Standardization of zygotic embryo culture from Nerium oleander L. and comparative analysis of biosynthesized cardiac glycosides within in vitro and acclimatized plants

The primary result of our experiment revealed that the germination percentage of N. oleander mature seeds is only 30%. From this observation, the concept of protocol standardization for zygotic embryo culture of this plant was originated. Zygotic embryo culture was proved an efficient in vitro multiplication system of N. oleander. The maximum germination percentage (96%) of zygotic embryos was observed on ¼ MS medium with 15 gm/L sucrose, whereas the best growth medium was optimized as ½ B5 with same sucrose concentration. The second part of this study was aimed to find out the cardiac glycoside accumulation pattern in both in vitro and acclimatized plants. For this purpose, one-month-old in vitro plantlets and acclimatized plants were subjected to LC-MS analysis and 09 cardiac glycosides were detected and quantified in both the systems. Most of the cardiac glycosides including odoroside A (32.71 mg/gm DW), odoroside H (4.69 mg/gm DW) and oleandrin (0.52 mg/gm DW) were found to be accumulated at maximum level within in vitro plantlets. CG 840b (1.89 mg/gm DW) is the only cardiac glycoside, which was maximally accumulated in acclimatized plants. From this study, it can be concluded that, zygotic embryo culture is a better choice for in vitro multiplication of N. oleander when compared to matured seeds and in vitro grown plantlets of this species favor cardiac glycosides biosynthesis in comparison to acclimatized plants. Therefore, all future research on the enrichment of cardiac glycosides from this plant may be conducted on zygotic embryos derived in vitro grown plantlets or cultures.

The Cardiac Glycoside Deslanoside Exerts Anticancer Activity in Prostate Cancer Cells by Modulating Multiple Signaling Pathways

Prostate cancer (PCa) is a leading cause of cancer-related deaths among men worldwide, and novel therapies for advanced PCa are urgently needed. Cardiac glycosides represent an attractive group of candidates for anticancer repurposing, but the cardiac glycoside deslanoside has not been tested for potential anticancer activity so far. We found that deslanoside effectively inhibited colony formation in vitro and tumor growth in nude mice of PCa cell lines 22Rv1, PC-3, and DU 145. Such an anticancer activity was mediated by both the cell cycle arrest at G2/M and the induction of apoptosis, as demonstrated by different functional assays and the expression status of regulatory proteins of cell cycle and apoptosis in cultured cells. Moreover, deslanoside suppressed the invasion and migration of PCa cell lines. Genome-wide expression profiling and bioinformatic analyses revealed that 130 genes were either upregulated or downregulated by deslanoside in both 22Rv1 and PC-3 cell lines. These genes enriched multiple cellular processes, such as response to steroid hormones, regulation of lipid metabolism, epithelial cell proliferation and its regulation, and negative regulation of cell migration. They also enriched multiple signaling pathways, such as necroptosis, MAPK, NOD-like receptor, and focal adhesion. Survival analyses of the 130 genes in the TCGA PCa database revealed that 10 of the deslanoside-downregulated genes (ITG2B, CNIH2, FBF1, PABPC1L, MMP11, DUSP9, TMEM121, SOX18, CMPK2, and MAMDC4) inversely correlated, while one deslanoside-upregulated gene (RASD1) positively correlated, with disease-free survival in PCa patients. In addition, one deslanoside-downregulated gene (ENG) inversely correlated, while three upregulated genes (JUN, MXD1, and AQP3) positively correlated with overall survival in PCa patients. Some of the 15 genes have not been implicated in cancer before. These findings provide another candidate for repurposing cardiac glycosides for anticancer drugs. They also suggest that a diverse range of molecular events underlie deslanoside’s anticancer activity in PCa cells.

10-4-4, A Cardiac Glycoside Block Growth in Cancer Cells via Nuclear Receptor Nur77 Mediated Na+/K+- ATPase Endocytosis

Background Cancer is second only to heart disease as a cause of death. Develop new and more effective treatment strategies for cancer remain a major challenge for human medicine today. Nur77, an orphan member for the nuclear receptor superfamily, inhibits growth in cancer cells by translocation to cytoplasmic. Small molecules that trigger Nur77 nuclear export may be an ideal anti-cancer candidate. Methods Cell proliferation was evaluated by 3-(4, 5-dimethylthiazol-2-y1)-2, 5-diphenyl tetrazolium bromide (MTT) assay. The protein expression level were detected by western blot analysis. Immunostaining and cell fractionation assays were used to assess subcelluar localization of Nur77. Cell apoptosis, cell cycle and calcium were detected by flow cytometry. Zebrafish liver cancer models were used to determine anti-cancer effect of 10-4-4 in vivo. Results In this study, we exhibit 10-4-4 a cardiac glycoside, extracted from Antiaris toxicaria lesch, has sensitivity to cancer cells. 10-4-4 induces apoptosis and G2/M cell cycle arrest in HepG2 cells. Consistently, 10-4-4 augments Nur77 expression and cytoplasmic localization, its restraint of cancer cells growth is Nur77 dependent. Meanwhile, as a cardiac glycoside, 10-4-4 inhibits Na+/K+- ATPase (NaK) activation. To further confirm the molecular mechanism of 10-4-4, we found the association between Nur77 and NaK. The suppression of NaK by 10-4-4 increases the level of intracellular Ca2+. Ca2+, as a second messenger, specific activates protein kinase C (PKC). PKC has been reported on the influence of Nur77 nuclear export. Identical conclusions are obtained in this studies that 10-4-4 induces PKC activation play an important role in Nur77 nuclear export. Notably, the cytoplasmic Nur77 induced by 10-4-4 interaction with NaK to induce NaK endocytosis, and then trigger G2/M cell cycle arrest and apoptosis. Studies in Zebrafish shows that 10-4-4 potently inhibits the growth of liver cancer cells in vivo. Conclusions Our results exhibit that 10-4-4 possesses an anti-cancer activity in vitro and in vivo via NaK-Nur77 signaling pathway and maybe offers a novel strategy in development of chemotherapeutic anti-cancer drug.

Interaction of (+)-Strebloside and Its Derivatives with Na+/K+-ATPase and Other Targets

Docking profiles for (+)-strebloside, a cytotoxic cardiac glycoside identified from Streblus asper, and some of its derivatives and Na+/K+-ATPase have been investigated. In addition, binding between (+)-strebloside and its aglycone, strophanthidin, and several of their other molecular targets, including FIH-1, HDAC, KEAP1 and MDM2 (negative regulators of Nrf2 and p53, respectively), NF-κB, and PI3K and Akt1, have been inspected and compared with those for digoxin and its aglycone, digoxigenin. The results showed that (+)-strebloside, digoxin, and their aglycones bind to KEAP1 and MDM2, while (+)-strebloside, strophanthidin, and digoxigenin dock to the active pocket of PI3K, and (+)-strebloside and digoxin interact with FIH-1. Thus, these cardiac glycosides could directly target HIF-1, Nrf2, and p53 protein–protein interactions, Na+/K+-ATPase, and PI3K to mediate their antitumor activity. Overall, (+)-strebloside seems more promising than digoxin for the development of potential anticancer agents.

Phytochemical Profile of Saribus rotundifolius (Anahaw) Crude Extract Found in the University of Eastern Philippines

Phytochemical profiling focuses on the determination of the secondary metabolite present on a sample. Aims: In this research, a phytochemical profiling was done in Saribus rotundifolius (Anahaw) leaves and fruit crude extracts. Methodology: This study determines the secondary metabolites; alkaloid, tannin, flavonoid, cardiac glycoside, saponin, and terpenoid. The samples were cut into pieces and was submerged in an ethanol solution for the extract of crude extract and undergo a rotary evaporator for the separation of the ethanol solvent. Results: The finding showed that the anahaw leaves and fruit crude extract contains secondary metabolites present in both leaves and fruit crude extracts, that can be used in pharmaceutical and drug development, this includes an anti-microbial potential.

Spathodea companulata (African tulip tree) stem and root barks extracts ameliorated N-nitroso diethylamine induced hepatic impairment in male rats

Spathodea campanulata Beauv. is an important plant widely used in traditional medicine for the treatment of hepatic disease. In the present study, we evaluate the phytochemical composition and hepatoprotective potential effect of the stem and root bark extracts of Spathodea companulata on dimethylnitrosamine (DEN) induced hepatic impairment in albino rats. Phytochemical compositions were analyzed using standard protocols. Forty-five male rats were grouped into 9 (A-I) of 5 rats each. Groups A-F were treated with stem bark extract, root bark extract, quercetin, DEN + stem bark extract, DEN + root bark extract, DEN + quercetin respectively while groups G-1 serve as control groups and were treated with corn oil only (Vehicle control), DEN only (Toxicant control), and distilled water (normal control) respectively. DEN was given at 25 mg/kg b.w., i.p on a weekly basis while all treatments were administered at 100 mg/kg BW orally for 60 days. Serum transaminase activities, body weight, and liver body weight ratio were evaluated. Results revealed the presence of alkaloids, cardiac glycoside, saponins, anthraquinone, steroids, and flavonoids in stem bark extract of Spathodea companulata, while the root bark contains cardiac glycoside, saponins, anthraquinonoid, steroids, and flavonoid. The DEN non-treated rat exhibited significant (p<0.05) weight loss and increase serum alanine transaminase (ALT) and aspartate transaminase (AST) activities when compared with the normal control. Treatment with stem and root bark extracts of Spathodea companulata significantly ameliorated the toxicant induce alterations in the transaminase activity and body weight loss when compared with the DEN nontreated rats. In conclusion, this study provides scientific validation of the use of this plant in traditional medicine for the management of hepatic disorders

Structural Insights into the Interactions of Digoxin and Na+/K+-ATPase and Other Targets for the Inhibition of Cancer Cell Proliferation

Digoxin is a cardiac glycoside long used to treat congestive heart failure and found recently to show antitumor potential. The hydroxy groups connected at the C-12, C-14, and C-3′a positions; the C-17 unsaturated lactone unit; the conformation of the steroid core; and the C-3 saccharide moiety have been demonstrated as being important for digoxin’s cytotoxicity and interactions with Na+/K+-ATPase. The docking profiles for digoxin and several derivatives and Na+/K+-ATPase were investigated; an additional small Asn130 side pocket was revealed, which could be useful in the design of novel digoxin-like antitumor agents. In addition, the docking scores for digoxin and its derivatives were found to correlate with their cytotoxicity, indicating a potential use of these values in the prediction of the cancer cell cytotoxicity of other cardiac glycosides. Moreover, in these docking studies, digoxin was found to bind to FIH-1 and NF-κB but not HDAC, IAP, and PI3K, suggesting that this cardiac glycoside directly targets FIH-1, Na+/K+-ATPase, and NF-κB to mediate its antitumor potential. Differentially, digoxigenin, the aglycon of digoxin, binds to HDAC and PI3K, but not FIH-1, IAP, Na+/K+-ATPase, and NF-κB, indicating that this compound may target tumor autophagy and metabolism to mediate its antitumor propensity.