Role of cystathionine β-synthase in human breast Cancer

Free Radical Biology and Medicine 86 (2015) 228–238

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Original Contribution

Role of cystathionine β-synthase in human breast Cancer Suvajit Sen a,b,n, Brian Kawahara a, Divya Gupta a, Rebecca Tsai a, Marine Khachatryan a, Sinchita Roy-Chowdhuri c, Shikha Bose d, Alexander Yoon e, Kym Faull b,e, Robin Farias-Eisner a, Gautam Chaudhuri a,b,f a

Department of Obstetrics and Gynecology, David Geffen School of Medicine at University of California at Los Angeles, Los Angeles, CA 90095, USA The Jonsson Cancer Center, David Geffen School of Medicine at University of California at Los Angeles, Los Angeles, CA 90095, USA c Department of Pathology at MD Anderson School of Medicine, Houston, TX 77054, USA d Pathology and Laboratory Medicine at Cedars–Sinai Medical Center, Los Angeles, CA 90048, USA e Semel Institute for Neuroscience and Human Behavior at University of California at Los Angeles, Los Angeles, CA 90095, USA f Molecular and Medical Pharmacology, David Geffen School of Medicine at University of California at Los Angeles, Los Angeles, CA 90095, USA b

art ic l e i nf o

a b s t r a c t

Article history: Received 25 March 2015 Received in revised form 15 May 2015 Accepted 16 May 2015 Available online 4 June 2015

Cystathionine β-synthase (CBS) is an enzyme in the transulfuration pathway that can catalyze the condensation of homocysteine (Hcy) and cysteine (Cys) to hydrogen sulfide (H2S) and cystathionine (CTH). CBS-derived H2S is important in angiogenesis and drug resistance in colon and ovarian cancers, respectively. However, the mechanisms by which cancer cell-derived H2S is utilized by cancer cells as a protective agent against host-derived activated macrophages are not yet investigated. This study investigated the mechanistic role of CBS-derived H2S in the protection of human breast cancer (HBC) cells against activated macrophages. HBC patient-derived tissue arrays and immunoblot analysis of HBC cells exhibited significantly increased levels of CBS when compared with their normal counterparts. This was associated with increased levels of H2S and CTH. Silencing of CBS in HBC cells caused a significant decrease in the levels of H2S and CTH but did not affect the growth of these cells per se, in in vitro cultures. However CBS-silenced cells exhibited significantly reduced growth in the presence of activated macrophages and in xenograft models. This was associated with an increase in the steady state levels of reactive aldehyde-derived protein adducts. Exogenous addition of H2S countered the effects of CBS silencing in the presence of macrophages. Conversely overexpression of CBS in human breast epithelial (HBE) cells (which do not naturally express CBS) protected them from activated macrophages, which were otherwise susceptible to the latter. & 2015 Elsevier Inc. All rights reserved.

Keywords: Cystathionine β-synthase Hydrogen sulfide Breast cancer 4-Hydroxynonenal

Introduction It is well known that CBS regulates the steady state levels of homocysteine (Hcy), a sulfur-containing amino acid, the accumulation of which leads to cellular toxicity associated with a variety of diseases including those of the cardiovascular and neurological systems [1–3]. CBS, which can catalyze the condensation of Hcy and cysteine (Cys) to hydrogen sulfide (H2S) and cystathionine (CTH) [4,5], may play an important role in breast cancer as well. Interestingly, gain of function mutations in the CBS gene have been shown to be associated with increased risks for breast cancer [6]. Moreover estrogen, an independent risk factor for breast cancer, activates the transcription factor SP1, which in turn upregulates n Corresponding author at: 650 Charles E Young Drive, University of Los Angeles, CHS 22115, CA 90095, USA. Fax: þ1 3102063670; E-mail address: [email protected] (S. Sen).

http://dx.doi.org/10.1016/j.freeradbiomed.2015.05.024 0891-5849/& 2015 Elsevier Inc. All rights reserved.

the transcription of CBS [7,8]. Furthermore, the enzyme activity of CBS is increased by lactate, the metabolite that is ubiquitously present in all cancers including HBC [9]. However the cellular mechanism by which CBS modulates breast cancer homeostasis is not yet known. Endogenous H2S derived from CBS is a reductant and has been shown to play important roles in the resolution of inflammation and immune modulation [10–12]. However its role in the downregulation of host immune surveillance especially with respect to the cross talk between cancer cells and macrophages has not been addressed. In this study we assessed the role of the HBC cell-derived CBS/H2S pathway in the mechanistic interaction between cancer cells and macrophages. This is of significant physiological relevance as macrophages comprise an important part of the host immune surveillance against cancers including breast cancer [13,14]. Infiltrated macrophages that compose a significant portion of the tumor mass exhibit both tumor-eliminating and

S. Sen et al. / Free Radical Biology and Medicine 86 (2015) 228–238

tumor-promoting properties [15–17]. HBC cells have evolved to counter the tumor-eliminating properties of macrophages as well as increasing the population of tumor-promoting macrophages [18,19]. One of the mechanisms by which tumoricidal macrophages induce cytotoxicity in tumor cells is by causing the formation of adducts between proteins and reactive aldehydes (formed due to oxidative damages on the plasma membrane lipids) [20–23]. Hence, we investigated a protective role of CBS in HBC cells on exposure to activated macrophages and the underlying mechanism(s).

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Immunohistochemistry Human breast tissue microarrays (NBP2-30212) were obtained from Novus Biologicals (Littleton, CO). Briefly, paraffin-embedded breast tissue sections were deparaffinized and washed with graded ethanol. Samples were incubated with CBS primary antibody (1:250). An Envision/HRP system was used for detection, and Gill’s hematoxylin was used for counterstaining. Slides were evaluated with a light microscope and a semiquantitative scoring system (scale 0–3þ : 0 being least intense, 3þ being most intense staining) was used to assess staining intensities as per published protocols [24] Three researchers that included a trained pathologist performed this blindly.

Experimental procedures Enrichment of plasma membrane fractions Materials L-Cysteine (W326305), DL-homocysteine (H4628), and protease inhibitor cocktail (P8340) and all other chemicals were purchased from Sigma-Aldrich (St. Louis, MO) unless stated otherwise. GYY 4137 (No. 13345), 4-hydroxynonenal or 4-HNE (No. 32100), and butathione sulfoxamine (BSO) (No. 83730-53-4) were purchased from Cayman Chemicals (Ann Arbor, MI). The following primary antibodies were used: anti CBS (sc-133208), anti CGL (sc100583), and anti GAPDH (sc-47724) from Santa Cruz Biotechnology (Santa Cruz, CA); Na þ /K þ ATPase (No. 3010 S) from Cell Signaling (Danvers, MA); and citrate synthase (No. 5771-1) from Epitomics (Burlingame, CA). Cell culture MDA-MB-468, MCF-7, MCF-10 A, and Hs 578 T were obtained from American Type Culture Collection (Manassas, VA). MDA-MB468, MCF-7, and Hs 578 T were grown in 1X DMEM supplemented with 10 mM nonessential amino acids, 2 mM L-glutamine, 1 μg/ml insulin, and 10% fetal bovine serum (FBS). MCF-10 A was grown in DMEM/F-12 1:1 supplemented with 10 μg/ml insulin, 20 ng/ml epidermal growth factor, 100 ng/ml cholera toxin, 0.5 μg/ml hydrocortisone, 5% horse serum, and 100 U/ml penicillin/streptomycin. Human mammary epithelial cells (HMEC) were obtained from Lonza (Cologne, Germany) [CC-2551]. HMEC were grown in the media recommended by Lonza: MEGM Bullet kit (CC-3151 and CC-4136). Cells were passaged no more than 10 times after being procured from the company and their genetic characteristics were tested regularly. We regularly checked for the presence of mycoplasma with the MycoAlert mycoplasma detection kit (LT07-318) from Lonza (Basel, Switzerland). For experimental purposes, cells were allowed to seed overnight prior to all treatments. RNA purification and quantitative reverse transcriptase-PCR Total RNA was extracted using TRIzol reagent according to the protocol provided by the manufacturer (Invitrogen: Carlsbad, CA). RNA concentrations were quantified with a NanoDrop 2000 c (Nanodrop: Wilmington, DE). Reverse transcription reaction was performed using 1 μg of total RNA with Maxima First Strand cDNA synthesis kit from Thermo Scientific (Pittsburgh, PA). We used validated CBS primer (PPH13484B-200) from Qiagen (Valencia, CA) and the sense and antisense primers: 5′-ATTGGCAATGAGCGGTTC-3′ and 5′-GGATGCCACAGGACTCCAT-3′ for β-actin. RT-qPCR was performed on the ABI (Applied Biosystems) 7900 HT Thermal cycler in standard mode using SYBR Select Master Mix for CFX from Life Technologies (Carlsbad, CA) for 40 cycles. Each reaction was run in triplicates and experiments were performed at least three times. Relative mRNA expression values (CBS mRNA versus β-actin) were calculated by the ΔΔCt method.

Plasma membrane fractions were isolated as published earlier [25]. Briefly, cultured cells (200  106) were harvested by scraping into phosphate-buffered saline (0.14 M NaCl, 0.05 M NaH2PO4, pH 7.5) with a cell scraper (Costar). The cells were concentrated by centrifugation for 10 min at 3000 rpm and were resuspended in 15 ml of 10 mM Hepes–KOH buffer, pH 7.5, that contained 15 mM KCl, 1.5 mM Mg acetate, 1 mM PMSF, and 1 mM dithiothreitol, and were allowed to swell for 10 min on ice. The mixture was centrifuged for 10 min at 3000 rpm and resuspended in 2 ml of the 10 mM Hepes–KOH buffer. The cells were ruptured, by passing thorough a 10 ml 25-gauge needle (20 strokes). The lysed suspension was centrifuged at 1000 g for 5 min to remove intact cells and the nucleus and then at 5000 g for 15 min to remove mitochondria. The relatively clear supernatant from this step was then centrifuged at 75,000 g for 40 min in an ultracentrifuge. The supernatant was drained out completely and the pellets were pooled together and washed two times with the Hepes–KOH buffer and suspended in subsequent assay buffers as per requirements. Protein concentration in the membrane preparation To determine the protein concentration, the membrane preparations were dissolved in 0.5% SDS and amount of protein was estimated using a BCA protein assay kit (from Pierce). Typically the protein concentration was in the range of 0.5 mg/ml. Membranes equivalent to approximately 15–20 mg protein per reaction were used as starting membrane concentration during vesicle preparation. Coculturing of human breast cells with macrophages Human breast cells (100,000) were seeded into 6-well plates and cocultured with 300,000 THP-1 on 0.4 μm pore membrane polycarbonate Transwell Permeable Support Inserts (No. 3412) from Corning (Corning, NY) in the presence of 25 ng/ml phorbol myristate acetate (PMA) to differentiate THP-1 monocytes into macrophages. Media were changed after 48 h, and cells continued to grow as per experimental conditions. Buthionine sulfoximine (BSO) was added at 0.2 mM concentrations to the HBC cells prior to culturing with activated macrophages, wherever relevant. Hydrogen sulfide (H2S) measurements Hydrogen sulfide production from human breast cells and plasma membrane protein was measured using HSN2, a compound developed and gifted by Prof. Pluth at the University of Oregon, as reported earlier [26]. Briefly cells (5  106) were incubated with 5 μM HSN2 for 30 min to allow development of florescence, which was read in a plate reader at (Ex/Em: 432/

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542 nm). All measurements were normalized against equal number of cells. For estimation of H2S, produced by plasma membrane fractions, 20 μg of purified plasma membrane fractions was incubated in 250 mM Tris–HCl, pH 8.6, with 20 mM L-Cys and DLHcys, 100 μM PLP, 0.5 mg/ml BSA, and 5 μM HSN2. The reaction was incubated at 37 °C for 30 min, and this was followed by the measurement of HSN2-generated fluorescence. The measurements were normalized against equal amounts of proteins in micrograms. Convincing evidence of the specific reactivity of HSN2 toward H2S has shown that the major intracellular thiol glutathione exhibits negligible reactivity toward HSN2 [26]. We too observed that while there was significant decrease in HSN2-derived fluorescent in HBC cells on silencing the gene for CBS, no significant decrease was observed when HBC cells were treated with BSO, a specific inhibitor of glutathione synthesis (Supplementary Fig. 1). This further confirmed the specificity of HSN2 toward H2S. Estimation of CTH Cells in the amount of 5  106 were suspended in 200 μl, 10 mM ammonium acetate, pH 7.4, and lysed via three cycles of freeze–thaw. Next, 800 μl methanol was added to lysates. Samples were immediately mixed vigorously, centrifuged (16,000 g, 5 min), and then supernatants were transferred to microcentrifuge tubes and dried in a vacuum centrifuge. Methanol (100 μl) was added to the dried samples. They were dried again under a stream of nitrogen and benzene (100 μl) was added. Then the samples were dried yet again under a stream of nitrogen. The samples were then treated with methanolic HCl (3 N, 50 μl, 60 min, 60 °C) and dried again under a nitrogen stream. Samples were redissolved in H2O (100 μl) and centrifuged (16,000 g, 5 min), and the supernatant was transferred to liquid chromatography (LC) injector vials. Aliquots of the solution (10 μl) were injected onto a reverse phase column (Phenomenex Kinetex XB-C18, 100  2.1 mm, 1.7 um particle size, 100 Å pore diameter) equilibrated with 85% eluant A (1 mM perfluorooctanoic acid in H2O) and 15% eluant B (1 mM perfluorooctanoic acid in acetonitrile) and eluted (100 ul/min) with increasing concentration of eluant B (min/% B; 0/15, 5/15, 35/ 50, 33/15, 45/15). The effluent from the column was directed to an Agilent Jet Stream electrospray ionization (ESI) source connected to a triple quadrupole mass spectrometer (Agilent 6460) operating in the positive ion tandem mass spectrometric multiple reactionmonitoring (MRM) mode in which the intensity of the CTH parent to fragment transition (251@ 481, rt 22.57 min) was recorded using previously optimized settings. With each set of samples, a set of standards was prepared containing increasing concentrations of CTH (0, 0.5, 2.5, 12.5, 25, 50 pmol/μl). The peak areas for CTH in the standards were used to construct calibration curves, and the amount of CTH in each biological sample was calculated by interpolation from the curves. Lentiviral transfection and generation of stable cell lines Four stable cell lines, silenced for either CBS or scrambled control, were generated in MCF-7 and MDA-MB-468 with shRNA, using lentiviral particles obtained from Santa Cruz Biotechnology (sc-60335- V and sc-108080, respectively) as per the manufacturer’s protocol. A set of four shRNAs was packed into each viral particle to counter off target effects. Cells were selected with 1 μg/ ml puromycin until resistant colonies could be identified and propagated. MCF-7 and MDA-MB-468 cells that survived puromycin selection after transfection with shRNA against CBS were given the names MCF7shCBS and 468shCBS, respectively. MCF-7 and MDA-MB-468 cells that survived puromycin selection after transfection with scrambled control shRNA were given the names MCF7scram and 468scram, respectively.

Overexpression of CBS Human CBS overexpression plasmids (Ad-h-CBS) and corresponding control plasmids (Ad-CMV-null), packaged in adenovirus, were obtained from Vector Biolabs (PA). Briefly, MCF-10 A cells were seeded into 100 mm dishes and grown to  95% confluency and then infected with the CBS overexpression or control viral stocks of 1  105  106 PFU/ml. After 24 h, the cells were washed of virus, fresh medium was added, and the cells were used for subsequent experiments. 4-HNE/MDA adduct assay Whole cell lysates were analyzed using the 4-HNE adduct ELISA kit from Cell Biolabs, Inc. (San Diego, CA). Cell pellet samples were sonicated for 15 s in alkaline lysis buffer (50 mM Tris, pH 8.6, 50 mM NaCl, protease inhibitor cocktail) and centrifuged at 10,000 g, 30 min, 4 °C. Assays were conducted as per the manufacturer’s protocols. Bovine serum albumin (BSA) standards and protein samples were adsorbed onto a 96-well plate overnight at 4 °C. The HNE/MDA–protein adducts present in the sample or standard were probed with an anti-HNE/MDA antibody, followed by horseradish peroxidase (HRP)-conjugated secondary antibody. The HNE/MDA–protein adduct content in an unknown sample was determined by comparing with a standard curve that is prepared from predetermined HNE/MDA–BSA standards. Xenograft studies Female Balb/c nude mice (were obtained from Charles River Laboratories), 4–6 weeks, 18–22 g weight were housed in a specific pathogen-free room. In our study, the models of xenografts of MCF-7 cells, scramble controls (MCF7scram), and MCF-7 silenced for CBS (MCF7shCBS) were grown to confluency and injected sc (5  106cells in 100 ml of PBS þ100 ml of Matrigel [(BD,USA)]) in nude mice (n ¼3) in the right and left flanks of each mice, respectively. The animal’s weight was measured every 2 days. We monitored tumor growth starting 2 days after injection up to 3 weeks. Tumors were measured twice a week with a caliper, and tumor volumes were calculated using the following formula: tumor volume ¼[length  width  (length þwidth/2)  0.56]. The Office of Animal Research Oversight at the University of California at Los Angeles approved all animal experiments. Glutathione assay Cells were plated at 1000 per well in white, opaque 96-well plates and allowed to attach overnight. After addition of BSO/ sulfasalazine, where applicable, plates were incubated for 3 h. Cells were then washed with PBS, and total and oxidized glutathionine levels were measured using GSH/GSSG-Glo, according to manufacturer’s instructions (Promega, Madison, WI). Luminescence was measured with a BioTek plate reader. Cell viability Cell viability was determined by the trypan blue exclusion method using a hemocytometer as well as the Vi-CELL XR cell viability analyzer from Beckman Coulter (Brea, CA). The number of viable cells for each treatment and time point was determined in triplicate. Treatment of HBCs with exogenous 4-HNE and GYY Briefly, 6-well plates were seeded with 100,000 MCF7scram and MCF7shCBS cells with 10 μM 4-HNE, 2 μM GYY, or vehicle

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controls. The cells were incubated overnight and then harvested and cell viability was assessed. The data were plotted as fold change from control. Western blot analysis Cytoplasmic extracts were prepared from cells after various treatments by lysis in buffer (50 mM Hepes, pH 7.5, 1 mM DTT, 150 mM NaCl, 1 mM EDTA, 0.1% Tween 20, 10% glycerol, 10 mM βglycerophosphate, 1 mM NaF, 0.1 mM orthovanadate, 10 mg/ml leupeptin, 10 mg/ml aprotinin, and 0.1 mM phenylmethylsulfonyl fluoride (PMSF) and kept at 4 °C for 30 min. Cytosolic protein concentrations were quantified by the Bradford assay. Equal amounts (20 μg protein) were separated on a 10% SDS-PAGE gel and transferred onto polyvinylidene difluoride (PVDF) membranes. Incubations with primary and HRP-conjugated secondary antibodies were done at 4 °C overnight and 1 h at RT, in 1:1000 and 1:10,000 dilutions, respectively. Immunoreactivity was detected by using enhanced chemiluminescence [Amersham Biosciences]. Cell cycle analysis We determined phases of cell cycle using flow cytometry. Cells were trypsinized, washed with 1  PBS, fixed, permeabilized with cold 70% ethanol, and finally incubated in the dark for 30 min with 1 ml of propidium iodide (containing NP-40) (Biosure, Grass Valley, CA, USA). The DNA content of these cells was measured based on the presence of propidium iodide (PI) staining. Flow cytometric analysis was done on at least 10,000 cells from each sample, and cell cycle data were analyzed using a FACSCalibur flow cytometer (BD BioSciences, San Jose, CA, USA) with an excitation wavelength of 488 nm and emission wavelength of 530 nm.

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Statistical analysis Data are presented as mean 7SEM. Student’s t test was performed for comparison between two groups. For statistical analysis of more than two groups, data were analyzed by ANOVA, and pairwise comparisons were performed by Tukey’s post hoc analysis. P values o0.05 (*) were considered statistically significant.

Results HBC is associated with an increased expression of CBS We first compared CBS expression in tissues obtained from breast cancer patients, and in HBC cells, with matched controls and human breast epithelial (HBE) cells, respectively. A tissue array analysis from 60 breast cancer patients demonstrated significantly increased expression of CBS in HBC tissues compared with adjacent normal controls. The scoring of the tissue array was performed by a total of three blinded researchers including one pathologist, according to the 0/1þ/2þ /3þ scoring scheme, 0 being least intense and 3þ being the highest. We observed that the average score for normal tissue sections was 1.2þ/– 0.43 compared to 3.0 þ/– 0.5 for the transformed sections (Fig. 1A,B). Moreover, the magnitude of CBS expression correlated with the progression of the disease. Immunohistochemical intensity was in the order: Normal oStage IIo Stage IIIolymph node metastasis (Fig. 1A,B). Expression of CBS was independent of the estrogen receptor (ER), progesterone receptor (PR), HER2, and p53 status of the HBC tissues studied (Fig. 1A). A representative panel of the tissue array staining is seen in Fig. 1A, while the complete panel along with annotations is included in supplementary figures (Figs. 2,3).

Fig. 1. HBC is associated with an increased expression of CBS. (A) Immunohistochemistry utilizing monoclonal antibody against CBS on a representative panel from a tissue array of 60 human samples showing receptor and p53 status and disease progression. (B) Mean 7SD of the scoring of CBS staining of the tissue array comprising 60 human samples. (C) mRNA expression of CBS in HMEC, MCF-10 A, Hs 578 T, MCF-7, and MDA-MB-468 by RT-qPCR. (D) Immunoblot of CBS in breast cell line panel; GAPDH was a loading control. (E) Comparison of steady levels of H2S among MCF-10 A, MCF-7, and MDA-MB-468 utilizing the florescent probe HSN2 normalized against equal number of cells. (F) Steady state levels of cystathionine in normal breast cells, MCF-10 A, and in breast cancer cells, MCF-7 and MDA-MB-468, measured by HPLC-MS (*Po 0.05).

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Fig. 2. Silencing of CBS-sensitized HBC cells to activated macrophages. (A) Immunoblot of CBS in the human breast cancer cells, MCF7 and MDA-MB-468, on silencing of CBS. MCF7shCBS and 468shCBS denote MCF7 and MDA-MB-468 cells, respectively, stably silenced for CBS utilizing shRNA-mediated gene knockout via lentiviral particles. MCF7scram and 468scram denote the corresponding scrambled controls for MCF7 and MDA-MB-468 cells, respectively. Rat liver extract was a positive control for CBS. GADPH was utilized as the loading control. (B) Comparison of intracellular H2S levels between CBS-silenced MCF7 and MDA-MB-468 cells and their corresponding scrambled controls shown by the fold differences in fluorescence measured by HSN2 normalized against equal number of cells (C) Cystathionine levels in the scrambled controls and CBS-silenced MCF7 and MDA-MB-468 cells measured by HPLC-MS. (D) Viable cell count of MCF7scrambled controls (MCF7scram) and CBS-silenced MCF7 cells (MCF7shCBS) over 5 days utilizing trypan blue exclusion methods. (E) Viable cell count of MDA-MB-468 scrambled controls (468scram) and CBS-silenced MDA-MB-468 cells (468shCBS) over 5 days utilizing trypan blue exclusion methods. (F) Viable cell count of MCF7scrambled controls (MCF7scram) and CBS-silenced MCF7 cells (MCF7shCBS) over 5 days in cocultures with activated macrophages, utilizing trypan blue exclusion methods. (G) Viable cell count of MDA-MB-468 scrambled controls (468scram) and CBS-silenced MDA-MB-468 cells (468shCBS) over 5 days in cocultures with activated macrophages, utilizing trypan blue exclusion methods. (H) Representative picture of tumors excised from mouse xenografts following injection of MCF7 scrambled controls (MCF7scram) (left panel) and CBS-silenced MCF7 (MCF7shCBS) cells (right panel), at 6 weeks following injection. The average volumes of the excised tumors (derived from MCF7scram and MCF7shCBS) from 3 experiments have been shown alongside (I) cell cycle distribution of MCF7scram/shCBS cells at 5 days in cocultures with activated macrophages. (J) Cell cycle distribution of 468scram/shCBS cells at 5 days in cocultures with activated macrophages (* Po 0.05).

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Fig. 3. Overexpression of CBS-protected HBE cells from activated macrophages. (A) Immunoblot utilizing antibody against CBS to show its overexpression in MCF-10 A cells following adenoviral transduction of plasmids carrying the CBS gene. (B) Fold change in the steady state levels of H2S production (detected by change in HSN2-derived fluorescence) in CBS overexpressed (10 AoxCBS) MCF10A cells in comparison with MCF10A empty vector controls (10Anull). (C) Cell viability of MCF-10A with or without the overexpression of CBS at 5 days on coculturing with activated macrophages (* Po 0.05).

We next assessed the basal expression of CBS in a panel of HBC cells along with two normal HBE cells. All three HBC cells, MCF-7 (ERþ ve), MDA-MB-468 (ER-ve), and Hs 578 T (ER-ve) expressed significantly increased levels of CBS, both at the level of mRNA and at the level of protein when compared with the HBE cells, HMEC and MCF-10 A (Fig. 1C,D). Fold increase in the mRNA expression of CBS was 6.2,  21.3, and 20.3 in MCF-7, MDA-MB-468, and Hs578T, respectively, when compared with HMEC. We observed that the increased CBS expression in cancer cells was associated with significantly elevated levels of endogenous H2S and CTH in MCF-7 and MDA-MB-468 cells, respectively, when compared to MCF-10 A (Fig.1E,F). HBC cells, MDA-MB-468, exhibited decreased levels of H2S when compared with MCF7cells but samples had similar levels of CTH. This could be due the production of CTH from other reactions catalyzed by CBS such as serine þ Hcy - CTH þH2O. Following this we proceeded to assess the effects of genetically silencing CBS in HBC cells. Silencing of CBS-sensitized HBC cells to activated macrophages We established a CBS-silenced stable cell lines to ascertain the role of CBS in HBC cell growth. For this purpose we utilized both ER þve, MCF-7 and ER-ve, MDA-MB-468 cells. Henceforth, the scrambled control and CBS-silenced MCF-7 and MDA-MB-468 cells would be denoted as MCF7scram and MCF7shCBS and 468scram and 468shCBS, respectively (Fig. 2A). Silencing of CBS in HBC cells per se did not elicit any changes either in the growth or in the viability in in vitro cell cultures despite a significant decrease in the steady state levels of H2S and CTH (Fig. 2B–E). On the other hand, CBS silencing significantly compromised the growth of these cells only in cocultures with activated macrophages and in in vivo xenograft models which indicated an average size of CBS silent tumors (derived from MCF7shCBS) to be  8 mm3 versus  125 mm3 for scrambled controls (derived from MCF7scram), following 3 weeks of growth in nude mice (Fig. 2F–H). Cell cycle analysis of HBC cells, cocultured with activated macrophages, indicated that MCF7shCBS exhibited a  9% decrease in the S-phase population as well as a  10% increase in the G0/G1 phase when compared to MCF7scram and 468shCBS exhibited a  11% increase in the sub-G1 population when compared to 468scram (Fig. 2I,J). This indicated that silencing of CBS inhibited cell cycle progression in both HBC cells, irrespective of their ER status. We further wanted to confirm the protective role of CBS and therefore overexpressed it in the HBE cells, MCF-10 A (that

otherwise exhibited undetectable CBS expression). The empty vector and CBS overexpressed MCF10A cells are indicated as 10Anull and 10AoxCBS, respectively. We utilized increasing concentrations (105 and 106 plaque forming units [PFU]/ml) of viral particles, overexpressing CBS to confirm specificity (Fig. 3A). This was associated with a  4.5-fold increase in steady state levels of H2S in these cells (Fig. 3B). Interestingly, MCF-10 A cells, which normally exhibited decreased growth in the presence of activated macrophages, became resistant to them once overexpressed with CBS. This was indicated by viable cell counting at 5 days following coculturing with activated macrophages. Equal numbers of 10Anull and 10AoxCBS cells were seeded in the presence of activated macrophages to start the experiment (Fig. 3C). CBS silencing increased the steady state levels of reactive aldehydederived adducts in HBC cells in the presence of activated macrophages Several lines of evidence have established that tumoricidal macrophages induce oxidative stress in target cells including cancer cells, to eliminate them [15,16]. One of the major mechanisms by which macrophages do so is by producing oxidants that eventually oxidizes lipids of the plasma membranes generating reactive aldehydes such as 4-hydroxynonenal (4-HNE) and malondialdehyde (MDA) [15,16,21]. These are oxidizing species that subsequently exert cellular toxicity by forming stable adducts with various biomolecules including proteins [20,21]. Hence, we evaluated whether the activated macrophage-induced cell growth inhibition of HBC cells was associated with the formation of adducts between cellular proteins and reactive lipid aldehydes such as 4-HNE and MDA in HBC cells and whether their steady state levels were dependent on the presence of CBS. We observed that HBC cells, silenced for CBS, exhibited significantly increased steady state levels of 4-HNE and MDA protein adducts compared to scrambled controls. MCF-7 and MDA-MB-468 cells exhibited 6- and  3-fold enhancement, respectively, in the levels of 4-HNE and 2.5- and2-fold enhancement, respectively, in the steady state levels of MDA adducts as evidenced from ELISAbased assays (Fig. 4). CBS-derived H2S protected HBC cells against reactive aldehyde-derived adduct formations in cocultures with activated macrophages We assessed whether exogenously administered H2S could compensate for the lack of its endogenous production by HBC cells silenced for CBS, in terms of regulating 4-HNE–protein adduct

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purified. Increased band intensity of Na þ /K þ ATPase (the typical plasma membrane residential protein) in plasma membrane fractions compared with whole cells validated the enrichment of the membrane fraction. Conversely the decreased intensity of the band intensity of citrate synthase (the typical mitochondrial residential protein), in plasma membrane fractions compared with whole cells, validated the absence of mitochondrial fractions in the plasma membrane preparation (Fig. 6A). We observed that purified plasma membrane fractions from HBC cells (MCF-7 [ER þve] and MDA-MB-468 [ER-ve]) not only expressed CBS but also produced H2S in the presence of Hcy and Cys. Interestingly, HBE cells, MCF-10 A, were not found to express CBS in their plasma membrane fractions and expectedly the latter did not produce H2S in the presence of Cys and Hcy (Fig. 6B,C). Exogenous H2S decreased growth inhibition in CBS-silenced HBC cells on exposure to 4-HNE We observed that the direct addition of 4-HNE (  10 μM for 48 h as reported to be toxic) [30] decreased the cell growth of CBSsilenced HBC cells by  1.7-fold when compared with  0.2-fold in scrambled controls (Fig. 6D). However, preincubation with GYY 4137, 2 μM (H2S donor), decreased the sensitivity of CBS-silenced HBC cells ( 2-fold recovery) toward the growth retarding effects of 4-HNE (Fig. 6E). Glutathione status was independent of CBS expression in HBC cells

Fig. 4. CBS silencing increased the steady state levels of reactive aldehyde-derived adducts in HBC cells in the presence of activated macrophages. (A) Comparison of protein–4-HNE adduct formations between MCF7 scrambled controls (MCF7scram) and MCF7 cells silenced for CBS (MCF7shCBS) and between MDA-MB-468 scrambled controls (468scram) and MDA-MB-468 silenced for CBS (468shCBS) at 5 days in cocultures with activated macrophages. (B) Protein–MDA-adduct formations in MCF7scram/shCBS and 468scram/shCBS at 5 days in cocultures with activated macrophages. (* Po 0.05).

formations following incubation with activated macrophages. We observed a significant decrease in the steady state levels of 4-HNE– protein adducts in CBS-silenced HBC cells when exposed to GYY4137 (a slow releaser of H2S) kept in cocultures with activated macrophages over a period of 5 days. There was a 40% and  50% decrease in the levels of 4-HNE–protein adducts in MCF7shCBS and 468shCBS, respectively (Fig. 5A,B). This was associated with a recovery of cell growth of 60% and  80% in MCF7shCBS and 468shCBS, respectively as assessed from viable cell counts at 5 days following coculturing with activated macrophages (Fig. 5C,D). The H2S/HS– system has been shown to reduce oxidants including reactive aldehydes directly [27]. Oxidants from macrophages initially accumulate at the vicinity of plasma membranes to cause oxidation of phospholipids [28]. Since H2S has a short halflife [29], we hypothesized it was being produced in the vicinity of the production of oxidants, i.e., the plasma membrane. We therefore examined whether the CBS–H2S system was associated with the plasma membrane fractions in HBC cells. The CBS/H2S machinery was detected on the plasma membrane fractions of HBC cells Plasma membrane fractions from HBC cells, MCF7 [ER þve] and MDA-MB-468 [ER-ve] along with HBE cells, MCF10A, were

In addition to catalyzing the production of H2S, CBS also produces CTH, which in turn is metabolized by cystathionine γ-lyase (CGL) to produce cysteine required for the endogenous production of glutathione GSH, an important component of the cellular antioxidant system. We therefore wanted to assess whether in our model system GSH in addition to H2S played a role toward regulating the levels of the 4-HNE-protein adducts. To our surprise, we observed that silencing of CBS did not affect the levels of either the total GSH or the ratio of oxidized /reduced GSH (Fig. 7A,B). Moreover, we observed that CGL was undetectable in tissues derived from breast cancer patients as well in HBC cells (Fig. 7C). Furthermore exposure of HBC cells both scrambled controls and those silenced for CBS, to 0.2 mM BSO, a specific inhibitor of glutathione synthesis as reported earlier [31], did not have any effect on cellular viability over time in the presence of activated macrophages (Fig. 7D,E).

Discussion Our primary objective of this study was to investigate whether CBS played an important role in the maintenance of HBC homeostasis. We also wanted to assess the mechanistic link between the downregulation of macrophage-induced oxidative stress and CBSderived H2S in breast cancer. The increased expression of CBS in tissues derived from HBC patients and in HBC cells, irrespective of their molecular subtype and receptor status (at least in the tissue samples that we studied), indicated an important role of CBS in this disease. Moreover, the undetectable levels of CBS mRNA and protein in normal HBE cells suggested that it catered to a specific cellular function that was associated only with the event of transformation. Similar to that reported for colon and ovarian cancers, we observed that CBS-catalyzed condensation of Hcy þCys to CTH and H2S, was important in breast cancer as well [32, 33]. This was evidenced by a significant decrease in the levels of H2S and CTH on silencing of CBS in HBC cells. The decrease in the steady state levels of H2S was more pronounced in MCF7 cells compared to

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Fig. 5. CBS-derived H2S protected HBC cells against reactive aldehyde-derived adduct formations in cocultures with activated macrophages. (A,B) Protein–4-HNE adduct formations in MCF7scram/shCBS and 468scram/shCBS, þ /– 2 mM GYY4137, at 5 days in the presence of activated macrophages. (C) Cell viability of MCF7shCBS þ 2 mM GYY4137 in the presence of macrophages versus scrambled controls. (D) Cell viability of 468shCBS þ 2 mM GYY4137 in the presence of macrophages versus scrambled controls (* P o 0.05).

MDA-MB-468 cells, suggesting that there could be other sources of endogenous H2S in these cells that were not investigated in this study. The decrease in CTH was also more pronounced in MCF7 cells compared with MDA-MB-468 following CBS silencing. This could be due to other reactions of CBS such as serine þhomocysteine -CTH þH2O [4] being more prevalent in MCF7 cells. Kinetic data utilizing purified CBS have also suggested the preferential condensation of Hcy and Cys in the production of H2S and CTH [4, 5]. Silencing of CBS in colon cancer cells significantly decreased the levels of CTH and H2S, which was associated with inhibition of cell growth in in vitro cultures. However, unlike that observed in colon cancer [32], CBS silencing per se did not affect the growth of HBC cells in in vitro cultures, although there was a decrease in the levels of H2S and CTH. This indicated that the mechanism of CBS action in breast cancer is likely to be different from that reported in colon cancer. As CBS is mechanistically linked to the antioxidant capacity of a cell [4,5], we hypothesized that HBC cells utilized CBS to protect themselves against oxidative insults induced exogenously by immune cells including infiltrated tumoricidal macrophages. In order to test our hypothesis, we cocultured HBC cells (both ER þve and ER-ve), with activated macrophages (that produce reactive oxygen and nitrogen species). Under these conditions silencing of CBS compromised the growth of HBC cells, confirming its protective role in HBC. The growth curves generated by viable cell counting over a period of time correlated with that of cell cycle analysis, indicating delayed cell cycle progression for CBS-silenced cells. ER þve, MCF-7 cells exhibited a decrease in S-phase population as well an increase in the GO/G1 phase, whereas the ER-ve,

MDA-MB-468 cells exhibited an increase in the sub-G1 population. This difference in the cell cycle population most likely suggested that MDA-MB-468 cells tended more toward apoptosis at the end of 5 days in cocultures with activated macrophages compared to MCF-7 cells. However both cell types indicated a general decrease in cell cycle progression. This difference may be attributed to their different ER status. HBE cells, MCF-10 A, did not express CBS and hence similar to CBS-silenced HBC cells were susceptible to growth inhibition induced by activated macrophages. As expected, overexpression of CBS in these cells made them resistant to macrophage-induced inhibition of cell growth. Our in vivo data utilizing the xenograft model that showed compromised growth of tumors on CBS silencing further confirmed our hypothesis, as nude mice that were injected with HBC cells were capable of producing macrophages. We next evaluated the mechanism by which CBS protected HBC cells, against activated macrophages. Our data demonstrated that silencing of CBS increased the steady state levels of adduct formed between reactive aldehydes and proteins in HBC cells when cultured in the presence of activated macrophages. It is well established that macrophages that inhibit tumor growth release oxidants that oxidize lipids on the plasma membrane to produce reactive aldehydes which in turn cause cytotoxicity by adduct formations with various biomolecules including proteins [27]. 4-HNE and MDA–protein adducts are stable compounds that represents footprints of oxidative damage and are known to be important in macrophage-induced oxidation [27]. We therefore monitored the changes in the levels of 4-HNE and MDA–protein adduct formations in HBC cells following cocultures with activated macrophages on silencing of CBS. We observed that the steady

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Fig. 6. The CBS/H2S machinery was detected on the plasma membrane fractions of HBC cells. (A) Immunoblot of Naþ/K þ ATPase and citrate synthase in plasma membrane fractions [20 mg] versus whole lysates [20 g] of MCF-7, MDA-MB-468, and MCF-10 A. (B) Cysteine/homocysteine-dependent H2S production by plasma membrane fractions of MCF-7, MDA-MB-468, and MCF-10 A. (C) Immunoblot of CBS in 20 mg plasma membrane fractions of MCF-7, MDA-MB-468, and MCF-10 A. Na K ATPase was used as loading controls for membrane fractions. (D) Viable cell count of MCF7scram at 48 h on exposure to 10 mM 4-HNE versus vehicle controls. (E) Viable cell count of MCF7shCBS at 48 h on exposure to 10 mM 4-HNE and/or 2 mM GYY4137 versus control (* Po 0.05).

state levels of these adducts in HBC cells were dependent on the expression of CBS. Several lines of evidences have reported a major role for 4-HNE in biology [34]. Hence we chose to monitor changes in 4-HNE–protein adducts in further investigations. Our observation that exogenous H2S could deplete the levels of 4-HNE–protein adducts in CBS-silenced HBC cells confirmed that the CBS-derived H2S was responsible for protection against reactive aldehydes. Our data indicated that the percentage decrease in the steady state levels of protein–4HNE adducts was 44% and 60% in MCF7shCBS and 468shCBS, respectively. This correlated to a lesser degree of recovery of cell growth on exogenous treatment of H2S in MCF7shCBS (i.e.,  40%) compared with  80% recovery in 468shCBS. Our results are in agreement with other studies performed in neuronal cells where exogenous H2S was shown to inactivate 4-HNE adducts as well to downregulate cytotoxicity induced by Aβ peptides [27]. Interestingly, we observed that the CBS/H2S machinery was associated with the plasma membrane in HBC cells. It is possible that CBS localizes to the plasma membrane during macrophagic attack to release H2S at the site of accumulation of oxidants released by macrophages. Our findings are supported by other studies that reported the localization of CBS in the plasma membrane of tissues from the gall bladder [35]. We, however, did not investigate the relative proportions of CBS distribution between the cytosol and the plasma membrane nor did we assess its localization in the mitochondrial fractions as is reported in colon cancer cells [32]. To assess the regulation of 4-HNE–protein adducts by H2S in a more direct manner, we exposed HBC cells with or without intact CBS to toxic concentrations of pure 4-HNE. As expected, CBS-silenced HBC cells were more sensitive to cell growth inhibition by

4-HNE when compared with scrambled controls. Moreover, preincubation with exogenous H2S partially rescued cell growth in CBS-silenced HBC cells. The fact that the rescue was not 100% could be due to the differential kinetics of the reaction of 4-HNE with cellular proteins compared with the initial kinetics of the reaction between 4-HNE and the slowly released H2S by its donor. Finally our data demonstrated that CBS was not a major regulator of the endogenous levels of GSH in HBC. The lack of CGL created a truncation in the transulfuration pathway, which inhibited the conversion of CTH to Cys and subsequently its incorporation into GSH. This phenomenon was also reported in neuroblastomas as well as in the early developing stages of the fetus [36,37]. Moreover our observation that viability of either CBS intact or silenced HBC cells (in the presence of activated macrophages) was not affected by BSO, a specific inhibitor of glutathione synthesis, further confirmed that glutathione did not play an important role in the regulation of macrophage-induced reactive aldehyde-mediated cytotoxicity in HBC cells. In this study we did not assess the role of other possible reducing agents such as Cys SSH (cysteine persulfides) that can be synthesized by CBS from cystines [38]. Thus the antioxidant capacity promoted by CBS may also include persulfides and H2S derived from persulfides, as recently reported in lung cancer cells [38]. This work may provide rationale for the utilization of tissuespecific CBS blockers as adjuvants in the treatment of breast cancer. Compromising CBS may significantly downregulate the cancer cell’s ability to counter oxidative stress induced by many chemotherapeutic drugs. This may thus increase drug efficacy in breast cancer treatment.

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6.0

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GSH/GSSG Rao M M C F7 CF sc 7s ram 46 hCB 46 8sc S 8s ram hC BS

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468scram 468scram+BSO 468shCBS 468shCBS+BSO

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Fig. 7. Glutathione status was independent of CBS expression in HBC cells. (A) Measurement of total glutathione (GSH) in MCF7scram/shCBS and 468scram/shCBS. (B) GSH/ GSSG ratio in MCF7scram/shCBS and 468scram/shCBS. (C) Immunoblot utilizing antibodies against cystathionine γ-lyase [CGL] in whole cell lysates of MCF-10 A, MCF-7, and MDA-MB-468. (D) Viable cell count of MCF7 cells either scrambled controls (MCF7scram) or silenced for CBS (MCF7shCBS) in cocultures with activated macrophages with or without pretreatment with buthione sulfoximine (BSO). (E) Viable cell count of MDA-MB-468 cells either scrambled controls (468scram) or silenced for CBS (468shCBS) in cocultures with activated macrophages with or without pretreatment with buthione sulfoximine (BSO) the inhibitor of glutathione synthesis (* P o 0.05).

Acknowledgments We acknowledge The Roberta Deutsch Foundation and Kelly Day for their financial support and Svetlana Roberts for technical help. We sincerely thank Prof. Pluth at the University of Oregon, for gifting us HSN2 for intracellular H2S measurements. We also acknowledge Ana Maria Cruz, M.S., for her contribution toward editing the language in this manuscript.

Appendix A. Supplementary material Supplementary data associated with this article can be found in the online version at http://dx.doi.org/10.1016/j.freeradbiomed. 2015.05.024

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