6-Shogaol from Dried Ginger Inhibits Growth of Prostate Cancer Cells Both in Vitro and in Vivo Through Inhibition of STAT3 and NF-Kb Signaling

Published OnlineFirst April 1, 2014; DOI: 10.1158/1940-6207.CAPR-13-0420

Cancer Prevention Research Article Research

6-Shogaol from Dried Ginger Inhibits Growth of Prostate Cancer Cells Both In Vitro and In Vivo through Inhibition of STAT3 and NF-kB Signaling

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

Abstract Despite much recent progress, prostate cancer continues to represent a major cause of cancer-related mortality and morbidity in men. Prostate cancer is the most common nonskin neoplasm and 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) and mouse (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 and NF-kB–regulated target genes at the protein level, including cyclin D1, survivin, and cMyc and modulated mRNA 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 and NF-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-SHO may 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.

Introduction Moreover, the toxicity associated with chemotherapy seri- Prostate cancer is the most common noncutaneous neo- ously compromises the quality of life of patients with plasm and is the second leading cause of cancer death in prostate cancer (7). The identification of agents that are American men (1). Approximately 1 in 6 men will be less toxic as well as effective against both androgen-depen- diagnosed with prostate cancer during their lifetime and dent and androgen-independent prostate cancer would about 1 in 36 will die of prostate cancer (2). It is estimated provide better options for prostate cancer management. that approximately 238,590 new cases of prostate cancer Furthermore, such compounds may also have value in the will be diagnosed and 29,720 prostate cancer-related prevention of prostate cancer. deaths will occur in the United States in 2013 (1, 3). During Ginger is a natural dietary ingredient with antioxidant, the early stages of the disease, prostate cancer cells are anti-inflammatory, and anticarcinogenic properties (8). dependent on androgen for their growth and can be suc- Ginger contains several pungent constituents such as gin- cessfully treated with androgen ablation therapy. However, gerols, shogaols, paradols, and gingerdiols (9). Gingerols in a majority of cases, the disease eventually progresses to an were identified as the major active components in fresh androgen-independent state and becomes unresponsive to ginger rhizome with 6-gingerol (6-GIN) being the most chemotherapy, radiotherapy, or hormonal therapy (4–6). abundant 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- Authors' Affiliations: 1Division of Pharmacology and Toxicology and nents for their potential chemopreventive effects (12). 2Department of Nutritional Sciences, Dell Pediatric Research Institute; and 3Department of Chemistry, The University of Texas at Austin, Austin, Texas For example, an ethanol extract of ginger was shown to decrease the incidence, size, number, and multiplicity Note: Supplementary data for this article are available at Cancer Prevention Research Online (http://cancerprevres.aacrjournals.org/). of skin papillomas in SENCAR mice (13). Lee and colleagues reported that 6-GIN inhibited cell proliferation, Corresponding Author: John DiGiovanni, Dell Pediatric Research Insti- tute, The University of Texas at Austin, 1400 Barbara Jordan Blvd, Austin, induced apoptosis, and G1 cell-cycle arrest in human colo- TX 78723. Phone: 512-495-4726; Fax: 512-495-4946; E-mail: rectal cancer cells (14). Several in vitro and in vivo studies [email protected] also showed that 6-Shogaol (6-SHO) has more potent anti- doi: 10.1158/1940-6207.CAPR-13-0420 inflammatory activity than 6-GIN and another widely stud- 2014 American Association for Cancer Research. ied phytochemical, curcumin (15, 16). Nonetheless, the

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beneficial effects of 6-SHO and its mechanism of action tive cells were measured by Guava-based flow cytometry have not been clearly evaluated in prostate cancer. according to the manufacturer’s instructions (Millipore). In the present study, we evaluated the efficacy of 6-SHO for inhibition of cell survival and induction of apoptosis Western blotting in both human and mouse prostate cancer cells. The Expression of phosphorylated and total protein levels was effects of 6-SHO were compared with the effects of two measured by Western blot analysis with slight modifica- other ginger compounds, 6-GIN and 6-paradol (6-PAR). tions as described previously (21). Protein lysates were 6-SHO was also evaluated for its efficacy at inhibiting prepared from cultured cells following treatment as indi- HMVP2 cell growth, a prostate tumor cell line derived cated. Proteins were visualized using a Commercial Chemi- from HiMyc mice (J. Blando and colleagues; unpublished luminescent Detection Kit (Thermo Scientific). data) in vivo. We report for the first time that 6-SHO produces significant anticancer activity against human Quantitative real-time PCR and mouse prostate cancer cells by inhibiting cell survival Cells were treated with 6-SHO for 24 hours. Total RNA and inducing apoptosis through reduction of STAT3 and was isolated using a Qiagen RNeasy Mini Kit (Qiagen). NF-kB activity. Collectively, the current results suggest Reverse transcription was performed as previously that 6-SHO may have a role to play as a chemopreventive described (21). Gene expression levels were quantitatively and/or therapeutic agent for prostate cancer. determined by the dCt method using a Viia7 Real-Time PCR System (Applied Biosystems) in conjunction with Materials and Methods SYBR Green PCR Master Mix (Qiagen). Reagents 6-SHO was synthesized from zingerone using a modifi- Immunofluorescence staining cation of published procedures (refs. 17, 18; see Supple- DU145 or LNCaP cells (1 x105) were plated on coverslips, mentary Information). Samples of 6-SHO, 6-GIN, and 6- exposed to vehicle or 6-SHO for 2 hours, stimulated with PAR were also purchased from Dalton Pharma. Media either 10 ng/mL interleukin (IL)-6 or TNF-a for 30 minutes, (RPMI-1640), FBS was purchased from Life Technologies. fixed with ice-cold methanol for 10 minutes at 20 C, and Antibodies were purchased as follows: STAT3, pSTAT3Y705, permeabilized with 0.05% Triton X-100 for 5 minutes. Cells pSTAT3S727, cyclin D1, survivin, cMyc, pNF-kBp65S536, NF- were then incubated with PBS (10% goat serum, 1% bovine kBp65, pJAK2Y1007/08, JAK2, pSrcY416, PARP, and caspase-7, serum albumin) for 1 hour followed by overnight incuba- Cell Signaling Technology; p21, p27, and pIkBaS32/36, tion with primary antibodies at 4 C, washed and treated Santa Cruz Biotechnology; and b-actin, Sigma-Aldrich. Sec- with 2 mg/mL of Alexa Fluor 568-conjugated secondary ondary antibodies were purchased from GE Healthcare. antibody (Molecular Probes) for 1 hour at room tempera- ture, and mounted with medium containing 40, 6-diami- Cell culture dino-2-phenylindole (22). Cells were visualized using a The human prostate cancer cells LNCaP, DU145, and PC- fluorescence microscope (Olympus Optical Co. Ltd.). 3 were purchased from American Type Culture Collection. Cells were maintained in RPMI-1640 medium with 10% Allograft tumor experiments FBS. The prostate tumor cell line, HMVP2, was derived from Syngeneic FVB/N male mice were obtained from an in- the ventral prostate of a one-year-old HiMyc mouse (19) house breeding colony and were fed a semipurified diet and cultured in RPMI-1640 medium containing 10% FBS. A (AIN76A, 10 Kcal% fat, Research Diets) and water ad nontumorigenic cell line, NMVP, was isolated from the libitum. All protocols were approved by the Institutional ventral prostate of 5.5-month-old FVB/N mice and cultured Animal Care and Use Committee of the University of Texas in DMEM/F12 (Lonza) medium containing 10% FBS, at Austin (Austin, TX). HMVP2 cells were plated in ultra-low bovine pituitary extract (28 mg/mL), EGF (10 ng/mL), attaching tissue culture dishes for 3 days to generate spher- insulin (8 mg/mL), transferrin (5 mg/mL), and gentamycin oids. Spheroids were harvested, mixed with Matrigel (1:1), (80 mg/mL; J. Blando and colleagues; unpublished data). and injected subcutaneously into the mouse flank. Mice were randomly divided into three groups of 5 mice and Cell survival assay treated with vehicle control or 6-SHO (50 and 100 mg/kg Cell viability was measured by MTT assay (20). Briefly, body weight) intraperitoneally (i.p.) every other day for 32 cells (5 x104/mL) in 96-well plates were treated with indi- days. The doses of 50 and 100 mg/kg were determined on cated concentrations of 6-SHO, 6-GIN, and 6-PAR. After the basis of the existing literature of 6-SHO (23), where 10 incubation, the cells were treated with MTT solution, incu- and 50 mg/kg daily doses of 6-SHO were previously used. bated for an additional 3 hours, and were dissolved in However, we decreased the frequency of treatments to every SDS solution, and the absorbance was measured at 570 nm other day to minimize the stress associated with injection using a microplate reader (Tecan Group Ltd.). and therefore a higher dose was utilized (100 mg/kg). Tumor size was measured twice a week using a digital caliper Apoptosis assay and tumor volume was calculated by the formula: 0.5236 2 The percentage of apoptotic cells was determined using a D1(D2) , where D1 and D2 are the long and short diameter, Guava Nexin apoptosis detection kit and Annexin V-posi- respectively. Food consumption and body weight of the

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mice were measured weekly. Allograft tumors were weighed we investigated whether this effect was due to the induction at the end of the study. of apoptosis. As shown in Fig. 1B, 6-SHO (40 mmol/L) significantly increased the number of apoptotic cells in all Statistical analyses three human prostate cancer cells as assessed by flow Data are representative of at least three independent cytometry. Furthermore, treatment with 6-SHO led to the experiments unless otherwise indicated. Statistical analyses cleavage of both caspase-7 and PARP(Fig. 1C). were performed using one-way ANOVA followed by Bon- ferroni multiple comparison test except for the single-dose Inhibition of constitutive and IL-6–induced STAT3 studies where a two tailed Student t test was used. Signif- activation by 6-SHO icance was set at P 0.05 in all cases. To further explore the mechanism for the effects of 6- SHO on cell survival, we examined its effects on consti- Results tutive and IL-6–induced activation of STAT3 in human prostate cancer cells. Previous studies have shown in both Inhibition of human prostate cancer cell survival by 6- LNCaP and DU145 cells that STAT3 is constitutively SHO phosphorylated on Ser727, whereas constitutive phos- The effect of 6-SHO on cell survival was evaluated using phorylation on Tyr705 is observed only in DU145 cells both androgen-dependent (LNCaP) and androgen-inde- (refs. 22, 24; see Fig. 2A). PC-3 cells did not express pendent (DU145, PC-3) human prostate cancer cells. As significant levels of either unphosphorylated or phos- shown in Fig. 1A, 6-SHO at concentrations of 10 to 40 phorylated STAT3 and therefore were not used for these m mol/L reduced the survival of LNCaP cells. Reductions in studies. As shown in Fig. 2B and C, 6-SHO inhibited the m survival of LNCaP cells at a concentration of 40 mol/L were phosphorylation of STAT3Ser727 in both LNCaP and 67%, 85%, and 96% at 24, 48, and 72 hours of treatment, DU145 cells and STAT3Tyr705 in DU145 cells in both a respectively. 6-SHO also decreased the survival of DU145 concentration- and time-dependent manner. and PC-3 cells at the same concentration by 64% and 66% Treatment with IL-6 induced phosphorylation of STAT3 after 24 hours, 80% and 78% after 48 hours, and 80% and at both Ser727 and Tyr705 in LNCaP cells (Fig 2D and E) 76% after 72 hours, respectively. and 6-SHO at concentrations of 20 and 40 mmol/L inhibited phosphorylation of STAT3 at both phosphorylation sites. 6-SHO induces apoptosis in human prostate cancer The inhibition of IL-6–induced levels of pSTAT3Ser727 and cells pSTAT3Tyr705 by 6-SHO in both cells was further confirmed Given the robust survival inhibition of prostate cancer by immunocytochemical staining (see Supplementary Fig. cells observed following treatment with 40 mmol/L 6-SHO, S1A and S1B). Following phosphorylation at Tyr705, STAT3

Figure 1. 6-SHO inhibits cell survival and induces apoptosis in human prostate cancer cells. A, LNCaP, DU145, and PC-3 cells were treated with the indicated concentrations of 6-SHO for 24, 48, and 72 hours and cell survival was measured by MTT assay. The data are presented as meanSEM. B, 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 meanSEM. C, cells were treated with vehicle or 6-SHO for 24 hours and Western blot analysis was performed for apoptosis markers. a, signiﬁcantly different (P < 0.05) compared with the control group and (b) compared with the other treated groups. Changes in relative band intensities (normalized to b-actin) for Western blot data in C are given at the top of each column and represent the average of two separate 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 in Materials and Methods. Western blot analysis was performed to determine STAT3 status and level (pSTAT3Y705, pSTAT3S727, and total STAT3) in whole-cell lysates collected after the indicated treatments. A, cell lysates were prepared from untreated cultures of LNCaP, DU145, and PC-3 cells. B, LNCaP and DU145 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 specific These results indicate that 6-SHO modulates expression of a STAT3-binding sites on nuclear target genes (25). As shown number of genes that may play an important role in its in Fig. 2F, 30 minutes after treatment of DU145 cells with IL- ability to alter the growth and survival of prostate cancer 6, STAT3 was observed primarily in the nucleus. Pretreat- cells. ment with 6-SHO prevented the nuclear translocation of STAT3. These results are consistent with the hypothesis that Effects of 6-SHO on HMVP2 cells 6-SHO inhibits nuclear translocation of STAT3 as a result of Recently, we developed a prostate tumor cell line from a decrease in pSTAT3Tyr705. We also examined the effect of 6- the ventral prostate of one-year-old HiMyc transgenic SHO on JAK2 and Src activity in LNCaP and DU145 cells mice. These cells (called HMVP2) display characteristics of and found that 6-SHO decreased the levels of IL-6–induced cancer stem cells in that they produce spheroids under pJAK2Tyr1007/1008 and pSrcTyr416 (Fig 2G). appropriate culture conditions and express stem cell markers (LINneg/Sca1high/CD49f high). These cells form well- Inhibition of constitutive and TNF-a–induced NF-kB differentiated adenocarcinomas upon subcutaneous injec- activation by 6-SHO tion into syngeneic mice (J. Blando and colleagues; un- As shown in Fig. 3A, treatment with 6-SHO (40 mmol/L) published data; see also Supplementary Fig. S5). Similar reduced the pNF-kBp65Ser536 in DU145 and PC-3 cells. 6- to the results observed in the human prostate cancer cells, SHO also decreased levels of pNF-kBp65Ser536 and its neg- 6-SHO decreased the survival of cultured HMVP2 cells, at ative regulator pIkBaS32/36 following treatment with TNF-a concentrations 10 to 40 mmol/L (Fig. 5A). 6-SHO (40 (Fig. 3B) in all three human prostate cancer cells. Further- mmol/L) also induced apoptosis in HMVP2 cells (Fig. 5B) more, we found that 6-SHO treatment decreased the level of and reduced phosphorylation of STAT3 as shown in Fig. 5C. nuclear NF-kBp65 in LNCaP and DU145 cells following Notably, 6-SHO inhibited both constitutive and IL-6– treatment with TNF-a (Fig. 3C). induced phosphorylation of STAT3Tyr705 in HMVP2 cells. In addition, 6-SHO also decreased both constitutive and Inhibition of STAT3 and NF-kB downstream targets by TNF-a–induced NF-kB activation (Fig. 5D). Furthermore, 6-SHO 6-SHO decreased both constitutive and IL-6–induced Both STAT3 and NF-kB regulate the expression of various expression of cyclin D1 and TNF-a–induced cMyc in cell cycle and apoptosis genes (25–27). As shown in Fig. 4A HMVP2 cells similar to that seen in human prostate cancer and B, treatment with 6-SHO reduced the levels of cyclin cells (see Fig. 5E and F). Thus, 6-SHO was capable of D1, survivin, and cMyc in both LNCaP and DU145 cells. 6- reducing cell survival while inducing apoptosis in HMVP2 SHO also inhibited the expression of cyclin D1, survivin, cells in a manner similar to that seen for human prostate and cMyc in both LNCaP and DU145 cells following cancer cells; again, these effects correlated with reduced treatment with IL-6 (Fig. 4C). Finally, 6-SHO also decreased activation of both NF-kB and STAT3 signaling. TNF-a–induced cMyc expression in all three human pros- As shown in Supplementary Fig. S2, 6-SHO, when used at tate cancer cells (Fig. 4D). Thus, inhibition of STAT3 and the same concentration range as above (i.e., 10–40 mmol/ NF-kB activation by 6-SHO was also accompanied by L), did not have a significant inhibitory effect on the survival downregulation of their target proteins cyclin D1, survivin, of a nontumorigenic mouse prostate cell line, NMVP. and cMyc. Comparison of the effects of 6-GIN and 6-PAR with 6-SHO modulates inflammatory cytokines, 6-SHO in human and mouse prostate cancer cells chemokines, cell cycle, and apoptosis-related genes in Because ginger contains several biologically active com- human prostate cancer cells ponents, we compared the effects of 6-SHO with those of 6- Inflammation is now considered one of the major com- GIN and 6-PAR in both human and mouse prostate cancer ponents in cancer development and progression, including cell lines. As shown in Fig. 5G, both 6-GIN and 6-PAR in prostate cancer (28), and various inflammatory cytokines reduced the survival of human and mouse prostate cancer and chemokines seem to be involved in this process (29). As cells. However, each of these compounds was less active on shown in Fig. 4E, 6-SHO decreased the mRNA expression of a molar basis than 6-SHO. For example, approximately 4- IL-7 and CCL5 in both DU145 and LNCaP cells. 6-SHO also fold higher concentrations of both 6-GIN and 6-PAR were increased expression of the apoptosis regulatory gene BAX required to produce equivalent reductions in cell survival as and decreased expression of BCL2. Interestingly, 6-SHO compared with what was seen for 40 mmol/L 6-SHO in both increased the expression of p21, p27, SOCS1, and IRF1. the human and mouse prostate cancer cell lines. Western

(Continued.) C, time course of the effect of 6-SHO on total and phosphorylated STAT3 in LNCaP and DU145 cells. Cells were treated with vehicle or 40 mmol/L 6-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 hours followed 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 by immunoﬂuorescence staining. G, LNCaP and DU145 cells were treated with vehicle or 40 mmol/L 6-SHO for 2 hours followed by IL-6 for 30 minutes. pJAK2 and pSrc was determined by Western blotting. 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 are signiﬁcant (P < 0.05) where a decrease in phosphorylation is 40%.

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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 with vehicle or 40 mmol/L 6-SHO and subjected to Western blot analyses. B, cells were treated with 20 and 40 mmol/L 6-SHO for 2 hours followed by TNF-a for 30 minutes and pNF-kBp65, NF-kBp65, and pIkBa levels were 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 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 localization of NF-kBp65 (green) was measured by immunoﬂuorescence staining.

blot analyses with 6-GIN and 6-PAR provided confirmation 6-SHO inhibits growth of HMVP2 cells in an allograft that pSTAT3Tyr705 was reduced by both compounds at a model concentration of 100 mmol/L (Supplementary Fig. S3A) in The finding that 6-SHO inhibits growth and survival of both LNCaP and DU145 cells. Both 6-GIN and 6-PAR both human and mouse prostate cancer cell lines prompted decreased the level of survivin and increased the expression us to look at the efficacy of this compound in vivo. Toward of cyclin-dependent kinase inhibitors p21 and p27 in all this end, HMVP2 cells, grown as spheroids, were used to three human prostate cancer cells, at the 100 mmol/L con- initiate tumors in FVB/N male mice following subcutane- centration (Supplementary Fig. S3B). TNF-a–induced ous injection as described in the Materials and Methods phosphorylation of NF-kBp65 and IkBa was also reduced section. Cells were allowed to grow for approximately 2 by 6-GIN and 6-PAR in both DU145 and PC-3 cells at the weeks, at which time small tumors at the injection site were 100 mmol/L concentration (see Supplementary Fig. S4A). 6- palpable. As shown in Fig. 6A, treatment with 6-SHO PAR also decreased the phosphorylation of STAT3, NF- produced statistically significant decreases in tumor volume kBp65, and the level of survivin in HMVP2 cells (Supple- at both the 50 and 100 mg/kg doses (62% and 73%, mentary Fig. S4B). Collectively, these results demonstrate respectively; P < 0.05) compared with vehicle control trea- that 6-GIN and 6-PAR have the ability to block growth and ted mice at the end of the study. In addition, tumor weights reduce survival of both human and mouse prostate cancer were also reduced at both doses of 6-SHO (48% and 65% cells but that they are both less potent than 6-SHO. reduction, respectively; Fig. 6B). The reduction in tumor

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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/L 6-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 3 hours and the level of cyclin D1, survivin, and cMyc was measured by Western blot analysis. C, LNCaP and DU145 cells were treated with vehicle or 40 mmol/L 6-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, and PC-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 to b-actin) for Western blot data are given at the top of each column. The results are signiﬁcant (P < 0.05) where a decrease in protein level is 40%. E, LNCaP and DU145 cells were treated with vehicle, 20 or 40 mmol/L 6-SHO for 24 hours. Gene expression for CCL5, 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.

weight at the 100 mg/kg dose of 6-SHO was statistically Discussion signiﬁcant (P < 0.05) compared with the vehicle-treated In the work presented here, we report for the ﬁrst time that control group. There were neither appreciable changes in 6-SHO inhibits the survival of both human and mouse body weight or the daily food consumption among the 6- prostate cancer cells in culture, a reduction that is accom- SHO-treated and control mice (Fig. 6C and D) nor did gross panied by the induction of apoptosis. Previously published observations at necropsy reveal tissue abnormalities. As studies have shown the potential antiproliferative and apo- shown in Fig. 6, Western blot analyses of protein lysates ptosis inducing activity of 6-SHO in lung, colorectal, liver from tumor tissue showed a decrease in pSTAT3Y705 (Fig. and, hematologic cancer cells (15, 30–32). In the present 6E) and both cyclin D1 and survivin levels (Fig. 6F) in 6- study, 6-SHO was also shown to be effective at inhibiting SHO–treated groups compared with the vehicle-treated the growth of HMVP2 cells in vivo. The effect of 6-SHO on control group. Taken together, these data demonstrate that survival of both human and mouse prostate cancer cells in 6-SHO has potent in vivo antitumor activity at the doses vitro was compared with two additional ginger constituents, tested and is free of apparent adverse effects. 6-GIN and 6-PAR. The results support the conclusion that

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Figure 5. 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, and 72 hours and cell survival was measured by MTT assay. The data are presented as meanSEM. 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 meanSEM. C, HMVP2 cells were treated with 40 mmol/L 6-SHO for 30 and 60 minutes and Western blot analysis was performed for pSTAT3Tyr705,STAT3,andb-actin (top two); HMVP2 cells were treated 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,and b-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 Western blot 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 followed by TNF-a for 30 minutes and Western blot analysis was performed for pNF-kBp65, NF-kBp65, pIkBa,andb-actin (bottom). E, HMVP2 cells were treated 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, HMVP2 cells 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 are signiﬁcant (P < 0.05) where a decrease in phosphorylation or protein level is 40%. G, LNCaP, DU145, PC-3, and HMVP2 cells were treated with the indicated 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 meanSEM. 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 the experiment. 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 meanSEM; n ¼ 5 mice per group; , P < 0.05 compared with the control 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 are given at the top of each column. The results are signiﬁcant (P < 0.05) where a decrease in phosphorylation or protein level is 40%.

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among the three compounds tested, 6-SHO was the most involved in proliferation, survival, and apoptosis, such as potent for inhibiting growth of the prostate cancer cells in cyclin D1, survivin, cMyc, and Bcl2 (22, 24, 40). Treatment vitro. More broadly, 6-SHO seems to possess characteristics of cultured human and mouse prostate cancer cells with 6- that make it worthy of further study as an agent for the SHO reduced the levels of cyclin D1, survivin, and cMyc prevention and/or treatment of prostate cancer. protein and altered mRNA expression of selected chemo- Accumulating evidence shows that STAT3 is constitu- kine, cytokine, cell cycle, and apoptosis regulatory genes tively activated in human prostate cancer and that STAT3 is (Fig. 4). Thus, inhibition of STAT3 and NF-kB activity by 6- involved in cell proliferation, metastasis, angiogenesis, SHO was associated with reduced expression of their target and resistance to apoptosis (33–35). Several studies have gene products. Given that 6-SHO effectively inhibited the also shown that inhibition of STAT3 activation results in growth of prostate cancer cells regardless of STAT3 expres- growth inhibition and induction of apoptosis in cultured sion or activity, multiple mechanisms are likely involved in prostate cancer cells (36, 37). Mechanistic studies per- its effects on prostate cancer cell survival, including effects formed as part of the current study revealed that 6-SHO on STAT3, NF-kB, and possibly other signaling pathways. In treatment of prostate cancer cells inhibits both constitutive this regard, it has been reported that 6-SHO induces apo- (DU145 and HMVP2) and IL-6–induced (LNCaP) phos- ptosis by generation of reactive oxygen species in human phorylation of STAT3Tyr705. 6-SHO treatment also colorectal cancer cells, by directly regulating Akt1/2 in non– decreases phosphorylation of STAT3Ser727 as assessed in small cell lung carcinoma cells and by modulation of LNCaP and DU145 cells. The activation of STAT3 is estrogen receptor stress in hepatocellular carcinoma cells regulated by upstream kinases, including receptor tyrosine (23, 32, 43). In addition, as shown in Fig. 2G, Src activa- kinases (e.g., EGF receptor) and non-receptor tyrosine tion was inhibited following treatment with 6-SHO. Thus, kinases such as Jak2 and Src (25). As shown in Fig. 2G, 6-SHO affected multiple signaling pathways that likely 6-SHO inhibited activation of both JAK2 and Src, suggest- contributed to its ability to inhibit cell growth and induce ing that inhibition of both JAK2 and Src kinases likely apoptosis in all of the prostate cancer cell lines examined plays a role in mediating the effects of 6-SHO on down- in the current study. stream signaling, including STAT3 activation. As part of the current study, the activity of two other Previous studies have shown that PC-3 cells do not compounds found in ginger (6-GIN and 6-PAR) were express significant amounts of STAT3 or pSTAT3 even after compared with 6-SHO. The potential chemopreventive prolonged exposure to IL-6 (22). This was also the case in effects of 6-GIN and 6-PAR have been previously reported. the current experiments where PC3 cells were devoid of For example, Jeong and colleagues reported that 6-GIN measurable STAT3 (Fig. 2A). However, because 6-SHO suppresses colon cancer cell growth by inhibiting leuko- produced significant growth inhibition and induced apo- triene A4 (44). 6-GIN was also found to inhibit COX-2 ptosis in PC-3 cells (Fig 1A and B), other mechanisms expression by blocking mitogen-activated protein kinase associated with 6-SHO action were investigated. In this and NF-kB in 12-O-tetradecanoylphorbol-13-acetate regard, 6-SHO has been shown to inhibit activation of (TPA)-induced mouse skin (45). In another report, both NF-kB in several cell types and in mouse skin in vivo after 6-GIN and 6-PAR were shown to induce apoptosis in HL-60 topical treatment (16, 38). NF-kB is constitutively active in leukemia cells (46). 6-PAR treatment also induced apopto- androgen-independent prostate cancer cells such as PC-3 sis and caspase-3 activation in an oral squamous carcinoma cells (39–41). As shown in Figs. 3 and 4, NF-kB was found to cell line and demonstrated chemopreventive and antioxi- be constitutively active in PC-3, DU145, and HMVP2 cells. dant activity in the 7,12-dimethylbenz(a)anthracene Treatment with 6-SHO inhibited both constitutive and (DMBA)-induced hamster buccal pouch carcinogenesis TNF-a–induced phosphorylation of NF-kBp65 in all three assay (47, 48). As shown in Fig. 5G and Supplementary human prostate cancer cells. Immunocytochemical data Fig. S3, both 6-GIN and 6-PAR inhibited survival of human also demonstrated that NF-kBp65 was located primarily in and mouse prostate cancer cells and reduced activation of the nucleus after treatment with TNF-a and that 6-SHO both STAT3 and NF-kB. However, higher concentrations blocked this nuclear localization (Fig. 3C). Inhibitors of IkB (2–3 times) were required to achieve effects comparable kinase (IKK) are the major upstream regulators of NF-kB to 6-SHO. These results are consistent with previously and are activated by a wide variety of stimuli including published reports that 6-SHO is more potent than 6-GIN proinflammatory cytokines (e.g., TNF-a and IL-1; ref. 40). (15, 16). Once IKKs are activated and phosphorylated, they in turn On the basis of this precedent and the cell culture data cause phosphorylation and proteasomal degradation of showing that 6-SHO was the most effective of the three IkBa, thus leaving the free active form of NF-kB for nuclear ginger-derived compounds that were considered in the localization (26, 39–42). As shown in Fig. 3B, 6-SHO present study, its effects on tumor growth in an in vivo treatment also decreased the TNF-a–induced phosphory- model system were evaluated (HMVP2 cells grown in syn- lation of IkBa, as would be expected for an inhibitory effect geneic FVB/N mice). As shown in Fig. 6, 6-SHO at two for NF-kB in prostate cancer cells that is mediated through different doses (50 and 100 mg/kg, i.p. every other day) the classical IKK/IkB complex (41). produced statistically significant reductions in the growth of The activation of both STAT3 and NF-kB signaling leads HMVP2 cells at both doses (tumor volume) and at the to alterations in the expression of multiple target genes higher dose (tumor weight) without any apparent toxic

636 Cancer Prev Res; 7(6) June 2014 Cancer Prevention Research

6-Shogaol Inhibits Prostate Cancer Cell Growth

effects. Analysis of protein lysates from tumor tissue con- Authors' Contributions ﬁrmed a reduction in STAT3 activation and altered expres- Conception and design: A. Saha, J.L. Sessler, J. DiGiovanni Development of methodology: A. Saha, J. DiGiovanni sion of several target proteins in the 6-SHO–treated groups. Acquisition of data (provided animals, acquired and managed patients, In conclusion, the results of the current study demon- provided facilities, etc.): A. Saha, J. Blando, E. Silver, J. DiGiovanni strate that treatment of androgen-dependent and -indepen- Analysis and interpretation of data (e.g., statistical analysis, biosta- tistics, computational analysis): A. Saha, J. Blando, J.L. Sessler, dent human prostate cancer cells in culture with 6-SHO J. DiGiovanni inhibits survival and induces apoptosis. 6-SHO also inhibits Writing, review, and/or revision of the manuscript: A. Saha, J. Blando, E. Silver, L. Beltran, J.L. Sessler, J. DiGiovanni survival and induces apoptosis in cultured mouse prostate Administrative, technical, or material support (i.e., reporting or orga- cancer cells derived from HiMyc mice. Importantly, 6-SHO nizing data, constructing databases): A. Saha, J.L. Sessler was highly effective at inhibiting the growth of HMVP2 cells Study supervision: J. DiGiovanni in an allograft tumor model. These effects of 6-SHO were associated with inhibition of both STAT3 and NF-kB signaling and possibly other signaling pathways (e.g., Src). On Grant Support the basis of our ﬁndings, we suggest that 6-SHO has a A. Saha and E. Silver were supported by Cancer Prevention Research Institute of Texas postdoctoral and predoctoral trainee awards under grant combination of activity, low toxicity, and biochemical RP101501 from the State of Texas. properties that makes it of potential utility as a naturally The costs of publication of this article were defrayed in part by the occurring chemopreventive and/or therapeutic agent in payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate prostate cancer. this fact.

Disclosure of Potential Conﬂicts of Interest Received December 5, 2013; revised February 24, 2014; accepted March No potential conﬂicts of interest were disclosed. 26, 2014; published OnlineFirst April 1, 2014.

References 1. American Cancer Society. Cancer facts & figures 2013. Atlanta: Amer- 15. Kim JS, Lee SI, Park HW, Yang JH, Shin TY, Kim YC, et al. Cytotoxic ican Cancer Society; 2013. components from the dried rhizomes of Zingiber officinale Roscoe. 2. Stangelberger A, Waldert M, Djavan B. Prostate cancer in elderly men. Arch Pharm Res 2008;31:415–8. Rev Urol 2008;10:111–9. 16. Wu H, Hsieh MC, Lo CY, Liu CB, Sang S, Ho CT, et al. 6-Shogaol is 3. Siegel R, Naishadham D, Jemal A. Cancer statistics, 2013. CA Cancer more effective than 6-gingerol and curcumin in inhibiting 12-O-tetra- J Clin 2013;63:11–30. decanoylphorbol 13-acetate-induced tumor promotion in mice. Mol 4. Grossmann ME, Huang H, Tindall DJ. Androgen receptor signaling in Nutr Food Res 2010;54:1296–306. androgen-refractory prostate cancer. J Natl Cancer Inst 2001;93: 17. Fleming SA, Dyer CW, Eggington J. A convenient one-step gingerol 1687–97. synthesis. Synth Commun 1999;29:1933–9. 5. Heinlein CA, Chang C. Androgen receptor in prostate cancer. Endocr 18. Kim DSHL, Kim JY. Side-chain length is important for shogaols in Rev 2004;25:276–308. protecting neuronal cells from beta-amyloid insult. Bioorg Med Chem 6. Sharifi N, Farrar WL. Androgen receptor as a therapeutic target Lett 2004;14:1287–9. for androgen independent prostate cancer. Am J Ther 2006;13: 19. Ellwood-Yen K, Graeber TG, Wongvipat J, Iruela-Arispe ML, Zhang 166–70. J, Matusik R, et al. Myc-driven murine prostate cancer shares 7. Shrotriya S, Gagan D, Ramasamy K, Raina K, Barbakadze V, Merlani molecular features with human prostate tumors. Cancer Cell 2003; M, et al. Poly[3-(3, 4-dihydroxyphenyl) glyceric acid] from Comfrey 4:223–38. exerts anti-cancer efficacy against human prostate cancer via target- 20. Saha A, Kuzuhara T, Echigo N, Suganuma M, Fujiki H. New role of ing androgen receptor, cell cycle arrest and apoptosis. Carcinogenesis (-)-epicatechin in enhancing the induction of growth inhibition and 2012;33:1572–80. apoptosis in human lung cancer cells by curcumin. Cancer Prev Res 8. Baliga MS, Haniadka R, Pereira MM, D'Souza JJ, Pallaty PL, Bhat 2010;3:953–62. HP, et al. Update on the chemopreventive effects of ginger and its 21. Blando J, Moore T, Hursting S, Jiang G, Saha A, Beltran L, et al. Dietary phytochemicals. Crit Rev Food Sci Nutr 2011;51:499–523. energy balance modulates prostate cancer progression in Hi-Myc 9. Shukla Y, Singh M. Cancer preventive properties of ginger: a brief mice. Cancer Prev Res 2011;4:2002–14. review. Food Chem Toxicol 2007;45:683–90. 22. Hahm ER, Singh SV. Sulforaphane inhibits constitutive and inter- 10. Karna P, Chagani S, Gundala SR, Rida PC, Asif G, Sharma V, et al. leukin-6-induced activation of signal transducer and activator of Benefits of whole ginger extract in prostate cancer. Br J Nutr 2012; transcription 3 in prostate cancer cells. Cancer Prev Res 2010;3: 107:473–84. 484–94. 11. Zick SM, Djuric Z, Ruffin MT, Litzinger AJ, Normolle DP, Alrawi S, et al. 23. Hu R, Zhou P, Peng YB, Xu X, Ma J, Liu Q, et al. 6-Shogaol induces Pharmacokinetics of 6-gingerol, 8-gingerol, 10-gingerol, and 6-sho- apoptosis in human hepatocellular carcinoma cells and exhibits anti- gaol and conjugate metabolites in healthy human subjects. Cancer tumor activity in vivo through endoplasmic reticulum stress. PLoS ONE Epidemiol Biomarkers Prev 2008;17:1930–6. 2012;7:e39664. 12. Kundu JK, Na HK, Surh YJ. Ginger-derived phenolic substances with 24. Sun M, Liu C, Nadiminty N, Lou W, Zhu Y, Yang J, et al. Inhibition of cancer preventive and therapeutic potential. Forum Nutr 2009;61: Stat3 activation by sanguinarine suppresses prostate cancer cell 182–92. growth and invasion. Prostate 2012;72:82–9. 13. Katiyar SK, Agarwal R, Mukhtar H. Inhibition of tumor promotion in 25. Johnston PA, Grandis JR. STAT3 signaling: anticancer strategies and SENCAR mouse skin by ethanol extract of Zingiber officinale rhizome. challenges. Mol Interv 2011;11:18–26. Cancer Res 1996;56:1023–30. 26. Ghosh S, Karin M. Missing pieces in the NF-kappaB puzzle. Cell 14. Lee SH, Cekanova M, Baek SJ. Multiple mechanisms are involved in 6- 2002;109 Suppl:S81–96. gingerol-induced cell growth arrest and apoptosis in human colorectal 27. Gilmore TD. Multiple mutations contribute to the oncogenicity of the cancer cells. Mol Carcinog 2008;47:197–208. retroviral oncoprotein v-Rel. Oncogene 1999;18:6925–37.

www.aacrjournals.org Cancer Prev Res; 7(6) June 2014 637

Saha et al.

28. Blando J, Saha A, Kigichi K, DiGiovanni J. Obesity, inflammation and matrix metalloproteinase-9 expression via blockade of nuclear factor- prostate cancer. In: Dannenberg AJ, Berger NA, editors. Obesity, inflam- kappaB activation. Br J Pharmacol 2010;161:1763–77. mation and cancer, energy balance and cancer. New York: Springer; 39. Gasparian AV, Yao YJ, Kowalczyk D, Lyakh LA, Karseladze A, Slaga 2013. TJ, et al. The role of IKK in constitutive activation of NF-kappaB 29. Clevers H. At the crossroads of inflammation and cancer. Cell transcription factor in prostate carcinoma cells. J Cell Sci 2002;115: 2004;118:671–4. 141–51. 30. ChenCY,LiuTZ,LiuYW,TsengWC,LiuRH,LuFJ,etal.6-shogaol 40. Suh J, Rabson AB. NF-kappaB activation in human prostate cancer: (alkanone from ginger) induces apoptotic cell death of human important mediator or epiphenomenon? J Cell Biochem 2004;91: hepatoma p53 mutant Mahlavu subline via an oxidative stress- 100–17. mediated caspase-dependent mechanism. J Agric Food Chem 41. Yemelyanov A, Gasparian A, Lindholm P, Dang L, Pierce JW, Kisseljov 2007;55:948–54. F, et al. Effects of IKK inhibitor PS1145 on NF-kappaB function, 31. Hung JY, Hsu YL, Li CT, Ko YC, Ni WC, Huang MS, et al. 6-Shogaol, an proliferation, apoptosis and invasion activity in prostate carcinoma active constituent of dietary ginger, induces autophagy by inhibiting cells. Oncogene 2006;25:387–98. the AKT/mTOR pathway in human non-small cell lung cancer A549 42. Palayoor ST, Youmell MY, Calderwood SK, Coleman CN, Price BD. cells. J Agric Food Chem 2009;57:9809–16. Constitutive activation of IkappaB kinase alpha and NF-kappaB in 32. Pan MH, Hsieh MC, Kuo JM, Lai CS, Wu H, Sang S, et al. 6-Shogaol prostate cancer cells is inhibited by ibuprofen. Oncogene 1999;18: induces apoptosis in human colorectal carcinoma cells via ROS 7389–94. production, caspase activation, and GADD 153 expression. Mol Nutr 43. Kim MO, Lee MH, Oi N, Kim SH, Bae KB, Huang Z, et al. [6]-Shogaol Food Res 2008;52:527–37. inhibits growth and induces apoptosis of non-small cell lung cancer 33. Barton BE, Karras JG, Murphy TF, Barton A, Huang HF. Signal cells by directly regulating Akt1/2. Carcinogenesis 2013;35:683–91. transducer and activator of transcription 3 (STAT3) activation in pros- 44. Jeong CH, Bode AM, Pugliese A, Cho YY, Kim HG, Shim JH, et al. [6]- tate cancer: Direct STAT3 inhibition induces apoptosis in prostate Gingerol suppresses colon cancer growth by targeting leukotriene A4 cancer lines. Mol Cancer Ther 2004;3:11–20. hydrolase. Cancer Res 2009;69:5584–91. 34. Dhir R, Ni Z, Lou W, DeMiguel F, Grandis JR, Gao AC. Stat3 activation 45. Kim SO, Kundu JK, Shin YK, Park JH, Cho MH, Kim TY, et al. [6]- in prostatic carcinomas. Prostate 2002;51:241–6. Gingerol inhibits COX-2 expression by blocking the activation of p38 35. Lee SO, Lou W, Hou M, de Miguel F, Gerber L, Gao AC. Interleukin-6 MAP kinase and NF-kappaB in phorbol ester-stimulated mouse skin. promotes androgen-independent growth in LNCaP human prostate Oncogene 2005;24:2558–67. cancer cells. Clin Cancer Res 2003;9:370–6. 46. Lee E, Surh YJ. Induction of apoptosis in HL-60 cells by pungent 36. Calvin DP, Nam S, Buettner R, Sekharam M, Torres-Roca J, Jove R. vanilloids, [6]-gingerol and [6]-paradol. Cancer Lett 1998;134:163–8. Inhibition of STAT3 activity with STAT3 antisense oligonucleotide 47. Keum YS, Kim J, Lee KH, Park KK, Surh YJ, Lee JM, et al. Induction of (STAT3-ASO) enhances radiation-induced apoptosis in DU145 pros- apoptosis and caspase-3 activation by chemopreventive [6]-paradol tate cancer cells. Int J Radiat Oncol Biol Phys; 2003. p. S297. and structurally related compounds in KB cells. Cancer Lett 2002; 37. Ni Z, Lou W, Leman ES, Gao AC. Inhibition of constitutively activated 177:41–7. Stat3 signaling pathway suppresses growth of prostate cancer cells. 48. Suresh K, Manoharan S, Vijayaanand MA, Sugunadevi G. Chemo- Cancer Res 2000;60:1225–8. preventive and antioxidant efficacy of (6)-paradol in 7,12-dimethylbenz 38. Ling H, Yang H, Tan SH, Chui WK, Chew EH. 6-Shogaol, an active (a)anthracene induced hamster buccal pouch carcinogenesis. Phar- constituent of ginger, inhibits breast cancer cell invasion by reducing macol Rep 2010;62:1178–85.

638 Cancer Prev Res; 7(6) June 2014 Cancer Prevention Research

6-Shogaol from Dried Ginger Inhibits Growth of Prostate Cancer Cells Both In Vitro and In Vivo through Inhibition of STAT3 and NF- κB Signaling

Achinto Saha, Jorge Blando, Eric Silver, et al.

Cancer Prev Res 2014;7:627-638. Published OnlineFirst April 1, 2014.

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