Chemopreventive Effects of Purple Rice (Oryza sativa L. indica) Extract on Prostate Carcinogenesis

Abstract

Purple rice (Oryza sativa L. indica), also called Khao Kum, is a glutinous pigmented rice that is widely distributed and consumed in the Northern region of Thailand. Our preliminary study showed that crude ethanolic extract of purple rice attenuated benign prostatic hyperplasia (BPH) in the rats induced by testosterone and suppressed the growth of human prostate cancer LNCaP cells. Purple rice extract promises to exert the biological activities through its phytochemicals. Here it was possible to initially clarify the phytochemical composition of purple rice fractions and its effects against BPH and prostate cancer in vivo, in addition to investigate the molecular mechanisms regarding to anti-carcinogenic activity in vitro. By comparing the phytochemicals between hexane soluble fraction (HSF) and hexane insoluble fraction (HIF) from the purple rice extract, HSF primarily contained lipophilic phytochemicals including gamma-oryzanol and vitamin E derivatives, but no phenolics. On the other hand, anthocyanins, including cyanidin-3-glucoside and peonidin- 3 - glucoside, were concentrated mostly in the HIF. After screening the safety, both fractions did not cause mutation toward Salmonella typhimurium strain TA98 and TA100 in either with or without metabolic activation. However, HSF exhibited higher toxic to a normal murine embryonic fibroblast NIH3T3 cells than HIF. To define the highly active fraction, the biological functions of each fraction were then compared. Both fractions possessed moderate to strong anti - mutagenicities against mutagenesis of standard carcinogens, including 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP), 2- amino-6-methyldipyrido[1,2-a:3',2'-d]-imidazole (Glu-P-1), and 2-amino-3- methylimidazo[4,5-f] quinolone (IQ), upon the presence of metabolic activation. Although the HSF had a more potent cytotoxic effect in LNCaP cells, the HIF exhibited a stronger modulating effect on androgen receptor (AR) expression, together with low toxicity to the normal mammalian cells. According to these results, the HIF is possible to be the most highly effective and less toxic fraction of the purple rice extract against BPH as well as prostate cancer. Thus, the molecular mechanisms responsible for anti-cancer property of the active fraction were then determined in vitro. According to the downregulation of AR, HIF also downregulated the expressions of AR downstream targets including prostate-specific antigen (PSA) and proteins involved in cell cycle progression, such as cyclin D1, cdk4, in LNCaP as compared to vehicle control cells. Consistently, HIF treatment resulted in the decreased clonogenic survival and cell cycle arrest at G0/G1 phase in LNCaP cells. Notably, the depletion of an AR-collaborative regulator, NKX3.1, and AR mRNA expression were observed in LNCaP after HIF treatment, indicating that HIF modulates AR expression at the level of transcription. In addition, exposure to HIF not only downregulated the expression of fatty acid synthase (FAS), but also attenuated the activation of AMP-activated protein kinase (AMPK)-α in the LNCaP cells. Moreover, HIF could inhibit rat microsomal 5α-reductase activity and suppressed 5α-reductase mRNA expression on DU-145 cells in a dosedependent manner. Altogether, these findings elucidate the potential mechanisms that can be responsible for anti-prostate cancer activity of HIF. The potential actions of the HIF on prostate carcinogenesis were then elucidated using the animal models. In benign prostatic hyperplasia (BPH) rat model, oral administration of HIF at least 0.1 g/kg.bw retarded prostate enlargement and improved histological changes induced by testosterone implantation, without any effects on the serum testosterone levels. A lower proliferating cell nuclear antigen (PCNA) labelling index and the downregulated expression of AR, cyclin D1, and FAS were clearly observed in the prostates of HIF-fed rats. Besides, the mRNA levels of inflammatory cytokines and enzyme, including IL-1β, IL-6, TNF-α, and iNOS, in the prostate tissues significantly decreased after HIF treatment. These findings suggest that anti-prostatic hyperplasia effects of HIF partly related to the modulation of AR expression and activation, together with its anti-inflammatory effects in the prostate tissues. Besides, the transgenic rat for adenocarcinoma of prostate (TRAP) rat model was also conducted in this study. 1% HIF mixed diet-fed TRAP rats showed a significantly higher percentage of low-grade prostatic intraepithelial neoplasia and obvious reduction in the incidence of adenocarcinoma in the lateral lobes of prostates, indicating that most acini persisted in a benign neoplastic stage. However, supplementation with HIF had no significant effect on the serum levels of testosterone and estrogen. Like LNCaP cells, the downregulation of AR, NKX3.1, cyclin D1, cdk4, and FAS expression together with attenuation of AMPKα and p38 mitogen-activated protein kinase (MAPK) activation were occurred in the prostate tissues from HIF-supplied groups. Meanwhile, HIF mixed diet failed to deactivate extracellular signal-regulated kinase (ERK)1/2 pathway in the rats’ prostate tissues. These results indicate that HIF blocks prostate cancer development mainly via regulating cell proliferation and metabolism through AR modulation. Taken together, the highly active and less toxic fraction isolated from purple rice extract in the present study consisted of hydrophilic bioactive components, such as anthocyanins. The potential protective effects of the highly active fraction from purple rice, HIF, against prostate enlargement and prostate carcinogenesis were partly related to the suppression of AR-mediated prostate cancer cell proliferation and metabolism. This observation therefore provides the rationale for the use of purple rice as a functional food in the prevention of BPH as well as prostate cancer in the future.