Betulinic acid and oleanolic acid, natural pentacyclic triterpenoids, interfere with N-linked glycan modifications to intercellular adhesion molecule-1, but not its intracellular transport to the cell surface.

Betulinic acid (3ß-hydroxy-20(29)-lupen-28-oic acid), oleanolic acid (3ß-hydroxy-olean-12-en-28-oic acid), and ursolic acid (3ß-hydroxy-urs-12-en-28-oic acid) are close structural isomers of natural pentacyclic triterpenoid carboxylic acids. We recently identified a unique biological effect of ursolic acid, its inhibition of the intracellular trafficking of glycoproteins. In the present study, we demonstrated that betulinic acid and oleanolic acid did not inhibit the interleukin-1α-induced expression of cell-surface intercellular adhesion molecule-1 (ICAM-1) in human lung carcinoma A549 cells. Nevertheless, betulinic acid and, to a lesser extent, oleanolic acid interfered with N-linked glycan modifications to ICAM-1 in a similar manner to castanospermine (an inhibitor of endoplasmic reticulum α-glucosidases I and II), but not swainsonine (an inhibitor of Golgi α-mannosidase II). Consistent with these results, betulinic acid and oleanolic acid inhibited yeast α-glucosidase activity, but not Jack bean α-mannosidase activity. Thus, to the best of our knowledge, this is the first study to show that betulinic acid and oleanolic acid interfere with N-linked glycan modifications to ICAM-1, but not its intracellular transport to the cell surface.

Cardenolide aglycones inhibit tumor necrosis factor α-induced expression of intercellular adhesion molecule-1 at the translation step by blocking Na⁺/K⁺-ATPase.

Cardiac glycosides, which are inhibitors of Na(+)/K(+)-ATPase, are classified into cardenolides and bufadienolides. We have recently shown that two cardenolide glycosides, ouabain and odoroside A, inhibit Na(+)/K(+)-ATPase, thereby preventing nuclear factor κB-inducible protein expression by blocking Na(+)-dependent amino acid transport. In this study, we investigated the mechanism of action of cardenolide aglycones in tumor necrosis factor α (TNF-α)-induced gene expression. Ouabagenin, digitoxigenin, and digoxigenin were found to inhibit the TNF-α-induced cell-surface expression of intercellular adhesion molecule-1 (ICAM-1) in human lung carcinoma A549 cells. Those cardenolide aglycones did not inhibit the TNF-α-induced expression of ICAM-1 mRNA, but strongly inhibited the TNF-α-induced expression of ICAM-1 as translation product. The inhibition of the TNF-α-induced ICAM-1 expression by ouabagenin, digitoxigenin, and digoxigenin was significantly reversed by the ectopic expression of ouabain-resistant rat Na(+)/K(+)-ATPase α1 isoform. Moreover, knockdown of Na(+)/K(+)-ATPase α1 isoform augmented the inhibition of the TNF-α-induced ICAM-1 expression by ouabagenin or ouabain. These results clearly indicate that cardenolide aglycones inhibit the TNF-α-induced ICAM-1 expression at the translation step by blocking Na(+)/K(+)-ATPase.

Ursolic acid, a natural pentacyclic triterpenoid, inhibits intracellular trafficking of proteins and induces accumulation of intercellular adhesion molecule-1 linked to high-mannose-type glycans in the endoplasmic reticulum.

Ursolic acid (3ß-hydroxy-urs-12-en-28-oic acid) is a natural pentacyclic triterpenoid that is present in many plants, including medicinal herbs, and foods. Ursolic acid was initially identified as an inhibitor of the expression of intercellular adhesion molecule-1 (ICAM-1) in response to interleukin-1α (IL-1α). We report here a novel biological activity: ursolic acid inhibits intracellular trafficking of proteins. Ursolic acid markedly inhibited the IL-1α-induced cell-surface ICAM-1 expression in human cancer cell lines and human umbilical vein endothelial cells. By contrast, ursolic acid exerted weak inhibitory effects on the IL-1α-induced ICAM-1 expression at the protein level. Surprisingly, we found that ursolic acid decreased the apparent molecular weight of ICAM-1 and altered the structures of N-linked oligosaccharides bound to ICAM-1. Ursolic acid induced the accumulation of ICAM-1 in the endoplasmic reticulum, which was linked mainly to high-mannose-type glycans. Moreover, in ursolic-acid-treated cells, the Golgi apparatus was fragmented into pieces and distributed over the cells. Thus, our results reveal that ursolic acid inhibits intracellular trafficking of proteins and induces the accumulation of ICAM-1 linked to high-mannose-type glycans in the endoplasmic reticulum.

Ursolic Acid Inhibits Na+/K+-ATPase Activity and Prevents TNF-α-Induced Gene Expression by Blocking Amino Acid Transport and Cellular Protein Synthesis.

Pro-inflammatory cytokines, such as tumor necrosis factor (TNF)-α, induce the expression of a wide variety of genes, including intercellular adhesion molecule-1 (ICAM-1). Ursolic acid (3ß-hydroxy-urs-12-en-28-oic acid) was identified to inhibit the cell-surface ICAM-1 expression induced by pro-inflammatory cytokines in human lung carcinoma A549 cells. Ursolic acid was found to inhibit the TNF-α-induced ICAM-1 protein expression almost completely, whereas the TNF-α-induced ICAM-1 mRNA expression and NF-κB signaling pathway were decreased only partially by ursolic acid. In line with these findings, ursolic acid prevented cellular protein synthesis as well as amino acid uptake, but did not obviously affect nucleoside uptake and the subsequent DNA/RNA syntheses. This inhibitory profile of ursolic acid was similar to that of the Na+/K+-ATPase inhibitor, ouabain, but not the translation inhibitor, cycloheximide. Consistent with this notion, ursolic acid was found to inhibit the catalytic activity of Na+/K+-ATPase. Thus, our present study reveals a novel molecular mechanism in which ursolic acid inhibits Na+/K+-ATPase activity and prevents the TNF-α-induced gene expression by blocking amino acid transport and cellular protein synthesis.