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Recent evidence suggests that oxidative stress contributes to the pathogenesis of prostate cancer. The present study focused on the effect of apocynin, an inhibitor of NADPH oxidase, on prostate carcinogenesis using the transgenic rat for adenocarcinoma of prostate (TRAP) model. There were no toxic effects with apocynin treatment. The percentages and numbers of carcinomas in both the ventral and lateral prostate were significantly reduced by apocynin treatment, with dose dependence. Reduction of reactive oxygen species by apocynin was confirmed by immunohistochemistry of 8-OHdG and dihydroethidium staining. Positivity of Ki67 was significantly reduced by apocynin treatment, and downregulation of clusterin expression, as well as inactivation of the MEK-ERK1/2 pathway, was a feature of the apocynin treated groups. In human prostate cancer cell line LNCaP, apocynin also inhibited reactive oxygen species production and blocked cell growth by inducing G0/G1 arrest with downregulation of clusterin and cyclin D1. These data suggest that apocynin possesses chemopreventive potential against prostate cancer.
Prostate cancer is the second most frequently diagnosed cancer in men in the world, with particularly high incidences in Oceania, Europe and North America. In Japan, incident and mortality rates for prostate cancer are relatively low are but increasing.[1, 2] There are potentially curative options, such as radical prostatectomy or radiotherapy, but once the disease is metastatic, the outlook is poor. Therefore, research into chemoprevention of prostate cancer is critical.
Reactive oxygen species (ROS) can be important factors for carcinogenesis and tumor progression, not only inducing DNA damage but also producing cellular alterations, such as upregulation of MAPK and protein kinase C.[3, 4] Recently, oxidative stress has been reported to contribute to cancer and progression in the prostate.[5, 6] Therefore, we have focused on inhibition of ROS production as an anti-carcinogenic approach. ROS is produced by mitochondria, peroxisomes, cytochrome P-450 and other cellular elements as a byproduct, and is generated by NADPH oxidase, which is also implicated in a variety of signaling events, including cell growth, cell survival and cell death. Apocynin, which belongs to the methoxy-substituted catechol family, inhibits NADPH oxidase activity by blocking the formation of NADPH oxidase complex and is commonly used as a standard NOX inhibitor for research purposes. In addition, apocynin can be converted by peroxidase-mediated oxidation to a dimer, which has been shown to be a more efficient inhibitor than apocynin itself. We previously presented evidence that apocynin reduced oxidative stress induced by arsenite treatment of rat urothelium in vivo.
In the present study we focus on NADPH oxidase and test whether its inhibitor, apocynin, is able to suppress prostate carcinogenesis in the transgenic rat for adenocarcinoma of prostate (TRAP) model, which was generated in our laboratory and features development of well-differentiated prostate adenocarcinomas in prostatic lobes within a short period. The TRAP rat has a transgene that encodes SV40 T antigen under probasin promoter, so that the carcinomas that develop are androgen-dependent.[11, 12]
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- Materials and Methods
- Disclosure Statement
- Supporting Information
In the present study, we demonstrated suppressive effects of apocynin treatment on prostatic carcinogenesis in the TRAP rat model. Because apocynin is known to be an NADPH oxidase inhibitor, we focused on ROS conditions in prostate tissue using immunohistochemistry for 8-OHdG and dihydroethidium staining. Some reports have indicated that antioxidants such as N-acetylcysteine and vitamin C clearly reduce oxidative stress in vitro, but exert less effects with oral dosing in vivo.[15, 16] ROS is generally produced from mitochondria, peroxisomes, cytochrome P450 and NADPH oxidase.[4, 6, 7] Apocynin only affects ROS reduction from NADPH oxidase, but it would be expected to be a good antioxidant in vivo. In addition, we did not detect any toxic effects of apocynin in this study.
Reactive oxygen species generation is not only considered associated with tissue injury and/or DNA damage, but also neoplastic transformation, aberrant growth and/or proliferation.[4, 7] Indeed, ROS may play broad roles in cellular processes associated with initiation and development of many cancers, including prostate cancer. In this study, apocynin reduced the Ki-67 labeling index and cyclin D1 expression, which indicates that ROS generation is related to cell proliferation in prostate lobes of TRAP rats. Furthermore, NADPH oxidase is reported to regulate plasma adiponectin, whose concentration may be negatively correlated with prostate cancer progression. Therefore, we investigated adiponectin concentration in serum, but did not find any differences among the groups.
In TRAP rats, we previously detected activation of p38 MAPK and ERK1/2 in prostate tissue, and inactivation by chemopreventive agents such as angiotensin II receptor blocker or purple corn color.[14, 19] These also reduced cell proliferation and/or induced apoptosis in prostate tissue. In this study, we also detected dephosphorylation of ERK1/2 in the prostate after apocynin treatment. To assess the relationship between ROS generation and phosphorylation of ERK1/2, microarray analysis was employed. The data obtained for upregulated and downregulated genes indicated associations with ROS generation, immune function and translation by gene ontology. We focused on clusterin, dysregulated in many types of cancer, including prostate cancer, with cellular functions such as a sulfated glycoprotein in regulation of apoptosis, cell cycle control, DNA repair, cell adhesion and tissue remodeling.[22, 23] It has two isoforms. Secretory clusterin (sCLU) has a chaperone action like that of small heat shock proteins, and is sized around 70–80 kDa. The other clusterin isoform is the nuclear form (nCLU) of 55 kDa, associated with cell death.[21-23] In this study, we detected significant reduction of clusterin protein around 70 kDa. Because sCLU is reported to be downregulated by p53 in some tumor cells, high expression of clusterin in the prostate would be expected given dysfunction of p53 under the influence of SV40 T antigen in TRAP rats.
In human prostate cancer, clusterin expression is initially low in contrast with the prostate cancer cells resistant to conventional chemotherapy or hormonal therapy which show upregulation.[21, 25] Meanwhile, we detected rather high expression of clusterin in all stages of prostate cancer development in TRAP rats. In te transgenic adenocarcinoma of mouse prostate (TRAMP) model, higher expression of clusterin in the prostate glands was detected compared to that in non-transgenic mice; however, analyses of overt carcinoma revealed no expression. These data suggest that SV40 T antigen supports to induce clusterin expression among the carcinogenic process in the prostate of both mice and rats. The difference in clusterin expression in carcinoma may be related to the different histology between mice (poorly-differentiated neuroendocrine-like carcinomas) and rats (moderately differentiated adenocarcinomas), because clusterin is mainly stained in the membrane.
Recently, sCLU was reported to induce phosphorylation of ERK1/2 in lung adenocarcinoma and pancreatic cancer cells.[27, 28] Therefore, we considered that apocynin reduced prostate carcinogenesis via clusterin, acting on the ERK1/2 pathway and reducing cell proliferation in TRAP rats. We also found that apocynin reduced cell growth and ROS generation, along with expression of clusterin and cyclin D1 in LNCaP cells (human prostate cell line). Although there is a possibility that the exact intracellular molecular pathways affected by apocynin treatment may be different between humans and rats, we suggest that there may be similar anti-neoplastic effects caused by apocynin, especially related to clusterin expression, in rat and human prostate.
Chemoprevention attempts are comprehensive in prostate cancer, and various agents are reported to prevent, reverse or delay tumor deveolpment and progression. In this study, we detected lower incidence of adenocarcinoma in the lateral lobes of 500 mg/L apocynin-treated rats, and reduction of cell proliferation was observed in both PIN and adenocarcinoma. Thus, we consider that apocynin has potential to prevent and delay the carcinogenic process, and is suitable as a chemopreventive drug. Recently, custirsen (OGX-011), a clusterin inhibitor, was selected for use as an anti-cancer drug in a randomized phase II study of patients with metastatic castration-resistant prostate cancer.[30, 31] We earlier reported that apocynin inhibited growth of androgen-independent prostate cancer with inhibition of phosphorylation of Rac1 and NF-κB and angiogenesis. Together with the present data, we confirm that apocynin has the potential to be a candidate anti-cancer drug for androgen-dependent/independent prostate cancer.
In conclusion, apocynin, an NADPH oxidase inhibitor, suppressed prostate carcinogenesis while reducing ROS in the TRAP rat model. The mechanism of prevention appears to involve regulation of cell proliferation via clusterin and the MEK-ERK1/2 pathway. Apocynin warrants further attention as a promising chemopreventive drug for prostate cancer.