Phytochemical Antioxidants Induce Membrane Lipid Signaling in Vascular

Endothelial Cells

THESIS

Presented in Partial Fulfillment of the Requirements for the Degree Master of Science in the Graduate School of The Ohio State University

Jordan Douglas Secor

Graduate Program in Pathology

The Ohio State University

2012

Master's Examination Committee:

Dr. Narasimham Parinandi, Co-advisor

Dr. W. James Waldman, Co-advisor

Jordan Douglas Secor

2012

Abstract

Isolated phytochemicals have recently been increasingly consumed worldwide for prevention of cancer, cardiovascular and cerebrovascular diseases, and other ailments.

Hence, the phytochemical nutraceuticals have been thoroughly investigated as anticancer chemotherapeutics and use as dietary antioxidant continues to grow. Phytochemical polyphenols including baicalein, myricetin, rutin, and resveratrol are among a number of plant extracts believed to possess potential therapeutic efficacies. The putative anticancer effects of these natural compounds are believed to result, in part, from prevention of eicosanoid production through inhibition of 12-lipoxygenase (12-LOX). However, the mechanisms underlying these observations are not entirely clear and the effects of these compounds on noncancerous cells such as the vascular endothelial cells (ECs) are largely yet to be investigated. Few preliminary studies have reported that some isolated phytochemical antioxidants may actually act through a prooxidant mechanism. Earlier we have reported that prooxidants activate phospholipase D (PLD) signaling in ECs through depletion of intracellular thiols, generation of reactive oxygen species (ROS), and lipid peroxidation. We have also shown that oxidant-induced activation of PLD, which generates the potent bioactive lipid signaling molecule phosphatidic acid (PA), results in mitogenesis, cellular trafficking, cytoskeletal rearrangements and, ultimately, cytotoxicity. Consequently, here we used our established, bovine pulmonary artery EC

(BPAEC) model to examine whether the phytochemical polyphenol treatment would

modulate lipid signaling in the vascular ECs. We hypothesized that baicalein, a

polyphenolic phytochemical derived from Scutellaria baicalensis, would induce

activation of PLD through the loss of intracellular thiols, generation of ROS, induction of

peroxidation of membrane phospholipids in a prooxidant mechanism in the vascular

endothelial cells in culture, ultimately leading to cytotoxicity regulated by the PLD-

derived PA. Hence, in this study, we investigated this potential mechanism through (i)

indexing PLD activity by measuring PA formation and PLD1 and PLD2 in situ translocation and phosphorylation (ii) measuring formation of intracellular ROS (iii) analyzing global and PLD2-specific tyrosine phosphorylation (iv) measuring loss of

intracellular thiols (both GSH and total thiols) (v) quantifying 8-isoprostane release as a

measure of lipid peroxidation and (vi) measuring LDH release as an index of cytotoxicity.

With the use of different specific pharmacological inhibitors, we investigated the

mechanism of PLD activation in BPAECs as quantified by the formation of PA.

Furthermore, we observed this mechanism to be dependent on and attenuable through

inhibition of the following: (i) PLD activation with the PLD-specific inhibitor FIPI (ii)

loss of thiols through the thiol-protectants DTT and NAC (iii) free radical formation

through propyl gallate, MnTBAP, and MnTMPyP (iv) labile iron through the iron

chelator desferal (v) calcium loss through BAPTA and (vi) protein tyrosine kinase

(PTyK) signaling by the PTyK-specific inhibitors erbstatin, damnacanthal, tyrophostin

AG34, and genistein. Cumulatively, this study revealed for the first time that the

phytochemical polyphenolic 12-LOX inhibitor, baicalein induced activation of PLD in

iii

the vascular ECs through thiol-redox-dependent oxidant signaling and protein tyrosine kinase leading to the bioactive lipid (PA)-mediated cytotoxicity. Consequently, the cancer-specific chemotherapeutic efficacy, anti-angiogenic action, and antioxidant potential of baicalein and other polyphenolic phytochemicals must be reconsidered.

Acknowledgments

I would like to thank Dr. Narasimham Parinandi for his guidance and support as a mentor

in my continuing development as a research scientist. My success is a result of his efforts.

Also, I would like to thank Dr. Jim Waldman for affording me the opportunity to pursue a

Master of Science degree. I am forever grateful. Finally, I would like to thank Dr. Sainath

Kotha for training me by example to be an independent and innovative scientist.

Attaining Dr. Kotha’s clinical acumen and research excellence are lifetime goals.

Vita

May 2006 ...... Ada High School

2010...... B.S. Biology, The Ohio State University

Publications

Secor, J., Kotha, S., Gurney, T., Patel, R., Kefauver, N., Gupta, N., Morris, A., Haley, B.,

and Parinandi, N. 2011. Novel lipid-soluble thiol-redox antioxidant and heavy metal

chelator, N,N'-bis(2-mercaptoethyl)isophthalamide (NBMI) and phospholipase D-

specific inhibitor, 5-fluoro-2-indolyl des-chlorohalopemide (FIPI) attenuate mercury-

induced lipid signaling leading to protection against cytotoxicity in aortic endothelial

cells. International Journal of Toxicology 30, 619-638.

Kotha, S., Secor, J., Abbott, J., Gurney, T., Patel, R., Morris, A., Elton, T., Natarajan, V.,

and Parinandi, N. 2012. Angiotensin II-Induced Vascular Smooth Muscle Cell

Proliferation is Mediated by Phosphatidic Acid: Role of ERK-regulated Phospholipase D

Signaling. Submitted for publication in the J. Biological Chemistry.

Reviews:

Malireddy, S., Kotha, S., Secor, J., Gurney, T., Abbott, J., Maulik, G., Maddipati, K.,

Parinandi, and N. 2012. Phytochemical Antioxidants Modulate Mammalian Cellular

Epigenome: Implications in Health and Disease. Antioxidants and Redox Signaling [epub ahead of print].

Book Chapters:

Kotha, S., Secor, J., Malireddy, S., and Parinandi, N. 2012. Bioactive Phospholipid

Mediators of Inflammation. Chronic Inflammation: Molecular Pathophysiology,

Nutritional and Therapeutic Interventions. Cenveo Publishers.

Fields of Study

Major Field: Pathology

vii

Table of Contents

Abstract ...... ii-iv

Acknowledgments...... v

Vita ...... vvi-vii

List of Figures ...... ixx-x

Chapter 1: Introduction ...... pp. 1-5

Chapter 2: Materials and Methods ...... pp. 6-13

Chapter 3: Results ...... pp. 14-23

Chapter 4: Figures ...... pp. 24-51

Chapter 5: Discussion ...... pp. 55-62

Chapter 7: References ...... pp. 63-65

viii

List of Figures

1. Baicalein Structure 2. Baicalein Induces PLD Activation in Endothelial Cells A. Time Course and Dose Response in BLMVECs B. Time Course and Dose Response in BPAECs C. Kaempferol Does Not Activate PLD in BPAECs D. FIPI Inhibits Baicalein-Induced PLD Activation 3. Thiol-Protectants Inhibit Baicalein-Induced PLD Activation in Endothelial Cells A. DTT Inhibits Baicalein-Induced PLD Activation B. NAC Inhibits Baicalein-Induced PLD Activation 4. Free Radical Quenchers and Antioxidants Inhibit Baicalein-Induced PLD Activation in Endothelial Cells A. Propyl Gallate Inhibits Baicalein-Induced PLD Activation B. MnTBAP Inhibits Baicalein-Induced PLD Activation C. MnTMPyp Inhibits Baicalein-Induced PLD Activation 5. The Iron Chelator Desferal, but Not DTPA or EDTA, Attenuates Baicalein- Induced PLD Activation in Endothelial Cells A. DTPA Fails to Attenuate Baicalein-Induced PLD Activation B. EDTA Fails to Attenuate Baicalein-Induced PLD Activation C. Desferal Inhibits Baicalein-Induced PLD Activation 6. Intracellular Calcium Chelators Inhibit Baicalein-Induced PLD Activation in Endothelial Cells A. BAPTA Inhibits Baicalein-Induced PLD Activation 7. Protein Tyrosine Kinase Inhibitors Attenuate Baicalein-Induced PLD Activation in Endothelial Cells A. Erbstatin and Damnacanthal Inhibit Baicalein-Induced PLD Activation B. Tyrophostin AG34 Inhibits Baicalein-Induced PLD Activation 8. Baicalein Induces Translocation of PLD in Endothelial Cells A. Baicalein Induces Transolcation of PLD1 (confocal) B. Baicalein Induces Transolcation of PLD2 (confocal) 9. Baicalein Induces Tyrosine Phosphorylation in Endothelial Cells A. Baicalein Induces Global Tyrosine Phosphorylation (western) B. Baicalein Induces PLD1 Tyrosine Phosphorylation (confocal) C. Baicalein Induces PLD2 Tyrosine Phosphorylation (confocal) 10. Baicalein Causes Generation of ROS in Endothelial Cells ix

A. Baicalein Causes Generation of DHE (confocal) 11. NAC Protects Against Baicalein-Induced Loss of Thiols A. NAC Protects Against Baicalein-Induced Loss of Soluble Thiols (GSH) B. NAC Protects Against Baicalein-Induced Loss of Total Thiols 12. Baicalein Induces 8-Isoprostane Release from Endothelial Cells 13. Attenuation of Baicalein-Induced Cytotoxicity in Endothelial Cells A. NAC Attenuates Baicalein-Induced LDH Release B. Genistein Attenuates Baicalein-Induced LDH Release C. FIPI Attenuates Baicalein-Induced LDH Release Schema: Overall Mechanism

Chapter 1: Introduction

Biological membranes, the phospholipid bilayers enclosing each mammalian cell

and nucleus, are essential, unique and often understudied components of animal biology.

Each phospholipid bilayer is composed of amphipathic outer and inner leaflets. In turn, each leaflet contains outward-facing, polar, phosphate head groups which, through ester linkages, are bound to two inward-facing, nonpolar long chain fatty acids. The long chain fatty acid tails, through a shared chemical aversion of water and an affinity for one another, align forming an inner lipophilic membrane region. The polar, negatively charged head groups of the outer leaflet coalesce to form the outer membrane barrier to the extracellular environment and, on the inner leaflet, form the inner membrane barrier facing the cytoplasm. Differences in fatty acid composition, types of polar head groups, associations with membrane proteins, lipid rafts, and interactions with the extracellular environment are critical factors that contribute to the diverse function of biological membranes. Furthermore, the nature of biological membranes can vary widely from cell to cell, or spatially and temporally within the same cell as a function of these factors.

Regulation of membrane phospholipids is achieved, in large part, by the class of enzymes known as phospholipases. Phospholipases are ubiquitous, mammalian enzymes

that each cleave at distinct positions along the phospholipid to influence membrane

composition and integrity, regulate associations with the membrane, and generate

bioactive lipid signaling mediators. Phospholipases are divided into classes according to

the hydrolysis of membrane phospholipids and are currently designated as

phospholipases A1, A2, C and D. Within each class are different isoforms which share a

hydrolysis site but are otherwise differently regulated, expressed, and intracellularly located and/or have unique affinities and associations. The generation of bioactive lipid signaling mediators through phospholipase enzymes is highly-regulated and essential to cellular signaling. Lipid signaling is a dynamic process involved in many significant cellular events including cellular proliferation, induction of apoptosis, vesicular trafficking, and mitochondrial activity. Moreover, phospholipase enzyme activity has been implicated as a physiological factor in diseases including cancer and cardiovascular disease and phospholipase-knockout animals are not viable. Of particular interest to our group is phospholipase D (PLD) which preferentially hydrolyzes phosphatidylcholine

(PC) at the head group of the phospholipid to generate phosphatidic acid (PA) and choline.

PA is a potent bioactive lipid signaling molecule that plays important roles in

MAP kinase and Rho kinase signaling and is a substrate for conversion to diacylglycerol

(DAG) by phosphatidic acid phosphohydrolase (PAP). Specifically, PLD-generated PA is essential for myocardial calcium regulation in the heart (Dai et al., 1992), COPI vesicle fission in golgi maintenance (Yang et al., 2008), and von Willebrand factor secretion

from endothelial cells (Disse et al., 2009). The activity of PLD is regulated through

phosphorylation, intracellular translocation, and to some extent expression and transcription. Important to our study is the mechanism of agonist-mediated induction of

PLD activation and the effectiveness of the pharmacological inhibitors which ameliorate such activation.

Among the myriad of agonists (physiological and pharmacological) known to activate PLD, oxidants display the most robust activation. Specifically phorbol esters, such as 12-O-tetradecanoylphorbol-13-acetate (TPA), and reactive oxygen species

(ROS), such as hydrogen peroxide (H2O2) and other oxidants, induce significant PLD

activation as demonstrated by PA formation, phosphorylation, and intracellular

translocation in mammalian cells including endothelial and smooth muscle cells. The role of ROS has been extensively studied in vascular systems including endothelial and smooth muscle cells. Not surprisingly, oxidants which disrupt essential redox signaling in vascular cells, by decreasing nitric oxide (NO), superoxide dismutase (SOD), and

glutathione peroxidase (GSH-PX) activity, ultimately induce cytotoxicity in cultured

cells. The vascular endothelium, the specialized inner monolayer lining the vascular

lumen, is uniquely sensitive to oxidant-induced redox destabilization through the actions

of ROS and the subsequent cytotoxicity. The mechanism by which ROS-destabilized

redox signaling in the endothelium culminates in cytotoxicity is multifaceted and not

entirely resolved. However, studies have shown that ROS-induced disruption of essential

lipid signaling, including dysregulation of PLD-generated PA formation, is involved and

redox-stabilizing antioxidants, such as dithiothreitol (DTT) and N-acetylcysteine (NAC), attenuate oxidant-induced PLD activity and associated cytotoxicity (Parinandi et al.,

1999). ROS-mediated PLD activation is also attenuated by protein tyrosine kinase inhibitors (Parinandi et al., 2001).

In addition to the chemically synthesized DTT and NAC, resveratrol and other phytochemical antioxidants have demonstrated the ability to ameliorate endothelial cell injury induced by cisplatin and other oxidants (Attia, 2012). Similarly, rhein, an anthraquinone found in rhubarb, has shown to protect against ROS production and LDH release in H2O2-treated human umbilical vein cells (HUVECs) (Zhong et al., 2012).

Quercetin, another plant-derived dietary polyphenol, exhibited inhibitory effects on other

ROS markers (mitochondrial swelling and glutathione depletion) in sodium deoxycholate-treated chicks (Rivoira et al., 2012). While the antioxidant effects of phytochemical polyphenols is apparent, the mechanism(s) by which the effects are conferred remain unclear. Many polyphenolic phytochemical compounds have exhibited inhibitory effects on lipoxygenases (LOX) 5, 10, and 12. As LOX inhibitors, phytochemical antioxidants antagonize production of leukotrienes, the eicosanoids which cause vascular dysfunction. In addition to serving as antioxidants and promising anticancer therapeutics, it appears that some phytochemical polyphenols are also prooxidants. Here, we hypothesized the widely used phytochemical antioxidant and

LOX-inhibiting drug, baicalein, would activate PLD signaling in vascular endothelial

cells through prooxidant mechanisms, thiol-redox dysregulation, and tyrosine kinase activation.

Chapter 2: Materials and Methods

Materials

Bovine pulmonary artery ECs (BPAECs) (passage 2) were obtained from Cell

Applications Inc. (San Diego, CA). Phosphate-buffered saline (PBS) was obtained from

Biofluids Inc. (Rockville, MD). Minimal essential medium (MEM), nonessential amino

acids, trypsin, fetal bovine serum (FBS), penicillin/streptomycin, DMEM phosphate-free

modified medium, aprotinin, leupeptin, BAICALEIN, nonessential amino acids, trypsin-

EDTA, disodium ethylenediaminetetraacetic acid (Na2-EDTA), diethylenetriaminepentaacetic acid (DTPA), N-acetylcysteine (NAC), propyl gallate, dimethyl sulfoxide (DMSO), lactate dehydrogenase cytotoxicity assay kit (LDH release assay kit), t-octylphenoxypolyethoxyethanol (Triton X-100), bovine serum albumin

(BSA), and analytical reagents of highest purity were all purchased from Sigma

Chemical Co. (St. Louis, MO). 5-Fluoro-2-indolyl des-clorohalopemide hydrochloride

hydrate (FIPI) was prepared as described earlier.5,20,21 GSH chemiluminescence assay kit

(GSH-Glo) was obtained from Promega Corporation (Madison, WI).Phosphatidylbutanol

(PBt) was obtained from Avanti Polar Lipids (Alabaster, AL). [32P] Orthophosphate

(carrier-free) was obtained from New England Nuclear (Wilmington, DE). Damnacanthal

was obtained from Biomol Research Laboratories (Plymouth, PA). Desferal, erbstatin,

genistein, tyrophostin AG 34, obtained from Calbiochem (San Diego, CA).

Dihydroethidine (DHE) was purchased from Molecular Probes (Eugene, OR).

Endothelial cell growth factor was obtained from Upstate Biotechnology (Lake Plack,

NY). Polyclonal primary rabbit antibodies against PLD1 and PLD2 were purchased from

Biosource (Camarillo, CA). Primary mouse anti-phosphotyrosine antibodies were obtained from Upstate Biotechnology (Waltham, MA). Secondary anti-rabbit Alexafluor

488-conjugated and anti-mouse Alexafluor 586-conjugated antibodies were obtained

from Molecular Probes (Eugene, OR). Kaempferol and the assay kits for measuring 8-

isoprostane were obtained from Cayman Chemical Company (Ann Arbor, MI). Protease

inhibitor cocktail tablets were obtained from Roche (Indianapolis, IN). HRP-conjugated

anti-rabbit and anti-mouse secondary antibodies and the enhanced chemiluminescence kit

(ECL) for the detection of proteins by Western blots were obtained from Amersham

Biosciences (Arlington Heights, IL).

Culture of Endothelial Cells

BPAECs were grown to confluence in MEM supplemented with 10% fetal bovine serum,

100 units/ml penicillin and streptomycin, 5 μg/ml endothelial cell growth factor and 1%

o nonessential amino acids at 37 C in a 95% air-5% CO2 atmosphere as described earlier

(53, 54 ). BPAECs, from passages 8 to 20 and bovine lung microvascular endothelial

cells (BLMVECs) were used in the experiments. ECs from each primary T-75 cm flask

were detached with 0.05% trypsin, resuspended in fresh medium, and sub-cultured in 35-

mm or 60-mm sterile dishes or T-75 cm sterile flasks in complete medium to ~95%

o confluence under 95% air-5% CO2 at 37 C for treatment with baicalein and desired

pharmacological agents. Stock solutions of baicalein and other water-insoluble

pharmacological inhibitors were dissolved in tissue culture grade DMSO and diluted in

MEM for treatment of cells, keeping the final DMSO concentration to 0.1%.

Assay of PLD activation in ECs

BPAECs in 35-mm dishes (5 x 105 cells/dish) were prelabeled with [32P]orthophosphate

(5 μCi/ml) in DMEM phosphate-free medium containing 2% fetal bovine serum for 12-

14 h (53, 54). Cells were washed with MEM and incubated at 37o C in 1 ml of MEM

containing 0.05% butanol in absence and presence of desired concentrations of baicalein

for different lengths of time under a humidified 95% air-5% CO2 atmosphere. In some

experiments, wherever required, ECs were pretreated for 1 h with selected

pharmacological inhibitors prior to exposure to baicalein alone or baicalein in presence of

pharmacological inhibitors for the desired length of incubation times. Wherever DMSO was used to prepare the stock solutions of the pharmacological agent to be used for cellular treatments, appropriate controls were established with treatments containing

identical concentrations of DMSO alone. The incubations were terminated by addition of

methanol:conc. HCl (100:1, by vol.). Lipids were extracted essentially according to the

method of Bligh and Dyer as described previously (53, 54). [32P]-labeled

phosphatidylbutanol (PBt) formed as a result of PLD activation and

transphosphatidylation reaction, an index of PLD activity in intact cells, was separated

utilizing the thin-layer chromatography (TLC) on 1% potassium oxalate-impregnated

silica gel H plates using the upper phase of ethyl acetate:2,2,4-trimethyl pentane:glacial acetic acid:water (65:10:15:50, by vol.) as the developing solvent system (53, 54).

Unlabeled PBt was added as a carrier during the lipid separation by TLC and was visualized under iodine vapors. Radioactivity associated with the [32P]-PBt was

quantified with the aid of liquid scintillation counting and data were expressed as DPM

normalized to 106 counts in the total cellular lipid extract or as % of control (vehicle-

treated control cells).

Preparation of cell lysates, sodium dodecyl sulfate-polyacrylamide gel

electrophoresis (SDS-PAGE) and western blotting

Preparation of cell lysates of proteins, SDS-PAGE and western blotting of proteins were

carried out according to our established procedures (54). BPAECs were treated with

MEM or MEM containing pharmacological inhibitors or baicalein or baicalein plus

pharmacological inhibitors at chosen concentrations for different lengths of time. Cells

were washed with ice-cold phosphate-buffered saline (PBS) containing 1 mM vanadate

and were lysed in a buffer containing 20 mM Tris (pH 7.4), 137 mM NaCl, 1 mM EDTA,

1% NP-40, 0.5% Triton X-100, 2 μg/ml leupeptin, 1 μg/ml aprotinin, 1 mM PMSF, 1 mM vanadate, and protease inhibitor cocktail (1 tablet/10 ml of lysis buffer). Cell lysates were sonicated thrice at a setting of 3 with a probe sonicator for 5 seconds and centrifuged at 10,000g for 15 min at 4o C. An aliquot of the supernatant was used for

protein determination utilizing the BCA protein assay (Pierce).

The extent of tyrosine phosphorylation (phosphotyrosine formation) in proteins was

detected, as described previously (36). For analysis of phosphotyrosine in total proteins,

the supernatants of cell lysates containing equal amounts of protein were mixed with 6X

Laemmli buffer and boiled for 5 min. Proteins were separated on 8% gels employing the

SDS-PAGE, transferred onto PVDF membranes, washed 3 times with Tris-buffered

saline containing 0.1% Tween-20 (TBST), and subjected to immunoblotting with mouse

anti-phosphotyrosine antibodies (1:1000 dilution) in TBST containing 3% milk overnight

at 4o C. The membranes were washed 3 times with TBST (20 min each washing), incubated with secondary anti-mouse IgG-HRP conjugate antibodies (1:5000 dilution) in

TBST containing 3% milk for 1 h at room temperature, washed 3 times with TBST (20

min each washing), and the immunoblots containing antiphosphotyrosine bands were developed with ECL Western Blot detection reagents according to the manufacturer’s

recommendations.

Confocal immunofluorescence microscopy

Immunofluorescence microscopy of PLD1, PLD2 and phosphotyrosine in intact BPAECs was carried out as described previously (54). Coverslips (Harvard Apparatus, 22 mm2) were sterilized in the laminar flow tissue culture hood for 30 min with 70% ethanol and were placed in the 6-well 35-mm sterile plates. Upon drying the coverslips in the tissue culture hood, BLMVECs were seeded in the 35-mm wells of the 6-well plate at a density of 104 cells/35-mm dish, 24 h preceding the experiment. BLMVECs, thus grown on

sterile coverslips were treated with MEM alone and MEM containing baicalein at

different concentrations for specified lengths of time under a humidified atmosphere of

o 95% air-5% CO2 at 37 C. At the end of the incubation period, cells attached to coverslips

were washed with 1X PBS and fixed with 3.7% of para-formaldehyde for 10 min,

permeabilized with 0.25% Triton X-100 in TBST containing 0.01% Tween-20 for 5 min,

and blocked for 30 min with 1% BSA in 0.01% TBST. Cells grown on coverslips were

incubated with (1) rabbit primary anti-PLD1 (N-terminal and Internal) or anti-PLD2 (N- terminal and Internal) antibodies at a dilution of 1:200 for visualization of PLD or (2) mouse primary anti-phosphotyrosine antibodies at a dilution of 1:200, in 0.01% TBST containing 1% BSA for 1 h at room temperature. Following treatment of cells with the chosen primary antibodies, they were incubated with secondary anti-rabbit AlexaFluor

488-conjugated and secondary anti-mouse AlexaFluor 586-conjugated antibodies (1:100 dilution), wherever necessary, for 1 h at room temperature. The coverslips with cells were then mounted on a glass slide with the antifade mounting medium, Fluoromount-G, viewed under Zeiss confocal microscope at a magnification of 60 X, and pictures were captured digitally. The digital images were quantitatively analyzed for fluorescence intensities with the ImageJ software.

Detection of intracellular ROS generation

Generation of superoxide in BPAECs was determined by DHE (dihydroethidium)

fluorescence (Khan M et al, JPET 2010). BPAECs grown in 35-mm dishes were washed once with phenol-red free MEM prior to exposure to baicalein (0-100 µM) for 2 h. After treatment, the cells were washed once with warm PBS and incubated with DHE (5 µM)

in warm PBS for 30 min. After incubation, cells were washed once with warm PBS and

examined under Zeiss LSM 510 Confocal/Multiphoton Microscope at 543-nm excitation

with a 560-nm long pass filter at 10X magnification. These images were captured digitally. Fluorescent intensity was determined using he NIH sponsored program ImageJ.

GSH Determination

Intracellular soluble thiol (GSH) levels were determined using the GSH-Glo GSH assay kit as reported earlier (Patel et al., 2011). BPAECs grown up to 90% confluence in 96

well plates were treated with MEM alone or MEM containing desired concentrations of

treatments under a humidified 95% air - 5% CO2 atmosphere. Following incubation, intracellular GSH levels were determined according to the manufacturer’s recommendations (Promega Corp. Madison, WI).

Lactate Dehydrogenase (LDH) Release Assay for Cytotoxicity

Cytotoxicity in BPAECs was determined by assaying the extent of release of LDH from cells according to our previously published method (Patel et al., 2011). At the end of

treatment, the medium was collected and LDH released into the medium was determined

spectrophotometrically by using the commercial LDH assay kit according to the

manufacturer’s recommendations (Sigma Chemical Co., St. Louis, MO).

Protein determination

Protein was determined employing the BCA protein assay (Pierce).

Statistical analysis

All experiments were done in triplicate. Data were expressed as mean ± standard

deviation (SD). Statistical analysis was carried out with ANOVA using SigmaStat

(Jandel). The level of statistical significance was taken as p < 0.05.

Chapter 3: Results

Baicalein, a Polyphenolic Phytochemical, Activates PLD in Endothelial Cells

Baicalein, a polycyclic, polyphenolic phytochemical, has been reported as an antioxidant

which inhibits cancer cell proliferation through inhibiting lipoxygenase (LOX) (Figure

1A). Baicalein is a flavone within the flavanoid class of compounds and is found in the

root of Scutellaria baicalensis or Baikal Skullcap. Phytochemical antioxidants similar in

structure and origin to baicalein have primarily been reported as antioxidants with

therapeutic potential but recent reports suggest some of these phytochemicals may act

through a prooxidant mechanism (REF). Also, earlier we have reported that prooxidants

activate PLD lipid signaling in vascular ECs (REF). Therefore, here we examined

whether baicalein treatment would induce activation of PLD in our BPAEC model. After

1 and 2 h of treatment, baicalein (10, 50, and 100 µM) induced significant and dose-

dependent activation of PLD ([32P]PBt formation) in BPAECs relative to untreated

control cells (Figure 2A). To determine if this enzyme activation was unique to our

BPAEC model, we also conducted the experiment with our bovine lung microvascular

endothelial cell (BLMVEC) model under identical conditions and found similar significant and dose-dependent activation of PLD following baicalein treatment relative

to untreated control cells (Figure 2B). Next, we examined whether the well-estbalished

LOX-specific inhibitor, kaempferol, would induce PLD activation in BPAECs (Figure

1B). Surprisingly, kaempferol, for the same duration of treatment (1 and 2 h) and

concentrations (10, 50, and 100 µM), did not induce significant activation of PLD in

BPAECs (Figure 2C). Following these experiments, we focused our investigation on

determining the mechanism by which baicalein induced activation of PLD in BPAECs.

All experiments from here onward were conducted using our BPAEC model with

baicalein as the agonist. In our earlier reports, we have shown that agonist-induced PLD

activation can be attenuated by the novel PLD-specific inhibitor 5-fluoro-2-indolyl des-

chlorohalopemide (FIPI). However, FIPI has not been evaluated as inhibitor to baicalein- induced PLD activity. Accordingly, we tested whether baicalein-induced activation of

PLD could be attenuated by FIPI treatment. BPAECs pre-treated with FIPI (250 nM) for

12 h prior to treatment with baicalein (100 µM) for 2 h showed significant attenuation of baicalein-induced PLD activation to levels equal to untreated control cells (Figure 2D).

These results revealed that baicalein significantly and uniquely activated PLD in endothelial cells and baicalein-induced PLD activation was attenuated by the PLD- specific inhibitor FIPI.

Thiol-Protectants Inhibit Baicalein-Induced PLD Activation in Endothelial Cells

Earlier, we have reported that agonist- and oxidant- induced activation of PLD in

endothelial cells, including BPAECs, occurs through thiol-redox dysregulation. We have

also shown that agonist- and oxidant-mediated PLD activation can be attenuated by

treatment with the known thiol-protectants, dithiothreitol (DTT) and N-acetylcysteine

(NAC). To determine if baicalein-induced activation of PLD would be mediated through thiol-redox dysregulation in endothelial cells, we pre-treated BPAECs with DTT (10 mM) or NAC (5 mM) for 1 h and challenged the cells with baicalein (100 µM) for 2 h.

DTT significantly attenuated baicalein-induced PLD activation to near control levels observed in untreated control cells (Figure 3A). Similarly, NAC significantly attenuated baicalein-induced activation of PLD to levels almost equal to untreated control BPAECs

(Figure 3B). From these experiments, we ascertained that baicalein, similar to other known oxidants and PLD agonists, activated PLD through thiol-redox dysregulation and baicalein-induced PLD activation was attenuated by the thiol-protecting agents DTT and

NAC.

Free Radical-Quenching Antioxidants Inhibit Baicalein-Induced PLD Activation in

Endothelial Cells

As the next step in elucidating the mechanism of PLD activation induced by baicalein, we analyzed the efficacy of free radical quenching antioxidants in inhibiting baicalein- induced PLD activation in BPAECs. We hypothesized that baicalein, because it caused induction of PLD through thiol-redox dysregulation, would also generate ROS in

BPAECs similar to other agonists which activate PLD through thiol-redox dysregulation.

Furthermore, we speculated that baicalein-induced PLD activation would be reduced by treatment with antioxidants. Earlier studies have revealed that antioxidants, presumably through quenching free radicals and preventing free radical generation, are effective inhibitors of oxidant-induced PLD activity. Propyl gallate (a polyphenolic ester used as a

preservative for foods containing oils), MnTBAP (a superoxide dismutase [SOD]

mimetic), and MnTMPyp (another SOD mimetic) are all antioxidants with proven

efficacy which we tested here. In BPAECs pre-treated with propyl gallate (500 µM) for 1 h prior to treatment with baicalein (100 µM) for 2 h, a significant decrease in the extent

of PLD activation was observed (Figure 4A). Similar attenuation in baicalein-induced

PLD activity was also observed in BPAECs pre-treated for 1 h with MnTBAP (5 µM)

and MnTMPyP (1 µM) (Figures 4B and C, respectively). These results revealed that

baicalein likely activated PLD, at least in part, through generation of ROS as witnessed

by the significant reductions in [32P]PBt formation in cells pre-treated with free radical-

quenching antioxidants.

Iron Chelator Desferal, but Not DTPA or EDTA, Attenuates Baicalein-Induced

PLD Activation in Endothelial Cells

After demonstrating that baicalein acted as a PLD agonist through generation of ROS, we

investigated the role of iron in facilitating the ROS-mediated baicalein induction of PLD

activity. Oxidant-mediated generation of intracellular ROS often occurs via the Fenton

reaction which requires labile iron to serve as a catalyst for free radical initiation (REF).

Here, we utilized three distinct iron-chelating compounds, DTPA, EDTA, and desferal, each with unique coordination chemistry and iron affinities, to determine the dependence of baicalein on intracellular iron in generating ROS to activate PLD. BPAECs pre-treated with DTPA (5 mM) and EDTA (5 mM) for 1 h prior to treatment with baicalein (100

µM) for 2 h showed no attenuation of baicalein-induced PLD activity relative to cells

treated with baicalein alone (Figures 5A and B, respectively). In fact, it appeared DTPA

and EDTA pre-treatment slightly enhanced baicalein-induced PLD activity relative to

BPAECs treated with baicalein alone. The mechanism underlying this observation is likely due to the coordination chemistry of DTPA and EDTA with labile iron but this matter was beyond the scope and outside the focus of our investigation. However, cells pre-treated with desferal (1 and 5 mM) for 1 h prior to treatment with baicalein (100 µM) for 1 h exhibited significant decreases in baicalein-induced PLD activity relative to cells treated with baicalein alone (Figure 5C). These results further substantiated the notion that baicalein induces PLD activation through ROS production involving iron.

Additionally, we observed that baicalein-induced PLD activation was dependent on the availability of labile iron as pre-treatment with the iron chelator desferal was effective in attenuating baicalein-induced PLD activity.

Intracellular Calcium Chelator, BAPTA, Inhibits Baicalein-Induced PLD Activity in Endothelial Cells

Earlier, we have reported the role of calcium in the agonist-induced PLD activation in

BPAECs (Peltz et al.). This report and our finding on the dependence of baicalein on intracellular iron led us to examine the dependence of baicalein-induced PLD activation on intracellular calcium. To determine this dependence, we used the intracellular calcium chelator, BAPTA. BAPTA is calcium-specific polyamino carboxylic acid which has demonstrated efficacy in use as a calcium chelator in vitro. BPAECs pre-treated with

BAPTA (5 µM) for 1 h before being treated with baicalein (100 µM) for 2 h showed a

significant attenuation in baicalein-induced PLD activity relative to cells treated with baicalein alone (Figure 6). These results revealed that PLD activation through baicalein treatment was dependent on intracellular calcium and the intracellular calcium chelator,

BAPTA, was effective at attenuating baicalein-induced PLD activity.

Protein Tyrosine Kinase Inhibitors Attenuate Baicalein-Induced PLD Activation

Molecular studies have demonstrated that PLD activity can be regulated by tyrosine phosphorylation (REF). This fact led us to examine if baicalein-induced activation of

PLD was dependent on protein tyrosine kinase signaling. We used the known tyrosine kinase-specific inhibitors, erbstatin, damnacanthal, and tyrophsotin AG34 to examine this effect. BPAECs pre-treated for 1 h with erbstatin (10 µM) before being treated with baicalein (100 µM) for 2 h displayed a significant reduction in PLD activity relative to cells treated with baicalein alone (Figure 7A). Additionally, cells pre-treated with damnacanthal (10 µM) for 1 h prior to treatment with baicalein (100 µM) for 2 h showed an even greater attenuation of baicalein-induced PLD activity to levels equal to untreated control cells. Pre-treament with tyrophostin AG34 (10, 50, and 100 µM) prior to treatment with baicalein (100 µM) for 2 h was also effective in inhibiting baicalein- induced PLD activity and tyrophostin AG34-mediated inhibition was demonstrated in a dose-dependent fashion (Figure 7B). For the first time, these results demonstrated that the effects induced by baicalein treatment, including PLD activation, were dependent on protein tyrosine kinase signaling and the PTyK inhibitors erbstatin, damnacanthal, and tyrophostin AG34 effectively attenuated baicalein-induced PLD activity.

Baicalein Induces in situ Translocation of PLD1 and PLD2 in Endothelial Cells

Previously, we have demonstrated that translocation of both isoforms of PLD, PLD1 and

32 PLD2, occurs concomitantly with agonist-induced PLD activity ([ P]PBt formation) in

endothelial cells (REF). Therefore, in this study we sought to establish whether baicalein-

induced PLD translocation in situ would precede the enzyme activation and tested

whether the novel PLD-specific inhibitor, FIPI, would attenuate enzyme translocation.

Untreated, control BPAECs exhibited very low levels of microscopically visible PLD1 and PLD2 but, upon treatment with baicalein (50 and 100 µM) for 1 h, vivid PLD1 and

PLD2 staining was visible (Figures 8A and B, respectively). Furthermore, BPAECs pre- treated with the PLD-specific inhibitor, FIPI (500 nM), for 4 h prior to treatment with baicalein (50 and 100 µM) for 1 h exhibited significantly less PLD1 and PLD2 translocation relative to cells treated with baicalein alone. These observations revealed that PLD1 and PLD2 translocation was involved in the mechanism by which baicalein

induces PLD activation in BPAECs and the PLD-specific inhibitor FIPI was effective in

attenuating baicalein-induced PLD1 and PLD2 in situ translocation.

Baicalein Stimulates Global and PLD1 and PLD2 Tyrosine Phosphorylation in

Endothelial Cells

Earlier in this study, we revealed that the protein tyrosine kinase inhibitors erbstatin,

damnacanthal, and tyrophostin were effective in attenuating baicalein-induced ([32P]PBt formation). This discovery led us to investigate the effect of baicalein on global tyrosine

phosphorylation and, specifically, PLD1 and PLD2 phosphorylation. BPAECs treated

with baicalein (10, 50, and 100 µM) for 1 h demonstrated significant, dose-dependent

increases in global tyrosine phosphorylation relative to untreated control cells as

visualized by western blotting with specific monoclonal anti-phosphotyrosine antibodies

(Figure 9A). Furthermore, BPAECs treated with baicalein (100 µM) for 30, 60, and 120

min exhibited significant increases in global tyrosine phosphorylation in a time-

dependent fashion with the greatest phosphorylation occurring at 30 min and decreasing

temporally in comparison to untreated control cells. PLD1 and PLD2 phosphorylation also

increased following baicalein (50 and 100 µM) treatment for 1 h relative to untreated

control cells as seen demonstrated by the immunofluorescence microscropy and staining

with antiPLD1-phosphotyrosine- and antiPLD2-phosphotyrosine-specifc antibodies

(Figures 9B and C, respectively). These results revealed, for the first time, that baicalein

induced global and PLD1- and PLD2-specific phosphorylation in BPAECs.

Baicalein Causes Generation of ROS in Endothelial Cells

Our earlier observation that the free radical-quenching antioxidants propyl gallate,

MnTBAP, and MnTPyP attenuated baicalein-induced PLD activity led us to determine baicalein-induced ROS production in BPAECs. In accordance with our earlier findings, we hypothesized that baicalein treatment would cause a generation of ROS in BPAECs.

To test this hypothesis, we used DHE staining to visualize superoxide production

following treatment with baicalein. BPAECs treated with baicalein (50 and 100 µM) for

2 h exhibited markedly greater DHE staining when compared to untreated control cells

with and without DHE which displayed little to no staining (Figure 10). These images further validated our hypothesis that baicalein caused activation of PLD through generation of ROS.

NAC Protects Against Baicalein-Induced Loss of Thiols in Endothelial Cells

Our earlier studies and studies by others have shown that oxidants deplete intracellular thiols, including glutathione (GSH), which cause redox-dependent activation of PLD

(REF). To further substantiate the notion that baicalein acts as a prooxidant molecule in our BPAEC model, we determined the effect of baicalein treatment on soluble thiols and total intracellular thiols. NAC, the thiol-protectant which has the ability to attenuate baicalein-induced PLD activation, was also tested here to attenuate baicalein-induced loss of thiols. In agreement with our earlier findings, BPAECs treated with baicalein (25 and

50 µM) for 2 h exhibited significant depletion of GSH and total intracellular thiols relative to untreated control cells (Figures 11A and 11B, respectively). Furthermore,

NAC (1mM) pre-treatment for 2 h prior to baicalein treatment prevented the losses in

GSH and total intracellular thiols exhibited by cells treated with baicalein alone. These data revealed that baicalein depleted both GSH and total intracellular thiols and these depletions were attenuated by pre-treatment with NAC.

Baicalein Generates 8-Isoprostane Release from Endothelial Cells

Next, we investigated if the putative intracellular oxidant, baicalein, would affect membrane phospholipids through lipid peroxidation. The rationale underlying this

investigation was the idea that as both an oxidant (thiol-depletion and ROS generation)

and PLD activator ([32P]PBt formation, translocation, phosphorylation) baicalein might

cause the peroxidation of membrane phospholipids. To examine this possibility, we measured the release of 8-isoprostane which is a product of non-enzymatic, radical oxygen-mediated oxidation of membrane phospholipids. BPAECs treated with baicalein

(25, 50, and 100 µM) displayed significant, dose-dependent increases in 8-isoprostane release relative to untreated control cells (Figure 12). These results revealed that baicalein caused lipid peroxidation of membrane phospholipids by generating ROS.

Baicalein-Induced Cytotoxicity Can Be Attenuated by Thiol Protection and

Inhibition of Protein Tyrosine Kinase and PLD in Endothelial Cells

The effects we observed on PLD activity, PLD translocation, PLD phosphorylation, ROS production, and 8-isoprostane release induced by baicalein in BPAECs directed us to evaluate the temporally downstream effects of baicalein on cytotoxicity. Here, we measured lactate dehydrogenase (LDH) release as index of cytotoxicity which is an established marker of cell death and damage. Further, we evaluated the ability of the thiol-protectant NAC, the tyrosine kinase inhibitor genistein, and the PLD-specific inhibitor FIPI to attenuate baicalein-induced LDH release. BPAECs treated with baicalein (25, 50, and 100 µM) for 12 h consistently exhibited significant increases in

LDH release relative to untreated control cells (Figure 13A). BPAECs pre-treated with

NAC (1 mM) and genistein (100 µM) exhibited significantly less baicalein-induced LDH release than cells treated with baicalein alone (Figure 13B). Additionally, BPAECs pre-

treated with FIPI (250 and 500 nM) exhibited significant, dose-dependent reductions in baicalein-induced LDH release relative to cells treated with baicalein alone (Figure 13C).

These results revealed, for the first time, that baicalein caused cytotoxicity in BPAECs

and these effects were mediated through ROS generation, thiol depletion, protein tyrosine

kinase signaling, and PLD activation as inhibitors to each facet effectively inhibited

baicalein-induced cytotoxicity.

Chapter 4: Figures

Fig. 2A 1h 2h

4500

4000 * *

3500 * 3000

2500 27 * 2000 * * 1500

1000

P] PBt Formation (DPM/dish) 500 32 [

0 Vehicle 10 50 100 Concentration of Baicalein (µM)

Fig. 2B 1h 2h

5000 * 4500

4000 * 3500 * 3000 *

2500 28 2000 * * 1500

P] PBt Formation (DPM/dish) Formation PBt P] 1000 32 [ 500

0 Vehicle 10 50 100 Concentration of Baicalein (µM)

Fig. 2C 1h 2h 4000

3500

3000 * * 2500 *

2000 29 * 1500

P] PBt Formation (DPM/dish) 1000 32 [

500

0 Vehicle 10 50 100 Concentration of Kaempferol (µM)

Fig. 2D Control FIPI (250 nM) 1600 * 1400

1200

1000

800 30 ** 600

400 P] PBt Formation (DPM/dish) 32 [ 200

0 Vehicle Baicalein (100 µM)

Fig. 3A Control DTT (10 mM) 4000 * 3500

3000

2500 **

2000 31

1500

P] PBt Formation (DPM/dish) 1000 32 [

500

0 Vehicle Baicalein (100 µM)

Fig. 3B Control NAC (5 mM) 5000 * 4500

4000

3500

3000

2500 32

2000

1500 P] PBt Formation (DPM/dish) 32 [ 1000 **

500

0 Vehicle Baicalein (100 µM)

Fig. 4A Control 5000 Propyl Gallate (500 µM)

4500

4000

3500 **

33 3000

2500

2000

1500 P] PBt Formation (DPM/dish) 32 [ 1000

500

0 Vehicle Baicalein (100 µM)

Fig. 4B Control

7000 MnTBAP (5 µM) * 6000 ** 5000

4000 34

3000

2000 P] PBt formation (DPM/dish) PBt formation P] 32 [

1000

0 Vehicle Baicalein (100 µM)

Fig. 4C Control 6000 MnTMPyp (1 µM) * 5000

4000 **

3000 35

2000 P] PBt Formation (DPM/dish) 32 [ 1000

0 Vehicle Baicalein (100 µM)

Fig. 5A Control

DTPA (5 mM) 7000 * 6000

5000 *

4000 36 3000

2000 P] PBt (DPM/dish) Formation 32 [ 1000

0 Vehicle Baicalein (100 µM)

Fig. 5B Control 5000 EDTA (5 mM) 4500 *

4000

3500 * 3000

2500 37

2000

P] PBt formation (DPM/dish) PBt formation P] 1500 32 [ 1000

500

0 Vehicle Baicalein (100 µM)

Fig. 5C Control Desferal (1 mM) 7000 Desferal (5 mM)

6000 *

5000 **

4000 ** 38 3000

P] PBt formation (DPM/dish) PBt formation P] 2000 32 [

1000

0 Vehicle Baicalein (100 µM)

Fig. 6 Control

7000 BAPTA (5 µM) * 6000

5000 **

4000 39 3000

2000 P] PBt Formation (DPM/dish) 32 [

1000

0 Vehicle Baicalein (100 µM)

Fig. 7A Control

6000 Erbstatin (10 µM)

* 5000

4000 **

3000 40

2000 P] PBt Formation (DPM/dish) P] PBt Formation 32 [ **

1000

0 Vehicle Baicalein (100 µM)

Fig. 7B Control Tyrophostin AG34 (10 µM)

3000 Tyrophostin AG34 (50 µM) * Tyrophostin AG34 (100 µM) 2500

2000 **

41 1500 ** 1000 ** P] PBt Formation (DPM/dish) 32 [ 500

0 Vehicle Baicalein (100 µM)

Fig. 11A 30

Vehicle 25 NAC (1 mM)

20 cells) 6 15 (µM/10

48 GSH 10

0 Control Baicalein (25 µM) Baicalein (50 µM)

Fig. 11B 0.74

0.72

0.7

0.68 Vehicle Thiols

49 Baicalein (50 µM)

Total 0.66

0.64

0.62

0.6 Control NAC (1 mM)

Fig. 12 30 min 60 min

450

400 *

350

300 *

250 50 200

150

100 * * 8-Isoprostane Release (pg/mL)

0 Vehicle 25 50 100 Cocentration of Baicalein (µM)

Chapter 5: Discussion

The results of the current study revealed that in BPAECs (i) baicalein

significantly induced activation of PLD in a dose- and time-dependent manner which was

attenuated by the PLD-specific inhibitor, FIPI; (ii) the thiol protectants DTT and NAC

and the free radical-quenching antioxidants propyl gallate, MnTBAP, and MnTMPyP

attenuated baicalein-induced PLD activation; (iii) chelation of labile intracellular iron

with desferal, but not DTPA or EDTA, and chelation of intracellular calcium with

BAPTA both attenuated baicalein-induced PLD activation; (iv) the protein tyrosine

kinase (PTyK) inhibitors erbstatin, damnacanthal, and tyrophostin AG34 prevented

baicalein-induced PLD activation; (v) baicalein induced in situ translocation of PLD1 and

PLD2 which occurred concomitantly with global protein tyrosine phosphorylation, PLD1 phosphorylation, and PLD2 tyrosine phosphorylation and were all attenuated by FIPI; (vi)

baicalein caused significant dose-dependent generation of ROS; (vii) the thiol-protectant

NAC attenuated baicalein-induced loss of soluble thiols (glutathione [GSH] and total

thiols); (viii) baicalein caused peroxidation of membrane lipids leading to 8-isoprsotane

formation; and (ix) baicalein induced significant dose-dependent cytotoxicity (as indexed

through LDH release) which was attenuated by thiol protection (NAC), tyrosine kinase

inhibition (genistein), and PLD inhibition (FIPI). Overall, for the first time, the current

study demonstrated that the plant polyphenolics LOX inhibitor, baicalein, induced PLD

activation through ROS generation, loss of cellular thiol-redox status, activation of

PTyKs, and protein tyrosine phosphorylation thus leading to the PLD signaling-mediated

cytotoxicity in ECs (Schema-1).

The essential lipid signaling enzyme phospholipase D (PLD) has been shown to

be activated by oxidants and regulated through phosphorylation and association with

MAP and tyrosine kinases. Reports have suggested that isolated phytochemical

antioxidants, typically polycyclic polyphenolic compounds such as resveratrol, quercetin,

rutin, myricetin, and baicalein, may act through a prooxidant mechanism in mammalian

cells (Malireddy et al. 2012). To investigate this possible mechanism and to evaluate the

effects of isolated phytochemical exposure on lipid signaling in mammalian cells, in the

current study, we chose the well-established and widely used phytochemical polyphenolics 12-LOX inhibitor, baicalein, and investigated its modulatory actions on the

PLD activity and associated signaling events in our established BPAEC model in culture.

Similar to known oxidants, baicalein exhibited time- and dose-dependent activation of

PLD. However, the current study showed that baicalein-induced PLD activation was

attenuated by the novel PLD-specific inhibitor FIPI confirming that baicalein in fact

activated the PA-generating PLD enzyme system in BPAECs. In earlier reports, we have

shown that oxidant-, mercury- and bleomycin-induced PLD activation in ECs is

dependent on intracellular thiol depletion and generation of ROS (Parinandi et al. 1999;

Hagele et al., 2007 and Patel et al., 2012). This led us to investigate this possible

mechanism of baicalein-induced activation of PLD in BPAECs. Through protecting intracellular thiols with DTT and NAC and inhibiting free radical formation with the antioxidants propyl gallate, MnTBAP, and MnTMPyP, our results confirmed that baicalein-induced PLD activation was also dependent on depletion of intracellular thiols and generation of ROS. Intracellular iron has also been implicated in oxidant-mediated generation of ROS including hydrogen peroxide-induced activation of PLD through oxidant (ROS) generation in BPAECs (Natarajan et al., 1993). Here, we demonstrated that baicalein-induced PLD activation was dependent on labile intracellular iron as the iron chelator desferal, but not DTPA or EDTA, attenuated baicalein-induced formation for PBt to control levels. Together, the current study demonstrated that baicalein, similar to other oxidants, caused the generation of ROS that led to depletion of intracellular thiols and activation of PLD which was attenuable through scavenging ROS and protecting the loss of thiol-redox status. Iron (Fe+2/Fe+3) is established to play a critical role in ROS-mediated biochemical reaction both in vitro and in vivo. The current study revealed that baicalein induced the formation of ROS in cells in situ but it was not confirmed up to what extent those ROS generated by baicalein in BPAECs were hydrogen peroxide. Nevertheless, the generation of hydrogen peroxide by baicalein in

BPAECs under the current experimental conditions and the role of intracellular labile iron as a Fenton catalyst were not ruled out. The loss of thiols caused by baicalein could have led to the formation of reactive thiol species (thiyl radicals) and/or thiol degradation products, which could have exacerbated the ROS-mediated PLD activation in BPAECs under baicalein exposure. Needless to mention, the fate of cellular thiols (both GSH and

protein thiols) under baicalein treatment in ECs needs further thorough investigation.

More noticeably, thiol-protectants such as NAC and DTT effectively attenuated the

baicalein-induced PLD activation (generation of the lipid signal mediator PA) confirming

that cellular thiols were apparently major targets for baicalein. Hence, the current study

established that baicalein-induced PLD signaling was closely associated with the oxidant

signaling and thiol-redox signaling pathways in ECs.

Our previous studies have shown the known oxidant diperoxivanadate (DPV)

activates PLD with concomitant increases in PLD phosphorylation and association of

PLD with the Src tyrosine kinase in BPAECs (Parinandi et al. 2001). Oxidants have been

also shown to activate PLD through PTyK activation and tyrosine phosphorylation and

also MAPK signaling in ECs (Parinandi et al 2001; Varadharaj et al. 2006). Also, we

have shown mercury induced-activation and threonine phosphorylation of PLD is inhibited by the MEK1-specific inhibitor PD 98059 in mouse aortic ECs (Secor et al.,

2011). These findings have led us to investigate the role of PTyK signaling in baicalein- induced PLD activation in BPAECs. In the current investigation, the well-established

PTyK-specific inhibitors ersbstatin, damnacanthal, and tyrophostin AG34 attenuated baicalein-induced PLD activation in BPAECs demonstrating that upstream activation of

PTyK was responsible for the downstream activation of PLD that was induced by baicalein. Also, global protein tyrosine phosphorylation induced by baicalein revealed that other proteins, in addition to PLD1 and PLD2, were also phosphorylated at the

tyrosine residues under baicalein treatment in BPAECs. This observation suggested that

tyrosine phosphorylation of proteins other than PLDs could also play important role(s) in

the baicalein-induced signaling outcomes in ECs. As mentioned above, oxidants (ROS)

and thiol-redox dysregulation leads to PTyK activation and tyrosine phosphorylation in

ECs that are tied up with PLD activation (Parinandi et al. 2001). Concurring with the reports made, the current study clearly revealed that baicalein caused the ROS generation and depletion of thiol-redox status in BPAECs which are associated with protein tyrosine phosphorylation. However, the exact species of PTyK that was involved in catalyzing the protein tyrosine phosphorylation and tyrosine phosphorylation of PLD in BPAECs as

observed in the current study were not identified. Yet, the role of Src kinase in catalyzing tyrosine phosphorylation of proteins induced by baicalein in BPAECs is not ruled out at

this juncture since Src kinase has been identified earlier to be activated by oxidants in

BPAECs leading to PLD activation (Parinandi et al. 2001). This study demonstrated that

PTyKs are oxidant- and thiol-redox-sensitive upon baicalein treatment in BPAECs and

led to the activation of PLD signaling.

One of the striking observations of the current study was the in situ

translocation/relocalization of PLD induced by baicalein exposure in BPAECs as

visualized by confocal immunofluorescence microscopy. These results clearly revealed

that baicalein induced the translocation of both the isoforms of PLD, PLD1 and PLD2, which was inhibited by the PLD-specific inhibitor, FIPI. Earlier, we have reported that oxidants and pro-oxidants cause relocalization of PLD1 and PLD2 in BPAECs

(Varadharaj et al. 2006; Secor et al., 2011). The fact that FIPI inhibited the baicalein-

induced relocalization of PLD1 and PLD2 in BPAECs as observed in the current study

emphasized that the induction of enzyme activity by baicalein was tightly associated with

the relocalization of the enzyme. This warrants further investigation to establish whether

ROS, thiol-redox dysregulation, and protein tyrosine phosphorylation were required for

the translocation/relocalization of PLD1 and PLD2 induced by baicalein in BPAECs. On

the other hand, it could be surmised that relocalization of PLD1/PLD2 in different cellular compartments (cytosol, nucleus, and membranes) could be required for the intracellular catalytic activity of the isoforms of the enzyme upon baicalein treatment by bringing the substrate and the enzyme together for proper catalysis. This sort of behavior of the enzyme isoforms displays the intracellular substrate specificities and availabilities for the actions of specific PLD isoforms in situ.

The ultimate goal of this current study was to link the upstream PLD activation and signaling induced by baicalein in ECs through ROS generation, thiol-redox dysregulation, PTyK activation, and protein tyrosine phosphorylation to an important cellular physiological outcome such as cytotoxicity of baicalein. The current study demonstrated that baicalein induced cytotoxicity (cell membrane damage) as evidenced by LDH leak that was protected by the thiol-protectant (NAC), PTyK-specific inhibitor

(genistein), and the PLD-specific inhibitor (FIPI). These results suggested the role of thiol-redox depletion, activation of PTyK, and activation of PLD in baicalein-induced cytotoxicity in BPAECs. Therefore, it was apparent that baicalein induced adverse cellular effects (e.g. cytotoxicity) in BPAECs through thiol-redox, PTyK, and PLD

signaling. Earlier, we have shown that the oxidant drug, bleomycin and methylmercury

induced cytotoxicity (LDH leak) through oxidant and PLD signaling and the generation

of PLD-catalyzed PA, the bioactive lipid mediator. Hence, it was apparent that the

bioactive lipid mediator, PA, generated by PLD activation played a critical role in the

baicalein-induced cytotoxicity in BPAECs (Schema).

Baicalein is regarded as an effective 12-LOX-specific inhibitor. However, it was

revealed by the current study that baicalein acted as a prooxidant, induced ROS and thiol-

redox signaling, activated PTyKs, caused protein tyrosine phosphorylation, activated

PLD, generated the bioactive lipid signal mediator (PA), and ultimately caused

cytotoxicity through ROS generation, thiol-redox destabilization, protein tyrosine

phosphorylation, and PLD activation. Surprisingly, a pharmacological inhibitor such as

baicalein which is purported to act specifically on 12-LOX to cause its inhibition acted differently in the BPAEC in vitro culture system and acted as a prooxidant leading to the

ROS generation, thiol-redox depletion, activation of PTyKs, and PLD activation. These

observations of the current study suggested that a pharmacological inhibitor considered

specific to 12-LOX such as baicalein, when administered singly as an isolated (pure)

compound to the in vitro cultured cells like BPAECs, causes adverse effects on cells

through oxidant-mediated lipid signaling. This also raises concerns not only with regards

to the use of baicalein alone but also with respect to other phytochemical phenolics and

natural products as isolated molecules, either in the in vitro cellular models or in the in

vivo animal models, as specific enzyme inhibitors or antioxidants or preventive

nutraceuticals for diseases such as cancer and cardiovascular diseases. The reports of

isolated phytochemical phenolics (nutraceuticals), including baicalein, acting as

prooxidant molecules which cause oxidative stress when administered alone appear to be

increasing in both cellular in vitro models and in animal in vivo models (Malireddy et al.,

2012).

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