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.