard JG, Gabbert HE. Rac affects invasion of human renal cell carcinomas by up-regulating tissue inhibitor of metalloproteinases (TIMP)-1 and TIMP-2 expression. J Biol Chem. 2001;276(45):41889-97. [PubMed ID: 11551917].

  • 14.

    Sander EE, van Delft S, ten Klooster JP, Reid T, van der Kammen RA, Michiels F, et al. Matrix-dependent Tiam1/Rac signaling in epithelial cells promotes either cell-cell adhesion or cell migration and is regulated by phosphatidylinositol 3-kinase. J Cell Biol. 1998;143(5):1385-98. [PubMed ID: 9832565].

  • 15.

    Wang CM, Xu SY, Lai S, Geng D, Huang JM, Huo XY. Curculigo orchioides (Xian Mao) modifies the activity and protein expression of CYP3A in normal and Kidney-Yang Deficiency model rats. J Ethnopharmacol. 2012;144(1):33-8. [PubMed ID: 22974543].

  • 16.

    Beato VM, Orgaz F, Mansilla F, Montano A. Changes in phenolic compounds in garlic (Allium sativum L.) owing to the cultivar and location of growth. Plant Foods Hum Nutr. 2011;66(3):218-23. [PubMed ID: 21667145].

  • 17.

    Moustapha B, Marina GA, Raul FO, Raquel CM, Mahinda M. Chemical constituents of the Mexican mistletoe (Psittacanthus calyculatus). Molecules. 2011;16(11):9397-403. [PubMed ID: 22071447]. https://doi.org/10.3390/molecules16119397.

  • 18.

    Ren W, Qiao Z, Wang H, Zhu L, Zhang L. Flavonoids: promising anticancer agents. Med Res Rev. 2003;23(4):519-34. [PubMed ID: 12710022].

  • 19.

    Prior RL, Cao G. Flavonoids: diet and health relationships. Nutr Clin Care. 2000;3(5):279-88.

  • 20.

    Cook NC, Samman S. Flavonoids—Chemistry, metabolism, cardioprotective effects, and dietary sources. Nutr Biochem. 1996;7(2):66-76.

  • 21.

    Bulzomi P, Galluzzo P, Bolli A, Leone S, Acconcia F, Marino M. The pro-apoptotic effect of quercetin in cancer cell lines requires ERbeta-dependent signals. J Cell Physiol. 2012;227(5):1891-8. [PubMed ID: 21732360].

  • 22.

    Justesen U, Knuthsen P. Composition of flavonoids in fresh herbs and calculation of flavonoid intake by use of herbs in traditional Danish dishes. Food Chem. 2001;73(2):245-50.

  • 23.

    Formica JV, Regelson W. Review of the biology of Quercetin and related bioflavonoids. Food Chem Toxicol. 1995;33(12):1061-80. [PubMed ID: 8847003].

  • 24.

    Zhang FL, Zhang W, Chen XM, Luo RY. [Effects of quercetin and quercetin in combination with cisplatin on adhesion, migration and invasion of HeLa cells]. Chinese. Zhonghua Fu Chan Ke Za Zhi. 2008;43(8):619-21. [PubMed ID: 19087500].

  • 25.

    Lee TJ, Kim OH, Kim YH, Lim JH, Kim S, Park JW, et al. Quercetin arrests G2/M phase and induces caspase-dependent cell death in U937 cells. Cancer Lett. 2006;240(2):234-42. [PubMed ID: 16274926].

  • 26.

    Yang JH, Hsia TC, Kuo HM, Chao PD, Chou CC, Wei YH, et al. Inhibition of lung cancer cell growth by quercetin glucuronides via G2/M arrest and induction of apoptosis. Drug Metab Dispos. 2006;34(2):296-304. [PubMed ID: 16280456]. https://doi.org/10.1124/dmd.105.005280.

  • 27.

    Ramos S. Cancer chemoprevention and chemotherapy: dietary polyphenols and signalling pathways. Mol Nutr Food Res. 2008;52(5):507-26. [PubMed ID: 18435439].

  • 28.

    Williams RJ, Spencer JP, Rice-Evans C. Flavonoids: antioxidants or signalling molecules? Free Radic Biol Med. 2004;36(7):838-49. [PubMed ID: 15019969].

  • 29.

    Vidya Priyadarsini R, Senthil Murugan R, Maitreyi S, Ramalingam K, Karunagaran D, Nagini S. The flavonoid quercetin induces cell cycle arrest and mitochondria-mediated apoptosis in human cervical cancer (HeLa) cells through p53 induction and NF-kappaB inhibition. Eur J Pharmacol. 2010;649(1-3):84-91. [PubMed ID: 20858478].

  • 30.

    Bradford MM. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem. 1976;72(1–2):248-54.

  • 31.

    Huang LQ, Zhang W, Yang Y, Tao L. [Effects and its mechanism of quercetin on cervical cancer HeLa cells]. Chinese. Zhonghua Fu Chan Ke Za Zhi. 2009;44(6):436-9. [PubMed ID: 19953944].

  • 32.

    Zhang Y, Han G, Fan B, Zhou Y, Zhou X, Wei L, et al. Green tea (-)-epigallocatechin-3-gallate down-regulates VASP expression and inhibits breast cancer cell migration and invasion by attenuating Rac1 activity. Eur J Pharmacol. 2009;606(1-3):172-9. [PubMed ID: 19171136].

  • 33.

    Liu S, Yu M, He Y, Xiao L, Wang F, Song C, et al. Melittin prevents liver cancer cell metastasis through inhibition of the Rac1-dependent pathway. Hepatology. 2008;47(6):1964-73. [PubMed ID: 18506888].

  • 34.

    Kamai T, Yamanishi T, Shirataki H, Takagi K, Asami H, Ito Y, et al. Overexpression of RhoA, Rac1, and Cdc42 GTPases is associated with progression in testicular cancer. Clin Cancer Res. 2004;10(14):4799-805. [PubMed ID: 15269155].

  • 35.

    Kamai T, Shirataki H, Nakanishi K, Furuya N, Kambara T, Abe H, et al. Increased Rac1 activity and Pak1 overexpression are associated with lymphovascular invasion and lymph node metastasis of upper urinary tract cancer. BMC Cancer. 2010;10:164. [PubMed ID: 20426825]. https://doi.org/10.1186/1471-2407-10-164.