营养失调与治疗杂志

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2015 年营养大会：槲皮素和绿茶结合可增强前列腺癌的化学预防效果 - 王丕文 - 查尔斯·R·德鲁医学和科学大学

王丕文

绿茶 (GT)绿茶多酚 (GTP)对前列腺癌的化学预防作用已在临床前细胞培养和动物模型中得到充分证明。然而，人体研究的结果并不一致。GT 多酚 (GTP) 在体内的低生物利用度和广泛代谢限制了 GT 的抗癌活性。我们确定了甲基化抑制剂槲皮素 (Q) 是否会增强体内前列腺腺癌的化学预防。我们发现在人类前列腺组织和小鼠异种移植前列腺肿瘤组织中，在 GT 消耗后，约 50% 的 GTP 处于甲基化形式，因此甲基化降低了 GTP 的抗癌活性。我们确定天然甲基化抑制剂槲皮素 (Q) 与 GT 的混合物可使前列腺腺癌 LNCaP 和 PC-3 细胞中的 GTPs 细胞浓度增加 4-10 倍并降低 GTPs 的甲基化。这种混合处理增强了对两种细胞系的细胞增殖的抑制和对细胞凋亡的诱导。然后我们进行了一项动物研究以验证 GT 和 Q 在体内的联合作用。重度联合免疫缺陷 (SCID) 小鼠被植入雄激素敏感的 LAPC-4 前列腺腺癌细胞并用 GT、Q、GT+Q 或对照治疗。在干预前一周将雄激素敏感的 LAPC-4 前列腺腺癌细胞皮下注射到重度联合免疫缺陷 (SCID) 小鼠体内。作为饮料饮用的冲泡茶中 GTPs 的浓度为 0.07%，饮食中添加的 Q 浓度为 0.2% 或 0.4%。干预6周后，与对照相比，肿瘤生长抑制了16%（Q）、21%（GT）和45%（GT+Q）。联合组非甲基化GTPs的组织浓度显著升高，并且与儿茶酚-O-甲基转移和多药耐药相关蛋白（MRP）-1的蛋白表达降低有关。联合治疗还与增殖、雄激素受体（AR）和磷脂酰肌醇3-激酶（PI3K）/Akt信号传导的抑制以及细胞凋亡的刺激有关。GT+0.4% Q对肿瘤抑制的联合作用在另一项在肿瘤接种前开始干预的实验中得到进一步证实。该组合增强了对雄激素受体、前列腺特异抗原和血管内皮生长因子蛋白表达的抑制。

Quercetin (Q) is a flavonoid that is found in most vegetables and fruits that are edible, particularly in onions, apples, and red wine. The inhibitory effect of Q on the actions of MRPs and COMT has been documented well. Q itself has been shown to exhibit chemo preventive activities specially in prostate cancer. 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. 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 immune deficiency (SCID) mice and to elucidate the mechanisms of the increased anticarcinogenic effect of the combination treatment.

The effect of the combination treatment was related to the concentration of GTPs in tumor tissue, which in turn was dependent on the Q dose. The dose of GT used in this study is equivalent to the consumption of 5-6 cups of green tea per day for an adult human. This estimate is based on the observation that the consumption of 5-6 cups of tea daily achieved similar tissue concentrations in human prostate compared to tissue in mice consuming the same brewed GT. Q dose would be equivalent to 1.0g (low dose) and 2.0g (high dose) per day for an adult based on blood concentrations of Q and its metabolites as observed in the present study (data not shown) relative to that from a human study. The consumption of 1000 mg of Q per day was not associated with any adverse effects in humans. A pilot clinical trial is on-going to determine the Q concentration necessary in humans to increase the bioavailability of EGCG. Our results showed that the combination treatment decreased the protein expression of MRP1 in tumor tissues. However, no changes of mRNA expression of MRP1 were observed, indicating that post-transcriptional regulation such as microRNA (miRNA) may be responsible. Many polyphenols including GT and Q have been shown to modulate the expression of miRNA, a class of small non-coding RNAs that interact with mRNA to regulate the gene expression post-transcriptionally. Several other investigators demonstrated the inhibitory effects of Q on the activities of transport-regulating proteins such as p-glycoprotein and MRPs, leading to an increased absorption of GTPs from the intestinal tract and retention in the tissues. Although Q is extensively methylated, sulfated, or glucuronidated upon uptake it has been demonstrated that these Q metabolites, such as isorhamnetin and 7-O-glucuronosyl quercetin exhibited equal or stronger inhibition on the activities of MRPs compared to Q. Considering the importance of MRPs in the development of chemo resistance during chemotherapy, GT and Q may also be good candidates to be combined with chemotherapy drugs to reduce drug resistance and enhance therapeutic efficacy.

The important role of catechol O-methylation of GTPs in cancer prevention has been demonstrated in several studies. Due to a common polymorphism of COMT its activity can vary by 3 to 4-fold. A case control study in Asian-American women provided evidence that the risk of breast cancer was significantly reduced only among tea drinkers possessing at least one low-activity COMT allele. We found earlier that EGCG was extensively methylated in human prostate tissues obtained from prostatectomy and in mouse tissues after GT consumption. In cell culture experiments methylation significantly decreased the anticancer activities of EGCG as shown by our laboratory and other investigators. Previously we demonstrated in vitro that the combination of GT and Q significantly decreased the activity and protein expression of COMT in various cancer cell lines. This inhibition of COMT was associated with a decrease in EGCG methylation and increase in the anti proliferative activity. Similarly, Landis-Piwowar et al. demonstrated that EGCG treatment in breast cancer cells of lower COMT activity led to stronger proteasome inhibition and apoptosis induction. The present study confirmed the inhibition of COMT in vivo both in mRNA and protein expression by the combination treatment of GT and Q, which may contribute to the increased concentrations of non-methylated EGCG in tumor tissues and supports the important role of COMT in GT chemoprevention.

Cancer results from a multistage process with distinct molecular and cellular alterations. Therefore, treatments targeting many concerted processes may be advantageous in cancer prevention, therapy and reducing resistance to the treatment. Natural compounds such as GT and Q target multiple events and signaling pathways throughout the stages of carcinogenesis. In combination these compounds may increase the anticarcinogenic activity by expanding the coverage of molecular targets. The androgen receptor (AR) signaling pathway plays a critical role in prostate tumor growth and progression, thus it is an important target in prostate cancer prevention and treatment. Nevertheless, there are other signaling pathways particularly the phosphatidylinositol 3-kinases/Akt/ mammalian target of rapamycin (PI3K/Akt/mTOR) pathway that crosstalk with AR signaling and may directly regulate the expression and activation of AR. The upregulation and activation of PI3K/Akt/mTOR pathway is thought to play an important role in prostate cancer due to the decreased expression or loss of the negative regulator, tumor suppressor phosphatase and tensin homolog (PTEN). Akt is activated after phosphorylation by phosphorylated PI3K and in turn activates its substrates, one being mTOR, which leads to increased cell proliferation and survival. Therefore, an effective intervention strategy in prostate cancer may need to target both AR and PI3K/Akt/mTOR signaling pathways. Both GT and Q inhibit AR signaling through multiple mechanisms including the decrease of AR expression and its nuclear translocation. The combined use of GT and Q in the present study demonstrated an increasing ability to inhibit AR expression compared to individual treatments. In addition, the phosphorylation of Akt was significantly inhibited by the combination treatment while only a slight but non-significant decrease by the individual treatments. Further evidence of a stronger inhibition of AR and PI3K/Akt signaling was also provided through increased inhibition of AR-mediated PSA expression in tumor tissues from mice treated with GT+Q. Similar effects were demonstrated by a recent study that combined mTOR inhibition (everolimus) with an anti-androgen (bicalutamide) to block both pathways, resulting in tumor growth was statistically significantly reduced. In addition to their applications in cancer prevention, GT and Q may be ideal candidates to be combined with anti-androgens to enhance the therapeutic efficacy in a less-toxic manner in the treatment of advanced prostate cancer.

This study provides a novel regimen by combining GT and Q to enhance the chemoprevention of prostate cancer in a non-toxic manner. This was associated with an increased bioavailability of non-methylated GTPs and enhanced anti proliferative and proapoptotic effect. These results warrant future human intervention studies to confirm the combined effect of GT and Q in prostate cancer prevention and treatment.

Biography

王丕文于 2000 年获得山东医科大学医学博士学位，2003 年获得硕士学位。他于 2008 年获得德克萨斯理工大学博士学位，并在加州大学洛杉矶分校人类营养中心从事博士后研究。他目前是查尔斯·德鲁医科大学内科系、癌症研究与培训部助理教授。他在知名期刊上发表了 20 多篇论文，并担任多家科学期刊的同行评审员。

注：该研究部分在 2015 年 10 月 26 日至 28 日在美国伊利诺伊州芝加哥举行的第四届国际营养会议和展览会上展示。