The effects of ROS in prostatic stromal cells under hypoxic environment

Abstract

Objective: The objective of this study is to explore the effects of reactive oxygen species (ROS) under hypoxic environment in prostatic stromal cells (PSC).

Methods and materials: To detect the expression of ROS in PSC and the tissues of benign prostatic hyperplasia (BPH) by flow cytometry; under hypoxic conditions, to observe the changes of cells growth and ROS in PSC; quantitative PCR was used to detect hypoxia inducible factor-1α (HIF-1α), androgen receptors (AR), vascular endothelial growth factor (VEGF), and interleukin-8 (IL-8) in PSC; After edaravone intervening, to examine the changes of cells growth, ROS, HIF-1α, AR, VEGF, and IL-8 under hypoxic conditions.

Results: The expression of ROS in tissues and cells which under hypoxic condition was significantly increased. 3% O₂ promoted the proliferation. The HIF-1α, AR, VEGF, and IL-8 were upregulated under 3% O₂. After edaravone intervening, ROS significantly decreased, HIF-1α and VEGF were downregulated, and cell proliferation declined.

Conclusions: Hypoxia stimulates the generation of ROS, and the ROS may play a key role in BPH.

• AR
• BPH
• edaravone
• HIF-1α
• hypoxia
• IL-8
• ROS
• VEGF

Introduction

A study by Briganti et al. showed that 75% of men >50 year of age have symptoms arising from BPH [1], the proportion will continue to grow with age. To some extent, BPH is an inevitable disease for the ageing male population, and results serious public health problem. The etiologies of BPH are still controversial; however, aging is recognized as an absolutely necessary condition for BPH. So far, there are few papers that from aging standpoint research the etiology of BPH. The effects of ROS in aging have attracted more and more attention, ROS were also known to be critical for aging [2,3]. The researchers found that hypoxia [4] and ischemia [5] have an important role in the development of BPH. The local hypoxia and inflammation were the two major doctrines of BPH [6,7], and ROS were the pivotal factor in the two doctrines. Oxidative stress is well known to be critical for producing ROS, and inflammatory stimulation can generate a large number of ROS by the nicotinamide adenine dinucleotide phosphate (NADPH) pathway within leukocyte [8]. HIF-1α is a very important factor in oxygen homeostasis, it could facilitate the organism to adapt to hypoxia through activating a series of downstream genes, including VEGF [9]. It has been confirmed that ROS has a role in regulating HIF-1α [10,11]. Taken together, we speculated that ROS may have some important effects in the development of BPH. AR is an important gene that regulates the proliferation of prostatic cell [12]. IL-8 has a strong chemotactic and activating to neutrophils and might be in the hub location of chemokines [13]. Further research found that IL-8 promoted mitosis and angiogenesis [14]. These characteristics determine that the IL-8 may play an important role in prostatic inflammation and proliferation. Inflammation, proliferation, and vascular tissues cannot be separated, and VEGF is a central factor in regulating angiogenesis. Therefore, the important role of VEGF in prostate inflammation and proliferation cannot be ignored. Histological studies displayed that the stromal cells were the major component of BPH [15]. So we want to explore the expression levels of ROS in PSC in the hypoxic environment, understand whether ROS affects HIF-1α, AR, IL-8, and VEGF in PSC, and finally, to clarify the effects of ROS in BPH.

Materials and methods

Collection of human BPH specimens

BPH specimens were obtained from patients (67–86 year, average age 72.5 year) who underwent transurethral resection of prostate, ruled out canceration strictly by the pathologist. Control tissues were obtained from younger patients (37–46 year, average age 43.5 year) with bladder cancer (four cases) who underwent transurethral resection of bladder tumor, ruled out canceration and BPH strictly by the pathologist. BPH specimens were divided into two groups, treated (30 cases) or not treated with 5α-reductase inhibitors (30 cases) in the 6 months preceding surgery. There was no significant difference in the age and the size of the glands for all BPH specimens. All patients gave their informed consent.

Primary human PSC cultures

Primary human PSC were obtained from ScienCell Research Laboratories (Carlsbad, CA), and cultured according to the papers described [16,17]. In short, cells recovery at 37 °C, cultured in 21% O₂, 5% CO₂, cells began to emerge within 1 week and were used within the fifth to seventh passage.

Detection of ROS

Based on the literature [18], flow cytometry with fluorescent probe, carboxy-H₂-DCFDA (Sigma, St. Louis, MO), was used to detect the ROS of BPH specimens and cells. Briefly, specimens were cut into 1 mm³ pieces, grinded, and pour through a 300 μm mesh, eventually became cells suspension. Cells suspension and commercial stromal cells were incubated with carboxy-H₂-DCFDA for 30 min at 37 °C. Fluorescence was measured at emission wavelengths of 480 nm.

Flow cytometry analysis of cells cycle and apoptosis

After the different treating, cells were fixed with 75% ethanol at −20 °C for 24 h, propidium iodide (PI) was added to the cells, and the proliferation index was represented by the percent of S + G2 phase. The apoptosis rates were determined by Annexin V and PI double staining.

Detection of HIF-1α, AR, VEGF, and IL-8

Real-time fluorescent quantitative PCR (qRT-PCR) was performed using TaqMan® Universal PCR and standard TaqMan® RNA Reverse Transcription kit protocol of the Applied Biosystems 7900HT Sequence Detection System (ABI Prism®, Applied Biosystems, Foster City, CA) to detect the mRNAs levels of HIF-1α, AR, VEGF, and IL-8 in PSC under different interventions. All RNA samples were normalized based on the TaqMan® Gene Expression Assays for human or mouse glyceraldehyde-3-phosphate dehydrogenase (GAPDH) endogenous controls, Gene Expression Assay Mix as a fluorescent probe of GAPDH, HIF-1α, AR, VEGF, and IL-8 were purchased from ABI Inc. (Foster City, CA). Following the relative quantitative 2^−ΔΔCt method, all reactions were run according to the instructions of the manufacturer.

Cell cultures under hypoxic conditions and treatments

We established a hypoxia workstation by using tri-gas cell culture incubators and anaerobic incubator (Science and Technology Co, Ltd., Mafulu, Tianjin, China), because the glove ports of anaerobic incubator could ensure that cultures and treatments were exposed minimum to ambient conditions. Based on our pre-experiment and the literatures [19,20], 3% and 1% oxygen concentrations were selected. Edaravone (Melone Pharmaceutical Co, Ltd., Dalian, China) is a commonly used oxygen-free radical scavenger, study showed that it had the anti-inflammatory effects [21] and could prevent the injury by ischemia–reperfusion [22]. It can be used to treat the hypoxic cells, according to the paper by Chen et al. [23] and our pre-experiment. 3% O₂ was the most suitable experimental condition, and the final concentrations of edaravone were 50 μM/L and 80 μΜ/L, added to the PSC in 3% O₂ for 48 h.

RNA interference (RNAi) of HIF-1α

The commercially available plasmids of siRNA-HIF-1α were purchased from Santa Cruz Biotechnology Inc. (Dallas, TX) (PN: sc-35562). Transfection of cells was performed using siRNA transfection reagent (Santa cruz Biotechnology Inc., Dallas, TX, PN: sc-29528), all reactions were run according to the instructions of the manufacturer. Based on the paper by Hänze et al. [24], hypoxic incubation started after a 4-h normoxic period to allow degradation of preformed HIF-1α.

Statistical analyses

All values were expressed as the mean ± SD. All the assays were conducted at least three times in triplicate. The statistical significance of the expression level of HIF-1α, AR, VEGF, IL-8, and ROS in different oxygen concentrations and treatments was determined by the paired-samples t test. Genetic expression among tissues, control, and cells were determined by the Kruskal–Wallis H test. p Value of <0.05 was considered statistically significant. All data were analyzed using SPSS software ver. 17.0 (SPSS Inc., Chicago, IL).

Results

Immunohistochemistry and morphological observation showed that the main components of PSC, after 5–7 generations passaged, were smooth muscle and fibroblasts cells, and compliance with the histological features of human PSC [15]. The ROS, HIF-1α, VEGF, and IL-8 of prostatic tissues were significantly increased, but these, in control and PSC, were significantly decreased under 21% O₂ (Table 1). The expression of ROS between the specimens treated and not treated with 5α-reductase inhibitors showed no statistical difference (Figure 1).

Figure 1. The expression of ROS in different treatments. 5a = treated with 5a-reductase inhibitors, No 5a = not treated with 5a-reductase inhibitors, 3% O2 + E = 3% O2 + Edaravone (80μM).*p < 0.05, #p > 0.05, by the paired-samples T test.

Table 1. The expression of genes in different specimens.

Groups	HIF-1α	AR	VEGF	IL-8	ROS
BPH specimens	2.926 ± 0.379	2.918 ± 0.264	3.035 ± 0.366	1.970 ± 1.010	82.727 ± 15.923
Controls	0.612 ± 0.568	1.236 ± 0.363	1.094 ± 0.311	1.201 ± 0.115	40.947 ± 10.079
Cell in 21% O2	0.121 ± 0.068	1.027 ± 0.158	0.976 ± 0.128	1.001 ± 0.118	31.680 ± 11.079
χ^2	62.651	61.653	52.281	15.627	59.480
p	<0.01	<0.01	<0.01	<0.01	<0.01

Kruskal–Wallis H Test.

The ROS were significantly increased in PSC in 3% O₂ and 1% O₂, but ROS in 3% O₂ were significantly less than 1% O, as shown in Figure 1. The proliferation index in 3% O₂ was better than in 21% O₂ (Table 2). The cells’ apoptosis in 1% O₂ was more than in 3% O₂ (Table 3). HIF-1α, AR, VEGF, and IL-8 were upregulated in 3% O₂ (Figure 2).

Figure 2. The changes of genes in different treatments: *p < 0.05, #p > 0.05, by the paired-samples T test.

Table 2. The proliferation index in different treatments.

Groups	21% O2	3% O2	3% O2 + edaravone	3% O2	siRNA-HIF-1α	3% O2	1% O2	3% O2
G2 + S(%)	15.193 ± 1.446	21.267 ± 2.866	18.362 ± 1.681	21.267 ± 2.866	19.022 ± 1.405	21.267 ± 2.866	3.481 ± 0.789	21.267 ± 2.866
t	7.809		3.523		2.767		24.695
p	<0.01		<0.01		<0.05		<0.01

The proliferation index was not significant between the 3% O₂+ Edaravone and siRNA-HIF-1α, by the paired-samples t test.

Table 3. The apoptosis in different treatments.

Groups	3% O2	1% O2	3% O2	3% O2 + edaravone	siRNA-HIF-1α	3% O2
apoptosis(%)	2.685 ± 0.351	27.919 ± 4.249	2.685 ± 0.351	12.657 ± 1.400	11.819 ± 0.763	2.685 ± 0.351
t	−23.458		24.783		53.127
p	<0.01		<0.01		<0.01

The apoptosis was not significant between the 3% O₂ + Edaravone and siRNA-HIF-1α, by the paired-samples t test.

After the intervention of edaravone (80 μΜ/L), ROS were significantly reduced in 3% O₂ (Figure 1), but the 50 μM/L has no effect. The expressions of HIF-1α and VEGF were significantly lower than those without interference, the AR and the IL-8 were decreased, but no significance (Figure 1), and apoptosis was increased (Table 3).

After the intervention of siRNA-HIF-1α, HIF-1α were significantly reduced, there was no significant change in the expression of ROS, AR, and IL-8 (Table 4). The proliferation index was decreased (Table 3).

Table 4. The expression of genes in different treatments.

Groups	HIF-1α	AR	VEGF	IL-8	ROS
3% O2	2.828 ± 0.318	2.335 ± 0.443	2.688 ± 0.402	1.390 ± 0.545	84.094 ± 10.994
siRNA-HIF-1α	0.826 ± 0.285	2.167 ± 0.398	2.537 ± 0.377	1.271 ± 0.433	82.120 ± 12.168
t	30.452	2.001	2.499	1.860	2.040
p	<0.01	>0.05	<0.05	>0.05	>0.05

By the paired-samples t test.

Discussion

There are many doctrines about the etiologies of BPH, but only these two doctrines were widely accepted: aging and functional testes. Anti-androgen therapy is the current focus of BPH treatment, but two types of 5α-reductase inhibitor therapy do not completely prevent the clinical progression of BPH [25], and conversely, some studies have shown a high prevalence of low testosterone in men with BPH [26,27]. Therefore, androgen disorder does not fully explain the nature of BPH. In our study, there were no significant changes in the ROS between the tissues treated or not with 5a-reductase inhibitors. Research also found that there was no difference in the expressions of inflammatory cytokines between the tissues treated and not treated with 5α-reductase inhibitors [16]. BPH is a recognized age-related disease, and it plays a key role in the aging system. Aging leads to the changes in the prostatic microenvironment and induced the second development of prostate (the first development in adolescence). Studies suggested that ROS was critical for aging [3]. Human tissues are normally exposed to a range of low oxygen concentrations (3–6% O₂), with no apparent toxicity for cells [28]. Vascular aging is one of the basic mechanisms of aging [29], leading to progressive increase of local ischemia and hypoxia. Ultimately, harmful ischemia and hypoxia are inevitable in local organizations. Studies have confirmed that temperate hypoxia can stimulate proliferation [30]. So we speculated that hypoxia may be one of the initial causes of BPH, and changes in hypoxia and ischemia may be the most important factors. To verify this hypothesis, we exposed PSC in hypoxic environment, observed cells proliferation, apoptosis, and the changes in the related genes and ROS. Hypoxia can produce large amounts of ROS by NADPH and xanthine oxidase in mitochondrial [31]. In this study, we found that ROS, HIF-1α, AR, VEGF, and IL-8 were significantly increased in tissues of BPH, but in cultured cells (21% O₂) and no-BPH tissues, these were significantly decreased. These data could indicate that the expression of genes and ROS in normoxic and hypoxic conditions was not the same. Studies found that the cells of pulmonary artery endothelial exposed to hypoxia and ROS increased 2.5-fold after 1 h [32], cells begin to proliferate, and present antioxidant enzymes and DNA repair factors under moderated ROS stimulation [33]. We also found that hypoxia and ROS could stimulate proliferation. Using edaravone to scavenge ROS and siRNA-HIF-1α to knockdown HIF-1α, cells apoptosis was promoted. The different tissue-derived cells’ response to the concentrations of ROS is inconsistent, but the common point is that it stimulates cell growth in low concentrations, high concentrations lead to apoptosis or death [30]. Our observation was in agreement with this point. ROS itself or further activate other proliferation-, apoptosis-related genes that may lead to proliferation or apoptosis. These involve in extremely broad field which includes the concentration of ROS, cell types, signal transduction pathways, specific enzyme activation, and expression of DNA in different cells. ROS and HIF-1α are all closely related to oxidative stress. We found that ROS decreasing accompanied by the fall of HIF-1α, but in turn, was not the same. ROS may be more fundamental in the development of BPH. Our study showed that HIF-1α, AR, IL-8, and VEGF genes’ expression was significantly increased in PSC under hypoxic environment, when the cells were in the proliferative phase. However, after interference of edaravone, ROS obviously decreased, the HIF-1α and VEGF were significantly downregulated, and cell proliferation was decreased. We did not perform the research of protein levels, but these data still illustrate that temperate hypoxia could affect prostatic proliferation through ROS and related genes. The relationship among ROS, HIF-1α, AR, IL-8, and VEGF yet needs further study, especially, the mutual influence of protein levels. Local inflammatory reaction in the role of BPH has drown researchers’ more and more concern and attention, inflammation can produce large amounts of ROS through the NADPH pathway in leukocyte [8]. The ROS generated by hypoxia and inflammation may play a key role in BPH. This study found that edaravone could decrease ROS, HIF-1α, and VEGF in PSC, and finally promote apoptosis. Thus, to improve oxygen supply and reduce the content of ROS in prostate may be a new direction for the BPH treatment, and the findings need a further study to confirm.

Declaration of interest

The author declare that they have no conflicts of interest. This work was supported by a grant from the Nature Science Foundation of Qinghai Province (Grant no. 2012-Z-939Q).