Cancer Research Article Prevention€¦ · Research Article 6-Shogaol from Dried Ginger Inhibits Growth of Prostate ... chemopreventive and/or therapeutic agent for prostate cancer

Research Article

6-Shogaol from Dried Ginger Inhibits Growth of ProstateCancer Cells Both In Vitro and In Vivo through Inhibition ofSTAT3 and NF-kB Signaling

Achinto Saha1, Jorge Blando1, Eric Silver3, Linda Beltran1, Jonathan Sessler3, and John DiGiovanni1,2

AbstractDespite much recent progress, prostate cancer continues to represent a major cause of cancer-related

mortality andmorbidity inmen. Prostate cancer is themost commonnonskinneoplasmand second leading

cause of death in men. 6-Shogaol (6-SHO), a potent bioactive compound in ginger (Zingiber officinale

Roscoe), has been shown to possess anti-inflammatory and anticancer activity. In the present study, the

effect of 6-SHO on the growth of prostate cancer cells was investigated. 6-SHO effectively reduced survival

and induced apoptosis of cultured human (LNCaP,DU145, and PC3) andmouse (HMVP2) prostate cancer

cells. Mechanistic studies revealed that 6-SHO reduced constitutive and interleukin (IL)-6–induced STAT3

activation and inhibited both constitutive and TNF-a–induced NF-kB activity in these cells. In addition,

6-SHO decreased the level of several STAT3 andNF-kB–regulated target genes at the protein level, including

cyclinD1, survivin, and cMyc andmodulatedmRNA levels of chemokine, cytokine, cell cycle, and apoptosis

regulatory genes (IL-7, CCL5, BAX, BCL2, p21, and p27). 6-SHO was more effective than two other

compounds found in ginger, 6-gingerol, and 6-paradol at reducing survival of prostate cancer cells and

reducing STAT3 andNF-kB signaling. 6-SHO also showed significant tumor growth inhibitory activity in an

allograft model using HMVP2 cells. Overall, the current results suggest that 6-SHOmay have potential as a

chemopreventive and/or therapeutic agent for prostate cancer and that further study of this compound is

warranted. Cancer Prev Res; 7(6); 627–38. �2014 AACR.

IntroductionProstate cancer is the most common noncutaneous neo-

plasm and is the second leading cause of cancer death inAmerican men (1). Approximately 1 in 6 men will bediagnosed with prostate cancer during their lifetime andabout 1 in 36 will die of prostate cancer (2). It is estimatedthat approximately 238,590 new cases of prostate cancerwill be diagnosed and 29,720 prostate cancer-relateddeaths will occur in the United States in 2013 (1, 3). Duringthe early stages of the disease, prostate cancer cells aredependent on androgen for their growth and can be suc-cessfully treated with androgen ablation therapy. However,in amajority of cases, the disease eventually progresses to anandrogen-independent state and becomes unresponsive tochemotherapy, radiotherapy, or hormonal therapy (4–6).

Moreover, the toxicity associated with chemotherapy seri-ously compromises the quality of life of patients withprostate cancer (7). The identification of agents that areless toxic as well as effective against both androgen-depen-dent and androgen-independent prostate cancer wouldprovide better options for prostate cancer management.Furthermore, such compounds may also have value in theprevention of prostate cancer.

Ginger is a natural dietary ingredient with antioxidant,anti-inflammatory, and anticarcinogenic properties (8).Ginger contains several pungent constituents such as gin-gerols, shogaols, paradols, and gingerdiols (9). Gingerolswere identified as the major active components in freshginger rhizome with 6-gingerol (6-GIN) being the mostabundant constituent (10). The shogaols are the predom-inant pungent constituents in dried ginger (11). Recently,there has been a growing interest in ginger and its compo-nents for their potential chemopreventive effects (12).For example, an ethanol extract of ginger was shownto decrease the incidence, size, number, and multiplicityof skin papillomas in SENCAR mice (13). Lee and collea-gues reported that 6-GIN inhibited cell proliferation,induced apoptosis, and G1 cell-cycle arrest in human colo-rectal cancer cells (14). Several in vitro and in vivo studiesalso showed that 6-Shogaol (6-SHO) has more potent anti-inflammatory activity than 6-GIN and another widely stud-ied phytochemical, curcumin (15, 16). Nonetheless, the

Authors' Affiliations: 1Division of Pharmacology and Toxicology and2Department of Nutritional Sciences, Dell Pediatric Research Institute; and3Department of Chemistry, The University of Texas at Austin, Austin, Texas

Note:Supplementary data for this article are available atCancer PreventionResearch Online (http://cancerprevres.aacrjournals.org/).

Corresponding Author: John DiGiovanni, Dell Pediatric Research Insti-tute, The University of Texas at Austin, 1400 Barbara Jordan Blvd, Austin,TX 78723. Phone: 512-495-4726; Fax: 512-495-4946; E-mail:[email protected]

doi: 10.1158/1940-6207.CAPR-13-0420

�2014 American Association for Cancer Research.

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beneficial effects of 6-SHO and its mechanism of actionhave not been clearly evaluated in prostate cancer.

In the present study, we evaluated the efficacy of 6-SHOfor inhibition of cell survival and induction of apoptosisin both human and mouse prostate cancer cells. Theeffects of 6-SHO were compared with the effects of twoother ginger compounds, 6-GIN and 6-paradol (6-PAR).6-SHO was also evaluated for its efficacy at inhibitingHMVP2 cell growth, a prostate tumor cell line derivedfrom HiMyc mice (J. Blando and colleagues; unpublisheddata) in vivo. We report for the first time that 6-SHOproduces significant anticancer activity against humanand mouse prostate cancer cells by inhibiting cell survivaland inducing apoptosis through reduction of STAT3 andNF-kB activity. Collectively, the current results suggestthat 6-SHO may have a role to play as a chemopreventiveand/or therapeutic agent for prostate cancer.

Materials and MethodsReagents

6-SHO was synthesized from zingerone using a modifi-cation of published procedures (refs. 17, 18; see Supple-mentary Information). Samples of 6-SHO, 6-GIN, and 6-PAR were also purchased from Dalton Pharma. Media(RPMI-1640), FBS was purchased from Life Technologies.Antibodies were purchased as follows: STAT3, pSTAT3Y705,pSTAT3S727, cyclin D1, survivin, cMyc, pNF-kBp65S536, NF-kBp65, pJAK2Y1007/08, JAK2, pSrcY416, PARP, and caspase-7,Cell Signaling Technology; p21, p27, and pIkBaS32/36,Santa Cruz Biotechnology; and b-actin, Sigma-Aldrich. Sec-ondary antibodies were purchased from GE Healthcare.

Cell cultureThe human prostate cancer cells LNCaP,DU145, and PC-

3 were purchased from American Type Culture Collection.Cells were maintained in RPMI-1640 medium with 10%FBS. The prostate tumor cell line, HMVP2, was derived fromthe ventral prostate of a one-year-old HiMyc mouse (19)and cultured in RPMI-1640medium containing 10%FBS. Anontumorigenic cell line, NMVP, was isolated from theventral prostate of 5.5-month-old FVB/Nmice and culturedin DMEM/F12 (Lonza) medium containing 10% FBS,bovine pituitary extract (28 mg/mL), EGF (10 ng/mL),insulin (8 mg/mL), transferrin (5 mg/mL), and gentamycin(80 mg/mL; J. Blando and colleagues; unpublished data).

Cell survival assayCell viability was measured by MTT assay (20). Briefly,

cells (5 x104/mL) in 96-well plates were treated with indi-cated concentrations of 6-SHO, 6-GIN, and 6-PAR. Afterincubation, the cells were treated with MTT solution, incu-bated for an additional 3 hours, and were dissolved inSDS solution, and the absorbance was measured at 570 nmusing a microplate reader (Tecan Group Ltd.).

Apoptosis assayThe percentage of apoptotic cells was determined using a

Guava Nexin apoptosis detection kit and Annexin V-posi-

tive cells were measured by Guava-based flow cytometryaccording to the manufacturer’s instructions (Millipore).

Western blottingExpressionof phosphorylated and total protein levelswas

measured by Western blot analysis with slight modifica-tions as described previously (21). Protein lysates wereprepared from cultured cells following treatment as indi-cated. Proteins were visualized using a Commercial Chemi-luminescent Detection Kit (Thermo Scientific).

Quantitative real-time PCRCells were treated with 6-SHO for 24 hours. Total RNA

was isolated using a Qiagen RNeasy Mini Kit (Qiagen).Reverse transcription was performed as previouslydescribed (21). Gene expression levels were quantitativelydetermined by the dCt method using a Viia7 Real-TimePCR System (Applied Biosystems) in conjunction withSYBR Green PCR Master Mix (Qiagen).

Immunofluorescence stainingDU145or LNCaP cells (1 x105)were plated on coverslips,

exposed to vehicle or 6-SHO for 2 hours, stimulated witheither 10 ng/mL interleukin (IL)-6 or TNF-a for 30minutes,fixed with ice-cold methanol for 10 minutes at �20�C, andpermeabilizedwith 0.05%TritonX-100 for 5minutes. Cellswere then incubated with PBS (10% goat serum, 1%bovineserum albumin) for 1 hour followed by overnight incuba-tion with primary antibodies at 4�C, washed and treatedwith 2 mg/mL of Alexa Fluor 568-conjugated secondaryantibody (Molecular Probes) for 1 hour at room tempera-ture, and mounted with medium containing 40, 6-diami-dino-2-phenylindole (22). Cells were visualized using afluorescence microscope (Olympus Optical Co. Ltd.).

Allograft tumor experimentsSyngeneic FVB/N male mice were obtained from an in-

house breeding colony and were fed a semipurified diet(AIN76A, 10 Kcal% fat, Research Diets) and water adlibitum. All protocols were approved by the InstitutionalAnimal Care and Use Committee of the University of Texasat Austin (Austin, TX).HMVP2 cellswere plated in ultra-lowattaching tissue culture dishes for 3 days to generate spher-oids. Spheroids were harvested, mixed with Matrigel (1:1),and injected subcutaneously into the mouse flank. Micewere randomly divided into three groups of 5 mice andtreated with vehicle control or 6-SHO (50 and 100 mg/kgbody weight) intraperitoneally (i.p.) every other day for 32days. The doses of 50 and 100 mg/kg were determined onthe basis of the existing literature of 6-SHO (23), where 10and 50 mg/kg daily doses of 6-SHO were previously used.However, we decreased the frequency of treatments to everyother day to minimize the stress associated with injectionand therefore a higher dose was utilized (100 mg/kg).Tumor sizewasmeasured twice aweekusing a digital caliperand tumor volume was calculated by the formula: 0.5236D1(D2)

2, where D1 and D2 are the long and short diameter,respectively. Food consumption and body weight of the

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mice weremeasuredweekly. Allograft tumors were weighedat the end of the study.

Statistical analysesData are representative of at least three independent

experiments unless otherwise indicated. Statistical analyseswere performed using one-way ANOVA followed by Bon-ferroni multiple comparison test except for the single-dosestudies where a two tailed Student t test was used. Signif-icance was set at P � 0.05 in all cases.

ResultsInhibition of human prostate cancer cell survival by 6-SHOThe effect of 6-SHO on cell survival was evaluated using

both androgen-dependent (LNCaP) and androgen-inde-pendent (DU145, PC-3) human prostate cancer cells. Asshown in Fig. 1A, 6-SHO at concentrations of 10 to 40mmol/L reduced the survival of LNCaP cells. Reductions insurvival of LNCaP cells at a concentrationof 40mmol/Lwere67%, 85%, and 96% at 24, 48, and 72 hours of treatment,respectively. 6-SHO also decreased the survival of DU145and PC-3 cells at the same concentration by 64% and 66%after 24 hours, 80% and 78% after 48 hours, and 80% and76% after 72 hours, respectively.

6-SHO induces apoptosis in human prostate cancercellsGiven the robust survival inhibition of prostate cancer

cells observed following treatment with 40 mmol/L 6-SHO,

we investigated whether this effect was due to the inductionof apoptosis. As shown in Fig. 1B, 6-SHO (40 mmol/L)significantly increased the number of apoptotic cells in allthree human prostate cancer cells as assessed by flowcytometry. Furthermore, treatment with 6-SHO led to thecleavage of both caspase-7 and PARP(Fig. 1C).

Inhibition of constitutive and IL-6–induced STAT3activation by 6-SHO

To further explore the mechanism for the effects of 6-SHO on cell survival, we examined its effects on consti-tutive and IL-6–induced activation of STAT3 in humanprostate cancer cells. Previous studies have shown in bothLNCaP and DU145 cells that STAT3 is constitutivelyphosphorylated on Ser727, whereas constitutive phos-phorylation on Tyr705 is observed only in DU145 cells(refs. 22, 24; see Fig. 2A). PC-3 cells did not expresssignificant levels of either unphosphorylated or phos-phorylated STAT3 and therefore were not used for thesestudies. As shown in Fig. 2B and C, 6-SHO inhibited thephosphorylation of STAT3Ser727 in both LNCaP andDU145 cells and STAT3Tyr705 in DU145 cells in both aconcentration- and time-dependent manner.

Treatment with IL-6 induced phosphorylation of STAT3at both Ser727 and Tyr705 in LNCaP cells (Fig 2D and E)and6-SHOat concentrations of 20 and40mmol/L inhibitedphosphorylation of STAT3 at both phosphorylation sites.The inhibition of IL-6–induced levels of pSTAT3Ser727 andpSTAT3Tyr705 by 6-SHO in both cells was further confirmedby immunocytochemical staining (see Supplementary Fig.S1A andS1B). Following phosphorylation at Tyr705, STAT3

Figure 1. 6-SHO inhibits cellsurvival and induces apoptosis inhuman prostate cancer cells. A,LNCaP, DU145, and PC-3 cellswere treated with the indicatedconcentrations of 6-SHO for 24,48, and 72 hours and cell survivalwas measured by MTT assay. Thedata are presented asmean�SEM.B, cells were treated with vehicle or40 mmol/L 6-SHO for 48 hours andapoptosis was measured byAnnexin V staining. The data arepresented as mean�SEM. C, cellswere treated with vehicle or 6-SHOfor 24 hours and Western blotanalysis was performed forapoptosis markers. a, significantlydifferent (P < 0.05) compared withthe control group and (b) comparedwith the other treated groups.Changes in relative bandintensities (normalized to b-actin)for Western blot data in C are givenat the top of each column andrepresent the average of twoseparate experiments.

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Figure 2. Reduction of constitutive and IL-6 induced STAT3 activation by 6-SHO in human prostate cancer cells. A–E, cell lines were cultured as indicated inMaterials and Methods. Western blot analysis was performed to determine STAT3 status and level (pSTAT3Y705, pSTAT3S727, and total STAT3) in whole-celllysates collected after the indicated treatments. A, cell lysates were prepared from untreated cultures of LNCaP, DU145, and PC-3 cells. B, LNCaP andDU145 cells were treated with 20 or 40 mmol/L 6-SHO and subjected to Western blot analysis. (Continued on the following page.)

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is translocated to the nucleus where it binds to specificSTAT3-binding sites on nuclear target genes (25). As showninFig. 2F, 30minutes after treatment ofDU145 cellswith IL-6, STAT3 was observed primarily in the nucleus. Pretreat-ment with 6-SHO prevented the nuclear translocation ofSTAT3. These results are consistent with the hypothesis that6-SHO inhibits nuclear translocation of STAT3 as a result ofa decrease in pSTAT3Tyr705.We also examined the effect of 6-SHO on JAK2 and Src activity in LNCaP and DU145 cellsand found that 6-SHO decreased the levels of IL-6–inducedpJAK2Tyr1007/1008 and pSrcTyr416 (Fig 2G).

Inhibition of constitutive and TNF-a–induced NF-kBactivation by 6-SHOAs shown in Fig. 3A, treatment with 6-SHO (40 mmol/L)

reduced the pNF-kBp65Ser536 in DU145 and PC-3 cells. 6-SHO also decreased levels of pNF-kBp65Ser536 and its neg-ative regulator pIkBaS32/36 following treatment with TNF-a(Fig. 3B) in all three human prostate cancer cells. Further-more, we found that 6-SHO treatment decreased the level ofnuclear NF-kBp65 in LNCaP and DU145 cells followingtreatment with TNF-a (Fig. 3C).

Inhibition of STAT3 and NF-kB downstream targets by6-SHOBoth STAT3 andNF-kB regulate the expression of various

cell cycle and apoptosis genes (25–27). As shown in Fig. 4Aand B, treatment with 6-SHO reduced the levels of cyclinD1, survivin, and cMyc in both LNCaP and DU145 cells. 6-SHO also inhibited the expression of cyclin D1, survivin,and cMyc in both LNCaP and DU145 cells followingtreatmentwith IL-6 (Fig. 4C). Finally, 6-SHO also decreasedTNF-a–induced cMyc expression in all three human pros-tate cancer cells (Fig. 4D). Thus, inhibition of STAT3 andNF-kB activation by 6-SHO was also accompanied bydownregulation of their target proteins cyclin D1, survivin,and cMyc.

6-SHO modulates inflammatory cytokines,chemokines, cell cycle, and apoptosis-related genes inhuman prostate cancer cellsInflammation is now considered one of the major com-

ponents in cancer development and progression, includingin prostate cancer (28), and various inflammatory cytokinesand chemokines seem to be involved in this process (29). Asshown in Fig. 4E, 6-SHOdecreased themRNA expression ofIL-7 andCCL5 in bothDU145 and LNCaP cells. 6-SHOalsoincreased expression of the apoptosis regulatory gene BAXand decreased expression of BCL2. Interestingly, 6-SHOincreased the expression of p21, p27, SOCS1, and IRF1.

These results indicate that 6-SHOmodulates expression of anumber of genes that may play an important role in itsability to alter the growth and survival of prostate cancercells.

Effects of 6-SHO on HMVP2 cellsRecently, we developed a prostate tumor cell line from

the ventral prostate of one-year-old HiMyc transgenicmice. These cells (called HMVP2) display characteristics ofcancer stem cells in that they produce spheroids underappropriate culture conditions and express stem cell mar-kers (LINneg/Sca1high/CD49f high). These cells form well-differentiated adenocarcinomas upon subcutaneous injec-tion into syngeneic mice (J. Blando and colleagues; un-published data; see also Supplementary Fig. S5). Similarto the results observed in the human prostate cancer cells,6-SHO decreased the survival of cultured HMVP2 cells, atconcentrations 10 to 40 mmol/L (Fig. 5A). 6-SHO (40mmol/L) also induced apoptosis in HMVP2 cells (Fig. 5B)and reduced phosphorylation of STAT3 as shown in Fig. 5C.Notably, 6-SHO inhibited both constitutive and IL-6–induced phosphorylation of STAT3Tyr705 in HMVP2 cells.In addition, 6-SHO also decreased both constitutive andTNF-a–induced NF-kB activation (Fig. 5D). Furthermore,6-SHO decreased both constitutive and IL-6–inducedexpression of cyclin D1 and TNF-a–induced cMyc inHMVP2 cells similar to that seen in human prostate cancercells (see Fig. 5E and F). Thus, 6-SHO was capable ofreducing cell survival while inducing apoptosis in HMVP2cells in a manner similar to that seen for human prostatecancer cells; again, these effects correlated with reducedactivation of both NF-kB and STAT3 signaling.

As shown in Supplementary Fig. S2, 6-SHO,when used atthe same concentration range as above (i.e., 10–40 mmol/L), did not have a significant inhibitory effect on the survivalof a nontumorigenic mouse prostate cell line, NMVP.

Comparison of the effects of 6-GIN and 6-PAR with6-SHO in human and mouse prostate cancer cells

Because ginger contains several biologically active com-ponents, we compared the effects of 6-SHOwith those of 6-GIN and 6-PAR in both human and mouse prostate cancercell lines. As shown in Fig. 5G, both 6-GIN and 6-PARreduced the survival of human and mouse prostate cancercells. However, each of these compounds was less active ona molar basis than 6-SHO. For example, approximately 4-fold higher concentrations of both 6-GIN and 6-PAR wererequired to produce equivalent reductions in cell survival ascomparedwithwhat was seen for 40 mmol/L 6-SHO in boththe human and mouse prostate cancer cell lines. Western

(Continued.) C, time course of the effect of 6-SHOon total and phosphorylated STAT3 in LNCaP andDU145 cells. Cells were treatedwith vehicle or 40mmol/L6-SHO for 1, 3, or 6 hours and Western blot analysis performed. D, LNCaP and DU145 cells were pretreated with 20 and 40 mmol/L 6-SHO for 2 hoursfollowed by IL-6 for 30 minutes. E, LNCaP and DU145 cells were treated with vehicle or 40 mmol/L 6-SHO for 2 hours followed by IL-6 treatment for 0.5, 1,or 3 hours. F, DU145 cells were treated with vehicle or 40 mmol/L 6-SHO for 2 hours and the nuclear localization of STAT3 (red) was measured byimmunofluorescencestaining.G, LNCaPandDU145cellswere treatedwith vehicle or 40mmol/L 6-SHO for 2hours followedby IL-6 for 30minutes. pJAK2andpSrc was determined by Western blotting. Changes in relative band intensities (normalized to b-actin and total protein) for Western blot data are givenat the top of each column. The results are significant (P < 0.05) where a decrease in phosphorylation is �40%.






blot analyses with 6-GIN and 6-PAR provided confirmationthat pSTAT3Tyr705 was reduced by both compounds at aconcentration of 100 mmol/L (Supplementary Fig. S3A) inboth LNCaP and DU145 cells. Both 6-GIN and 6-PARdecreased the level of survivin and increased the expressionof cyclin-dependent kinase inhibitors p21 and p27 in allthree human prostate cancer cells, at the 100 mmol/L con-centration (Supplementary Fig. S3B). TNF-a–inducedphosphorylation of NF-kBp65 and IkBa was also reducedby 6-GIN and 6-PAR in both DU145 and PC-3 cells at the100 mmol/L concentration (see Supplementary Fig. S4A). 6-PAR also decreased the phosphorylation of STAT3, NF-kBp65, and the level of survivin in HMVP2 cells (Supple-mentary Fig. S4B). Collectively, these results demonstratethat 6-GIN and 6-PAR have the ability to block growth andreduce survival of both human and mouse prostate cancercells but that they are both less potent than 6-SHO.

6-SHO inhibits growth of HMVP2 cells in an allograftmodel

The finding that 6-SHO inhibits growth and survival ofboth human andmouse prostate cancer cell lines promptedus to look at the efficacy of this compound in vivo. Towardthis end, HMVP2 cells, grown as spheroids, were used toinitiate tumors in FVB/N male mice following subcutane-ous injection as described in the Materials and Methodssection. Cells were allowed to grow for approximately 2weeks, at which time small tumors at the injection site werepalpable. As shown in Fig. 6A, treatment with 6-SHOproduced statistically significant decreases in tumor volumeat both the 50 and 100 mg/kg doses (62% and 73%,respectively; P < 0.05) compared with vehicle control trea-ted mice at the end of the study. In addition, tumor weightswere also reduced at both doses of 6-SHO (48% and 65%reduction, respectively; Fig. 6B). The reduction in tumor

Figure 3. Inhibition of constitutive and TNF-a induced NF-kB activity by 6-SHO in human prostate cancer cells. A, DU145 and PC-3 cells were treated withvehicle or 40 mmol/L 6-SHO and subjected toWestern blot analyses. B, cells were treated with 20 and 40 mmol/L 6-SHO for 2 hours followed by TNF-a for 30minutes and pNF-kBp65, NF-kBp65, and pIkBa levels were measured by Western blot analysis. Changes in relative band intensities (normalized to b-actinand total protein) forWestern blot data are given at the top of each column. The results are significant (P < 0.05) where a decrease in phosphorylation is�40%.C, LNCaP and DU145 cells were treated with vehicle or 40 mmol/L 6-SHO for 2 hours followed by TNF-a for 30 minutes and the nuclear localizationof NF-kBp65 (green) was measured by immunofluorescence staining.

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weight at the 100 mg/kg dose of 6-SHO was statisticallysignificant (P < 0.05) compared with the vehicle-treatedcontrol group. There were neither appreciable changes inbody weight or the daily food consumption among the 6-SHO-treated and controlmice (Fig. 6C andD) nor did grossobservations at necropsy reveal tissue abnormalities. Asshown in Fig. 6, Western blot analyses of protein lysatesfrom tumor tissue showed a decrease in pSTAT3Y705 (Fig.6E) and both cyclin D1 and survivin levels (Fig. 6F) in 6-SHO–treated groups compared with the vehicle-treatedcontrol group. Taken together, these data demonstrate that6-SHO has potent in vivo antitumor activity at the dosestested and is free of apparent adverse effects.

DiscussionIn theworkpresentedhere,we report for thefirst time that

6-SHO inhibits the survival of both human and mouseprostate cancer cells in culture, a reduction that is accom-panied by the induction of apoptosis. Previously publishedstudies have shown the potential antiproliferative and apo-ptosis inducing activity of 6-SHO in lung, colorectal, liverand, hematologic cancer cells (15, 30–32). In the presentstudy, 6-SHO was also shown to be effective at inhibitingthe growth of HMVP2 cells in vivo. The effect of 6-SHO onsurvival of both human and mouse prostate cancer cells invitrowas comparedwith two additional ginger constituents,6-GIN and 6-PAR. The results support the conclusion that

Figure 4. Effect of 6-SHO on STAT3 and NF-kB targets in human prostate cancer cells. A, LNCaP and DU145 cells were treated with vehicle, 20 or 40 mmol/L6-SHO and the level of survivin was measured by Western blot analysis. B, LNCaP and DU145 cells were treated with vehicle or 40 mmol/L 6-SHO for 1 or 3hours and the level of cyclin D1, survivin, and cMycwasmeasured byWestern blot analysis. C, LNCaP andDU145 cells were treatedwith vehicle or 40mmol/L6-SHO for 2 hours followed by IL-6 and the level of cyclin D1, survivin, and cMyc was measured by Western blot analysis. D, LNCaP, DU145, andPC-3 cells were treated with vehicle or 40 mmol/L 6-SHO for 2 hours followed by TNF-a and were subjected to Western blot analysis for cMyc protein.Changes in relative band intensities (normalized tob-actin) forWestern blot data are given at the topof each column. The results are significant (P<0.05)whereadecrease in protein level is�40%.E, LNCaPandDU145cellswere treatedwith vehicle, 20or 40mmol/L 6-SHO for 24hours.Geneexpression forCCL5, IL-7,BCL2, BAX, p21, p27, SOCS1, and IRF-1 was measured by quantitative PCR. a, significantly different (P < 0.05) compared with the control group and b,compared with the other treated groups.






Figure5. Effect of 6-SHO on mouse prostate cancer HMVP2 cells. A, HMVP2 cells were treated with the indicated concentrations of 6-SHO for 24, 48, and72 hours and cell survival was measured by MTT assay. The data are presented as mean�SEM. B, HMVP2 cells were treated with vehicle or 40 mmol/L 6-SHO for 48 hours and apoptosis was measured by Annexin V staining. The data are presented as mean�SEM. C, HMVP2 cells were treated with40 mmol/L 6-SHO for 30 and 60 minutes and Western blot analysis was performed for pSTAT3Tyr705, STAT3, and b-actin (top two); HMVP2 cells weretreated with 20 and 40 mmol/L 6-SHO for 2 hours followed by IL-6 for 30 minutes and Western blot analysis was performed for pSTAT3Tyr705, STAT3, andb-actin (bottom two). D, HMVP2 cells were treated with vehicle or 40 mmol/L 6-SHO followed by TNF-a treatment for 30 minutes and Westernblot analysis was performed for pNF-kBp65, NF-kBp65, and b-actin (top); HMVP2 cells were treated with 20 and 40 mmol/L 6-SHO for 2 hours followedby TNF-a for 30 minutes and Western blot analysis was performed for pNF-kBp65, NF-kBp65, pIkBa, and b-actin (bottom). E, HMVP2 cells weretreated with vehicle, 20 or 40 mmol/L 6-SHO for 2 hours followed by IL-6 and the level of cyclin D1 was measured by Western blot analysis. F, HMVP2cells were treated with vehicle or 40 mmol/L 6-SHO for 2 hours followed by TNF-a and the level of cMyc was measured by Western blot analysis.Changes in relative band intensities (normalized to b-actin and total protein) for Western blot data are given at the top of each column. The results aresignificant (P < 0.05) where a decrease in phosphorylation or protein level is �40%. G, LNCaP, DU145, PC-3, and HMVP2 cells were treated with theindicated concentrations of 6-PAR, 6-GIN, or 6-SHO for 48 hours and cell survival was measured by MTT assay. The data are presented as mean�SEM.a, significantly different (P < 0.05) compared with the control group and b, compared with the other treated groups of the same compound.

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Figure 6. 6-SHO reduces HMVP2 allograft tumor growth in vivo. Spheroids from HMVP2 cells were injected subcutaneously into the flank of male FVB mice.Two weeks after injection, mice were treated intraperitoneally with 6-SHO (50 or 100 mg/kg body weight, BW) every other day for the duration of theexperiment. A, tumor volume is expressed as average tumor volume/mouse (mm3). B, tumor weight is presented as average tumor weight (mg)/mouse. C,average mouse BW (g) for each group. D, average feed consumption/mouse/day. Data are mean�SEM; n¼ 5 mice per group; �, P < 0.05 compared with thecontrol group. Lysates of allograft tumor tissues were subjected to Western blot analysis for pSTAT3Tyr705 and STAT3 (E) and cyclinD1 and survivin (F)and b-actin was used as control for both sets of blots. Changes in relative band intensities (normalized to b-actin and total protein) for Western blot data aregiven at the top of each column. The results are significant (P < 0.05) where a decrease in phosphorylation or protein level is �40%.






among the three compounds tested, 6-SHO was the mostpotent for inhibiting growth of the prostate cancer cells invitro. More broadly, 6-SHO seems to possess characteristicsthat make it worthy of further study as an agent for theprevention and/or treatment of prostate cancer.

Accumulating evidence shows that STAT3 is constitu-tively activated in human prostate cancer and that STAT3 isinvolved in cell proliferation, metastasis, angiogenesis,and resistance to apoptosis (33–35). Several studies havealso shown that inhibition of STAT3 activation results ingrowth inhibition and induction of apoptosis in culturedprostate cancer cells (36, 37). Mechanistic studies per-formed as part of the current study revealed that 6-SHOtreatment of prostate cancer cells inhibits both constitutive(DU145 and HMVP2) and IL-6–induced (LNCaP) phos-phorylation of STAT3Tyr705. 6-SHO treatment alsodecreases phosphorylation of STAT3Ser727 as assessed inLNCaP and DU145 cells. The activation of STAT3 isregulated by upstream kinases, including receptor tyrosinekinases (e.g., EGF receptor) and non-receptor tyrosinekinases such as Jak2 and Src (25). As shown in Fig. 2G,6-SHO inhibited activation of both JAK2 and Src, suggest-ing that inhibition of both JAK2 and Src kinases likelyplays a role in mediating the effects of 6-SHO on down-stream signaling, including STAT3 activation.

Previous studies have shown that PC-3 cells do notexpress significant amounts of STAT3 or pSTAT3 even afterprolonged exposure to IL-6 (22). This was also the case inthe current experiments where PC3 cells were devoid ofmeasurable STAT3 (Fig. 2A). However, because 6-SHOproduced significant growth inhibition and induced apo-ptosis in PC-3 cells (Fig 1A and B), other mechanismsassociated with 6-SHO action were investigated. In thisregard, 6-SHO has been shown to inhibit activation ofNF-kB in several cell types and in mouse skin in vivo aftertopical treatment (16, 38). NF-kB is constitutively active inandrogen-independent prostate cancer cells such as PC-3cells (39–41). As shown inFigs. 3 and4,NF-kBwas found tobe constitutively active in PC-3, DU145, and HMVP2 cells.Treatment with 6-SHO inhibited both constitutive andTNF-a–induced phosphorylation of NF-kBp65 in all threehuman prostate cancer cells. Immunocytochemical dataalso demonstrated that NF-kBp65 was located primarily inthe nucleus after treatment with TNF-a and that 6-SHOblocked this nuclear localization (Fig. 3C). Inhibitors of IkBkinase (IKK) are the major upstream regulators of NF-kBand are activated by a wide variety of stimuli includingproinflammatory cytokines (e.g., TNF-a and IL-1; ref. 40).Once IKKs are activated and phosphorylated, they in turncause phosphorylation and proteasomal degradation ofIkBa, thus leaving the free active form of NF-kB for nuclearlocalization (26, 39–42). As shown in Fig. 3B, 6-SHOtreatment also decreased the TNF-a–induced phosphory-lation of IkBa, as would be expected for an inhibitory effectfor NF-kB in prostate cancer cells that is mediated throughthe classical IKK/IkB complex (41).

The activation of both STAT3 and NF-kB signaling leadsto alterations in the expression of multiple target genes

involved in proliferation, survival, and apoptosis, such ascyclin D1, survivin, cMyc, and Bcl2 (22, 24, 40). Treatmentof cultured human and mouse prostate cancer cells with 6-SHO reduced the levels of cyclin D1, survivin, and cMycprotein and altered mRNA expression of selected chemo-kine, cytokine, cell cycle, and apoptosis regulatory genes(Fig. 4). Thus, inhibition of STAT3 and NF-kB activity by 6-SHO was associated with reduced expression of their targetgene products. Given that 6-SHO effectively inhibited thegrowth of prostate cancer cells regardless of STAT3 expres-sion or activity, multiple mechanisms are likely involved inits effects on prostate cancer cell survival, including effectson STAT3,NF-kB, andpossibly other signaling pathways. Inthis regard, it has been reported that 6-SHO induces apo-ptosis by generation of reactive oxygen species in humancolorectal cancer cells, by directly regulating Akt1/2 in non–small cell lung carcinoma cells and by modulation ofestrogen receptor stress in hepatocellular carcinoma cells(23, 32, 43). In addition, as shown in Fig. 2G, Src activa-tion was inhibited following treatment with 6-SHO. Thus,6-SHO affected multiple signaling pathways that likelycontributed to its ability to inhibit cell growth and induceapoptosis in all of the prostate cancer cell lines examinedin the current study.

As part of the current study, the activity of two othercompounds found in ginger (6-GIN and 6-PAR) werecompared with 6-SHO. The potential chemopreventiveeffects of 6-GIN and 6-PAR have been previously reported.For example, Jeong and colleagues reported that 6-GINsuppresses colon cancer cell growth by inhibiting leuko-triene A4 (44). 6-GIN was also found to inhibit COX-2expression by blocking mitogen-activated protein kinaseand NF-kB in 12-O-tetradecanoylphorbol-13-acetate(TPA)-induced mouse skin (45). In another report, both6-GIN and 6-PARwere shown to induce apoptosis inHL-60leukemia cells (46). 6-PAR treatment also induced apopto-sis and caspase-3 activation in an oral squamous carcinomacell line and demonstrated chemopreventive and antioxi-dant activity in the 7,12-dimethylbenz(a)anthracene(DMBA)-induced hamster buccal pouch carcinogenesisassay (47, 48). As shown in Fig. 5G and SupplementaryFig. S3, both 6-GIN and 6-PAR inhibited survival of humanand mouse prostate cancer cells and reduced activation ofboth STAT3 and NF-kB. However, higher concentrations(�2–3 times) were required to achieve effects comparableto 6-SHO. These results are consistent with previouslypublished reports that 6-SHO is more potent than 6-GIN(15, 16).

On the basis of this precedent and the cell culture datashowing that 6-SHO was the most effective of the threeginger-derived compounds that were considered in thepresent study, its effects on tumor growth in an in vivomodel system were evaluated (HMVP2 cells grown in syn-geneic FVB/N mice). As shown in Fig. 6, 6-SHO at twodifferent doses (50 and 100 mg/kg, i.p. every other day)produced statistically significant reductions in the growthofHMVP2 cells at both doses (tumor volume) and at thehigher dose (tumor weight) without any apparent toxic


Saha et al.




effects. Analysis of protein lysates from tumor tissue con-firmed a reduction in STAT3 activation and altered expres-sion of several target proteins in the 6-SHO–treated groups.In conclusion, the results of the current study demon-

strate that treatment of androgen-dependent and -indepen-dent human prostate cancer cells in culture with 6-SHOinhibits survival and induces apoptosis. 6-SHOalso inhibitssurvival and induces apoptosis in cultured mouse prostatecancer cells derived from HiMyc mice. Importantly, 6-SHOwas highly effective at inhibiting the growth ofHMVP2 cellsin an allograft tumor model. These effects of 6-SHO wereassociated with inhibition of both STAT3 and NF-kB sig-naling and possibly other signaling pathways (e.g., Src). Onthe basis of our findings, we suggest that 6-SHO has acombination of activity, low toxicity, and biochemicalproperties that makes it of potential utility as a naturallyoccurring chemopreventive and/or therapeutic agent inprostate cancer.

Disclosure of Potential Conflicts of InterestNo potential conflicts of interest were disclosed.

Authors' ContributionsConception and design: A. Saha, J.L. Sessler, J. DiGiovanniDevelopment of methodology: A. Saha, J. DiGiovanniAcquisitionofdata (provided animals, acquired andmanagedpatients,provided facilities, etc.): A. Saha, J. Blando, E. Silver, J. DiGiovanniAnalysis and interpretation of data (e.g., statistical analysis, biosta-tistics, computational analysis): A. Saha, J. Blando, J.L. Sessler,J. DiGiovanniWriting, review, and/or revision of the manuscript: A. Saha, J. Blando,E. Silver, L. Beltran, J.L. Sessler, J. DiGiovanniAdministrative, technical, or material support (i.e., reporting or orga-nizing data, constructing databases): A. Saha, J.L. SesslerStudy supervision: J. DiGiovanni

Grant SupportA. Saha and E. Silver were supported by Cancer Prevention Research

Institute of Texas postdoctoral and predoctoral trainee awards under grantRP101501 from the State of Texas.

The costs of publication of this article were defrayed in part by thepayment of page charges. This article must therefore be hereby markedadvertisement in accordance with 18 U.S.C. Section 1734 solely to indicatethis fact.

Received December 5, 2013; revised February 24, 2014; accepted March26, 2014; published OnlineFirst April 1, 2014.

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2014;7:627-638. Published OnlineFirst April 1, 2014.Cancer Prev Res Achinto Saha, Jorge Blando, Eric Silver, et al. B Signalingκ

through Inhibition of STAT3 and NF-In Vivo and In VitroCells Both 6-Shogaol from Dried Ginger Inhibits Growth of Prostate Cancer

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