Breast cancer drugs dampen vascular functions by interfering with nitric oxide signaling in endothelium

Toxicology and Applied Pharmacology 269 (2013) 121–131

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Breast cancer drugs dampen vascular functions by interfering with nitric oxide signaling in endothelium Palanivel Gajalakshmi, Mani Krishna Priya, Thangaraj Pradeep, Jyotirmaya Behera, Kandasamy Muthumani 1, Srinivasan Madhuwanti 2, Uttara Saran, Suvro Chatterjee ⁎ Vascular Biology Lab, AU-KBC Research Centre, Anna University, Chennai, India

a r t i c l e

i n f o

Article history: Received 25 October 2012 Revised 24 January 2013 Accepted 1 March 2013 Available online 24 March 2013 Keywords: Nitric oxide Endothelium Tamoxifen citrate Capecitabine Epirubicin cGMP

a b s t r a c t Widely used chemotherapeutic breast cancer drugs such as Tamoxifen citrate (TC), Capecitabine (CP) and Epirubicin (EP) are known to cause various cardiovascular side-effects among long term cancer survivors. Vascular modulation warrants nitric oxide (NO) signal transduction, which targets the vascular endothelium. We hypothesize that TC, CP and EP interference with the nitric oxide downstream signaling specifically, could lead to cardiovascular dysfunctions. The results demonstrate that while all three drugs attenuate NO and cyclic guanosine mono-phosphate (cGMP) production in endothelial cells, they caused elevated levels of NO in the plasma and RBC. However, PBMC and platelets did not show any significant changes under treatment. This implies that the drug effects are specific to the endothelium. Altered eNOS and phosphorylated eNOS (Ser-1177) localization patterns in endothelial cells were observed following drug treatments. Similarly, the expression of phosphorylated eNOS (Ser-1177) protein was decreased under the treatment of drugs. Altered actin polymerization was also observed following drug treatment, while addition of SpNO and 8Br-cGMP reversed this effect. Incubation with the drugs decreased endothelial cell migration whereas addition of YC-1, SC and 8Br-cGMP recovered the effect. Additionally molecular docking studies showed that all three drugs exhibited a strong binding affinity with the catalytic domain of human sGC. In conclusion, results indicate that TC, CP and EP cause endothelial dysfunctions via the NO–sGC–cGMP pathway and these effects could be recovered using pharmaceutical agonists of NO signaling pathway. Further, the study proposes a combination therapy of chemotherapeutic drugs and cGMP analogs, which would confer protection against chemotherapy mediated vascular dysfunctions in cancer patients. © 2013 Elsevier Inc. All rights reserved.

Introduction It has been recognized in recent years that, majority of the chemotherapeutic drugs in use have resulted in the development of unfavorable cardiovascular complications, particularly in long term cancer survivors (Schmitz et al., 2012). As cancer survivors are at a greater risk of developing cardiovascular disease (CVD) than a malignancy relapse, understanding cardio-oncology is crucial when devising strategies for effective preventive care (Hong et al., 2010). Considering the mode of action of anti-angiogenesis drugs such as bevacizumab, sorafenib, sunitinib and pazopanib, one could expect a degree of negative side-effects of these drugs on cardiovascular functions as discussed in previous reviews (Isenberg et al., 2009). Interestingly, cardiovascular problems are not restricted to anti-angiogenesis drugs alone, but are

⁎ Corresponding author at: Vascular Biology Lab, AU-KBC Research Centre, MIT Campus, Anna University, Chennai 600 044, Tamil Nadu, India. Fax: +91 44 2223 1034. E-mail address: [email protected] (S. Chatterjee). 1 M.Sc project student from Bharathidasan University, Tiruchy, India. 2 B.Tech project student from Sathyabama University, Chennai, India. 0041-008X/$ – see front matter © 2013 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.taap.2013.03.011

documented with cytotoxic drugs as well (Schimmel et al., 2004). About 1/4th of testicular cancer patients developed diastolic arterial hypertension following cisplatin treatment (Strumberg et al., 2002). Similarly, the cancer drug paclitaxel, which targets microtubules, has been known to cause thrombosis (Sevelda et al., 1994). TC, CP and EP are the most commonly used primary and secondary chemotherapeutic drugs for breast cancer treatment in India, and all three drugs have been reported to cause cardiovascular problems in patients following chemotherapy. Studies have reported that TC treatment increased serum triglyceride levels among breast cancer patients and was associated with higher rates of venous thromboembolic disease (Fisher et al., 1998; Hozumi et al., 1998). The cardiotoxic effects of CP include incidence of myocardial ischemia (Bathina and Yusuf, 2010; Sentürk et al., 2009), the development of aortic dissection (Sclafani et al., 2010) and acute coronary syndrome (Cardinale et al., 2006) in 1.2–18% of patients following chemotherapy. Other studies have reported a significant reduction in the left ventricular systolic parameters in contrast to the diastolic parameters following treatment with EP, predisposing cancer patients to cardiotoxic effects (Appel et al., 2010; Okura et al., 2012).

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Cardiovascular homeostasis in turn, is heavily linked with nitric oxide (NO) signaling (Papapetropoulos et al., 1999). NO, a gaseous molecule is produced by the action of endothelial nitric oxide synthase enzyme (eNOS) in the transformation of L-arginine to L-citrulline (Sessa, 1994). Studies have reported that eNOS knockout mice demonstrate a significant increase in hypertension and decreased heart rate compared to wild type controls (Yang et al., 1999). NO has been previously shown to facilitate the relaxation of pre-contracted bovine coronary arteries (Gruetter et al., 1979) via the activation of sGC and cGMP (Ignarro, 1989). Bevacizumab, an anti-VEGF therapeutic drug against cancer induces hypertension through the suppression of NO production (Robinson et al., 2010). The interaction of the breast cancer drugs (TC, CP and EP) and NO signaling pathway is still unknown. We postulate that these drugs induce cardiovascular dysfunctions by interfering with NO signaling of the endothelium. The present study elaborates the implications of these breast cancer drugs on vascular functions and the mechanistic interaction of these drugs with cGMP mediated NO signaling pathway.

analysis) using a grid cell counter software. EC10 concentration for all the three drugs, TC, CP, and EP was formulated using OriginPro.8 software. Measurement of NO production in cells i) NO measurement using DAF-2DA: EAhy926 and HDMEC cells were grown in 12-well plates to 70% confluence. The cells were then treated with EC10 concentration of TC (1 μM), CP (4 mM) and EP (250 μM) for 1 h. L-Arginine and cPTIO were used as positive and negative controls respectively. Following treatment, the cells were washed with PBS before being incubated with 5 μM of DAF-2DA for 20 min. The cells were then scraped and centrifuged. The supernatant was measured for fluorescence at 495/515 excitation/ emission using a Varian Cary Eclipse UV–Vis Fluorescence spectrophotometer. ii) Griess assay: EAhy926 cells were treated with EC10 concentration of the cancer drugs for 1 h. Nitrite was then measured using Griess protocol, as described by Nims et al. (1996).

Materials & methods Measurement of NO in solution Materials Tamoxifen citrate (Fresenius Kabi Oncology Limited), Capecitabine (Roche pharmaceuticals) and Epirubicin (Alkem Laboratories Limited) were purchased commercially. DMEM, FBS, trypsin, penicillin/ streptomycin were purchased from PAN Biotech. MTT reagent, sildenafil citrate (SC), 3-(5-hydroxymethyl-2-furyl)-1-benzyl indazole (YC-1), and guanosine-3,5-monophosphate 8-bromo-sodium salt (8BrcGMP) were from Sigma Chemical Co., St. Louis, MO. DAF-2DA and DAF-FM were purchased from Invitrogen, NY, US. SpNO was obtained from Cayman Chemicals. All other chemicals were of laboratory grade and obtained commercially. Cell culture Immortalized endothelial hybrid cell lines EAhy926 and ECV304, stably transfected with eNOS–GFP constructs were gifts from Dr. C.J.S. Edgell (University of North Carolina, Chapel Hill) and Dr. Vijay Shah (Mayo Clinic, Rochester, USA) respectively. These cells were cultured in DMEM media supplemented with 10% FBS (v/v) and 1% penicillin (w/v) and streptomycin (w/v). Primary cell culture Bovine aortic endothelial cells (BAECs) were isolated from aorta of freshly slaughtered bovine obtained from a government-authorized abattoir. The aorta was collected aseptically and placed in phosphatebuffered saline (PBS; pH 7.4) for transport to the laboratory. In the laboratory, the vessels were washed with PBS, and connective tissue and fat were removed aseptically. Next, ECs were harvested as described elsewhere (Ryan, 1984). Isolated cells were confirmed as ECs by using antibodies against endothelial marker eNOS. The cells were used for experiments until passage 6. Primary dermal micro vascular endothelial cells (HDMEC) were also used and were obtained from ATCC, USA.

The direct effect of the cancer drugs on NO was determined using cell-free 1 × PBS buffer. SpNO (10 μM) was added as the source of NO to cell-free PBS following which EC10 concentration of the cancer drugs were added and incubated for 1 h. cPTIO was used as the negative control. Next, NO specific fluorescent probe, DAF-FM (5 μM) was added to the solution for 10 min. The fluorescence of the solution was then measured at 495/515 excitation/emission using a Varian Cary Eclipse UV–Vis Fluorescence Spectrophotometer. Measurement of NO in human blood components Human blood (about 20 ml) was collected from healthy volunteers in EDTA coated vacutainer tubes. Following collection, 2 ml of blood was treated with EC10 concentration of each cancer drug for 1 h. Next, the blood was centrifuged, and the supernatant (plasma layer) was subjected to Griess assay (Nims et al., 1996). Additionally, the remaining blood sample (about 10 ml) was subjected to Ficoll–Hypaque density gradient centrifugation as described elsewhere (Kanof et al., 1993). Peripheral Blood Mononuclear Cells (PBMC) and platelets obtained were seeded overnight on fibronectin coated 24-well plates. The cells were then treated with the cancer drugs for 1 h and NO production was measured using DAF-FM assay as described earlier. The remaining RBCs were treated with three cancer drugs for 1 h and subjected to DAF-FM assay. L-Arginine and L-NAME were used as positive and negative controls respectively. The study was performed in accordance with guidelines set by the Institutional Biosafety and Ethical Committee. Imaging eNOS–GFP localisation pattern ECV 304 cells stably transfected with eNOS–GFP were treated with all the three cancer drugs for 1, 6 and 12 h respectively. The cells were then fixed and the nuclei were stained with DAPI. Fluorescent images were taken using an Olympus IX71 microscope adapted with a DP71 camera.

Cell viability assay

Immunofluorescence

EAhy926 cells were seeded overnight in a 24-well plate at 70% confluence. The cells were treated for 1 h with different concentrations of TC (0 to 100 μM), CP (0 to 100 mM) and EP (0 to 25 mM) respectively. The cells were incubated with Trypan blue (0.04 mg/mL) for further 10 min. The cells were then washed with PBS before imaging. Bright field images were taken using Olympus inverted microscope. Live, round and dead cell images were counted manually (double blind

EAhy926 cells were seeded in 12-well plates and treated with TC, CP and EP (EC10 concentration) for 1 h. The cells were then fixed and permeabilised as per protocol described elsewhere (Mukhopadhyay et al., 2007). The cells were then incubated with phosphorylated eNOS at Ser-1177 anti-rabbit primary antibody (dilution 1:1000) and corresponding goat anti-rabbit secondary antibody (dilution 1:2000) tagged with TRITC. The nucleus was stained with DAPI.

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Fig. 1. Dose–response curves for the three breast cancer drugs. EAhy926 cells were treated with varying concentrations of TC, CP and EP and a cell viability assay was performed to determine the EC10 of each cancer drug. The percentage of live, round and dead cells in each concentration of drugs was calculated and the dose–response curve was plotted using OriginPro (version 8) software. The EC10 was found to be 1 μM, 4 mM and 250 μM for TC, CP and EP respectively.

Images were taken using an Olympus IX71 epifluorescence microscopy system equipped with a DP71 camera.

blots were developed by using TMB/H2O2 as substrate. Loading control was the expression level of β-actin gene (ab8229, Abcam, USA).

Western blot analysis Phalloidin staining EAhy926 cells were treated with EC10 concentration of TC, CP and EP for 1 h and harvested for Western blot analysis (Laemmli, 1970). Total protein was normalized using the Lowry's method. Proteins were detected by using 1:1000 dilution of eNOS antibody (SC-654, Santa Cruz, CA, USA), 1:1000 dilution of P-eNOS Ser-1177 (ab75639, Abcam, USA) and 1:2000 dilution of goat anti-rabbit IgG conjugated to horseradish peroxidase (Bangalore GENEI, Bangalore, India). The

EAhy926 cells were treated with the three anti-cancer drugs, SpNO, 8Br-cGMP and their combinations for 1 h. The cells were then stained with phalloidin as per protocol described elsewhere (Kolluru et al., 2008). The nucleus was stained with DAPI. Images were captured using the Olympus IX71 epifluorescence microscopy system equipped with a DP71 camera.

Fig. 2. NO production is decreased in endothelial cells under TC, CP and EP treatments. (A) EC10 of the cancer drugs significantly decreased NO production in EAhy926 cells by both DAF-2DA measurement and Griess assay. ** indicates p b 0.001 versus control. (B) Primary HDMEC cells also showed decreased NO levels under TC, CP and EP treatments by DAF-2DA measurement. * indicates p b 0.05 versus control. (C) Results of DAF-FM fluorimetry in cell-free solution showed that all the three drugs are effective quenchers of NO containing SpNO. TC was observed to be the most effective quencher of NO in cell free solution. ** indicates p b 0.001 versus control.

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Isolation of RNA and semi-quantitative PCR EAhy926 cells were treated with cancer drugs for 1and 6 h and total RNA was isolated using a total RNA Isolation kit (Medox Inc.). The RNA was then quantified using Nanodrop 2000 spectrophotometer (Thermo Scientific). cDNA synthesis was performed on 200 ng of RNA using reverse transcriptase (New-England Biolabs). PCR's were performed on cDNA using human eNOS (5′-AAGATCTCCGCCTCGCTCA-3′; 5′-GCT GTTGAAGCGGATCTTA-3′) and GAPDH primers (5′-TCAACGGATTTGGT CGTATT-3′; 5′-CTGTGGTCATGAGTCCTTCC-3′) respectively (Kong et al., 2004; Yang et al., 2010). PCR conditions consisted of 30 cycles at 92 °C denaturation for 1 min, 57 °C annealing for 2 min and 72 °C elongation for 3 min. PCR products were resolved on 1% agarose gel, the specific band for human eNOS was at 336 bp and GAPDH was at 510 bp. BIO-RAD gel quantification software was used to quantify the density of each band. Scratch wound assay EAhy926 cells were cultured on collagen coated 24-well plates at 100% confluence. A wound was created on the EC monolayer using a 100-μl sterile pipette tip. The monolayers were gently washed to remove cell debris before being incubated with the three cancer drugs, SC, 8Br-cGMP, YC-1 and their combinations. Images of the wound area were taken at 0, 2 and 4 h using a Nikon digital camera and the area of the cell-free wound at selected time points (0 and 4 h) was analyzed using Image J image software (Release α 4.0 3.2). Chemotactic assay using Boyden's chamber EAhy926 and BAECs were used for migration studies using Boyden's chamber, as per protocol described elsewhere (Tamilarasan et al.,

2006). Cells were treated with TC, CP and EP, as well as NO donor SpNO (10 μM), 50 μM 8Br-cGMP (an analog of cGMP), 1 μM Sildenafil Citrate (SC; an inhibitor of phosphodiesterase PDE-5), 10 μM YC-1(NO independent activator of sGC) and their combinations. Molecular docking and inter atomic contact analysis The crystal structure of the catalytic domain of the heterodimeric human soluble guanylate cyclase1 (sGC) protein (http://www.rcsb. org/pdb/explore/explore.do?structureId=3uvj) was downloaded from Protein Data Bank (PDB ID: 3uvj). The 3D conformer structure of TC (CID: 2733526), CP (CID: 60953), and EP (CID: 65348) was retrieved from PubChem compound database. These PDB structures of sGC and the cancer drugs were used for docking by AutoDock Vina software (Trott and Olson, 2010). Inter atomic contact analysis was performed to ascertain the contacts between sGC and the cancer drugs using Discovery studio 3.1 (Accelrys Software Inc., 2011). Transfection EAhy926 cells grown in 24-well plates were transfected with FlincG plasmid (addgene-pcDNA3.1 (+)-deltaFlincG) encoding cGMP–GFP using standard electroporation protocol prescribed in BTX Harvard electroporator. Briefly, the cells were electroporated at 1000 UF capacitance, 25 Ω resistance with 2 μg of FlincG plasmid for 8 ms. At 40 h post transfection, cells were treated with the cancer drugs, NO donor DEA NONOate and ODQ (inhibitor of sGC) followed by live cell imaging at 0, 30 s and 1 min time intervals using a DP71 camera attached to the Olympus IX71 fluorescence microscope. The fluorescence intensity of the cells was calculated by using the Adobe Photoshop cs4 portable version.

Fig. 3. Effect of drugs on the level of NO in human blood components. (A) Freshly collected whole blood was treated with drugs for 1 h, plasma was isolated and the NO level was measured using Griess assay. The level of NO was found to be increased under CP and EP treatments, while TC did not interfere with the level of NO. ** indicates p b 0.001 versus control, # indicates p b 0.05 versus control. (B) RBCs were treated with drugs for 1 h and subjected to DAF-FM fluorimetry. TC and CP could increase the NO level whereas EP did not interfere with the level of NO. ** indicates p b 0.001 versus control. (C) & (D) PBMC and Platelets showed no difference in the level of NO under the drug treatments when compared to control.

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Fig. 4. Effect of drugs on eNOS protein. (A) Fluorescence microscopy images of ECV304 (stably transfected with eNOS–GFP) cells show the presence of eNOS protein in the plasma membrane (yellow arrows) and in the perinuclear region (red arrows) at different time points. Under the treatment of drugs, the localization of eNOS protein in the plasma membrane decreased significantly while it is still found in the perinuclear region at all-time points. (B) Drugs interfere in the localization of phosphorylated eNOS at Ser-1177. Immunofluorescence was carried out on EAhy926 cells treated with TC, CP and EP for 1 h. The arrows in the images show the TRITC-positive spots originating from antibodies bound to phosphorylated eNOS. There is a significant decrease in the number of membrane bound phosphorylated eNOS under the treatment of drugs. Bar graph represents the number of spots/cell in control and under the drug treatment. ** indicates p b 0.001 versus control. (C) Western blots for eNOS, P-eNOS at Ser-1177 and β-actin were performed using protein samples prepared from cells treated with drugs for 1 h. Immuno-detection of eNOS and phosphorylated eNOS (Ser-1177) revealed that cancer drugs decreased the P-eNOS expression while it had no effect on the expression of eNOS in EAhy926 cells (n = 3). (D) EAhy926 cells were treated with EC10 concentration of TC, CP and EP for 1 h and 6 h. Total RNA was harvested from the cells and evaluated for eNOS expression profile. cDNA was primed with human eNOS primers. GAPDH mRNA was measured in parallel as an internal control. Densitometric analysis of blots was done relative to GAPDH expression. No statistically significant difference in the expression of eNOS mRNA was found between the control and the cells treated with drugs.

Vasodilation assay Fertilized fifth day eggs were collected from the Centre for Animal Health Studies, TANUVAS, Madhavaram Milk Colony, Chennai. CAM based vasodilation model was performed with slight modifications (Siamwala et al., 2013). Briefly, the egg shell of fifth day eggs was gently cut open to expose the area vasculosa for video recording. Paper discs soaked in EC10 concentration of TC, CP, EP cancer drugs, SpNO and 8Br-cGMP, crude extract of beetroot juice, fennel seed extract and their combinations were placed directly on the blood vessel and recorded for 20 min. The 0, 5, 10, 15, and 20 min images were used to calculate the area of a fixed portion of blood vessel. The change in area of the blood vessel for each treatment was calculated with respect to its 0 min. Statistical analysis All experiments were carried out in triplicate (n = 3). The data are presented as mean ± SEM. The data were analyzed using one-way

ANOVA test, Student's t-test and Tukey post-hoc tests using the SIGMASTAT software package. Data analysis and graphing workspace were prepared using OriginPro.8 software. p ≤ 0.05 was considered as statistically significant.

Results Establishing a dose–response curve for the breast cancer drugs To determine the effective concentration (EC10) of the three breast cancer drugs TC, CP and EP, we treated EAhy926 cells with a wide range of concentrations for each drug and performed cell viability assays. Live, round and dead cells were counted and OriginPro.8 software was used to determine the EC10 concentration of each drug by plotting a dose–response curve. The dose that resulted in 90% of live cells and in 10% of dead cells was selected as EC10. Results showed that 1 μM, 4 mM and 250 μM were the EC10 for TC, CP and EP (Fig. 1; Supplementary Figs.

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Fig. 5. Effect of drugs on cellular cGMP levels. EAhy926 cells were transfected with FlincG plasmid (containing cGMP–GFP) by using electroporation method. (A) Representative images (both bright field and fluorescent cells) of transfected EAhy926 cells are shown. (B) Fluorescence intensity of the cells was calculated by using the image analysis module of Adobe Photoshop cs4 portable version. A decrease in cGMP expression under all the three drug treatments was observed. ** indicates p b 0.001 versus control. # indicates p b 0.05 versus control.

1A, B and C) respectively. These doses were used in all subsequent experiments.

(Fig. 3B). The cancer drugs did not show any variation of NO levels in the PBMC and platelets (Figs. 3C and D).

Effect of cancer drugs on NO production

Cancer drugs affect the localization pattern of eNOS and phosphorylated eNOS (Ser-1177)

DAF-2DA and Griess assays were used to determine the effect of the breast cancer drugs on NO production in EAhy926 cells. L-Arginine (precursor of NO) and cPTIO (NO specific quencher) were used as positive and negative controls respectively. Results showed that each of the breast cancer drugs significantly decreased NO production of EAhy926 compared to control (Fig. 2A). Primary HDMEC cells also showed a significant decrease in NO levels as determined by DAF-2DA measurement. Interestingly, the primary endothelial cells generated less NO when treated with the three cancer drugs than with cPTIO (Fig. 2B). To further illustrate the NO quenching property of these drugs, we measured the NO levels of cell-free PBS containing SpNO following treatment with TC, CP and EP. Results showed that all three drugs significantly decreased NO levels compared to control (Fig. 2C). Additionally TC was found to be the most effective in decreasing the NO levels in the cells as well as in the cell-free solutions. We also observed the effect of these drugs on the NO levels of isolated components (Plasma, RBC, PBMC and Platelets) of whole blood collected from healthy volunteers. Examination of the plasma component following treatment showed that only CP and EP increased NO levels compared to controls (Fig. 3A). Similarly, both TC and CP increased NO levels of RBC's while EP did not show any increase

The endothelial cell line, ECV304 stably transfected with eNOS– GFP was used to study the eNOS localization pattern under TC, CP and EP treatments. Live cell images of control cells taken at 0, 1, 6 and 12 h showed eNOS localized at both the plasma membrane as well as around the peri nuclear region. In contrast, all three cancer drugs demonstrated a change in the pattern of eNOS localization particularly at the plasma membrane at 1, 6 and 12 h post treatment (Fig. 4A). Immunofluorescence technique was used to detect phosphorylated eNOS at Ser-1177 expression in EAhy926 cells following TC, CP and EP treatments for 1 h. In control cells, the focal points of phosphorylated eNOS spots were observed around the plasma membrane region whereas this population was decreased following treatment with the cancer drugs (Fig. 4B). Bar graph shows the decreased number of phosphorylated eNOS (Ser-1177) spots in drug treated cells compared to the control (Fig. 4B). EP was found to cause maximum reduction of the phosphorylated eNOS (Ser-1177) spots compared to TC and CP. Similarly Western blot analysis for phosphorylated eNOS also showed decreased expression in cells subjected to drug treatment for 1 h when compared to control (Fig. 4C). However Western blot and mRNA analysis for total eNOS did not demonstrate any changes in eNOS expression following drug

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Fig. 6. Drugs inhibit the migration of EAhy926 and BAECs. (A) EAhy926 cells were subjected to wound healing assay and treated with the cancer drugs individually and in combination with YC-1, SC and 8Br-cGMP. Results show that TC, CP and EP alone blocked endothelial wound healing while addition of YC-1, SC and 8Br-cGMP reversed the inhibition of wound healing. ** and # indicates p b 0.001 and p b 0.05 respectively versus control. (B) Representative images of wounded endothelial monolayer photographed at 0 and 4 h. (C) Drugs inhibit the actin cytoskeleton structure of EAhy926 cells. The image panel shows the phalloidin stained EAhy926 cells under TC, CP and EP treatments with and without SpNO and 8Br-cGMP. Images show the significant changes in the pattern of actin polymerization in EAhy926 cells treated with TC, CP and EP. Addition of SpNO and 8Br-cGMP leads to the partial recovery of actin polymerization pattern in the cells. (D) & (E) bar graphs represent the results obtained by chemotactic assay on EAhy926 and BAECs respectively. The graph shows that the three cancer drugs prevented the migration of cells while addition of YC-1, SC and 8Br-cGMP recovered the cells from drug induced dysfunctional migration. ** and # indicate p b 0.001 and p b 0.05 respectively versus control.

treatment for 1 h and 6 h respectively when compared to the control cells (Figs. 4C and D).

Cancer drugs decreased cellular cGMP levels EAhy926 cells were transfected with FlincG plasmid (cGMP–GFP construct) and the cellular cGMP level was measured under drug treatments. Representative images (bright field and fluorescence) of the cells transfected with cGMP–GFP under various treatments are shown in Fig. 5A. Decreased cellular cGMP expression was observed under all three drug treatments. It was noted that CP was the most effective in decreasing the cGMP levels of EAhy926 cells (Fig. 5B).

Cancer drugs inhibit migration of EAhy926 and BAECs A wound healing model was used to estimate the collective migration potential of the EC monolayer under drug treatments. All three drugs significantly decreased wound healing of the EAhy926 cells compared to control. Addition of YC-1, SC and 8Br-cGMP leads to an increase in wound healing and restored the cells from drug induced effects (Figs. 6A and B). Phalloidin staining was performed to study the cytoskeleton arrangements under drug treatments. All three cancer drugs demonstrated disturbed actin polymerization compared to control. Results also showed that addition of both exogenous NO (via NO donor SpNO) and 8Br-cGMP (analogue of cGMP) to cells exposed to the cancer

Table 1 This table shows the interacting amino acid residues between sGC and the three cancer drugs using AutoDock Vina and Discovery studio 3.1. S. No

Types of interaction

Drugs TC

CP

EP

Asp C 486, Ile C 528, Ala C 531, Asn D 548 Ile C 487, Gly C 529, Asp C 530, Asn D 548, Arg D 552, Glu C 608 Val D 475, Thr C 491, Thr c 527, Leu C 596, Val C 601 and Glu C 626

Ile B 427, Lys B 478, Glu B 554, Gly A 598, Asn A 605 Asn B 431 and Thr B 555

1

Hydrogen bond

Nil

2

Polar contacts

Thr B 474

3

Non-polar contacts

Gln A 470, Val B 472, Cys B 476, Met B 480, Lys A 524, Glu A 526, Thr A 527, Ile A 528, Gly A 529, Ser B 541, Leu B 542 Phe B543, and Val A 587

Gly B 426, Phe B 430, Asp B 477, Arg B 552, Phe A 597, Thr A 602

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Fig. 7. Drugs interact with the catalytic domain of sGC. (A–C) 3D images are depicting the docking structure of sGC with TC (orange), CP (green) and EP (yellow) respectively using AutoDock Vina software. Hydrogen bond interactions between the ligand and the protein are shown in pale yellow dotted lines with respective amino acid residues in red color. (D–F) 2D images depicting the results of interatomic contact analysis performed using Discovery studio 3.1 (Accelrys) software. The distance between the ligand and the sGC is shown in Å. Hydrogen bonds are shown in dotted lines. The results showed that TC did not have any hydrogen bond interaction with sGC protein but had polar and non-polar contacts. Both CP and EP showed hydrogen, polar and non-polar interactions with sGC. The binding affinity between the drugs and the sGC protein was found to be very high as evidenced by negative energy values. Polar contacts are shown in magenta caret and nonpolar contacts are shown in green caret.

drugs were able to partially recover and restore actin polymerization (Fig. 6C). Analysis of Boyden's chamber experiments showed that all three drugs arrested the migration of EAhy926 (Fig. 6D) and BAECs (Fig. 6E) compared to control cells. Addition of YC-1, SC and 8Br-cGMP, all showed recovery of cell migration from the inhibitory effect of the cancer drugs (Figs. 6D and E).

In silico binding of cancer drugs to the catalytic region of sGC We predicted an efficient binding between sGC protein and the three breast cancer drugs using AutoDock Vina software. The energy required for binding of sGC with TC was as low as − 7.7 (kcal/mol), − 7.8 (kcal/mol) for CP and − 9.5 (kcal/mol) for EP. The results of the inter atomic contact analysis showed that there are hydrogen bonds, and polar and non-polar contacts of the drugs with the alpha-3 (ranging from 468 to 690 amino acid residues) and beta-1 (ranging from 408 to 619 amino acid residues) chains of sGC protein. These results are summarized in Table 1 and in Fig. 7. Figs. 7A–C represent the interaction between sGC and the three (TC — orange, CP — green, EP — yellow) drugs in 3D image form using AutoDock Vina software. The results of the inter atomic contact analysis between sGC and the cancer drugs using Discovery studio 3.1 (Accelrys) software are shown in Figs. 7D–F.

Cancer drugs induce vasoconstriction In the CAM based vasodilation model, we observed a significant vasoconstriction of the blood vessels when treated with TC, CP and EP for 20 min. Representative bright field images (200×) of 0 and 20 min for control, 8Br-cGMP, SpNO, beetroot juice (BJ), fennel seed extract (FSE), and adrenaline on chick embryo vascular bed are shown in Fig. 8A. Results presented in Fig. 8B show that addition of SpNO and 8Br-cGMP significantly abolished the drug induced vasoconstriction and leads to relaxation of the blood vessels. Similarly, natural nitrate sources such as crude extracts of BJ and FSE also showed partial recovery from drug induced vasoconstriction. Discussion Cardiovascular toxicity of chemotherapeutic drugs has become a major concern among cancer survivors (Floyd et al., 2005). Anthracyclines are well known for its cardiac toxicity leading to congestive heart failure and left ventricular dysfunction (Slordal and Spigset, 2006). However, the specific mechanism by which chemotherapeutic drugs interfere with the cardiovascular functions of cancer patients is still not known. The results of the present study indicate that TC, CP and EP cause endothelial dysfunctions through NO/sGC/cGMP dependent pathway. The concentration of the three cancer drugs chosen for the present study was selected using cell

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Fig. 8. Drugs induce vasoconstriction of blood vessels. (A) Representative bright field images (200×) of 0 and 20 min for control, 8Br-cGMP, SpNO, BJ, FSE, and adrenaline on chick embryo vascular bed. (B) Bar diagram shows the vasodilation of blood vessels under 8Br-cGMP, SpNO, BJ, FSE, and adrenaline at 20 min. TC treated blood vessel was significantly constricted compared to control. Additional treatment with 8Br-cGMP, SpNO, BJ and FSE recovered the blood vessels from constriction. ** indicates p b 0.001 versus control. CP treated blood vessel leads to vasoconstriction while the addition of 8Br-cGMP, SpNO, FSE and BJ reversed the effect. EP caused vasoconstriction of blood vessels whereas addition of 8Br-cGMP, SpNO, FSE and BJ leads to vasodilation. # indicates p b 0.05 versus control.

viability assays. EAhy926 cells were treated with a wide range of concentration for each drug (TC — 0 to 100 μM; CP — 0 to 100 mM; EP — 0 to 25 mM) and cell viability was tracked. We then selected the concentration (EC10) at which 90% of healthy cells and 10% of unhealthy, rounded cells, were found as the final concentration (TC — 1 μM; CP — 4 mM; EP — 250 μM), used in subsequent experiments (Fig. 1 and Supplementary Fig. 1). We observed a significant reduction of NO levels in endothelial cells following treatment with the cancer drugs (Fig. 2). Interestingly, we also observed that

addition of each drug to cell-free solution containing exogenous NO mimicked the NO specific scavenger cPTIO and decreased NO levels significantly compared to control (Fig. 2C). Furthermore, even very low doses of these drugs were shown to quench NO levels in cell free solution (Supplementary Fig. 2). As these drugs are administered either orally or intravenously, the bioavailability of the drugs in the serum could potentially have varied effects on the cardiovascular system. We postulated that these drugs might exert differential effects on plasma and other

Fig. 9. A schematic representation showing the effect of breast cancer drugs on NO/sGC/cGMP pathway.

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blood components compared to the endothelium. Our results showed that these drugs indeed have differential effects on plasma and RBC, with both components demonstrating marginally increased NO levels in contrast to the decreased NO levels observed from treated endothelium (Figs. 3A and B). Previous reports by Atakisi et al. (2009) and Guven et al. (2007) have shown that acute TC and EP treatment was capable of increasing the NO levels of rabbit plasma and serum respectively. The increase in the plasma NO levels may be attributed to the nitrite reductase (NR) activity of RBC hemoglobin (Gladwin and Kim-Shapiro, 2008). Furthermore drug metabolizing enzymes such as cytochrome P450, are not only known for its activation of anti-cancer drugs but are also known to increase the level of NO through nitrate and nitrite reductions (Chang et al., 1993; Li et al., 2006). The results of our study clearly tell us that the effects of the cancer drugs were specific to the endothelium. We confer from the results of the present study that specific inhibitory effects of the drugs on NO production improvises endothelial dysfunctions. However, we did not know why the NO production by endothelial cells was particularly low when exposed to the cancer drugs. It has been shown in earlier studies that the bioavailability of NO is heavily controlled at various points of the NO signaling pathways (Sessa, 1994). The multi-step controls of eNOS are 1) expression level of NOS protein, 2) kinase dependent signaling such as Akt/PKA associated phosphorylation of NOS, and 3) sub-cellular trafficking and localization of NOS. In the present work, we categorically checked the interactions of the selected drugs with each of these NO controlling steps and results indicate that these drugs interfered at all these points. The sub-cellular localization of eNOS, eNOS enzyme activity and NO production are intimately controlled by posttranslational modifications (Liu et al., 1996; Sessa et al, 1995). Results of fluorescence microscopy analysis showed that the three cancer drugs used in the study affected the localization pattern of eNOS in ECV304 endothelial cells stably transfected with eNOS–GFP. However analysis of total eNOS expression in EAhy926 cells did not demonstrate any changes in mRNA nor protein expressions following drug treatment compared to controls. In contrast immunofluorescence and Western blot analysis for phosphorylated eNOS (Ser-1177) showed that all three cancer drugs significantly decreased phosphorylated eNOS expression when compared to control cells (Fig. 4). Although eNOS is a constitutive protein, it is possible that these drugs interfered with the post translational palmitoylation and phosphorylation of eNOS, which are critical to NOS localization and activation. The work of Gong et al. (2005) elaborated that bleomycin induces pulmonary hypertension in rabbits along with decreased eNOS mRNA expression in the endothelium. Adriamycin, another drug in the series, is also known for its severe interference with vascular systems. Vasquez-Vivar et al. (1997) have reported that NO production is decreased as a consequence of reductive activation of adriamycin by the reductase domain of eNOS enzyme. The importance of studying the NO downstream pathway involving sGC and cGMP in cardiovascular health under cancer treatments was reviewed by Isenberg et al. (2009) who suggested that cancer drugs possibly interfered with sGC activity. NO binds sGC to produce cGMP, which, in turn, induces vascular functions by inciting PKG downstream signaling (Ignarro, 1991). The present study elaborates NO downstream signaling under drug treatments. We observed that cellular cGMP levels decreased significantly following treatments (Fig. 5). These results indicate that the drugs interfere with downstream signaling of NO. Data from the in silico analysis also predicts the same, with results demonstrating that these drugs exhibiting strong binding affinity with the catalytic region of the sGC protein (Fig. 7 and Table 1). sGC is a heterodimer composed of alpha and beta (heme-binding) subunits of one each. Binding of NO to the heme domain results in activation of the C-terminal catalytic domain, which produces cGMP from GTP (Rodgers, 1999). Although the crystal structure of the catalytic domain of the heterodimeric human soluble guanylate cyclase 1 is deposited in Protein Data Bank (ID: 3uvj),

it is yet to be published. Amino acid residues from 408 to 690 of the catalytic region of the alpha subunit of human sGC were used for molecular docking in the current study. It is speculated that these drugs bind with sGC to attenuate cGMP production in addition to reducing the NO level. Thalidomide, an anticancer drug for multiple myeloma has been shown to inhibit angiogenesis by interacting with the NO/sGC/cGMP pathway (Majumder et al., 2009; Tamilarasan et al., 2006). Many studies have suggested that endothelial cells are being affected by chemotherapy. Mikaelian et al. (2010) reported of an upregulation of genes associated with vascular damage and downregulation of endothelial cell specific molecules in the transcriptomic profiling of the myocardium on exposure to anticancer agents such as colchicine, vinblastine, and vincristine. Based on this, it was identified that endothelial cell cycle arrest was the prime causative factor for cardiotoxicity of inhibitors of tubulin assembly. NO is known to mediate the migration and actin polymerization of endothelial cells (Disanza et al., 2005; Isenberg et al., 2006). Our observations demonstrate that the migration and actin cytoskeleton structure of endothelial cells were inhibited under drug treatments while the addition of YC-1, SC, 8Br-cGMP and SpNO respectively, recovered and rescued these cells from the drug-induced inhibition (Fig. 6). TC is known to significantly reduce the growth of endothelium in implanted tumors in nude mice (Haran et al., 1994). A recent study on doxorubicin, camptothecin, and thapsigargin revealed that the origin of endothelial cells determines the susceptibility to mitochondrial dependent cell death. Endocardial endothelial cells are, in particular, resistant to anticancer agents (Maney et al., 2011). Specifically, we observed that TC is more effective in decreasing the NO production in endothelial cells when compared to CP and EP. This could be because of its nongenomic action through its binding to ERα in endothelial cells. ERα is known to activate the eNOS and NO production in endothelial cells (Chen et al., 1999; Mendelsohn, 2000). We also demonstrated that these cancer drugs induced vasoconstriction in ex-ovo CAM model, which was subsequently blunted by the addition of NO donor, SpNO and 8Br-cGMP respectively. Furthermore, we also postulated whether naturally occurring nitrates in food could show similar drug-mediated recovery as observed with synthetic modulators (SpNO and 8Br-cGMP). Interestingly, addition of beetroot juice and fennel seed extracts demonstrated partial recovery from drug-mediated effects (Fig. 8). The study of Mosseri et al. (1993) indicated that vasoconstriction by 5-flurouracil (metabolite of CP) was reversed by increasing the levels of cGMP in rings of aorta isolated from rabbits. The findings of our study suggest that breast cancer drugs target the cGMP that might restrict the cardiovascular functions of the cancer patients. In summary, this work partially dissected the mechanism of endothelial dysfunctions by showing the decreased level of NO and inhibition of the downstream targets under the breast cancer drug treatment. Since there was a clear reversal of drug mediated effects by 8Br-cGMP, it is proposed that a combination therapy with 8BrcGMP along with chemotherapy drugs may prevent cancer patients from developing cardiovascular complications under these drug treatments (Fig. 9). However, as the NO signaling candidates are pro-angiogenic in nature, a cautious and balanced approach should be taken during taking such strategy. Supplementary data to this article can be found online at http:// dx.doi.org/10.1016/j.taap.2013.03.011. Conflict of interest The authors declare that there are no conflicts of interest. Acknowledgments This work is financially supported by DST (File No. SR/WOS-A/ LS-14/2011(G), Government of India).

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