Prostate cancer is the second most common cancer in males worldwide [1]. Although, the growth and development of prostate gland can be controlled by androgen but estrogen can also play an important role in its development and carcinogenesis [2, 3]. Not only the estrogen but its metabolites such as 16$\alpha$-hydroxyestrone (16$\alpha$-OHE${}_1$) and 2-hydroxyestrone (2-OHE${}_1$) can also be related with PC [4]. These are predominant metabolites that are produced during oxidative metabolism of estrogen [5]. 16$\alpha$-OHE${}_1$ is an active estrogen metabolite that is bound to its receptor with high affinity and can function as estrogen agonist to induce various responses [6]. In contrast, 2-OHE${}_1$ has weak estrogen activity and inhibits angiogenesis [7]. Their ratio (i.e., 2-OHE${}_1$/16$\alpha$-OHE${}_1$) has been more related to breast cancer [8] than PC. In prostate cancer, patients with high urinary 2-OHE${}_1$/16$\alpha$-OHE${}_1$ ratio had a 40% non-significant reduction in the risk of PC, with a condition in which prostate-specific antigen (PSA) concentration higher than 4 ng/mL, was excluded from control subjects [9]. PSA levels were closely linked to prostate size and larger prostate gland was also related with estrogen levels [10]. Later study on the PC showed that there is no difference in the concentration of estrogen metabolites rather DNA adducts formation by estrogen, were found to be more active in PC patients than control subjects [11]. Recently, to probe the role of estrogen in PC, 15 urinary estrogen metabolites were determined in different PC patients and it was found a modest difference in the estrogen metabolites concentration between the cancer patients and control subjects [12]. 4-hydroxyestrone (4-OHE${}_1$) ranked higher in abundance among cases than control groups. Concentration of estriol (E${}_3$), estrone (E${}_1$), 16-ketoestradiol (16-kE2), 2-hydroxyestrone (2-OHE${}_1$) and estradiol (E${}_2$) were the highest among all groups, about 60–70% of the total urinary metabolites and E${}_3$ was the dominant estrogen in all study groups. On the other hand, 4-methoxyestradiol (4-MeOE${}_2$) was least abundant metabolites detected in the urine of all the groups. In another study, same urinary estrogen metabolites and their ratio were determined in PC patients. This study showed that oxidative metabolism of estrogen favoring 2-hydroxylation over 16$\alpha$-hydroxylation, was associated with reduced risk of PC [13]. Previous studies from the lab also showed an important role of catechol estrogen modified DNA in the etiopathogenesis of PC [14]. Recent study showed that 16$\alpha$-hydroxyestrone is responsible for causing breast cancer [15]. Infect, these estrogen metabolites play an important role in cancer as well as in autoimmune diseases [15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26].

The importance of estrogen in PC can be explained by two important facts. One is the presence of estrogen receptor (ER) in the prostate tissues in these patients [27] and the other is the response to estrogen therapy by the PC patients [28]. The effect of estrogen is mediated by two receptors: ER$\alpha$ and ER$\beta$. These two receptors are expressed in prostate tissues. ER$\beta$ is the most prevalent and mainly expressed in basal-epithelial cells of prostate while ER$\alpha$ is mainly confined to stromal cells occasionally found in basal-epithelial [29]. Although, PC specimen express both ER$\alpha$ and ER$\beta$ but their concentration remains unknown at different stages of the cancer [30]. The expression and functions of both the receptors mediate the development and growth of PC. The loss of ER$\beta$ is linked to the development from normal tissues to PC, whereas, those cancers retaining their expression might have a chance for recurrence [31]. In addition, expression of isoform of ER$\beta$ (ER$\beta$2 and ER$\beta$5) showed a prognostic biomarker in PC patients [32]. Estrogen plays important role in prostate carcinogenesis [33]. Circulating levels of estradiol (estrone) were slightly higher in African American men than in Caucasian men, whereas, these males have twice the prostate cancer risk of Caucasian [34]. Therefore, increased circulating estrogen might increase prostate cancer risk. Epidemiological data from various source showed mixed results. One study showed an association of increased plasma estrogen with an elevated risk of prostate cancer [35] and another corelating risk with elevated estrogens [36]. While other studies showed opposite finding i.e., increasing prostate cancer risk associated with decreasing levels of estradiol [37]. Other results showed that estrogen metabolic pathway favoring 2-hydroxylation over 16$\alpha$-hydroxylation might reduce the risk of clinically evident prostate cancer [38]. The study also confirmed an association between the risk of PC and higher urinary levels of 16$\alpha$-OHE${}_1$ and protective effect of higher 2-OHE${}_1$ to 16$\alpha$-OHE${}_1$ ratio [39].

Estrone and estradiol are oxidatively metabolized via two major pathways: formation of catechol estrogen and 16$\alpha$-hydroxylation. The catechol estrogens are 2-hydroxy and 4-hydroxyestrogens that are further inactivated in liver by conjugation reaction such as glucuronidation and sulfation. The other pathway of inactivation occurs in extra hepatic tissues by O-methylation catalyzed by catechol-o-methyl transferase [40].

There are significant evidences that showed that estrogen metabolites play an important role in PC. One study showed a negative relation between the risk of PC and elevated ratio of 2-OHE${}_1$/16$\alpha$-OHE${}_1$ and positive relation between PC and 16$\alpha$-OHE${}_1$ [9]. Most of the studies on PC either explain the role of urinary estrogen metabolites in PC or expression of ER in PC but none of them could explain the combined effects of estrogen metabolite and its receptor on PC. To test this important hypothesis, we investigate antibodies from PC to 16$\alpha$-OHE${}_1$-ER because estrogen metabolite directly involved in PC and ER had been expressed in prostate tissues. This gives us opportunity to screen PC patient’s sera with 16$\alpha$-OHE${}_1$-ER to probe their role. Furthermore, antibodies induced against this complex can also be used as an immunological probe for the determination of 16$\alpha$-OHE${}_1$ concentration and 2-OHE${}_1$/16$\alpha$-OHE${}_1$ ratio in the PC patients.

Estrogen (and its metabolites) can function as potential agent in the progression and development of PC [45]. They play a causative role in PC but the exact mechanism remains unknown. The potential mechanism that can explain the role of estrogen in PC includes epigenetic modification and estrogenic imprinting hyperprolectinemia, direct genotoxicity, inflammation and receptor-mediated actions. Although, estrogen can be used as potential hormonal therapy in PC but it can also cause this cancer [46]. Estrogen mediated its effect through the binding to its receptor (ER$\alpha$ and ER$\beta$) in the cells. Both the receptors for estrogen are expressed in normal prostate. ER$\alpha$ is expressed in the stromal cells and ER$\beta$ is found in the basal cells of prostate. ER$\beta$ has tumor suppression role in which its expression is decreased leading to methylation of CpG dinucleotide in the gene [47]. Moreover, polymorphism in codon 10 of ER$\alpha$ is a risk factor for PC [48]. Estrogen act as causative factor not only through their receptors but also through their role as genotoxic agent [49]. Estrogen can be oxidized to active catechol-estrogen metabolites by P450-mediated hydroxylation [50]. These metabolites lead to the generation of ROS that can damaged DNA and make DNA adducts [49]. Once DNA get damaged it alters its antigenicity leading to the generation of autoantibodies in autoimmune diseases [15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 44]. P450-mediated hydroxylation also produced 16$\alpha$-hydroxyestrone metabolites that exert its effect through binding to its receptor [51]. Elevated levels of urinary 16$\alpha$-OHE${}_1$ were associated with increased risk of prostate cancer [9] and ER had been expressed in prostate, so there might be a good opportunity to know the combed effect of 16$\alpha$-OHE${}_1$ and ER on PC. To study this important phenomenon, the binding affinity of the antibodies from PC with 16$\alpha$-OHE${}_1$-ER was measured to check whether this complex (16$\alpha$-OHE${}_1$-ER) has any affinity with antibodies from PC. The binding specificity of antibodies from sera of 60 PC patients and 40 controls to 16$\alpha$-OHE${}_1$-ER was checked with direct binding and inhibition ELISA. This complex showed high binding with almost all the chosen sera compared to controls (p 0.001). The combination of 16$\alpha$-OHE${}_1$ and ER exposed specific groups/molecules that can function as good epitopes for the prostate cancer IgGs. These results showed that 16$\alpha$-OHE${}_1$-ER can acts as better inhibitor showing a substantial difference in the binding of 16$\alpha$-OHE${}_1$-ER over ER (p 0.05) or 16$\alpha$-OHE${}_1$ (p 0.001). This data is similar to our previous studies from the lab that showed high binding of 16$\alpha$-OHE${}_1$ and ER adduct by breast cancer IgGs [44]. Various therapeutic approaches targeting the use of monoclonal antibodies (Mabs) to prostate specific antigen in PC had been taken into consideration [52]. These approaches include early detection of PC with the use of monoclonal antibodies with hormone and chemotherapy [53]. These Mabs in PC include anti-human epidermal growth factor receptor-2 (HER2) Mabtrastuzumab, anti-epidermal growth factor receptor (EGFR) Mabscetuximab and panituzumab and the anti-vascular endothelial growth factor (VEGF) Mabbevacizumab [54]. In animal model, anti-IL-20 monoclonal antibodies suppress PC growth and therefore, can be a novel target for the treatment of PC [55]. The presence or high level of serum antibodies against genitouring pathogens was not associated with PC [55].

The specificities of antibodies from PC were also tested according to various clinical characteristics in PC patients. Among them, those PC patients who expressed ER showed the highest inhibition followed by those cancer patients who had history of smoking, low 2-OHE${}_1$/16$\alpha$-OHE${}_1$ ratio and PSA level $\ge$4. As mention already, ER$\alpha$ and ER$\beta$ are expressed in prostate tissues and they are present in prostate during carcinogenesis [27]. Such binding might be observed because these patients already had antibodies against ER, which in combination of 16$\alpha$-OHE${}_1$, generate more immunological response. Cigarette smoking may increase the risk of PC by effecting circulatory hormone or through exposure to various carcinogens [56]. It might be possible that smoking increases the concentration of 16$\alpha$-OHE${}_1$ in these cancer patients that come in contact with ER, makes a complex and thus generate antibodies against this complex and showed high binding. As far as the high binding of patients with low 2-OHE${}_1$/16$\alpha$-OHE${}_1$ ratio is concern, low ratio means high concentration of 16$\alpha$-OHE${}_1$, which is an active estrogen metabolite and elevated urinary level have been associated with increased risk for PC [9]. Estrogen metabolites such as 16$\alpha$-OHE${}_1$ involved in the release of inflammatory mediators from the human amnion-derived cells [57] and somehow linked to inflammation. Again, high binding is due to the autoantibodies produced during inflammatory conditions. High binding in patients with PSA level $\ge$4 might be due to prostatitis or urinary tract infections, in which its concentration has been dramatically increased. The inhibition values gradually increased according to different stages and grades of PC indicating that more antibodies are produced as the PC progresses [58]. In addition, depression augments the production of antibodies against 16$\alpha$-OHE${}_1$-ER complex in prostate cancer patients [58]. Depression increased the production of antibodies through the generation of pro-inflammatory conditions in these patients.

To further confirmed the recognition of 16$\alpha$-OHE${}_1$-ER complex by antibodies from PC, we determined the affinity of antibodies by quantitative precipitin titration. The affinity constant of the order of 10${}^{-7}$ M clearly demonstrates high recognition of this complex by PC antibodies. The high recognition of 16$\alpha$-OHE${}_1$-ER complex by PC IgGs indicates possible participation of 16$\alpha$-OHE${}_1$-ER complex in prostate cancer pathogenesis. Studies have shown that estrogen metabolites (including 16$\alpha$-OHE${}_1$) are present in tissues, bile, urine and blood [7]. The production of antibodies in prostate cancer might arise as a result of formation of 16$\alpha$-OHE${}_1$-ER complex. Therefore, it could be possible that 16$\alpha$-OHE${}_1$-ER complex might be one of the important factors toward the generation of antibodies in prostate cancer. The induced antibodies showed cross-reactivity towards other antigens (i.e., anti-16$\alpha$-OHE${}_1$-ER with 16$\alpha$-OHE${}_1$ and anti-16$\alpha$-OHE${}_1$ antibodies with 16$\alpha$-OHE${}_1$-ER). Because anti-16$\alpha$-OHE${}_1$-ER antibodies showed cross-reactivity with 16$\alpha$-OHE${}_1$, these antibodies can be used as immunochemical probe for the estimation of 16$\alpha$-OHE${}_1$ in PC patients. These antibodies (i.e., anti-16$\alpha$-OHE${}_1$-ER antibodies) were also used to determined 2-OHE${}_1$/16$\alpha$-OHE${}_1$ ratio in PC patients.

The proposed mechanism for the generation of antibodies in PC includes the production of antibodies against 16$\alpha$-OHE${}_1$-ER through the formation of 16$\alpha$-OHE${}_1$-ER complex. 16$\alpha$-OHE${}_1$ and ER bind to each other to formed 16$\alpha$-OHE${}_1$-ER complex in prostate tissues. Formation of complex modified its immunogenicity leading to the generation and elevated levels of PC antibodies (Fig. 6). We strongly recommended to use other techniques like western blotting and immunohistochemistry of the cancer patient’s samples to confirmed their systemic level and in the tissue. This would help us to know their levels in the serum to compare with in the tissues of the cancer patients.

Fig. 6.

The proposed mechanism for the production of high affinity antibodies in prostate cancer (PC) patients.

In conclusion, our data clearly demonstrates the possible antigenic role of 16$\alpha$-OHE${}_1$-ER in the generation of antibodies in PC patients. Results imply that combination of 16$\alpha$-OHE${}_1$ and ER generates discriminating epitopes, which were highly recognized by PC IgG. Anti-16$\alpha$-OHE${}_1$-ER antibodies shown to represent an alternate immunological probe for the estimation of 16$\alpha$-OHE${}_1$ and 2-OHE${}_1$/16$\alpha$-OHE${}_1$ ratio in the urine of different PC patients, although we recommended to generate monoclonal antibodies-based probe for more specificity and accuracy.

16$\alpha$-OHE${}_1$, 16$\alpha$-hydroxyestrone; PC, prostate cancer; ER, estrogen receptor; 2-OHE${}_1$, 2-hydroxyestrone; 16$\alpha$-OHE${}_1$-ER, 16$\alpha$-hydroxyestrone-estrogen receptor; ELISA, enzyme linked immunosorbent assay; PSA, prostate-specific antigen; SDS-PAGE, sodium dodecyl sulfate-polyacrylamide gel electrophoresis.

WAK conceived, designed, performed the experiments and wrote the paper; MWAK performed the experiments and analyzed the data.

Prior consent from all the subjects was taken and finally, this study was approved by the Institutional Ethical Review Board (Deanship of scientific Research, KKU, 1/53/39).

The authors thank Sam A. Mark for his help and support. We are greatly thankful to him for reviewing the English language for this manuscript work.

The research was funded by Deanship of Scientific Research at King Khalid University, grant number R.G.P.1/53/39.

The authors declare no conflict of interest.