Basic and Translational Science Zinc Induces a Bell-shaped Proliferative Dose-response Effect in Cultured Smooth Muscle Cells From Benign Prostatic Hyperplasia €rn Bloth, Staffan Ha €gg, and Samuel P. S. Svensson Per I. Adolfsson, Bjo OBJECTIVE METHODS
RESULTS
CONCLUSION
To investigate the effects of zinc (Zn2þ) concentrations on cultured benign prostatic hyperplasia (BPH) smooth muscle cell (SMC) proliferation. The effects of Zn2þ were studied in primary cultures of human BPH SMC, stimulated with either 10-mM lysophosphatidic acid (LPA) or LPA in combination with 100-nM testosterone. Deoxyribonucleic acid replication and protein synthesis using [3H]-thymidine and [35S]-methionine incorporation were measured. Furthermore, studies were performed to evaluate if Zn2þ could potentiate the inhibitory effect of phosphodiesterase-5 blockers, on BPH SMC proliferation. Zn2þ generated a bell-shaped concentration response, both regarding deoxyribonucleic acid replication and protein synthesis in cultured BPH SMC. Below a threshold value (approximately 200 mM), a significant mitogenic effect was seen, whereas higher concentrations inhibited SMC proliferation after stimulation with LPA. This effect was even more pronounced after stimulation of LPA in combination with testosterone. Moreover, phosphodiesterase-5 inhibitors, that is, sildenafil blocked LPA-stimulated BPH SMC proliferation. This antiproliferative effect, was significantly potentiated by coincubation with Zn2þ in an additative manner. The bell-shaped concentration response of Zn2þ on cultured BPH SMC proliferation suggests that changes in prostate Zn2þ concentrations, during aging, diet, or inflammatory conditions, may be of importance in the pathogenesis of BPH. UROLOGY 85: 704.e15e704.e19, 2015. 2015 Elsevier Inc.
Z
inc (Zn2þ) is an essential micronutrient required for many cellular processes and is a regulatory cofactor for approximately 300 enzymes.1 The prostate contains high levels of Zn2þ, and the concentration of Zn2þ in normal prostate is >3 times higher compared with other soft tissues and 500 times higher than in other body fluids.1 Three zinc-regulated enzymes that influence smooth muscle cell (SMC) proliferation of the stromal part of the prostate gland are important targets in pharmacologic treatment of benign prostatic hyperplasia (BPH) and lower urinary tract symptoms (LUTS); the adenylyl cyclase, phosphodiesterase 5 (PDE5),2 and 5a-reductase.3-6 Adenylyl cyclase stimulation or
Financial Disclosure: SPSS and BB are supported by Karin & Sten Mortstedt CBD Solutions. The other authors declare that they have no relevant financial interests. From the Department of Drug Research, Faculty of Health Sciences, Link€oping University, Link€oping, Sweden; and the Department of Clinical Neuroscience, Laboratory of Translational Neuropharmacology, Center of Molecular Medicine, Karolinska Institute, Stockholm, Sweden; Department of Clinical Pharmacology and Department of Medical and Health Sciences, Link€oping University, Link€oping, Sweden; Futurum Academy for Health and Care, J€onk€oping County Council, J€onk€oping, Sweden Address correspondence to: Samuel P. S. Svensson, Department of Drug Research/ Clinical Pharmacology, Faculty of Health Sciences, Link€oping University, SE-581 85 Link€oping, Sweden. E-mail: [email protected] Submitted: September 3, 2014, accepted (with revisions): November 25, 2014
704.e15 ª 2015 Elsevier Inc. All Rights Reserved
PDE-5 inhibition, both increase intracellular cyclic nucleotide levels generating prostate SMC proliferation inhibition,2 whereas 5a-reductase promotes prostate SMC proliferation by converting testosterone to its 10fold more potent form, dihydrotestosterone.6 Zn2þ has been suggested to have a role in the development of prostate cancer (PCa),7 but the role of Zn2þ in BPH is unclear. However, data suggest that total Zn2þ uptake declines during aging because of an age-related shifted diet pattern and reduced gastrointestinal Zn2þ uptake. An age-related nondietary coupled diminished uptake has also been demonstrated for cobolamine.8 Regarding the plasma Zn2þ levels, there is a 27% decrease in PCa patients compared with controls and an 18% reduction in regarding BPH, whereas in dry tissue weight, the prostate Zn2þ levels in PCa tissue were reduced by 83%, respectively, and in BPH tissue, Zn2þ levels were decreased by 61%.9-12 Furthermore, a shifted zinc transporter expression has been demonstrated in PCa tissue generating reduced intracellular Zn2þ levels in prostatic cancer.13-15 The prostate-specific antigen (PSA) may also be influenced by the prostate Zn2þ content. PSA is a kallikrein protease, and its activity is strongly suppressed http://dx.doi.org/10.1016/j.urology.2014.11.026 0090-4295/15
Figure 1. Dose-response of Zn2þ on cultured primary benign prostatic hyperplasia smooth muscle cell deoxyribonucleic acid replication and protein synthesis, measured by [3H]-thymidine incorporation (A) or [35S]-methionine incorporation (B), respectively. The control column set to 100% corresponds to 10-mM lysophosphatidic acid stimulation (generating approximately 100% increased proliferation compared with Dulbecco’s modified Eagle’s medium, not shown). All further columns represent additional to 10-mM lysophosphatidic acid, coincubation with increasing Zn2þ.
by Zn2þ16,17 suggesting that prostate Zn2þ may suppress invasion and metastasis of PCa by regulating PSA activity.16 Moreover, reduction in urine Zn2þ content has been suggested to be superior to PSA for separating high-risk from low-risk malignancies in PCa.18 Data published in earlier referred articles regarding changed Zn2þ levels in normal prostate tissue compared with BPH and PCa, respectively, could imply that Zn2þ may have an antiproliferative effect at higher concentrations. In the present study, we have studied the concentration-response of Zn2þ in cultured BPH SMCs. As Zn2þ is an essential cofactor for enzymes integrated in BPH SMC proliferation, that is, PDE-52 and 5a-reductase,3 the proliferative effect of Zn2þ per se and also in combination with testosterone, or in combination with sildenafil (PDE-5 inhibitor), was analyzed in cultured BPH SMCs by measuring the deoxyribonucleic acid (DNA) replication and protein synthesis.
METHODS BPH was studied in vitro by using the DNA replication marker [3H]-thymidine and the protein synthesis marker [35S]-methionine for incorporation in cultured prostate SMCs as previously described,2 both markers having obtained from Amersham Biosciences (Uppsala, Sweden). Furthermore, all chemicals and drugs were obtained from Sigma Aldrich (Stockholm, Sweden). The cell culture medium, Dulbecco’s modified Eagle’s medium (DMEM), nonessential amino acids (100), sodium pyruvate (100 mM), penicillin-streptomycin (1000 U/mL), fetal bovine serum (FBS), and trypsin were all purchased from Gibco Life Technologies (Stockholm, Sweden).
Tissue Collection BPH stromal SMC culture obtained from tissue collection biopsies from 5 men subjected to transurethral resection of the prostate due to LUTS and BPH SMC obtained from PromoCell (HPrSMC) were used in the study. Surgery was made at the Division of Urology, University Hospital, Link€oping, Sweden. The BPH biopsies were immediately placed in sterile tubes containing phosphate-buffered saline solution (pH ¼ 7.3) and transported on ice to the laboratory. Tissue not used directly was UROLOGY 85 (3), 2015
frozen in liquid nitrogen and stored at 80 C. The study was approved by the Research Ethical Committee, Link€ oping, Sweden.
Establishment of SMC Culture From BPH Tissue Biopsies were briefly washed in 70% EtOH, transferred to a continuous laminar flow hood and placed on 150-mm dishes containing culture medium with the following components: DMEM, 1% nonessential amino acids, 1% sodium pyruvate, and 1% penicillin-streptomycin. Thereafter, biopsies were sliced into 3-mm3 fragments and subsequently digested with 2mg/ml collagenase at 37 C. Fragments were treated in 30 minutes, whereon the cell suspension was transferred to new sterile tubes and centrifuged for 5 minutes at 500g to pellet the cell fraction. Cells were separated from collagenase by resuspending the pellet in DMEM containing 10% FBS and thereafter centrifuged once more. The pelleted cells were resuspended in fresh DMEM containing 10% FBS and thereafter spread out on new culture dishes. In parallel, an equivalent enzyme mixture was added to the remaining tissue fragments for a new period of incubation. Two equivalent enzyme solutions were used for 6 periods of time. The first pelleted cell fraction was not used for cell culturing. Subsequently, cells were incubated for 2-3 weeks before trypsinization and split into new culture dishes. Incubation was carried out in an incubator at 37 C in a humidified atmosphere (95%) with 5% CO2 and 95% air.
Establishment of HPrSMC Cell Culture Starting up HPrSMC culturing was followed precisely according to the instructions of the manufacturer (PromoCell, United Kingdom).
[3H]-thymidine and [35S]-methionine Incorporation in BPH SMC The [3H]-thymidine and [35S]-methionine incorporation was made as follows: initially, the BPH SMCs were spliced into 96well plates, with 104 SMCs per well cultured in DMEM (10% FBS) for 48 hours and thereafter 48 hours in starvation medium without FBS to generate cell cycleephase synchronization. Hereafter, in the first 2 studies, the cells were incubated with 10 mM of lysophosphatidic acid (LPA) and [3H]-thymidine or [35S]-methionine, respectively, with increasing concentrations of Zn2þ in 24 hours (Figs. 1, 2). With exception of the 704.e16
Figure 2. Representative study of 3 separate experiments showing the mean and standard error of the mean of mitogenic dose-response of Zn2þ and 100 nM of testosterone on cultured primary benign prostatic hyperplasia smooth muscle cells. The proliferation was measured by analyzing the deoxyribonucleic acid replication by [3H]-thymidine incorporation. Experimentally, everything is identical with what was described regarding the Figure 1 studies, with one extra component consisting of a coincubation of 100 nM of testosterone. The control column deprived of Zn2þ represents 100 nM of testosterone and 10 mM of lysophosphatidic acid. testosterone effect (Fig. 2), the controls correspond to BPH SMCs only treated with 10 mM of LPA, whereas in Figure 2, the control also included 100 nM of testosterone. Additional samples also contained Zn2þ and/or sildenafil in marked concentrations. After 24 hours of incubation, the SMCs were rinsed once with phosphate-buffered saline solution and subsequently treated with 5% (vol/vol) ice-cold trichloroacetic acid for 30 minutes to terminate the incubation. Subsequently, each well was added 200 mL of scintillation liquid (OptiPhase SuperMix Wallac; Perkin Elmer, Stockholm, Sweden) and analyzed using a Wallac 1450 TriLux (Perkin Elmer) plate reader. In Figure 1A,B, increasing concentrations of Zn2þ in relation to [3H]-thymidine and [35S]-methionine incorporation, respectively, in primary cultured BPH SMCs stimulated with 10 mM LPA. In Figure 2, increasing concentrations of Zn2þ in relation to [3H]-thymidine incorporation in BPH SMCs that were coincubated with 100-nM testosterone and 10-mM LPA and finally, in Figure 3, [3H]-thymidine incorporation in BPH SMC, besides 10-mM LPA cells were treated with increasing concentrations of sildenafil with or without 225-mM Zn2þ.
RESULTS In cultured BPH SMCs, Zn2þ induced a biphasic response in 10-mM LPA-stimulated BPH SMC both regarding DNA replication and protein synthesis analyzed by measuring [3H]-thymidine and [35S]-methionine incorporation, respectively (Fig. 1A,B). This biphasic effect of Zn2þ was detected both in primary BPH SMCs as well as in commercial available cultured HPrSMC (PromoCell, United Kingdom; results not shown). Lower concentrations of Zn2þ (50-200 mM) induced a mitogenic effect on BPH SMCs, whereas higher concentrations of Zn2þ 704.e17
Figure 3. This is a representative study of 3 separate experiments showing the mean and standard error of the mean of the antimitogenic dose-response of sildenafil (specific phosphodiesterase-5 inhibitor) with or without coincubation of 225-mM Zn2þ on cultured benign prostatic hyperplasia smooth muscle cells treated with 10-mM lysophosphatidic acid. The proliferation was measured by analyzing the deoxyribonucleic acid replication by [3H]-thymidine incorporation as described in Figure 1. LPA, lysophosphatidic acid.
(200 mM) significantly reduced BPH SMC proliferation (Fig. 1A,B). The EC50 for the mitogen effect was approximately 75 mM and the IC50 for the inhibitory effect was 275 mM for [3H]-thymidine incorporation, whereas the corresponding [35S]-methionine values were approximately 125 mM and 325 mM, respectively. The highest concentrations of Zn2þ showed an antiproliferative effect, which was well below the baseline value of 10-mM LPA, which by itself, both here and in previous data2 generated approximately 100% increased proliferation. The mitogenic effect of zinc was even more pronounced when Zn2þ was combined with 100-nM testosterone (Fig. 2). Similarly to results previously described, higher concentrations of Zn2þ (>225 mM) efficiently inhibited proliferation stimulated with testosterone. Taken together, these results show that Zn2þ have a bell-shaped concentration-response curve, stimulating proliferation at lower concentrations (225 mM). We have previously shown that selective PDE-5 inhibitors such as sildenafil have an antiproliferative effect in LPA-stimulated BPH SMCs.2 In the present study, we further investigated the antiproliferative effect of sildenafil combined with Zn2þ. BPH SMCs were stimulated with 10-mM LPA, in the presence of increasing concentrations of sildenafil. Two hundred twenty-five micromolar Zn2þ potentiated the inhibitory response of sildenafil in an additive manner and induced a potency shift in IC50 from 6 to 3 mM (Fig. 3).
COMMENT Reduced levels of Zn2þ have been implicated in PCa genesis. Worldwide epidemiology studies indicate that dietary parameters are of high importance for the development of PCa.19,20 In Japan and China, PCa is a lesscommon disease, and the frequency of PCa is 26 times lower in China than that in the United States.15 UROLOGY 85 (3), 2015
However, after one generation, US-emigrated Japanese get PCa in the same frequency as inborn Caucasians indicating that dietary patterns could have a potential role affecting the development of PCa.21 Daily intake of Zn2þ might be one of these dietary parameters. In Japan, there is an extremely high daily intake of Zn2þ as the dietary tradition comprises a lot of shellfish, oysters, cartilage fish, beans, lentils, and rice, which all have a high Zn2þ content.22 The role of Zn2þ in the pathogenesis of BPH is however unclear, and we have in the present study focused on how Zn2þ regulates BPH SMC growth by measuring DNA replication and protein synthesis in vitro. Our result shows that Zn2þ induces a biphasic doseresponse regarding both DNA replication and protein synthesis of BPH SMCs in a way that could be described as shifting from an agonistic mitogenic agent to an antagonistic mediator in a very sharp and well-defined Zn2þ borderline concentration segment. The divergent effect of Zn2þ in BPH SMC proliferation has to our knowledge not previously been described. The effect was seen in primary BPH SMCs isolated in our laboratory and in commercially available BPH SMCs (results not shown). The antiproliferative or cytotoxic effect of Zn2þ in prostate epithelial cancer cell lines23 PC3, LNCaP, and DU145 are well known24 and consistent with tumor growth inhibition by intratumorally injected Zn2þ.25 Furthermore, in a Swedish cohort, a highly dietary intake of Zn2þ was associated with a reduced risk of PCaspecific mortality providing medical impact regarding Zn2þ and prostate pathogenesis.26 Interestingly, in proliferative studies using rat vascular SMCs, a long-term Zn2þ deprivation generates accelerated proliferation,27 whereas Zn2þ supplement increased human airway SMC apoptosis,28 which supports our results. Thus, we envision that present results may be evaluated in a clinical setting to understand if and how dietary Zn2þ supplement may influence BPH. Important parameters that will add value to such a study are Zn2þ content in the urine, blood plasma, and seminal plasma, in combination with more conventional markers such as PSA and dihydrotestosterone levels, prostate imaging, and function. The main limitation of the present study is the fact that the results are obtained from SMCs in culture. Although the cells are isolated from BPH patients, the cultured conditions may have influenced the cell phenotype and physiology.
CONCLUSION The bell-shaped concentration-response curve for Zn2þ, shown in the present study, suggests that small changes in prostate Zn2þ levels, due to aging, dietary patterns, or inflammatory conditions, may influence the pathogenesis of BPH in opposite effects. These findings warrant further
UROLOGY 85 (3), 2015
preclinical and clinical studies to evaluate if dietary Zn2þ supplement could be of beneficial effect regarding LUTS generated by BPH. References 1. Kelleher SL, McCormick NH, Velasquez V, et al. Zinc in specialized secretory tissues: roles in the pancreas, prostate, and mammary gland. Adv Nutr. 2011;2:101-111. 2. Adolfsson PI, Ahlstrand C, Varenhorst E, et al. Lysophosphatidic acid stimulates proliferation of cultured smooth muscle cells from human BPH tissue: sildenafil and papaverin generate inhibition. Prostate. 2002;51:50-58. 3. Azzouni F, Mohler J. Role of 5a-reductase inhibitors in prostate cancer prevention and treatment. Urology. 2012;79:1197-1205. 4. Klein C, Heyduk T, Sunahara RK. Zinc inhibition of adenylyl cyclase correlates with conformational changes in the enzyme. Cell Signal. 2004;16:1177-1185. 5. He F, Seryshev AB, Cowan CW, et al. Multiple zinc binding sites in retinal rod cGMP phosphodiesterase, PDE6alpha beta. J Biol Chem. 2000;275:20572-20577. 6. Leake A, Gripsholm GD, Habib FK. The effect of zinc on the 5 alpha-reduction of testosterone by the hyperplastic human prostate gland. J Steroid Biochem. 1984;20:651-655. 7. Kolenko V, Teper E, Kutikov A, et al. Zinc and zinc transporters in prostate carcinogenesis. Nat Rev Urol. 2013;10:219-226. 8. Howard JM, Azen C, Jacobsen DW, et al. Dietary intake of cobalamin in elderly people who have abnormal serum cobalamin, methylmalonic acid and homocysteine levels. Eur J Clin Nutr. 1998; 52:582-587. 9. Bales CW, Steinman LC, Freeland-Graves JH, et al. The effect of age on plasma zinc uptake and taste acuity. Am J Clin Nutr. 1986;44: 664-669. 10. Mocchegiani E. Zinc and ageing: third Zincage conference. Immun Ageing. 2007;4:1-7. 11. Crawford ED. Epidemiology of prostate cancer. Urology. 2003;62: 3-12. 12. Christudoss P, Selvakumar R, Fleming JJ, et al. Zinc status of patients with benign prostatic hyperplasia and prostate carcinoma. Indian J Urol. 2011;27:14-18. 13. Rishi I, Baidouri H, Abbasi JA, et al. Prostate cancer in African American men is associated with downregulation of zinc transporters. Appl Immunohistochem Mol Morphol. 2003;11:253-260. 14. Golovine K, Makhov P, Uzzo RG, et al. Overexpression of the zinc uptake transporter hZIP1 inhibits nuclear factor-kappaB and reduces the malignant potential of the prostate cancer cells in vitro and in vivo. Clin Cancer Res. 2008;14:5376-5384. 15. Franklin RB, Milon B, Feng P, et al. Zinc and zinc transporters in normal prostate and the pathogenesis of prostate cancer. Front Biosci. 2005;1:2230-2239. 16. Ishii K, Otsuka T, Iguchi K, et al. Evidence that the prostate-specific antigen (PSA)/Zn2þ axis may play a role in human prostate cancer cell invasion. Cancer Lett. 2004;15:79-87. 17. Medarova Z, Ghosh SK, Vangel M, et al. Risk stratification of prostate cancer patients based on EPS-urine zinc content. Am J Cancer Res. 2014;4:385-393. 18. Satheesh Babu AK, Vijayalakshmi MA, Smith GJ, Chadha KC. Thiophilic-interaction chromatography of enzymatically active tissue prostate-specific antigen (T-PSA) and its modulation by zinc ions. J Chromatogr B Analyt Technol Biomed Life Sci. 2008;861: 227-235. 19. Costello LC, Franklin RB. Zinc is decreased in prostate cancer: an established relationship of prostate cancer! J Biol Inorg Chem. 2011; 16:3-8. 20. Yu H, Harris RE, Gao YT, et al. Comparative epidemiology of cancers of the colon, rectum, prostate and breast in Shanghai, China versus the United States. Int J Epidemiol. 1991;20: 76-81.
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21. Brawley OW, Knopf K, Thomson I. The epidemiology of prostate cancer part II: the risk factors. Semin Urol Oncol. 1998;16: 193-201. 22. Sarukura N, Kogirima M, Takai S, et al. Dietary zinc intake and its effects on zinc nutrition in healthy Japanese living in the central area of Japan. J Med Invest. 2011;58:203-209. 23. Rumsby M, Schmitt J, Rodrigues G, et al. Human prostate cell lines from normal and tumourigenic epithelia differ in the pattern and the control of choline lipid headgroups released into the medium on stimulation of protein kinase C. Br J Cancer. 2011;104:673-684. 24. Kriedt CL, Baldassare J, Shah M, et al. Zinc functions as a cytotoxic agent for prostate cancer cells independent of culture growth conditions. J Exp Ther Oncol. 2010;8:287-295.
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25. Shah MR, Kriedt CL, Lents NH, et al. Direct intra-tumoral injection of zinc-acetate halts tumor growth in a xenograft model of prostate cancer. J Exp Clin Cancer Res. 2009;28:84-94. 26. Epstein MM, Kasperzyk JL, Andren O, et al. Dietary zinc and prostate cancer survival in a Swedish cohort. Am J Clin Nutr. 2011; 93:86-93. 27. Alcantara EH, Shin MY, Feldman J, et al. Long-term zinc deprivation accelerates rat vascular smooth muscle cell proliferation involving the down-regulation of JNK1/2 in MAPK signaling. Atherosclerosis. 2013;228:6-52. 28. Chiou YL. The supplementation of zinc increased the apoptosis of airway smooth muscle cells by increasing p38 phosphorylation. Environ Toxicol Pharmacol. 2012;33:70-77.
UROLOGY 85 (3), 2015
RESULTS
CONCLUSION
To investigate the effects of zinc (Zn2þ) concentrations on cultured benign prostatic hyperplasia (BPH) smooth muscle cell (SMC) proliferation. The effects of Zn2þ were studied in primary cultures of human BPH SMC, stimulated with either 10-mM lysophosphatidic acid (LPA) or LPA in combination with 100-nM testosterone. Deoxyribonucleic acid replication and protein synthesis using [3H]-thymidine and [35S]-methionine incorporation were measured. Furthermore, studies were performed to evaluate if Zn2þ could potentiate the inhibitory effect of phosphodiesterase-5 blockers, on BPH SMC proliferation. Zn2þ generated a bell-shaped concentration response, both regarding deoxyribonucleic acid replication and protein synthesis in cultured BPH SMC. Below a threshold value (approximately 200 mM), a significant mitogenic effect was seen, whereas higher concentrations inhibited SMC proliferation after stimulation with LPA. This effect was even more pronounced after stimulation of LPA in combination with testosterone. Moreover, phosphodiesterase-5 inhibitors, that is, sildenafil blocked LPA-stimulated BPH SMC proliferation. This antiproliferative effect, was significantly potentiated by coincubation with Zn2þ in an additative manner. The bell-shaped concentration response of Zn2þ on cultured BPH SMC proliferation suggests that changes in prostate Zn2þ concentrations, during aging, diet, or inflammatory conditions, may be of importance in the pathogenesis of BPH. UROLOGY 85: 704.e15e704.e19, 2015. 2015 Elsevier Inc.
Z
inc (Zn2þ) is an essential micronutrient required for many cellular processes and is a regulatory cofactor for approximately 300 enzymes.1 The prostate contains high levels of Zn2þ, and the concentration of Zn2þ in normal prostate is >3 times higher compared with other soft tissues and 500 times higher than in other body fluids.1 Three zinc-regulated enzymes that influence smooth muscle cell (SMC) proliferation of the stromal part of the prostate gland are important targets in pharmacologic treatment of benign prostatic hyperplasia (BPH) and lower urinary tract symptoms (LUTS); the adenylyl cyclase, phosphodiesterase 5 (PDE5),2 and 5a-reductase.3-6 Adenylyl cyclase stimulation or
Financial Disclosure: SPSS and BB are supported by Karin & Sten Mortstedt CBD Solutions. The other authors declare that they have no relevant financial interests. From the Department of Drug Research, Faculty of Health Sciences, Link€oping University, Link€oping, Sweden; and the Department of Clinical Neuroscience, Laboratory of Translational Neuropharmacology, Center of Molecular Medicine, Karolinska Institute, Stockholm, Sweden; Department of Clinical Pharmacology and Department of Medical and Health Sciences, Link€oping University, Link€oping, Sweden; Futurum Academy for Health and Care, J€onk€oping County Council, J€onk€oping, Sweden Address correspondence to: Samuel P. S. Svensson, Department of Drug Research/ Clinical Pharmacology, Faculty of Health Sciences, Link€oping University, SE-581 85 Link€oping, Sweden. E-mail: [email protected] Submitted: September 3, 2014, accepted (with revisions): November 25, 2014
704.e15 ª 2015 Elsevier Inc. All Rights Reserved
PDE-5 inhibition, both increase intracellular cyclic nucleotide levels generating prostate SMC proliferation inhibition,2 whereas 5a-reductase promotes prostate SMC proliferation by converting testosterone to its 10fold more potent form, dihydrotestosterone.6 Zn2þ has been suggested to have a role in the development of prostate cancer (PCa),7 but the role of Zn2þ in BPH is unclear. However, data suggest that total Zn2þ uptake declines during aging because of an age-related shifted diet pattern and reduced gastrointestinal Zn2þ uptake. An age-related nondietary coupled diminished uptake has also been demonstrated for cobolamine.8 Regarding the plasma Zn2þ levels, there is a 27% decrease in PCa patients compared with controls and an 18% reduction in regarding BPH, whereas in dry tissue weight, the prostate Zn2þ levels in PCa tissue were reduced by 83%, respectively, and in BPH tissue, Zn2þ levels were decreased by 61%.9-12 Furthermore, a shifted zinc transporter expression has been demonstrated in PCa tissue generating reduced intracellular Zn2þ levels in prostatic cancer.13-15 The prostate-specific antigen (PSA) may also be influenced by the prostate Zn2þ content. PSA is a kallikrein protease, and its activity is strongly suppressed http://dx.doi.org/10.1016/j.urology.2014.11.026 0090-4295/15
Figure 1. Dose-response of Zn2þ on cultured primary benign prostatic hyperplasia smooth muscle cell deoxyribonucleic acid replication and protein synthesis, measured by [3H]-thymidine incorporation (A) or [35S]-methionine incorporation (B), respectively. The control column set to 100% corresponds to 10-mM lysophosphatidic acid stimulation (generating approximately 100% increased proliferation compared with Dulbecco’s modified Eagle’s medium, not shown). All further columns represent additional to 10-mM lysophosphatidic acid, coincubation with increasing Zn2þ.
by Zn2þ16,17 suggesting that prostate Zn2þ may suppress invasion and metastasis of PCa by regulating PSA activity.16 Moreover, reduction in urine Zn2þ content has been suggested to be superior to PSA for separating high-risk from low-risk malignancies in PCa.18 Data published in earlier referred articles regarding changed Zn2þ levels in normal prostate tissue compared with BPH and PCa, respectively, could imply that Zn2þ may have an antiproliferative effect at higher concentrations. In the present study, we have studied the concentration-response of Zn2þ in cultured BPH SMCs. As Zn2þ is an essential cofactor for enzymes integrated in BPH SMC proliferation, that is, PDE-52 and 5a-reductase,3 the proliferative effect of Zn2þ per se and also in combination with testosterone, or in combination with sildenafil (PDE-5 inhibitor), was analyzed in cultured BPH SMCs by measuring the deoxyribonucleic acid (DNA) replication and protein synthesis.
METHODS BPH was studied in vitro by using the DNA replication marker [3H]-thymidine and the protein synthesis marker [35S]-methionine for incorporation in cultured prostate SMCs as previously described,2 both markers having obtained from Amersham Biosciences (Uppsala, Sweden). Furthermore, all chemicals and drugs were obtained from Sigma Aldrich (Stockholm, Sweden). The cell culture medium, Dulbecco’s modified Eagle’s medium (DMEM), nonessential amino acids (100), sodium pyruvate (100 mM), penicillin-streptomycin (1000 U/mL), fetal bovine serum (FBS), and trypsin were all purchased from Gibco Life Technologies (Stockholm, Sweden).
Tissue Collection BPH stromal SMC culture obtained from tissue collection biopsies from 5 men subjected to transurethral resection of the prostate due to LUTS and BPH SMC obtained from PromoCell (HPrSMC) were used in the study. Surgery was made at the Division of Urology, University Hospital, Link€oping, Sweden. The BPH biopsies were immediately placed in sterile tubes containing phosphate-buffered saline solution (pH ¼ 7.3) and transported on ice to the laboratory. Tissue not used directly was UROLOGY 85 (3), 2015
frozen in liquid nitrogen and stored at 80 C. The study was approved by the Research Ethical Committee, Link€ oping, Sweden.
Establishment of SMC Culture From BPH Tissue Biopsies were briefly washed in 70% EtOH, transferred to a continuous laminar flow hood and placed on 150-mm dishes containing culture medium with the following components: DMEM, 1% nonessential amino acids, 1% sodium pyruvate, and 1% penicillin-streptomycin. Thereafter, biopsies were sliced into 3-mm3 fragments and subsequently digested with 2mg/ml collagenase at 37 C. Fragments were treated in 30 minutes, whereon the cell suspension was transferred to new sterile tubes and centrifuged for 5 minutes at 500g to pellet the cell fraction. Cells were separated from collagenase by resuspending the pellet in DMEM containing 10% FBS and thereafter centrifuged once more. The pelleted cells were resuspended in fresh DMEM containing 10% FBS and thereafter spread out on new culture dishes. In parallel, an equivalent enzyme mixture was added to the remaining tissue fragments for a new period of incubation. Two equivalent enzyme solutions were used for 6 periods of time. The first pelleted cell fraction was not used for cell culturing. Subsequently, cells were incubated for 2-3 weeks before trypsinization and split into new culture dishes. Incubation was carried out in an incubator at 37 C in a humidified atmosphere (95%) with 5% CO2 and 95% air.
Establishment of HPrSMC Cell Culture Starting up HPrSMC culturing was followed precisely according to the instructions of the manufacturer (PromoCell, United Kingdom).
[3H]-thymidine and [35S]-methionine Incorporation in BPH SMC The [3H]-thymidine and [35S]-methionine incorporation was made as follows: initially, the BPH SMCs were spliced into 96well plates, with 104 SMCs per well cultured in DMEM (10% FBS) for 48 hours and thereafter 48 hours in starvation medium without FBS to generate cell cycleephase synchronization. Hereafter, in the first 2 studies, the cells were incubated with 10 mM of lysophosphatidic acid (LPA) and [3H]-thymidine or [35S]-methionine, respectively, with increasing concentrations of Zn2þ in 24 hours (Figs. 1, 2). With exception of the 704.e16
Figure 2. Representative study of 3 separate experiments showing the mean and standard error of the mean of mitogenic dose-response of Zn2þ and 100 nM of testosterone on cultured primary benign prostatic hyperplasia smooth muscle cells. The proliferation was measured by analyzing the deoxyribonucleic acid replication by [3H]-thymidine incorporation. Experimentally, everything is identical with what was described regarding the Figure 1 studies, with one extra component consisting of a coincubation of 100 nM of testosterone. The control column deprived of Zn2þ represents 100 nM of testosterone and 10 mM of lysophosphatidic acid. testosterone effect (Fig. 2), the controls correspond to BPH SMCs only treated with 10 mM of LPA, whereas in Figure 2, the control also included 100 nM of testosterone. Additional samples also contained Zn2þ and/or sildenafil in marked concentrations. After 24 hours of incubation, the SMCs were rinsed once with phosphate-buffered saline solution and subsequently treated with 5% (vol/vol) ice-cold trichloroacetic acid for 30 minutes to terminate the incubation. Subsequently, each well was added 200 mL of scintillation liquid (OptiPhase SuperMix Wallac; Perkin Elmer, Stockholm, Sweden) and analyzed using a Wallac 1450 TriLux (Perkin Elmer) plate reader. In Figure 1A,B, increasing concentrations of Zn2þ in relation to [3H]-thymidine and [35S]-methionine incorporation, respectively, in primary cultured BPH SMCs stimulated with 10 mM LPA. In Figure 2, increasing concentrations of Zn2þ in relation to [3H]-thymidine incorporation in BPH SMCs that were coincubated with 100-nM testosterone and 10-mM LPA and finally, in Figure 3, [3H]-thymidine incorporation in BPH SMC, besides 10-mM LPA cells were treated with increasing concentrations of sildenafil with or without 225-mM Zn2þ.
RESULTS In cultured BPH SMCs, Zn2þ induced a biphasic response in 10-mM LPA-stimulated BPH SMC both regarding DNA replication and protein synthesis analyzed by measuring [3H]-thymidine and [35S]-methionine incorporation, respectively (Fig. 1A,B). This biphasic effect of Zn2þ was detected both in primary BPH SMCs as well as in commercial available cultured HPrSMC (PromoCell, United Kingdom; results not shown). Lower concentrations of Zn2þ (50-200 mM) induced a mitogenic effect on BPH SMCs, whereas higher concentrations of Zn2þ 704.e17
Figure 3. This is a representative study of 3 separate experiments showing the mean and standard error of the mean of the antimitogenic dose-response of sildenafil (specific phosphodiesterase-5 inhibitor) with or without coincubation of 225-mM Zn2þ on cultured benign prostatic hyperplasia smooth muscle cells treated with 10-mM lysophosphatidic acid. The proliferation was measured by analyzing the deoxyribonucleic acid replication by [3H]-thymidine incorporation as described in Figure 1. LPA, lysophosphatidic acid.
(200 mM) significantly reduced BPH SMC proliferation (Fig. 1A,B). The EC50 for the mitogen effect was approximately 75 mM and the IC50 for the inhibitory effect was 275 mM for [3H]-thymidine incorporation, whereas the corresponding [35S]-methionine values were approximately 125 mM and 325 mM, respectively. The highest concentrations of Zn2þ showed an antiproliferative effect, which was well below the baseline value of 10-mM LPA, which by itself, both here and in previous data2 generated approximately 100% increased proliferation. The mitogenic effect of zinc was even more pronounced when Zn2þ was combined with 100-nM testosterone (Fig. 2). Similarly to results previously described, higher concentrations of Zn2þ (>225 mM) efficiently inhibited proliferation stimulated with testosterone. Taken together, these results show that Zn2þ have a bell-shaped concentration-response curve, stimulating proliferation at lower concentrations (225 mM). We have previously shown that selective PDE-5 inhibitors such as sildenafil have an antiproliferative effect in LPA-stimulated BPH SMCs.2 In the present study, we further investigated the antiproliferative effect of sildenafil combined with Zn2þ. BPH SMCs were stimulated with 10-mM LPA, in the presence of increasing concentrations of sildenafil. Two hundred twenty-five micromolar Zn2þ potentiated the inhibitory response of sildenafil in an additive manner and induced a potency shift in IC50 from 6 to 3 mM (Fig. 3).
COMMENT Reduced levels of Zn2þ have been implicated in PCa genesis. Worldwide epidemiology studies indicate that dietary parameters are of high importance for the development of PCa.19,20 In Japan and China, PCa is a lesscommon disease, and the frequency of PCa is 26 times lower in China than that in the United States.15 UROLOGY 85 (3), 2015
However, after one generation, US-emigrated Japanese get PCa in the same frequency as inborn Caucasians indicating that dietary patterns could have a potential role affecting the development of PCa.21 Daily intake of Zn2þ might be one of these dietary parameters. In Japan, there is an extremely high daily intake of Zn2þ as the dietary tradition comprises a lot of shellfish, oysters, cartilage fish, beans, lentils, and rice, which all have a high Zn2þ content.22 The role of Zn2þ in the pathogenesis of BPH is however unclear, and we have in the present study focused on how Zn2þ regulates BPH SMC growth by measuring DNA replication and protein synthesis in vitro. Our result shows that Zn2þ induces a biphasic doseresponse regarding both DNA replication and protein synthesis of BPH SMCs in a way that could be described as shifting from an agonistic mitogenic agent to an antagonistic mediator in a very sharp and well-defined Zn2þ borderline concentration segment. The divergent effect of Zn2þ in BPH SMC proliferation has to our knowledge not previously been described. The effect was seen in primary BPH SMCs isolated in our laboratory and in commercially available BPH SMCs (results not shown). The antiproliferative or cytotoxic effect of Zn2þ in prostate epithelial cancer cell lines23 PC3, LNCaP, and DU145 are well known24 and consistent with tumor growth inhibition by intratumorally injected Zn2þ.25 Furthermore, in a Swedish cohort, a highly dietary intake of Zn2þ was associated with a reduced risk of PCaspecific mortality providing medical impact regarding Zn2þ and prostate pathogenesis.26 Interestingly, in proliferative studies using rat vascular SMCs, a long-term Zn2þ deprivation generates accelerated proliferation,27 whereas Zn2þ supplement increased human airway SMC apoptosis,28 which supports our results. Thus, we envision that present results may be evaluated in a clinical setting to understand if and how dietary Zn2þ supplement may influence BPH. Important parameters that will add value to such a study are Zn2þ content in the urine, blood plasma, and seminal plasma, in combination with more conventional markers such as PSA and dihydrotestosterone levels, prostate imaging, and function. The main limitation of the present study is the fact that the results are obtained from SMCs in culture. Although the cells are isolated from BPH patients, the cultured conditions may have influenced the cell phenotype and physiology.
CONCLUSION The bell-shaped concentration-response curve for Zn2þ, shown in the present study, suggests that small changes in prostate Zn2þ levels, due to aging, dietary patterns, or inflammatory conditions, may influence the pathogenesis of BPH in opposite effects. These findings warrant further
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