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.
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.
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.
Citation: Arriazu R, Durán E, Pozuelo JM, Santamaria L (2013) 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. PLoS ONE 8(2): e57742. https://doi.org/10.1371/journal.pone.0057742
Editor: Syed A. Aziz, Health Canada, Canada
Received: October 17, 2012; Accepted: January 24, 2013; Published: February 22, 2013
Copyright: © 2013 Arriazu et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: This study was supported in part by a financial grant from San Pablo-CEU University and Santander Bank (USP-BS-PPC04/2011). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing interests: The authors have declared that no competing interests exist.
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 , . 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 –.
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 –. Initially, LPA was thought to be secreted primarily by platelets during wound healing ; 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 , . Also, it is known that LPA-1 is detected in many tumors, such as those in the lung, breast, stomach, kidney, and prostate . It is well established that LPA signals various events through its G protein-coupled receptors (GPCRs), namely, LPA-1 to LPA-6 –.
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 1-phosphate. The switching expression of LPA-1 receptors is found to be associated with prostate cancer development , , . 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 . In normal tissues, LPA-1 is broadly expressed, whereas expression of LPA-2 and LPA-3 is more restricted . The physiological role of LPA-4 has not been investigated  and LPA-5 was found to be highly expressed on cells associated with the immune system , and LPA-6 is a novel receptor implicated in human hair growth , .
In the case of prostate cancer, LPA is reported to induce proliferation and survival of androgen-independent prostate cancer cells . Prostate cancer is the second leading cause of male deaths in the majority of Western countries , . Information regarding the expression profile of LPA-1 in human biopsies is limited .
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 , . Angiogenesis is defined as the process leading to the formation of new blood vessels and is essential for normal growth and development . Recently, LPA was demonstrated to promote ovarian cancer growth by inducing angiogenic factors , but this relation in prostate dysplastic lesions needs further investigation.
A useful experimental model for human prostate cancer is the rat –, and morphological similarities between human PIN and dysplastic changes experimentally promoted in rodent prostate have been reported . Our group designed an experimental model based on the administration of low doses of cadmium chloride –, . 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).
Materials and Methods
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 CO2 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 , J Histochem Cytochem 54: 981–90 , and Hormonal Carcinogenesis IV, Springer pp. 522–528 .
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.
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-mm-width 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 µm-thickness and stained with hematoxylin-eosin or used for immunohistochemical techniques.
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 (Histostain® Bulk Kit, Invitrogen Corporation, Carlsbad, California) and subsequently incubated overnight with primary antibodies in a moist chamber at 4°C. Information on primary antibodies is provided in Table 1.
On the second day, immunohistochemistry was performed with standard procedures using Histostain® Bulk Kit (Invitrogen Corporation, Carlsbad, California). The immunostaining reaction product was developed using 0.1 g diaminobenzidine (DAB) (3,3′,4,4′–Tetraminobiphenyl, Sigma, St Louis, USA) in 200 ml of PBS, plus 40 µl hydrogen peroxide. After immunoreactions, sections were counterstained with acetic carmine, Harris hematoxylin, or methyl green, dehydrated in ethanol and mounted in a synthetic resin (Depex, Serva, Heidelberg, Germany). The specificity of the immunohistochemical procedures was checked by incubation of sections with nonimmune serum instead of the primary antibody.
DNA fragmentation detection
To detect the apoptotic fragmentation of DNA, a TUNEL (TdT-mediated dUTP-biotin nick end labeling) technique (Boehringer Mannheim) was used , . 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 µl/ml in TRIS/EDTA pH 8) for 30 min at 37°C 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 37°C 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 37°C. The reaction was detected using 0.1 g DAB in PBS (200 ml), plus 40 µl 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).
Quantitative evaluation of LPA-1, cell proliferation, apoptosis, ubiquitin, and p53
The percentages of LPA-1–immunostained nuclei (LPA-1 labeling index, LILPA1), PCNA-immunopositive nuclei (PCNA labeling index, LIPCNA), labeled apoptotic nuclei (apoptotic labeling index, LIAPO), ubiquitin-immunostained nuclei (UBI labeling index, LIUBI), and p53-immunostained nuclei (p53 labeling index, LIp53) were calculated in each selected section for control rats, nondysplastic acini of cadmium-treated (Cd-treated) rats, and dysplastic acini of Cd-treated animals, using the following formula: number of labeled nuclei×100/total number (labeled + unlabeled) of nuclei , , , .
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 (VF) was measured and expressed as percentage of immunostained epithelium. Estimation of the VF 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 VF . The VF 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 (LVMV)
The length of microvessels per unit of volume (LVMV/mm3) of prostate tissue was evaluated in normal acini of control rats, nondysplastic acini of Cd-treated rats, and dysplastic acini of Cd-treated 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 . The LVMV was a calculated by the formula: LV = (2×_Q-)/_A, where Q- = number of immunopositive vascular profiles and _A = total area sampled, that is, area of dissector frame (1482 µm2) multiplied by the number of selected frames . These measurements were performed using the CAST-GRID program (Stereology Software Package, Silkeborg, Denmark).
Statistical evaluation was performed using GraphPad Prism5 (La Jolla, USA). Data were presented as means ± 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.
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).
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.
A: Immunoreactive nuclei for LPA-1 are observed in the epithelium (arrowheads) of control rat. B,C: LPA-1 immunostained dysplastic acini (B) and nondysplastic acini (C) from a cadmium-exposed rat. Many LPA-1–positive nuclei are observed (arrowheads). The number of immunoreactive nuclei observed in B and C is higher than that in A. D,E: PCNA immunoexpression in dysplastic (D) and nondysplastic acini of Cd-treated rats (E). Positive nuclei are detected (arrowheads); nevertheless, dysplastic acini show a huge number. F,G: The positive MCM7 nuclei (arrowheads) are evident in dysplastic (F) and nondysplastic acini (G). H,I: Ubiquitin expression, in H control acini has immunopositive expression in the cytoplasm (black arrowhead) and in the nucleus (white arrowheads). The nucleus interior is not uniform. Dysplastic lesions (I) present positive nucleus for ubiquitin (arrowheads). J,K: The localization of Bcl-2 immunoreactivity is similar in normal and dysplastic epithelium. Normal acini of Cd-treated rats are homogeneous in the cytoplasm (arrowheads in J). Dysplastic acinus shows mainly granular immunoreactivity in the apical border of the epithelium. L,M: DNA fragmentation test. Control acinus with abundant positive nuclei (arrowheads). Dysplastic acini show few apoptotic nuclei (arrowheads). N,O: p53 immunoreactive nuclei (black arrowheads) and cytoplasm (white arrowheads) cells are detected in the dysplastic lesions (L) and in normal epithelium (O). P,Q: Endothelial positive cells to FVIII are recognized in dysplastic stromal tissue (P) and normal acini (Q). Sections counter stained: Harris hematoxylin (A,B,C,P,Q), methyl green (D,E,H,I), and acetic carmine (F,G,J,K,L,M,N,O).
Nuclear immunoreactivity to PCNA (Figs. 1D, 1E) and MCM7 (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).
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).
A significant increase for LILPA1 (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 LIPCNA was significantly increased in dysplastic lesions (p<0.05), whereas the control acini and cadmium nondysplastic acini did not show significant variations (Fig. 3A). LIMCM7 was significantly increased in the animals exposed to cadmium (normal acini and dysplastic acini) in comparison to control animals (Fig. 3B).
Lines and asterisks indicate statistically significant differences (**p<0.01).
Results are expressed as mean ± SD. Lines and asterisks indicate statistically significant differences (*p<0.05).
There was no significant difference in the VF Bcl-2 between the control and cadmium-treated animals (Fig. 4A). A significant decrease of LIAPO 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 normal-acini of Cd-treated group was not detected (Fig. 4B). For LIp53, no significant differences between the normal epithelium of the control and Cd-treated rats were observed; however LIp53 of dysplastic acini of rats treated with cadmium was significantly increased (p<0.05) compared with cadmium normal acini (Fig. 4C). For LIUBI, a significant decrease (p<0.01) was observed in dysplastic acini compared with the control and normal cadmium acini (Fig. 4D).
Bars showing lines and asterisks on top of the error bars differ significantly (*p<0.05, **p<0.01, ***p<0.001).
The density of microvessel length (LVMV) did not differ considerably among control and normal acini of Cd-treated group (Fig. 5). Nevertheless, a significant increase of LVMV (p<0.01) in dysplastic acini was identified (Fig. 5).
Statistical differences are indicated by lines and asterisks on top of the error bars (**p<0.01).
When the relationship between LILPA1 and proliferative, apoptotic, or angiogenesis markers in dysplastic acini of Cd-treated rats was investigated, statistically significant correlations were found only between LILPA1 and LIUbi (Table 2).
Animal and occupational studies have strongly suggested that cadmium is carcinogenic to the prostate , –. Waalkes et al. (1989)  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 –, .
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 –. In our study, we found that expression of LPA-1 (LILPA1) 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 LIPCNA in dysplastic acini was observed, as previously described by authors , , –. PCNA is a nuclear protein that plays a significant role in DNA replication. PCNA is a good marker for tumor growth and prognosis . 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 –. As PCNA, LIMCM7 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 .
Not significant variation in VFBcl-2 between controls and Cd-treated rats was detected, in contrast to those observed in other studies . Bcl-2 is a small intracellular nonglycosylated protein that is able to inhibit the apoptotic pathway when overexpressed in cells . By the TUNEL technique, we viewed a significant decrease in dysplastic acini in comparison to control acini and a representative increase of LIp53. p53 is a tumor suppressor protein that regulates the cell cycle and, thus, is involved in preventing cancer . 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 , . 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) . Ubiquitin is a protein implicated in extra-lysosomal proteolysis and importantly associated with apoptosis . LIUBI 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 LVMV 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 , . 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 , .
When correlation between LILPA1 with the other markers were studied in dysplastic acini of Cd-treated rats, only a significant negative correlation were observed with LIUBI. Nevertheless, a positive correlation with LIPCNA, LIMCM7, LIp53, and LVMV and a negative correlation with LIAPO and VFBcl-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 , . Further studies are needed to determine the role of LPA-1 in prostate cancer.
Conceived and designed the experiments: RA JMP LS. Performed the experiments: RA ED. Analyzed the data: RA. Contributed reagents/materials/analysis tools: RA JMP LS. Wrote the paper: RA JMP.
- 1. Lin CE, Chen SU, Lin CC, Chang CH, Lin YC, et al. (2012) Lysophosphatidic acid enhances vascular endothelial growth factor-C expression in human prostate cancer PC-3 cells. PLoS One 7: e41096.
- 2. Aimola P, Carmignani M, Volpe AR, Di Benedetto A, Claudio L, et al. (2012) Cadmium induces p53-dependent apoptosis in human prostate epithelial cells. PLoS One 7: e33647.
- 3. Bonkhoff H, Remberger K (1998) Diagnostic criteria and differential diagnosis of prostatic intraepithelial neoplasia. Pathologe 19: 33–41.
- 4. Marshall JR (2001) High-grade prostatic intraepithelial neoplasia as an exposure biomarker for prostate cancer chemoprevention research. IARC Sci Publ 154: 191–198.
- 5. Nicholson B, Theodorescu D (2003) Molecular therapeutics in prostate cancer. Histol Histopathol 18: 275–298.
- 6. Arriazu R, Pozuelo JM, Martin R, Rodriguez R, Santamaria L (2005) Quantitative and Immunohistochemical Evaluation of PCNA, Androgen Receptor, Apoptosis and Glutathione-S-Transferase P1 on Preneoplastic Changes Induced by Cadmium and Zinc Chloride in the Rat Ventral Prostate. Prostate 63: 347–357.
- 7. Arriazu R, Pozuelo JM, Rodriguez R, Henriques-Gil N, Perucho T, et al. (2005) Cadmium and zinc chloride-induced preneoplastic changes in the rat ventral prostate: An immunohistochemical and molecular study. In: Jonathan J Li, Sara A LI, Antonio LLombart-Bosch, editors. Hormonal Carcinogenesis IV. Springer. pp. 522–528.
- 8. Arriazu R, Pozuelo JM, Henriques-Gil N, Perucho T, Martín R, et al. (2006) Immunohistochemical Study of Cell Proliferation, Bcl-2, p53 and Caspase-3 Expression on Preneoplastic Changes Induced by Cadmium and Zinc Chloride in the Ventral Rat Prostate. J Histochem Cytochem 54: 981–90.
- 9. Yamashita H, Tripathi M, Jourquin J, Kam Y, Liu S, et al. (2010) Lysophosphatidic Acid Upregulates Laminin-332 Expression during A431 Cell Colony Dispersal. J Oncol 2010. pii: 107075. Epub; 2010 Aug 25 107075.
- 10. Chen SU, Lee H, Chang DY, Chou CH, Chang CY, et al. (2008) Lysophosphatidic acid mediates interleukin-8 expression in human endometrial stromal cells through its receptor and nuclear factor-kappaB-dependent pathway: a possible role in angiogenesis of endometrium and placenta. Endocrinology 149: 5888–5896.
- 11. Guo R, Kasbohm EA, Arora P, Sample CJ, Baban B, et al. (2006) Expression and function of lysophosphatidic acid LPA1 receptor in prostate cancer cells. Endocrinology 147: 4883–4892.
- 12. Murph MM, Nguyen GH, Radhakrishna H, Mills GB (2008) Sharpening the edges of understanding the structure/function of the LPA1 receptor: expression in cancer and mechanisms of regulation. Biochim Biophys Acta 1781: 547–557.
- 13. Yanagida K, Ishii S (2011) Non-Edg family LPA receptors: the cutting edge of LPA research. J Biochem 150: 223–232.
- 14. Yanagida K, Masago K, Nakanishi H, Kihara Y, Hamano F, et al. (2009) Identification and characterization of a novel lysophosphatidic acid receptor, p2y5/LPA6. J Biol Chem 284: 17731–17741.
- 15. Choi JW, Herr DR, Noguchi K, Yung YC, Lee CW, et al. (2010) LPA receptors: subtypes and biological actions. Annu Rev Pharmacol Toxicol 50: 157–86
- 16. Chen M, Towers LN, O'Connor KL (2007) LPA2 (EDG4) mediates Rho-dependent chemotaxis with lower efficacy than LPA1 (EDG2) in breast carcinoma cells. Am J Physiol Cell Physiol 292: C1927–C1933.
- 17. Sumida H, Noguchi K, Kihara Y, Abe M, Yanagida K, et al. (2010) LPA4 regulates blood and lymphatic vessel formation during mouse embryogenesis. Blood 116: 5060–5070.
- 18. Lundequist A, Boyce JA (2011) LPA5 is abundantly expressed by human mast cells and important for lysophosphatidic acid induced MIP-1beta release. PLoS One 6: e18192.
- 19. Martens-Uzunova ES, Jalava SE, Dits NF, van Leenders GJ, Moller S, et al. (2012) Diagnostic and prognostic signatures from the small non-coding RNA transcriptome in prostate cancer. Oncogene 31: 978–991.
- 20. Zhu G, Wang X, Yang Z, Cao H, Meng Z, et al. (2011) Effects of TRPM8 on the proliferation and angiogenesis of prostate cancer PC-3 cells in vivo. Oncol Lett 2: 1213–1217.
- 21. Arakawa A, Soh S, Chakraborty S, Scardino PT, Wheeler TM (1997) Prognostic significance of angiogenesis in clinically localized prostate cancer (staining for Factor VIII-related antigen and CD34 Antigen. Prostate Cancer Prostatic Dis 1: 32–38.
- 22. O'Brien TS, Harris AL (1995) Angiogenesis in urological malignancy. Br J Urol 76: 675–682.
- 23. Pollard M, Wolter W, Sun L (2001) Diet and the duration of testosterone-dependent prostate cancer in Lobund-Wistar rats. Cancer Lett 173: 127–131.
- 24. Morrissey C, Buser A, Scolaro J, O'Sullivan J, Moquin A, et al. (2002) Changes in hormone sensitivity in the ventral prostate of aging Sprague-Dawley rats. J Androl 23: 341–351.
- 25. Arunkumar A, Vijayababu MR, Venkataraman P, Senthilkumar K, Arunakaran J (2006) Chemoprevention of rat prostate carcinogenesis by diallyl disulfide, an organosulfur compound of garlic. Biol Pharm Bull 29: 375–379.
- 26. Bosland MC (1999) Use of animal models in defining efficacy of chemoprevention agents against prostate cancer. Eur Urol 35: 459–463.
- 27. Martin JJ, Martin R, Codesal J, Fraile B, Paniagua R, et al. (2001) Cadmium chloride-induced dysplastic changes in the ventral rat prostate: an immunohistochemical and quantitative study. Prostate 46: 11–20.
- 28. Gavrieli Y, Sherman Y, Ben Sasson SA (1992) Identification of programmed cell death in situ via specific labeling of nuclear DNA fragmentation. J Cell Biol 119: 493–501.
- 29. Rittmaster RS, Manning AP, Wright AS, Thomas LN, Whitefield S, et al. (1995) Evidence for atrophy and apoptosis in the ventral prostate of rats given the 5 alpha-reductase inhibitor finasteride. Endocrinology 136: 741–748.
- 30. Sterio DC (1984) The unbiased estimation of number and sizes of arbitrary particles using the disector. J Microsc 134: 127–136.
- 31. Santamaria L, Ingelmo I, Ruiz J, Teba F, Herranz LM, et al. (2008) Stereological estimate of microvessels and the number and proliferation of epithelial and endothelial cells in human normal and pathologic prostate. The Open Prostate Cancer Journal 2: 46–53.
- 32. Kolonel L, Winkelstein W Jr (1977) Cadmium and prostatic carcinoma. Lancet 2: 566–567.
- 33. Aronson KJ, Siemiatycki J, Dewar R, Gerin M (1996) Occupational risk factors for prostate cancer: results from a case-control study in Montreal, Quebec, Canada. Am J Epidemiol 143: 363–373.
- 34. Lauwerys RR, Bernard AM, Roels HA, Buchet JP (1994) Cadmium: exposure markers as predictors of nephrotoxic effects. Clin Chem 40: 1391–1394.
- 35. Waalkes MP, Rehm S, Riggs LW, Bare RM, Devor DE, et al. (1989) Cadmium carcinogenesis in the male Wistar (Crl(WI)BR) rats: Dose-response analysis of effects of zinc on tumor induction in the prostate and in the testes and at the injection site. Cancer Res 49: 4282–4288.
- 36. Waalkes MP (2000) Cadmium carcinogenesis in review. J Inorg Biochem 79: 241–244.
- 37. Waalkes MP (2003) Cadmium carcinogenesis. Mutat Res 533: 107–120.
- 38. Tang DG, Li L, Chopra DP, Porter AT (1998) Extended survivability of prostate cancer cells in the absence of trophic factors: increased proliferation, evasion of apoptosis, and the role of apoptosis proteins. Cancer Res 58: 3466–3479.
- 39. Luo JH (2011) Oncogenic activity of MCM7 transforming cluster. World J Clin Oncol 2: 120–124.
- 40. Ota T, Clayton AC, Minot DM, Shridhar V, Hartmann LC, et al. (2011) Minichromosome maintenance protein 7 as a potential prognostic factor for progression-free survival in high-grade serous carcinomas of the ovary. Mod Pathol 24: 277–287.
- 41. Ishino H, Hara Y, Takekoshi S, Teshima T, Teramoto A, et al. (2011) Ki-67 and minichromosome maintenance-7 (MCM7) expression in canine pituitary corticotroph adenomas. Domest Anim Endocrinol 41: 207–213.
- 42. Knillova J, Kolar Z (2003) The significance of key regulators of apoptosis in the development and prognosis of prostate carcinoma. I. Proteins of the Bcl-2 family and protein p53. Biomed Pap Med Fac Univ Palacky Olomouc Czech Repub 147: 3–10.
- 43. Reisman D, Takahashi P, Polson A, Boggs K (2012) Transcriptional Regulation of the p53 Tumor Suppressor Gene in S-Phase of the Cell-Cycle and the Cellular Response to DNA Damage. Biochem Res Int 2012:808934. Epub; 2012 Jul 11 808934.
- 44. Silvestrini R, Benini E, Daidone MG, Veneroni S, Boracchi P, et al. (1993) p53 as an independent prognostic marker in lymph node-negative breast cancer patients. J Natl Cancer Inst 85: 965–970.
- 45. Bosari S, Lee AK, Viale G, Heatley GJ, Coggi G (1992) Abnormal p53 immunoreactivity and prognosis in node-negative breast carcinomas with long-term follow-up. Virchows Arch A Pathol Anat Histopathol 421: 291–295.
- 46. Kwon J, Wang YL, Setsuie R, Sekiguchi S, Sato Y, et al. (2004) Two closely related ubiquitin C-terminal hydrolase isozymes function as reciprocal modulators of germ cell apoptosis in cryptorchid testis. Am J Pathol 165: 1367–1374.
- 47. Delves GH, Stewart AB, Cooper AJ, Lwaleed BA (2007) Prostasomes, angiogenesis, and tissue factor. Semin Thromb Hemost 33: 75–79.
- 48. Kong D, Li Y, Wang Z, Banerjee S, Sarkar FH (2007) Inhibition of angiogenesis and invasion by 3,3'-diindolylmethane is mediated by the nuclear factor-kappaB downstream target genes MMP-9 and uPA that regulated bioavailability of vascular endothelial growth factor in prostate cancer. Cancer Res 67: 3310–3319.
- 49. Zeng Y, Kakehi Y, Nouh MA, Tsunemori H, Sugimoto M, et al. (2009) Gene expression profiles of lysophosphatidic acid-related molecules in the prostate: relevance to prostate cancer and benign hyperplasia. Prostate 69: 283–292.