Immunohistochemical study of Rho-kinase pathway expression in a mouse model of prostate hypertrophy for preliminary study of progressive prostate cancer model establishment
Original Article

Immunohistochemical study of Rho-kinase pathway expression in a mouse model of prostate hypertrophy for preliminary study of progressive prostate cancer model establishment

Young-Min Yang1, Koichi Kitagawa2, Yuto Hirata3, Michika Moriwaki3, Tenei Ono4, Yi-Te Chiang5, Kuan-Chou Chen5, Katsumi Shigemura4,6

1Department of Urology, Kobe University Graduate School of Medicine, Kobe, Japan; 2Department of Advanced Medical Science, Graduate School of Science, Technology and Innovation, Kobe University, Kobe, Japan; 3Department of International Health, Kobe University Graduate School of Health Sciences, Kobe, Japan; 4Department of Urology, Teikyo University Hospital, Tokyo, Japan; 5Department of Urology, Shuang Ho Hospital, Taipei Medical University, New Taipei City; 6Department of Urology, Kurashiki Medical Center, Kurashiki, Japan

Contributions: (I) Conception and design: K Shigemura; (II) Administrative support: KC Chen; (III) Provision of study materials or patients: Y Hirata, M Moriwaki, T Ono; (IV) Collection and assembly of data: YM Yang, K Kitagawa; (V) Data analysis and interpretation: YT Chiang; (VI) Manuscript writing: All authors; (VII) Final approval of manuscript: All authors.

Correspondence to: Katsumi Shigemura, MD, PhD. Department of Urology, Kurashiki Medical Center, 250 Bakuro-cho, Kurashiki 710-8522, Japan; Department of Urology, Teikyo University Hospital, Tokyo, Japan. Email: yutoshunta@gmail.com.

Background: For aggressive prostate research, a suitable orthotopic animal model is in demand. This study aims to future establishment of mice prostate cancer model via testosterone dosing after castration, and, as a preliminary experiment, evaluate the changes in prostate and bladder caused by benign prostate hyperplasia (BPH). An animal prostate enlargement model was used to assess bladder smooth muscle changes via immunohistochemical (IHC) markers of the prostate and bladder.

Methods: Injection of testosterone propionate into 12-week-old C57BL/6 mice stimulated prostatic hypertrophy in a mouse model. Three groups of mice were divided into control, castrated mice with a single subcutaneous dose of 50 μL testosterone administration, castrated mice without testosterone administration, and sacrificed 4 weeks later. α-1 adrenoceptor and transforming growth factor-beta (TGF-β) expression in the prostate ventral lobe and β-3 adrenoceptor and Rho-kinase expression in the bladder mucosa were evaluated by IHC staining analyses and compared among the 3 groups.

Results: Prostate weights for the control, castrated, and castrated + testosterone groups were 75±27, 101.9±17, and 130±19 mg, respectively, showing a significant difference in prostate weight between the subject and castration plus treatment groups (P<0.05). IHC data showed that the castrated + testosterone group had significantly higher expression of α-1 adrenoceptor antibody (0±0 vs. 1.25±0.433, P=0.02) in the prostate ventral lobe and β-3 adrenoceptor antibody (0±0 vs. 1±1.41, P=0.0003) and Rho-kinase antibody (0±0 vs. 2±0, P=0.0001) in the bladder mucosa, compared with the control groups.

Conclusions: For our establishment of a prostate cancer model for mice, our in vivo experiments using IHC study demonstrated that the overexpression of α-1 adrenoceptor (prostate enlargement), β-3 adrenoceptor and Rho-kinase (bladder) biomarkers indicated that prostate enlargement can cause bladder muscle atrophy. Further research could be undertaken using this prostate enlargement model for cancer research.

Keywords: Progressive prostate cancer model establishment; prostatic hyperplasia; animal study; bladder muscle atrophy

Submitted May 31, 2025. Accepted for publication Sep 07, 2025. Published online Dec 26, 2025.

doi: 10.21037/tau-2025-384

Highlight box

Key findings

• Prostate enlargement model may cause overactive bladder (β-3 adrenoceptor) and bladder muscle extension (Rho-kinase).

What is known and what is new?

• Clinically benign prostate hyperplasia may cause overactive bladder.

• Our results may offer data on overactive bladder (β-3 adrenoceptor) and bladder muscle extension (Rho-kinase) caused by prostate enlargement.

What is the implication, and what should change now?

• An aggressive prostate cancer mouse model needs to be explored.

Introduction

In prostate cancer patients, some simultaneously have prostate enlargement, and hypertrophic glandular proliferation in the transitional area near the urethra compresses the urethra to cause a variety of urinary symptoms, such as difficulty in passing urine and weak urinary output, residual urine, and frequent urination (1). An analysis of 185 consecutive patients with benign prostate hyperplasia (BPH) has reported that approximately 20% had concomitant detrusor dysfunction (2), suggesting that BPH and bladder dysfunction are related and may be associated with dysuria.

In fact, early bladder changes in hypertrophic phases, such as hypertrophy and hyperplasia of smooth muscle, are followed by compensatory and non-compensatory phases (3).

Recently, for the basic story for urinary tract epithelial or smooth muscle change, the β-3 adrenoceptor and the Rho-kinase pathway are attracting attention as new markers for bladder smooth muscle extension, a possible cause of bladder dysfunction and/or neurogenic bladder (3,4). The β-3 adrenoceptor is a receptor that is abundant in white and brown adipose tissue (5). In humans, it is present in bladder smooth muscle and is involved in smooth muscle relaxation of the bladder (6). Rho is a low molecular weight GTPase known to control the formation of stress fibers and focal adhesions and smooth muscle contraction downstream of extracellular signals such as lysophosphatidic acid, mainly via the actin cytoskeleton (7-9). Regulation of phosphorylation levels of myosin light chain (MLC) by Rho-kinase, an effector molecule of Rho, and a myosin-binding subunit is central to stress fiber formation and smooth muscle contraction control by Rho-kinase (10). Rho-kinase also plays an important role in maintaining bladder compliance, as there are reports of a link between β-3 adrenergic receptor activity and inhibition of the Rho-kinase pathway (7), and β-3 adrenergic receptors are increased by bladder hyperactivity. Thus, these proteins play a very important role in confirming bladder activity. Therefore, it is possible that β-3 adrenoceptor and Rho-kinase are associated with prostate enlargement and bladder changes in patients who are castrated and have dysuria. Thus, for establishment of mice prostate cancer model, in order to examine the relationship between prostate enlargement and bladder changes, we performed a study in a mouse prostate enlargement model focusing on how prostate enlargement causes bladder changes by the expression of β-3 adrenoceptor and Rho-kinase biomarkers.

However, the Rho-kinase pathway is also known to be activated in vasculitis. We decided to use transforming growth factor-beta (TGF-β) and α-1 adrenoceptor to rule out the possibility of vasculitis. TGF-β is a multifunctional cytokine released at wound healing sites and is involved in proliferation, differentiation, and stromal chemotaxis (11). It is also a cytokine released during inflammation and has an inhibitory effect on inflammation (12). They are because cancer especially progressive status, can cause tissue inflammation and cytokine release owing to local invasion into other or surrounding tissues or organs. α-1 adrenoreceptors are present in the prostate, urethra, bladder (urothelium, smooth muscle, and afferent nerves), ureters, vas deferens, peripheral ganglia, nerve endings, vascular tissue, and the central nervous system and stimulate sympathetic tone in prostate smooth muscle in BPH patients, causing compression of the prostate against the urethra (13,14). If the Rho-kinase pathway can be used as an independent marker of prostatic enlargement, it could be a new indicator of prostatic enlargement for understanding prostate cancer with prostate enlargement.

This study aimed to see which markers and what kinds of changes will happen in the mouse prostate enlargement model for the future establishment of a mouse prostate cancer model and to see, as a future study, the urinary condition evaluation in prostate cancer. We present this article in accordance with the ARRIVE reporting checklist (available at https://tau.amegroups.com/article/view/10.21037/tau-2025-384/rc).

Methods

Animal study

Ten 7-week-old C57BL/6 and Balb/c nu-nu mice (Charles River Laboratories, Wilmington, Massachusetts, USA), aged 12 weeks, were divided into 3 groups: 4 mice in a control group without castration, and 6 mice castrated and separated into an experimental group and a castrated group under the appropriate housing and husbandry conditions under the institutional approval. The experimental group was injected with 50 µL (12.5 mg) testosterone propionate subcutaneously once daily for 4 weeks while the others (control group and castrated + testosterone group) were injected with 50 µL sesame oil subcutaneously once daily for 4 weeks. The bladder and prostate were removed after euthanasia, after 4 weeks of administration, and the prostates were weighed. The measured weight was used to calculate the prostate index using the following formula: (prostate index) = [prostate wight of mice (g)]/[body weight of mice (g)] × 1,000 (15). Immunostaining was performed using anti-α-1 adrenoceptor antibody (1:2,000; GeneTex, Irvine, USA; #GTX100190) and anti-TGF-β antibody (1:250; Abcam, Cambridge, UK; #ab215715) for the prostate and anti-β-3 adrenoceptor antibody (1:200; Abcam; #ab94506) and anti-Rho-kinase antibody (1:20,000; Abcam; #ab40673) for the bladder.

All procedures in the animal study were approved by the institutional ethics and animal welfare committee of Kobe University (approval No. P200712), in compliance with institutional guidelines for the care and use of animals. A protocol was prepared before the study without registration.

Immunohistochemical (IHC) analysis

IHC scoring was based on the percentage of positive cells. The staining intensity was scored as 0 (negative), 1+ (weak), 2+ (medium) or 3+ (strong). The percentage of stained cells was categorized as: 1, 0–10%; 2, 11–50%; and 3, more than 50% stained cells. The total IHC score was determined by multiplying the frequency and intensity scores10 (16).

Statistical analysis

Comparisons between two different groups were performed using Student’s t-test. Comparisons between multiple groups were performed using one-way analysis of variance (ANOVA) followed by the Tukey-Kramer method. Statistical differences among means were considered significant when P<0.05.

Results

Animal model

All mice in each group were evaluated. The pre- and post-treatment body weights and Prostate index of the mice are summarized in Table 1. There was no difference in body weight among individual mice. At 4 weeks of administration of vehicle or testosterone propionate, the mean prostate weight was 75±27 mg in the control group, 101.9±1.7 mg in the castrated group, and 130±19 mg in the castrated + testosterone group. When a significant difference test was performed, there was no significant difference between the castrated group and the control group (P=0.10), but the difference between the castrated + testosterone group and the control group was significant (P<0.05). There were no dead mice during the experiments and the period.

Table 1

Body weight and prostate index of mice

Groups Body weight (g) Prostate index
Before treatment After treatment
Control group 37.8 42.3 1.788
Castrated group 38 41 2.487
Castrated + testosteron group 38.2 43 3.056

IHC staining and score analyses

The IHC score of the α-1 adrenoceptor antibody in the control group (prostate ventral lobe) was 0, the TGF-β antibody (prostate ventral lobe) was 0.5, the β-3 adrenoceptor antibody (bladder mucosa) was 0, and the Rho-kinase antibody (bladder mucosa) was also 0. The scores of the castrated group were 1.66, 0, 0.66, and 1.66, respectively. The castrated + testosterone group scored 1.25, 1, 1, and 2, respectively. The α-1 adrenoceptor expressions (P=0.12) and TGF-β antibody (P=0.07) in the prostate ventral lobe, and the β-3 adrenoceptor expressions (P=0.17) and Rho-kinase expressions (P=0.06) in the bladder mucosa did not show any significant difference between the control group and the castrated group. The TGF-β expressions in the prostate ventral lobe did not show a significant difference between the control group and the castrated + testosterone group (P=0.44), while the α-1 adrenoceptor expressions in the prostate ventral lobe (P=0.02), β-3 adrenoceptor expressions in the bladder mucosa (P=0.0003), and Rho-kinase expressions in the bladder mucosa (P=0.0001) were significantly different (Table 2, shown in Figures 1-4). Taken together, the α-1 adrenoceptor, TGF-β, β-3 adrenoceptor and Rho-kinase expressions did not show a significant difference between the control group and the castrated group. Only the TGF-β expressions in the prostate ventral lobe showed no significant difference between the control group and the castrated + testosterone group, and the α-1 adrenoceptor in the prostate ventral lobe, β-3 adrenoceptor and Rho-kinase antibody in the bladder mucosa all showed a significant difference (Table 2).

Table 2

IHC scores in mouse groups

Groups α-1 adrenoreceptor TGF-β β-3 adrenoreceptor Rho-kinase
Scores P value Scores P value Scores P value Scores P value
Control group 0±0 0.5±0.70 0±0 0±0
Castrated group 1.66±1.15 0.12 0±0 0.07 0.66 0.17 1.66±1.15 0.06
Castrated + testosteron group 1.25±0.433 0.02 1±0.81 0.44 1±1.41 0.0003 2±0 0.0001

Data are presented as mean ± standard deviation. IHC, immunohistochemical; TGF-β, transforming growth factor-beta.

Figure 1 Immunohistochemical staining of α-1 adrenoceptor in the group of a control group (A), sham group (castrated mice without testosterone administration) (B) and testosterone-treated group (castrated mice with testosterone administration) (C). Brown collar parts shows positive staining.
Figure 2 Immunohistochemical staining of TGF-β in the group of a control group (A), sham group (B) and testosterone-treated group (C). Brown collar parts shows positive staining. TGF-β, transforming growth factor-beta.
Figure 3 Immunohistochemical staining of β-3 adrenoceptor in the group of a control group (A), sham group (B) and testosterone-treated group (C). Brown collar parts shows positive staining.
Figure 4 Immunohistochemical staining of Rho-kinase in the group of a control group (A), sham group (B) and testosterone-treated group (C). Brown collar parts shows positive staining.

Discussion

Prostate cancer patients often have prostate enlargement and some suffer prostate enlargement-related urinary disturbance, such as difficulty urinating. In our study, we explored what biomarkers are altered in the tissue in such cases for evaluating how the urinary condition, including bladder, is changed in the prostate enlargement status, such as progressive prostate cancer.

As a standard for further evaluation, an index that can directly judge bladder compliance is required. We used Rho-kinase as a biomarker for this purpose. Since Rho-kinase responds to the extension of bladder smooth muscle and acts to maintain bladder compliance (9,17-20), it may be possible to evaluate bladder compliance from an early stage by observing the strength of expression of this pathway.

In our experimental model, anti-α-1 adrenoreceptor antibody was more expressed in the castrated + testosterone group in the prostate ventral lobe. These results indicate that a model of prostate enlargement was successfully created. Also, anti-β-3 adrenoceptor antibody expression in the bladder mucosa was significantly more in the castrated + testosterone group. Accumulation of urine in the bladder stimulates β-3 adrenoceptors, relaxes bladder smooth muscle and increases bladder capacity (21). Expression of β-3 adrenoceptor antibody is thought to reflect this pathway. This pathway has been clinically applied and β-3 adrenoceptor agonists have been adopted as therapeutic agents for overactive bladder, which could be more happened in the progressive prostate cancer status as well (22). In addition, the expression level of anti-TGF-β antibody, which is an inflammation mediator, was low, suggesting that no other factors, such as inflammation, affected our results and prostate enlargement does not necessarily relate to or involve inflammation, which could be mediated in progressive cancer status. The Rho pathway is known to occur not only in bladder wall extension but also in peri-cystic vasculitis (23). We showed that the Rho pathway was activated in this model without the effects of vasculitis.

It is noteworthy that the anti-Rho-kinase antibody was strongly expressed in the castrated + testosterone group versus the control group. This is because the Rho-kinase pathway might respond to the extension of bladder smooth muscle more sensitively than expected. We discovered that there is a strong expression of Rho kinase antibody in the prostate enlargement group at a very early stage (4 weeks). Further evaluation with a longer period of treatment is demanded.

This is the study to confirm, by bladder smooth muscle staining, the hypothesis that prostate enlargement, assuming progressive prostate cancer, may cause bladder dysfunction. This animal study showed that prostate enlargement leads to overactive bladder (β-3 adrenoceptor) and bladder muscle extension (Rho-kinase) in an IHC study. Thus, we have shown that Rho-kinase may be a biomarker that can sensitively determine the possibility of bladder muscle dysfunction by collecting a small amount of bladder muscle tissue and evaluating its expression, assuming progressive prostate cancer status.

There are some limitations in this study. The animal prostate enlargement model was created by subcutaneous injection of testosterone propionate and caused glandular cell-dominant proliferation. This differs from human prostate enlargement and also progressive prostate cancer status. Next, we did not include real prostate cancer with prostate enlargement model because this is a kind of preliminary study. Next, since we did not examine bladder function in this case, we cannot say that urinary obstruction strictly affects bladder function. Therefore, a study is needed to rigorously examine the relationship. Next, since the purpose of this study was to examine the expression of the Rho-kinase pathway to evaluate the bladder or urinary tract system only by immunostaining, we examined adrenergic receptor antibodies and Rho-kinase antibodies. Therefore, protein expression needs to be confirmed in future experiments. Next, since the absence of TGF-β expression does not completely rule out an inflammatory process, other markers should also be examined. These limitations will be overcome in our future study.

Conclusions

As a first step study for progressive prostate cancer with prostate enlargement model establishment, our animal study of testosterone administration demonstrated that prostate enlargement also resulted in overactive bladder indicated by highly expressed β-3 adrenoceptor and bladder muscle atrophy indicated by highly expressed Rho-kinase. In summary, prostate enlargement influenced bladder function in our study. Future experiments will be conducted using real orthotopic prostate cancer bearing model with prostate enlargement to evaluate which kind of change occurs in urinary tract and bladder.

Acknowledgments

None.

Footnote

Reporting Checklist: The authors have completed the ARRIVE reporting checklist. Available at https://tau.amegroups.com/article/view/10.21037/tau-2025-384/rc

Data Sharing Statement: Available at https://tau.amegroups.com/article/view/10.21037/tau-2025-384/dss

Peer Review File: Available at https://tau.amegroups.com/article/view/10.21037/tau-2025-384/prf

Funding: This work was supported by JSPS Bilateral Collaborations 2025.

Conflicts of Interest: All authors have completed the ICMJE uniform disclosure form (available at https://tau.amegroups.com/article/view/10.21037/tau-2025-384/coif). The authors have no conflicts of interest to declare.

Ethical Statement: The authors are accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved. All procedures in the animal study were approved by the institutional ethics and animal welfare committee of Kobe University (approval No. P200712), in compliance with institutional guidelines for the care and use of animals.

Open Access Statement: This is an Open Access article distributed in accordance with the Creative Commons Attribution-NonCommercial-NoDerivs 4.0 International License (CC BY-NC-ND 4.0), which permits the non-commercial replication and distribution of the article with the strict proviso that no changes or edits are made and the original work is properly cited (including links to both the formal publication through the relevant DOI and the license). See: https://creativecommons.org/licenses/by-nc-nd/4.0/.

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Cite this article as: Yang YM, Kitagawa K, Hirata Y, Moriwaki M, Ono T, Chiang YT, Chen KC, Shigemura K. Immunohistochemical study of Rho-kinase pathway expression in a mouse model of prostate hypertrophy for preliminary study of progressive prostate cancer model establishment. Transl Androl Urol 2025;14(12):3897-3903. doi: 10.21037/tau-2025-384

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