RESEARCH ARTICLE Open Access The effects of 1,4-dimethylpyridine in metastatic prostate cancer in mice Agnieszka Denslow1, Marta Switalska1, Marcin Nowak2, Magdalena Maciejewska1, Stefan Chlopicki3,4, Andrzej Marcinek5, Jerzy Gebicki5 and Joanna Wietrzyk1* Abstract Background: We previouslyshowedthat 1-methylnicotinamide(1-MNA) anditsanalog1,4-dimethylpyridine(1,4-DMP) couldinhibittheformationoflung metastasesandenhancetheefficacyofcyclophosphamide-based chemotherapyin themodelofspontaneouslymetastasizing4T1mousemammaryglandtumors.Inthepresentstudy,we aimedto investigatewhetherthepreviouslyobservedactivity ofpyridinecompounds pertains alsotothepreventionandthe treatmentof metastaticprostatetumors, inacombinedchemotherapy withdocetaxel. Methods: Cancer-preventingactivityof1,4-DMP wasstudiedinthemodelof prostate tumors spontaneouslyarisingin C57BL/6-Tg (TRAMP)8247Ng/J(TRAMP) mice.Theefficacyofthecombinedchemotherapy, comprising simultaneous use of1,4-DMP anddocetaxel,wasevaluatedinthe orthotopic mousemodelofhumanPC-3M-luc2prostatecancer.The toxicity of the applied treatment was also determined. Results: Thedevelopmentofprostate tumorsin TRAMPmiceremainedunaffected after administrationof1,4-DMP. Similarly,no effectof1,4-DMPwasfound onthegrowthof orthotopicallytransplantedPC-3M-luc2 tumors.However, when1,4-DMPwasadministeredalongwithdocetaxel,it enhancedthe anticanceractivityofthechemotherapy.Asa result,inPC-3M-luc2-bearing micestatisticallysignificantinhibitionofthe tumorgrowth andlowermetastasesincidence wereobserved.Thedecreasedmetastaticyieldisprobably relatedtothe diminishedplateletactivity observedinmice treatedwith combinedtherapeuticregimen. Finally,thecombinedtreatmentexhibitedloweredside effects accompanying docetaxel administration. Conclusions: Resultspresented herein confirmpreviouslypublished dataontheanticanceractivityofpyridine compoundsanddemonstratethat1,4-DMPmaybebeneficiallyimplementedinto chemotherapyutilizing various cytotoxic agents, directed against multiple metastatic tumor types. Keywords: Prostate cancer,Metastasis, Prevention,Combinedtherapy,1-methylnicotinamide, 1,4-dimethylpyridine, Docetaxel Background Prostate cancer is the second most common cancer of men, affecting approximately14% of patients [1]. While the riskofdevelopingprostate cancer mightbebeneficiallyin­fluencedby proper diet andphysical activity[2],thereare no confirmedpharmacological meansfor thepreventionof thesetypesof tumors.Themajorityof prostatecancercases arediagnosed at thelocalized stageenablingeffective treat­ment; however, a significant fraction of patients develops * Correspondence: wietrzyk@iitd.pan.wroc.pl 1Hirszfeld Institute of Immunology and Experimental Therapy, Polish Academy of Sciences, Weigla 12, Wroclaw 53-114, Poland Full list of author information is available at the end of the article metastatic disease that often progresses into treatment-irresponsive, ultimately resulting in patient’sdeath[3]. Initial treatment of prostate cancer usually comprises hormonetherapy;however,whentumors are irrespon­sive to hormonal treatment (i.e., in case of castrate­resistant prostate cancer), the most common first-line treatment includes the simultaneous use of docetaxel and prednisone. Docetaxel is a semi-synthetic taxane that inhibits microtubular depolymerization and block bcl-2 and bcl-xl gene expression [4, 5]. Prednisone, in turn, is a glucocorticoid that is used to improve symp­toms such as pain [6]. It was also shown to inhibit cell proliferation and induce apoptosis in prostate cancer ©The Author(s). 2017Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons. org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. cells [7, 8], and thus decrease the level of prostate­specific antigen [9]. Accordingly, in multiple studies, prednisone was shown to promote anticancer activity of docetaxel [10–14].However,the useofglucocorti­costeroids in patients with prostate cancer is associated with theriskof adverse sideeffects(as reviewed,for ex­ample, by Dorff and Crowford [15]), and it eventually leads to the development of resistance to chemotherapy [16]. Therefore, there is still an urgent need for new treatment regimens that would enable efficient yet safe means for the therapy of patients suffering from pros-tate cancer. 1-methylnicotinamide (1-MNA) is an endogenous me-tabolite of nicotinamide (NA) that has recently gained attention due to its anti-inflammatory [17] and anti­thrombotic [18] activity drivenbymechanisms dependent on prostacyclin (PGI2) release [18, 19]. Another compound that has been shown to modulate thrombus formation based on the PGI2-related mechanisms is 1,4­dimethylpyridine (1,4-DMP) – a structural analog of 1­MNA that arises naturally in roasted coffee seeds[20]. In addition, it has been recently shown that both 1-MNA and 1,4-DMP could inhibit metastases formation in the modelof experimental andspontaneous metastasis of4T1 murine mammarygland cancer [21]. The present work is aimed to establish whether 1,4­DMP may have an anti-oncogenic effect in the prophy­laxis and the treatment of prostate tumors. Methods Drugs 1,4-DMP and 1-MNA were usedin the form of chlorides providedbytheInstituteofApplied RadiationChemistry, Technical University of Lodz, Poland. Prior to use, both salts were diluted in drinking water such that mice re-ceived the predetermined dose of the drugs. Docetaxel (DTX) was purchased at Ak Scientific (USA). All drugs were administrated at the doses and according to the schedulespresentedinTable 1. Mice Eight-to twelve-weeks-old male C57BL/6-Tg(TRAMP) 8247 Ng/J (TRAMP) mice were purchased from the Jackson Laboratory (USA). Seven-to eight-weeks-old BALB/c Nude male mice were provided by Charles Riv­ers Laboratories (Germany) (Table 2). All experiments were performed according to the Interdisciplinary Prin­ciples and Guidelines for the Use of Animals in Research, Marketing and Education issued by the New York Acad­emy of Sciences’ Ad Hoc Committee on Animal Re-search and were approved by the 1st Local Committee for Experiments with the Use of Laboratory Animals, Wroclaw, Poland. Cell culture and transplantation Human prostate cancer PC-3M-luc2 cell line stably ex-pressing the firefly luciferase gene (luc) was obtained from Caliper Life Sciences Inc. (USA). Cells were cul­tured in RPMI 1640+Gluta-MAX™ medium (Life Tech-nologies, USA) supplemented with 10% fetal bovine serum (Sigma-Aldrich, Germany) and antibiotics (peni­cillin and streptomycin—Polfa Tarchomin, Poland). Cell line cultures were maintained at 37 °C in a humidified atmosphere with 5% CO2. Prior to the transplantation, cells were trypsinized (IIET, Poland), centrifuged (200×g, 4 °C, 5 min) and counted. Then, cells were resuspended in Hank’s Bal-anced Salt Solution (HBSS; IIET, Poland). Male BALB/c Nude mice wereintraperitoneallyinjected with ketamine at a dose of 50 mg/kg(VET-AGRO Sp. z o.o., Poland) and anesthetized with the mixture of air and isoflurane (3% v/v). 1.0 cm wide abdominal wall incision wasmadejust above the bladder,in thelower part of ab­domen, andtheprostategland was exposedfortheinjec­tion.Then,5×106 PC-3M-luc2 cells in 0.05 mlof HBSS wereinoculatedinto the dorsalprostate lobes ofmice.Im-mediatelyafter the transplantation,incised abdominal wall andskin were sewedwith soluble surgical suture. Estimation of the antitumor activity The development of prostate tumors in TRAMP mice was monitored weekly by physical examination. Adeno-carcinoma formation was confirmed by histological examination of the tumors isolated from mice during necropsy carried out in animals with clear physiological (e.g., body weight or body temperature decrease, body posture, ruffled fur) and behavioral symptoms (e.g., de-creased movement) of an advanced disease. Briefly, Table1 Drugs, doses and therapeutic regimens applied in the presented studies Experimental model Drug Route of administration Dose Treatment regimen Spontaneous tumor 1-MNA per os in drinking water 100 mg/kg/day continuously from the age of 8 to 12 weeks formation (Fig. 1) to the day of the necropsy 1,4-DMP per os in drinking water 100 mg/kg/day continuously from the age of 8 to 12 weeks to the day of the necropsy PC-3 M-luc2 tumors 1,4-DMP per os in drinking water 100 mg/kg/day continuously from the day 1 to the end (Fig. 2) of the experiment DTX intraperitoneally 10 mg/kg 2doses at days 15 and 22 Table2 Strains and number of mice used in the experiments Experimental model Mouse No of mice/ Total No of strain group used mice Spontaneous tumor formation (Fig. 1) TRAMP 15 45 PC-3M-luc2 tumors (Figs. 2, 3and 4) C57BL/6 9 36 prostate tumors were isolated and fixed in buffered for­malin, and then cut into 4-µm-thick sections that were subsequently dewaxed with xylene. Following rehydra­tion in a gradient of ethanol, the sections were washed in distilled water, cytoplasm was stained with eosin while nuclei were counterstained in hematoxylin. Finally, the preparations were dehydrated in an alcohol gradient and coverslip mounted. The histological appearance of the tissue was examined at 50× or 100× magnitude. Using an In vivo MS FX PRO system (Carestream Health INC., USA), in vivo visualizations of PC-3 M­luc2 tumors growing in prostate gland of BALB/c Nude mice were performed no more often than every 4 days starting from the 15th day of the experiment. In brief, about 10 min before imaging, D-luciferin potassium salt (Synchem INC., Germany) was administered to each mouse intraperitoneally at a dose of 150 mg/kg. Then, animals were anesthetized with a 3–5% (v/v) mixture of isoflurane (Forane, Abbott Laboratories, USA) in syn-thetic air (200 ml/min). Anesthesia was maintained with 1.5–2% (v/v) mixture of isoflurane and synthetic air de-livered via individual masks. Visualization was carried out using the following settings: for X-ray — t=2 min, f-stop = 5.57, FOV = 198.6; for luminescence capture — t =3 min, binning 2×2, f-stop= 5.57, FOV = 198.6. Im-ages were analyzed with Carestream MI SE software (Carestream Health INC., USA). The intensity of the lu-minescent signal is presented as the sum intensity of the region of interest and expressed in arbitrary units (a.u.). Tumor tissue was also excised and weighted on the last day of the experiment (day 46). Evaluation of the antimetastatic effect Livers, lungs, kidneys, bones and axillary as well as in-guinal lymph nodes were isolated and fixed in buffered formalin on the day of the necropsy, in order to detect metastases in the mice bearing prostate tumors. Then, tissue samples were cut into 4-µm-thick sections and stained as described hereinabove. The number of metas­tases in isolated tissues was counted at 50× or 400× magnitude. Platelet activation status Blood samples were collected on days 87, 122, 213 and during animal’s necropsy in the model of the spontan-eously formed prostate tumors or on the last day of the experiment (day 46) in case of mice bearingPC-3M-luc2 tumors. Samples were collected in tubes containing 0.05 ml of 5% ethylenediaminetetraacetic acid (EDTA) solution (Sigma-Aldrich, Germany). Platelet-related morphology analyzes were performed using Mythic 18 analyzer (C2 Diagnostics, France). Then, blood plasma was obtainedby centrifugation (2000×g,15 min,4°C) and stored at -80 °C until further analyzes. Prostacyclin generation in the treated mice was determined by the quantification of plasma 6-keto-prostaglandin F1. (6­keto-PGF1.) levels. Based on thromboxane B2 (TXB2), von Willebrand factor (vWF) and soluble P-selectin plasma concentrations, platelet activation status was es-timated. Using commercial kits available from Cusabio Biotech Co. Ltd. (Wuhan, China), all analyzes were con-ducted via the ELISA technique. In addition, plasma concentration of transforming growth factor ß1 (TGF­ß1) was determined with ELISA kit from Boster Bio­logical Technology (USA). All ELISA-based analyzes were conducted according to the manufacturer’s instructions. Protein expression in tumor tissue Protein expression in prostate tumor tissue was analyzed according to the standard Western blot procedure [22]. In brief, using a FastPrep®-24 MP Bio device (Mp Bio­medicals LLC., USA), samples of tumor tissue that were collected and immediately frozen on the last day of the experiments were homogenized in RIPA Buffer (Sigma-Aldrich, Germany) with the following settings: CP 24× 2,6 m/s, 40 s. According to the manufacturer’s protocol, protein content in all samples was analyzed using a Bio-Rad Protein Assay (Bio-Rad Laboratories Inc., USA). Samples containing 100 µg of protein were separated on the pre-cast 4–20% gradient gels (Bio-Rad Laboratories, Inc., USA) and transferred onto 0.45 µm polyvinylidene fluoride (PVDF) membranes (Merck Millipore, USA). Next, the membranes were probed with primary rabbit polyclonal anti-E-cadherin (1:1000), anti-N-cadherin (1:1000), anti-VEGFR-1 (1:200) antibodies (all from Pro-teintech Group, USA) or mouse anti-ß-actin (1:1000, Sigma-Aldrich, Germany) antibody. Finally, according to the manufacturer’s instruction, the analyzed proteins were detected with IRDye® 800CW Goat anti-Rabbit IgG or IRDye® 680RD Donkey anti-Mouse IgG (both from LI-COR, USA). Blots were visualized in ODDYSEY® CLx Imager (LI-COR, USA) and analyzed with ImageJ Soft­ware as follows. The total E-cadherin cellular content comprising truncated and unprocessed E-cadherin (with a molecular weight of approximately 100 and 130 kDa, respectively) was calculated. Similarly, total N-cadherin cellular content comprising mature and unprocessed N-cadherin (with a molecular weight of approximately 70 and 100 kDa, respectively) was determined. Then, E­cadherin and N-cadherin contents were normalized to ß-actin. Finally, E-cadherin to N-cadherin ratios in indi­vidual samples were calculated and presentedas mean± SD values. Toxicity of the anticancer treatment The toxicity of the proposed anticancer treatment strat-egy and its influence on the overall health condition were estimated based on body weight changes as well as morphological and biochemical blood analyzes. The body weight of experimental animals was measured thrice each week throughout the course of all studies. Blood morphology was performed with Mythic 18 analyzer (C2 Diagnostics, France). Using reagents and procedures provided by the manufacturer, biochemical analyzes were performed in Cobas C 111 analyzer (Roche Diagnostics, Switzerland). Statisticalanalysis Data normality was estimated using the Shapiro-Wilk test with a predetermined value of p<0.05. The Tukey-Kramer multiple comparison test for parametric data or the Kruskal–Wallis Test for non-parametric data was applied; p values lower than 0.05 were considered sig­nificant. All calculations were performed using Graph-Pad Prism7(GraphPad Software, Inc., USA) software. Unless stated otherwise, all data presented on graphs correspond to mean±SD values. Results The influence of 1,4-DMP on the onset and metastasis of spontaneously formed prostate tumors To establish whether 1,4-DMPmightpreventthedevel­opment of prostate tumors, the compound was con-tinuously given to male TRAMP mice that during their life span spontaneously develop mild intraepithelial hyperplasia to malignant neoplasia within prostate gland. For comparative purposes, another group of the animals was treated with 1-MNA, a primary analog of 1,4-DMP that was proven to possess significant anti­thrombotic and anti-inflammatory activity. 1-MNA, and to a lesser extent also 1,4-DMP, delayed the onset ofprostate lesionsinTRAMP mice(Fig.1a).However, none of the given compounds prolonged the life span of treated animals (Fig. 1b). Histopathological analysis of the tumor tissues excised during the necropsy con-firmed the development of malignant adenocarcinomas in approximately 80% of the mice in all experimental groups (Fig.1c andd). Histopathological analysis demonstrated that metas­tases in TRAMP mice prostate adenocarcinomas were developed in lymph nodes, lungs, liver and kidneys (Fig.1d).Metastatic lesions were diagnosed in around 30% of untreated animals. Similarly, metastases were found in 30% of animals treated with 1,4-DMP. In contrast, when treated with 1-MNA, 50% of mice de-veloped metastases (Fig. 1c, not statistically significant difference). The analysis of morphological features of blood platelets did not reveal changes in platelet mean vol-ume and platelet distribution width(PDW) occurring during the study; however, we noted an increase in platelet count anddecreasedlevelof PDWinanimals during thenecropsy(AdditionalFile:FigureS1c).The treatment either with 1-MNA or 1,4-DMP did not affect theplateletmorphological parameters(Fig.1e– g, Additional File 1: Figure S1a-c). Similarly to plate­lets, red blood cell parameters also remained un­affectedby the tumorprogression,with anexception of the time preceding necropsy where a significant drop in red blood count was observed in all experi­mental groups. However, none of the studied com-pounds influenced the red blood cell parameters (AdditionalFile1:FigureS1d). While there was no obvious change in the white blood cellcountinTRAMP micedevelopingprostate tumors that wasobservedinthe course of thestudy, we observed that 1,4-DMP and to a lesser extent 1­MNAtendedtodecreasethe numberofall oflympho­cytes, monocytes and granulocytes in the treated animals, when compared to the control group (Fig. 1h–j, Additional File 1: Figure S1e–g). This effect mightbeattributed to prostacyclin-dependentsplenic dilation in thetreated mice that leadstowhite blood cells poolinginthe spleen,andin consequence, asys­temic decrease in the white blood cell count [23]. The analysis of thebiochemical parameters of platelet ac-tivity revealed no effect of the studied compounds on platelet activity in TRAMP mice (Fig. 1l–o). Finally, dur-ing the tumor development process, we observed that in TRAMP mice, the plasma level of TGF-ß1 was not af­fectedeitherby1,4-DMPorby 1-MNA(AdditionalFile 1: FigureS1h). Anticancer activity of the combined treatment of prostate cancer comprising simultaneous application of 1,4-DMP anddocetaxel Thegrowthofprimary tumorslocalizedinprostateglands of BALB/c Nude mice was monitored throughout the ex-periment by in vivo imaging ofthe luminescence generated by PC-3M-luc2 cells. The analysis of the luminescence in-tensity indicated that 1,4-DMP when administered alone did not inhibitthegrowthofPC-3M-luc2prostate tumors. On thecontrary, marked tumorgrowthinhibition was ob-served when mice were treated with docetaxel alone or ad-ministered with 1,4-DMP (Fig. 2a and b). These observations wereconfirmedby theanalysis of the tumor mass isolated from the mice on day 46 of the experiment. Fig.1 The influence of 1,4-DMP and 1-MNA on the development and the progression of prostate cancer. a The onset of the prostate gland le-sions; bsurvival of the TRAMP mice continuously treated with 1-MNA and 1,4-DMP. c Summarized results of the frequency of the neoplasia, adenocarcinomas and metastases in TRAMP mice determined by the means of histopathological analysis of the tissues isolated during the nec-ropsy. dImages of primary tumors (neoplasia and adenocarcinomas) identifiedin non-treated and drug-receiving animals and of the metastases localized in lungs of the control animals, kidney of the animal treated with 1-MNA, or liver of the mouse receiving 1,4-DMP. Results of the mor-phological analysis carried out on blood samples collected during the necropsy of the animals: e platelet count; fmean platelet volume (MPV); g platelet distribution width (PDW); hlymphocyte count; imonocyte count; and jgranulocyte count. Plasma concentrations of kTXB2, l6-keto­PGF1., m vWF, n soluble P-selectin determinedby ELISA. All data are presented as mean±SD Docetaxel, when administered alone, inhibited the growth of PC-3M-luc2 tumors in around 50% when compared to thecontrolgroupof animals (0.40g vs.0.81g,respectively). Antitumor activity of docetaxel was additionally enhanced when the drug was administrated with 1,4-DMP and reached approx.80% tumor growthinhibition (0.17g vs. 0.81g, p<0.05) (Fig.2c). Histopathological analysis of tissues collected from mice led to the identification of PC-3M-luc2 metastatic lesions in such tissues as lymph nodes, liver and lungs (Fig. 2d). Weobserved that thefrequencyof metastases formationin mice treated with 1,4-DMP decreased by almost 50%. Similarly, the number of metastases-bearing animals de-creased in case of single-drug treatment with docetaxel. Most interestingly, none of the mice treated with 1,4-DMP and docetaxel developed PC-3M-luc2 metastases during the study(seeTable3). We also decided to investigate the influence of ap-plied treatment on the metastatic potential of tumor-forming cancer cells. To this end, we evaluated the expression of E-cadherin, N-cadherin and vascular endothelial growth factor receptor 1 (VEGFR1) in tumor tissue. The results of the Western blot analysis show that 1,4-DMP had no significant effect on E­ Fig.2 Anticancer activity of the combined treatment comprisingthe use of docetaxel (DTX) and 1,4-DMP in the model of human prostate cancer PC-3M-luc2 xenografted into prostate glands of BALB/c Nude mice. a Results of in vivo imaging of PC-3M-luc2 tumors performed on day 41 of the experiment. bKinetics of the PC-3M-luc2 tumor growth in mice treated with docetaxel (DTX) and 1,4-DMP given either alone and in a comparison to the control group of animals. Days of drug administration are indicated with gray arrows for docetaxel (DTX) and dotted arrow for 1,4-DMP. c PC-3M-luc2 tumor weight measured on the last day of the experiment (day 46) (*p<0.05 vs. control and 1,4-DMP). dImages of metas­tases localized in liver of the control animal, lymph node of docetaxel(DTX)-treated mouse and lungs of the 1,4-DMP-treated mouse (from left to right). e Images of bands obtained during Western blot analysis of protein expression in tumor tissue of (I) control animals and animals treated with (II) docetaxel (DTX), (III) 1,4-DMP and (IV) docetaxel (DTX) with 1,4-DMP. fE-cadherin : N-cadherin expression ratios in the samples of tumor tissue collected on the last day of the experiment. The total cellular content of E-cadherin (comprising protein characterizedby the molecular weight of 130 and 100 kDa) and N-cadherin (comprising protein characterized by the molecular weight of 100 and 70 kDa) was first normalized to the content of ß-actin and then used to determine E-cadherin to N-cadherin expression ratios. gThe level of low molecular weight fragment of E-cadherin in PC-3M-luc2 tumors normalized to the content of ß-actin. hThe expression of VEGFR-1 in PC-3M-luc2 tumors normalized to the content of ß-actin. iPlasma concentration of TGF-ß1in mice bearing PC-3M-luc2 tumors. All data are presented as mean±SD values Table3 Frequency of metastases formation and localization of new lesions in mice bearing PC-3M-luc2 tumors Treatment Animals with metastases/all Metastases location animals tested Control 7/9 Lungs, liver, lymph nodes 1,4-DMP 4/9 Lungs, liver DTX 1/6 Lymph nodes 1,4-DMP + 0/8 — DTX cadherin to N-cadherin expression ratio. In contrast, when mice were treated with docetaxel given alone, over twofold increase in E-cadherin to N-cadherin ex-pression ratio was observed (not statistically signifi-cant). Such a phenomenon was additionally enhanced by simultaneous application of 1,4-DMP that allowed to reach over threefold enhancement of E-cadherin to N-cadherinexpression ratio(Fig. 2f). In addition, in PC-3M-luc2 tumor-bearing mice, we have observed the appearance of low molecular weight fragments of E-cadherin(40 kDa).The concentrationofthese pro­tein fragments was lowered in both groups treated with docetaxel, given either alone or with 1,4-DMP, while it was unaltered in mice receiving 1,4-DMP alone (Fig. 2g). The expression of VEGFR-1 was decreased in about 30% in mice treated with com-bined therapy but was not affected either by docetaxel orby 1,4-DMPgiven alone(Fig. 2h). Increased E-cadherin to N-cadherin expression ratio was accompanied by the decreased plasma concentra­tion of TGFß-1 in mice receiving the combined treat­ment consisting of docetaxel and 1,4-DMP (20.5± 5.1ng/ml vs. 28.13±3.7 ng/mlinthe controlgroupof mice, Fig. 2i). The analysis of the morphological parameters of blood platelets revealed that while docetaxel given aloneslightlylowered theplatelet count,1,4-DMP did not influence the platelet number when given alone but restored the number in docetaxel-treated animals (Fig. 3a and b). In addition, we observed that in ani-mals receiving 1,4-DMP together withdocetaxel,the mean platelet volume (MPV) and PDW were lowered when compared to the untreated animals (6.02±0.4 and 36.6±4.8 vs. 6.42±0.4 and 40.19±5.2 fL in the controlgroup of animals, Fig. 3c andd). In addition, when analyzing biochemical parameters of platelet activity, we observed that both docetaxel given alone as well as administered with 1,4-DMP significantly reduced plasma concentrations of TXB2 (49.18± 24.0pg/ml,36.55±19.6pg/ml,respectively, vs. 147.4 ±43.1 pg/ml in the control, p<0.05), soluble P-selectin (158.3±46.3 ng/ml, 157.6±36.7 ng/ml, respectively, vs. 256±53.9 ng/ml in the control, p< 0.05),and vWF(2479±764 ng/ml, 2785±432ng/ml, respectively, vs. 4134±753 ng/ml in the control) (Fig. 3f–h). However, we also observed a significant drop in 6-keto-PGF1. plasma concentration in mice treated with both docetaxel and 1,4-DMP (70.36 vs. 130.7pg/ml, p<0.05)(Fig.3e). Toxicity of the combined treatment of prostate cancer comprising simultaneous application of 1,4-DMP and docetaxel In the control and 1,4-DMP-treated groups of animals, there were no cases of deaths recorded. On the contrary, administration of docetaxel resulted in 3 incidences of treatment-related deaths (effect was not statistically Fig.3 Theinfluenceofthe combinedtreatment comprisingtheuseofdocetaxel(DTX) and1,4-DMP onplatelet morphology and activityinBALB/c Nude mice bearingPC-3M-luc2tumors. a Platelet count; bplateletcrit (PCT); c meanplatelet volume (MPV); dplatelet distribution width(PDW)deter­mined onthelastdayofthe experiment(day46).Plasma concentrationof: e 6-keto-prostaglandin F1.(6-keto-PGF1.)(*p<0.05 vs. control and 1,4­DMP); fthromboxaneB2 (TXB2)(*p<0.05vs. control and1,4-DMP); gsolubleP-selectin (*p<0.05 vs. control and 1,4-DMP); andhvon Willebrand Factor (vWF)(*p<0.05 vs. controland1,4-DMP),determined onthelastdayofthe experiment(day46).Alldataarepresentedasmean±SD values significant). Surprisingly, docetaxel-induced toxicity was lowered when cytotoxic drug was given simultaneously with 1,4-DMP. In case of animals treated with the com-bined regimen, only 1 incidence of death was recorded (Fig. 4a). Regardless of the treatment applied, the body weight of all tumor-bearing BALB/c Nude mice was de-creasing throughout the study with the most prominent body loss observed among control and 1,4-DMP-treated animals (approx. 10–12% body weight loss). Among the mice treated with docetaxel, a reduced bodyloss was ob-served (approx. 6% body weight loss), which was nearly abolished among animals treated with the combined treatment (approx. 3% bodyweight loss) (Fig. 4b). The analysis of blood morphology of the treated ani-mals revealed that similar to the platelet count described above, red blood cell count was lower in mice receiving docetaxel alone (8.30±0.8×106 vs. 9.1±0.2×106). However, in contrast to platelet count, red blood cell count was not restored when docetaxel was adminis­tered simultaneously with 1,4-DMP (8.4±0.6×106)and corresponded to non-significant change in hemoglobin content that was observed in mice given docetaxel either alone or in combination with 1,4-DMP (14.17±1.3 g/dl and 14.34±0.9 g/dl vs. 14.81±2.2 g/dl in the control). Blood biochemistry analysis revealed that in groups re-ceiving docetaxel, levels of plasma concentrations for creatinine (5.7±2.2 vs. 7.59±2.3 µmol/l in the control) and urea (6.98±1.3 vs. 7.33±1.1 mmol/l in the control) were not significantly changed. However, when docetaxel was concurrently administered with 1,4-DMP, it resulted ina significantly lowered creatinine (4.99±1.5 µmol/l, p <0.05 vs. control and 1,4-DMP-treated group) and urea (5.67±0.9 mmol/l, p<0.05 vs. control and 1,4-DMP­treated group) concentrations (Fig. 4e–f). Fig.4 Toxicity of the combined treatment of prostate cancer comprising simultaneous application of 1,4-DMP and docetaxel (DTX). a Survival of BALB/c Nude mice bearing PC-3 M-luc2 tumors, treated with docetaxel (DTX) and 1,4-DMP either alone or in combination. bBody weight of BALB/c Nude mice bearing PC-3M-luc2 tumors, treated with docetaxel (DTX) and 1,4-DMP either alone or in combination. In graphs a and b, days of drug administration are indicated with gray arrows for docetaxel (DTX) and dotted arrow for 1,4-DMP. c Red blood cell (RBC) count (*p<0.05 vs. 14-DMP; **p<0.05 vs. control and 1,4-DMP); and dhemoglobin concentration in blood samples taken from BALB/c Nude mice bearing PC­3M-luc2 tumors on the last day of the experiment (day 46). Plasma concentrations of: e creatinine (*p<0.05 vs. control and 1,4-DMP) and furea (*p<0.05 vs. control and 1,4-DMP) determinedfor plasma samples obtained from BALB/c Nude mice bearing PC-3M-luc2 tumors on the last day of the experiment (day 46). The activity of: glactate dehydrogenase (LDH) (*p<0.05 vs. control, DTX and 1,4-DMP); haspartate aminotransferase (AST) (*p<0.05 vs. control, DTX and 1,4-DMP); and ialanine aminotransferase (ALT) (*p<0.05 vs. control and 1,4-DMP) determined in blood plasma samples obtained from BALB/c Nude mice bearing PC-3 M-luc2 tumors on the last day of the experiment (day 46). All data are presented as mean±SD values On the contrary, as it was determined for the blood plasma samples taken from the treated mice bearing PC-3M-luc2tumors, administrationof docetaxel hadnoinflu­ence onthe activity oflactatedehydrogenase(LDH)(887.1 ±251.5 vs.929.6±202.5U/linthecontrol,Fig.4g)while resulted in a slightly increased activity of aspartate amino­transferase(AST)(209.1±62.4 vs. 178.3±51.7U/lin the control) and alanine aminotransferase (ALT)(82.72±31.8 vs.75.23±34.1U/linthecontrol group) (Fig.4h andi). However, when docetaxel was given simultaneously with 1,4-DMP to mice, significantly lower activity of all studied liverenzymeswasobserved(LDH:494.2±73.9 U/l;AST: 96.36±22.6U/l;for bothenzymes p<0.05 vs. control,do­cetaxel and1,4-DMP-treated group; ALT: 36.16±10.9U/l, p<0.05vs.controland DTX-treated group) (Fig.4g–i). Discussion 1-MNAisanendogenousmetaboliteofNAthatwas previ­ously shown to possess significant anti-inflammatory and anti-thrombotic activity[17,18]. 1-MNAissynthetizedby nicotinamide N-methyltransferase (NMMT), an enzyme expressed primarily in liver cells where it participates in methylation of NA and other pyridine compounds [24]. Concurrently, the expression of NMMT was reported in multiple types of cancer in which it was associated with tumor-promoting activity[25–27]that couldbefurtherat­tributed to1-MNA[28].Onthecontrary,someofthe pub-lished reports show the beneficial correlation between NMMT expression and cancersurvival[29,30].Along the lineswith suchdatainarecently publishedstudy,wehave shown that exogenous 1-MNA does not enhance the growth of cancer cells neither in vitro nor in vivo but, in contrary, may possess antimetastatic activity, most likely resulting from its PGI2-releasing capacity. We have also shown that 1,4-DMP, a structural analog of 1-MNA, pos­sesses similar antimetastatic activity; however, both com-poundsseemedtohavedifferent mechanismsof actionthat ultimately resulted in platelet-dependent metastasis inhib­ition. Importantly, pyridine compounds, but particularly 1,4-DMP, when givenina combination with cyclophospha­mide contributed to its anticancer activity enhancing both antitumor and antimetastatic activity of cytostatic drug [21].Referredstudies were,however,carried outexclusively in the mouse model of breast cancer, and the report did not mention any activity of 1-MNA or 1,4-DMP in other types of malignant tumors or with different anticancer agents. Inthepresent work, weinvestigatedthe activity ofboth compounds in the model of TRAMP mice that spontan-eously develop prostate tumors. Similar to our previously reported studies [21], both compounds, when adminis­trated alone to mice developing prostate tumors, revealed no significant anticancer activity;however, to some extent these compounds delayed the disease onset (Fig. 1a–c). Such a delayed disease onset might be attributed to the prostacyclin-dependent activity of the compounds, as prostacyclin was shown to inhibit lung tumor develop­ment in PPAR.-dependentmechanism[31]that wasalso shown to be involved in tumor growth arrest in prostate tumors[32, 33].Lack of the significant tumorpreventing activity ofboth pyridine compounds while being somehow disappointing in terms of the possible application of 1­MNA, andits analogin the prevention of prostate cancer, is important for theirimplementationin anticancer treat­ments, in general, as once again we demonstrated that neither 1-MNA nor 1,4-DMP promoted the growth of solid tumors. On the contrary, we have not observed any antimetastatic activity of neither of the studied com-pounds. In contrast, among 1-MNA-treated animals, we even observed a slight increase of metastases frequency (Fig.1candd).Sucha surprising resultmightbethe con-sequenceofprostacyclin-relatedinhibitionof naturalkiller cells[34] that, in turn, was shown to stimulate prostate tumor metastasis [35]. Another explanation of the ob-servedlimited antimetastatic activityofboth1-MNA and 1,4-DMP might be associated with previously reported re-lationship between thrombin generation and the growth and metastasis of prostate tumorsin TRAMP mice[36]. Possibly, thrombin as a potent coagulation andplatelet ac-tivator that was proven to facilitate metastasis[37] coun­teracts a possible antiplatelet activity of pyridine compounds in this model. Importantly, in this study, we noted that during the prostate tumor development, TGF­ß1 plasma concentration in TRAMP mice increased (Fig. 1p), which remains consistent with the previous re-portsindicatingthe usefulnessofthis molecule asaprog­nosticfactor inprostate tumors[38]. When investigating the anticancer activity of therapeutic regimen including simultaneous use of docetaxel and 1,4­DMP(that seemedtobe more potentinthemodelof pros-tate tumor as compared with 1-MNA) in the therapy of metastatic human prostate cancer PC-3M-luc2, we ob-served 60%enhancement of the antitumoractivityofdoce­taxel given alone (Fig. 2c) and complete abolition of metastases formation(Table3) inmicetreated with doce­taxel administrated with 1,4-DMP. These results confirm that 1,4-DMP may promote anticancer activity of various cytotoxic drugs. Beneficial therapy outcome was also reflected in the decreased plasma level of TGFß-1, a mol-ecule often acknowledged as a prognostic marker in pros-tate cancer (Fig.2i). TGF-ß1is commonly recognized as a molecule inducing epithelial-to-mesenchymal transition (EMT) in tumor-forming cells. EMT is a phenomenon in result of which non-invasive tumor cells of epithelial phenotype acquire mesenchymal properties and become able to migrate and invade distant tissues [39]. Therefore, in our study, lower plasma concentrationofTGF-ß1, andbyimplicationlower metastatic capacity, was associated with higher expression ratioofE-cadherintoN-cadherin (Fig. 2f),cell adhesion molecules commonly accepted as important markers of EMTincancer cells, includingthoseofprostateorigin [40]. Lower metastatic capacity of tumor-forming cells was add­itionally accompanied by the lower level of short E-cadherin fragments (40 kDa) observed in the tumor mass ofmicelackingmetastases. Such shortintracellularprotein fragments arise because of full-length E-cadherin cleavage resultinginthe releaseintoextracellular matrix andnextto bloodstream of 80 kDa E-cadherin extracellular domain [41].Indeed, 80 kDafragments identified in metastatic sites or serum were previously discussed as potential prostate cancer progression markers [42, 43]. Accordingly, in our study, we observed that 40 kDa intracellular domain of E-cadherin was abundant in tumor mass isolated from mice bearing PC-3M-luc2 tumors diagnosed with metastases (Fig. 2g). Finally, the prominent efficacy of the combined treatmentcomprisingthe useofdocetaxel and1,4-DMPis additionally confirmed by the decreased expression of VEGFR-1 (Fig. 2h), another prognostic marker that has been previously linked to enhanced metastatic potential of prostatetumors [44]. We have previouslyshown that observed enhanced anti-tumor and antimetastatic activity of cytotoxic drugs when ina combinationwith1,4-DMPmightbea resultof anti­platelet activity of thelatter compound[21]. Platelets,in turn, contribute to metastases formation by several mech-anisms as comprehensively reviewedin the literature[45, 46].To confirm thatincreased anticancer efficacy of doce­taxel observed when cytotoxic drug was given with 1,4­DMP was associated with diminished platelets activity, we have analyzedmorphological andbiochemicalparameters reflectingplateletactivationstatus.Inthis regard,inmice receiving the studied combined treatment, we observed lowered values of mean platelet volume and PDW (Fig. 3a–d) that may suggest decreased platelet activity [47, 48]. Additionally, in mice treated with docetaxel and 1,4-DMP, we also noted a marked reduction in plasma concentrations of TXB2, vWF and soluble P-selectin (Fig. 3e–h), constituting biochemical markers, further confirming diminished platelet activity. Interestingly, increased antitumor activity of docetaxel when administrated simultaneously with 1,4-DMP was accompanied by its reduced toxicity manifested in the decreased incidence of treatment-related deaths and im-proved liver function (Fig. 4). Although we are currently investigating these phenomena, our initial results indi­cate that the observed protective activity of 1,4-DMP may involve acetylcholinesterase and consequently histamine-dependent pathways. It seems possible that in response to the treatment with 1,4-DMP, the level of his-tamine is increased that, in turn, may prevent liver injury [49]. This novel and unexpected feature of the 1,4-DMP treatment might not only be of a great value for possible improvement of side effects in patients undergoing chemotherapy, but may also allow to increase dosages in patients with drug-resistant tumors to induce desired re-sponse while maintaining acceptable treatment toxicity. Conclusions Theresults of thepresented studyprove that neither1­MNA nor 1,4-DMPwhenadministrated alonedonot influ­ence the development and growth of the primary prostate tumors supporting our previous findings in the murine model of metastaticbreast cancer. However, pyridine com-pounds, such as 1,4-DMP, may beneficially influence the antitumor and antimetastatic activity of docetaxel and add­itionally limit the side effects accompanying chemotherapy. Such findings allow us to believe that pyridine compound endowed withPGI2 releasingproperties[18, 20] maybe­ come a promising agent for the adjuvant therapy of meta-static cancer. Additional file Additional file 1: Figure S1. Changes in blood morphology and TGF­ß1 plasma concentration in TRAMP mice over time. Analysis were carried out on blood samples taken on days 87, 122, 213 of the experiment and during the necropsy of the animals: a platelet count (*p<0.05 vs. control D213, **p<0.05 vs. 1,4-DMP D122); bmean platelet volume (MPV); c platelet distribution width (PDW) (*p<0.05 vs. Control D87, D122, D213; **p<0.05 vs. 1,4-DMP D87, D122); dred blood cell count (*p<0.05 vs. Control D87, D213, ** p<0.05 vs. 1-MNA D87, ***p<0.05 vs. 1,4-DMP D87, D213); e lymphocyte count (*p<0.05 vs. Control D87; **p<0.05 vs. 1,4-DMP D87, D213); fmonocyte count; ggranulocyte count and hTGF-ß1 determined by ELISA (*p<0.05 vs. Control D122, D213’ **p<0.05 vs. 1-MNA D87, D213; ***p<0.05 vs. 1,4-DMP D87, D213). All data are pre­sented as mean±SD. Data significantly different(p<0.05) are marked with stars. (TIFF 888 kb) Abbreviations %BW:%of body weight; 1,4-DMP: 1,4-dimethylpyridine; 1-MNA: 1­methylnicotinamide; 6-keto-PGF1.: 6-keto-prostaglandin F1.;a.u.: Arbitrary units; ALT: Alanine aminotransferase; AST: Aspartate aminotransferase; DTX: Docetaxel; EDTA: Ethylenediaminetetraacetic acid; EMT: Epithelial to mesenchymal transition; HBSS: Hanks Balanced Salt Solution; LDH: Lactate dehydrogenase;MPV: Mean platelet volume; NA: Nicotinamide; NNMT: Nicotinamide N-methyltransferase; PCT: Platelet crit; PDW: Platelet distribution width; PGI2: Prostacyclin; PVDF: Polyvinylidene fluoride; RBC: Red blood cells; rpm: Round per minute; SD: Standard deviation; SDS: Sodium dodecyl sulfate; TGF-ß1: Transforming growth factor ß1; TGI: Tumor growth inhibition; TNF-.: Tumor necrosis factor .;TV: Tumor volume; TXB2: ThromboxaneB2;VEGFR-1: Vascular endothelial growth factor receptor 1; vWF: von Willebrand Factor Acknowledgements Not applicable. Funding This study was supported by the European Union from the resources of the European Regional Development Fund within the Innovative Economy Program (grant coordinated by the JCET-UJ, No. POIG.01.01.02–00–069/09) and The National Center for Research and Development under the Polish Strategic Framework Program STRATEGMED (grant coordinated by JCET-UJ No. STRATEGMED1/233226/11/NCBR/2015). The publication was supported by Wroclaw Center of Biotechnology within a Program The Leading National Research Center (KNOW)for years 2014–2018. The funding bodies did not participate in the design of the study and collection, analysis and interpretation of data and in writing the manuscript. Availability of data and materials All data generated or analyzed duringthis study are included in this published article [and its supplementary information files]. Authors’ contributions AD, JW and SC conceived and designed the experiments; AM and JG synthetized and provided pyridinium salts; AD, JW, MS, MM and MN performed the experiments; AD analyzed the data and wrote the paper. All authors read and approved the final manuscript. Competing interest The authors declare that they have no competing interests. Consent for publication Not applicable. Ethics approval and consent to participate All animal experiments were performed according to the Interdisciplinary Principles and Guidelines for the Use of Animals in Research, Marketingand Education issued by the New York Academy of Sciences’ Ad Hoc Committee on Animal Research and were approved by the 1st Local Committee for Experiments with the Use of Laboratory Animals, Wroclaw, Poland. Author details 1Hirszfeld Institute of Immunology and Experimental Therapy, Polish Academy of Sciences, Weigla 12, Wroclaw 53-114, Poland. 2Wroclaw University of Environmental and Life Sciences, Norwida 31, Wroclaw 50-375, Poland. 3Chair of Pharmacology, Jagiellonian University, Medical College, Grzegorzecka 16, Krakow 31-531, Poland. 4Jagiellonian Center for Experimental Therapeutics (JCET), Jagiellonian University, Bobrzynskiego 14, Krakow 30-348, Poland. 5Lodz University of Technology, Zeromskiego 116, Lodz 90-924, Poland. Received:9 November 2016 Accepted:1 March 2017 References 1. Torre LA, Bray F, Siegel RL, Ferlay J, Lortet-Tieulent J, Jemal A. Global cancer statistics, 2012. CA CancerJClin. 2015;65:87–108. 2. Cuzick J, Thorat MA, Andriole G, Brawley OW, Brown PH, Culig Z, Eeles RA, Ford LG, Hamdy FC, Holmberg L, et al. Prevention and early detection of prostate cancer. Lancet Oncol. 2014;15:e484–92. 3. Hwang C. Overcoming docetaxel resistance in prostate cancer: a perspective review. Ther Adv Med Oncol. 2012;4:329–40. 4. Herbst RS, Khuri FR. Mode of action of docetaxel -a basis for combination with novel anticancer agents. Cancer Treat Rev. 2003;29:407–15. 5. Pienta KJ. Preclinical mechanisms of action of docetaxel and docetaxel combinations in prostate cancer. Semin Oncol. 2001;28:3–7. 6. Tannock I, Gospodarowicz M, Meakin W, Panzarella T, Stewart L, Rider W. Treatment of metastatic prostatic cancer with low-dose prednisone: evaluation of pain and quality of life as pragmatic indices of response.JClin Oncol. 1989;7:590–7. 7. Herr I, Pfitzenmaier J. Glucocorticoid use in prostate cancer and other solid tumours: implications for effectiveness of cytotoxic treatment and metastases. Lancet Oncol. 2006;7:425–30. 8. Fakih M, Johnson CS, Trump DL. Glucocorticoids and treatment of prostate cancer: a preclinical and clinical review. Urology. 2002;60:553–61. 9. Sartor O, Weinberger M, Moore A, Li A, Figg WD. Effect of prednisone on prostate-specific antigen in patients with hormone-refractory prostate cancer. Urology. 1998;52:252–6. 10. Teply BA, Luber B, Denmeade SR, Antonarakis ES. The influence of prednisone on the efficacy of docetaxel in men with metastatic castration­resistant prostate cancer. Prostate Cancer Prostatic Dis. 2016;19:72–8. 11. GeifmanN, ButteAJ.Apatient-level data meta-analysisof standard-of-care treatments from eight prostate cancer clinical trials. Sci Data. 2016;3:160027. 12. Hotte SJ, Saad F. Current management of castrate-resistant prostate cancer. Curr Oncol. 2010;17 Suppl 2:S72–79. 13. TannockIF,deWitR,BerryWR,HortiJ,PluzanskaA,ChiKN,OudardS,Theodore C,JamesND,TuressonI,etal.Docetaxelplus prednisoneor mitoxantroneplus prednisoneforadvancedprostatecancer.NEnglJMed. 2004;351:1502–12. 14. Lam ET, Flaig TW. Upfront chemotherapy for metastatic prostate cancer. Oncology. 2015;29:956. Williston Park. 15. Dorff TB, Crawford ED. Management and challenges of corticosteroid therapy in men with metastatic castrate-resistant prostate cancer. Ann Oncol. 2013;24:31–8. 16. Zhang C, Wenger T, Mattern J, Ilea S, Frey C, Gutwein P, Altevogt P, Bodenmuller W, Gassler N, Schnabel PA, et al. Clinical and mechanistic aspects of glucocorticoid-induced chemotherapy resistance in the majority of solid tumors. Cancer Biol Ther. 2007;6:278–87. 17. Gebicki J, Sysa-Jedrzejowska A, Adamus J, Wozniacka A, Rybak M, Zielonka J. 1-Methylnicotinamide: a potent anti-inflammatory agent of vitamin origin. PolJPharmacol. 2003;55:109–12. 18. Chlopicki S, Swies J, Mogielnicki A, Buczko W, Bartus M, Lomnicka M, Adamus J, Gebicki J. 1-Methylnicotinamide (MNA), a primary metabolite of nicotinamide, exerts anti-thrombotic activity mediated by a cyclooxygenase-2/prostacyclin pathway. BrJPharmacol. 2007;152:230–9. 19. BryniarskiK,BiedronR,JakubowskiA,ChlopickiS,MarcinkiewiczJ.Anti­inflammatoryeffectof 1-methylnicotinamidein contacthypersensitivityto oxazoloneinmice;involvementofprostacyclin.EurJPharmacol. 2008;578:332–8. 20. Kalaska B, Piotrowski L, Leszczynska A, Michalowski B, Kramkowski K, Kaminski T, Adamus J, Marcinek A, Gebicki J, Mogielnicki A, Buczko W. Antithrombotic effects of pyridinium compounds formed from trigonelline upon coffee roasting.JAgric Food Chem. 2014;62:2853–60. 21. Blazejczyk A, Switalska M, Chlopicki S, Marcinek A, Gebicki J, Nowak M, Nasulewicz-Goldeman A, Wietrzyk J. 1-methylnicotinamide and its structural analog 1,4-dimethylpyridine for the prevention of cancer metastasis.JExp Clin Cancer Res. 2016;35:110. 22. MilczarekM, Filip-PsurskaB, SwietnickiW, KutnerA, WietrzykJ. VitaminD analogs combined with 5-fluorouracil in human HT-29 colon cancer treatment. Oncol Rep. 2014;32:491–504. 23. Noguchi K, Matsuzaki T, Ojiri Y, Sakanashi M, Nakasone J, Sakanashi M. Prostacyclin causes splenic dilation and haematological change in dogs. Clin Exp Pharmacol Physiol. 2006;33:81–8. 24. AlstonTA,AbelesRH.Substratespecificityofnicotinamidemethyltransferase isolatedfromporcineliver.ArchBiochem Biophys.1988;260:601–8. 25. Yu T, Wang YT, Chen P, Li YH, Chen YX, Zeng H, Yu AM, Huang M, Bi HC. Effects of nicotinamide N-methyltransferase on PANC-1 cells proliferation, metastatic potential and survival under metabolic stress. Cell Physiol Biochem. 2015;35:710–21. 26. Zhang J, Wang Y, Li G, Yu H, Xie X. Down-regulation of nicotinamide N-methyltransferase induces apoptosis in human breast cancer cells via the mitochondria-mediated pathway. PLoS One. 2014;9:e89202. 27. Chen C, Wang X, Huang X, Yong H, Shen J, Tang Q, Zhu J, Ni J, Feng Z. Nicotinamide N-methyltransferase: a potential biomarker for worse prognosis in gastric carcinoma. AmJCancer Res. 2016;6:649–63. 28. ParsonsRB, AravindanS,KadampeswaranA,EvansEA, SandhuKK,LevyER, ThomasMG,AustenBM,RamsdenDB.Theexpressionof nicotinamideN­methyltransferaseincreases ATPsynthesis andprotectsSH-SY5Y neuroblastoma cellsagainstthetoxicityofComplexIinhibitors.BiochemJ.2011;436:145–55. 29. ZhouW,GuiM, ZhuM,LongZ, HuangL,ZhouJ,HeL,ZhongK.Nicotinamide N-methyltransferaseisoverexpressedinprostatecancer andcorrelateswith prolongedprogression-freeandoverall survivaltimes.OncolLett.2014;8:1175–80. 30. Sartini D, Santarelli A, Rossi V, Goteri G, Rubini C, Ciavarella D, Lo Muzio L, Emanuelli M. Nicotinamide N-methyltransferase upregulation inversely correlates with lymph node metastasis in oral squamous cell carcinoma. Mol Med. 2007;13:415–21. 31. Nemenoff R, Meyer AM, Hudish TM, Mozer AB, Snee A, Narumiya S, Stearman RS, Winn RA, Weiser-Evans M, Geraci MW, Keith RL. Prostacyclin prevents murine lung cancer independent of the membrane receptor by activation of peroxisomal proliferator–activated receptor gamma. Cancer Prev Res. 2008;1:349–56. Phila. 32. Subbarayan V, Sabichi AL, Kim J, Llansa N, Logothetis CJ, Lippman SM, Menter DG. Differential peroxisome proliferator-activated receptor-gamma isoform expression and agonist effects in normal and malignant prostate cells. Cancer Epidemiol Biomarkers Prev. 2004;13:1710–6. 33. Sikka S, Chen L, Sethi G, Kumar AP. Targeting PPARgamma signaling cascade for the prevention and treatment of prostate cancer. PPAR Res. 2012;2012:968040. 34. Lanefelt F, Ullberg M, Jondal M, Fredholm BB. PGE1 and prostacyclin suppression of NK-cell mediated cytotoxicity and its relation to cyclic AMP. Med Biol. 1983;61:324–30. 35. Liu G, Lu S, Wang X, Page ST, Higano CS, Plymate SR, Greenberg NM, Sun S, Li Z, Wu JD. Perturbation of NK cell peripheral homeostasis accelerates prostate carcinoma metastasis.JClin Invest. 2013;123:4410–22. 36. Hu L, Ibrahim S, Liu C, Skaar J, Pagano M, Karpatkin S. Thrombin induces tumor cell cycle activation and spontaneous growth by down-regulation of p27(Kip1), in association with the Up-regulation of Skp2 and MiR-222. Cancer Res. 2009;69:3374–81. 37. Nierodzik ML, Kajumo F, Karpatkin S. Effect of thrombin treatment of tumor cells on adhesion of tumor cells to platelets in vitro and tumor metastasis in vivo. Cancer Res. 1992;52:3267–72. 38. Adler HL, McCurdyMA, Kattan MW, Timme TL, Scardino PT, Thompson TC. Elevatedlevels ofcirculatinginterleukin-6and transforminggrowthfactor­beta1inpatientswithmetastaticprostatic carcinoma.JUrol. 1999;161:182–7. 39. Moustakas A, Heldin CH. Mechanisms of TGFbeta-Induced Epithelial-Mesenchymal Transition.JClin Med. 2016;5(7):63. 40. Huo C, Kao YH, Chuu CP. Androgen receptor inhibits epithelial­mesenchymal transition, migration, and invasion of PC-3 prostate cancer cells. Cancer Lett. 2015;369:103–11. 41. David JM, Rajasekaran AK. Dishonorable discharge: the oncogenic roles of cleaved E-cadherin fragments. Cancer Res. 2012;72:2917–23. 42. Kuefer R, Hofer MD, Zorn CSM, Engel O, Volkmer BG, Juarez-Brito MA, Eggel M, Gschwend JE, Rubin MA, Day ML. Assessment of a fragment of e-cadherin as a serum biomarker with predictive value for prostate cancer. Br JCancer. 2005;92:2018–23. 43. Kuefer R, Hofer MD, Gschwend JE, Pienta KJ, Sanda MG, Chinnaiyan AM, Rubin MA, Day ML. The role of an 80 kDa fragment of E-cadherin in the metastatic progression of prostate cancer. Clin Cancer Res. 2003;9:6447–52. 44. Tsourlakis MC, Khosrawi P, Weigand P, Kluth M, Hube-Magg C, Minner S, Koop C, Graefen M, Heinzer H, Wittmer C, et al. VEGFR-1 overexpression identifies a small subgroup of aggressive prostate cancers in patients treatedby prostatectomy. IntJMol Sci. 2015;16:8591–606. 45. Blazejczyk A, Papiernik D, Porshneva K, Sadowska J, Wietrzyk J. Endothelium and cancer metastasis: perspectives for antimetastatic therapy. Pharmacol Rep. 2015;67:711–8. 46. Leblanc R, Peyruchaud O. Metastasis: new functional implications of platelets and megakaryocytes. Blood. 2016;128:24–31. 47. ChuSG,BeckerRC,BergerPB,BhattDL,EikelboomJW,KonkleB,MohlerER, ReillyMP,BergerJS.Meanplatelet volume asapredictorof cardiovascularrisk: asystematic reviewandmeta-analysis.JThrombHaemost.2010;8:148–56. 48. Vagdatli E, Gounari E, Lazaridou E, Katsibourlia E, Tsikopoulou F, Labrianou I. Platelet distribution width: a simple, practical and specific marker of activation of coagulation. Hippokratia. 2010;14:28–32. 49. Masaki T, Chiba S, Tatsukawa H, Noguchi H, Kakuma T, Endo M, Seike M, Watanabe T, Yoshimatsu H. The role of histamine H1 receptor and H2 receptor in LPS-induced liver injury. FASEB J. 2005;19:1245–52. Submit your next manuscript to BioMed Central and we will help you at every step: • We accept pre-submission inquiries  Our selector tool helps you to .nd the most relevant journal  We provide round the clock customer support  Convenient online submission  Thorough peer review  Inclusion in PubMed and all major indexing services  Maximum visibility for your research Submit your manuscript at www.biomedcentral.com/submit