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Repurposing mechanistic insight of PDE-5 inhibitor in cancer chemoprevention through mitochondrial-oxidative stress intervention and blockade of DuCLOX signalling

Repurposing mechanistic insight of PDE-5 inhibitor in cancer chemoprevention through... Background: This study evaluates the anti-cancer effects of Tadalafil (potent PDE-5 inhibitor) in female albino wistar rats against n-methyl n-nitrosourea induced mammary gland carcinogenesis. Methods: The animals were selected and randomly divided among four groups and each group contains six animals per group. The animal tissue and serum samples were evaluated for the presence of antioxidant parameters and the cellular morphology was studied using carminic staining, haematoxylin staining and scanning electron microscopy followed by immunoblotting analysis. Results: On the grounds of hemodynamic recordings and morphology, n-methyl n-nitrosourea treated group showed distorted changes along with distorted morphological parameters. For morphological analysis, the mammary gland tissues were evaluated using scanning electron microscopy, whole mount carmine staining, haematoxylin and eosin staining. The serum samples were evaluated for the evaluation of oxidative stress markers and inflammatory markers. The level of caspase 3 and 8 were also evaluated for the estimation of apoptosis. The fatty acid profiling of mammary gland tissue was evaluated using fatty acid methyl esters formation. The mitochondrial mediated apoptosis and inflammatory markers were evaluated using immunoblotting assay. Conclusion: The results confirm that Tadalafil treatment restored all the biological markers to the normal and its involvement in mitochondrial mediated death apoptosis pathway along with inhibition of inflammatory markers. Keywords: Tadalafil, ER+ mammary gland cancer, Mitochondrial stress, PDE-5 inhibitor, N-methyl-n-nitrosourea, DuCLOX Background nowadays [1]. Tadalafil is a known phosphodiesterase-5 Distinct metastasis and resistance development by self- (PDE-5) inhibitor, which have anti-cancer effects in vari- renewing nascent cancer cells in response with the ous types of cancer [2]. In the search of molecularly tar- chemotherapy drug regimen and simultaneous radiations geted compounds that have the structure similarity with are still a main obstacle in cancer treatment for patients the known anti-cancerous drugs like viscristine, vinblas- tin, vindesine, vinorelbine, leuprolide and geserelin got tremendous attention due to the expression and regula- * Correspondence: gauravpharm@hotmail.com; gauravk@bbau.ac.in tion of the important signalling pathways [3]. In the last Manjari Singh and Sweta Kasna contributed equally to this work. decade, Tadalafil used to inhibit the myeloid derived Department of Pharmaceutical Sciences, Babasaheb Bhimrao Ambedkar University, (A Central University), Vidya Vihar, Raebareli road, Lucknow, UP suppressor cells (MDSCs) and restore the T cells in can- 226 025, India cer patients as compared to the normal peoples [4]. Full list of author information is available at the end of the article © The Author(s). 2019 Open 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. Singh et al. BMC Cancer (2019) 19:996 Page 2 of 15 PDE-5, PDE-6, PDE-9 are the predominant active iso- Sciences, Naini Allahabad, India. All animals were ran- form of ubiquitously distributed metallohydrolases con- domized among four groups having eight animals each. stitute of 11 distinct gene families, which initiate the Carcinogenesis was induced by single tail vein injection of st sequential lcleavage of cyclic adenosine monophosphate MNU on day 1 in each animal. MNU was dissolved im- (cAMP) and cyclic guanosine monophosphate (cGMP) mediately before use in glacial acetic acid and water (pH into their intermediate inactive 50 derivatives and 5- 4.5–5). The experimental groups were randomized as fol- GMP. It ultimately regulating the amplitude and dur- lows: group I (normal control, saline 3 ml/kg, p.o.; group ation of their intracellular downstream signaling mech- II (toxic control, MNU 47 mg/kg, i.v.); group III (Tadalafil anism [5]. This inhibition of cGMP conversion leads to 2 mg/kg, p.o. + MNU 47 mg/kg, i.v.) and group IV (Tada- activate protein kinase G and map kinase pathway of lafil 4 mg/kg, p.o. + MNU 47 mg/kg, i.v). Tadalafil was ad- apoptosis and also simultaneously affect cancer cell ministered from 7th to 110th day at the dose specified growth and adhesion, mitochondrial energy homeostasis, above. At the end of study, animals were sacrificed for the neuronal signaling and muscle relaxation in systemic collection of mammary gland tissue under ketamine hydro- vasculature, prostate, heart, brain, lungs and platelets chloride (100 mg/kg, i.m.) and diazepam (5 mg/kg, i.m.) [6]. In previous literature, it was reported that PDE-5 in- combination as anaesthesia. hibition produced caspase dependent apoptosis of B-cell All the animal experimental procedures follow the AR- chronic lymphocytic leukemic cells [7]. Various prelim- RIVE guidelines. inarily studies also demonstrated that PDE-5 inhibitors can induce apoptosis by activation of cGMP and nitric Changes in hemodynamic recordings oxide (NO) in cancerous cell [8]. In recent findings, in- The hemodynamic changes were recorded using the Lab dole derivatives have the potency to treat mammary Chart Pro-8 (AD Instruments, Australia) instrument accord- gland cancer and Tadalafil also have the indole moiety ing to the methods elaborated by us previously [11, 12]. in its structure, which ultimately reveals its molecular mechanism [9]. It was reported that the expression of Effect of Tadalafil upon morphological changes PDE-5 was significantly high in the malignant breast tu- Whole mount alum stain mors as compare with normal breast tissues and benign Rats were sacrificed after completions of study and forth tumors [10]. Consistently increased PDE-5 expression mammary gland tissues were excised and fixed for a has also been reported in a variety of mammary gland minimum of 2 days in Carnoy’s fixative solution followed cancer cell lines (MCF-7, T47D, HTB-26, MDA-MB- by staining with carmine solution (1 g carmine and 2.5 g 231), which gives the rationale to assess the anticancer aluminum potassium sulfate in 500 ml water). The fixed effects of PDE-5 inhibitors in carcinogen induced animal glands were hydrated with 90, 70, 35 and 15% ethyl alco- model and check the involvement of reactive oxygen hol and then rinsed with water for 5 min. The glands species (ROS), death receptor and mitochondrial signal- were then dehydrated progressively in 35, 70, 95 and ling as part of the combinatorial apoptosis mechanism. 100% ethanol, dipped in xylene for 2 days and mounted Based on the previous literature, we hypothesize to on glass slides. The sections were examined for the iden- evaluate the effect of tadalafil upon carcinogen induced tification of angiogenesis [13, 14]. ER+ mammary gland carcinoma in vivo model. Histopathological studies Methods Thin section (5 μm) of mammary glands tissue was cut Drugs and chemicals using microtome and fixed overnight in 10% hank’s bal- Tadalafil (API) was procured from the Sanofi Aventis as a anced salt solution (HBSS) buffered formalin which fur- gift sample followed by FTIR analysis to establish its purity. ther used for representative gross morphological and N-methyl-n-nitrosourea (MNU) (Sigma-Aldrich, N1517) histological features of haematoxylin and eosin (H&E) was procured from Sigma Life Science Aldrich Co. 3050 stained paraffin sections of the mammary glands tissue Spruce Street, St. Louis, USA. Other chemicals were pur- [15, 16]. chased from Himedia Pvt. Ltd., Sigma Aldrich and Amresco. Scanning electron microscopy (SEM) In vivo study Sodium cacodylate and HBSS buffer based HCl collage- The study protocol was approved from CPCSEA guide- nase hyaluronic acid enzymatic digestion method was lines for laboratory Animals and Ethics, Government of used for preparation of tissue before SEM analysis. The India (IAEC/SHIATS/PA16III/SSPG19). Female albino entire methodology was performed according to wistar rats of 100-120 g were used for this study and methods elaborated by us previously [17]. The sample collected from the central animal house facility of Sam was dried with increasing grade of acetone (70, 80, 90 Higginbottom University of Agriculture, Technology and and 100%). All the acetone specimens were dehydrated Singh et al. BMC Cancer (2019) 19:996 Page 3 of 15 and replaced by exchange of soak/flash liquid CO fluids Gas chromatographic (GC) analysis of mammary gland with ethanol or acetone by critical point method (31 °C tissue and 74 bar). Samples were further processed by platinum The fatty acid methyl esters (FAME) analysis was per- coating, which has been examined under SEM(X1000) formed for the estimation of free fatty acids in the mam- (JEOL JSM-6490LV) [15, 18]. mary gland tissue. The methodology of FAME was elaborated elsewhere [25]. Effect of Tadalafil upon oxidative stress markers Western blotting The antioxidant parameters were evaluated using tissue The mammary gland tissue sample was homogenized homogenate (10%w/v). The homogenate was prepared in using RIPA lysis buffer, isolated by acetone precipitation 0.15 M KCl and the same was centrifuged for 15 min at and total protein concentration was assayed by using 4 °C. In thiobarbituric acid reactive substances (TBARs), Bradford method [26]. All the protein samples were re- malondialdehyde (MDA) forms a 1:2 adduct with TBAR solved on SDS-PAGE gels with Tris buffer system. The and produce the reaction which can be measured by proteins were transferred to PVDF membrane by elec- spectrophotometer at 540 nm. For superoxide dismutase troblotting by semidry system. Prestained protein ladder (SOD), the inhibition of pyrogallol auto-oxidation was was used as a molecular weight identification marker. measured. The absorbance was measured at 420 nm. After 2 h of blocking with blocking buffer at room Catalase catalyzes the decomposition of hydrogen perox- temperature, membranes were processed for primary ide to water and oxygen. The absorbance was measured antibody binding (1: 2000 dilutions) against Bcl-xl (MA- at 250 nm. Glutathione was measured using Ellamanis 5-15,142), BAD (SC-8044), NFκBp65 (MA5–1616), reagent (DTNB), when DTNB reacts with GSH then 5 UCHL-1 (MA1–83428) and COX (MA5–14568) in 4 °C thionitrobenzoic acid (TNB) and GS-TNB was formed. followed by respective secondary antibody (anti-rabbit The absorbance was measured at 410 nm [19, 20]. (SC-2030), anti-goat (SC-2020) and anti-mouse (31,430, Pierce Thermo Scientific, USA) in 1:5000 dilutions. β- actin (MA5–15739-HRP) was used as loading control. Serum NO level The PVDF blotting membranes were processed at final For NO estimation, same proportion of serum isolated steps by using an enhanced ECL substrate (Western from animal blood and Griess reagent were mixed and Bright ECL HRP substrate, Advansta, Melanopark, Cali- incubated at 37 °C for 30 min. The reaction was pro- fornia, US) in Geldock system. The proteins bands were ceeded at room temperature, and the absorbance was quantified using ImageJ software [27]. read at 540 nm using UV-visible spectrophotometer (Cary 60, Agilent Technologies, CA, 95051, US). The standard curve was plot using serial dilutions of sodium Digital image analysis nitrite [21]. For microscopic analysis data obtained from H&E and SEM micrographs, six randomly chosen regions were se- lected from all group and analysed independently for Estimation of serum hydrogen sulphide (H S) level each respective sample. To identify the luminescence For H S estimation, 500 μl of tissue supernatant and from experimental images and convert them into the 75 μl of serum sample was added to the premixed zinc heights to the final surface plot,3D interactive surface acetate (1%w/v) solution, respectively. The concentration plot experiments was used to identify the pure H&E of H S in the serum sample was measured using the stained and SEM areas nearest neighbouring sampling in method elaborated by us previously. The absorbance was a square plot. This unmixing ultimately helps to distin- read spectrophotometrically at 670 nm [22]. guish between nuclear, ECM and background stains in a single complimentary image. Automated score was assigned by observing and measuring the pure H&E Cyclooxygenase (COX) and lipoxygenase (LOX) estimation staining pattern and SEM micrograph in Image J stand- The COX and LOX were estimated in the rat serum sam- ard program feature. ple followed by the method elaborated elsewhere [23]. Protein–protein interaction and gene ontological studies Caspase3 and caspase8 estimation To identify the various functional relations between dif- 96-well amber colored plate has been used for the col- ferent gene and protein protein-protein interactions orimetric identification of caspase-3/8. The estimation were studied using STRING 10.0. Different biological was performed on the basis of methodology elaborated process, cellular component and biological functions has by us previously [24]. been identified by gene ontological study (Search tool Singh et al. BMC Cancer (2019) 19:996 Page 4 of 15 for the Retrieval of Interacting Genes/Proteins) software declined in toxic control group (0.05 ± 0.01) which was (http://string-db.org). restored after Tadalafil treatment (0.06 ± 0.01) (Fig. 1). In accordance with HRV parameters, the frequency Statistical analysis domain parameters like average RR, median RR, SDRR All data were presented as mean ± SD and analyzed by and SD rate were elevated after MNU treatment. Tadala- one way ANOVA followed by Bonferroni’s multiple fil treatment helped to restore the above said parameters comparison tests for the possible significance identifica- about to normal (Fig. 2). tion between the various groups *p < 0.05, **p < 0.01, ***p < 0.001 were considered statistically significant. Stat- Morphological analysis of rat mammary gland tissue istical analysis was carried out using Graph Pad Prism Carmine staining (5.01), San Diego, California. The two main markers for angiogenesis are alveolar bud (ABs) count and differentiation (DF) score. In carmine Results staining, the no. of ABs and DF score were calculated. The Effect of Tadalafil upon hemodynamic changes ABs (both AB1 and AB2), lobules and DF were elevated Heart rate variability (HRV) is the noninvasive measures after MNU treatment (17 ± 5.65, 12 ± 5.65, 12 ± 2.82 and of autonomic nervous system (ANS) regulation. It was 41 ± 14.12) and after Tadalafil treatment; all the parameters previously reported that altered and dysfunctional ANS of cell proliferation were restored to normal (Fig. 3a-d). has been reported cancer patients. The RR interval was elevated in MNU treated group (0.18 ± 0.02) and Tadala- H&E staining fil in low dose (2 mg/kg, p.o.) restored RR interval about H&E staining provide the cellular and morphological alter- to normal (0.16 ± 0.01). On the other hand, heart rate ations occur in the tissue. In normal control tissues, all the (HR) was increased in MNU treated group (331.95 ± cell organelles were clearly visible (Fig. 4a). MNU treatment 0.68) which was diminished after Tadalafil treatment in was evident in scattered pattern of cuboidal epithelial cells dose dependent manner. QT interval was considerably (CEC), myoepithelial cells (MEC), lymphocytes, adipocytes Fig. 1 Effect of Tadalafil treatment on ECG recording. Representative box-cum-whisker plots showing quantitative variations of relative signal integrals for autonomic dysfunction relevant in the context of pathophysiology of mammary gland cancer. Groups were differentiated as: 1- Control (Normal saline, 3 ml/kg, p.o.), 2-Toxic control (MNU 47 mg/kg, i.v.), 3- MNU + Tadalafil (47 mg/kg i.v. + 2 mg/kg p.o.), 4- MNU + Tadalafil (47 mg/kg i.v. + 4 mg/kg p.o.). For presented ECG recordings, the VIP score > 1 and statistical significance is at the level of p ≤ 0.05. In the box plots, th the boxes denote interquartile ranges, horizontal line inside the box denote the median, and bottom and top boundaries of boxes are 25 and th 75th percentiles, respectively. Lower and upper whiskers are 5 and 95th percentiles, respectively Singh et al. BMC Cancer (2019) 19:996 Page 5 of 15 Fig. 2 Effects of Tadalafil treatment on HRV. Representative box-cum-whisker plots showing quantitative variations of relative signal integrals for HRV parameters relevant in the context of pathophysiology of mammary gland cancer. Groups were differentiated as: 1-Control (Normal saline, 3 ml/kg, p.o.), 2-Toxic control (MNU 47 mg/kg, i.v.), 3- MNU + Tadalafil (47 mg/kg i.v. + 2 mg/kg p.o.), 4- MNU + Tadalafil (47 mg/kg i.v. + 4 mg/kg p.o.). For presented heart rate variability, the VIP score > 1 and statistical significance is at the level of p ≤ 0.05. In the box plots, the boxes denote th interquartile ranges, horizontal line inside the box denote the median, and bottom and top boundaries of boxes are 25 and 75th percentiles, th respectively. Lower and upper whiskers are 5 and 95th percentiles, respectively andductalong with looseconnectivetissue(LCT) and (499.79 ± 109.2298 nM of MDA/μg of protein and dense connective tissues (DCT) (Fig. 4b). Tadalafil treat- 7.28 ± 0.82 nM/ml). GSH level in MNU treated group ment restored all the parameters in dose dependent man- (0.61 ± 0.04 mg %) was restored to almost control group ner (Fig. 4cand d). after Tadalafil treatment (1.39 ± 0.10 mg %). The level of SOD and catalase was increased after MNU treatment SEM analysis (0.33 ± 0.07 units of SOD/mg of protein and 2.46 ± 0.48 SEM analysis was used to analyse the internal surface of nM of H O /min/mg of protein) which was decreased 2 2 whole cells and its organelles. In normal control tissue, after Tadalafil treatment to a significant level (i.e. 0.17 ± collagen layer, duct and intra-arterial cushion/collage- 0.13 units of SOD and 1.43 ± 0.01 nM of H O ) (Fig. 6). 2 2 nous covering, were seen clearly (Fig. 5a). All the cellular organelles were absent in MNU treated group (Fig. 5b). Effect of inflammatory markers upon mammary gland Tadalafil treatment perceived decrease in tumor micro- carcinoma vessel formation with restoration of intra-arterial cush- Inflammatory markers produced reactive nitrogen spe- ion (Fig. 5c and d). cies (RNS) and ROS from epithelial and inflammatory cells, which leads to DNA damage. The inflammatory Effect of antioxidant markers upon mammary gland markers (NO, H S, COX and LOX) were upregulated carcinoma after MNU treatment. After Tadalafil treatment, the Reactive oxygen species (ROS) are the byproducts of level of all the inflammatory markers was downregulated normal metabolism through the electron transport (Fig. 7). chain. ROS and other oxidative stress are considered as a driving force for cancer progression. Tadalafil treat- ment helps to restore the diminished antioxidant profile, Effect of Tadalafil upon caspase assay when compared with MNU treated toxic group. The Caspases cysteinyl protease family members and they phenomena of protein and lipid peroxidation were very play a crucial role in apoptosis. The serum caspase 3 and well evident after MNU treatment which correlates with caspase 8 was measured using fluorometric assay kits. the high level of TBARs and PC after MNU treatment The caspase 3 and caspase 8 levels were decreased in (872.12 ± 72.98 nM of MDA/μg of protein and 12.24 ± MNU treated group and after Tadalafil treatment, the 1.79 nM/ml respectively). Tadalafil treatment decreased levels of casapse 3 and caspase 8 were upregulated in a the level of TBARs and PC in dose dependent manner dose dependent manner (Fig. 7). Singh et al. BMC Cancer (2019) 19:996 Page 6 of 15 Fig. 3 (See legend on next page.) Singh et al. BMC Cancer (2019) 19:996 Page 7 of 15 (See figure on previous page.) Fig. 3 Carmine staining of mammary gland whole mount tissue. Whole mount carmine alum staining of ductal epithelium reveals the presence of AB1 (1), AB2 (2) and lobules (3) in respective groups [a-control (normal saline, 3 ml/kg, p.o.); b-toxic control (MNU 47 mg/kg, i.v.); c (MNU + Tadalafil; 47 mg/kg i.v. + 2 mg/kg p.o.) and d (MNU + Tadalafil; 47 mg/kg i.v. + 4 mg/kg p.o.)]. The extent of AB and lobules formation is excessive in the toxic group (b) which was subsided after Tadalafil treatment (c & d). The extent of AB and lobules formation is excessive in the toxic group (b) which has been subsided with respective treatment groups (c & d). The ABs score, lobules and DF scores were also higher in toxic control which was subsided after Tadalafil treatment in a dose dependent manner Fig. 4 H&E analysis and digital image analysis of mammary gland tissue. H&E staining of four individual groups [a-control (normal saline, 3 ml/kg, p.o.); b-toxic control (MNU 47 mg/kg, i.v.); c (MNU + Tadalafil; 47 mg/kg i.v. + 2 mg/kg p.o.) and d (MNU + Tadalafil; 47 mg/kg i.v. + 4 mg/kg p.o.)] revealed presence of adipocytes (4), duct (5) LCT (6), DCT (7), lymphocytes (8), CEC (9) and MEC (10) in control as well as Tadalafil treated groups (a, c and d). In the toxic control group (b), the cell morphology was distorted, and cell organelles were absent. 3D image reconstruction and software-based analysis dataset of constructs representing score was done by using Image J (NIH) software by thresh holding of stained zones of H&E images followed by pixel vs intensity determination by the 3D interactive surface plot and log-histogram analysis (e,f,g,h and i,j,k,l). Singh et al. BMC Cancer (2019) 19:996 Page 8 of 15 Fig. 5 SEM and digital image analysis of mammary gland tissue. SEM analysis of four individual groups [a-control (normal saline, 3 ml/kg, p.o.); b- toxic control (MNU 47 mg/kg, i.v.); c (MNU + Tadalafil; 47 mg/kg i.v. + 2 mg/kg p.o.) and d (MNU + Tadalafil; 47 mg/kg i.v. + 4 mg/kg p.o.)] was performed. Control (a) demonstrated presence of collagenous layers (11), duct (12), nodules (13) and small capillary network (14). MNU (b) administration perceived loss of collagenous covering (11) and formation of tumormicrovessels/small capillary network (14) and nodules (13). Tadalafil treatment restores all the cell organelles close to normal. 3D image reconstruction and software-based analysis dataset of constructs representing score was done by using Image J (NIH) software by thresh holding of stained zones of H&E images followed by pixel vs intensity determination by the 3D interactive surface plot and log-histogram analysis (e,f,g,h and i,j,k,l) Effect of Tadalafil upon FAME analysis MNU treated animals (Additional file 1:FigureS1 FAME assay converts cellular lipids into fatty acid and Additional file 2:Table S1). methyl esters, resolved and identified by gas chroma- tography. After MNU treatment, there was substan- Immunoblotting analysis tial increase in saturated fatty acid, monounsaturated Western blotting is used to separate and identify proteins. fatty acid along with decrease in the unsaturated In this technique a mixture of proteins is separated on the fatty acid and polyunsaturated fatty acid content. basis of molecular weight through gel electrophoresis. Bcl- Tadalafil treatment restored the lipid content in xl (anti-apoptotic) expression was increased after MNU Singh et al. BMC Cancer (2019) 19:996 Page 9 of 15 Fig. 6 Effect of Tadalafil on the antioxidant markers. Bar diagram represents the difference between the control, toxic and Tadalafil treated groups. The markers of protein and lipid (PC and TBARs) were increased in MNU treated group and reduced after Tadalafil treatment. GSH was decreased after MNU treatment whereas the level of SOD and catalase were increased after MNU treatment which was subsided after Tadalafil treatment. All the data was presented as mean ± SD. Comparisons were made on the basis of one-way ANOVA followed by Bonferroni multiple test and all groups are compared to the toxic control group (*p < 0.05, **p < 0.01, ***p < 0.001) treatment with diminished level of BAD (proapoptotic). are to target different key proteomic marker that plays a Tadalafil treatment helped to restore the same and sug- crucial role in downstream pathways of carcinogenesis gesting mitochondrial dysfunction (Fig. 8). MNU treat- and distinct tissue metastasis. Thus it will be worthy to ment validates the inflammation when perceived through identify the proteomic check point for the development increased expression of COX, NFκBp65 and UCHL-1. of innovative and safe therapeutic outcome to control Tadalafil treatment dose-dependently curtails down the and curing of malignancy. The present study demon- expression of COX, NFκBp65 and UCHL-1(Fig. 8). strated the repurposing effect of the PDE-5 inhibitor (Tadalafil) upon MNU induced mammary gland carcin- Discussion oma in albino wistar rats. MNU is one of the classical la- Despite vast development in the cumulative survival rate boratory carcinogens of alkyl-nitrosourea family which of hormone dependent breast cancer patients but post- has the ability to directly alkylate DNA [28]. MNU is a operative or chemotherapy received advanced metastatic widely used chemical carcinogenic model for induction stage still a positive life-threatening for cancer patient. of mammary gland carcinoma in albino wistar rats [14]. Most primitive challenges in mammary gland carcinoma Cumulative cardiotoxicity and autonomic dysfunction is Singh et al. BMC Cancer (2019) 19:996 Page 10 of 15 Fig. 7 Effect of Tadalafil upon various inflammatory and apoptotic markers. Groups are defined as: Control (Normal saline, 3 ml/kg, p.o.); Toxic control (MNU 47 mg/kg, i.v.); MNU + Tadalafil (47 mg/kg i.v. + 2 mg/kg p.o.) and MNU + Tadalafil (47 mg/kg i.v. + 4 mg/kg p.o.).Values were expressed as mean ± SD. Comparisons were made on the basis of one-way ANOVA followed by Bonferroni multiple test and all groups are compared to the toxic control group (*p < 0.05, **p < 0.01, ***p < 0.001) a well evident side effect of long term chemotherapy inflammation, coronary microcirculation it was an ur- regimen of cancer patients [29]. It ultimately hinders the gent need to evaluate the cardiological parameter during application of so many potent active chemotherapeutic the study and report safety of Tadalafil dose used agents. Being evidently well effective in pulmonary throughout the study. Reduced HRV constitutes an inde- arterial hypertension, pulmonary vasodilatation, cardiac pendent prognostic factor for autonomic dysfunction. It remodelling, diastolic function, interstitial fibrosis and was previously reported that HR was increased in breast Singh et al. BMC Cancer (2019) 19:996 Page 11 of 15 Fig. 8 Tadalafil mediated activation of mitochondrial mediated apoptosis pathway. Protein extracted from individual groups [Control (Normal saline, 3 ml/kg, p.o.); Toxic control (MNU 47 mg/kg, i.v.); MNU + Tadalafil (47 mg/kg i.v. + 2 mg/kg p.o.) and MNU + Tadalafil (47 mg/kg i.v. + 4 mg/ kg p.o.)] were subjected to immunoblotting of proapoptotic (BAD) and anti-apoptotic (Bcl-xl) marker along with COX, NFκBp65 and UCHL-1. β- actin was used as internal loading control. Each experiment was performed in triplicate. The data was represented as mean ± SD. The groups were significantly different by one-way ANOVA followed by Bonferroni multiple tests. All groups were compared to the toxic control group (*p < 0.05, **p < 0.01, ***p < 0.001) cancer patients [30].RR interval, R waves and HR are the distorted after MNU treatment, LCT and DCT were parameters which represent autonomic dysfunction. In hard to identify. After Tadalafil treatment, all the cell or- the present study, the RR interval, R waves and HR were ganelles were restored in dose dependent manner increased in MNU treated group which was decreased (Fig. 4a-d). Mammary gland tissues were also evaluated after Tadalafil treatment (Fig. 1). In the time domain, we for its surface texture analysis using SEM. After MNU analysed average RR, median RR, SDRR, CVRR and SD treatment, marked proliferation was observed with in- rate. All the cardiological parameters were significantly creased in micro vessel formation, loss of intra-arterial increased in MNU treated group and Tadalafil treatment cushion and vascular conglomeration. Tadalafil treat- helps normalised it. The reason to follow the main spec- ment restored the cellular architecture after SEM ana- tral components of LF is to analyse the frequency do- lysis (Fig. 5a-d). The finding from the morphological main and measure sympathetic and parasympathetic analysis revealed that Tadalafil in dose dependent man- activities. On the other hand, vagal parasympathetic activ- ner restored the features of cellular proliferation and ity and LF/HF ratio which indicates the sympathovagal warrants its role as an anti-carcinogenic drug. balance also examined. In MNU treated group; LF, HF Oxidative stress is a well-known fact in cancer pro- and LF/HF were increased which were subsided after gression. It is defined as a physiological state in which Tadalafil treatment (Fig. 2). In summary, PDE-5 inhibitor the level of ROS is higher and generation of free radical selectively exhibit pleiotropic actions through pulmonary is occurred. In mitochondria of the cell, ROS is gener- vasodilatation, LV remodelling, improved diastolic func- ated through adenosine triphosphate (ATP) where elec- tion, negative inotropic effects in naive cardiac myocytes, trons react with oxygen (O ) and results in the antiarrhythmic properties which ensure its long term formation of superoxide anion (O )[31]. Downstream safety in chemotherapeutic regimen. experimental condition reveals high level of NO in toxic The anti-carcinogenic activity of Tadalafil was again group which may be endogenous NO which inhibited evaluated on the grounds of morphological architecture cytochrome c oxidase, causing generation of excess ROS. using carmine staining, H&E staining and SEM analysis. Previous studies validated that oxidative stress is linked The results of carmine staining were in line with the with many of the pathophysiological diseases. Oxidative previous reported results [14]. Increased ABs, lobules stress leads to damaging of the DNA molecule and regu- and DF score is the sign for cellular proliferation and lates the progression of many cancers [32]. The markers the same was reported in MNU treated group [27]. The of oxidative stress are TBARs, PC, SOD, catalase and number of ABs, lobules and DF score were decreased GSH. In the present study, protein markers (TBARs and after Tadalafil treatment in dose dependent manner PC) were increased after MNU treatment and these (Fig. 3a-d). Histopathogical analysis of control group re- markers decreased after Tadalafil treatment in dose vealed the presence of CEC, MEC, lymphocytes, adipo- dependent manner (Fig. 6). The level of GSH was de- cytes, duct, LCT and DCT. All the cell organelles were creased in MNU treated group which was restored after Singh et al. BMC Cancer (2019) 19:996 Page 12 of 15 Tadalafil treatment. SOD and catalase are the major To establish possible mechanisms of the anticancer ef- component of antioxidant defence system and the level fects via PDE-5 inhibition and role in caspase-dependent of SOD and catalase was increased after MNU treatment apoptosis and cell growth arrest we have checked the which was normalised after Tadalafil treatment (Fig. 6). caspase group of protein. Caspase 3 and caspase 8 are Inflammatory markers also have a profound effect upon key regulators of the apoptotic response in which cas- cancer prognosis. Tadalafil is a well-known specific inhibi- pase 3 is executioner caspase and caspase 8 is initiator tor of the NO/cGMP pathway which has been perceived caspase. Caspase 8 is activated through self-cleavage and by NO [33]. Significant downregulation has been observed after that it activates downstream effector caspase like in the treatment groups which revealed Tadalafil mediated caspase 3 which [39] may be linked to concomitant in- inhibition of NO synthesis in tissue and inhibition of creases in regulation of downstream pathways through intracellular ROS generation [34]. Overexpression of JNK, mitogen-activated protein kinase 1, decreased cystathionine-gamma-lyase (CSE), acystathionine-beta- Wnt/β-catenin expression and down-regulation of cyclin synthase (CBS), and 3-mercaptopyruvate sulfurtransferase D, inhibition kinases 1/2 (ERK1/2) and alterations in the (3-MST) which ultimately leads to increased amounts of regulation of p42/p44 (MAPK) and p21 pathways. In the H S, which augmented tumour growth and distinct me- present study, the results from caspase assay revealed tastasis by activating cellular bioenergetics, proliferative, that after MNU treatment the level of caspase 3 and 8 migratory, and invasive signalling pathways [35]. Involve- was decreased and the same was restored after Tadalafil ment of NO-H S dual signalling is responsible for the cur- treatment (Fig. 7). tailment of ABC transported mediated drug resistance FAME analysis revealed the participation of saturated and inhibition of carcinogenesis via c-GMP blockade [36]. fatty acids, monounsaturated fatty acid, unsaturated fatty Tadalafil treatment downregulates the level of NO and acid and polyunsaturated fatty acids. It was reported that H S in MNU treated animals (Fig. 7). women’s consumed saturated fatty acid in diet have a Dual inhibition strategies for COX and LOX in cancer higher risk of breast cancer and the same observations are one of the established areas of research and our la- were found in the present study [40]. The sharp peaks of boratory is very much focused in the specific area of saturated fatty acid were seen in MNU group which was interest. Previously, we have shown that dual inhibition subsided after Tadalafil treatment. Monounsaturated (specific combinations of COX and LOX metabolites) fatty acid has both effects depending upon the source of has been considered for future best possible chemother- food. In MNU treated group, the level of monounsatu- apeutic regime [24]. Tadalafil treated group shows the rated fatty was increased and the same was decreased reduction of COX and LOX when examined through after Tadalafil treatment. It was previously reported that biochemical and immunoblotting assay in comparison unsaturated Trans fatty acid may increase the risk of in- with MNU treated group. Several research reports sug- vasive breast cancer and the same observations were gested that the expression of COX is associated with in- found in FAME analysis after MNU treatment [41]. cidence of breast cancer. It includes large tumour size, Various research reports validated the promoting as well positive axillary lymph node metastasis and HER2- as inhibitory effect of polyunsaturated fatty acid and positive tumour status [37]. Previous study on mice and mammary gland tumorigenesis [41]. In the resent piece rats that moderate to high COX expression is related to of work, inhibitory effects of polyunsaturated fatty acids generation of mammary gland tumours. The result of were seen after Tadalafil treatment (Additional file 1: present study also indicates the same (Fig. 7). The ex- Figure S1 and Additional file 2: Table S1). pression of COX in MNU treated group was increased Cell apoptosis is a process of programmed cell death which was subsided after Tadalafil treatment. Modula- and it is regulated by two major pathways: intrinsic and tion of downstream metabolite like PGE2, TXA2 and extrinsic pathways. The intrinsic pathway and Mitochon- TXB2 and LTA4, LTB4, LTC4, LTD4, LTE4 can ex- drial Outer Membrane Permeabilization (MOMP) are ploit multidirectional signalling cascade to exert their regulated through the balance of pro- and anti-apoptotic anticancer effects and the same have been explored in proteins of Bcl-2 family [42, 43]. In the present study, the present study with wider prospects to enlighten the expression of anti-apoptotic protein (Bcl-xl) was in- the chemotherapeutic strategies by repurposing the creased after MNU treatment and the vice versa effect mechanism of PDE-5 inhibitor. Dual inhibition en- was found upon pro-apoptotic protein (BAD). Collab- sures the alteration of AA metabolism in cancer cells orative result from pro-apoptotic and anti-apoptotic pro- and exhibits subsided inflammation, cell proliferation tein with downstream caspase cascade validate the and neo-angiogenesis. Identification of specific sub- participation of Tadalafil in mitochondrial mediated type of COX and LOX responsible for apotogenic po- death apoptosis pathway (Figs. 8 and 9). tential of Tadalafil is the future direction of our Previous reports revealed that the 26S centre of research [38]. UCHL-1 is regulated by 20S immunoproteasome subunit Singh et al. BMC Cancer (2019) 19:996 Page 13 of 15 Fig. 9 Protein-Protein interaction and Co-expression network analysis. Protein-protein interaction and co-expression analysis was performed using STRING V 10.0 with respect to differentially expressed proteins as identified from the Western blot analysis in relation to mitochondrial and hypoxic proteomic checkpoints which inhibits the degradation of the ubiquitinated pro- Conclusion teins [21]. In MNU treated group the expression of Authors would like to conclude that PDE-5 inhibitor UCHL-1 was increased which was subsided after Tadala- (Tadalafil) had a marked effect upon MNU induced fil treatment. The same relation has been linked with the mammary gland cancer. The mechanism of action of expression of NFκB/p50 precursor p105 and IκBα as it is Tadalafil is validated through mitochondria mediated essential for activity of NFκBp65. In line with to above death apoptosis pathway and Tadalafil also decreased results, the expression of NFκBp65 was increased in the inflammatory markers. Earlier, it was also reported MNU treated group which was decreased after Tadalafil that combination of sildenafil with celecoxib was found treatment (Figs. 8 and 9). to be cytotoxic in breast, hepatoma, colorectal cancer, Singh et al. BMC Cancer (2019) 19:996 Page 14 of 15 glioblastoma and medulloblastoma cell lines. It would be Author details Department of Pharmaceutical Sciences, Babasaheb Bhimrao Ambedkar helpful if well directed future preclinical and clinical University, (A Central University), Vidya Vihar, Raebareli road, Lucknow, UP study targeting the repurposing effect of PDE5 inhibitor, 2 226 025, India. Department of Pharmaceutical Sciences, King Faisal to explore its additional mechanism. Tadalafil has low- University, Al-Ahsa, Saudi Arabia. Department of Pharmacology, College of Pharmacy, Prince Sattam Bin Abdulaziz University, Al-Kharj, Kingdom of Saudi cost and low-toxicity with great preclinical potential Arabia. which can be further explored through well directed clinical trial in near future to include it with current and Received: 11 February 2019 Accepted: 11 September 2019 emerging standard of care treatments in breast cancer. References Supplementary information 1. Chang JC. Cancer stem cells: role in tumor growth, recurrence, metastasis, Supplementary information accompanies this paper at https://doi.org/10. and treatment resistance. Medicine. 2016;95(Suppl 1):S20–5. 1186/s12885-019-6152-9. 2. Kumazoe M, Kim Y, Bae J, Takai M, Murata M, Suemasu Y, Sugihara K, Yamashita S, Tsukamoto S, Huang Y. Phosphodiesterase 5 inhibitor acts as a Additional file 1: Figure S1. FAME analysis of the mammary gland potent agent sensitizing acute myeloid leukemia cells to 67-kDa laminin tissue subjected to MNU and Tadalafil. receptor-dependent apoptosis. FEBS Lett. 2013;587(18):3052–7. 3. Catalano S, Campana A, Giordano C, Győrffy B, Tarallo R, Rinaldi A, Bruno G, Additional file 2: Table S1. Fatty acid profiling of mammary gland Ferraro A, Romeo F, Lanzino M. Expression and function of tissue treated with MNU and Tadalafil. phosphodiesterase type 5 in human breast cancer cell lines and tissues: implications for targeted therapy. Clin Cancer Res. 2016;22(9):2271–82. Abbreviations 4. Weed DT, Vella JL, Reis IM, Adriana C, Gomez C, Sargi Z, Nazarian R, Califano 3-MST: 3-mercaptopyruvate sulfurtransferase; AA: Arachidonic acid; J, Borrello I, Serafini P. Tadalafil reduces myeloid-derived suppressor cells ABs: Alveolar buds; ATP: Adinosine triphosphate; BSA: Bovine serum albumin; and regulatory T cells and promotes tumor immunity in patients with head cAMP: Cyclic adenosine monophosphate; cGMP: Cyclic guanosine and neck squamous cell carcinoma. Clin Cancer Res. 2015;21(1):39–48. monophosphate; CBS: Acystathionine-beta-synthase; CEC: Cuboidal epithelial 5. Shi Z, Tiwari AK, Ashby Jr CR, Chen Z-S, Fu L-W: Anti-cancer Role of cell; COX: Cycloxygenase; CSE: Cystathionine-gamma-lyase; DCT: Dense Phosphodiesterase-5 Inhibitors. connective tissue; DF: Differentiation score; DTT: Dithiothreitol; 6. Thornton TM, Rincon M. Non-classical p38 map kinase functions: cell cycle ECG: Electrocardiogram; ERK: Extracellular-signal regulated kinases; checkpoints and survival. Int J Biol Sci. 2009;5(1):44. FAME: Fatty acid methyl esters; FTC: Ferrithiocyanate; GSH: Glutathione; 7. Serafini P, Meckel K, Kelso M, Noonan K, Califano J, Koch W, Dolcetti L, H&E: Hematoxyline and eosin; H S: Hydrogen sulphide; HBSS: Hank’s Bronte V, Borrello I. Phosphodiesterase-5 inhibition augments endogenous balanced salt solution; HR: Heart rate; HRV: Heart rate variability; antitumor immunity by reducing myeloid-derived suppressor cell function. J LOX: Lipoxygenase; MAPK: Mitogen activated-protin kinase; MDSCs: Myeloid Exp Med. 2006;203(12):2691–702. derived suppressor cells; MEC: Myoepithelail cell; MNU-n: Methyl-n- 8. Barone I, Giordano C, Bonofiglio D, Andò S, Catalano S. Phosphodiesterase nitrosourea; MOMP: Mitochondrial membrane permeabilization; NO: Nitric type 5 and cancers: progress and challenges. Oncotarget. 2017;8(58):99179. oxide; PC: Protein carbonyl; PDE: Phosphodiesterase; ROS: Reactive oxygen 9. Tiwari AK, Chen SZ. Repurposing phosphodiesterase-5 inhibitors as species; SEM: Scanning electron microscopy; SOD: Superoxide dismutase; chemoadjuvants. Front Pharmacol. 2013;4:82. https://doi.org/10.3389/fphar. TBARs: Thiobarbituric acid reactive substances; TMPD- N,N,N′N′: Tetramethyl- 2013.00082. p-phenylenediamine 10. Saravani R, Galavi HR, Shahraki A. Inhibition of Phosphodiesterase 5 and Increasing the Level of Cyclic Guanosine 3′,5′ Monophosphate by Hydroalcoholic Achillea wilhelmsii C. Koch Extract in Human Breast Cancer Acknowledgements Cell Lines MCF-7 and MDA-Mb-468. Breast Cancer Basic Clin Res. 2017;11: We appreciate the hard work of the colleagues at the university who provide us all the facilities for completion of the study. 11. Mishra RK, Sammi SR, Rawat JK, Roy S, Singh M, Gautam S, Yadav RK, Devi U, Ansari MN, Saeedan AS. Palonosetron attenuates 1, 2-dimethyl hydrazine Authors’ contributions induced preneoplastic colon damage through downregulating MS and SK carried out the bench work; SR performed the immunoblotting acetylcholinesterase expression and up-regulating synaptic acetylcholine assay; SA, ASS and NA perform the statistical analysis of the data; GK perceived concentration. RSC Adv. 2016;6(46):40527–38. the idea, designed and supervised the whole study, prepared and proof read 12. Kaithwas G, RAWAT JK, Roy S, Singh M, Gautam S, Yadav R, Ansari MN, the final manuscript. All authors have read and approved the manuscript. Aldossary SA, Saeedan AS. Transcutaneous vagus nerve stimulation regulates the cholinergic anti-inflammatory pathway to counteract 1, 2- Funding dimethyl hydrazine induced colon carcinogenesis in albino wistar rats. Front MS received fellowship from University Grants Commission New Delhi Pharmacol. 2019;10:353. (RGNF-2013-14-SC-UTT-38150). The fellowship helped MS to purchase all the 13. De Assis S, Warri A, Cruz MI, Hilakivi-Clarke L. Changes in mammary gland required chemicals and drugs specified in the present study. morphology and breast cancer risk in rats. J Vis Exp. 2010;44. https://doi.org/ 10.3791/2260. Availability of data and materials 14. Singh M, Devi U, Roy S, Gupta PS, Kaithwas G. Chemical activation of prolyl The datasets used and analysed during the current study are available from hydroxylase-2 by BBAP-1 down regulates hypoxia inducible factor-1α and the corresponding author on reasonable request. fatty acid synthase for mammary gland chemoprevention. RSC Adv. 2018; 8(23):12848–60. Ethics approval and consent to participate 15. Gautam S, Singh P, Singh M, Roy S, Rawat JK, Yadav RK, Devi U, Gupta PS, The study has been conducted according to the guidelines of CPCSEA for Saraf SA, Kaithwas G. Rifaximin, a pregnane X receptor (PXR) activator laboratory Animals and Ethics, Government of India (IAEC/SHIATS/PA16III/ regulates apoptosis in a murine model of breast cancer. RSC Adv. 2018;8(7): SSPG19). 3512–21. 16. Devi U, Singh M, Roy S, Tripathi AC, Gupta PS, Saraf SK, Ansari MN, Saeedan Consent for publication AS, Kaithwas G. PHD-2 activation: a novel strategy to control HIF-1α and Not applicable. mitochondrial stress to modulate mammary gland pathophysiology in ER+ subtype. Naunyn Schmiedeberg's Arch Pharmacol. 2019;392:1–18. Competing interests 17. Roy S, Rawat AK, Sammi SR, Devi U, Singh M, Gautam S, Yadav RK, Rawat JK, The authors declare that they have no competing interests. Singh L, Ansari MN. Alpha-linolenic acid stabilizes HIF-1 α and Singh et al. BMC Cancer (2019) 19:996 Page 15 of 15 downregulates FASN to promote mitochondrial apoptosis for mammary 40. Xia H, Ma S, Wang S, Sun G. Meta-analysis of saturated fatty acid intake and gland chemoprevention. Oncotarget. 2017;8(41):70049. breast cancer risk. Medicine. 2015;94(52):e2391. 18. Devi U, Singh M, Roy S, Gupta PS, Ansari MN, Saeedan AS, Kaithwas G. 41. MacLennan M, Ma DW. Role of dietary fatty acids in mammary gland Activation of prolyl hydroxylase-2 for stabilization of mitochondrial stress development and breast cancer. Breast Cancer Res. 2010;12(5):211. along with simultaneous downregulation of HIF-1α/FASN in ER+ breast 42. Klutzny S, Anurin A, Nicke B, Regan JL, Lange M, Schulze L, Parczyk K, cancer subtype. Cell Biochem Funct. 2019;37(4):216–27. Steigemann P. PDE5 inhibition eliminates cancer stem cells via induction of PKA signaling. Cell Death Dis. 2018;9(2):192. 19. Rani V, Gautam S, Rawat JK, Singh M, Devi U, Yadav RK, Roy S, Kaithwas G. Effects of minocycline and doxycycline against terbutaline induced early 43. Roy S, Singh M, Sammi SR, Pandey R, Kaithwas G. ALA-mediated biphasic downregulation of alpha-7nAchR/HIF-1alpha along with mitochondrial postnatal autistic changes in albino rats. Physiol Behav. 2018;183:49–56. stress modulation strategy in mammary gland chemoprevention. J Cell 20. Kaithwas G, Majumdar DK. In vitro antioxidant and in vivo antidiabetic, Physiol. 2019;234(4):4015–29. antihyperlipidemic activity of linseed oil against streptozotocin-induced toxicity in albino rats. Eur J Lipid Sci Technol. 2012;114(11):1237–45. 21. Rani A, Roy S, Singh M, Devi U, Yadav RK, Gautam S, Rawat JK, Ansari MN, Publisher’sNote Saeedan AS, Prakash A. α-Chymotrypsin regulates free fatty acids and UCHL- Springer Nature remains neutral with regard to jurisdictional claims in 1 to ameliorate N-methyl nitrosourea induced mammary gland carcinoma published maps and institutional affiliations. in albino wistar rats. Inflammopharmacology. 2016;24(5):277–86. 22. Tiwari V, Singh M, Rawat JK, Devi U, Yadav RK, Roy S, Gautam S, Saraf SA, Kumar V, Ansari N. Redefining the role of peripheral LPS as a neuroinflammatory agent and evaluating the role of hydrogen sulphide through metformin intervention. Inflammopharmacology. 2016;24(5):253–64. 23. Manral C, Roy S, Singh M, Gautam S, Yadav RK, Rawat JK, Devi U, Ansari MN, Saeedan AS, Kaithwas G. Effect of β-sitosterol against methyl nitrosourea- induced mammary gland carcinoma in albino rats. BMC Complement Altern Med. 2016;16(1):260. 24. Gautam S, Rawat AK, Sammi SR, Roy S, Singh M, Devi U, Yadav RK, Singh L, Rawat JK, Ansari MN. DuCLOX-2/5 inhibition attenuates inflammatory response and induces mitochondrial apoptosis for mammary gland chemoprevention. Front Pharmacol. 2018;9:314. 25. Yadav S, Tiwari V, Singh M, Yadav RK, Roy S, Devi U, Gautam S, Rawat JK, Ansari MN, Saeedan AS. Comparative efficacy of alpha-linolenic acid and gamma-linolenic acid to attenuate valproic acid-induced autism-like features. J Physiol Biochem. 2017;73(2):187–98. 26. Kruger NJ. 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Tseng H-S, Chen L-S, Kuo S-J, Chen S-T, Wang Y-F, Chen D-R. Tumor characteristics of breast cancer in predicting axillary lymph node metastasis. Med Sci Monit. 2014;20:1155. 38. Gautam S, Roy S, Ansari MN, Saeedan AS, Saraf SA, Kaithwas G. DuCLOX-2/5 inhibition: a promising target for cancer chemoprevention. Breast Cancer. 2017;24(2):180–90. 39. Bell RA, Megeney LA. Evolution of caspase-mediated cell death and differentiation: twins separated at birth. Cell Death Differ. 2017;24(8):1359. http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png BMC Cancer Springer Journals

Repurposing mechanistic insight of PDE-5 inhibitor in cancer chemoprevention through mitochondrial-oxidative stress intervention and blockade of DuCLOX signalling

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Springer Journals
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Copyright © The Author(s). 2019
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Biomedicine; Cancer Research; Oncology; Surgical Oncology; Health Promotion and Disease Prevention; Biomedicine, general; Medicine/Public Health, general
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Abstract

Background: This study evaluates the anti-cancer effects of Tadalafil (potent PDE-5 inhibitor) in female albino wistar rats against n-methyl n-nitrosourea induced mammary gland carcinogenesis. Methods: The animals were selected and randomly divided among four groups and each group contains six animals per group. The animal tissue and serum samples were evaluated for the presence of antioxidant parameters and the cellular morphology was studied using carminic staining, haematoxylin staining and scanning electron microscopy followed by immunoblotting analysis. Results: On the grounds of hemodynamic recordings and morphology, n-methyl n-nitrosourea treated group showed distorted changes along with distorted morphological parameters. For morphological analysis, the mammary gland tissues were evaluated using scanning electron microscopy, whole mount carmine staining, haematoxylin and eosin staining. The serum samples were evaluated for the evaluation of oxidative stress markers and inflammatory markers. The level of caspase 3 and 8 were also evaluated for the estimation of apoptosis. The fatty acid profiling of mammary gland tissue was evaluated using fatty acid methyl esters formation. The mitochondrial mediated apoptosis and inflammatory markers were evaluated using immunoblotting assay. Conclusion: The results confirm that Tadalafil treatment restored all the biological markers to the normal and its involvement in mitochondrial mediated death apoptosis pathway along with inhibition of inflammatory markers. Keywords: Tadalafil, ER+ mammary gland cancer, Mitochondrial stress, PDE-5 inhibitor, N-methyl-n-nitrosourea, DuCLOX Background nowadays [1]. Tadalafil is a known phosphodiesterase-5 Distinct metastasis and resistance development by self- (PDE-5) inhibitor, which have anti-cancer effects in vari- renewing nascent cancer cells in response with the ous types of cancer [2]. In the search of molecularly tar- chemotherapy drug regimen and simultaneous radiations geted compounds that have the structure similarity with are still a main obstacle in cancer treatment for patients the known anti-cancerous drugs like viscristine, vinblas- tin, vindesine, vinorelbine, leuprolide and geserelin got tremendous attention due to the expression and regula- * Correspondence: gauravpharm@hotmail.com; gauravk@bbau.ac.in tion of the important signalling pathways [3]. In the last Manjari Singh and Sweta Kasna contributed equally to this work. decade, Tadalafil used to inhibit the myeloid derived Department of Pharmaceutical Sciences, Babasaheb Bhimrao Ambedkar University, (A Central University), Vidya Vihar, Raebareli road, Lucknow, UP suppressor cells (MDSCs) and restore the T cells in can- 226 025, India cer patients as compared to the normal peoples [4]. Full list of author information is available at the end of the article © The Author(s). 2019 Open 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. Singh et al. BMC Cancer (2019) 19:996 Page 2 of 15 PDE-5, PDE-6, PDE-9 are the predominant active iso- Sciences, Naini Allahabad, India. All animals were ran- form of ubiquitously distributed metallohydrolases con- domized among four groups having eight animals each. stitute of 11 distinct gene families, which initiate the Carcinogenesis was induced by single tail vein injection of st sequential lcleavage of cyclic adenosine monophosphate MNU on day 1 in each animal. MNU was dissolved im- (cAMP) and cyclic guanosine monophosphate (cGMP) mediately before use in glacial acetic acid and water (pH into their intermediate inactive 50 derivatives and 5- 4.5–5). The experimental groups were randomized as fol- GMP. It ultimately regulating the amplitude and dur- lows: group I (normal control, saline 3 ml/kg, p.o.; group ation of their intracellular downstream signaling mech- II (toxic control, MNU 47 mg/kg, i.v.); group III (Tadalafil anism [5]. This inhibition of cGMP conversion leads to 2 mg/kg, p.o. + MNU 47 mg/kg, i.v.) and group IV (Tada- activate protein kinase G and map kinase pathway of lafil 4 mg/kg, p.o. + MNU 47 mg/kg, i.v). Tadalafil was ad- apoptosis and also simultaneously affect cancer cell ministered from 7th to 110th day at the dose specified growth and adhesion, mitochondrial energy homeostasis, above. At the end of study, animals were sacrificed for the neuronal signaling and muscle relaxation in systemic collection of mammary gland tissue under ketamine hydro- vasculature, prostate, heart, brain, lungs and platelets chloride (100 mg/kg, i.m.) and diazepam (5 mg/kg, i.m.) [6]. In previous literature, it was reported that PDE-5 in- combination as anaesthesia. hibition produced caspase dependent apoptosis of B-cell All the animal experimental procedures follow the AR- chronic lymphocytic leukemic cells [7]. Various prelim- RIVE guidelines. inarily studies also demonstrated that PDE-5 inhibitors can induce apoptosis by activation of cGMP and nitric Changes in hemodynamic recordings oxide (NO) in cancerous cell [8]. In recent findings, in- The hemodynamic changes were recorded using the Lab dole derivatives have the potency to treat mammary Chart Pro-8 (AD Instruments, Australia) instrument accord- gland cancer and Tadalafil also have the indole moiety ing to the methods elaborated by us previously [11, 12]. in its structure, which ultimately reveals its molecular mechanism [9]. It was reported that the expression of Effect of Tadalafil upon morphological changes PDE-5 was significantly high in the malignant breast tu- Whole mount alum stain mors as compare with normal breast tissues and benign Rats were sacrificed after completions of study and forth tumors [10]. Consistently increased PDE-5 expression mammary gland tissues were excised and fixed for a has also been reported in a variety of mammary gland minimum of 2 days in Carnoy’s fixative solution followed cancer cell lines (MCF-7, T47D, HTB-26, MDA-MB- by staining with carmine solution (1 g carmine and 2.5 g 231), which gives the rationale to assess the anticancer aluminum potassium sulfate in 500 ml water). The fixed effects of PDE-5 inhibitors in carcinogen induced animal glands were hydrated with 90, 70, 35 and 15% ethyl alco- model and check the involvement of reactive oxygen hol and then rinsed with water for 5 min. The glands species (ROS), death receptor and mitochondrial signal- were then dehydrated progressively in 35, 70, 95 and ling as part of the combinatorial apoptosis mechanism. 100% ethanol, dipped in xylene for 2 days and mounted Based on the previous literature, we hypothesize to on glass slides. The sections were examined for the iden- evaluate the effect of tadalafil upon carcinogen induced tification of angiogenesis [13, 14]. ER+ mammary gland carcinoma in vivo model. Histopathological studies Methods Thin section (5 μm) of mammary glands tissue was cut Drugs and chemicals using microtome and fixed overnight in 10% hank’s bal- Tadalafil (API) was procured from the Sanofi Aventis as a anced salt solution (HBSS) buffered formalin which fur- gift sample followed by FTIR analysis to establish its purity. ther used for representative gross morphological and N-methyl-n-nitrosourea (MNU) (Sigma-Aldrich, N1517) histological features of haematoxylin and eosin (H&E) was procured from Sigma Life Science Aldrich Co. 3050 stained paraffin sections of the mammary glands tissue Spruce Street, St. Louis, USA. Other chemicals were pur- [15, 16]. chased from Himedia Pvt. Ltd., Sigma Aldrich and Amresco. Scanning electron microscopy (SEM) In vivo study Sodium cacodylate and HBSS buffer based HCl collage- The study protocol was approved from CPCSEA guide- nase hyaluronic acid enzymatic digestion method was lines for laboratory Animals and Ethics, Government of used for preparation of tissue before SEM analysis. The India (IAEC/SHIATS/PA16III/SSPG19). Female albino entire methodology was performed according to wistar rats of 100-120 g were used for this study and methods elaborated by us previously [17]. The sample collected from the central animal house facility of Sam was dried with increasing grade of acetone (70, 80, 90 Higginbottom University of Agriculture, Technology and and 100%). All the acetone specimens were dehydrated Singh et al. BMC Cancer (2019) 19:996 Page 3 of 15 and replaced by exchange of soak/flash liquid CO fluids Gas chromatographic (GC) analysis of mammary gland with ethanol or acetone by critical point method (31 °C tissue and 74 bar). Samples were further processed by platinum The fatty acid methyl esters (FAME) analysis was per- coating, which has been examined under SEM(X1000) formed for the estimation of free fatty acids in the mam- (JEOL JSM-6490LV) [15, 18]. mary gland tissue. The methodology of FAME was elaborated elsewhere [25]. Effect of Tadalafil upon oxidative stress markers Western blotting The antioxidant parameters were evaluated using tissue The mammary gland tissue sample was homogenized homogenate (10%w/v). The homogenate was prepared in using RIPA lysis buffer, isolated by acetone precipitation 0.15 M KCl and the same was centrifuged for 15 min at and total protein concentration was assayed by using 4 °C. In thiobarbituric acid reactive substances (TBARs), Bradford method [26]. All the protein samples were re- malondialdehyde (MDA) forms a 1:2 adduct with TBAR solved on SDS-PAGE gels with Tris buffer system. The and produce the reaction which can be measured by proteins were transferred to PVDF membrane by elec- spectrophotometer at 540 nm. For superoxide dismutase troblotting by semidry system. Prestained protein ladder (SOD), the inhibition of pyrogallol auto-oxidation was was used as a molecular weight identification marker. measured. The absorbance was measured at 420 nm. After 2 h of blocking with blocking buffer at room Catalase catalyzes the decomposition of hydrogen perox- temperature, membranes were processed for primary ide to water and oxygen. The absorbance was measured antibody binding (1: 2000 dilutions) against Bcl-xl (MA- at 250 nm. Glutathione was measured using Ellamanis 5-15,142), BAD (SC-8044), NFκBp65 (MA5–1616), reagent (DTNB), when DTNB reacts with GSH then 5 UCHL-1 (MA1–83428) and COX (MA5–14568) in 4 °C thionitrobenzoic acid (TNB) and GS-TNB was formed. followed by respective secondary antibody (anti-rabbit The absorbance was measured at 410 nm [19, 20]. (SC-2030), anti-goat (SC-2020) and anti-mouse (31,430, Pierce Thermo Scientific, USA) in 1:5000 dilutions. β- actin (MA5–15739-HRP) was used as loading control. Serum NO level The PVDF blotting membranes were processed at final For NO estimation, same proportion of serum isolated steps by using an enhanced ECL substrate (Western from animal blood and Griess reagent were mixed and Bright ECL HRP substrate, Advansta, Melanopark, Cali- incubated at 37 °C for 30 min. The reaction was pro- fornia, US) in Geldock system. The proteins bands were ceeded at room temperature, and the absorbance was quantified using ImageJ software [27]. read at 540 nm using UV-visible spectrophotometer (Cary 60, Agilent Technologies, CA, 95051, US). The standard curve was plot using serial dilutions of sodium Digital image analysis nitrite [21]. For microscopic analysis data obtained from H&E and SEM micrographs, six randomly chosen regions were se- lected from all group and analysed independently for Estimation of serum hydrogen sulphide (H S) level each respective sample. To identify the luminescence For H S estimation, 500 μl of tissue supernatant and from experimental images and convert them into the 75 μl of serum sample was added to the premixed zinc heights to the final surface plot,3D interactive surface acetate (1%w/v) solution, respectively. The concentration plot experiments was used to identify the pure H&E of H S in the serum sample was measured using the stained and SEM areas nearest neighbouring sampling in method elaborated by us previously. The absorbance was a square plot. This unmixing ultimately helps to distin- read spectrophotometrically at 670 nm [22]. guish between nuclear, ECM and background stains in a single complimentary image. Automated score was assigned by observing and measuring the pure H&E Cyclooxygenase (COX) and lipoxygenase (LOX) estimation staining pattern and SEM micrograph in Image J stand- The COX and LOX were estimated in the rat serum sam- ard program feature. ple followed by the method elaborated elsewhere [23]. Protein–protein interaction and gene ontological studies Caspase3 and caspase8 estimation To identify the various functional relations between dif- 96-well amber colored plate has been used for the col- ferent gene and protein protein-protein interactions orimetric identification of caspase-3/8. The estimation were studied using STRING 10.0. Different biological was performed on the basis of methodology elaborated process, cellular component and biological functions has by us previously [24]. been identified by gene ontological study (Search tool Singh et al. BMC Cancer (2019) 19:996 Page 4 of 15 for the Retrieval of Interacting Genes/Proteins) software declined in toxic control group (0.05 ± 0.01) which was (http://string-db.org). restored after Tadalafil treatment (0.06 ± 0.01) (Fig. 1). In accordance with HRV parameters, the frequency Statistical analysis domain parameters like average RR, median RR, SDRR All data were presented as mean ± SD and analyzed by and SD rate were elevated after MNU treatment. Tadala- one way ANOVA followed by Bonferroni’s multiple fil treatment helped to restore the above said parameters comparison tests for the possible significance identifica- about to normal (Fig. 2). tion between the various groups *p < 0.05, **p < 0.01, ***p < 0.001 were considered statistically significant. Stat- Morphological analysis of rat mammary gland tissue istical analysis was carried out using Graph Pad Prism Carmine staining (5.01), San Diego, California. The two main markers for angiogenesis are alveolar bud (ABs) count and differentiation (DF) score. In carmine Results staining, the no. of ABs and DF score were calculated. The Effect of Tadalafil upon hemodynamic changes ABs (both AB1 and AB2), lobules and DF were elevated Heart rate variability (HRV) is the noninvasive measures after MNU treatment (17 ± 5.65, 12 ± 5.65, 12 ± 2.82 and of autonomic nervous system (ANS) regulation. It was 41 ± 14.12) and after Tadalafil treatment; all the parameters previously reported that altered and dysfunctional ANS of cell proliferation were restored to normal (Fig. 3a-d). has been reported cancer patients. The RR interval was elevated in MNU treated group (0.18 ± 0.02) and Tadala- H&E staining fil in low dose (2 mg/kg, p.o.) restored RR interval about H&E staining provide the cellular and morphological alter- to normal (0.16 ± 0.01). On the other hand, heart rate ations occur in the tissue. In normal control tissues, all the (HR) was increased in MNU treated group (331.95 ± cell organelles were clearly visible (Fig. 4a). MNU treatment 0.68) which was diminished after Tadalafil treatment in was evident in scattered pattern of cuboidal epithelial cells dose dependent manner. QT interval was considerably (CEC), myoepithelial cells (MEC), lymphocytes, adipocytes Fig. 1 Effect of Tadalafil treatment on ECG recording. Representative box-cum-whisker plots showing quantitative variations of relative signal integrals for autonomic dysfunction relevant in the context of pathophysiology of mammary gland cancer. Groups were differentiated as: 1- Control (Normal saline, 3 ml/kg, p.o.), 2-Toxic control (MNU 47 mg/kg, i.v.), 3- MNU + Tadalafil (47 mg/kg i.v. + 2 mg/kg p.o.), 4- MNU + Tadalafil (47 mg/kg i.v. + 4 mg/kg p.o.). For presented ECG recordings, the VIP score > 1 and statistical significance is at the level of p ≤ 0.05. In the box plots, th the boxes denote interquartile ranges, horizontal line inside the box denote the median, and bottom and top boundaries of boxes are 25 and th 75th percentiles, respectively. Lower and upper whiskers are 5 and 95th percentiles, respectively Singh et al. BMC Cancer (2019) 19:996 Page 5 of 15 Fig. 2 Effects of Tadalafil treatment on HRV. Representative box-cum-whisker plots showing quantitative variations of relative signal integrals for HRV parameters relevant in the context of pathophysiology of mammary gland cancer. Groups were differentiated as: 1-Control (Normal saline, 3 ml/kg, p.o.), 2-Toxic control (MNU 47 mg/kg, i.v.), 3- MNU + Tadalafil (47 mg/kg i.v. + 2 mg/kg p.o.), 4- MNU + Tadalafil (47 mg/kg i.v. + 4 mg/kg p.o.). For presented heart rate variability, the VIP score > 1 and statistical significance is at the level of p ≤ 0.05. In the box plots, the boxes denote th interquartile ranges, horizontal line inside the box denote the median, and bottom and top boundaries of boxes are 25 and 75th percentiles, th respectively. Lower and upper whiskers are 5 and 95th percentiles, respectively andductalong with looseconnectivetissue(LCT) and (499.79 ± 109.2298 nM of MDA/μg of protein and dense connective tissues (DCT) (Fig. 4b). Tadalafil treat- 7.28 ± 0.82 nM/ml). GSH level in MNU treated group ment restored all the parameters in dose dependent man- (0.61 ± 0.04 mg %) was restored to almost control group ner (Fig. 4cand d). after Tadalafil treatment (1.39 ± 0.10 mg %). The level of SOD and catalase was increased after MNU treatment SEM analysis (0.33 ± 0.07 units of SOD/mg of protein and 2.46 ± 0.48 SEM analysis was used to analyse the internal surface of nM of H O /min/mg of protein) which was decreased 2 2 whole cells and its organelles. In normal control tissue, after Tadalafil treatment to a significant level (i.e. 0.17 ± collagen layer, duct and intra-arterial cushion/collage- 0.13 units of SOD and 1.43 ± 0.01 nM of H O ) (Fig. 6). 2 2 nous covering, were seen clearly (Fig. 5a). All the cellular organelles were absent in MNU treated group (Fig. 5b). Effect of inflammatory markers upon mammary gland Tadalafil treatment perceived decrease in tumor micro- carcinoma vessel formation with restoration of intra-arterial cush- Inflammatory markers produced reactive nitrogen spe- ion (Fig. 5c and d). cies (RNS) and ROS from epithelial and inflammatory cells, which leads to DNA damage. The inflammatory Effect of antioxidant markers upon mammary gland markers (NO, H S, COX and LOX) were upregulated carcinoma after MNU treatment. After Tadalafil treatment, the Reactive oxygen species (ROS) are the byproducts of level of all the inflammatory markers was downregulated normal metabolism through the electron transport (Fig. 7). chain. ROS and other oxidative stress are considered as a driving force for cancer progression. Tadalafil treat- ment helps to restore the diminished antioxidant profile, Effect of Tadalafil upon caspase assay when compared with MNU treated toxic group. The Caspases cysteinyl protease family members and they phenomena of protein and lipid peroxidation were very play a crucial role in apoptosis. The serum caspase 3 and well evident after MNU treatment which correlates with caspase 8 was measured using fluorometric assay kits. the high level of TBARs and PC after MNU treatment The caspase 3 and caspase 8 levels were decreased in (872.12 ± 72.98 nM of MDA/μg of protein and 12.24 ± MNU treated group and after Tadalafil treatment, the 1.79 nM/ml respectively). Tadalafil treatment decreased levels of casapse 3 and caspase 8 were upregulated in a the level of TBARs and PC in dose dependent manner dose dependent manner (Fig. 7). Singh et al. BMC Cancer (2019) 19:996 Page 6 of 15 Fig. 3 (See legend on next page.) Singh et al. BMC Cancer (2019) 19:996 Page 7 of 15 (See figure on previous page.) Fig. 3 Carmine staining of mammary gland whole mount tissue. Whole mount carmine alum staining of ductal epithelium reveals the presence of AB1 (1), AB2 (2) and lobules (3) in respective groups [a-control (normal saline, 3 ml/kg, p.o.); b-toxic control (MNU 47 mg/kg, i.v.); c (MNU + Tadalafil; 47 mg/kg i.v. + 2 mg/kg p.o.) and d (MNU + Tadalafil; 47 mg/kg i.v. + 4 mg/kg p.o.)]. The extent of AB and lobules formation is excessive in the toxic group (b) which was subsided after Tadalafil treatment (c & d). The extent of AB and lobules formation is excessive in the toxic group (b) which has been subsided with respective treatment groups (c & d). The ABs score, lobules and DF scores were also higher in toxic control which was subsided after Tadalafil treatment in a dose dependent manner Fig. 4 H&E analysis and digital image analysis of mammary gland tissue. H&E staining of four individual groups [a-control (normal saline, 3 ml/kg, p.o.); b-toxic control (MNU 47 mg/kg, i.v.); c (MNU + Tadalafil; 47 mg/kg i.v. + 2 mg/kg p.o.) and d (MNU + Tadalafil; 47 mg/kg i.v. + 4 mg/kg p.o.)] revealed presence of adipocytes (4), duct (5) LCT (6), DCT (7), lymphocytes (8), CEC (9) and MEC (10) in control as well as Tadalafil treated groups (a, c and d). In the toxic control group (b), the cell morphology was distorted, and cell organelles were absent. 3D image reconstruction and software-based analysis dataset of constructs representing score was done by using Image J (NIH) software by thresh holding of stained zones of H&E images followed by pixel vs intensity determination by the 3D interactive surface plot and log-histogram analysis (e,f,g,h and i,j,k,l). Singh et al. BMC Cancer (2019) 19:996 Page 8 of 15 Fig. 5 SEM and digital image analysis of mammary gland tissue. SEM analysis of four individual groups [a-control (normal saline, 3 ml/kg, p.o.); b- toxic control (MNU 47 mg/kg, i.v.); c (MNU + Tadalafil; 47 mg/kg i.v. + 2 mg/kg p.o.) and d (MNU + Tadalafil; 47 mg/kg i.v. + 4 mg/kg p.o.)] was performed. Control (a) demonstrated presence of collagenous layers (11), duct (12), nodules (13) and small capillary network (14). MNU (b) administration perceived loss of collagenous covering (11) and formation of tumormicrovessels/small capillary network (14) and nodules (13). Tadalafil treatment restores all the cell organelles close to normal. 3D image reconstruction and software-based analysis dataset of constructs representing score was done by using Image J (NIH) software by thresh holding of stained zones of H&E images followed by pixel vs intensity determination by the 3D interactive surface plot and log-histogram analysis (e,f,g,h and i,j,k,l) Effect of Tadalafil upon FAME analysis MNU treated animals (Additional file 1:FigureS1 FAME assay converts cellular lipids into fatty acid and Additional file 2:Table S1). methyl esters, resolved and identified by gas chroma- tography. After MNU treatment, there was substan- Immunoblotting analysis tial increase in saturated fatty acid, monounsaturated Western blotting is used to separate and identify proteins. fatty acid along with decrease in the unsaturated In this technique a mixture of proteins is separated on the fatty acid and polyunsaturated fatty acid content. basis of molecular weight through gel electrophoresis. Bcl- Tadalafil treatment restored the lipid content in xl (anti-apoptotic) expression was increased after MNU Singh et al. BMC Cancer (2019) 19:996 Page 9 of 15 Fig. 6 Effect of Tadalafil on the antioxidant markers. Bar diagram represents the difference between the control, toxic and Tadalafil treated groups. The markers of protein and lipid (PC and TBARs) were increased in MNU treated group and reduced after Tadalafil treatment. GSH was decreased after MNU treatment whereas the level of SOD and catalase were increased after MNU treatment which was subsided after Tadalafil treatment. All the data was presented as mean ± SD. Comparisons were made on the basis of one-way ANOVA followed by Bonferroni multiple test and all groups are compared to the toxic control group (*p < 0.05, **p < 0.01, ***p < 0.001) treatment with diminished level of BAD (proapoptotic). are to target different key proteomic marker that plays a Tadalafil treatment helped to restore the same and sug- crucial role in downstream pathways of carcinogenesis gesting mitochondrial dysfunction (Fig. 8). MNU treat- and distinct tissue metastasis. Thus it will be worthy to ment validates the inflammation when perceived through identify the proteomic check point for the development increased expression of COX, NFκBp65 and UCHL-1. of innovative and safe therapeutic outcome to control Tadalafil treatment dose-dependently curtails down the and curing of malignancy. The present study demon- expression of COX, NFκBp65 and UCHL-1(Fig. 8). strated the repurposing effect of the PDE-5 inhibitor (Tadalafil) upon MNU induced mammary gland carcin- Discussion oma in albino wistar rats. MNU is one of the classical la- Despite vast development in the cumulative survival rate boratory carcinogens of alkyl-nitrosourea family which of hormone dependent breast cancer patients but post- has the ability to directly alkylate DNA [28]. MNU is a operative or chemotherapy received advanced metastatic widely used chemical carcinogenic model for induction stage still a positive life-threatening for cancer patient. of mammary gland carcinoma in albino wistar rats [14]. Most primitive challenges in mammary gland carcinoma Cumulative cardiotoxicity and autonomic dysfunction is Singh et al. BMC Cancer (2019) 19:996 Page 10 of 15 Fig. 7 Effect of Tadalafil upon various inflammatory and apoptotic markers. Groups are defined as: Control (Normal saline, 3 ml/kg, p.o.); Toxic control (MNU 47 mg/kg, i.v.); MNU + Tadalafil (47 mg/kg i.v. + 2 mg/kg p.o.) and MNU + Tadalafil (47 mg/kg i.v. + 4 mg/kg p.o.).Values were expressed as mean ± SD. Comparisons were made on the basis of one-way ANOVA followed by Bonferroni multiple test and all groups are compared to the toxic control group (*p < 0.05, **p < 0.01, ***p < 0.001) a well evident side effect of long term chemotherapy inflammation, coronary microcirculation it was an ur- regimen of cancer patients [29]. It ultimately hinders the gent need to evaluate the cardiological parameter during application of so many potent active chemotherapeutic the study and report safety of Tadalafil dose used agents. Being evidently well effective in pulmonary throughout the study. Reduced HRV constitutes an inde- arterial hypertension, pulmonary vasodilatation, cardiac pendent prognostic factor for autonomic dysfunction. It remodelling, diastolic function, interstitial fibrosis and was previously reported that HR was increased in breast Singh et al. BMC Cancer (2019) 19:996 Page 11 of 15 Fig. 8 Tadalafil mediated activation of mitochondrial mediated apoptosis pathway. Protein extracted from individual groups [Control (Normal saline, 3 ml/kg, p.o.); Toxic control (MNU 47 mg/kg, i.v.); MNU + Tadalafil (47 mg/kg i.v. + 2 mg/kg p.o.) and MNU + Tadalafil (47 mg/kg i.v. + 4 mg/ kg p.o.)] were subjected to immunoblotting of proapoptotic (BAD) and anti-apoptotic (Bcl-xl) marker along with COX, NFκBp65 and UCHL-1. β- actin was used as internal loading control. Each experiment was performed in triplicate. The data was represented as mean ± SD. The groups were significantly different by one-way ANOVA followed by Bonferroni multiple tests. All groups were compared to the toxic control group (*p < 0.05, **p < 0.01, ***p < 0.001) cancer patients [30].RR interval, R waves and HR are the distorted after MNU treatment, LCT and DCT were parameters which represent autonomic dysfunction. In hard to identify. After Tadalafil treatment, all the cell or- the present study, the RR interval, R waves and HR were ganelles were restored in dose dependent manner increased in MNU treated group which was decreased (Fig. 4a-d). Mammary gland tissues were also evaluated after Tadalafil treatment (Fig. 1). In the time domain, we for its surface texture analysis using SEM. After MNU analysed average RR, median RR, SDRR, CVRR and SD treatment, marked proliferation was observed with in- rate. All the cardiological parameters were significantly creased in micro vessel formation, loss of intra-arterial increased in MNU treated group and Tadalafil treatment cushion and vascular conglomeration. Tadalafil treat- helps normalised it. The reason to follow the main spec- ment restored the cellular architecture after SEM ana- tral components of LF is to analyse the frequency do- lysis (Fig. 5a-d). The finding from the morphological main and measure sympathetic and parasympathetic analysis revealed that Tadalafil in dose dependent man- activities. On the other hand, vagal parasympathetic activ- ner restored the features of cellular proliferation and ity and LF/HF ratio which indicates the sympathovagal warrants its role as an anti-carcinogenic drug. balance also examined. In MNU treated group; LF, HF Oxidative stress is a well-known fact in cancer pro- and LF/HF were increased which were subsided after gression. It is defined as a physiological state in which Tadalafil treatment (Fig. 2). In summary, PDE-5 inhibitor the level of ROS is higher and generation of free radical selectively exhibit pleiotropic actions through pulmonary is occurred. In mitochondria of the cell, ROS is gener- vasodilatation, LV remodelling, improved diastolic func- ated through adenosine triphosphate (ATP) where elec- tion, negative inotropic effects in naive cardiac myocytes, trons react with oxygen (O ) and results in the antiarrhythmic properties which ensure its long term formation of superoxide anion (O )[31]. Downstream safety in chemotherapeutic regimen. experimental condition reveals high level of NO in toxic The anti-carcinogenic activity of Tadalafil was again group which may be endogenous NO which inhibited evaluated on the grounds of morphological architecture cytochrome c oxidase, causing generation of excess ROS. using carmine staining, H&E staining and SEM analysis. Previous studies validated that oxidative stress is linked The results of carmine staining were in line with the with many of the pathophysiological diseases. Oxidative previous reported results [14]. Increased ABs, lobules stress leads to damaging of the DNA molecule and regu- and DF score is the sign for cellular proliferation and lates the progression of many cancers [32]. The markers the same was reported in MNU treated group [27]. The of oxidative stress are TBARs, PC, SOD, catalase and number of ABs, lobules and DF score were decreased GSH. In the present study, protein markers (TBARs and after Tadalafil treatment in dose dependent manner PC) were increased after MNU treatment and these (Fig. 3a-d). Histopathogical analysis of control group re- markers decreased after Tadalafil treatment in dose vealed the presence of CEC, MEC, lymphocytes, adipo- dependent manner (Fig. 6). The level of GSH was de- cytes, duct, LCT and DCT. All the cell organelles were creased in MNU treated group which was restored after Singh et al. BMC Cancer (2019) 19:996 Page 12 of 15 Tadalafil treatment. SOD and catalase are the major To establish possible mechanisms of the anticancer ef- component of antioxidant defence system and the level fects via PDE-5 inhibition and role in caspase-dependent of SOD and catalase was increased after MNU treatment apoptosis and cell growth arrest we have checked the which was normalised after Tadalafil treatment (Fig. 6). caspase group of protein. Caspase 3 and caspase 8 are Inflammatory markers also have a profound effect upon key regulators of the apoptotic response in which cas- cancer prognosis. Tadalafil is a well-known specific inhibi- pase 3 is executioner caspase and caspase 8 is initiator tor of the NO/cGMP pathway which has been perceived caspase. Caspase 8 is activated through self-cleavage and by NO [33]. Significant downregulation has been observed after that it activates downstream effector caspase like in the treatment groups which revealed Tadalafil mediated caspase 3 which [39] may be linked to concomitant in- inhibition of NO synthesis in tissue and inhibition of creases in regulation of downstream pathways through intracellular ROS generation [34]. Overexpression of JNK, mitogen-activated protein kinase 1, decreased cystathionine-gamma-lyase (CSE), acystathionine-beta- Wnt/β-catenin expression and down-regulation of cyclin synthase (CBS), and 3-mercaptopyruvate sulfurtransferase D, inhibition kinases 1/2 (ERK1/2) and alterations in the (3-MST) which ultimately leads to increased amounts of regulation of p42/p44 (MAPK) and p21 pathways. In the H S, which augmented tumour growth and distinct me- present study, the results from caspase assay revealed tastasis by activating cellular bioenergetics, proliferative, that after MNU treatment the level of caspase 3 and 8 migratory, and invasive signalling pathways [35]. Involve- was decreased and the same was restored after Tadalafil ment of NO-H S dual signalling is responsible for the cur- treatment (Fig. 7). tailment of ABC transported mediated drug resistance FAME analysis revealed the participation of saturated and inhibition of carcinogenesis via c-GMP blockade [36]. fatty acids, monounsaturated fatty acid, unsaturated fatty Tadalafil treatment downregulates the level of NO and acid and polyunsaturated fatty acids. It was reported that H S in MNU treated animals (Fig. 7). women’s consumed saturated fatty acid in diet have a Dual inhibition strategies for COX and LOX in cancer higher risk of breast cancer and the same observations are one of the established areas of research and our la- were found in the present study [40]. The sharp peaks of boratory is very much focused in the specific area of saturated fatty acid were seen in MNU group which was interest. Previously, we have shown that dual inhibition subsided after Tadalafil treatment. Monounsaturated (specific combinations of COX and LOX metabolites) fatty acid has both effects depending upon the source of has been considered for future best possible chemother- food. In MNU treated group, the level of monounsatu- apeutic regime [24]. Tadalafil treated group shows the rated fatty was increased and the same was decreased reduction of COX and LOX when examined through after Tadalafil treatment. It was previously reported that biochemical and immunoblotting assay in comparison unsaturated Trans fatty acid may increase the risk of in- with MNU treated group. Several research reports sug- vasive breast cancer and the same observations were gested that the expression of COX is associated with in- found in FAME analysis after MNU treatment [41]. cidence of breast cancer. It includes large tumour size, Various research reports validated the promoting as well positive axillary lymph node metastasis and HER2- as inhibitory effect of polyunsaturated fatty acid and positive tumour status [37]. Previous study on mice and mammary gland tumorigenesis [41]. In the resent piece rats that moderate to high COX expression is related to of work, inhibitory effects of polyunsaturated fatty acids generation of mammary gland tumours. The result of were seen after Tadalafil treatment (Additional file 1: present study also indicates the same (Fig. 7). The ex- Figure S1 and Additional file 2: Table S1). pression of COX in MNU treated group was increased Cell apoptosis is a process of programmed cell death which was subsided after Tadalafil treatment. Modula- and it is regulated by two major pathways: intrinsic and tion of downstream metabolite like PGE2, TXA2 and extrinsic pathways. The intrinsic pathway and Mitochon- TXB2 and LTA4, LTB4, LTC4, LTD4, LTE4 can ex- drial Outer Membrane Permeabilization (MOMP) are ploit multidirectional signalling cascade to exert their regulated through the balance of pro- and anti-apoptotic anticancer effects and the same have been explored in proteins of Bcl-2 family [42, 43]. In the present study, the present study with wider prospects to enlighten the expression of anti-apoptotic protein (Bcl-xl) was in- the chemotherapeutic strategies by repurposing the creased after MNU treatment and the vice versa effect mechanism of PDE-5 inhibitor. Dual inhibition en- was found upon pro-apoptotic protein (BAD). Collab- sures the alteration of AA metabolism in cancer cells orative result from pro-apoptotic and anti-apoptotic pro- and exhibits subsided inflammation, cell proliferation tein with downstream caspase cascade validate the and neo-angiogenesis. Identification of specific sub- participation of Tadalafil in mitochondrial mediated type of COX and LOX responsible for apotogenic po- death apoptosis pathway (Figs. 8 and 9). tential of Tadalafil is the future direction of our Previous reports revealed that the 26S centre of research [38]. UCHL-1 is regulated by 20S immunoproteasome subunit Singh et al. BMC Cancer (2019) 19:996 Page 13 of 15 Fig. 9 Protein-Protein interaction and Co-expression network analysis. Protein-protein interaction and co-expression analysis was performed using STRING V 10.0 with respect to differentially expressed proteins as identified from the Western blot analysis in relation to mitochondrial and hypoxic proteomic checkpoints which inhibits the degradation of the ubiquitinated pro- Conclusion teins [21]. In MNU treated group the expression of Authors would like to conclude that PDE-5 inhibitor UCHL-1 was increased which was subsided after Tadala- (Tadalafil) had a marked effect upon MNU induced fil treatment. The same relation has been linked with the mammary gland cancer. The mechanism of action of expression of NFκB/p50 precursor p105 and IκBα as it is Tadalafil is validated through mitochondria mediated essential for activity of NFκBp65. In line with to above death apoptosis pathway and Tadalafil also decreased results, the expression of NFκBp65 was increased in the inflammatory markers. Earlier, it was also reported MNU treated group which was decreased after Tadalafil that combination of sildenafil with celecoxib was found treatment (Figs. 8 and 9). to be cytotoxic in breast, hepatoma, colorectal cancer, Singh et al. BMC Cancer (2019) 19:996 Page 14 of 15 glioblastoma and medulloblastoma cell lines. It would be Author details Department of Pharmaceutical Sciences, Babasaheb Bhimrao Ambedkar helpful if well directed future preclinical and clinical University, (A Central University), Vidya Vihar, Raebareli road, Lucknow, UP study targeting the repurposing effect of PDE5 inhibitor, 2 226 025, India. Department of Pharmaceutical Sciences, King Faisal to explore its additional mechanism. Tadalafil has low- University, Al-Ahsa, Saudi Arabia. Department of Pharmacology, College of Pharmacy, Prince Sattam Bin Abdulaziz University, Al-Kharj, Kingdom of Saudi cost and low-toxicity with great preclinical potential Arabia. which can be further explored through well directed clinical trial in near future to include it with current and Received: 11 February 2019 Accepted: 11 September 2019 emerging standard of care treatments in breast cancer. References Supplementary information 1. Chang JC. Cancer stem cells: role in tumor growth, recurrence, metastasis, Supplementary information accompanies this paper at https://doi.org/10. and treatment resistance. Medicine. 2016;95(Suppl 1):S20–5. 1186/s12885-019-6152-9. 2. Kumazoe M, Kim Y, Bae J, Takai M, Murata M, Suemasu Y, Sugihara K, Yamashita S, Tsukamoto S, Huang Y. Phosphodiesterase 5 inhibitor acts as a Additional file 1: Figure S1. FAME analysis of the mammary gland potent agent sensitizing acute myeloid leukemia cells to 67-kDa laminin tissue subjected to MNU and Tadalafil. receptor-dependent apoptosis. FEBS Lett. 2013;587(18):3052–7. 3. Catalano S, Campana A, Giordano C, Győrffy B, Tarallo R, Rinaldi A, Bruno G, Additional file 2: Table S1. Fatty acid profiling of mammary gland Ferraro A, Romeo F, Lanzino M. Expression and function of tissue treated with MNU and Tadalafil. phosphodiesterase type 5 in human breast cancer cell lines and tissues: implications for targeted therapy. Clin Cancer Res. 2016;22(9):2271–82. Abbreviations 4. Weed DT, Vella JL, Reis IM, Adriana C, Gomez C, Sargi Z, Nazarian R, Califano 3-MST: 3-mercaptopyruvate sulfurtransferase; AA: Arachidonic acid; J, Borrello I, Serafini P. Tadalafil reduces myeloid-derived suppressor cells ABs: Alveolar buds; ATP: Adinosine triphosphate; BSA: Bovine serum albumin; and regulatory T cells and promotes tumor immunity in patients with head cAMP: Cyclic adenosine monophosphate; cGMP: Cyclic guanosine and neck squamous cell carcinoma. Clin Cancer Res. 2015;21(1):39–48. monophosphate; CBS: Acystathionine-beta-synthase; CEC: Cuboidal epithelial 5. Shi Z, Tiwari AK, Ashby Jr CR, Chen Z-S, Fu L-W: Anti-cancer Role of cell; COX: Cycloxygenase; CSE: Cystathionine-gamma-lyase; DCT: Dense Phosphodiesterase-5 Inhibitors. connective tissue; DF: Differentiation score; DTT: Dithiothreitol; 6. Thornton TM, Rincon M. Non-classical p38 map kinase functions: cell cycle ECG: Electrocardiogram; ERK: Extracellular-signal regulated kinases; checkpoints and survival. Int J Biol Sci. 2009;5(1):44. FAME: Fatty acid methyl esters; FTC: Ferrithiocyanate; GSH: Glutathione; 7. 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Palonosetron attenuates 1, 2-dimethyl hydrazine Authors’ contributions induced preneoplastic colon damage through downregulating MS and SK carried out the bench work; SR performed the immunoblotting acetylcholinesterase expression and up-regulating synaptic acetylcholine assay; SA, ASS and NA perform the statistical analysis of the data; GK perceived concentration. RSC Adv. 2016;6(46):40527–38. the idea, designed and supervised the whole study, prepared and proof read 12. Kaithwas G, RAWAT JK, Roy S, Singh M, Gautam S, Yadav R, Ansari MN, the final manuscript. All authors have read and approved the manuscript. Aldossary SA, Saeedan AS. Transcutaneous vagus nerve stimulation regulates the cholinergic anti-inflammatory pathway to counteract 1, 2- Funding dimethyl hydrazine induced colon carcinogenesis in albino wistar rats. Front MS received fellowship from University Grants Commission New Delhi Pharmacol. 2019;10:353. (RGNF-2013-14-SC-UTT-38150). 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Rifaximin, a pregnane X receptor (PXR) activator laboratory Animals and Ethics, Government of India (IAEC/SHIATS/PA16III/ regulates apoptosis in a murine model of breast cancer. RSC Adv. 2018;8(7): SSPG19). 3512–21. 16. Devi U, Singh M, Roy S, Tripathi AC, Gupta PS, Saraf SK, Ansari MN, Saeedan Consent for publication AS, Kaithwas G. PHD-2 activation: a novel strategy to control HIF-1α and Not applicable. mitochondrial stress to modulate mammary gland pathophysiology in ER+ subtype. Naunyn Schmiedeberg's Arch Pharmacol. 2019;392:1–18. Competing interests 17. Roy S, Rawat AK, Sammi SR, Devi U, Singh M, Gautam S, Yadav RK, Rawat JK, The authors declare that they have no competing interests. Singh L, Ansari MN. Alpha-linolenic acid stabilizes HIF-1 α and Singh et al. BMC Cancer (2019) 19:996 Page 15 of 15 downregulates FASN to promote mitochondrial apoptosis for mammary 40. Xia H, Ma S, Wang S, Sun G. Meta-analysis of saturated fatty acid intake and gland chemoprevention. 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Journal

BMC Cancer – Springer Journals

Published: Oct 24, 2019

Keywords: Tadalafil; ER+ mammary gland cancer; Mitochondrial stress; PDE-5 inhibitor; N-methyl-n-nitrosourea; DuCLOX

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