Microbial Synthesis of Non-Natural Anthraquinone Glucosides Displaying Superior Antiproliferative Properties
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molecules Article Microbial Synthesis of Non-Natural Anthraquinone Glucosides Displaying Superior Antiproliferative Properties Trang Thi Huyen Nguyen 1,†, Ramesh Prasad Pandey 1,2,† ID , Prakash Parajuli 1 ID , Jang Mi Han 1, Hye Jin Jung 1,2, Yong Il Park 3 and Jae Kyung Sohng 1,2,* ID 1 Department of Life Science and Biochemical Engineering, Sun Moon University, 70 Sunmoon-ro 221, Tangjeong-myeon, Asan-si, Chungnam 31460, Korea; [email protected] (T.T.H.N.); [email protected] (R.P.P.); [email protected] (P.P.); [email protected] (J.M.H.); [email protected] (H.J.J.) 2 Department of BT-Convergent Pharmaceutical Engineering, Sun Moon University, 70 Sunmoon-ro 221, Tangjeong-myeon, Asan-si, Chungnam 31460, Korea 3 Department of Biotechnology, The Catholic University of Korea, Bucheon, Gyeonggi-do 14662, Korea; [email protected] * Correspondence: [email protected]; Tel: +82-(41)-530-2246; Fax: +82-(41)-530-8229 † These authors contributed equally to this work. Received: 17 July 2018; Accepted: 21 August 2018; Published: 28 August 2018 Abstract: Anthraquinones, naturally occurring bioactive compounds, have been reported to exhibit various biological activities, including anti-inflammatory, antiviral, antimicrobial, and anticancer effects. In this study, we biotransformed three selected anthraquinones into their novel O-glucoside derivatives, expressing a versatile glycosyltransferase (YjiC) from Bacillus licheniformis DSM 13 in Escherichia coli. Anthraflavic acid, alizarin, and 2-amino-3-hydroxyanthraquinone were exogenously fed to recombinant E. coli as substrate for biotransformation. The products anthraflavic acid-O-glucoside, alizarin 2-O-b-D-glucoside, and 2-amino-3-O-glucosyl anthraquinone produced in the culture broths were characterized by various chromatographic and spectroscopic analyses. The comparative anti-proliferative assay against various cancer cells (gastric cancer-AGS, uterine cervical cancer-HeLa, and liver cancer-HepG2) were remarkable, since the synthesized glucoside compounds showed more than 60% of cell growth inhibition at concentrations ranging from ~50 µM to 100 µM. Importantly, one of the synthesized glucoside derivatives, alizarin 2-O-glucoside inhibited more than 90% of cell growth in all the cancer cell lines tested. Keywords: anti-cancer agents; anthraquinones; glycosyltransferase 1. Introduction Anthraquinones are naturally occurring phenolic compounds based on the 9,10-anthraquinone skeleton. They are widely available from plants, microbes, fungi, and lichens [1]. Anthraquinones have various biological benefits [2,3]. Anthraquinones of the Rubiaceae family exhibit interesting in vivo biological activities such as antimicrobial [4], antifungal [5], hypotensive and analgesic [6], anti-malarial [7], anti-oxidant [8], antileukemic, and mutagenic functions [9]. Several anthraquinones are widely used in the treatment of cancer. They display cytotoxic activities through interaction with DNA, preferentially at guanine/cytosine-rich sites [10]. Emodin was studied as an agent that could reduce the impact of type 2 diabetes [11] and could inhibit human cytomegalovirus development [12]. Aloe emodin has strong stimulant-laxative action [13] and is found in the gel, soap or leaves of Aloe vera and the rhizome of rhubarb (Rheum rhaponticum). Chrysazin (known as Dantron) is mainly used in Molecules 2018, 23, 2171; doi:10.3390/molecules23092171 www.mdpi.com/journal/molecules Molecules 2018, 23, x FOR PEER REVIEW 2 of 14 Moleculesfound in2018 the, 23 ,gel, 2171 soap or leaves of Aloe vera and the rhizome of rhubarb (Rheum rhaponticum2 of). 13 Chrysazin (known as Dantron) is mainly used in palliative care to counteract the constipating effects of opioids [14]. Quinizarin is an inexpensive dye used to color gasoline and heating oil [15]. palliativeIt is also careused to as counteract an intermediate the constipating for the synt effectshesis of of opioids indanthrene- [14]. Quinizarin and alizarin-derived is an inexpensive dyes. dyeAlizarin used tohas color been gasoline used andas heatinga prominent oil [15]. red It is alsodye, used principally as an intermediate for dyeing for thetextile synthesis fabrics. of indanthrene-Anthraquinone and glycosides alizarin-derived upon hydrolysis dyes. Alizarin yield has the been aglycone, used as awhich prominent is usually red dye, a di-, principally tri- or fortetrahydroxyanthraquinone dyeing textile fabrics. Anthraquinone or a derivative glycosides of these upon compounds. hydrolysis yieldWhile the free aglycone, anthraquinone which is usuallyaglycones a di-,exhibit tri- ortherapeutic tetrahydroxyanthraquinone activity, glycosides that or a enhance derivative solubility of these are compounds. accepted as Whilekey active free anthraquinonecomponents with aglycones various exhibitpharmacolo therapeuticgical actions activity, such glycosides as antileukemic, that enhance antiseptic, solubility anti-cancer, are accepted and asantitumor key active activity components [16,17]. with various pharmacological actions such as antileukemic, antiseptic, anti-cancer,Recently, and the antitumor synthesis activity of anthraquinone [16,17]. derivatives has attracted significant interest. Various synthesisRecently, methods the synthesis have been of anthraquinone reported, the derivatives most common has attracted of which significant is the interest.intramolecular Various synthesiscondensation methods of aryl have and been O reported,-arylbenzoic the most acids, common using offuming which issulfuric the intramolecular acid, benzoyl condensation chloride, ofconcentrated aryl and O -arylbenzoicsulfuric acid, acids, benzoyl using chloride, fuming zinc sulfuric chloride, acid, benzoyland POCl chloride,3/P2O3C concentratedl4 as catalyst, sulfuricwhich acid,produces benzoyl anthraquinone chloride, zinc derivatives chloride, and [18]. POCl Anthra3/P2Oquinone3Cl4 as catalyst,and its which associated produces derivatives anthraquinone were derivativespreviously synthesized [18]. Anthraquinone by a chemical and pathway, its associated although derivatives this method were was previously expensive synthesized and involved by a chemicalseveral hurdles pathway, [19]. although This report this method is based was on expensive the utilization and involved of indigenous several hurdles E. coli [sugar19]. This (uridine report isdiphosphate based on the(UDP)-glucose) utilization of as indigenous a sugar donorE. coli whichsugar will (uridine be utilized diphosphate by the glycosyltransferase (UDP)-glucose) as to a sugarconjugate donor to whichdifferent will anthraquione be utilized by derivatives. the glycosyltransferase This process to is conjugate inexpensive todifferent and environmentally anthraquione derivatives.friendly. We designed This process a scheme is inexpensive to consider and wild environmentally type E. coli BL21 friendly. (DE3) strain. We designedWe then expressed a scheme toglycosyltransferases consider wild type fromE. coli BacillusBL21 (DE3)licheniformis strain. WeDSM13 then expressed(YjiC) and glycosyltransferases biotransformed three from Bacillusanthraquionones licheniformis intoDSM13 their (YjiC)respective and biotransformed glucosides (Figure three anthraquionones1). The anticancer into activities their respective of all glucosidesderivatives (Figurewere 1).assessed The anticancer and the activities results of were all derivatives significant were compared assessed andwith the those results of werethe significantcorresponding compared aglycones. with those of the corresponding aglycones. FigureFigure 1.1. SchemeScheme showing pathway and utilizing Escherichia coli indigenousindigenous UDP-glucose UDP-glucose by by BacillusBacillus glycosyltransferaseglycosyltransferase forfor modificationmodification of selected anthraquinones into respective glucosides. 2. Results 2.1. Biotransformation of Anthraquinones The approach was to biosynthesize anthraquinone glucoside utilizing E. coli indigenous UDP-glucose as a sugar donor to the expressing glycosyltransefrase (Figure1). Cultures were prepared for biotransformation reactions with recombinant strain expressing pET28a-YjiC, as explained in the Molecules 2018, 23, x FOR PEER REVIEW 3 of 14 2. Results 2.1. Biotransformation of Anthraquinones The approach was to biosynthesize anthraquinone glucoside utilizing E. coli indigenous UDP- glucose as a sugar donor to the expressing glycosyltransefrase (Figure 1). Cultures were prepared forMolecules biotransformation2018, 23, 2171 reactions with recombinant strain expressing pET28a-YjiC, as explained3 of in 13 the Materials and Methods section. Three anthraquinones: 2-amino-3-hydroxyanthraquinone, anthraflavicMaterials and acid, Methods and alizarin section. were Three chosen anthraquinones: for biotransformation. 2-amino-3-hydroxyanthraquinone, anthraflavic acid,Anthraquinones and alizarin were were chosen supplied for biotransformation. exogenously to each flask of E. coli BL21 (DE3) harboring pET28a-YjiCAnthraquinones culture after were 20 suppliedh of IPTG exogenously induction at toa final each concentration flask of E. coli of 0.2BL21 mM. (DE3) Cultures harboring were allowedpET28a-YjiC to undergo culture afterbiotransformation 20 h of IPTG inductionfor up to at60 a finalh and concentration were then harvested of 0.2 mM. using Cultures a double were volumeallowed of to undergoethyl acetate biotransformation and analyzed for by up analytical to 60 h and HPLC-PDA, were then harvestedas described using in athe double Materials volume and of Methodsethyl acetate