Rao Gollapudi

Cytotoxic withanolide constituents of Physalis longifolia.

Fourteen new withanolides, 1-14, named withalongolides A-N, respectively, were isolated from the aerial parts of Physalis longifolia together with eight known compounds (15-22). The structures of compounds 1-14 were elucidated through spectroscopic techniques and chemical methods. In addition, the structures of withanolides 1, 2, 3, and 6 were confirmed by X-ray crystallographic analysis. Using a MTS viability assay, eight withanolides (1, 2, 3, 7, 8, 15, 16, and 19) and four acetylated derivatives (1a, 1b, 2a, and 2b) showed potent cytotoxicity against human head and neck squamous cell carcinoma (JMAR and MDA-1986), melanoma (B16F10 and SKMEL-28), and normal fetal fibroblast (MRC-5) cells with IC₅₀ values in the range between 0.067 and 9.3 μM.

Novel withanolides target medullary thyroid cancer through inhibition of both RET phosphorylation and the mammalian target of rapamycin pathway

BACKGROUND Despite development of current targeted therapies for medullary thyroid cancer (MTC), long-term survival remains unchanged. Recently isolated novel withanolide compounds from Solanaceae physalis are highly potent against MTCs. We hypothesize that these withanolides uniquely inhibit RET phosphorylation and the mammalian target of rapamycin (mTOR) pathway in MTC cells as a mechanism of antiproliferation and apoptosis. METHODS MTC cells were treated with novel withanolides and MTC-targeted drugs. In vitro studies assessed cell viability and proliferation (MTS; trypan blue assays), apoptosis (flow cytometry with Annexin V/PI staining; confirmed by Western blot analysis), long-term cytotoxic effects (clonogenic assay), and suppression of key regulatory proteins such as RET, Akt, and mTOR (by Western blot analysis). RESULTS The novel withanolides potently reduced MTC cell viability (half maximal inhibitory concentration [IC(50)], 270-2,850 nmol/L; 250-1,380 nmol/L for vandetanib; 360-1,640 nmol/L for cabozantinib) with induction of apoptosis at <1,000 nmol/L of drug. Unique from other targeted therapies, withanolides suppressed RET and Akt phosphorylation and protein expression (in a concentration- and time-dependent manner) as well as mTOR activity and translational activity of 4E-BP1 and protein synthesis mediated by p70S6kinase activation at IC(50) concentrations. CONCLUSION Novel withanolides from Physalis selectively and potently inhibit MTC cells in vitro. Unlike other MTC-targeted therapies, these compounds uniquely inhibit both RET kinase activity and the Akt/mTOR prosurvival pathway. Further translational studies are warranted to evaluate their clinical potential.

Co-enzyme Q10 (Ubiquinone): It's Implication in Improving the Life Style of the Elderly

Coenzyme Q10 (CoQ10) (1) is lipid-soluble and an important mitochondrial redox component, endogenously produced antioxidant in the human organisms. CoQ10 (1) plays an important role in the production of cellular energy, strengthens the immune system and acts as a free radical scavenger. Aging, poor eating habits, infections and stress affect the amounts of CoQ10 (1) in the humans. As human beings age, they begin to lose the ability to synthesize CoQ10 (1) from food resulting in its deficiency. CoQ10 (1) facilitates the production of adenosine triphosphate (ATP) in the mitochondria by participating in redox reactions within the electron transport chain. Cardiovascular disease deaths in elders are 80% in males and 75% in females. The average age of death from cardiovascular diseases in the developing world is 68 years and in developed world it is 80 years. Cardiovascular disease onset is 7-10 years earlier in men as compared to women. A low level of myocardial CoQ10 (1) is related to the severity of heart failure. Long-term CoQ10 (1) treatment of patients with chronic heart failure is safe and reduces major adverse cardiovascular complications.

Translational application of novel withanolides for cancer treatment from Physalis species and other Solanaceae

Natural products have been the most significant source of drugs and drug leads in history. Plant biodiversity provides a resource of unlimited structural diversity for drug lead and molecular probe discovery programs such as those currently underway at the University of Kansas. Technology is gradually overcoming the traditional difficulties encountered in natural products research by improving access to biodiverse resources and ensuring compatibility of samples with high throughput procedures. Our drug lead discovery effort is aimed at uncovering bioactive molecules from plants of the U.S. Great Plains. Approaches utilized in the selection of samples consist of biodiversity-based collections and ethnobotanical information on the medicinal uses in the areas of study. In order to identify antiproliferative compounds, a library of over 1,200 plant extracts was tested for antiproliferative activity against an array of cell lines [melanoma cell lines (B16F10, SKMEL28); human head and neck squamous cell carcinomas (HNSCC) cell lines (MDA1986, JMAR, UM-SCC-2, JHU011); medullary thyroid carcinoma (MTC) cell lines (TT and DRO81–1), and a non-malignant cell line (MRC5)]. In vitro experiments included evaluation of anticancer activity and effects on cell proliferation by MTS assay and trypan blue exclusion. Apoptosis and cell cycle arrest were characterized by annexin V-propidium iodide (PI) flow cytometry and confirmed by Western Blot analysis for activation of caspase 3 and cleavage of PARP as well as cell-cycle specific proteins. Analysis of specific pathways including modulation of Akt, MAP-kinase and RET phosphorylation were evaluated in vitro as well as ex vivo in treated mice using Western blot analysis. Physalis longifolia Nutt. and several related members in the Solanaceae were identified as the most active of the species tested in this study. This led to the discovery of over 30 withanolide-type steroidal lactones, 16 of which are new compounds. In addition to withaferin A (WA), 14 withanolides showed IC50 values in the range between 0.067 and 9.3µM in vitro by MTC with selectivity of 3 to 20 fold compared to normal cells. A mechanistic study showed that WA induced apoptosis and cell death in HNSCC and MTC cells as well as modulation of the cell cycle from G0/G1 arrest to a shift to G2/M and S-phase. Western blot analysis demonstrated that cancer cells treated with WA exhibited inhibition of Akt and caspase-3 expression, and cleavage of PARP. WA was further evaluated in vivo in a metastaic murine model of MTC. All treated animals were alive, showing tumor regression and growth delay without toxicity or weight loss at six weeks post-treatment. Tumor cells treated with WA demonstrated inhibition of total and phospho-RET levels by Western blot analysis in a dose dependent manner (almost complete inhibition with treatment of 5µM WA) as well as potent inhibition of phosphor-ERK and phosphor-Akt levels. WA was shown to be a novel natural-product RET-inhibitor with efficacy in a metastatic murine model of MTC. This investigation constitutes the first report of the antiproliferative activity of WA and other withanolides against MTC and HNSCC. Procurement and screening protocols, isolation and structure determination, lead optimization, and antiproliferative activities of withanolides will be presented.