Proscillaridin A Sensitizes Human Colon Cancer Cells to TRAIL-Induced Cell Death.

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is a cytotoxic cytokine that induces cancer cell death by binding to TRAIL receptors. Because of its selective cytotoxicity toward cancer cells, TRAIL therapeutics, such as recombinant TRAIL and agonistic antibodies targeting TRAIL receptors, have garnered attention as promising cancer treatment agents. However, many cancer cells acquire resistance to TRAIL-induced cell death. To overcome this issue, we searched for agents to sensitize cancer cells to TRAIL-induced cell death by screening a small-molecule chemical library consisting of diverse compounds. We identified a cardiac glycoside, proscillaridin A, as the most effective TRAIL sensitizer in colon cancer cells. Proscillaridin A synergistically enhanced TRAIL-induced cell death in TRAIL-sensitive and -resistant colon cancer cells. Additionally, proscillaridin A enhanced cell death in cells treated with TRAIL and TRAIL sensitizer, the second mitochondria-derived activator of caspase mimetic. Proscillaridin A upregulated TRAIL receptor expression, while downregulating the levels of the anti-cell death molecules, cellular FADD-like IL-1ß converting enzyme-like inhibitor protein and Mcl1, in a cell type-dependent manner. Furthermore, proscillaridin A enhanced TRAIL-induced cell death partly via O-glycosylation. Taken together, our findings suggest that proscillaridin A is a promising agent that enhances the anti-cancer efficacy of TRAIL therapeutics.

Repurposing proscillaridin A in combination with decitabine against embryonal rhabdomyosarcoma RD cells.

PURPOSE: Embryonal rhabdomyosarcoma (eRMS) is the most common type of rhabdomyosarcoma in children. eRMS is characterized by malignant skeletal muscle cells driven by hyperactivation of several oncogenic pathways including the MYC pathway. Targeting MYC in cancer has been extremely challenging. Recently, we have demonstrated that the heart failure drug, proscillaridin A, produced anticancer effects with specificity toward MYC expressing leukemia cells. We also reported that decitabine, a hypomethylating drug, synergizes with proscillaridin A in colon cancer cells. Here, we investigated whether proscillaridin A exhibits epigenetic and anticancer activity against eRMS RD cells, overexpressing MYC oncogene, and its combination with decitabine. METHODS: We investigated the anticancer effects of proscillaridin A in eRMS RD cells in vitro. In response to drug treatment, we measured growth inhibition, cell cycle arrest, loss of clonogenicity and self-renewal capacity. We further evaluated the impact of proscillaridin A on MYC expression and its downstream transcriptomic effects by RNA sequencing. Then, we measured protein expression of epigenetic regulators and their associated chromatin post-translational modifications in response to drug treatment. Chromatin immunoprecipitation sequencing data sets were coupled with transcriptomic results to pinpoint the impact of proscillaridin A on gene pathways associated with specific chromatin modifications. Lastly, we evaluated the effect of the combination of proscillaridin A and the DNA demethylating drug decitabine on eRMS RD cell growth and clonogenic potential. RESULTS: Clinically relevant concentration of proscillaridin A (5 nM) produced growth inhibition, cell cycle arrest and loss of clonogenicity in eRMS RD cells. Proscillaridin A produced a significant downregulation of MYC protein expression and inhibition of oncogenic transcriptional programs controlled by MYC, involved in cell replication. Interestingly, significant reduction in total histone 3 acetylation and on specific lysine residues (lysine 9, 14, 18, and 27 on histone 3) was associated with significant protein downregulation of a series of lysine acetyltransferases (KAT3A, KAT3B, KAT2A, KAT2B, and KAT5). In addition, proscillaridin A produced synergistic growth inhibition and loss of clonogenicity when combined with the approved DNA demethylating drug decitabine. CONCLUSION: Proscillaridin A produces anticancer and epigenetic effects in the low nanomolar range and its combination with decitabine warrants further investigation for the treatment of eRMS.

PL201, a Reported Rhamnoside Against Alzheimer's Disease Pathology, Alleviates Neuroinflammation and Stimulates Nrf2 Signaling.

Neuroinflammation induced by overactivated glia cells is believed to be a major hallmark of Alzheimer's disease (AD) and a hopeful target against AD. A rhamnoside PL201 was previously reported to promote neurogenesis and ameliorate AD, and in this study, we revealed that PL201 also significantly reduced accumulation of the activated microglia and proinflammatory cytokines in APP/PS1 mice. In vitro, PL201 consistently suppressed the microglia induction of proinflammatory cytokines after stimulation with lipopolysaccharides and Aß42. Further mechanistic studies demonstrated that PL201 considerably enhanced the expression level and the nuclear translocation of Nrf2, a key regulator of neuroinflammation. Moreover, PL201 effectively stimulated Nrf2 signaling cascade, including upregulation of HO-1 and downregulation of NF-κB pathway. Thus, our findings indicated the anti-neuroinflammatory effect by PL201 in vivo and suggested that PL201 or the like, with multiple functions such as neurogenesis, mitochondria maintenance, and anti-neuroinflammation, could be a promising candidate in AD treatment.

The bioavailability of methylepoxyproscillaridin (P35): a new semisynthetic cardiac glycoside.

The pharmacokinetic data of methylepoxyproscillaridin (3'-methyl-4'-5'-epoxyproscillaridin) (P35) after 1 mg i.v. or oral administration in two different galenic preparations (hard and soft gelatin capsules) in randomized succession were studied in 9 healthy volunteers, showing a bioavailability of 88 +/- 10.5% (soft gelatin capsule) and 81 +/- 12% (hard gelatin capsule). The half-life of P35 was calculated to be 80 +/- 13.3 h.

[Threshold of action of meproscillarin following intravenous administration]. / Wirkungseintritt von Meproscillarin nach intravenöser Applikation.

A placebo-controlled double-blind trial was conducted, to determine the onset of effect of Meproscillarin (injected intravenously). 28 days apart six healthy volunteers were given double-blind 1,0 mg Meproscillarin or placebo and 60 days later 0,6 mg Digoxin, each drug as short infusion over four minutes. The total electromechanical systole corrected for heart rate (QS2c), was used as criterion of the inotropic effect of the glycoside. After placebo there no changes were observed over a period of two hours. Both glycosides shortened QS2c for about--16 ms. From Meproscillarin a 40% higher dosis was required, to achieve the same effect as with digoxin. After use of Meproscillarin the peak effect was reached after ten minutes; after digoxin a period of 60--120 minutes were required. The results show that intravenously given Meproscillarin is a fast acting glycoside.

Disposition rate of proscillaridin A in man after multiple oral doses.

After multiple oral doses, the disposition rate constant (beta) of proscillaridin was studied in 4 young healthy volunteers and 33 elderly patients with congestive heart failure. Glycoside activity in plasma was assayed by the 86Rb-technique. In the volunteers the beta averaged 0.0299 corresponding to a half-life (t 1/2) of 23 h. beta could be determined in 24 patients and was 0.0139 +/- 0.0077 (mean +/- SD). The SD of beta due to biological factors was estimated to be 0.0072. The total variation of beta was 10fold. The mean beta corresponded to a t 1/2 of 49 h with a range from 19 to 209 h. It is concluded that the great variation of beta means difficulty in obtaining adequate plasma levels of proscillaridin and that a rapid elimination of the glycoside cannot be presumed.

Biliary excretion and enterochepatic recycling of proscillaridin A after oral adminstration to man.

A single oral dose of proscillaridin A (1.0-1.5 mg) was given to six patients with T-tube drainage of the common bile duct, and simultaneous samples of bile and plasma were collected at various times during the following 24 hours. Glycoside activity was assayed by the 86Rb-uptake inhibition technique. Peak activities in plasma (mean 0.80 ng/ml) were attained after 0.5-2h, and in bile (mean 6.9 ng/ml) after 1-4h. Subsequently, proscillaridin activity in bile was less than 5 ng/ml for the remainder of the sampling period, and 10-100 times higher than that in plasma. Bile samples treated with beta-glucuronidase and sulphatase showed 100-200 fold increase in glycoside activity. Deconjugation was also produced by treatment with enteric contents. The results suggest that conjugation of unchanged proscillaridin is a major metabolic route. After excretion in the bile, the conjugates may be split in the intestine and reabsorbed as active glycoside.

Proscillaridin activity in portal and peripheral venous blood after oral administration to man.

The absorption of proscillaridin A was studied in four patients undergoing catheterization of the portal vein for diagnostic purposes. Proscillaridin 1.5 mg was given as a single oral dose and plasma glycoside activity was analyzed by the 86Rb-uptake inhibition technique. Proscillaridin appeared rapidly in the portal blood, peak activity being found after 15 min in three and after 30 min in one patient. In peripheral blood the peak activity occurred after approximately 35 min. Despite rapid passage across the gut wall, porto-peripheral differences in glycoside activity were small; they were zero after 4h. The mean amount absorbed as active porscillaridin during the first 4h after the dose was calculated to be only 7.1% of the given amount. Late porto-peripheral differences, probably due to enterohepatic recycling, appeared after 6h in three patients; The results suggest that proscillaridin undergoes first pass inactivation in the gut wall. Enterohepatic recirculation may contribute to the amounts of active glycoside that reach the systemic circulation.

Activities of proscillaridin A in thoracic duct lymph after single oral doses in man.

In order to study the possibility that orally administered proscillaridin after absorption is transported by the lymph to the systemic circulation, the concentrations of the glycoside in thoracic duct lymph were analyzed in two patients with thoracic duct drainage. They received the drug as a single oral dose; plasma and lymph concentrations were measured by 86Rb-technique. Lymph was collected at various intervals for 24 hrs. The proscillaridin activity in thoracic duct lymph was low and followed closely that the plasma. During the sampling period, a total of 300 ng and 240 ng, respectively, was recovered in the lymph, corresponding to less than 0.03% of the administered dose. The results indicate that proscillaridin is not transported by the thoracic duct lymph.

On the absorption of proscillaridin A after single oral doses to normal and achlorhydric subjects.

Proscillaridin A was given in single oral doses (1.5-2.5 mg) to normal and achlorhydric subjects. Plasma activities of the glycoside were analysed by 86Rb-technique. The absorption pattern was similar in both groups. A marked first peak of proscillaridin activity was seen after about 30 min. After a first minimum, a second peak of activity was registered within 6-12 hrs. An estimate of the amount of active glycoside absorbed during the first 12 hrs after the administration was obtained by calculating the areas under the plasma activity curves (AUC). When corrected for differences in dose per kg body weight, the mean AUC in the achlorhydric group was about 60 per cent greater than in the normal group. The results suggest that proscillaridin is rapidly absorbed; gastric acidity seems to contribute to inter-individual differences in the bio-availability of the glycoside.

[Enteral availability and therapeutic activity of proscillaridin-4'-methyl ether (author's transl)]. / Enterale Verfügbarkeit und therapeutische Wirksamkeit von Proscillaridin-4' methyläther

20 cardiac patients with the clinical and radiological signs of heart failure were treated alternately intravenously and orally with proscillaridin-4'-methyl ether in a 2-period change-over procedure. With medium rate full saturation a steady state was maintained under observation of electrocardiographic parameters. The maintenance dose was determined to be 1.00 mg intravenously and 1.71 mg orally. Thus, relative enteral availability was 60%. In 70% of patients cardiac recompensation could be reached only by treatment with proscillaridin-4'-methyl ether. In 9 of 20 patients undesirable side effects consisted of laxative effects.

Plasma concentrations during repeated intravenous and oral methyl-proscillaridin application in man.

The aim of this study was to establish data on the plasma levels of the glycoside derivative methyl-proscillaridin (MP) following repeated intravenous and oral doses. The study was carried out on healthy male volunteers. Each received 0.5 mg of MP daily at 8 a.m. for 7 days. 6 volunteers received the drug i.v., 6 p.o. as tablets and 5 p.o. as elixir. Plasma glycoside concentrations were measured utilizing a 86Rb-erythrocyte assay. The mean plasma concentrations on the 6th and 7th days of application were: 752.9 (Sx = 303.5) pg/ml for the i.v. route, 432.9 (Sx = 115.5) pg/ml for the tablets and 473.1 (Sx = 321.5) pg/ml for the elixir. The mean ratio between plasma concentrations with tablets and i.v. injection averaged 63%. It is concluded that the therapeutic activity of oral MP is about 60 to 70% that of the i.v. application.

[Investigations on the Stability of Proscillaridine, Proscillaridine-3'-methylether and Proscillaridine-4'-methylether in Artificial Gastric and Intestinal Fluids (author's transl)]. / Untersuchungen zur Stabilität von Proscillaridin, Proscillaridin-3'-methyläther und Proscillaridin-4'-methyläther in künstlichen Verdauungsflüssigkeiten

The stability of bufadienolides in artificial gastric and intestinal fluids was investigated at different pH-values as a function of incubation time using proscillaridine, proscillaridine-3'-methylether and proscillaridine-4'-methylether. These glycosides were completely stable on pH 3.0--8.5 during the investigated time interval of 1 h. At lower pH-values cleavage of the glycosidic bond occurred. At pH 1.0 73% of proscillaridine, 33% of proscillaridine-3'-methylether and 41% of proscillaridine-4'-methylether were decomposed. At pH 2.0 only about 10% decomposition of these compounds could be detected. These experiments show that the stability of proscillaridine in gastric juice is comparable with the stability of digitalis glycosides and digoxin derivatives. The stability of methylated proscillaridine derivatives in artificial digestion fluids, however, proved to be superior to any orally administered cardiac glycoside.

On the pharmacokinetics of proscillaridin A in man.

The plasma concentration of proscillaridin was measured by a modified 86Rb method during treatment with multiple doses of a commercial preparation of proscillaridin. Despite high doses, very low plasma levels were found, and there were only minute amounts of glycoside activity in urine and faeces. Administration of an enteric-coated proscillaridin preparation gave higher plasma levels, which raises the possibility of inactivation of the glycoside by acid gastric juice. The results suggest that proscillaridin has low biological availability when given orally, and that it is extensively metabolised in the body.