Rutinosides-derived from Sarocladium strictum 6-O-α-rhamnosyl-ß-glucosidase show enhanced anti-tumoral activity in pancreatic cancer cells.

BACKGROUND: Low targeting efficacy and high toxicity continue to be challenges in Oncology. A promising strategy is the glycosylation of chemotherapeutic agents to improve their pharmacodynamics and anti-tumoral activity. Herein, we provide evidence of a novel approach using diglycosidases from fungi of the Hypocreales order to obtain novel rutinose-conjugates therapeutic agents with enhanced anti-tumoral capacity. RESULTS: Screening for diglycosidase activity in twenty-eight strains of the genetically related genera Acremonium and Sarocladium identified 6-O-α-rhamnosyl-ß-glucosidase (αRßG) of Sarocladium strictum DMic 093557 as candidate enzyme for our studies. Biochemically characterization shows that αRßG has the ability to transglycosylate bulky OH-acceptors, including bioactive compounds. Interestingly, rutinoside-derivatives of phloroglucinol (PR) resorcinol (RR) and 4-methylumbelliferone (4MUR) displayed higher growth inhibitory activity on pancreatic cancer cells than the respective aglycones without significant affecting normal pancreatic epithelial cells. PR exhibited the highest efficacy with an IC50 of 0.89 mM, followed by RR with an IC50 of 1.67 mM, and 4MUR with an IC50 of 2.4 mM, whereas the respective aglycones displayed higher IC50 values: 4.69 mM for phloroglucinol, 5.90 mM for resorcinol, and 4.8 mM for 4-methylumbelliferone. Further, glycoconjugates significantly sensitized pancreatic cancer cells to the standard of care chemotherapy agent gemcitabine. CONCLUSIONS: αRßG from S. strictum transglycosylate-based approach to synthesize rutinosides represents a suitable option to enhance the anti-proliferative effect of bioactive compounds. This finding opens up new possibilities for developing more effective therapies for pancreatic cancer and other solid malignancies.

Glycosylated 4-methylumbelliferone as a targeted therapy for hepatocellular carcinoma.

BACKGROUND & AIMS: Reaching efficacious drug delivery to target cells/tissues represents a major obstacle in the current treatment of solid malignancies including hepatocellular carcinoma (HCC). In this study, we developed a pipeline to selective add complex-sugars to the aglycone 4-methylumbelliferone (4MU) to help their bioavailability and tumour cell intake. METHODS: The therapeutic efficacy of sugar-modified rutinosyl-4-methylumbelliferone (4MUR) and 4MU were compared in vitro and in an orthotopic HCC model established in fibrotic livers. The mechanistic bases of its selective target to liver tumour cells were evaluated by the interaction with asialoglycoprotein receptor (ASGPR), the mRNA expression of hyaluronan synthases (HAS2 or HAS3) and hyaluronan deposition. RESULTS: 4MUR showed a significant antiproliferative effect on liver tumoural cells as compared to non-tumoural cells in a dose-dependent manner. Further analysis showed that 4MUR is incorporated mostly into HCC cells by interaction with ASGPR, a receptor commonly overexpressed in HCC cells. 4MUR-treatment decreased the levels of HAS2 and HAS3 and the cytoplasmic deposition of hyaluronan. Moreover, 4MUR reduced CFSC-2G activation, hence reducing the fibrosis. In vivo efficacy showed that 4MUR treatment displayed a greater tumour growth inhibition and increased survival in comparison to 4MU. 4MUR administration was associated with a significant reduction of liver fibrosis without any signs of tissue damage. Further, 60% of 4MUR treated mice did not present macroscopically tumour mass post-treatment. CONCLUSION: Our results provide evidence that 4MUR may be used as an effective HCC therapy, without damaging non-tumoural cells or other organs, most probably due to the specific targeting.

Glycoconjugation: An approach to cancer therapeutics.

Cancer constitutes the second leading cause of death globally and is considered to have been responsible for an estimated 9.6 million fatalities in 2018. Although treatments against gastrointestinal tumors have recently advanced, those interventions can only be applied to a minority of patients at the time of diagnosis. Therefore, new therapeutic options are necessary for advanced stages of the disease. Glycosylation of antitumor agents, has been found to improve pharmacokinetic parameters, reduce side effects, and expand drug half-life in comparison with the parent compounds. In addition, glycosylation of therapeutic agents has been proven to be an effective strategy for their targeting tumor tissue, thereby reducing the doses of the glycodrugs administered to patients. This review focusses on the effect of the targeting properties of glycosylated antitumor agents on gastrointestinal tumors.

The flavonoid degrading fungus Acremonium sp. DSM 24697 produces two diglycosidases with different specificities.

AbstractDiglycosidases hydrolyze the heterosidic linkage of diglycoconjugates, releasing the disaccharide and the aglycone. Usually, these enzymes do not hydrolyze or present only low activities towards monoglycosylated compounds. The flavonoid degrading fungus Acremonium sp. DSM 24697 produced two diglycosidases, which were termed 6-O-α-rhamnosyl-ß-glucosidase I and II (αRßG I and II) because of their function of releasing the disaccharide rutinose (6-O-α-L-rhamnosyl-ß-D-glucose) from the diglycoconjugates hesperidin or rutin. In this work, the genome of Acremonium sp. DSM 24697 was sequenced and assembled with a size of ~ 27 Mb. The genes encoding αRßG I and II were expressed in Pichia pastoris KM71 and the protein products were purified with apparent molecular masses of 42 and 82 kDa, respectively. A phylogenetic analysis showed that αRßG I grouped in glycoside hydrolase family 5, subfamily 23 (GH5), together with other fungal diglycosidases whose substrate specificities had been reported to be different from αRßG I. On the other hand, αRßG II grouped in glycoside hydrolase family 3 (GH3) and thus is the first GH3 member that hydrolyzes the heterosidic linkage of rutinosylated compounds. The substrate scopes of the enzymes were different: αRßG I showed exclusive specificity toward 7-O-ß-rutinosyl flavonoids, whereas αRßG II hydrolyzed both 7-O-ß-rutinosyl- and 3-O-ß-rutinosyl- flavonoids. None of the enzymes displayed activity toward 7-O-ß-neohesperidosyl- flavonoids. The recombinant enzymes also exhibited transglycosylation activities, transferring rutinose from hesperidin or rutin onto various alcoholic acceptors. The different substrate scopes of αRßG I and II may be part of an optimized strategy of the original microorganism to utilize different carbon sources.

Enzyme-mediated transglycosylation of rutinose (6-O-α-l-rhamnosyl-d-glucose) to phenolic compounds by a diglycosidase from Acremonium sp. DSM 24697.

The structure of the carbohydrate moiety of a natural phenolic glycoside can have a significant effect on the molecular interactions and physicochemical and pharmacokinetic properties of the entire compound, which may include anti-inflammatory and anticancer activities. The enzyme 6-O-α-rhamnosyl-ß-glucosidase (EC 3.2.1.168) has the capacity to transfer the rutinosyl moiety (6-O-α-l-rhamnopyranosyl-ß-d-glucopyranose) from 7-O-rutinosylated flavonoids to hydroxylated organic compounds. This transglycosylation reaction was optimized using hydroquinone (HQ) and hesperidin as rutinose acceptor and donor, respectively. Since HQ undergoes oxidation in a neutral to alkaline aqueous environment, the transglycosylation process was carried out at pH values ≤6.0. The structure of 4-hydroxyphenyl-ß-rutinoside was confirmed by NMR, that is, a single glycosylated product with a free hydroxyl group was formed. The highest yield of 4-hydroxyphenyl-ß-rutinoside (38%, regarding hesperidin) was achieved in a 2-h process at pH 5.0 and 30 °C, with 36 mM OH-acceptor and 5% (v/v) cosolvent. Under the same conditions, the enzyme synthesized glycoconjugates of various phenolic compounds (phloroglucinol, resorcinol, pyrogallol, catechol), with yields between 12% and 28% and an apparent direct linear relationship between the yield and the pKa value of the aglycon. This work is a contribution to the development of convenient and sustainable processes for the glycosylation of small phenolic compounds.

Screening and quantification of the enzymatic deglycosylation of the plant flavonoid rutin by UV-visible spectrometry.

The enzymatic deglycosylation of the plant flavonoid rutin (quercetin-3-O-(6-O-α-l-rhamnopyranosyl-ß-d-glucopyranoside) is usually assessed by means of high performance liquid chromatography (HPLC). We have developed a spectrophotometric method for the quantification of the released quercetin. After the enzymatic reaction, quercetin is extracted with ethyl acetate, and subsequently oxidized under basic conditions. The absorbance of quercetin autooxidation products at 320nm was correlated with the quercetin concentration by linear regression (molar extinction coefficient 23.2 (±0.3)×103M-1cm-1). With this method, rutin-deglycosylation activity in buckwheat flour and a commercial naringinase was measured, and showed no significant differences with the results obtained by HPLC. The convenience of this method resides on the enzymatic activity quantification using the natural substrate by UV-visible spectrometry. Moreover, the simplicity and speed of analysis allows its application for a large number of samples.