Borneol and Luteolin from Chrysanthemum morifolium Regulates

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Bioactive Constituents, Metabolites, and Functions

Borneol and Luteolin from Chrysanthemum morifolium Regulates Ubiquitin-Signal Degradation Tsui-Ling Chang, Pei-Shin Liou, Pei-Yuan Cheng, Hsiang-Ning Chang, and Pei-Jane Tsai J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.8b01972 • Publication Date (Web): 11 Jul 2018 Downloaded from http://pubs.acs.org on July 13, 2018

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Journal of Agricultural and Food Chemistry

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Borneol and Luteolin from Chrysanthemum morifolium Regulates Ubiquitin-Signal

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Degradation

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Tsui-Ling Chang*1, Pei-Shin Liou1, Pei-Yuan Cheng1, Hsiang-Ning Chang1, and Pei-Jane Tsai2

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Authors' Affiliations: 1Department of Biological Sciences and Technology, National University of

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Tainan, No 33, sec. 2, Shu-Lin Street, Tainan 70005, and 2Department of Medical Laboratory

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Science and Biotechnology, National Cheng Kung University, 138 Sheng-Li Road, Tainan 70428,

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TAIWAN

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Author affiliations:

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To whom correspondence should be addressed [(T.-L.C.) telephone (886) 6-2602587, fax (886)

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6-2606153, e-mail [email protected]]

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Running title: Ubiquitin-Proteasome Pathway and Autophagy Affected by luteolin and Borneol

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Pei-Shin Liou and Pei-Yuan Cheng contributed equally to the work

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20S, 20S proteasome; DUB, deubiquitinating enzymes; E1, ubiquitin-activating enzyme; USP47,

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ubiquitin carboxy-terminal hydrolase 47; Ub, ubiquitin; Poly-ub, polyubiquitin; Pol β, Pol β, DNA

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polymerase; 3-MA, 3-methyladenine; Bo + Lu, borneol and luteolin

Abbreviations:

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ACS Paragon Plus Environment


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ABSTRACT

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Targeting the two degradation systems, ubiquitin-proteasome pathway and ubiquitin-signal

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autophagy-lysosome system, plays an important function in cancer prevention. Borneol is

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called an “upper guiding drug”. Luteolin has demonstrated anti-cancer activity. That borneol

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regulates luteolin can be sufficient to serve as an alternative strategy. Borneol activates

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luteolin to inhibit E1 and 20S activity (IC50 = 118.8 ± 15.7 µM) and perturb the 26S

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proteasome structure in vitro. Borneol regulates luteolin to inhibit 26S activity (IC50 = 157 ±

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19 µM), induces apoptosis (LC50 = 134 ± 4 µM), and causes pre-G1 and G0/G1 arrest in

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HepG2 cells. Borneol regulates luteolin to induce ubiquitin signal autophagic degradation,

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resulting in induction of E1, reduction of USP47 and accumulation of p62 in HepG2 reporter

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cells. Interestingly, luteolin decreased Ub-conjugates while borneol increased the

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accumulation of Ub-conjugates in HepG2 reporter cells. E1, p62, and ubiquitin levels were

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down regulated in borneol-treated HepG2 reporter cells at 24 hours. These observations

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suggest a potential autophagic inhibitor of borneol that may guide luteolin in the

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ubiquitin-proteasome pathway and the ubiquitin-signal autophagic degradation.

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Key words: Luteolin; Borneol; Proteasome; E1 enzyme; Autophagy 2


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INTRODUCTION

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Three molecular mechanisms of polyphenols, flavonoids, as antioxidants on oxidative

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processes was considered to prevent the damaging effects of the metabolic syndrome, reduce

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in cardiovascular disease risk, and modulate of superoxide and uric acid for type 2 diabetes

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(1). Flavonoids from plants are poorly absorbed from the gut and subject to degradation by

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intestinal micro-organisms (2). Chrysanthemum morifolium is a well-known food source, tea

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material, and herbal medicine. Decreasing irascibility is one of the functions of

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Chrysanthemum morifolium. The major bioactive components of C. morifolium flower are

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abundant in caffeoylquinic acids and flavonoids. Luteolin (3’,4’,5,7-tetrahydroxyflavone, one

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(5.22 mg/g) of the abundant flavonoids) and borneol ((C10H18O, BO), major constituent

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(15.5%) of the oil of the flower) are present in Chrysanthemum morifolium (3, 4). The C.

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morifolium flowers have been widely used in the drug and food industries. The strong

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anti-proliferative activity of luteolin can act against different cancer cells, including breast,

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gastric and prostate cancers (5-7). Luteolin shows anti-hepatoma cell function by inducing

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cell cycle arrest and apoptosis (8). Luteolin negatively modulates the JAK/STAT pathway

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underlying intestinal anti-inflammatory action (9). Borneol, a highly lipid-soluble bicyclic

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monoterpenoid compound, is a classical aromatic refreshing traditional Chinese medicine.

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Borneol is called an “upper guiding drug” and is used as an adjuvant component and can

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guide other components to the targeted tissues or organs in the upper part of the body,

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especially in the brain (10). Borneol is administered against exogenous oxidative DNA

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damage to protect primary rat hepatocytes (11). Natural borneol enhances cellular

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ROS-mediated DNA damage- to potentiate selenocystine-induced apoptosis in human liver

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cells (12). Liver cancer, the cause of cancer deaths, is tightly linked to two major protein

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degradation pathways in eukaryotic cells - ubiquitin-proteasome pathway (UPP) and

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ubiquitin-signal autophagy-lysosome system (13, 14). A recent report shows a new therapeutic

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approach to target liver cancer was synergistic to inhibit autophagy and neddylation pathways 3



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(14). However, little attention was focused on the effects of the active component of

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Chrysanthemum morifolium on the subunits of the UPP and autophagy-lysosome system in

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liver cancer.

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The function of the ubiquitin-proteasome pathway is an energy-dependent molecular machine

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to degrade the majority of damaged, misfolding, and ubiquitin-modified intracellular proteins

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in normal cellular function in eukaryotes (15-17). ubiquitin (Ub, 76-amino-acids) is a

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conserved small protein in all eukaryotic cells. Three enzymes, ubiquitin-activating enzyme

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(E1), ubiquitin-conjugating enzyme (E2), and ubiquitin-ligating enzyme (E3), are involved in

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the multistep process for ubiquitin conjugation to target proteins. The E1 catalyzes ATP to

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bind ubiquitin and release pyrophosphate (PPi). Finally, E1 binds to two ubiquitins by a

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thioester bond of C-terminal ubiquitin and a adenylate bond of C-terminal ubiquitin (18).

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Ubiquitin conjugates was cleavage at the carboxy-terminal glycine residue to ubiquitin

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monomers or polyubiquitin (poly-ub). One of deubiquitinating enzymes (DUB), ubiquitin

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carboxy-terminal hydrolase 47 (USP47), is to remove polyubiquitin chains from target

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proteins. USP47 deubiquitylated DNA polymerase (Pol β) and involved in DNA repair (19).

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USP47 serves as a source of nuclear Pol β in base excision repair to stabilize newly synthesized

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cytoplasmic Pol β (19).

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Tagged proteins, polyubiquitin attached to target proteins, are selected for destruction by 26S

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proteasomes (18, 20). The proteasome (EC3.4.99.46, Mr = 2,400,000), a large, multi-subunit

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enzyme, found at a high amount in all mammalian cells. The eukaryotic 26S proteasome

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composes of the 20S core particle (21) and the 19S regulatory particle (19S cap, PA700, Rps)

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(22, 23). ATPase (Rpt1-6) and non-ATPase (Rpn) subunits are in the 19S regulatory particle.

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The 20S core particles have four stacked rings. Only denatured proteins can pass through a

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narrow channel between the two outer rings (called α rings) and the 19S regulatory particles.

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Two inner β rings form the catalytic chamber, a multicatalytic protease, each of which

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contains

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post-glutamyl peptide hydrolase-like hydrolytic active sites (21). Proteins are degraded by the

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20S core generating peptides that are 3–25 amino acids in length (24).

three

well-characterized

peptidase:

chymotrypsin-like,

trypsin-like,

and

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Degrading nonspecific bulk cytoplasmic components is responsible for autophagy (25). The

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processing of neurodegenerative disease showed that the specific autophagic degradation of

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polyubiquitinated protein aggregates. Mono-ubiquitination of normally long-lived cytoplasmic

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substrates is for autophagic degradation involving p62 binding in mammalian cells (26). The

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proteasome inhibitor with the autophagy inhibitor induce apoptosis and suppress proliferation

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in hepatocellular carcinoma (27). When the proteasome is inhibited, the ubiquitinated protein

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can also be degraded by autophagy (26). Inhibition of autophagy increases proteasome

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inhibitor-induced endoplasmic reticulum stress and accumulation of polyubiquitinated

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proteins (28). Simultaneous administration of proteasome and autophagic inhibitors may

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increase the accumulation of toxic aggregates, induce apoptosis and prevent the development

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of drug resistant cells (29). Deubiquitinating enzymes-related autophagy are involved in

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disease and health (30).

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Cancer could be more preventable under increasing knowledge of the effects of natural

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products on the mechanisms of the UPP and the ubiquitin-signal autophagic degradation. In

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this study, we showed that borneol enhances luteolin to inhibit 26S proteasome activity and

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cause pre-G1 and G0/G1 arrest in HepG2 cells. Borneol, a potential autophagic inhibitor,

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increased the accumulation of UbG76V–YFP in HepG2 cells. The combination of borneol and

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luteolin regulating the UPP and autophagic degradation system may play an important role in

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mediating apoptosis. Evaluating the combination of borneol and luteolin as an oral drug to 5



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prevent cancer may provide a pharmacological rationale to pursue preclinical trials of this

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combination.

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MATERIALS AND METHODS

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Chemicals

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Chrysanthemum morifolium was purchased from a traditional Chinese medicine store (Tainan,

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Taiwan). Borneol was obtained from Alfa Aesar (Ward Hill, MA, USA). Luteolin, G418

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disulfate salt, rabbit anti-ubiquitin antiserum, anti-p62, and anti-α-tubulin were purchased

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from Sigma-Aldrich (St. Louis, MO, USA). Tannic acid was purchased from Acros Organics

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(Geel, Belgium). A fluorogenic peptide linked to aminomethylcoumarin (AMC),

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Suc-Leu-Leu-Val-Tyr-AMC (Suc-LLVY-AMC), was purchased from Bachem (Bubendorf,

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Switzerland). Mouse monoclonal anti-USP47 (MW 150-250 KDa) (Abcam, Cambridge, UK),

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Anti-RFP (dsRed) and anti-UBE1 (GeneTex, Irvine, CA, USA), anti-rabbit IgG (H + L)

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alkaline phosphatase conjugate (Promega, Madison, WI, USA), ubiquitin-activating enzyme

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(UBE1) (Boston Biochem, Cambridge, MA, USA), lactacystin and 3-methyladenine (Cayman

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Chemicals, Ann Arbor, USA) were used as received. Secondary antibodies anti-rabbit

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HRP-conjugated or anti-mouse-HRP, MG132, and rabbit polyclonal antibodies against human

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20S core subunits were purchased from Calbiochem (La Jolla, CA, USA). DEAE Affi-Gel®

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Blue Gel (Bio-Rad, Hercules, CA, USA), UbG76V-YFP (Addgene, Cambridge, MA, USA),

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PolyJetTM (SignaGen Laboratories, Ijamsville, MD), Cell Counting Kit-8 (CCK-8) (Dojindo

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Molecular Technologies, Inc., Gaithersburg, MA), DMEM (Cellgro, Manassas, VA) and

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BCIP/NPT (nitro-blue tetrazolium chloride/5-bromo-4-30- indoyl phosphate p-toluidine salt)

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stock solution (Roche, Nutley, NJ, USA) were used as received.

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Purification of 26S or 20S proteasome from pig red blood cells (RBCs)

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Fresh pig blood was obtained from an abattoir (Tainan, Taiwan). To remove white blood cells

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(WBCs) and plasma from samples, fresh pig blood was centrifuged at 500×g for 10 min at

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4°C. Red blood cells (RBCs) were re-suspended 1:1 (v/v) with cold phosphate buffer saline

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(PBS). The RBCs were centrifuged at 3700×g for 17 min at 4°C. The purification of 20S or

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26S proteasomes from pig RBCs as previously described (31).

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Proteolysis measurement

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The measurement of proteasome activity was determined by our previous report with slight

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modifications (32). The extraction of Chrysanthemum morifolium was dissolved in ethanol.

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Borneol and luteolin were dissolved in DMSO. The activity pig 20S and 26S proteasomes

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toward Suc-LLVY-AMC was determined by 1 µl of Chrysanthemum morifolium, luteolin,

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and borneol in 100 µl of reaction buffer (1 mM ATP, 0.5 mM DTT, 5 mM MgCl2, 30 mM

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Tris-HCl (pH 8) with 50 µM Suc-LLVY-AMC) at 37°C for 15 min. The assuming

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molecular mass of 20S and 26S proteasomes are 0.8 and 2.4 MDa. The final concentration of

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20S and 26S proteasomes in the assay was 349 µg/ml (or 436.8 nM) and 210.7 µg/ml (or 87.8

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nM), respectively. AMC was quantified by 360 nm/460 nm (excitation/emission wavelengths)

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in a BioTek FL800 plate reader (Winooski, VT) at 37°C for 15 min. 20S or 26S proteasome

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with 1 µl of DMSO was served as control.

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Glycerol Density Gradient Centrifugation

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Purified 26S (87.8 nM) from pig RBCs were treated with either 1 µl of borneol, luteolin,

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borneol + luteolin, or DMSO for 15 min at 25°C. The linear glycerol gradients was 10–40%.

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Gradients (136 µl) contained 50 mM Tris-HCl, pH 7.6, 1 mM ATP, 1 mM dithiothreitol, and

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5 mM MgCl2. Samples using a Type 42.2 Ti rotor (Fullerton, CA, USA) were centrifuged at

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39,300 rpm for 3 h in a Beckman Coulter OptimaTM L-90K ultracentrifuge. In each fraction, 8

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µl was collected for appropriate analysis in the individual experiments.

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Expression and purification of the recombinant plasmid

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The mRFP-Ub was amplified from a plasmid and subcloned into T&A cloning vector.

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expressed in Escherichia coli DH5α according to previously described methods (33). Primers

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used were as follows: 5’- GCTAGCACCACCATGGCCT -3’ and

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5’-TAGATCCGGTGGATCCCGG. The mRFP-Ub protein was eluted from DEAE Affi-Gel®

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Blue Gel.

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Ubiquitination

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The E1 activity was carried out as previously described with slight modifications (33). 0.2 µl

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of borneol, luteolin, Bo+Lu or tannic acid was added to E1 (222 nM) to

incubate at room

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temperature. After 15 minutes, 119 nM mRFP-Ub was added to a total volume of 5 µl

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ubiquitinating reaction buffer (20 mM Tris-HCl, pH 7.6, 1 mM ATP, and 5 mM MgCl2) and

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incubated at 37°C for 5 minutes. The samples in non-reducing SDS-PAGE sample buffer

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were loaded on 10% SDS-PAGE at 4 °C and electrophoretically transfer onto nitrocellulose

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membrane. Next, the membranes were blotted with anti-RFP (1:1500). The secondary was

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used by an alkaline phosphatase-conjugated anti-rabbit IgG (H+L) (1:2000). The

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nitrocellulose membrane was detected by BCIP/NPT stock solution.

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Cell culture and treatment

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Human HepG2 cells were maintained using DMEM with 10% (v/v) fetal bovine serum, 4.5

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g/L glucose, 0.1 g/L kanamycin/streptomycin, and 3.7 g/L sodium bicarbonate at 37°C under

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5% CO2. Cell suspension from the flask was centrifuged at 1200 rpm for 5 min and the cell

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pellet was re-suspended in the medium.

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Cell viability

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HepG2 (5x103 cells) were plated in 96-well microtiter plates in 100 µl of medium per well for

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24 hours. HepG2 cells were incubated with borneol, luteolin (0, 0.005, 0.01, 0.05, 0.1, 0.5, 1 9



1

mM), Bo+Lu (0, 0.0025, 0.005, 0.025, 0.05, 0.25, 0.5 mM) or DMSO for 24 hours. CCK-8

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(10 µl) was added to each well of the plate and returned to an incubator for 3 hours. For cell

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viability, the production of WST-8 was measured at 450 nm using a Tecan sunrise plate

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reader (Männedorf, Switzerland).

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Measurement of chymotrypsin-like activity of 26S in human cells

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HepG2 (5x103 cells) were dispensed into 96-well microtiter plates in 100 µl of medium per

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well and incubated for 24 hours. HepG2 incubated with Borneol, luteolin (0, 0.005, 0.01,

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0.05, 0.1, 0.2, 0.3, 0.5, 1 mM), Bo+Lu (0, 0.0025, 0.005, 0.025, 0.05, 0.01, 0.15, 0.25, 0.5

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mM) or DMSO for 5 hours. Suc-LLVY-AMC (1 µL) was added to each well and incubated

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for 2 hours in an incubator. AMC was quantified by 360 nm/460 nm (excitation/emission

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wavelengths) in a BioTek FL800 plate reader at 37°C for 15 min.

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Transfection

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HepG2 (0.12x106 or 1x106 cells) were grown in 24- or 6-well microtiter plates in 500 or 1000

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µl of medium per well for 24 h. Cells were transfected with 1 µg UbG76V-YFP plasmid DNA

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using PolyJetTM according to the manufacturer’s instructions. After 18 h post-transfection, the

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medium of the transfected cells was changed to 0.5 mg/ml G418 disulfate salt.

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Fluorescence microscopy

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HepG2 UbG76V-YFP cells on a 6-well plate were individually treated with 20 µM MG132, 4

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µM lactacystin, 44 µM 3-methyladenine, 10 µM MG132 + 22 µM 3-methyladenine, 200 µM

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borneol, 200 µM luteolin, and 100 µM Bo+Lu. The fluorescence generated due to the UPP

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system or autophagic degradation blocked by these compounds. The accumulation of

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UbG76V-YFP in HepG2 cells was determined in an inverted microscope (Nikon ECLIPSE

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TE2000-U) at different times. Ten fluorescence photographs at different positions and at 10


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different time point in each well were randomly taken to quantify the optical density by

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ImageJ software.

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Western Blot Analysis

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HepG2 UbG76V-YFP cells in a 6-well plate were individually treated with borneol, luteolin,

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Bo+Lu, or DMSO for 24 hours. Twenty micrograms of cell lysates was loaded on 10%

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SDS-PAGE and electrophoretically transferred onto nitrocellulose membranes. After blocking

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with milk, the membranes were probed with the relevant antibodies overnight at 4°C. The

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primary antibodies anti-ubiquitin (1:2000), anti-20S core subunits (1:2000), anti-USP47

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(1:1000), or anti-α-tubulin (1:2000) were used following by secondary antibodies (the

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standard HRP-conjugated anti-rabbit or anti-mouse antibody-HRP (1:2000)). Proteins on the

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nitrocellulose membrane were detected by enhanced chemiluminescence (ECL).

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Flow Cytometry

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The measurement of cell cycle distribution was determined by our previous report with slight

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modifications (34). HepG2 cells in a 6-well plate were individually treated with 100 µM or

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500 µM borneol, luteolin, Bo+Lu, or DMSO for 24 hours. HepG2 cells were harvested and

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washed twice with ice cold PBS. HepG2 cells (3x106) were re-suspended in 1 ml of PBS,

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immobilized with 0.5 ml of 70% ethanol overnight at -20°C. Cells were stained with 1 ml of

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propidium iodide staining solution (50 µg of propidium iodide, 100 units of RNase A, 0.2%

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Triton X-100, 0.5 ml PBS), and then incubated at room temperature for 30 min. Cell apoptosis

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were analyzed by FACScan (Becton Dickinson Immunocytometry Systems, San Jose, CA).

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Statistical Analysis

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The measured data expressed are the means of three different experiments ± standard error

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(SE) and statistically analyzed using one-way analysis of variance (ANOVA). The differences

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between two sets of values was characterized by Student’s unpaired t-test. The standard

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p

Borneol and Luteolin from Chrysanthemum morifolium Regulates

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