Influence of the Novel Anticancer Agents on the Activity of Outward Rectifier Potassium Currents in Human Prostate Cancer Cell Line - LNCaP

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1 ORIGINAL ARTICLE Influence of the Novel Anticncer Agents on the Activity of Outwrd Rectifier Potssium Currents in Humn Prostte Cncer Cell Line - LNCP Kirn George, R. Mlthi Deprtment of Instrumenttion Engineering, Bioengineering Group, Annmli University, Chidmrm, Tmil Ndu, Indi Astrct Bckground: Phenolic compounds re uiquitous in dietry fruits nd vegetles nd hve een proposed to hve ntioxidnt, nticteril, nti-inflmmtory, nd nticncer effects. Voltge gted K + chnnels (I K ) contriute to vrious cellulr mechnisms involved in the tumor cell progression. Therefore, the modultion of I K current my e potentil trget for cncer therpy. In this study, we electrophysiologiclly chrcterized four phenolic compounds for their modultory ctivities on I K currents. Mterils nd Methods: The whole cell configurtion of the ptch clmp recording technique ws used to record the I K currents from humn prostte cncer line (LNCP). In ddition, 200 µm of phenolic compounds (curcumin, resvertrol, rutin, nd troxerutin) were externlly perfused to the cells. Results: Otined results provide evidence tht resvertrol t 200 µm inhiited more thn hlf of the I K current mplitude. On the other hnd, curcumin, rutin, nd troxerutin did not show ny modultory effect on I K. Conclusion: This study demonstrtes tht not ll the nticncer compounds re effective inhiitors of I K current, ut only few of them. The I K current inhiitors might exhiit effective ntimetsttic properties. Key words: I K current, phenolic compounds, prostte cncer INTRODUCTION Ion chnnels re involved in lrge vriety of physiologicl functions, including excitility, contrction, cell volume regultion, migrtion, cell cycle progression, nd hormone secretion. [1] Most of these ctivities re directly or indirectly proposed to promote the complex process of cncer formtion y vrious mechnisms. [2] Among different types of ion chnnels, norml expression of voltge gted K + chnnels (I K ) is reported in mny cncer cells including prostte, [3] rest, [4] colon, [5] nd glil. [6] In prostte cncer cells, the vrying metsttic properties re distinguished y their ion chnnel chrcteristics. [7] However, it is well documented tht I K chnnel lockers inhiit cell prolifertion in prostte cncer cells. [8] Therefore, I K chnnel is one of the most interesting nd promising therpeutic trgets or prognostic mrker in the fight ginst cncer, ut selective I K chnnel modultors hve hindered criticl verifiction for mny yers. In these spects, investigtion on the influence of novel compounds on the ctivity of I K chnnels seems to e new potentil trget for prostte cncer tretment. Diets rich in phytochemicls hve een ssocited with reduced risk in prostte cncer. [9] Plnt-derived phenolic compounds re secondry metolites in plnts with common romtic ring with one or more hydroxyl groups. These phenolic compounds hve een shown to exhiit ntioxidnt, nti-inflmmtory, ntithromogenic, nd nticncer ctivities. The nticncer ctivities of phenolic compounds hve shown to e extended towrd the inhiition of tumor initition nd progression y its complex moleculr Address for correspondence: R. Mlthi, Deprtment of Instrumenttion Engineering, Bioengineering Group, Annmli University, Chidmrm, Tmil Ndu, Indi. E-mil: vsmlu@gmil.com Received: Revised: Accepted: Asin Journl of Phrmceutics Jul-Sep 2017 (Suppl) 11 (3) S603

2 structure nd chemicl fetures, s well s its ility to interct with severl signling pthwys. [10] Few studies emphsized tht phenolic compounds ffect vriety of ion chnnels nd trnsporters. Especilly curcumin nd resvertrol exerts modultory effect on severl ion chnnels, including voltge gted sodium nd potssium chnnels. [11-13] However, to dte, no studies investigted the modultory effect of phenolic compounds curcumin, resvertrol, rutin, nd troxerutin on voltge gted K + chnnel in humn prostte cncer cell line LNCP. Therefore, in this study, we hve mde n ttempt to investigte the modultory effect of curcumin, resvertrol, rutin, nd troxerutin on I K current in humn prostte cncer cell line LNCP. Regents MATERIALS AND METHODS Curcumin, resvertrol, rutin, nd troxerutin were purchsed from Sigm (St. Louis, MO, USA). The stock solutions were prepred in dimethyl sulfoxide (DMSO) nd were stored in 20 C. All the drug solutions were freshly prepred from stock solutions efore ech set of experiments. The finl concentrtion of DMSO ws <0.1%. Cell culture LNCP cells were sourced from the Ntionl Center for Cell Science (Pune, Indi). LNCP cells were cultured in RPMI medium (HiMedi Lortories, Indi) supplemented with 10% fetl ovine serum nd with 1% ntiiotics (penicillin 100 IU/ml nd streptomycin 100 mg/ml) in humidified incutor t 37 C supplemented with 5% CO 2. Electrophysiology Whole-cell ptch recordings were performed on LNCP cells. Recordings were mde t room temperture. Pipettes were pulled from orosilicte glss cpillries with resistnces of 2-3 MΩ when filled with internl solution. Currents were recorded using n Axon ptch the Axoptch 200B ptch-clmp mplifier (Axon Instruments, Sunnyvle, CA), Digidt 1322A (Axon Instruments), nd PClmp softwre (version 6.0.3, Axon Instruments). The ccess resistnce in our experiments ws pproximtely 5-10 MΩ, nd 40-60% series resistnce compenstion ws chieved. Current records were cquired t 5 khz nd filtered t 2 khz. The externl solution used to record K + currents contined the following (in mm): NCl 140, KCl 5, MgCl 2 1, D-glucose 10, nd HEPES 10, djusted to ph 7.4 with 1 M NOH. The internl solution contined the following (in mm): KCl 140, NCl 5, CCl 2 2, MgCl 2 1, D-glucose 10, nd HEPES 10, djusted to ph 7.2 with 1 M KOH. To evlute the effect of phenolic compounds on the I K currents, the cells were held t voltge of 80 mv, nd memrne potentil ws stepped from 120 mv to +70 mv for 200 ms t 30 s intervls. All the recordings were performed with lek sutrction. The cell under investigtion ws continuously perfused with the externl nd drug solutions using the Octflow (ALA instruments) perfusion system. Sttisticl nlysis The current-voltge curves were nlyzed on ClmpFit ( ), IgorPro (5.04B), nd Microsoft Excel All dt vlues were clculted s men ± stndrd error of men. Sttisticl significnce of pired t-test nd P < 0.05 ws considered. RESULTS The effects of curcumin on I K We chrcterized four phenolic compounds for their modultory ctivities on I K current in humn prostte cncer cell line LNCP. These compounds included flvonoids (curcumin, rutin, nd troxerutin) nd stilenes (resvertrol). We first identified LNCP cell lines tht were expressing I K current. Then, we investigted the effect of curcumin on I K current in LNCP cell line y depolrizing step pulse from 120 to +70 mv for 200 ms t 30 s ws used to record the whole cell I K currents in LNCP cells [Figure 1 - top left]. The representtive current trces of efore nd fter the exposure of 200 µm of curcumin re shown in Figure 1. Current-voltge (I-V) curves for I K currents re constructed from the ctive currents [Figure 1]. The I-V curve confirms tht 200 µm of curcumin did not cuse ny effect on LNCP cells. The effects of rutin on I K We hve screened the effect of rutin on I K current in LNCP cell line y depolrizing step pulse from 120 to +70 mv for 200 ms t 30 s ws used to record the whole cell I K currents in LNCP cells [Figure 2 - top left]. The representtive current trces of efore nd fter the exposure of 200 µm of rutin re shown in Figure 2. The I-V reltionship in the sence nd presence of rutin is constructed [Figure 2]. The I-V curve shows tht 200 µm of rutin did not exert ny effect on LNCP cells. The effects of troxerutin on I K We further exmined the effect of troxerutin on I K current in LNCP cell line y depolrizing step pulse from 120 to +70 mv for 200 ms t 30 s ws used to record the whole cell I K currents in LNCP cells [Figure 3 - top left]. The Asin Journl of Phrmceutics Jul-Sep 2017 (Suppl) 11 (3) S604

3 Figure 1: The effects of curcumin on I K. () Representtive trces of I K currents recorded in the presence nd sence of curcumin 200 µm in LNCP cells. () The current voltge (I-V) reltionships of I K currents in the sence nd presence of curcumin in LNCP cells. Dt re plotted s men± stndrd error of men (n > 7) Figure 2: The effects of rutin on I K. () Representtive trces of I K currents recorded in the presence nd sence of rutin 200 µm in LNCP cells. () The current voltge (I-V) reltionships of I K currents in the sence nd presence of rutin in LNCP cells. Dt re plotted s men ± stndrd error of men (n > 7) representtive current trces of efore nd fter the exposure of 200 µm of troxerutin re shown in Figure 3. Current voltge (I-V) curves for I K currents re plotted from the ctive currents [Figure 3]. However, 200 µm of troxerutin lso did not show ny sign of I K current inhiition. The effects of resvertrol on I K All the three flvonoids (curcumin, rutin, nd troxerutin) did not cuse ny inhiitory ction on I K currents in LNCP cells. Therefore, we further investigted the effect of resvertrol stilenes on I K current in LNCP cell line y depolrizing step pulse from 120 to +70 mv for 200 ms t 30 s ws used to record the whole cell I K currents in LNCP cells [Figure 4 - top left]. The superimposed current trces of efore nd fter the perfusion of 200 µm of resvertrol re shown in Figure 4. Current-voltge (I-V) curves for I K currents re plotted from the ctive currents [Figure 4]. The I-V curve confirms tht 200 µm of resvertrol induce lockde in LNCP cells. The pek current density plot lso confirms tht 200 µm resvertrol locked I K currents in LNCP cells [Figure 4c]. However, this effect ws reversile immedite fter the perfusion of externl solution. Altogether, these results show tht 200 µm of resvertrol locked I K currents to more thn hlf of the I K. DISCUSSION In this study, we investigted the effect of phenolic compounds (curcumin, resvertrol, rutin, nd troxerutin) on the I K chnnel in humn prostte cncer line-lncp. Our mjor findings were tht mong the four phenolic compounds, resvertrol inhiited I K current nd the other three compounds curcumin, rutin, nd troxerutin did not show ny effect on I K. These findings suggest tht the I K current modultory effect of ny nticncer compounds firmly depends on their structurl ffinity with I K chnnel proteins. Flvonoids re the most importnt clss of phenolic compounds. The nticncer properties of flvonoids hve een reported in different types of cell lines. Despite lrge numer of flvonoids, we hve selected curcumin, rutin, nd troxerutin for investigting the I K chnnel ctivity Asin Journl of Phrmceutics Jul-Sep 2017 (Suppl) 11 (3) S605

4 Figure 3: The effects of troxerutin on I K. () Representtive trces of I K currents recorded in the presence nd sence of troxerutin 200 µm in LNCP cells. () The current-voltge (I-V) reltionships of I K currents in the sence nd presence of troxerutin in LNCP cells. Dt re plotted s men ± stndrd error of men (n > 7) c Figure 4: The effects of resvertrol on I K. () Representtive trces of I K currents recorded in the presence nd sence of resvertrol 200 µm in LNCP cells. () The current voltge (I-V) reltionships of I K currents in the sence nd presence of resvertrol in LNCP cells. (c) Reltive pek current density in the sence nd presence of resvertrol in LNCP cells. *P < 0.05 compred with the pek current mplitudes in the sence of resvertrol. Dt re plotted s men ± stndrd error of men (n > 7) in LNCP cells. Among these three flvonoids, curcumin hs shown to exert nticncer properties in mny different types of cncer y modulting multiple signling pthwys. Bsed on its complex moleculr structure, curcumin modulte severl ion chnnels including voltge-gted potssium chnnels. [14] Curcumin locked I K current in rit coronry Asin Journl of Phrmceutics Jul-Sep 2017 (Suppl) 11 (3) S606

5 rteril smooth muscle cells, [15] inhiits Kv 1.3 in effector memory T cells, [16] nd locked Kv1.4 in ovine drenl zon fscicult cells. [12] However, our otined results show tht 200 µm of curcumin did not cuse ny modultory effect on I K current in humn prostte LNCP cells. The other two flvonoids rutin nd troxerutin exerted nticncer properties in severl cncer cell lines through modulting multiple signling pthwys. [17] However, in prostte cncer cells rutin exhiited very low level of cell prolifertion inhiition. [18] Our present study results revel tht rutin nd troxerutin did not cuse ny chnges in the I K current properties in humn prostte LNCP cells. Altogether these results suggest tht the I K current inhiition depends on the interction etween the I K chnnel protein nd the chemicl structure of the compound. Resvertrol (3,4,5-trihydroxystillene), nturl polyphenolic compound commonly present in nturl products such s grpes, erries, penuts, nd soyens hs een suggested to tret vriety of cncer types including prostte cncer. [19] It hs een reported to modulte severl signling pthwys to control the growth of cncer cells. [20] In ddition, resvertrol hs shown to ttenute severl voltge gted ion chnnels in severl cell lines. Minly resvertrol inhiited voltge gted sodium current in rt prostte cncer cell line (MAT-LyLu) [11] nd Kv1.3 in humn lymphocytes. [21] Our otined result shows tht perfusion of 200 µm of resvertrol inhiited I K current in LNCP cells. When compred to control, the I-V curve shows tht 200 µm of resvertrol inhiited more thn hlf of the I K current. The pek current lso shows significnt inhiition of I K. However, this inhiitory effect ws reversile immedite fter the perfusion of externl solution. These results suggest tht resvertrol effectively inhiit the I K current in humn prostte cncer cell line LNCP. CONCLUSION The results provide evidence tht not ll the tested phenolic compounds pplied t concentrtion of 200 µm ppered to e n inhiitor of I K. This confirms tht not ll the nticncer compounds hve the ility to inhiit or interct with I K chnnel ut only few of them. Our otined results suggest tht, when compred to flvonoids (curcumin, rutin, nd troxerutin), stilenes (resvertrol) re effective inhiitor of I K current in humn prostte cncer cell line LNCP. However, we cnnot ignore tht these tested compounds re incompetent in locking ion chnnels. The tested compounds my inhiit I K current t higher dosges. Further, studies re required on higher dosges of these compounds nd other types of cell lines. ACKNOWLEDGMENT We grtefully cknowledge the funding support from University Grnt Commission, New Delhi, for providing grnt in the form of Bsic Scientific Reserch Fellowship for meritorious students (JRF) (No.F.7-376/2012(BSR)). REFERENCES 1. Cmcho J. Ether à go-go potssium chnnels nd cncer. Cncer Lett 2006;233: Schönherr R. Clinicl relevnce of ion chnnels for dignosis nd therpy of cncer. J Memr Biol 2005;205: Lnido ME, Frser SP, Djmgoz MB. Voltgegted K(+) chnnel ctivity in humn prostte cncer cell lines of mrkedly different metsttic potentil: Distinguishing chrcteristics of PC-3 nd LNCP cells. Prostte 2001;46: Sun T, Song ZG, Jing DQ, Nie HG, Hn DY. Docetxel modultes the delyed rectifier potssium current (IK) nd ATP-sensitive potssium current (IKATP) in humn rest cncer cells. J Memr Biol 2015;248: Ousingswt J, Spitzner M, Puntheernurk S, Terrccino L, Tornillo L, Buendorf L, et l. Expression of voltge-gted potssium chnnels in humn nd mouse colonic crcinom. Clin Cncer Res 2007;13: Chin LS, Prk CC, Zitny KM, Sinh M, DiPtri AJ Jr, Perillán P, et l. 4-minopyridine cuses poptosis nd locks n outwrd rectifier K + chnnel in mlignnt strocytom cell lines. J Neurosci Res 1997;48: Prevrsky N, Skrym R, Bidux G, Flourkis M, Shu Y. Ion chnnels in deth nd differentition of prostte cncer cells. Cell Deth Differ 2007;14: Adul M, Hoosein N. Expression nd ctivity of potssium ion chnnels in humn prostte cncer. Cncer Lett 2002;186: Bommreddy A, Eggleston W, Prelewicz S, Antl A, Witczk Z, McCune DF, et l. Chemoprevention of prostte cncer y mjor dietry phytochemicls. Anticncer Res 2013;33: Annthrju PG, Gowd PC, Vimlmike MG, Mdhunpntul SV. An overview on the role of dietry phenolics for the tretment of cncers. Nutr J 2016;15: Frser SP, Peters A, Fleming-Jones S, Mukhey D, Djmgoz MB. Resvertrol: Inhiitory effects on metsttic cell ehviors nd voltge-gted N(+) chnnel ctivity in rt prostte cncer in vitro. Nutr Cncer 2014;66: Liu H, Dnthi SJ, Enyert JJ. Curcumin potently locks Kv1.4 potssium chnnels. Biochem Biophys Res Commun 2006;344: Djmgoz M, Isilen B. Dietry compounds s nti-cncer gents: A preliminry evlution of ion chnnels nd memrne excitility s possile trget mechnisms. Turk Biochem 2006;31: Zhng X, Chen Q, Wng Y, Peng W, Ci H. Effects of curcumin on ion chnnels nd trnsporters. Front Physiol 2014;5: Hong DH, Son YK, Choi IW, Prk WS. The inhiitory Asin Journl of Phrmceutics Jul-Sep 2017 (Suppl) 11 (3) S607

6 effect of curcumin on voltge-dependent K+ chnnels in rit coronry rteril smooth muscle cells. Biochem Biophys Res Commun 2013;430: Lin YT, Yng XF, Wng ZH, Yng Y, Yng Y, Shu YW, et l. Curcumin serves s humn kv1.3 locker to inhiit effector memory T lymphocyte ctivities. Phytother Res 2013;27: Thoms NS, George K, Arivlgn S, Mni V, Siddique AI, Nmsivym N. The in vivo ntineoplstic nd therpeutic efficcy of troxerutin on rt preneoplstic liver: Biochemicl, histologicl nd cellulr spects. Eur J Nutr 2016;113: Knowles LM, Zigrossi DA, Tuer RA, Hightower C, Milner JA. Flvonoids suppress ndrogen-independent humn prostte tumor prolifertion. Nutr Cncer 2000;38: Singh SK, Bnerjee S, Acost EP, Lillrd JW, Singh R. Resvertrol induces cell cycle rrest nd poptosis with docetxel in prostte cncer cells vi p53/ p21waf1/cip1 nd p27kip1 pthwy. Oncotrget 2017;8: Selvrj S, Sun Y, Sukumrn P, Singh BB. Resvertrol ctivtes utophgic cell deth in prostte cncer cells vi downregultion of STIM1 nd the mtor pthwy. Mol Crcinog 2016;55: Teisseyre A, Michlk K. Inhiition of the ctivity of humn lymphocyte Kv1.3 potssium chnnels y resvertrol. J Memr Biol 2006;214: Source of Support: UGC BSR SRF Fellowship, New Delhi, Indi. Conflict of Interest: None declred. Asin Journl of Phrmceutics Jul-Sep 2017 (Suppl) 11 (3) S608