Research article
Enhanced inhibition of prostate cancer xenograft tumor growth by combining quercetin and green tea,☆☆

https://doi.org/10.1016/j.jnutbio.2013.09.005 Get rights and content

Abstract

The chemopreventive activity of green tea (GT) is limited by the low bioavailability and extensive methylation of GT polyphenols (GTPs) in vivo. We determined whether a methylation inhibitor quercetin (Q) will enhance the chemoprevention of prostate cancer in vivo. Androgen-sensitive LAPC-4 prostate cancer cells were injected subcutaneously into severe combined immunodeficiency (SCID) mice one week before the intervention. The concentration of GTPs in brewed tea administered as drinking water was 0.07% and Q was supplemented in diet at 0.2% or 0.4%. After 6-weeks of intervention tumor growth was inhibited by 3% (0.2% Q), 15% (0.4% Q), 21% (GT), 28% (GT+0.2% Q) and 45% (GT+0.4% Q) compared to control. The concentration of non-methylated GTPs was significantly increased in tumor tissue with GT+0.4% Q treatment compared to GT alone, and was associated with a decreased protein expression of catechol-O-methyltransferase and multidrug resistance-associated protein (MRP)-1. The combination treatment was also associated with a significant increase in the inhibition of proliferation, androgen receptor and phosphatidylinositol 3-kinase/Akt signaling, and stimulation of apoptosis. The combined effect of GT+0.4% Q on tumor inhibition was further confirmed in another experiment where the intervention started prior to tumor inoculation. These results provide a novel regimen by combining GT and Q to improve chemoprevention in a non-toxic manner and warrant future studies in humans.

Introduction

Green tea (GT) is produced from the leaves of the plant Camellia sinensis. The major bioactive components of GT are GT polyphenols (GTPs) mainly including (−)-epigallocatechin (EGC), (−)-epigallocatechin-3-gallate (EGCG), (−)-epicatechin (EC), and (−)-epicatechin-3-gallate (ECG), with EGCG as the major component [1]. The chemopreventive activity of GT has been well documented in cell culture studies in vitro and in animal models against several types of cancers including prostate cancer [2], [3]. However, evidence from human studies is inconsistent [2]. Several clinical trial studies have suggested a promising role of GT in the chemoprevention of prostate cancer. Bettuzzi et al. demonstrated a reduced incidence of prostate cancer in men with prostatic intraepithelial neoplasia after a 1-year GT supplement intervention compared to a group of men receiving placebo [4]. Likewise, in a single-arm pre-prostatectomy trial of a GT supplement, McLarty et al. demonstrated a decrease in serum prostate-specific antigen (PSA) levels and decreased prostate tissue vascular endothelial growth factor and hepatocyte growth factor concentrations [5]. However, several other studies failed to find an association between GT consumption and risk of prostate cancer [2].

The anti-cancer potency of GT is limited by the low bioavailability of GTPs. The high doses of GTPs necessary in laboratory studies can hardly be achieved in humans by the consumption of GT alone. The absorption from the intestine and retention of GTPs in tissues is regulated by several transporters including the multidrug resistance-associated proteins (MRPs). Modulation of the MRP activity with combination treatments provides an opportunity to enhance the bioavailability of GTPs [6]. The anti-cancer potency of GT is also limited by the rapid biotransformation of GTPs in the body leading to enhanced excretion and reduced bioactivity [7], [8]. Upon uptake, the non-gallated GTPs such as EGC and EC undergo extensive glucuronidation and sulfation while the gallated GTPs EGCG and ECG are mainly present in the free form [9]. All GTPs are readily methylated by catechol-O-methyltransferase (COMT) leading to an increase in urine excretion [10]. COMT is a widely distributed intracellular enzyme [11]. Previously we found that approximately 50 percent of EGCG was present in methylated form (4″-O-methyl EGCG, 4″-MeEGCG) in human prostate tissues obtained at prostatectomy after consumption of 6 cups of GT daily for 3–5 weeks [12]. Methylation significantly decreased the anticarcinogenic activity of EGCG in cultured LNCaP prostate cancer cells and Jurkat cells [12], [13].

Quercetin (Q) is a flavonoid found in most edible vegetables and fruits particularly in onions, apples, and red wine. The inhibitory effect of Q on the activities of MRPs and COMT has been well documented [14], [15], [16], [17]. Q itself has been shown to exhibit chemopreventive activities especially in prostate cancer [18]. We were able to demonstrate in vitro that the combined use of Q with GT significantly increased the cellular concentrations of non-methylated EGCG in prostate cancer LNCaP and PC-3 cells, leading to enhanced anti-proliferative effects [19]. The present study was designed to test the hypothesis that the combined effect of Q and GT in vivo leads to an increased anticarcinogenic effect in a xenograft prostate tumor mouse model using severe combined immunodecificency (SCID) mice and to elucidate the mechanisms of the increased anticarcinogenic effect of the combination treatment.

Section snippets

Preparation of green tea and quercetin diet

GT was freshly prepared thrice a week on Monday, Wednesday and Friday by brewing one tea bag in 240mL of boiling water (pH 3) for 5 minutes. Tea bags (authentic GT) were generously provided by Celestial Seasonings (Boulder, CO, USA). The composition of GTPs in the brewed tea in mg/L was: EGC 204±4, EGCG 388±12, EC 44±2, ECG 64±7 and catechin 7±1. GT was administered as drinking water ad libitum. Q (Sigma-Aldrich, St Louis, MO, USA) was supplemented in AIN-93G diet at a concentration of 0.2% or

GT and Q in combination enhanced the inhibition of tumor growth

In the first experiment the intervention treatment started one week post tumor inoculation when the tumors reached a size of 10 mm3, and lasted for 6 weeks. A significant inhibition of tumor growth by 38% was observed 5weeks after the tumor inoculation (4 weeks post intervention) by the co-treatment of GT and 0.4% Q as compared to control (Fig. 1A). During the next 2 weeks GT+0.4% Q treatment demonstrated a significantly stronger inhibition of tumor growth than GT or Q alone. The tumor growth

Discussion

Our results presented here demonstrated that the combined treatment with GT and Q enhanced their chemopreventive activities in vivo. Natural compounds are promising sources for the development of non-toxic antitumor agents. However, due to low bioavailability, we may not be able to translate the in vitro findings to clinical application [25]. As demonstrated in our recently completed pre-prostatectomy GT trial, only a small amount of EGCG was detectable in prostate tissue after daily

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    Funding: This work was supported by the Department of Defense [W81XWH-10-1-0298 to P.W.]; and the National Institutes of Health [RO3 CA150047-01 and RO1 CA116242 to S.H., U54 CA143931-01 to J.V., and P50CA092131 to J.S.].

    ☆☆

    Conflict of Interest Statement: None declared.

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