Anticancer and Antiangiogenic Activities of Novel α-Mangostin Glycosides in Human Hepatocellular Carcinoma Cells via Downregulation of c-Met and HIF-1α.

Hepatocellular carcinoma (HCC) is the most common type of primary liver cancer and is a leading cause of cancer-related death worldwide. Therefore, exploring effective anticancer agents and their modes of action is essential for the prevention and treatment of HCC. Glycosylation can significantly improve the physicochemical and biological properties of small molecules, such as high solubility, stability increase, and lower toxicity. In the present study, for the first time, we evaluated the anticancer and antiangiogenic activities of α-mangostin-3-O-ß-D-2-deoxyglucopyranoside (Man-3DG) and α-mangostin 6-O-ß-D-2-deoxyglucopyranoside (Man-6DG), glycosides of α-mangostin, against human HCC cells. Our results demonstrated that Man-3DG and Man-6DG significantly suppressed the growth of three different HCC cells (Hep3B, Huh7, and HepG2) as well as the migration of Hep3B cells. Furthermore, they induced cell cycle arrest in the G0/G1 phases and apoptotic cell death by regulating apoptosis-related proteins of mitochondria in Hep3B cells. Noticeably, Man-3DG and Man-6DG also caused autophagy, while co-treatment of the α-mangostin glycosides with an autophagy inhibitor 3-MA enhanced the inhibitory effect on Hep3B cell growth in comparison to single agent treatment. Moreover, Man-3DG and Man-6DG inhibited the c-Met signaling pathway that plays a critical role in the pathogenesis of HCC. Furthermore, the α-mangostin glycosides decreased Hep3B cell-induced angiogenesis in vitro through the downregulation of hypoxia-inducible factor-1α (HIF-1α) and vascular endothelial growth factor (VEGF). Notably, Man-6DG more effectively inhibited the growth, tumorsphere formation, and expression of cancer stemness regulators compared to α-mangostin and Man-3DG in 3D spheroid-cultured Hep3B cells. These findings suggest that the α-mangostin glycosides might be promising anticancer agents for HCC treatment with superior pharmacological properties than the parent molecule α-mangostin.

Efficient enzymatic systems for synthesis of novel α-mangostin glycosides exhibiting antibacterial activity against Gram-positive bacteria.

Two enzymatic systems were developed for the efficient synthesis of glycoside products of α-mangostin, a natural xanthonoid exhibiting anti-oxidant, antibacterial, anti-inflammatory, and anticancer activities. In these systems, one-pot reactions for the synthesis of UDP-α-D-glucose and UDP-α-D-2-deoxyglucose were modified and combined with a glycosyltransferase (GT) from Bacillus licheniformis DSM-13 to afford C-3 and C-6 position modified glucose and 2-deoxyglucose conjugated novel α-mangostin derivatives. α-Mangostin 3-O-ß-D-glucopyranoside, α-mangostin 6-O-ß-D-glucopyranoside, α-mangostin 3,6-di-O-ß-D-glucopyranoside, α-mangostin 3-O-ß-D-2-deoxyglucopyranoside, α-mangostin 6-O-ß-D-2-deoxyglucopyranoside, and α-mangostin 3,6-di-O-ß-D-2-deoxyglucopyranoside were successfully produced in practical quantities and characterized by high-resolution quadruple time-of-flight electrospray ionization-mass spectrometry (HR-QTOF ESI/MS), (1)H and (13)C NMR analyses. In excess of the substrate, the maximum productions of three α-mangostin glucopyranosides (4.8 mg/mL, 86.5 % overall conversion of α-mangostin) and three α-mangostin 2-deoxyglucopyronosides (4.0 mg/mL, 79 % overall conversion of α-mangostin) were achieved at 4-h incubation period. All the α-mangostin glycosides exhibited improved water solubility, and their antibacterial activity against three Gram-positive bacteria Micrococcus luteus, Bacillus subtilis, and Staphylococcus aureus was drastically enhanced by the glucosylation at C-3 position. In this study, diverse glycosylated α-mangostin were produced in significant quantities by using inexpensive starting materials and recycling co-factors within a reaction vessel without use of expensive NDP-sugars in the glycosylation reactions.

Bioactivities of ß-mangostin and its new glycoside derivatives synthesized by enzymatic reactions.

Beta-mangostin is a xanthone commonly found in the genus Garcinia. Unlike α-mangostin, to date, there have only been a few studies on the biological activity and derivatization of ß-mangostin. In this study, two novel glycosylated derivatives of ß-mangostin were successfully synthesized via a one-pot enzymatic reaction. These derivatives were characterized as ß-mangostin 6-O-ß-d-glucopyranoside and ß-mangostin 6-O-ß-d-2-deoxyglucopyranoside by TOF ESI/MS and 1H and 13C NMR analyses. Beta-mangostin showed cytotoxicity against KB, MCF7, A549 and HepG2 cancer cell lines, with IC50 values ranging from 15.42 to 21.13 µM. The acetylcholinesterase and α-glucosidase inhibitory activities of ß-mangostin were determined with IC50 values of 2.17 and 27.61 µM, respectively. A strong anti-microbial activity of ß-mangostin against Gram-positive strains (Bacillus subtilis, Lactobacillus fermentum and Staphylococcus aureus) was observed, with IC50 values of 0.16, 0.18 and 1.24 µg ml-1, respectively. Beta-mangostin showed weaker activity against Gram-negative strains (Salmonella enterica, Escherichia coli and Pseudomonas aeruginosa) as well as Candida albicans fungus, with IC50 and MIC values greater than the tested concentration (greater than 32 µg ml-1). The new derivatives of ß-mangostin showed weaker activities than those of ß-mangostin, demonstrating the important role of the hydroxyl group at C-6 of ß-mangostin in its bioactivity.

Altering UDP-glucose Donor Substrate Specificity of Bacillus licheniformis Glycosyltransferase towards TDP-glucose.

The specificity of a Bacillus licheniformis uridine diphosphate (UDP) glycosyltransferase, YjiC, was increased towards thymidine diphosphate (TDP)-sugar by site-directed mutagenesis. The Arg-282 of YjiC was identified and investigated by substituting with Trp. Conversion rate and kinetic parameters were compared between YjiC and its variants with several acceptor substrates such as 7-hydroxyflavone (7-HF), 4',7-dihydroxyisoflavone, 7,8-dihydroxyflavone and curcumin. Molecular docking of TDP-glucose and 7-HF with YjiC model showed pi-alkyl interaction with Arg-282 and His-14, and pi-pi interaction with His14 and thymine ring. YjiC (H14A) variant lost its glucosylation activity with TDP-glucose validating significance of His-14 in binding of TDP-sugars.

Mutational analyses for product specificity of YjiC towards α-mangostin mono-glucoside.

Glycosyltransferases (GTs) are key enzymes for the post-modification of secondary metabolites in drug development processes. In our prior research, an one-pot enzymatic system produced α-mangostin 3,6-di-O-ß-D-glucopyranoside (Mg1) at a higher proportion using wild-type glycosyltransferase (YjiC) but α-mangostin 3-O-ß-D-glucopyranoside (Mg2) exhibited markedly higher anti-bacterial activities. This study focuses on a Bacillus licheniformis-originated flexible glycosyltransferase by mutagenesis to examine the active site residues involved in glycosylation for a product specificity towards Mg2. The generated H298A, H298S, and H298C mutants of YjiC exhibited a regiospecificity towards glycosylated product (Mg2) and were targeted in this study. The production pattern of Mg1 decreased to 63 (H298A), 85 (H298S) and 95% (H298C) yields compared to the wild-type YjiC. The increase of uridine 5'-diphosphate (UDP) leading to the inhibition of enzyme activity and production of uridine 5'-diphosphate glucose (UDP-glucose) in overall system was critical for the specific glycosylated product formation rate. H298A, H298S, and H298C mutants and YjiC exhibited 244, 251, and 186% increases in Mg2 production yields, respectively. And also H298A, H298S, and H298C showed 281, 279, and 251% increases in yield of Mg3 compared with wild type YjiC, respectively. There was improved conversion of both mono-glucosides product (Mg2a and Mg3) than di-glucosides products. The H298 mutants were found to overcome the limitation of the wild-type YjiC for regioselective synthesis of Mg2 by an enzymatic system.

Erratum to: Synthesis of Curcumin Glycosides with Enhanced Anticancer Properties Using One-Pot Multienzyme Glycosylation Technique.

This erratum is being published to correct the author's contribution and name of above manuscript by Gurung et al. that was published in Journal of Microbiology and Biotechnology (2017, 27: 1639-1648). The first author (Rit Bahadur Gurung) and the second author (So Youn Gong) contributed equally to this article. And the seventh author (Tae Jin Oh) should appear as 'Tae-Jin Oh'.

Synthesis of Curcumin Glycosides with Enhanced Anticancer Properties Using One-Pot Multienzyme Glycosylation Technique.

Curcumin is a natural polyphenolic compound, widely acclaimed for its antioxidant, antiinflammatory, antibacterial, and anticancerous properties. However, its use has been limited due to its low-aqueous solubility and poor bioavailability, rapid clearance, and low cellular uptake. In order to assess the effect of glycosylation on the pharmacological properties of curcumin, one-pot multienzyme (OPME) chemoenzymatic glycosylation reactions with UDP- α-D-glucose or UDP-α-D-2-deoxyglucose as donor substrate were employed. The result indicated significant conversion of curcumin to its glycosylated derivatives: curcumin 4'-O-ß- glucoside, curcumin 4',4''-di-O-ß-glucoside, curcumin 4'-O-ß-2-deoxyglucoside, and curcumin 4',4''-di-O-ß-2-deoxyglucoside. The products were characterized by ultra-fast performance liquid chromatography, high-resolution quadruple-time-of-flight electrospray ionization-mass spectrometry, and NMR analyses. All the products showed improved water solubility and comparable antibacterial activities. Additionally, the curcumin 4'-O-ß-glucoside and curcumin 4'-O-ß-2-deoxyglucoside showed enhanced anticancer activities compared with the parent aglycone and diglycoside derivatives. This result indicates that glycosylation can be an effective approach for enhancing the pharmaceutical properties of different natural products, such as curcumin.

Overexpression of a pathway specific negative regulator enhances production of daunorubicin in bldA deficient Streptomyces peucetius ATCC 27952.

The dnrO gene is the first regulator to be activated in the daunorubicin (DNR) biosynthesis pathway of Streptomyces peucetius ATCC 27952. DnrO is known for its self-repression capability while it activates rest of the DNR biosynthesis pathway through cascades of regulatory events. S. peucetius was found to contain no functional copy of bldA-tRNA while a detailed examination of dnrO codons reveals the presence of TTA codon, which is rarely encoded by bldA-tRNA. Therefore, for evaluating the role of dnrO in DNR production, multiple engineered strains of S. peucetius were generated by heterologously expressing bldA, dnrO and combination of bldA and dnrO. Using these strains, the effects of heterologously expressed bldA and overexpressed dnrO were evaluated on pathway specific regulators, mycelial densities and production of DNR. The results showed that the transcription level of dnrO and master regulator dnrI, was found to be elevated in bldA containing strain in comparison to dnrO overexpressed strain. The bldA containing strain produces 45.7% higher DNR than bldA deficient wild type strain from culture broth with OD600 of 1.45 at 72h. Heterologous expression of bldA-tRNA is accounted for increased transcription levels of the DNR pathway specific regulators and enhanced DNR production.

Enhanced production of nargenicin A(1) and generation of novel glycosylated derivatives.

Nargenicin A1, an antibacterial polyketide macrolide produced by Nocardia sp. CS682, was enhanced by increasing the pool of precursors using different sources. Furthermore, by using engineered strain Nocardia sp. ACC18 and supplementation of glucose and glycerol, enhancement was ~7.1 fold in comparison to Nocardia sp. CS682 without supplementation of any precursors. The overproduced compound was validated by mass spectrometry and nuclear magnetic resonance analyses. The novel glycosylated derivatives of purified nargenicin A1 were generated by efficient one-pot reaction systems in which the syntheses of uridine diphosphate (UDP)-α-D-glucose and UDP-α-D-2-deoxyglucose were modified and combined with glycosyltransferase (GT) from Bacillus licheniformis. Nargenicin A1 11-O-ß- D-glucopyranoside, nargenicin A1 18-O-ß-D-glucopyranoside, nargenicin A111 18-O-ß-D- diglucopyranoside, and nargenicin 11-O-ß-D-2-deoxyglucopyranoside were generated. Nargenicin A1 11-O-ß-D-glucopyranoside was structurally elucidated by ultra-high performance liquid chromatography-photodiode array (UPLC-PDA) conjugated with high-resolution quantitative time-of-flight-electrospray ionization mass spectroscopy (HR-QTOF ESI-MS/MS), supported by one- and two-dimensional nuclear magnetic resonance studies, whereas other nargenicin A1 glycosides were characterized by UPLC-PDA and HR-QTOF ESI-MS/MS analyses. The overall conversion studies indicated that the one-pot synthesis system is a highly efficient strategy for production of glycosylated derivatives of compounds like macrolides as well. Furthermore, assessment of solubility indicated that there was enhanced solubility in the case of glycoside, although a substantial increase in activity was not observed.