Betel Nut Arecoline Induces Different Phases of Growth Arrest Between

International Journal of Molecular Sciences

Article Betel Nut Arecoline Induces Diﬀerent Phases of Growth Arrest between Normal and Cancerous Prostate Cells through the Reactive Oxygen Species Pathway

Li-Jane Shih 1,2,3, Jia-Yu Wang 1, Jing-Yao Jheng 1, An-Ci Siao 1 , Yen-Yue Lin 1,4,5,* , Yi-Wei Tsuei 4, Yow-Chii Kuo 6, Chih-Pin Chuu 7 and Yung-Hsi Kao 1,* 1 Department of Life Sciences, National Central University, Jhongli, Taoyuan 320, Taiwan; [email protected] (L.-J.S.); [email protected] (J.-Y.W.); [email protected] (J.-Y.J.); [email protected] (A.-C.S.) 2 Graduate Institute of Medical Science, National Defense Medical Center, Taipei 114, Taiwan 3 Department of Medical Laboratory, Taoyuan Armed Forces General Hospital, Longtan, Taoyuan 325, Taiwan 4 Department of Emergency, Taoyuan Armed Forces General Hospital, Longtan, Taoyuan 325, Taiwan; [email protected] 5 Department of Emergency Medicine, Tri-Service General Hospital, National Defense Medical Center, Taipei 114, Taiwan 6 Department of Gastroenterology, Taiwan Landseed Hospital, Taoyuan 320, Taiwan; [email protected] 7 Institute of Cellular and System Medicine, National Health Research Institutes, Miaoli 350, Taiwan; [email protected] * Correspondence: [email protected] (Y.-Y.L.); [email protected] (Y.-H.K.); Tel.: +886-3-480-1604 (Y.-Y.L.); +886-3-426-0839 (Y.-H.K.); Fax: +886-3-479-9595 (Y.-Y.L.); +886-3-422-8482 (Y.-H.K.) Received: 4 November 2020; Accepted: 2 December 2020; Published: 3 December 2020 Abstract: Prostate cancer (PCa) is a reproductive system cancer in elderly men. We investigated the effects of betel nut arecoline on the growth of normal and cancerous prostate cells. Normal RWPE-1 prostate epithelial cells, androgen-independent PC-3 PCa cells, and androgen-dependent LNCaP PCa cells were used. Arecoline inhibited their growth in dose- and time-dependent manners. Arecoline caused RWPE-1 and PC-3 cell cycle arrest in the G2/M phase and LNCaP cell arrest in the G0/G1 phase. In RWPE-1 cells, arecoline increased the expression of cyclin-dependent kinase (CDK)-1, p21, and cyclins B1 and D3, decreased the expression of CDK2, and had no effects on CDK4 and cyclin D1 expression. In PC-3 cells, arecoline decreased CDK1, CDK2, CDK4, p21, p27, and cyclin D1 and D3 protein expression and increased cyclin B1 protein expression. In LNCaP cells, arecoline decreased CDK2, CDK4, and cyclin D1 expression; increased p21, p27, and cyclin D3 expression; had no effects on CDK1 and cyclin B1 expression. The antioxidant N-acetylcysteine blocked the arecoline-induced increase in reactive oxygen species production, decreased cell viability, altered the cell cycle, and changed the cell cycle regulatory protein levels. Thus, arecoline oxidant exerts differential effects on the cell cycle through modulations of regulatory proteins.

Keywords: areca nut; prostate cancer; cell cycle; cyclin; cyclin-dependent kinase; p21; reactive oxygen species

1. Introduction Prostate cancer (PCa) is the most common reproductive system cancer in elderly men. The American Cancer Society estimated that 180,890 new cases of PCa occurred in the US in 2016

Int. J. Mol. Sci. 2020, 21, 9219; doi:10.3390/ijms21239219 www.mdpi.com/journal/ijms Int. J. Mol. Sci. 2020, 21, 9219 2 of 18 and that approximately 26,120 deaths occurred that same year from PCa-specific disease. Generally, early PCa has no clear symptoms, while advanced PCa commonly develops and spreads from the prostate to the bones and causes bone pain and urinary dysfunction [1]. Although androgen deprivation, which was discovered by Charles Huggins in 1941, can cause the regression of androgen-responsive metastatic PCa, hormone therapy eventually induces the development of castration-resistant PCa within 3 years [2]. As the growth and development of PCa are regulated by food compounds [2,3], it is worthwhile to carefully examine the signaling pathways through which such compounds differentially affect the growth of normal prostate cells, androgen-independent PCa cells, and androgen-dependent PCa cells. Betel nut alkaloids (BNAs), especially arecoline, have been reported to be regulatory agents of the prostate gland in male rats [4]. Specifically, in one study, arecoline treatment increased the weight of the prostate gland and serum levels of gonadotropin and testosterone hormones. Elevated androgen levels induced by arecoline may lead to increased cell proliferation in the prostate gland, as indicated by increased levels of Ki-67 protein and regulatory proteins of the G1-to-S cell cycle, such as cyclin D1 and cyclin-dependent kinase (CDK) 4. Various in vivo studies in rats have shown that arecoline may also stimulate Leydig cell activity and the release of testosterone possibly via the inhibition of pineal activity [5]. Despite this evidence that arecoline has an indirect impact on the rat prostate gland through the modulation of testosterone levels, it is still unknown whether arecoline exerts direct, differential effects on normal and cancerous human prostate cells. Unfortunately, the results of these studies [4,5] did not demonstrate the effect of either guvacine or arecaidine on the growth of normal and cancerous prostate cells. Notably, arecoline differed from guvacine and arecaidine in inhibiting preadipocyte growth with reactive oxygen species (ROS) dependency [6]. Therefore, it is worthwhile to explore whether arecoline or other BNAs differentially affect the growth of normal prostate cells, androgen-independent PCa cells, and androgen-dependent PCa cells. Arecoline regulates the growth of non-prostate cells in part through cell cycle regulatory proteins and reactive oxygen species (ROS) production [6–10]. Specifically, arecoline was found to induce dysregulation of the G1 or G2 phase of the cell cycle in non-PCa cells through the modulation of cell cycle-related proteins (e.g., p21 and CDKs) and ROS. Despite these findings, no studies have demonstrated whether any cell cycle regulatory proteins, such as CDK1, CDK2, CDK inhibitors (CKIs), and cyclins, can transduce anti-growth signals induced by arecoline to regulate the cell cycle in human PCa cells. Further studies are therefore necessary to determine whether the oxidant activity of arecoline is required for its differential effects on the growth of normal prostate cells, androgen-independent PCa cells, and androgen-dependent PCa cells. These studies may help elucidate the mechanism that underlies the actions of arecoline on the prostate. This study was designed to increase our understanding of the mechanism by which betel nut arecoline differentially regulates the growth of normal RWPE-1 prostate epithelial cells, androgen- independent human PC-3 PCa cells, and androgen-dependent human LNCaP PCa cells. We confirmed that arecoline, but not arecaidine or guvacine, significantly reduced the viability of all three cell lines. We showed that high doses of arecoline differentially induced cell cycle dysregulation among RWPE-1, PC-3, and LNCaP cells, and selectively affected specific proteins in the CDK, CKI, and cyclin families in a cell type-dependent manner. We also found that the effects of arecoline are dependent on pathways that require intracellular ROS production.

2. Results and Discussion

2.1. Effects of BNAs on Cell Viability Significant variations were observed in the measured cell viability (Figure1). In RWPE-1, PC-3, and LNCaP cells, arecoline was generally more effective in reducing cell viability than arecaidine or guvacine (Figure1A–C), and the e ffects depended on the dosage and duration of treatment. For example, the IC50 values of arecaidine and guvacine for all three cell lines were all greater than 1 mM during Int.Int. J. Mol. Sci. 2020, 21, x 9219 FOR PEER REVIEW 33 ofof 1818

example, the IC50 values of arecaidine and guvacine for all three cell lines were all greater than 1 mM 48during h of treatment,48 h of treatment, except for except the approximate for the approximat value ofe 0.45 value mM of of 0.45 arecoline mM of in arecoline the treatment in the of treatment RWPE-1 andof RWPE-1 PC-3 cells and at PC-3 48 h. cells The at IC 5048 valuesh. The ofIC arecoline50 values of in thearecoline treatment in the of treatment LNCaP cells of LNCaP were 0.8–1 cells mM were at 480.8–1 h. ThesemM at observations 48 h. These suggestobservations the alkaloid-speciﬁc suggest the alkaloid-specific eﬀect of betel nuteffect on of the betel growth nut on of normalthe growth and cancerousof normal prostateand cancerous cells. prostate cells.

FigureFigure 1. ReducingReducing effect effect of of betel betel nut nut alkaloids alkaloids on on the the viability viability of normal of normal RWPE-1 RWPE-1 (A),( cancerousA), cancerous PC- B C PC-33 (B), ( and), and LNCaP LNCaP (C) ( prostate) prostate cells cells with with various various concentrations concentrations (0–1 (0–1 mM), mM), duration duration (24–48 (24–48 h) ofof treatment, and with different types of alkaloids. Data are expressed as the mean SEM from triplicate treatment, and with different types of alkaloids. Data are expressed as the mean± ± SEM from triplicate experiments. For clarity, statistical significance is not shown. experiments. For clarity, statistical significance is not shown. 2.2. Arecoline-Induced Differential Growth Arrest in Normal and Cancerous Prostate Cells 2.2. Arecoline-Induced Differential Growth Arrest in Normal and Cancerous Prostate Cells Alterations in the cell cycle can modulate cell growth [11]. We found that arecoline changed the percentagesAlterations of normal in the RWPE-1cell cycle cells can modulate in the G0/ G1,cell S,gr andowth G2 [11]./M phasesWe found of thethat cell arecoline cycle (Figure changed2 and the Supplementarypercentages of normal Materials). RWPE-1 Generally, cells in treatment the G0/G1, with S, and arecoline G2/M atphases a concentration of the cell cycle of 0.4 (Figure mM for 2 48and h tendedSupplementary to decrease Materials). the percentage Generally, of cells treatment in the G1 with and arecoline S phases andat a tendedconcentration to increase of 0.4 the mM percentage for 48 h oftended cells into the decrease G2/M phase the percentage (Figure2A). of Similar cells einff ectsthe wereG1 and observed S phases when and PC-3 tended cells were to increase treated with the arecoline,percentage in of that cells the in percentage the G2/M of phase cells in(Figure the G1 2A). phase Similar tended effects to decrease, were observed whereas thewhen percentage PC-3 cells of cellswere in treated the G2 /withM phase arecoline, tended in to that increase the percentage (Figure2B). However,of cells in inthe LNCaP G1 phase cells, tended arecoline to increaseddecrease, thewhereas percentage the percentage of cells in of the cells G1 in phase the G2/M and phase decreased tended the to percentage increase (Figure of cells 2B). in theHowever, S phase, in but LNCaP had nocells, significant arecoline eincreasedffect on the the percentage percentage ofof cellscells in the the G1 G2 phase/M phase and (Figuredecreased2C). the These percentage observations of cells suggestin the S thatphase, the but effects had of no arecoline significant on theeffect cell on cycle the ofpercentage normal and of cells cancerous in the prostateG2/M phase cells are(Figure cell type2C). dependent.These observations This induced suggest dysregulation that the effects of the of cellarecoline cycle byon arecoline, the cell cycle which of isnormal supported and bycancerous altered percentagesprostate cells of are cells cell in type the G1,dependen S, andt. G2 This/M induced growth phases, dysregulation may explain of the thecell observed cycle by arecoline, decreases which in the viabilityis supported of RWPE-1, by altered PC-3, percentages and LNCaP of cells in treated the G1, with S, and arecoline. G2/M Thegrowth results phases, obtained may explain in RWPE-1 the andobserved PC-3 cellsdecreases are similar in the toviability those reported of RWPE-1, for thePC-3, G2 /andM-dependent LNCaP cells eff ecttreated of arecoline with arecoline. on mucosal The fibroblasts,results obtained vascular in RWPE-1 endothelial and PC-3 cells, cells adipose are simila cells,r andto those basal reported cell carcinoma for the G2/M-dependent cells [6,7,9,12], but effect the resultsof arecoline obtained on mucosal in LNCaP fibroblasts, cells are consistentvascular endothelial with those cells, reported adipose for the cells, G0 /andG1-dependent basal cell carcinoma effects of arecolinecells [6,7,9,12], on normal but the hepatocytes results obtained and keratinocytes in LNCaP [8cells,10]. are consistent with those reported for the G0/G1-dependent effects of arecoline on normal hepatocytes and keratinocytes [8,10].

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Figure 2. A Figure 2. Arecoline-inducedArecoline-induced growth arrest at different different phases of the cell cycle cycle in in normal normal RWPE-1 RWPE-1 ( (A),), cancerous PC-3 (B), and LNCaP (C) prostate cells, as examined by flow cytometry after 24 and 48 h cancerous PC-3 (B), and LNCaP (C) prostate cells, as examined by flow cytometry after 24 and 48 h of treatment. The induction of growth arrest was affected by N-acetylcysteine (NAC) pretreatment. of treatment. The induction of growth arrest was affected by N-acetylcysteine (NAC) pretreatment. Cells were pretreated with 1.5 mM NAC for 1 h and then exposed to 0.4 mM arecoline for 24 and Cells were pretreated with 1.5 mM NAC for 1 h and then exposed to 0.4 mM arecoline for 24 and 48 48 h. Data are expressed as the mean SEM of triplicate experiments. Groups with different letters h. Data are expressed as the mean ± SEM± of triplicate experiments. Groups with different letters were were significant (p < 0.05) in a given phase. In some groups, the standard error bars are too small to significant (p < 0.05) in a given phase. In some groups, the standard error bars are too small to be seen. be seen. The symbols of “+” and “-” were presented with or without addition of either arecoline or The symbols of “+” and “–“ were presented with or without addition of either arecoline or NAC, NAC, respectively. respectively. 2.3. Arecoline Selectively Affected Specific Types of Cell Cycle-Regulating Proteins 2.3. Arecoline Selectively Affected Specific Types of Cell Cycle-Regulating Proteins The cell cycle can be regulated by proteins in the CDK, CKI, and cyclin families [11]. For example, The cell cycle can be regulated by proteins in the CDK, CKI, and cyclin families [11]. For example, CDK2 and p21 were found to regulate PCa cell growth in the absence of arecoline [13]. In other CDK2 and p21 were found to regulate PCa cell growth in the absence of arecoline [13]. In other experiments found in non-prostate cells, p21 mediated areoline-induced growth arrest of hepatocytes [8] experiments found in non-prostate cells, p21 mediated areoline-induced growth arrest of hepatocytes and keratinocyte [10], CDK1 mediated arecoline-induced G2/M arrest of basal cell carcinoma cells [12], [8] and keratinocyte [10], CDK1 mediated arecoline-induced G2/M arrest of basal cell carcinoma cells and CDK1 and cyclin B2 acted as signaling components in pathways through which arecoline mediates [12], and CDK1 and cyclin B2 acted as signaling components in pathways through which arecoline the cell cycle of 3T3-L1 preadipocytes [6]. In our experiment, RWPE-1 cells treated with arecoline at a mediates the cell cycle of 3T3-L1 preadipocytes [6]. In our experiment, RWPE-1 cells treated with concentration of 0.4 mM, but not at concentrations of 0.05 and 0.2 mM, exhibited a significant increase arecoline at a concentration of 0.4 mM, but not at concentrations of 0.05 and 0.2 mM, exhibited a in the CDK1 protein expression levels and exhibited a tendency for decreased CDK2 protein expression, significant increase in the CDK1 protein expression levels and exhibited a tendency for decreased whereas arecoline had no effect on CDK4 protein expression after 24 h of treatment (Figure3A). CDK2 protein expression, whereas arecoline had no effect on CDK4 protein expression after 24 h of In PC-3 cells, arecoline tended to decrease the expression of CDK1, CDK2, and CDK4 proteins in treatment (Figure 3A). In PC-3 cells, arecoline tended to decrease the expression of CDK1, CDK2, and a dose-dependent manner (Figure3B). In LNCaP cells, arecoline did not alter the CDK1 protein CDK4 proteins in a dose-dependent manner (Figure 3B). In LNCaP cells, arecoline did not alter the level, but it reduced the CDK2 and CDK4 protein levels in a dose-dependent manner (Figure3C). CDK1 protein level, but it reduced the CDK2 and CDK4 protein levels in a dose-dependent manner Since RWPE-1, PC-3, and LNCaP cells represent normal prostate epithelial cells, androgen-independent (Figure 3C). Since RWPE-1, PC-3, and LNCaP cells represent normal prostate epithelial cells, PCa cells, and androgen-dependent PCa cells, respectively, these observations suggest that the selective androgen-independent PCa cells, and androgen-dependent PCa cells, respectively, these effects of arecoline on specific proteins of the CDK family are differentially induced among these observations suggest that the selective effects of arecoline on specific proteins of the CDK family are cell types. differentially induced among these cell types.

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Figure 3. Differential effects of arecoline on proteins in the cyclin-dependent kinase (CDK) family Figure 3. Differential effects of arecoline on proteins in the cyclin-dependent kinase (CDK) family between normal RWPE-1 (A) and cancerous PC-3 (B) and LNCaP (C) prostate cells after 24 h of between normal RWPE-1 (A) and cancerous PC-3 (B) and LNCaP (C) prostate cells after 24 h of treatment. Data are expressed as the mean SEM from triplicate experiments after Western blot treatment. Data are expressed as the mean± ± SEM from triplicate experiments after Western blot analysis; each result was pooled from the data of four 10 cm culture plates. * p < 0.05 vs. the control. + analysis; each result was pooled from the data of four 10 cm culture plates. * p < 0.05 vs. the control. + p < 0.10 vs. the control. p < 0.10 vs. the control. When the CKI family was examined, we found that arecoline at concentrations of 0.05 and 0.2 mM had noWhen effect onthe p21 CKI protein family expression was examined, in RWPE-1 we found cells. that However, arecoline when at concentrations 0.4 mM arecoline of 0.05 was and used, 0.2 a significantmM had no increase effect on in p21 the protein p21 expression expression level in wasRWPE-1 observed cells. (FigureHowever,4A), when whereas 0.4 mM treatment arecoline with was 0.4used, mM arecolinea significant did increase not alter in the the p27 p21 protein expression expression level was level. observed In PC-3 (Figure cells, 0.2 4A), and whereas 0.4 mM, treatment but not 0.05with mM, 0.4 arecoline mM arecoline tended did to decreasenot alter the the p21 p27 and protein p27 protein expression expression level. In levels PC-3 (Figure cells, 40.2B). and In LNCaP 0.4 mM, cells,but treatmentnot 0.05 mM, with arecoline 0.4 mM arecoline tended to induced decrease increases the p21 in and the p27 expression protein ofexpressi both p21on andlevels p27 (Figure proteins 4B). (FigureIn LNCaP4C). cells, treatment with 0.4 mM arecoline induced increases in the expression of both p21 and p27When proteins the cyclin(Figure family 4C). was examined, we observed that arecoline at concentrations of 0.05 and 0.2 mMWhen had no the e ffcyclinect on family the levels was ofexamined, cyclins B1, we D1, observed and D3, that but arecoline that 0.4 mMat concentrations arecoline significantly of 0.05 and increased0.2 mM thehad levels no effect of cyclins on the B1levels and of D3 cyclins (Figure B1,5A). D1, In and PC-3 D3, cells, but treatmentthat 0.4 mM with arecoline 0.4 mM significantly arecoline increasedincreased the the levels levels of cyclin of cyclins B1 protein B1 and and D3 decreased (Figure 5A the). levelsIn PC-3 of cells, cyclin treatment D1 and D3 with proteins 0.4 mM (Figure arecoline5B). Inincreased LNCaP cells, the levels treatment of cyclin with B1 0.4 protein mM arecoline and decreased did not the induce levels significant of cyclin D1 changes and D3 in proteins the levels (Figure of cyclin5B). B1In LNCaP protein, cells, but the treatment levels of with cyclin 0.4 D1 mM were arecoline significantly did not decreased, induce significant while the changes levels of in cyclin the levels D3 wereof cyclin increased B1 protein, (Figure but5C). the levels of cyclin D1 were significantly decreased, while the levels of cyclin D3 were increased (Figure 5C).

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Figure 4. Differential effects of arecoline on proteins in the cyclin-dependent kinase inhibitor (CKI) Figure 4. Diﬀerential eﬀects of arecoline on proteins in the cyclin-dependent kinase inhibitor (CKI) family between normal RWPE-1 (A) and cancerous PC-3 (B) and LNCaP (C) prostate cells after 24 h familyFigure between4. Differential normal effects RWPE-1 of arecoline (A) and cancerouson proteins PC-3 in the (B) cyclin-dependent and LNCaP (C) prostate kinase cellsinhibitor after (CKI) 24 h of treatment. Data are expressed as the mean ± SEM from triplicate experiments after Western blot offamily treatment. between Data normal are expressed RWPE-1 ( asA) the and mean cancerousSEM PC-3 from (B triplicate) and LNCaP experiments (C) prostate after cells Western after blot24 h analysis; each result was pooled from the data± of four 10 cm culture plates. * p < 0.05 vs. the control. analysis;of treatment. each resultData are was expressed pooled from as the the mean data of± fourSEM 10from cm triplicate culture plates. experiments * p < 0.05 after vs. Western the control. blot analysis; each result was pooled from the data of four 10 cm culture plates. * p < 0.05 vs. the control.

Figure 5. Diﬀerential eﬀects of arecoline on the proteins in the cyclin family between normal RWPE-1

(AFigure) and cancerous5. Differential PC-3 effects (B) and of LNCaParecoline (C )on prostate the proteins cells after in the 24 cyclin h of treatment. family between Data are normal expressed RWPE- as theFigure1 (A mean) and 5. Differential cancerousSEM from PC-3 effects triplicate (B )of and arecoline experiments LNCaP on (C the) after prostate proteins Western cells in blot afterthe analysis;cyclin 24 h of family treatment. each between result Data was normal are pooled expressed RWPE- from ± the1as (A datathe) and mean of cancerous four ± SEM 10 cm PC-3from culture (triplicateB) and plates. LNCaP experiments * p < (0.05C) prostate vs. afte ther control.cellsWestern after blot24 h analysis;of treatment. each Data result are was expressed pooled asfrom the the mean data ± ofSEM four from 10 cm triplicate culture experimentsplates. * p < 0.05 afte vs.r Western the control. blot analysis; each result was pooled from the data of four 10 cm culture plates. * p < 0.05 vs. the control.

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2.4.2.4. The EEffectsffects of Arecoline on NormalNormal andand CancerousCancerous ProstateProstate CellCell GrowthGrowth Waswas Dependent on the ROS ROSPathway Pathway AlthoughAlthough the anti-growth eeffectffect of arecolinearecoline onon humanhuman cellscells hashas beenbeen reportedreported toto bebe ROSROS dependentdependent [[7–10],7–10], nono publishedpublished studies were found on normalnormal human prostate and PCa cells. Thus, wewe examinedexamined whetherwhether thethe arecoline-inducedarecoline-induced changes in cell viability, growthgrowth arrestarrest phase,phase, andand cellcell cyclecycle regulatoryregulatory moleculesmolecules inin RWPE-1, PC-3,PC-3, andand LNCaP cells were dependentdependent on the ROS pathway (Figures(Figures6 6–8).–8). Indeed,Indeed, wewe firstfirst found found that that arecoline arecoline inducedinduced aa significantsignificant increaseincrease in in ROS ROS production production inin allall threethree cellcell typestypes afterafter 2424 andand 4848 hh ofof treatmenttreatment (Figure(Figure6 ).6). Pretreatment Pretreatment with with N-acetylcysteine N-acetylcysteine (NAC)(NAC) blockedblocked thethe arecoline-inducedarecoline-induced increases in the levels of ROS production in all cells (Figure 66).). InIn addition, NAC NAC antagonized antagonized the the arecoline-induced arecoline-induced decreases decreases in the in the viability viability of RWPE-1, of RWPE-1, PC-3, PC-3, and andLNCaP LNCaP cells cells (Figure (Figure 7).7 ).In Inthe the presence presence of of ar arecoline,ecoline, NAC antagonizedantagonized thethe arecoline-alteredarecoline-altered percentagespercentages ofof RWPE-1, RWPE-1, PC-3, PC-3, and and LNCaP LNCaP cells incells the G1,in the S, and G1, G2 S,/M and phases G2/M (Figure phases2). Interestingly, (Figure 2). NACInterestingly, alone induced NAC alone increases induced in the increases percentage in the of RWPE-1 percentage cells of in RWPE-1 the G1 phase,cells in decreases the G1 phase, in the percentagedecreases in of the those percentage cells in the of those S phase, cells and in inducedthe S phase, no changesand induced in the no percentage changes in of the those percentage cells in the of G2those/M phasecells in (Figure the G2/M2A). Inphase PC-3 (Figure and LNCaP 2A). In cells, PC NAC-3 and alone LNCaP did notcells, a ffNACect the alone percentages did not ofaffect cells the in thepercentages G1, S, and of G2 cells/M in phases. the G1, S, and G2/M phases.

FigureFigure 6.6. The eeffectﬀect ofof arecolinearecoline onon thethe productionproduction ofof reactivereactive oxygenoxygen speciesspecies (ROS)(ROS) waswas observedobserved inin normalnormal RWPE-1RWPE-1 ( A(A) and) and cancerous cancerous PC-3 PC-3 (B) and(B) LNCaPand LNCaP (C) prostate (C) prostate cells after cells 24 after and 48 24 h and of treatment. 48 h of Thetreatment. induction The of induction ROS production of ROS was production reduced by was pretreatment reduced by with pretreatment N-acetylcysteine with (NAC). N-acetylcysteine Cells were pretreated(NAC). Cells with were 1.5 mMpretreated NAC andwith then 1.5 exposedmM NAC to and arecoline then exposed for 24 or to 48 arecoline h. The ROS for production24 or 48 h. wasThe measuredROS production using thewas 2 measured0,70-dichloroﬂuorescein using the 2’,7’-dichlorofluorescein diacetate method. * pdiacetate< 0.05 vs. method. the control; * p < 0.05 # p vs.< 0.05 the arecolinecontrol; # vs.p < arecoline0.05 arecoline+ NAC. vs. arecoline + NAC.

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Figure 7. N-acetylcysteine (NAC) blocked the arecoline-induced changes in the viability of normal Figure 7. N-acetylcysteine (NAC) blocked the arecoline-induced changes in the viability of normal FigureRWPE-1 7. N-acetylcysteine(A) and cancerous (NAC) PC-3 blocked(B) and LNCaPthe arecoline- (C) prostateinduced cells. changes Cells werein the pretreated viability of with normal NAC RWPE-1 (A) and cancerous PC-3 (B) and LNCaP (C) prostate cells. Cells were pretreated with NAC for RWPE-1for 1 h and (A) then and exposedcancerous to PC-30.4 mM (B) arecoline. and LNCaP After (C) 48 prostate h of treatment, cells. Cells we measuredwere pretreated cell viability with NAC using 1for h 1 and h and then then exposed exposed to 0.4to 0.4 mM mM arecoline. arecoline. After After 48 48 h h of of treatment, treatment, we we measured measuredcell cell viabilityviabilitya-c using an MTT assay. Data are expressed as the mean ± SEM from triplicate experiments.a–c Groups with an MTT assay. Data are expressed as the mean SEM from triplicate experiments. a-c Groups with andifferent MTT assay. letters Data differ are significantly expressed as ( pthe < 0.05).mean ± The± SEM symbols from triplicateof “+” and experiments. “-“ were presented Groups with with or different letters differ significantly (p < 0.05). The symbols of “+” and “-” were presented with or differentwithout lettersaddition differ of either significantly arecoline ( por < NAC,0.05). respectively.The symbols of “+” and “-“ were presented with or without additionaddition ofof eithereither arecolinearecoline oror NAC,NAC, respectively.respectively.

Figure 8. N-acetylcysteine (NAC) blocked the arecoline-induced alterations in the levels of cell cycle Figure 8. N-acetylcysteine (NAC) blocked the arecoline-induced alterations in the levels of cell cycle regulatory proteins, such as CDK1 (A), CDK2 (B), p21 (C), Cyclin B1 (D) and Cyclin D3 (E), in normal Figureregulatory 8. N-acetylcysteine proteins, such as(NAC) CDK1 blocked (A), CDK2 the arecoline-in (B), p21 (C),duced Cyclin alterations B1 (D) and in Cyclin the levels D3 ( Eof), cell in normal cycle RWPE-1 prostate cells. Cells were pretreated with NAC for 1 h and then exposed to 0.4 mM arecoline. regulatoryRWPE-1 prostate proteins, cells. such Cells as CDK1 were (pretreatedA), CDK2 (withB), p21 NAC (C), for Cyclin 1 h and B1 then(D) and exposed Cyclin to D3 0.4 ( mME), in arecoline. normal After 24 h of treatment, proteins were examined by Western blot analysis, and their levels were then RWPE-1After 24 prostate h of treatment, cells. Cells proteins were pretreatedwere examined with NACby Western for 1 h blotand analysis,then exposed and theirto 0.4 levels mM arecoline. were then expressed after normalization to actin expression. Data are expressed as the mean SEM from triplicate Afterexpressed 24 h of after treatment, normalization proteins wereto actin examined expression by Western. Data are blot expressed analysis, andas the their± mean levels ± wereSEM thenfrom experiments; each result was pooled from the data of four 10 cm culture plates. * p < 0.05 vs. the control; expressedtriplicate experiments;after normalization each result to actin was pooledexpression from. theData data are of expressed four 10 cm as culture the mean plates. ± *SEM p < 0.05 from vs. + p < 0.05 vs. the control; # p < 0.05 arecoline vs. NAC + arecoline. triplicatethe control; experiments; + p < 0.05 vs.each the result control; was # pooled p < 0.05 from arecoline the data vs. ofNAC four + 10 arecoline. cm culture plates. * p < 0.05 vs. the control; + p < 0.05 vs. the control; # p < 0.05 arecoline vs. NAC + arecoline. Next, we examined whether the arecoline-induced alterations in the levels of cell cycle regulatory proteinsNext, were we aﬀ ectedexamined by NAC whether (Figures the8 –arecoline-induced10). In RWPE-1 cells, alterations pretreatment in the with levels NAC of prevented cell cycle regulatoryNext, we proteins examined were affectedwhether bythe NAC arecoline-induced (Figures 8–10). Inalterations RWPE-1 cells,in the pretreatment levels of cellwith cycle NAC regulatoryprevented proteins arecoline-induced were affected alterations by NAC in(Figures the levels 8–10). of In CDK1, RWPE-1 CDK2, cells, cyclinpretreatment B1, and with cyclin NAC D3 prevented arecoline-induced alterations in the levels of CDK1, CDK2, cyclin B1, and cyclin D3

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Int. J. Mol. Sci. 2020, 21, x FOR PEER REVIEW 9 of 18 arecoline-induced alterations in the levels of CDK1, CDK2, cyclin B1, and cyclin D3 proteins (Figure8). Althoughproteins (Figure NAC did 8). notAlthough significantly NAC blockdid not arecoline-induced significantly block increases arecoline-induced in p21 protein increases levels, it slightlyin p21 proteinantagonized levels, the it eslightlyffect of antagonized arecoline. Interestingly, the effect of in arecoline. RWPE-1 Interestingly, cells, NAC alone in RWPE-1 tended tocells, decrease NAC alonethe levels tended of CDK1,to decrease cyclin the B1, levels and of cyclin CDK1, D3 cyclin proteins B1, and and cyclin increased D3 proteins the levels and of increased p21, while the itlevels had ofno p21, effect while on the it had levels no ofeffect CDK2 on (Figurethe levels8). of In CDK2 PC-3 cells,(Figure NAC 8). In alone PC-3 did cells, not NAC have alone any e didffects not on have the anylevels effects of CDK1, on the CDK2, levels p21, of CDK1, and cyclin CDK2, B1 proteinsp21, and (Figure cyclin9 B1). However,proteins (Figure arecoline 9). treatment However, prevented arecoline treatmentarecoline-induced prevented alterations arecoline-induced in the levels alterations of CDK2, in p21, the andlevels cyclin of CDK2, B1 proteins p21, and and cyclin had noB1 eproteinsffect on andthe arecoline-increasedhad no effect on the levels arecoline-increased of CDK1 protein. levels In LNCaP of CDK1 cells, protein. NAC In alone LNCaP did notcells, induce NAC significant alone did notchanges induce in significant the levels ofchanges CDK2, in CDK4, the levels p21, of p27, CDK2, cyclin CDK4, D1, orp21, cyclin p27, D3cyclin proteins D1, or (Figurecyclin D3 10 proteins). In the presence(Figure 10). of In arecoline, the presence NAC of prevented arecoline, the NAC arecoline-induced prevented the arecolin decreasee-induced in the levels decrease of CDK2, in the CDK4, levels ofand CDK2, cyclin CDK4, D1 proteins and cyclin and D1 the proteins arecoline-induced and the arecoline-induced increase in the levelsincrease of in p21, the p27, levels and of cyclinp21, p27, D3 proteins.and cyclin Taken D3 proteins. together, Taken these together, observations these suggest observ thatations the suggest cell type-dependent that the cell type-dependent effect of arecoline effect on prostateof arecoline cell cycleon prostate regulatory cell proteinscycle regulatory is mediated prot througheins is amediated pathway through that requires a pathway the induction that requires of ROS production.the induction It of was ROS evident production. that arecoline It was evident could participate that arecoline in the could generation participate of di inff erentthe generation metabolites of differentand free radicalmetabolites groups, and such free as arecolineradical N-oxide,groups, arecolinesuch as N-oxidearecoline mercapturic N-oxide, arecoline acid, nitrosamines, N-oxide mercapturic3-methylnitrosaminopropionate, acid, nitrosamines, and3-methylnitrosaminopropionate, N-nitrosoguvacoline [14,15]. Whetherand N-nitrosoguvacoline this mechanism explains [14,15]. Whetherthe ROS-dependent this mechanism effect ofexplains arecoline the on ROS-dependen normal and canceroust effect of prostate arecoline cell on cycle normal regulatory and cancerous proteins wasprostate not demonstratedcell cycle regulatory in this proteins study. was not demonstrated in this study.

Figure 9. N-acetylcysteine (NAC) blocked the arecoline-induced alterations in the levels of cell cycle Figure 9. N-acetylcysteine (NAC) blocked the arecoline-induced alterations in the levels of cell cycle regulatory proteins, such as CDK1(A), CDK2(B), p21(C) and Cyclin B1(D), in androgen-independent regulatory proteins, such as CDK1(A), CDK2(B), p21(C) and Cyclin B1(D), in androgen-independent human PC-3 prostate cancer cells. Cells were pretreated with NAC for 1 h and then exposed to human PC-3 prostate cancer cells. Cells were pretreated with NAC for 1 h and then exposed to 0.4 0.4 mM arecoline. After 24 h of treatment, proteins were examined by Western blot analysis and then mM arecoline. After 24 h of treatment, proteins were examined by Western blot analysis and then expressed after normalization to actin expression. Data are expressed as the mean SEM from triplicate expressed after normalization to actin expression. Data are expressed as the± mean ± SEM from experiments; each result was pooled from the data of four 10 cm culture plates. * p < 0.05 vs. the control; triplicate experiments; each result was pooled from the data of four 10 cm culture plates. * p < 0.05 vs. # p < 0.05 arecoline vs. NAC + arecoline. the control; # p < 0.05 arecoline vs. NAC + arecoline.

Int. J. Mol. Sci. 2020, 21, 9219 10 of 18 Int. J. Mol. Sci. 2020, 21, x FOR PEER REVIEW 10 of 18

Figure 10. N-acetylcysteine (NAC) blocked the arecoline-induced alterations in the levels of cell cycle Figure 10. N-acetylcysteine (NAC) blocked the arecoline-induced alterations in the levels of cell cycle regulatory proteins, such as CDK2 (A), CDK4 (B), p21 (C), p27 (D), Cyclin D1 (E) and Cyclin D3 (F) in regulatory proteins, such as CDK2 (A), CDK4 (B), p21 (C), p27 (D), Cyclin D1 (E) and Cyclin D3 (F) androgen-dependent human LNCaP prostate cancer cells. Cells were pretreated with NAC for 1 h and in androgen-dependent human LNCaP prostate cancer cells. Cells were pretreated with NAC for 1 h then exposed to 0.4 mM arecoline. After 24 h of treatment, the protein levels were examined by Western and then exposed to 0.4 mM arecoline. After 24 h of treatment, the protein levels were examined by blot analysis and were then expressed after normalization to actin expression. Data are expressed as the meanWesternSEM blot from analysis triplicate and experiments; were then expressed each result af waster poolednormalization from the to data actin of expression. four 10 cm cultureData are ± plates.expressed * p < 0.05 as the vs. mean the control; ± SEM # fromp < 0.05 triplicate arecoline experiments; vs. NAC + eacharecoline. result was pooled from the data of four 10 cm culture plates. * p < 0.05 vs. the control; # p < 0.05 arecoline vs. NAC + arecoline. Generally, CDK1 and CDK2/CDK4/CDK6 control the checkpoints of the G2/M phase and the G0/G1 phase,Generally, respectively, CDK1 and p21 CDK2/CDK4 and p27 act as/CDK6 endogenous control inhibitors the checkpoints of CDKs, of and the cyclinG2/MB1 phase and cyclinand the G0/G1 phase, respectively, p21 and p27 act as endogenous inhibitors of CDKs, and cyclin B1 and cyclin D function as the endogenous activators of CDK1 and CDK4/6, respectively [16,17]. Increased

Int. J. Mol. Sci. 2020, 21, 9219 11 of 18

D function as the endogenous activators of CDK1 and CDK4/6, respectively [16,17]. Increased p21 protein expression in RWPE-1 cells may inhibit CDK2 activity and lead to significant growth arrest in the G2/M phase. Cyclin B1 is a G2/M cyclin that is associated with the CDK1 protein and, when it accumulates, favors M-phase arrest at the G2/M checkpoint [11,18]. Therefore, the observed increase in cyclin B1 protein levels after treatment with 0.4 mM arecoline for 24 h strengthens the possibility of CDK1-related effects of arecoline on RWPE-1 growth arrest at the G2/M phase. The blocking effect of NAC on arecoline-induced increases in both CDK1 and cyclin B1 proteins helps explain the progression of cells from the G2/M phase to the G1 phase. Concurrently, the high p21 protein levels observed in the combined NAC and arecoline treatment group favor RWPE-1 cell arrest in the G1 phase, which was observed at 24 and 48 h. As p21 and p27 are endogenous proteins that favor cell cycle arrest of human PCa cells at the G1 checkpoint [13], decreased expression levels of p21 and p27 proteins in PC-3 cells by arecoline treatment may lead to significant decreases in the percentage of cells in the G0/G1 phase. Compared with arecoline alone, the blocking effect of NAC on the arecoline-induced decreases in CDK2 and p21 proteins may help rescue the percentage of PC-3 cells in the G1 phase after 24 and 48 h of treatment. Since CDK1 becomes the predominant CDK in the early G2/M transition of the cell cycle, and since cyclin B1 is the M-phase arrest protein [11,16–18], decreased CDK1 protein levels and increased cyclin B1 protein levels may result in the G2/M growth arrest in PC-3 cells, which was observed 24 and 48 h after arecoline treatment. The blocking effect of NAC on the arecoline-altered levels of cyclin B1, but not CDK1, may explain the lower percentage of cells in the G2/M phase in the combined NAC and arecoline group relative to the arecoline-only group. This phenomenon may also indicate the involvement of high cyclin B1 protein levels as arecoline exerts its effects. These findings are consistent with those reported for the arecoline-induced G2/M-phase arrest in KB human epidermoid carcinoma cells [18,19]. However, in LNCaP cells, decreased CDK2, CDK4, and cyclin D1 protein levels and increased p21, p27, and cyclin D3 protein levels induced by arecoline appear to favor cell arrest in the G1 phase. The finding that arecoline treatment did not lead to alterations in the CDK1 and cyclin B1 protein levels helps explain the lack of significant changes in the percentage of cells in the G2/M phase. The blocking effect of NAC on the arecoline-altered levels of CDK2, CDK4, p21, p27, cyclin D1, and cyclin D3 helps explain the lower percentage of cells in the G1 phase in the combined NAC and arecoline treatment group relative to the arecoline-only group; this also indicates the involvement of these proteins in the effects of arecoline on the G1 growth arrest of androgen-dependent PCa cells. Arecoline was found to possess multiple functions, including effects on endocrine, nervous, gastrointestinal, and cardiovascular systems [20]. This could be explained by various molecule targets of arecoline, including muscarinic receptors, anti-atherogenic factors (e.g., nitric oxide), inflammatory cytokines, calcium channels, steroid dehydrogenase, pancreas-duodenum homeobox-1, heme oxygenase-1, cell cycle-related proteins (e.g., CDK, CKI, and cyclins), the phosphatidylinositol-3- kinase (PI3K)–mammalian target of rapamycin (mTOR)–p53 pathway, catenin, and so forth. Although the exact target of arecoline in normal and cancerous prostate cells was not demonstrated in this study, we provided a certain in-depth understanding of its effects on the protein expression of particular types of CDK, CKI, and cyclin family members with ROS dependency. It is worthwhile to explore further whether any of those reported arecoline target molecules are necessary for its stimulating ROS production and altering levels of CDK, CKI, and cyclin proteins in normal and cancerous prostate cells. Differences of androgen sensitivity and tumorigenic activity occur among RWPE-1, PC-3, and LNCaP cell lines [21,22]. Although RWPE-1 cells were immortalized with human papilloma virus, they are non-tumorigenic and androgen sensitive with positive androgen receptor (AR) and prostate-specific antigen (PSA) expression. The cell line is positive for phosphatase and tensin homolog (PTEN), retinoblastoma (Rb), and p53 proteins, as well as response to epidermal growth factor (EGF) and transforming growth factor. PC-3 cells isolated from a vertebral metastatic prostate tumor are androgen insensitive without AR or PSA expression, and they are PTEN deficient and express high levels of EGF receptor (EGFR), Rb protein, and aberrant p53. LNCaP cells isolated from a human lymph node metastatic lesion of prostate adenocarcinoma are androgen sensitive with AR and PSA Int. J. Mol. Sci. 2020, 21, 9219 12 of 18 expression, and they are PTEN deficient and express EGFR protein and a low level of Rb protein. In our study, arecoline induced different phases of growth arrest between normal and cancerous prostate cells through the ROS pathway, as well as causing different alteration patterns of CDK, CKI, and cyclin protein levels. As AR, PTEN, p53, and Rb proteins can function as regulators of cell cycle progression and proliferation of prostate cells and they are differentially expressed among RWPE-1, PC-3, and LNCaP cells [21,22], we could not exclude the possibility that any of them or other protein signaling cascades (e.g., EGFR and AKT) might be involved in the distinct effects of arecoline on the growth and cell cycle proteins of RWPE-1, PC-3, and LNCaP cells. Notably, androgen-dependent LNCaP cells exhibited the different protein expression profiles and chemotherapy drug responses from androgen-independent PC-3 cells [22]. In vivo, arecoline was reported to increase the weight of the prostate gland and serum levels of gonadotropin and testosterone hormones and thereby leading to increased cell proliferation in the prostate gland [4]. It was evident by increased levels of Ki-67 protein, cyclin D1, and CDK4 and by increased activity of Leydig cells [4,5]. Interestingly, our study indicated that arecoline inhibited the growth of normal prostate cells, androgen-independent PCa cells, and androgen-dependent PCa cells. These observations were supported by an increased percentage of the G2/M phase in the RWPE-1 and PC-3 cell cycle and by an increased percentage of the G0/G1 phase arrest in the LNCaP cell cycle. In addition, in RWPE-1 cells, arecoline increased levels of CDK-1, p21, and cyclins B1 and D3, decreased CDK2 protein levels, and had no effects on CDK4 and cyclin D1 protein levels. In PC-3 cells, arecoline decreased CDK1, CDK2, CDK4, p21, p27, and cyclin D1 and D3 protein expression and increased cyclin B1 protein expression. In LNCaP cells, arecoline decreased CDK2, CDK4, and cyclin D1 expression, increased p21, p27, and cyclin D3 expression, and had no effects on CDK1 and cyclin B1 expression. A possible explanation for the in vivo and in vitro discrepancy is that the action of arecoline on prostate cell growth varies with the presence of testosterone and other serum factors, the varying levels and types of downstream signaling proteins (e.g., CDKs, CKI, and cyclins), and prostate cell types. Thus, arecoline may not only have an indirect impact on the growth of prostate cells through the modulation of serum factor levels, but also exert a direct effect on the proliferation of normal and cancerous human prostate cells. BNAs have numerous biological properties that can potentially result in various biological effects [4–10,18,19,23–34]. In most cases, but not all, arecoline is more active than other alkaloids. This contention is supported by our findings in RWPE-1, PC-3, and LNCaP cells because at the same dose and duration of treatment, arecoline was generally more effective than arecaidine and guvacine in altering the viability of these cells. The observed alkaloid-specific effects of the betel nut suggest that arecoline may act differently from arecaidine and guvacine in regulating the growth of normal and cancerous prostate cells. According to the nature of the unique structures of these three alkaloids tested [29], arecoline, but not arecaidine or guvacine, contains a methyl ester group on its N-containing aromatic ring. This methyl ester group of arecoline may be important for some conformational flexibility and in its interactions with other molecules.

3. Materials and Methods

3.1. Chemical Reagents All active reagents (e.g., arecoline hydrobromide, arecaidine hydrochloride, guvacine hydrochloride, and others) were purchased from Sigma Chemical (St. Louis, MO, USA), unless otherwise stated. BNAs were dissolved in sterile medium for cell treatment. RPMI-1640, Keratinocyte- SFM, fetal bovine serum (FBS), trypsin, and protein markers were purchased from Gibco-Invitrogen (Grand Island, NY, USA). The CDK4, CDK6, cyclin B1, cyclin D1, cyclin D3, p21, p27, and actin antibodies were obtained from Cell Signaling Technology (Billerica, MA, USA), while other antibodies (i.e., CDK1, CDK2, and all others) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA) (Table1). Int. J. Mol. Sci. 2020, 21, 9219 13 of 18

Table 1. Antibodies used in the experiments.

Species Raised in; Manufacturer, Catalog Number (#), and/or Name of Antibody Monoclonal or Polyclonal Name of Individual Providing the Antibody Cell Signaling Technology, Inc. Anti-β-actin Rabbit; Monoclonal (Billerica, MA, USA), #12620 Anti-CDC2 Rabbit; Polyclonal IgG Santa Cruz (Santa Cruz, CA, USA), sc-53 Anti-CDK2 Rabbit; Polyclonal IgG Santa Cruz (Santa Cruz, CA, USA), sc-163 Cell Signaling Technology, Inc. Anti-CDK4 Rabbit; Monoclonal (Billerica, MA, USA), #12790T Cell Signaling Technology, Inc. Anti-cyclin B1 Rabbit; Monoclonal (Billerica, MA, USA), #4138S Cell Signaling Technology, Inc. Anti-cyclin D1 Rabbit; Monoclonal (Billerica, MA, USA), #2978T Cell Signaling Technology, Inc. Anti-cyclin D3 Mouse; Monoclonal (Billerica, MA, USA), #2936T Cell Signaling Technology, Inc. Anti-p21 Rabbit; Monoclonal (Billerica, MA, USA), #2947T Cell Signaling Technology, Inc. Anti-p27 Rabbit; Monoclonal (Billerica, MA, USA), #3686T Donkey anti-rabbit IgG-HRP Donkey; Polyclonal Santa Cruz (Santa Cruz, CA, USA), sc-2313 Goat anti-mouse IgG-HRP Goat; Polyclonal Santa Cruz (Santa Cruz, CA, USA), sc-2005

3.2. Cell Culture According to a previously published method [13], RWPE-1 cells (American Type Culture Collection, Manassas, VA, USA; CRL-11609), PC-3 (ATCC, CRL-1435), and LNCaP-FGC (ATCC, CRL-1740) were seeded at a density of approximately 10,000–20,000 cells/cm2. RWPE-1 cells were grown in Keratinocyte-SFM (pH 7.4) containing 0.05 mg/mL bovine pituitary extract (Gibco), 5 ng/mL human epidermal growth factor (EGF), 100 µg/mL penicillin, and 100 µg/mL streptomycin in a humidiﬁed atmosphere of 95% air and 5% CO2 at 37 ◦C. PC-3 and LNCaP-FGC PCa cells were grown in RPMI-1640 supplemented with 10% FBS, 100 µg/mL penicillin, and 100 µg/mL streptomycin (GibcoBRL) in a humidiﬁed atmosphere of 95% air and 5% CO2 at 37 ◦C.

3.3. Cell Viability To determine whether BNAs exert their eﬀects on RWPE-1, PC-3, and LNCaP cell viability in a dose- or time-dependent manner, cells (5000 cells/well in a 96-well plate) were treated with various concentrations (0–1 mM) of arecoline, arecaidine, or guvacine in the presence of 10% FBS-supplemented medium for the indicated time periods [6,35]. After a particular duration in culture, 0.5 µg/mL of the tetrazolium dye (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazlium bromide [MTT]) were added to the cells, which were incubated in the dark at 37 ◦C for 3 h. Then, an aliquot of 100 µL of 100% dimethyl sulfoxide (DMSO) was added to stop the reaction; this step allowed the insoluble formazan to dissolve in DMSO. The absorbance was then read at 570 nm.

3.4. Experimental Treatment We followed the methods reported by Tien et al. [6] and Wang et al. [36] to investigate the ROS-dependent eﬀect of arecoline. RWPE-1, PC-3, and LNCaP cells were pretreated with the antioxidant N-acetylcysteine (NAC; 1.5 mM) for 1 h and then exposed to 0.4 mM arecoline. After 24 or 48 h of treatment, we measured cell viability, the percentage of cells in diﬀerent phases of the cell cycle, the expression of cell cycle-related proteins, and ROS production.

3.5. Flow Cytometric Analysis Changes in cell cycle kinetics were analyzed by flow cytometry, as described by Hung et al. [37]. RWPE-1, PC-3, and LNCaP cells were seeded at a density of 6 105 cells/plate in a 10 cm dish, × Int. J. Mol. Sci. 2020, 21, 9219 14 of 18 were treated with or without NAC for 1 h, and then exposed to 0.4 mM arecoline. After 24 and 48 h, cell pellets were harvested, fixed in 70% ethanol, and stored at 20 C until analysis. For analysis, − ◦ the cell pellets were washed with 10 mM cold phosphate-buffered saline (PBS) (pH 7.4), incubated at 37 ◦C for 30 min with 100 µg/mL RNase A, and then stained with 200 µg/mL propidium iodide in PBS containing 1% Triton X-100. The cell cycle profiles and distributions were determined by flow cytometric analysis of 104 cells using the CELLQuest program on a FACSCalibur™ flow cytometer (Becton Dickinson, San Jose, CA, USA). Gating was used to exclude clumped cells from the cell cycle distribution analysis.

3.6. Western Blot Analysis Western blot analysis was performed on supernatant fractions of RWPE-1, PC-3, and LNCaP cells, as described by Shih et al. [38]. An aliquot of approximately 50–75 µg of supernatant protein was separated by 12% sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis with 2 × gel-loading buffer (100 mM Tris-HCl, pH 6.8, 4% SDS, 20% glycerol, 0.2% bromophenol blue, and 10% β-mercaptoethanol) and then blotted onto Immobilon-NC transfer membranes (Millipore, Bedford, MA, USA). The immunoblots were blocked for 1 h at room temperature with 10 mM PBS containing 0.1% Tween 20 and 5% skim milk. The primary and secondary antibodies were used at dilutions of 1:1000 (~0.2 µg/mL) and 1:2000 (~0.2 µg/mL), respectively. The immunoblots were visualized using the Western Lightning™ Chemiluminescence Reagent Plus kit (PerkinElmer Life Science, Boston, MA, USA) for 3 min, followed by exposure to Fuji film for 2–3 min. Blots were quantified using the FX Pro Plus Molecular Imager® (Bio-Rad Laboratories, Hercules, CA, USA). After normalization to β-actin protein expression, the levels of these intracellular proteins were expressed as a percent of the control, unless noted otherwise.

3.7. ROS For the ROS assay, we followed the methods reported by Tien et al. [6] and Wang et al. [36]. RWPE-1, PC-3, and LNCaP cells (1.2 105 cells/well in a six-well plate) were pretreated with NAC for 1 h and × then incubated with 0.4 mM arecoline. After 24 or 48 h of incubation, 30 µM of 20,70-dichlorofluorescein diacetate (DCFDA) was added. After a 30 min incubation in the dark at 37 ◦C, dichlorofluorescein generated from the reaction of DCFDA with ROS was detected by fluorescence emission using a fluorescence spectrophotometer (F-4500, HITACHI) under an excitation wavelength of 504 nm and emission wavelength of 529 nm. Based on the absorbance value, the ROS levels were normalized to the number of cells and then expressed as multiples relative to the control. Cells were trypsinized and counted using the 0.4% trypan blue exclusion method. Only live cells were represented in this study.

3.8. Statistical Analysis We presented the data as the mean SEM. A statistical analysis was performed, as described by ± Shih et al. [39].

4. Conclusions We concluded that the effects of arecoline on the growth of normal RWPE-1 and cancerous PC-3 and LNCaP prostate cells are likely mediated through cell cycle dysregulation (Figure 11). The effects are related to the induction of different phases of growth arrest as well as alterations in the levels of specific members of the CDK, CKI, and cyclin protein families and are induced in a ROS-dependent manner. Arecoline is more effective than arecaidine and guvacine in the induction of prostate cell growth changes. Since the acetylcholine receptor (AchR) can function as an arecoline receptor [24,29], further studies are needed to determine whether AchR is responsible for the observed effects of arecoline on normal and cancerous prostate cells. Int. J. Mol. Sci. 2020, 21, x FOR PEER REVIEW 15 of 18

4. Conclusions We concluded that the effects of arecoline on the growth of normal RWPE-1 and cancerous PC- 3 and LNCaP prostate cells are likely mediated through cell cycle dysregulation (Figure 11). The effects are related to the induction of different phases of growth arrest as well as alterations in the levels of specific members of the CDK, CKI, and cyclin protein families and are induced in a ROS- dependent manner. Arecoline is more effective than arecaidine and guvacine in the induction of prostate cell growth changes. Since the acetylcholine receptor (AchR) can function as an arecoline receptor [24,29], further studies are needed to determine whether AchR is responsible for the Int. J. Mol. Sci. 2020, 21, 9219 15 of 18 observed effects of arecoline on normal and cancerous prostate cells.

FigureFigure 11. 11. AA proposed proposed mechanism mechanism of of the the differential diﬀerential acti actionsons of arecoline on cell growth among RWPE-1RWPE- 1( A(A),), PC-3 PC-3 (B ),(B and), and LNCaP LNCaP (C) cells.(C) cells. The arecolineThe arecoline signaling signaling was dependent was dependent on the cell on cycle-controlling the cell cycle- controllingprotein pathway, protein such pathway, as CDK1, such CDK4, as CDK1, p21, p27, CDK4, cyclin p21, B1, p27, or cyclin cyclin D3 B1, proteins, or cyclin in D3 the proteins, reactive oxygenin the reactivespecies (ROS)-dependentoxygen species (ROS)-dependent way. Thus, arecoline way. arrestedThus, arecoline RWPE-1 arrested and PC-3 RWPE-1 cells at and the PC-3 G2/M cells phase at andthe G2/MLNCaP phase cells and at the LNCaP G0/G1 cells phase, at the leading G0/G1 to phase, growth leadin inhibitiong to growth of all inhibition cells. The of symbols all cells. of The “ ”symbols and “ ” → a ofwere “→”and“┤” presented withwere either presented stimulatory with either or inhibitory stimulatory eﬀect or of inhibitory arecoline oneffect protein of arecoline expression. on protein expression. Supplementary Materials: Supplementary materials can be found at http://www.mdpi.com/1422-0067/21/23/9219/s1. SupplementaryAuthor Contributions: Materials:L.-J.S.: Supplementary Writing—Original materials draft, can be Formal found at Analysis, www.mdpi.com/xxx/s1. Investigation, Visualization, Data Curation, Resources; J.-Y.W.: Methodology, Validation, Formal Analysis, Investigation, Data Curation; J.-Y.J.: Validation, Formal Analysis, Investigation, Data Curation; A.-C.S.: Visualization; Y.-Y.L.: Conceptualization, Resources, Writing—Review and editing, Funding acquisition; Y.-W.T.: Resources, Funding acquisition; Y.-C.K.: Resources, Funding acquisition; C.-P.C.: Project administration, Resources; Y.-H.K.: Conceptualization, Methodology, Resources, Writing—Review and editing, Supervision, Project administration, Funding acquisition. All authors have read and agreed to the published version of the manuscript. Funding: This work was supported by grants from the Ministry of Science and Technology, Taiwan (MOST106- 2320-B-008-008-MY3; MOST109-2320-B-008-001-MY3), the Taoyuan Arm and Forces General Hospital, Taiwan (AFTYGH-106-11 and MAB-108-094), and Taiwan Landseed Hospital (NCU-LSH-105-B-010) to Y.-H.K. Acknowledgments: We are grateful to Tsu-Wen Tai at the Academia Sinica, Taiwan for her technical assistance. Int. J. Mol. Sci. 2020, 21, 9219 16 of 18

Conﬂicts of Interest: The authors declare no conﬂict of interest.

Abbreviations AchR, acetylcholine receptor; BNA, betel nut alkaloid; CDK, cyclin-dependent kinase; CKI, CDK inhibitor; CRPCa, castration-resistant prostate cancer; DCFDA, 20,70-dichloroﬂuorescein diacetate; DMSO, dimethyl sulfoxide; EGF, epidermal growth factor; FBS, fetal bovine serum; MTT, 3-(4,5-dimethylthiazol- 2-yl)-2,5-diphenyltetrazlium bromide; NAC, N-acetylcysteine; PBS, phosphate-buﬀered saline; PCa, prostate cancer; ROS, reactive oxygen species; SDS-PAGE, sodium dodecyl sulfate-polyacrylamide gel electrophoresis.

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