Digitoxigenin monodigitoxoside

Cytotoxic and cytostatic effects of digitoxigenin monodigitoxoside (DGX) in human lung cancer cells and its link to Na,K-ATPase.[Pubmed:29101813]

Biomed Pharmacother. 2018 Jan;97:684-696.

Cardiac glycosides (CGs) are natural compounds widely used to treat several cardiac conditions and more recently have been recognized as potential antitumor agents. They are known as Na,K-ATPases ligands, which is a promising drug target in cancer. In this study, the short and long-lasting cytotoxic effects of the natural cardenolide Digitoxigenin monodigitoxoside (DGX) were evaluated against two non-small cell lung cancer lines (A549 and H460 cells). It was found that DGX induced cytotoxic effects in both cells and the apoptotic effects were more pronounced on H460 cells. In long-term analysis, using the clonogenic and the cumulative population doubling (CPD) assays, DGX showed a reduction of cell survival, after 15days without re-treatment. To better understand DGX effects in A549 cells, several assays were conducted. In cell cycle analysis, DGX caused an arrest in S and G2/M phases. This compound also increased the number of cells in subG1 phase in a concentration- and time-dependent manner. The presence of beta-galactosidase positive cells, large nucleus and flattened cells indicated senescence. Additionally, DGX inhibited Na,K-ATPase activity in A549 cells, as well as in purified pig kidney and in human red blood cell membrane preparations, at nanomolar range. Moreover, results of molecular docking showed that DGX binds with high efficiency (-11.4Kcal/mol) to the Na,K-ATPase (PDB:4HYT). Taken together, our results highlight the potent effects of DGX both in A549 and H460 cells, and disclose its link with Na,K-ATPase inhibition.

Inhibition of cell proliferation, invasion and migration by the cardenolides digitoxigenin monodigitoxoside and convallatoxin in human lung cancer cell line.[Pubmed:26252521]

Nat Prod Res. 2016 Jun;30(11):1327-31.

Cardiac glycosides consist of a large family of naturally derived compounds that are clinically used to treat congestive heart failure, and also present anticancer properties. In this study, the cytotoxic effects of two cardenolides, Digitoxigenin monodigitoxoside (DGX) and convallatoxin (CON) were screened in four human tumour cell lines. Both compounds showed anti-proliferative effects in all tumour cells, at nanomolar concentrations. Since the human lung cancer cell line A549 was the most sensitive, we investigated the anti-proliferative, anti-migratory and anti-invasive effects of these cardenolides. DGX and CON reduced A549 cell migration, being able to reduce more than 90% of cell invasion. Their effects on the expression of key regulators of metastatic mechanism showed decreased levels of MMP-2, MMP-9 and p-FAK. Both compounds also presented low toxicity for healthy cells. Finally, this work provides the first insights into the effects of these cardenolides on key steps of lung cancer metastasis.

Effects of digitoxigenin, digoxigenin, and various cardiac glycosides on cardenolide accumulation in shoot cultures of Digitalis lanata.[Pubmed:17253315]

Planta Med. 1998 Dec;64(8):705-10.

Various cardenolide genins and cardenolide glycosides were administered to light-grown and dark-grown Digitalis lanata shoot cultures to investigate conversion reactions related to the formation and rearrangement of the sugar side chain of Digitalis glycosides. Digitoxigenin was converted to digitoxigen-3-one, 3-epidigitoxigenin, and digoxigenin. In addition, various cardiac glycosides were formed, including mono-glycosides with glucose, glucomethylose, fucose, and digitalose, as well as the corresponding diglycosides, all containing a terminal glucose. Digitoxosylated cardenolides were not formed, although the light-grown shoot cultures were capable of producing these compounds. Exogenous cardenolide fucosides were not converted into cardenolide digitoxosides. Administration of evatromonoside (Digitoxigenin monodigitoxoside) did not force the formation of cardenolide di- or tridigitoxosides. Our results support the hypothesis that cardenolide fucosides and digitoxosides are formed via different biosynthetic routes and that cardenolide genins can be fucosylated but not digitoxosylated, indicating that digitoxosylation may only occur at an earlier stage in the cardenolide pathway.

Preparation and antigenic properties of digitoxin-bovine serum albumin conjugates linked at the digitoxose C-3' and C-3" positions.[Pubmed:8069249]

Biol Pharm Bull. 1994 Apr;17(4):467-71.

In order to obtain specific antisera to digitoxin, four new types of hapten-bovine serum albumin (BSA) conjugates were synthesized from digitoxin. The haptens were linked to the carrier protein through hemisuccinate and hemisuccinylglycine bridges at the C-3' and C-3" positions in the digitoxose chain. The antisera were prepared by immunizing rabbits with each digitoxin-BSA conjugate and the properties of the antisera were investigated by RIA with 3H-labeled digitoxin. Among these antisera, the antiserum raised against digitoxin 3'-hemisuccinate-BSA conjugate possessed high specificity for digitoxin, exhibiting only minor cross-reactions with digitoxigenin bisdigitoxoside (4.0%), dihydrodigitoxin (2.8%), digoxin (2.4%), Digitoxigenin monodigitoxoside (0.23%) and digitoxigenin (< 0.05%).

Formation of a beta-glucuronidase-resistant glucuronide conjugate of digitoxin by dog liver microsomes.[Pubmed:7905397]

Drug Metab Dispos. 1993 Nov-Dec;21(6):1147-50.

The aim of these studies was to characterize the glucuronide conjugates of digitoxin and Digitoxigenin monodigitoxoside (DMD) produced by liver microsomes from the dog with respect to hydrolysis by beta-glucuronidase and to behavior on HPLC. These results have been compared with studies of conjugates produced by liver microsomes from the rat. Glucuronidation was similar with both substrates with dog microsomes, whereas rat microsomes formed the glucuronide with DMD but not with digitoxin. The DMD glucuronide from both species was completely hydrolyzed by beta-glucuronidase, but no hydrolysis of digitoxin glucuronide was detected. The digitoxin glucuronide was hydrolyzed by a buffer at pH 1.5 but not at pH 10. After acid hydrolysis, the major products appear to be digitoxigenin and DMD glucuronide. These results suggest that glucuronidation of certain drugs by the dog is quite different from that of other species and that the dog may be the only species that possesses a glucuronosyltransferase capable of forming a glucuronide conjugate with digitoxin. The dog also has a glucuronosyltransferase, similar to that in the rat, which is responsible for glucuronidation of DMD. Whether this represents a single glucuronosyltransferase or two different enzymes remains to be elucidated.

UDP-glucuronosyltransferase activity toward digitoxigenin monodigitoxoside in human liver microsomes.[Pubmed:8097706]

Drug Metab Dispos. 1993 Mar-Apr;21(2):338-41.

The properties of UDP-glucuronosyltransferase (UDPGT) toward digitoxigenin-monodigitoxoside (DT1) have been studied in human liver microsomes. The enzyme activity determined in nonactivated microsomes was very low (20 pmol/min/mg protein) compared with previously published values in rats (104 pmol/min/mg protein) or mice (379 pmol/min/mg protein) DT1-UDPGT activity was increased (180 to 220% of control activity) by Lubrol PX, Triton X-100, or (3-[3-cholamidopropyl]dimethylammonio)-1-propane sulfonic acid. The rate of DT1 glucuronidation determined for 29 different human liver microsomes was variable (18 to 87 pmol/min/mg protein). The KM found was approximately 4.5 microM. DT1-UDPGT activity was tentatively correlated with other known UDPGT activities. No significant correlations were found between DT1 and p-nitrophenol or 4-hydroxybiphenyl UDPGT activities. On the other hand, a strong correlation (r = 0.64, p < 0.05) was observed between DT1-UDPGT activity and digoxigenin-monodigitoxoside (DG1, another cardiac glycoside) glucuronidation rate.

Differential stability of drug-metabolizing enzyme activities in primary rat hepatocytes, cultured in the absence or presence of dexamethasone.[Pubmed:2067545]

Mutat Res. 1991 Jul;249(1):81-92.

The effects of primary hepatocyte culture on the rat cytochrome P450-dependent monooxygenase system and several conjugating enzyme activities were examined using a culture system similar to those used for evaluation of chemicals as potential genotoxins. Cytochrome P450 and cytochrome b5 contents progressively decreased throughout the 72-h culture period to less than 25% of initial values, whereas cytochrome P450 reductase rapidly decreased by 50% during attachment, but then remained stable. Cytochrome P450-dependent testosterone hydroxylase activities decreased more rapidly in culture than did cytochrome P450 content reaching less than 50% of attachment levels by 24 h. Cytochrome P450IIIA immunoreactive protein decreased at a similar rate to testosterone-6 beta-hydroxylase. Activated UDP-glucuronyltransferase activities towards 1-naphthol and testosterone declined more slowly over the 72 h than cytochrome P450 and remained at 50-60% of initial values at 72 h. UDP-glucuronyltransferase activity towards Digitoxigenin monodigitoxoside (DIG) did not decrease during culture. Glutathione-S-transferase and sulfotransferase activities also declined during the 72 h at rates which appeared to be isozyme-dependent. Addition of 1 microM dexamethasone (DEX) to the culture medium increased UDP-glucuronyltransferase activity towards DIG, cytochrome P450 reductase and testosterone-6 beta-hydroxylase activities up to 2.5-, 2.0- and 7-fold, respectively and induced cytochrome P450IIIA immunoreactive protein(s) in the hepatocytes after 24 and 48 h of culture; DEX was less effective at the 72 h time-point. DEX treatment also significantly accelerated the decreases in glutathione-S-transferase activities and in sulfotransferase activities towards 1-naphthol and estrone. Thus, it appears that primary rat hepatocytes cultured under standard conditions, not only rapidly lose their monooxygenase capabilities, but also some of their capacity for conjugation. Furthermore, the use of DEX in cell culture medium to enhance cell survival does not maintain total drug-metabolizing enzyme capability, but appears to transiently and selectively increase expression of certain isozymes at the expense of others.

Characterization of paracetamol UDP-glucuronosyltransferase activity in human liver microsomes.[Pubmed:2116803]

Biochem Pharmacol. 1990 Aug 1;40(3):595-600.

A specific high performance liquid chromatographic assay has been developed for the measurement of paracetamol glucuronide formation by the microsomal fraction of human liver. The procedure has been used to characterize paracetamol glucuronidation kinetics in human livers microsomes and to assess the substrate specificity of the paracetamol UDP-glucuronosyltransferase (UDPGT) activity. Paracetamol glucuronidation followed Michaelis-Menten kinetics, suggesting the involvement of a single form of UDPGT, or possibly two or more forms of UDPGT with similar affinities for paracetamol, in this reaction. Mean apparent Km and Vmax values were 7.37 +/- 0.99 mM and 4.76 +/- 1.35 nmol/min/mg, respectively. Addition of the non-ionic detergent Brij 58 to microsomal incubations resulted in approximately 50% activation of microsomal paracetamol UDPGT-activity. This contrasts to the approximately three-fold activation of 4-methylumbelliferone, morphine and 4-nitrophenol glucuronidation observed following Brij 58 treatment of human liver microsomes. The glucuronidated xenobiotics chloramphenicol, Digitoxigenin monodigitoxoside, 4-hydroxybiphenyl, 4-methylumbelliferone, morphine, 1-naphthol and 4-nitrophenol were screened for inhibitory effects on paracetamol glucuronidation. Of these compounds, only Digitoxigenin monodigitoxoside and 1-naphthol were found to cause significant inhibition of paracetamol UDPGT activity. Along with the results of previous studies of the kinetics and inhibitor profile of human liver glucuronidation reactions (Miners et al., Biochem Pharmacol 37: 665-671, 1988 and 37: 2839-2845, 1988), these data indicate that the model glucuronidated substrates paracetamol, morphine and 4-methyllumbelliferone may be used to differentiate at least four human liver UDPGT isozyme activities.

Kinetic and inhibitor studies of 4-methylumbelliferone and 1-naphthol glucuronidation in human liver microsomes.[Pubmed:3124857]

Biochem Pharmacol. 1988 Feb 15;37(4):665-71.

The glucuronidation kinetics of 4-methylumbelliferone (4MU) and 1-naphthol (1NP) have been investigated in human liver microsomes to determine the validity of using these compounds as probes for specific UDP-glucuronosyltransferase (GT) activities in human liver. 4MU glucuronidation followed Michaelis-Menten kinetics, whereas 1NP glucuronidation kinetics were biphasic. Cross inhibition studies were performed with 4MU and 1NP to determine the relationship between 4MU glucuronidation and the two phases of 1NP glucuronidation. 4MU glucuronidation was competitively inhibited by 1NP but 4MU inhibited only the high affinity component of 1NP glucuronidation. There was good agreement between the apparent Km values for 4MU and the high affinity component of 1NP glucuronidation and their respective apparent K1 values determined in the cross inhibition studies. These data suggest that the same form(s) of human liver GT is involved in 4MU glucuronidation and the high affinity component of 1NP glucuronidation. A number of compounds known to be specific substrates for purified rat liver GTs were screened for inhibitory effects on 4MU glucuronidation in human liver microsomes. 4-Nitrophenol, 2-aminophenol and androsterone inhibited 4MU glucuronidation whereas bilirubin, chloramphenicol, Digitoxigenin monodigitoxoside, morphine, oestrone and testosterone had no effect. 4-Nitrophenol and 2-aminophenol were competitive inhibitors of 4MU glucuronidation but the inhibition of 4MU glucuronidation by androsterone followed atypical kinetics. Overall, the substrate specificity of the human liver 4MU/high affinity 1NP-GT activity appears to be broadly similar to that of the 3-methylcholanthrene inducible rat hepatic microsomal GT.