Glycosylated 4-methylumbelliferone as a Targeted Therapy for Hepatocellular Carcinoma

Gisela Weiz; Maria I. Molejon; Mariana Malvicini; Caecilia H. C. Sukowati; Claudio Tiribelli; Guillermo Mazzolini; Javier D. Breccia


Liver International. 2022;42(2):444-457. 

In This Article

Abstract and Introduction


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.


Hepatocellular carcinoma (HCC) is the fourth leading cause of cancer-related death worldwide and is particularly refractory to the available therapeutic drugs.[1] Unfortunately, curative treatments such as surgery, liver transplantation, or ablation are reserved for early stages and can only be applied in less than 30% of the patients.[2] The current therapeutic options for advanced HCC are tyrosine kinase inhibitors (TKI) such as first-line sorafenib and lenvatinib or regorafenib, cabozantinib, or second-line ramucirumab.[3] Novel therapeutic strategies such as gene and cell-based therapies, and more recently immunotherapy strategies based on immune checkpoint inhibitors in different combinations have improved patient's survival.[4–7] However, many patients are resistant to these therapies. Hence, new, more powerful and more specific therapeutic approaches are required.

During hepatocarcinogenesis, the accumulation of extracellular matrix (ECM) occurs in the liver parenchyma. This excessive deposition of ECM culminates in major changes in liver architecture acting as a reservoir for pro-inflammatory and pro-fibrogenic mediators leading to liver fibrosis. Hepatic stellate cells (HSCs) are the key fibrogenic effector cell type in liver and the main ECM-producing cells. When hepatic injury persists, HSCs become activated and are transformed into myofibroblast-like cells.[8,9]

Hyaluronic acid (hyaluronan, HA) is one of the markers of fibrosis.[10,11] It is a linear, large and ubiquitous nonsulphated glycosaminoglycan of the ECM[12] produced by three hyaluronan synthases (HAS1, 2 and 3).[13] The coumarin 4-Methylumbelliferone (4MU) has been described as an inhibitor of HA synthesis in many studies.[14–17] It is a commercially available drug approved for use in humans as a cholagogue in several countries called 'hymecromone'.[18] While it has a long and relatively reassuring safety record, questions remain about the high dosage of hymecromone required for cancer treatment.

Glycosylation is an interesting strategy to improve drugs' selective cells/tissue targeting. Due to the wide spectrum of cell receptors and the specific interactions to which carbohydrates can be specifically recognized.[19–21] The surface of liver cells contains a significant number of asialoglycoprotein receptors (ASGPR) which are overexpressed in HCC.[22] ASGPR exhibits a high affinity for galactose and rhamnose residues,[23] in particular, the L-rhamnosyl ligands that are useful for drug targeting.[24–26] However, genes responsible for its synthesis had not been described in mammals, as well as the glycoside hydrolases which release L-rhamnose from glycoconjugates. Thus, rhamnosylated drugs may not be deglycosylated by endogenous enzymes, which guarantees that the active drugs are specifically released at the sites where L-rhamnose receptors are located.[27,28]

Recently, we synthesized a diglycosylated derivative of the coumarin 4MU, namely the rutinosyl-4-methylumbelliferone (4MUR).[29] 4MUR differs from 4MU in the disaccharide moiety rutinose (6-O-α-L-rhamnosyl-β-D-glucose) being L-rhamnose the terminal unit (Figure 1A).

Figure 1.

Poptimisation of 4MUR using the recombinant 6-O-α-rhamnosyl-β-glucosidase. (A) Rutinose transglycosylation from hesperidin to 4-methylumbelliferone performed by the enzyme α-rhamnosyl-β-glucosidase to obtain rutinosyl-4-methylumbelliferone. Effect on the yield of (B) rutinose donor concentration (hesperidin) (C) acceptor concentration (4MU) and (D) reaction time. This experiment is representative of three independent experiments. Data are expressed as the mean ± SD of 4MUR concentration (measured in triplicate)

In this work, we report 4MUR antitumoural effect in a targeted manner to liver cancer type, without damaging non-tumoural cells or other organs. This study opens a new perspective for drugs development, repurposing 4MU as antitumoural agent for future HCC treatments.