Effects of TRPM8 on the proliferation and angiogenesis of prostate cancer PC-3 cells in vivo

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1 ONCOLOGY LETTERS 2: , 2011 Effects of TRPM8 on the prolifertion nd ngiogenesis of prostte cncer PC-3 cells in vivo GUANGBIN ZHU, XINGHUAN WANG, ZHONGHUA YANG, HONG CAO, ZHE MENG, YONGZHI WANG nd DONG CHEN Deprtment of Urology, Zhongnn Hospitl, Wuhn University, Wuhn, Hubei , P.R. Chin Received My 16, 2011; Accepted August 30, 2011 DOI: /ol Abstrct. Prostte cncer is significnt helth concern. In the erly stges, prostte cncer cells depend on ndrogens for growth nd survivl, hence ndrogen-bltion therpy t this time my be effective in cusing tumor regression. However, tretment options for dvnced hormone-refrctory prostte cncers re still reltively inefficient. This study imed to investigte the possible effects of TRPM8 on the prolifertion nd ngiogenesis of ndrogen-independent cncer PC-3 cells in vivo. Thirty mle nude mice were divided into three groups: the PC-3, nd groups. PC-3, PC-3- vector nd cells were respectively inoculted in the right flnk to estblish trnsplnted tumor model. The mice were treted dily for four weeks nd ech group ws exmined by histology nd immunohistochemicl stining for CD34, FAK nd PCNA. A CD34 mrked microvsculr density (MVD) test ws performed. Western blot nlysis ws used to detect the VEGF protein expression level. Compred to the PC-3 nd groups, the group reveled decrese in tumor volume (P=0.000 nd P=0.000, respectively), MVD (P=0.045 nd P=0.041, respectively), VEGF (P=0.000 nd P=0.000, respectively), FAK nd PCNA. The correltion between MVD nd VEGF ws positive (r=0.419; P=0.021). These dt show tht the overexpression of TRPM8 hd negtive effect on the prolifertion nd ngiogenesis progression of PC-3 cells in vivo. Introduction Prostte cncer (PC) is mjor helth problem, ccounting for qurter of the new cncer cses dignosed in dult mles in Americ ech yer, nd ccounting for pproximtely 9% of cncer-relted mortlity in the sme popultion (1). In the erly stges, prostte cncer cells depend on ndrogens for growth nd survivl, hence ndrogen-bltion therpy t this Correspondence to: Dr Xinghun Wng, Deprtment of Urology, Zhongnn Hospitl, Wuhn University, 169 Donghu Rod, Wuhn, Hubei , P.R. Chin E-mil: urologistwxh@gmil.com Key words: TRPM8, prolifertion, ngiogenesis, prostte cncer time my be effective in cusing tumor regression. However, tretment options for dvnced hormone-refrctory prostte cncers (HRPC) re still reltively inefficient (2). The role of C 2+ is well estblished in the mjority of the cell signling pthwys involved in crcinogenesis (3). Clciumpermeble chnnels re potentil cndidtes for involvement in C 2+ homeostsis in prostte cncer cells. One trnsient receptor potentil (TRP) superfmily of ction chnnels is of prticulr interest. The humn trpm8 gene, initilly known s trp-p8, hs been shown to be minly expressed in the prostte nd is overexpressed in prostte cncer (4). The precise physiologicl function of the TRPM8 chnnel in norml nd cncer prostte tissue remins unknown. TRPM8 expression is mrkedly upregulted in PC nd in other tumors, suggesting significnt role in crcinogenesis (4). It hs been shown tht nti-ndrogen therpy gretly reduces the expression of TRPM8, suggesting tht TRPM8 is regulted by ndrogens (5). TRPM8 expression-silencing experiments using smll interfering RNA (sirna) suggested tht C 2+ influx through this chnnel plys n essentil role in cellulr C 2+ homeostsis in prostte epithelil cells nd is involved in cell survivl (6). Our previous study reveled tht PC-3 cells express n extremely low level of TRPM8, nd tht overexpression of TRPM8 hs negtive effect on the prolifertion nd mlignnt progression of PC-3 cells in vitro (7). However, upon dministrtion of nti-ndrogen therpy, the prostte epithelil cells downregulte the expression of ndrogen receptor (AR) nd, consequently, tht of TRPM8 mrna. Prostte cncer nd metstsis then progress into n ndrogen-independent (AI) stge, resulting in cncer relpse with more ggressive phenotype. It is well known tht ngiogenesis is essentil for tumor progression nd metstsis (8). In reltion to PC, it hs lso been suggested tht the degree of tumor ngiogenesis is correlted to clinicl stge (9). Vrious endothelil growth fctors hve been shown to ply crucil roles in tumor ngiogenesis. Vsculr endothelil growth fctor (VEGF) is one of the most potent nd specific ngiogenic fctors. Immunohistochemicl studies hve reveled tht PC cells produce VEGF (10,11) nd tht VEGF expression correltes with microvessel density (MVD) nd tumor progression (12). This study ws designed to investigte the possible effects of TRPM8 on the prolifertion nd ngiogenesis of ndrogenindependent cncer PC-3 cells in vivo.

2 1214 ZHU et l: EFFECTS OF TRPM8 ON PROLIFERATION AND ANGIOGENESIS OF PC-3 CELLS Mterils nd methods Cell culture. PC-3 cells were purchsed from the Americn Type Culture Collection (ATCC, Mnsss, VA, USA). PC-3-m8 cells were previously estblished in our lbortory. Cells were cultured s previously described (7). Animls. Thirty 5-week old mle nude mice (weight rnge g) were obtined from the Hubei Provincil Experimentl Animl Center, Chin. All niml study protocols were pproved by interntionlly ccepted principles nd the Guidelines for the Cre nd Use of Lbortory Animls of Wuhn University. Animl grouping. The nimls were rndomized into 3 groups: A (PC-3 cell group), group B ( group) nd group C ( group). Tumor models. PC-3, nd cells growing exponentilly were ech implnted into 10 mle nude mice by subcutneous (SC) injection of 1x10 6 cells (in 200 µl phosphte-buffered sline) into the right flnk. Observtion on growing condition of mice. The inoculted mice were fed in the Experimentl Animl Center of Wuhn University nd monitored dily for clinicl signs. Tumor mesurements were performed every three dys nd the tumor volume ws clculted ccording to the formul: V=(π/6)(d1 d2) 3/2 (13), where d1 nd d2 re perpendiculr tumor dimeters. Smple collection. Twenty-eight dys fter the inocultion of cells, ech mouse ws injected with 10% chlorl hydrte for hypernesthesi. The mice were scrificed by decpittion nd tumors were removed from the body. One section of the tumor ws fixed in formlin for prffin embedding nd one section ws snp-frozen in liquid nitrogen nd stored t -80 C. H&E stining ssy. Prffin-embedded tissues were cut into 4 µm slices nd deprffinted in dimethylbenzene for 5-10 min. Then the tissues were put into 100, 95, 85 nd 70% lcohol for 2-5 min in turn nd finlly wshed with distilled wter nd immersed in stining solution. Following hemtoxylin stining for 5-15 min, the excess stin solution on the slides ws wshed off, nd color seprtion with 0.5-1% hydrochloride lcohol ws performed for pproximtely 10 sec. After wshing in running wter for min, the tissues were stined by % eosin for 1-5 min. The tissues were then dehydrted with 75, 85, 95 nd 100% lcohol for 2-3 min in turn prior to hyliniztion with dimethylbenzene twice for pproximtely 10 min in totl. Finlly, neutrl gum ws dropped onto the slip, nd then the slip ws covered by slide. As result, nuclei were stined blue nd cytoplsm nd collgen fibers were stined vrious shdes of red or pink. Immunohistochemicl ssy. The streptvidin-peroxidsebiotin (SP) method ws used for immunohistochemistry. The slides were deprffinized conventionlly nd were immersed in 3% H 2 O 2 for 10 min to block endogenous peroxidse. Following ntigen retrievl by microwve, newborn clf serum ws dded for blocking for 10 min. The primry ntibody (1:50) ws then dded for incubtion overnight (4 C) nd secondry ntibodies were dded for incubtion for 20 min t room temperture. Then streptvidin-biotin-peroxidse solution ws used for incubtion for 30 min nd 3,3'-diminobenzidine (DAB) ws dded to the chlorte for 15 min. This ws followed by hemtoxylin stining, dehydrtion nd hyliniztion, nd the slip ws then covered. CD34 mrked MVD test. CD34 is expressed in vsculr endothelil cells, tumor cytoplsm or membrne nd is used s specific mrker of vsculr endothelil cells. By immunohistochemicl stining it revels distribution of brown or light brown solid bud-like or cord-like blood vessels. Low mgnifiction (x100) ws used to review the microvsculr stining in ech section nd determine the mximum microvsculr stining regions, nd then the vsculr endothelil cells or cell groups which ppered brown t high mgnifiction (x200) were counted. Ech cell group counted s independent micrngium on condition of n obvious distinction from neighboring micrngium nd tumorous cell. Five counts of micrngium of ech slide t high mgnifiction vision were recorded, nd the verge ws tken s the MVD. Detection of VEGF protein expression by Western blotting. One hundred milligrms of tumor tissue ws obtined. The protein lyste ws dded ccording to qulity/volume (w/v, mg/µl) t concentrtion of 1:5, fully homogenized, plced on ice for 30 min nd then centrifuged for 5 min t 260 x g. The totl protein content ws mesured using bicinchoninic cid (BCA) kit. The protein expression of VEGF nd β-ctin ws ssyed using Western blot nlysis using nti-vegfspecific nd nti-β-ctin-specific ntibodies (Snt Cruz Biotechnology, Snt Cruz, CA, USA). Sttisticl nlysis. Mesurement dt were shown s the men ± SD. s were compred with the one-wy ANOVA nlysis. The Spermn coefficent ws used to nlyse the correltion between MVD nd VEGF. P<0.05 ws considered to be sttisticlly significnt. All of the dt were nlyzed with SPSS Results Observtion on mice growing conditions nd behvior. In group C, the response to stimuli, ctivity level, nd ppetite of ech mouse ws similr to tht in groups A nd B, nd the body weight did not chnge significntly (dt not shown). Observtion on tumor growth. The tumor formtion rte in ech group ws 100%. The tumor growth ws infiltrtive with round or ovl shpe. The tumor volume in group C ws less thn tht of groups A nd B, nd there ws difference in the volume of the grft between group C nd group A or B (P=0.000 nd P=0.000, respectively; Fig. 1). Histopthologic observtion. Following H&E stining, the trnsplnted tumors in ech group reveled glnds of unequl size, which integrted to become lmellr, solid-like or cnelike ccompnied with diffuse infiltrtion when observed

3 ONCOLOGY LETTERS 2: , Tble I. Comprison of Gleson score in ech group (vlues re presented s the men ± SD). Gleson score PC-3 8.7± ± ±0.52 b Compred with PC-3 group, P>0.05 (P=1); b Compred with PC-3 nd group, P>0.05 (P=0.186). Tble II. MVD vlues of tumor in ech group (vlues re presented s the men ± SD). MVD in tumor PC ± ± ± 6.04 b,c Compred with PC-3 group, P>0.05 (P=0.983); b Compred with PC-3 group, P<0.05 (P=0.045); c Compred with group, P<0.05 (P=0.041). Tble III. Expression of VEGF protein in ech group (vlues re presented s the men ± SD). VEGF expression in tumor PC ± ± ± 0.11 b,c Compred with PC-3 group, P>0.05 (P=0.614); b Compred with PC-3 group, P<0.01 (P=0.000); c Compred with group, P<0.01 (P=0.000). under light microscope. Pthologicl cryokinesis nd lowdegree differentition implied high degree of mlignncy. There ws no difference in the Gleson score between the three groups (P>0.05; Tble I). MVD count of the different groups. To evlute tumor neovsculriztion, we immunostined tissue smples using CD34 ntibody (Abcm, Cmbridge, UK). CD34 is cell surfce silomucin widely used s mrker of most vsculr endothelil cells, including those of cpillries in the mjority of tissues. In the present study, the MVD in groups A nd B ws found to be higher thn tht in group C (P=0.045 nd P=0.041, respectively; Fig. 2G, H nd I; Tble II). Expression of VEGF protein in ech group. All groups reveled VEGF protein expression. The expression of VEGF protein in groups A nd B ws higher thn tht in group C (P=0.000 nd P=0.000, respectively; Fig. 3). Figure 1. Time course chnges of tumor growth in ech group. The tumor volume in the PC-3 nd groups ws higher thn tht in the PC-3- TRPM8 group. *P<0.05. Reltionship between MVD nd VEGF protein expression in ech group. MVD nd VEGF expression in tumor tissue hd correltion coefficient of r=0.419 (P=0.021) for ll three groups. Expression of focl dhesion kinse (FAK) nd proliferting cell nucler ntigen (PCNA) in ech group. FAK is non-receptor protein tyrosine kinse tht regultes dhesiondependent cell signling (14). In prostte cncer, FAK is known primrily for its role in cell motility nd cytoskeletl rerrngement, s supported by in vivo nd in vitro evidence (15). PCNA is nucler protein synthesized in the G1/S phse nd plys significnt role in DNA repliction. PCNA expression is low in non-dividing cells, but increses gretly in proliferting nd trnsformed cells. In the present study, nti-fak-py397-specific (Biosource, Cmrillo, CA, USA) nd nti-pcna-specific (Abcm) ntibodies were used. The expression of FAK nd PCNA in groups A nd B ws higher thn tht in group C (Fig. 2A, B, C, J, K nd L). Discussion Recent studies hve focused on the role of TRPM8, rendering it novel moleculr trget potentilly useful in the dignosis nd tretment of PC. The chnnel is ctivted by voltge, cold tempertures nd cooling compounds, such s menthol nd icilin (16). Our previous results indicted tht the overexpression of TRPM8 hs negtive effect on the prolifertion nd mlignnt progression of PC-3 cells in vitro (7). Similrly, Gkik et l (17) hve demonstrted tht PC-3 cells rtificilly overexpressing TRPM8 hve reduced motility, suggesting possible connection between TRPM8 ctivity nd reduced metsttic potentil. In this study, the tumor volume in group C ws less thn tht in groups A nd B, suggesting tht the overexpression of TRPM8 possibly hs negtive effect on the prolifertion of PC-3 cells in vivo. It is well estblished tht the growth nd dissemintion of solid tumors is dependent on ngiogenesis (18). Humn VEGF mrna is trnscribed from eight exons of single gene nd is

1216 ZHU et l: EFFECTS OF TRPM8 ON PROLIFERATION AND ANGIOGENESIS OF PC-3 CELLS Figure 2. H&E, PCNA, CD34 nd FAK stining of tumor tissue (mgnifiction, x200).

There ws no difference in Gleson score mong the three groups. (G-I) Blood vessels were stined using nti-cd34 ntibody; MVD of the PC-3 nd groups (G nd H) ws higher thn tht of the group (I) (P<0.05).

Expression of VEGF protein in the PC-3 nd groups ws higher thn tht in the group (P<0.05).

4 1216 ZHU et l: EFFECTS OF TRPM8 ON PROLIFERATION AND ANGIOGENESIS OF PC-3 CELLS Figure 2. H&E, PCNA, CD34 nd FAK stining of tumor tissue (mgnifiction, x200). (A-C) PCNA expression in tumor tissue; compred to the PC-3 nd groups (A nd B), PCNA expression ws decresed in the group (C). (D-F) H&E stining of tumor tissue. There ws no difference in Gleson score mong the three groups. (G-I) Blood vessels were stined using nti-cd34 ntibody; MVD of the PC-3 nd groups (G nd H) ws higher thn tht of the group (I) (P<0.05). (J-L) FAK expression in tumor tissue; compred to the PC-3 nd groups (J nd K), FAK expression ws decresed in the group (L). Figure 3. The PC-3, nd groups exhibit VEGF protein expression. Expression of VEGF protein in the PC-3 nd groups ws higher thn tht in the group (P<0.05). lterntively spliced into t lest six mrnas, which give rise to the mture proteins of 121, 145, 165, 183, 189 nd 206 mino cids. VEGF 121 nd VEGF 165 re the best chrcterized nd re the most bundnt in norml tissues, including blood vessels. As with most tumors, prostte tumors overexpress VEGF, thereby promoting the development of tumor neovsculriztion (19). Certin studies using immunohistochemistry hve reported n incresed expression of totl hvegf protein in humn prostte tumors, when compred with norml tissue or preinvsive prostte lesions (20). Our results reveled protein expression of VEGF 165 in ech group, but VEGF 121 ws not detected. This lck of detection my be becuse VEGF 165 hs greter moleculr weight nd is expressed more widely. Furthermore, in this study, the VEGF expression level of group C ws lower thn tht of groups A nd B (P=0.00 nd P=0.00, respectively). MVD is prognostic mrker for vrious tumors, including prostte cncer (21). In prostte cncer, MVD is correlted with the development of metstses, clinicl stge nd overll ptient survivl (22). In ddition, the progression of prostte cncer into the AI stte hs been shown to be ssocited with incresed ngiogenesis (23); thus, ntingiogenic therpy my

5 ONCOLOGY LETTERS 2: , be possible mens of improving tretment for ptients with HRPC. MVD is quntittive description of ngiogenesis. In this study, the MVD of group C ws significntly decresed compred to tht of groups A nd B (P=0.045 nd P=0.041, respectively; Fig. 2G, H nd I; Tble II), which, coupled with the results of the expression of VEGF, indicted tht TRPM8 my hve negtive effect on the ngiogenesis of PC-3 cells in vivo. PCNA is nucler protein nd plys significnt role in DNA repliction. The expression level of PCNA is closely correlted to the cell stte, which mens tht the level of PCNA expression correltes with the degree of mlignncy, invsion nd metstsis in cncer cells. Thus, PCNA is significnt evlutive mrker for tumor growth nd prognosis (24). FAK is non-receptor protein tyrosine kinse tht regultes dhesion-dependent cell signling (14). FAK expression is incresed in prostte cncer cell lines (25), nd n incresed expression correltes with enhnced motility nd tumorigenicity (26). Our previous study indicted tht overexpression of TRPM8, through inctivtion of FAK, reduced the motility of PC-3 cells in vitro (7). In this study, we hve further shown tht TRPM8 inhibits the expression of FAK in vivo. The results of this study hve shown tht the expression of FAK nd PCNA in group C ws lower thn tht in groups A nd B (Fig. 2J, K nd L). Coupled with the results of tumor volume in ech group, the TRPM8 chnnel my medite the repir nd synthesis of DNA, nd my, through inctivtion of FAK, hve negtive effect on prolifertion. However, there ws no difference in the Gleson score of ech group, nd the reson for this remins to be determined, but possibly lies in the limited time tken for the grft to grow. In conclusion, this study demonstrtes tht the overexpression of TRPM8 hd negtive effect on the prolifertion nd ngiogenesis progression of PC-3 cells in vivo. Therefore, for ptients in the AI stge, lthough there is currently no successful therpy, the ctivtion of the existing chnnels or the overexpression of the chnnel my serve s potentil lterntive tretment nd should be further investigted. Acknowledgements This work ws supported by the Fundmentl Reserch Funds for the Centrl Universities (no ). References 1. Jeml A, Siegel R, Wrd E, Ho Y, Xu J nd Thun MJ: Cncer sttistics, CA Cncer J Clin 59: , Mrtel CL, Gumerlock PH, Meyers FJ nd Lr PN: Current strtegies in the mngement of hormone refrctory prostte cncer. Cncer Tret Rev 29: , Berridge MJ, Lipp P nd Bootmn MD: The verstility nd universlity of clcium signlling. Nt Rev Mol Cell Biol 1: 11-21, Tsvler L, Shpero MH, Morkowski S nd Lus R: Trp-p8, novel prostte-specific gene, is up-regulted in prostte cncer nd other mlignncies nd shres high homology with trnsient receptor potentil clcium chnnel proteins. Cncer Res 61: , Henshll SM, Afr DE, Hiller J, et l: Survivl nlysis of genome-wide gene expression profiles of prostte cncers identifies new prognostic trgets of disese relpse. Cncer Res 63: , Zhng L nd Brritt GJ: Evidence tht TRPM8 is n ndrogendependent C 2+ chnnel required for the survivl of prostte cncer cells. Cncer Res 64: , Yng ZH, Wng XH, Wng HP nd Hu LQ: Effects of TRPM8 on the prolifertion nd motility of prostte cncer PC-3 cells. Asin J Androl 11: , Folkmn J: Tumor ngiogenesis: therpeutic implictions. N Engl J Med 285: , Brwer MK, Deering RE, Brown M, Preston SD nd Bigler SA: Predictors of pthologic stge in prosttic crcinom. The role of neovsculrity. Cncer 73: , Ferrer FA, Miller LJ, Andrwis RI, et l: Vsculr endothelil growth fctor (VEGF) expression in humn prostte cncer: in situ nd in vitro expression of VEGF by humn prostte cncer cells. J Urol 157: , Jckson MW, Bentel JM nd Tilley WD: Vsculr endothelil growth fctor (VEGF) expression in prostte cncer nd benign prosttic hyperplsi. J Urol 157: , Borre M, Nerstrom B nd Overgrd J: Assocition between immunohistochemicl expression of vsculr endothelil growth fctor (VEGF), VEGF-expressing neuroendocrine-differentited tumor cells, nd outcome in prostte cncer ptients subjected to wtchful witing. Clin Cncer Res 6: , Wrri AM, Huovinen RL, Line AM, Mrtikinen PM nd Hrkonen PL: Apoptosis in toremifene-induced growth inhibition of humn brest cncer cells in vivo nd in vitro. J Ntl Cncer Inst 85: , Prsons JT, Slck-Dvis J, Tilghmn R nd Roberts WG: Focl dhesion kinse: trgeting dhesion signling pthwys for therpeutic intervention. Clin Cncer Res 14: , Chng YM, Kung HJ nd Evns CP: Nonreceptor tyrosine kinses in prostte cncer. Neoplsi 9: , Voets T, Owsinik G nd Nilius B: Trpm8. Hndb Exp Phrmcol , Gkik D, Flourkis M, Lemonnier L nd Prevrsky N: PSA reduces prostte cncer cell motility by stimulting TRPM8 ctivity nd plsm membrne expression. Oncogene 29: , Liott LA, Steeg PS nd Stetler-Stevenson WG: Cncer metstsis nd ngiogenesis: n imblnce of positive nd negtive regultion. Cell 64: , Ferrer FA, Miller LJ, Lindquist R, et l: Expression of vsculr endothelil growth fctor receptors in humn prostte cncer. Urology 54: , Mzzucchelli R, Montironi R, Sntinelli A, Lucrini G, Pugnloni A nd Bigini G: Vsculr endothelil growth fctor expression nd cpillry rchitecture in high-grde PIN nd prostte cncer in untreted nd ndrogen-blted ptients. Prostte 45: 72-79, Hollingsworth HC, Kohn EC, Steinberg SM, Rothenberg ML nd Merino MJ: Tumor ngiogenesis in dvnced stge ovrin crcinom. Am J Pthol 147: 33-41, Weidner N, Crroll PR, Flx J, Blumenfeld W nd Folkmn J: Tumor ngiogenesis correltes with metstsis in invsive prostte crcinom. Am J Pthol 143: , Kosk T, Miyjim A, Tkym E, et l: Angiotensin II type 1 receptor ntgonist s n ngiogenic inhibitor in prostte cncer. Prostte 67: 41-49, Tnk S, Hrum K, Ttsut S, et l: Proliferting cell nucler ntigen expression correltes with the metsttic potentil of submucosl invsive colorectl crcinom. Oncology 52: , Lcoste J, Aprikin AG nd Chevlier S: Focl dhesion kinse is required for bombesin-induced prostte cncer cell motility. Mol Cell Endocrinol 235: 51-61, Slck JK, Adms RB, Rovin JD, Bissonette EA, Stoker CE nd Prsons JT: Altertions in the focl dhesion kinse/src signl trnsduction pthwy correlte with incresed migrtory cpcity of prostte crcinom cells. Oncogene 20: , 2001.

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