Oxidative stress marker 8-hydroxyguanosine is more highly expressed in prostate cancer than in benign prostatic hyperplasia

Introduction

Oxidative stress is a chief cause of vascular endothelial damage. It increases the levels of reactive oxygen species, which subsequently induces vascular endothelial dysfunction via the inhibition of nitric oxide production (1).

In the prostate, ischemia increases the levels of reactive oxygen species, growth factors, and cytokines, and induces the development of angiogenesis, resulting in cancer progression (2). Recent studies, using a prostate hyperplasia mouse model have demonstrated that ischemia in the prostatic epithelium induces oxidative stress, resulting in cell proliferation involved by inflammatory cytokines and several growth factors (3,4).

These findings indicate that oxidative stress plays an important role in the prostate. No report has described the tissue 8-hydroxyguanosine (8-OHdG) expression in prostatic specimens. In the present study, we compared the expression levels of an oxidative stress marker 8-hydroxyguanosine between prostate adenocarcinoma and non-neoplastic prostate tissues.

Materials and methods

Immunohistochemistry was performed, as described previously (5) in sections from a prostatic tissue microarray composed of 10 cases of prostatic adenocarcinoma and 70 cases of non-neoplastic prostate (PR804; US Biomax, Rockville, MD, USA). This company obtained informed consent (https://www.biomax.us/FAQs,Q10) Briefly, 5-µm-thick slides were dewaxed with xylene, hydrated with gradient ethanol, and microwaved at high level for 2 min., followed by 30 min. microwaved at middle-low levels for heat antigen retrieval (Target Retrieval Solution pH9, Dako, Carpinteria, CA, USA). After 3% hydrogen peroxidase block, the samples were incubated overnight at 4°C with a primary antibody to anti-8-OHdG (ab48508, diluted to 1:200; Abcam, Cambridge, MA, USA) and a broad-spectrum secondary antibody (Histostain-SP kit; Invitrogen, Grand Island, NY, USA) for 2 h (Table I). The slides were then examined by a single pathologist (HM) blinded to sample identify. The score of immunoreactivity was determined by multiplying the percentage of immunoreactive cells (0% = 0; 1–10% = 1; 11–50% = 2; 51–80% = 3; 81–100% = 4) by staining intensity (negative = 0; weak = 1; moderate = 2; strong = 3). The immunohistochemical scores (ranging from 0 to 12) were considered negative (0; 0–1), weakly positive (1+; 2–4), moderately positive (2+; 6–8), and strongly positive (3+; 9–12) for 8-OHdG expression (5).

Table I. First and second antibody for immunohistochemistry analysis.

Table I.

First and second antibody for immunohistochemistry analysis.

Name	Cat. no.	Company
First antibody
8-Hydroxyguanosine polyclonal antibody	ab48508	Abcam
Second antibody and staining kit
Histostain-SP kit	95–9943	Invitrogen

The 8-OHdG expression was analyzed by chi-square test using the Graph Pad Prism software program (Graph Pad Software, La Jolla, CA, USA).

Results

We immunohistochemically stained for 8-OHdG in a prostate tissue microarray. Positive signals were detected predominantly in cytoplasm of epithelial cells as well as in nucleus of epithelial cells only in a few cases (Fig. 1). 8-OHdG was immunoreactive in all 10 prostatic adenocarcinomas, including 3 (30%) cases with moderate (2+) expression and 7 (70%) cases with strong (3+) expression. Similarly, 8-OHdG was moderately and strongly positive in 50 (71.4%) and 20 (28.6%) cases of benign prostatic hyperplasia (Table II). Thus, the levels of 8-OHdG expression in prostatic adenocarcinoma tissue specimens were significantly higher than those in non-neoplastic prostate tissue specimens (P<0.01).

Figure 1. Immunohistochemistry of 8-hydroxyguanosine. Strong and moderate expression was observed in (A) non-neoplastic prostate and (B) prostatic adenocarcinoma tissue specimens, respectively.

Table II. Immunohistochemical staining of 8-hydroxyguanosine (P<0.01).

Table II.

Immunohistochemical staining of 8-hydroxyguanosine (P<0.01).

Immunoreactive score	0	1+	2+	3+
Prostate adenocarcinoma	0 (0.0%)	0 (0.0%)	3 (30.0%)	7 (70.0%)
Non-neoplastic tissue	0 (0.0%)	0 (0.0%)	50 (71.4%)	20 (28.6%)

Discussion

Our results showed that prostate adenocarcinoma showed higher 8-OHdG expression than non-neoplastic prostate tissue. Miyake et al showed that prostate cancer patients had significantly higher levels of urine 8-OHdG/Cre than those without cancer (6). In patients with prostate cancer, urine 8-OHdG/Cre levels were not markedly different before and after radical prostatectomy. In contrast, their levels were decreased significantly after androgen deprivation therapy. These findings indicate that androgen deprivation therapy reduces oxidative stress.

The detailed mechanism underlying the cancer progression induced by 8-OHdG is still unknown. Guanine is easily damaged by anti-oxidant stress, resulting in a change to 8-OHdG. 8-OHdG is structurally stable and widely used as an oxidant-stress biomarker (6,7). H2O2 has also been shown to increase 8-OHdG in a time-dependent manner in prostatic epithelial cells, which is one potential mechanism underlying cancer progression, presumably due to continuous anti-oxidant damage (8,9). Based on these findings, oxidant stress produced 8-OHdG in prostate tissue, resulting in the time-dependent accumulation of 8-OHdG and subsequent carcinogenesis due to the damage of DNA. Wu and Ni showed the correlation between 8-OHdg and carcinogenesis and speculated that hypomethylation and regional hypermethylation contribute to carcinogenesis (10). In gastric cancer, a correlation has been reported between gastric cancer and cytotoxin-associated genes that induces cancer progression. However, the detailed mechanisms are still unknown (11). While further studies are needed, the findings from the present study suggest that 8-OHdG might be a candidate tumor marker for detecting prostate cancer.

Tadalafil, a PDE-5 inhibitor, inhibits cGMP in smooth muscle cells and induces reflexes smooth muscle (12,13). Gotoh et al demonstrated that tadalafil improved the bladder blood flow and increased the level of 8-OHdG in tissue (14). Tadalafil has also been reported to show anti-inflammatory activity in the prostate and might be a prevention effect from prostate cancer (15,16).

The limitations of this study include no staining in completely normal prostatic tissue. Further studies, using normal prostate tissues and/or cell lines, are also required to reveal the underlying mechanism for the involvement of 8-OHdG in prostate carcinogenesis and cancer progression.

We found that 8-OHdG expression was elevated in prostate cancer tissues compared with non-neoplastic prostate tissues. 8-OHdG may therefore play an important role in prostate cancer development.

Acknowledgements

Not applicable.

Funding

Funding was received from Kakenhi grants (grant no. 16K20152) for the Ministry of Education, Culture, Sports, Science and Technology of Japan.

Availability of data and materials

The datasets used and/or analyzed during the current study are available from the corresponding author upon request

Authors' contributions

SO, TK and HU conceived and designed the experiments. SO, TK, YI, TT, SK, KI, HM and HU performed the experiments. SO, TK and HM wrote the manuscript. All authors have read and approved the manuscript

Ethics approval and consent to participate

Not applicable.

Patient consent for publication

Not applicable.

Competing interests

The authors declare that they have no competing interests.

References