Expression of Lysophosphatidic Acid Receptor 1 and Relation with Cell Proliferation, Apoptosis, and Angiogenesis on Preneoplastic Changes Induced by Cadmium Chloride in the Rat Ventral Prostate

Background Lysophosphatidic acid (LPA) is a phospholipid growth factor involved in cell proliferation, differentiation, migration, inflammation, angiogenesis, wound healing, cancer invasion, and survival. This study was directed to evaluate the immunoexpression of LPA-1, cell proliferation, apoptosis, and angiogenesis markers in preneoplastic lesions induced with cadmium chloride in rat prostate. Methods The following parameters were calculated in ventral prostate of normal rats and rats that received Cd in drinking water during 24 months: percentages of cells immunoreactive to LPA-1 (LILPA1), PCNA (LIPCNA), MCM7 (LIMCM7), ubiquitin (LIUBI), apoptotic cells (LIAPO), and p53 (LIp53); volume fraction of Bcl-2 (VFBcl-2); and length of microvessels per unit of volume (LVMV/mm3). Data were analyzed using Student's t-test and Pearson correlation test. Results The LILPA1 in dysplastic lesions and normal epithelium of Cd-treated rats was significantly higher than those in the control group. Markers of proliferation were significantly increased in dysplastic lesions, whereas some apoptotic markers were significantly decreased. No significant differences between groups were found in VFBcl-2. Dysplastic lesions showed a significant increase of LIp53. The length of microvessels per unit of volume was elevated in dysplastic acini. Statistically significant correlations were found only between LILPA1 and LIUBI. Conclusions Our results suggest that LPA-1 might be implicated in dysplastic lesions induced by cadmium chloride development. More studies are needed to confirm its potential contribution to the disease.


Introduction
Prostate cancer is one of the most frequently occurring cancers in male patients. The etiology of the disease is complex, and it has a multitude of potential contributing factors [1,2]. It is thought that prostatic intraepithelial neoplasia (PIN) is the histologic abnormality most commonly associated with prostate cancer and is proposed as a precursor of invasive prostate cancer. A number of studies concluded that high-grade PIN represents the most likely precursor to invasive adenocarcinoma [3][4][5][6][7][8].
Lysophosphatidic acid (LPA) is a phospholipid growth factor involved in a variety of physiological and pathological process such as cellular proliferation, differentiation, migration, inflammation, angiogenesis, wound healing, cancer invasion, and survival [9][10][11]. Initially, LPA was thought to be secreted primarily by platelets during wound healing [11]; however, recently, it was believed, that LPA may be produced by many cell types such as fibroblasts and adipocytes, and it can be expressed in different tissues, including the brain, ovary, and kidney [9,11]. Also, it is known that LPA-1 is detected in many tumors, such as those in the lung, breast, stomach, kidney, and prostate [12]. It is well established that LPA signals various events through its G protein-coupled receptors (GPCRs), namely, LPA-1 to LPA-6 [9][10][11][12][13][14].
LPA-1, LPA-2, and LPA-3 share about 50-57% amino acid sequence identities and form the Edg (Endothelial differentiation gene) family together with the GPCRs for sphingosine 1phosphate. The switching expression of LPA-1 receptors is found to be associated with prostate cancer development [1,13,15]. LPA-4, LPA-5, and LPA-6 were classified as 'non-Edg family' LPA receptors and provide a new framework for understanding different LPA functions [13]. In normal tissues, LPA-1 is broadly expressed, whereas expression of LPA-2 and LPA-3 is more restricted [16]. The physiological role of LPA-4 has not been investigated [17] and LPA-5 was found to be highly expressed on cells associated with the immune system [18], and LPA-6 is a novel receptor implicated in human hair growth [13,14].
In the case of prostate cancer, LPA is reported to induce proliferation and survival of androgen-independent prostate cancer cells [9]. Prostate cancer is the second leading cause of male deaths in the majority of Western countries [11,19]. Information regarding the expression profile of LPA-1 in human biopsies is limited [11].
The rates of proliferation and apoptosis should be determined to have a better knowledge of the dynamism of the cell population in normal and pathological conditions and to establish the relationship with LPA-1 expression.
On the other hand, angiogenesis is a critical feature of many diseases, including cancers and their precursors [20,21]. Angiogenesis is defined as the process leading to the formation of new blood vessels and is essential for normal growth and development [22]. Recently, LPA was demonstrated to promote ovarian cancer growth by inducing angiogenic factors [10], but this relation in prostate dysplastic lesions needs further investigation. A useful experimental model for human prostate cancer is the rat [23][24][25], and morphological similarities between human PIN and dysplastic changes experimentally promoted in rodent prostate have been reported [26]. Our group designed an experimental model based on the administration of low doses of cadmium chloride [6][7][8]27]. This model induces higher incidence of prostate carcinogenesis in Sprague-Dawley rats in a manner similar to those in humans.
In the present study, we estimated immunoexpression and quantification of LPA-1 in epithelial cells. Cell proliferation was determined by the quantification of proliferative cell nuclear antigen (PCNA) and the miniature chromosome maintenance (MCM7). The apoptosis was quantified using Bcl-2, ubiquitin, and p53 and by measuring the ratio of apoptotic nuclei to the total nuclei of epithelial cells using the TUNEL assay. The angiogenesis was observed by quantification of Von Willebrand factor (Factor VIII).
The aims of this study are as follows: a) to determine the LPA-1 immunoexpression in preneoplastic lesions induced with cadmium chloride; b) the evaluation of cell proliferation, apoptosis, and angiogenesis markers in these lesions; and c) to determine the correlation between LPA-1 immunoexpression and cell proliferation, apoptosis, and angiogenesis markers studied in b).

Ethics statement
This experiment and animal care were conducted in compliance with the guidelines established by the 'Guide for the Care and Use of Laboratory Animals' CEU-San Pablo University, Madrid, Spain. All rats were housed, five per cage, under controlled temperatures in a 12-h light/dark cycle with easy access to food Panlab Lab Chow (Panlab, Barcelona, Spain) and water. All the animals were euthanized using CO 2 narcosis 24 months after the beginning of the experiment, and all efforts were made to minimize suffering. All in vivo experiments were previously published in Prostate 63: 347-357 [6], J Histochem Cytochem 54: 981-90 [8], and Hormonal Carcinogenesis IV, Springer pp. 522-528 [7].
Care and handling of the animals and the storage of the samples All animals used had an attending veterinarian available, and health status was monitored at least once daily. Clinical specific signs or objective measurements of organ dysfunction were monitored.
All animal rooms were checked daily, including room conditions, animals with health problems, food and water levels, and proper cage conditions. Animal rooms were kept clean, quiet, and uncluttered. Lighting conditions were adequate for the animals to behave normally and for the animal caregivers to perform their duties. The light cycle was 12-h light/12-h dark, as we previously mentioned. Ventilation was adequate to provide oxygen and remove chemical, biological, and heat waste. Room temperatures should be maintained in a range suitable for the animal species, and the animals should be protected from abrupt changes. Noise in animal rooms was minimized whenever possible. Room surfaces were constructed of material that is easily sanitized; floors, counters, and sinks were cleaned daily, and other room surfaces, including cage racks, were sanitized monthly.
Checking was done every day, including weekends and holidays, and ventilation ducts and filters were cleaned at least monthly.

Treatment groups and experimental design
Ten adult male Sprague-Dawley rats, 30 days old at the beginning of the study, were used. Sprague-Dawley rats were randomly assigned into two groups, according to treatment (5 rats per group). Cadmium chloride (Panreac, Madrid, Spain) was added to the drinking water of the first group at a concentration of 60 ppm, during the time course of the experiment (24 months). The second group was used as control and received drinking water that was shown to be free of this metal.
After sacrifice, the organic remains from the animals and the residua of drinking water were adequately processed according to the guidelines in relation to the safety in the use of heavy metals established by the communitarian normative of the European Union.

Tissue preparation
The prostate complex was dissected from the abdominal cavity of each animal. Dorsolateral prostate lobes were routinely examined in all groups being studied, and no dysplastic changes were detected. Then, only the ventral lobe was used in the study.
Afterward, the ventral prostate was cut exhaustively into 2-mmwidth slices. The section plane was perpendicular to the sagittal axis of the gland. All specimens were fixed by immersion in 4% paraformaldehyde in phosphate-buffered saline (PBS) pH 7.4, for 24 h. Thereafter, the slices were embedded in paraffin, and the paraffin blocks were then serially sectioned at 5 mm-thickness and stained with hematoxylin-eosin or used for immunohistochemical techniques.

Immunohistochemical methods
Deparaffinized and rehydrated tissue sections were treated for 30 min with hydrogen peroxide 0.3% in phosphate-buffered saline (PBS), pH 7.4, to block endogenous peroxidase. Antigen unmasking was performed with pepsin (15 min, Sigma, St Louis, USA). To minimize nonspecific binding, sections were incubated with serum blocking solution (HistostainH Bulk Kit, Invitrogen Corporation, Carlsbad, California) and subsequently incubated overnight with primary antibodies in a moist chamber at 4uC. Information on primary antibodies is provided in Table 1.
On the second day, immunohistochemistry was performed with standard procedures using HistostainH Bulk Kit (Invitrogen Corporation, Carlsbad, California). The immunostaining reaction product was developed using 0.1 g diaminobenzidine (DAB) (

DNA fragmentation detection
To detect the apoptotic fragmentation of DNA, a TUNEL (TdT-mediated dUTP-biotin nick end labeling) technique (Boehringer Mannheim) was used [6,28]. This method involves the insertion of labeled nucleotides into broken ends of DNA strands. A brief description of the method follows: sections were deparaffinized and rehydrated and then incubated with proteinase K (10 ml/ml in TRIS/EDTA pH 8) for 30 min at 37uC for digestion of nuclear proteins. Endogenous peroxidase was inactivated with 0.3% hydrogen peroxide in distillate water during 30 min. The sections were incubated for 1 hr at 37uC in the TdT (terminal deoxynucleotide transferase) mixture with addition of labeled nucleotides (2:1), and after that, they were incubated in converter POD during 30 min at 37uC. The reaction was detected using 0.1 g DAB in PBS (200 ml), plus 40 ml hydrogen peroxide. After the TUNEL technique, sections were counterstained with acetic carmine. All slides were dehydrated in ethanol and mounted in Depex (Serva, Heidelberg, Germany).
Measurements were performed using a NIKON Eclipse E400 microscope, with a NIKON Digital Camera DXM1200; ten digital images were acquired for each slide. For counting the number of cells, freeware ImageJ v1.45 was used. Software was downloaded from NIH web site (http://rsb.info.nih.gov/ij).
In all cases (LPA-1, PCNA, MCM7, ubiquitin, and p53), nuclei were considered positive regardless of staining intensity. Apoptotic nuclei were considered positive when the stain was uniform and intense.

Quantitative evaluation of Bcl-2 immunostaining
To quantify the immunoreactivity of Bcl-2 protein, its volume fraction (V F ) was measured and expressed as percentage of immunostained epithelium. Estimation of the V F was performed using the ImageJ v1.45 program. The selected fields were photographed, thresholded, and binarized, and the percentage of epithelial area was automatically measured by the program to obtain the V F [7]. The V F of Bcl-2 immunoreactivity was estimated in ventral prostates from the control and experimental animals to ascertain if treatment changes the total amount of Bcl-2 immunostaining in the epithelium. This estimation was measured in each selected section from control rats, nondysplastic acini of Cd-treated rats, and dysplastic acini of Cd-treated rats.

Quantitative evaluation of length of microvessels (L V MV)
The length of microvessels per unit of volume (L V MV/mm 3 ) of prostate tissue was evaluated in normal acini of control rats, nondysplastic acini of Cd-treated rats, and dysplastic acini of Cdtreated rats. The stromal compartment was considered as the reference space. The Factor VIII vascular profiles immunostained to be eligible for counting were those sampled by the dissector frame and fulfilling the Sterio rule [30]. The L V MV was a calculated by the formula: L V = (26_Q-)/_A, where Q-= number of immunopositive vascular profiles and _A = total area sampled, that is, area of dissector frame (1482 mm2) multiplied by the number of selected frames [31]. These measurements were performed using the CAST-GRID program (Stereology Software Package, Silkeborg, Denmark).

Statistical analysis
Statistical evaluation was performed using GraphPad Prism5 (La Jolla, USA). Data were presented as means 6 standard deviations (SDs). The differences between groups were evaluated using Student's t test for parametric data and Mann-Whitney U test for nonparametric data. The correlation study was performed using the Pearson correlation test.

Results
Dysplastic changes in the acinar epithelium of the ventral prostate were detected to the end of the experiment in all rats treated by cadmium (5 rats).

Qualitative results
The expression of LPA-1 was detectable in the cytoplasm and nucleus (Figs. 1A, 1B, and 1C). Some interstitial cells also stained slightly, but the intensity was qualitatively weaker than that in epithelial cells.  (Figs. 1F, 1G) was detected in epithelial cells from the prostate acini throughout all the groups studied. PCNA immunostaining was also remarkable in the acini with dysplastic changes (Fig. 1D), as well as MCM7 (Fig. 1F).
Ubiquitin expression was observable in the cytoplasm and in the nucleus of the epithelial cells, mainly in the peripheral acini (Figs. 1H, 1I). Dysplastic acini showed nucleus immunostaining for ubiquitin, but no cytoplasm staining was detected (Fig. 1I).
Immunoreactivity to Bcl-2 protein was observed in control and Cd-treated rats. It was granular and expressed in the apical border of the epithelium in the control group and nondysplastic acini. In dysplastic glands, Bcl-2 immunostaining was more widely extended throughout all the cytoplasm, although it was more prominent near the lumen of the cribriform structures (Fig. 1J, 1K).
Apoptotic nuclei were observed in epithelial cells from the control group and treated animals (Figs. 1L). Scarce apoptotic nuclei were also visualized in dysplastic lesions (Fig. 1M).
Immunoreactivity to p53 was detected in cytoplasm and nuclear epithelial cells from the prostate acini throughout all the groups studied. Nuclear p53 immunostaining was also remarkable in the acini with dysplastic changes (Fig. 1N, 1O).
Microvessels immunostained to Factor VIII were observed in both control and Cd-treated rat. More capillaries were apparently seen in both dysplastic acini (Fig. 1P, 1Q).

Quantitative results
A significant increase for LI LPA1 (p,0.01) in the normal acini and dysplastic acini of Cd-treated rats was detected in comparison to the control acini (Fig. 2). The LI PCNA was significantly increased in dysplastic lesions (p,0.05), whereas the control acini and cadmium nondysplastic acini did not show significant variations (Fig. 3A). LI MCM7 was significantly increased in the animals exposed to cadmium (normal acini and dysplastic acini) in comparison to control animals (Fig. 3B).
There was no significant difference in the V F Bcl-2 between the control and cadmium-treated animals (Fig. 4A). A significant decrease of LI APO was observed in dysplastic acini of rats treated with cadmium compared with the control (p,0.01) or Cd-acini (p,0.001), but any difference between the control and normalacini of Cd-treated group was not detected (Fig. 4B). For LI p53 , no significant differences between the normal epithelium of the control and Cd-treated rats were observed; however LI p53 of dysplastic acini of rats treated with cadmium was significantly increased (p,0.05) compared with cadmium normal acini (Fig. 4C). For LI UBI , a significant decrease (p,0.01) was observed in dysplastic acini compared with the control and normal cadmium acini (Fig. 4D).
The density of microvessel length (L V MV) did not differ considerably among control and normal acini of Cd-treated group (Fig. 5). Nevertheless, a significant increase of L V MV (p,0.01) in dysplastic acini was identified (Fig. 5).
When the relationship between LI LPA1 and proliferative, apoptotic, or angiogenesis markers in dysplastic acini of Cdtreated rats was investigated, statistically significant correlations were found only between LI LPA1 and LI Ubi ( Table 2).

Discussion
Animal and occupational studies have strongly suggested that cadmium is carcinogenic to the prostate [2,[32][33][34][35]. Waalkes et al. (1989) [35] demonstrated a dose-response relation in a rodent model when tumors were induced by injecting cadmium subcutaneously. Our experimental model based on the administration of low doses of cadmium chloride induces higher incidence of prostate carcinogenesis in Sprague-Dawley rats in a manner similar to that of humans [6][7][8]27].
In the present study, we first demonstrate that LPA-1 is expressed in dysplastic acini of Cd-treated rats. LPA-1 regulates cell proliferation, survival, angiogenesis, or migration [9][10][11]. In our study, we found that expression of LPA-1 (LI LPA1 ) was significantly higher in dysplastic acini compared with benign tissue.
Cell proliferation was studied by the expression of PCNA and MCM7. A significant difference of LI PCNA in dysplastic acini was observed, as previously described by authors [8,27,[36][37][38]. PCNA is a nuclear protein that plays a significant role in DNA replication. PCNA is a good marker for tumor growth and prognosis [20]. The miniature chromosome maintenance (MCM) complex is a group of proteins that are essential for DNA replication licensing and control of cell cycle progression from G1 to S phase. Recent studies suggest that MCM7 is overexpressed and amplified in a variety of human malignancies. Most of these studies used MCM7 as a proliferation marker to compare with proliferating cell nuclear antigen (PCNA) or Ki-67 [39][40][41]. As PCNA, LI MCM7 was significantly incremented in dysplastic acini; the nondysplastic acini of Cd-treated rats showed a significant increase. MCM7 is expressed in more cells than the PCNA because it is expressed in cells licensed to proliferate in addition to those that are already proliferating [40]. Not significant variation in V F Bcl-2 between controls and Cdtreated rats was detected, in contrast to those observed in other studies [27]. Bcl-2 is a small intracellular nonglycosylated protein that is able to inhibit the apoptotic pathway when overexpressed in cells [42]. By the TUNEL technique, we viewed a significant decrease in dysplastic acini in comparison to control acini and a representative increase of LI p53 . p53 is a tumor suppressor protein that regulates the cell cycle and, thus, is involved in preventing cancer [43]. p53 protein mutation is more stable and have a longer half-life than functional p53 because it is easy to detect in cell nucleus using immunohistochemistry [44,45]. p53 mutation allows cell replication without correction of DNA mutations and can be a contributing factor for the acquisition of apoptotic resistance in cadmium prostatic carcinogenesis, as described by Aimola P et al. (2012) [2]. Ubiquitin is a protein implicated in extra-lysosomal proteolysis and importantly associated with apoptosis [46]. LI UBI decreased significantly in dysplastic lesions; this reduction is implicated with an apoptosis diminution, in relation to the results that we observed.
There is a remarkable increase of the L V MV on dysplastic lesions induced by cadmium chloride in comparison with controls  and normal epithelium of Cd-treated rats; this might be related to the increase of angiogenesis indicated in cancer and preinvasive lesions by other authors [47,48]. Angiogenesis is an important factor in growth and progression of solid neoplasms. Tumor metastasis is also angiogenesis dependent; new capillaries supply a doorway for entry of metastases to the circulatory system [20,21].
When correlation between LI LPA1 with the other markers were studied in dysplastic acini of Cd-treated rats, only a significant negative correlation were observed with LI UBI . Nevertheless, a positive correlation with LI PCNA , LI MCM7 , LI p53 , and L V MV and a negative correlation with LI APO and V F Bcl-2 were found. It can be possible that LPA-1 regulates cell proliferation and apoptosis through ubiquitin.
In summary, our study showed an immunoexpression increase of LPA-1 receptor in normal and dysplastic acini of Cd-treated rats, an increment of cell proliferation, a decrease of apoptosis, and an increase of angiogenesis in dysplastic lesions. Finally, it is possible that LPA-1 could attenuate ubiquitin immunoexpression and modify cell proliferation and apoptosis. However, the small sample size used in our work is a limitation to draw conclusions.
Overall, our findings support other reports that emphasize that LPA-1 is associated with prostate cancer development [13,49]. Further studies are needed to determine the role of LPA-1 in prostate cancer.