Abstract B24: Natural withanolides: A new group of anticancer drugs that selectively target the PI3K-mTOR pathway as novel potent therapeutics against colon cancers in vitro and in vivo
Authors: Subramanian Chitra , White Peter , Gingrich Alicia , Timmermann Barbara N. , Cohen Mark Steven 
Published: October 2015

Abstract
Background: Current targeted inhibitors for colon cancer can reduce tumor burdens or stabilize disease, but are limited by their toxicity and lack of durable efficacy. Efforts continue to identify novel agents for this disease and newer multi-kinase inhibitors are currently being evaluated in clinical trials. Novel natural withanolides have been shown to selectively inhibit the PI3K-Akt-mTOR pathway in vitro and in vivo in adrenal cancer and medullary thyroid cancers. We hypothesize that this new class of drug compounds will be potent, selectively targeted inhibitors of this pathway network in colon cancers in vitro as well as in vivo as a promising novel therapeutics for this disease.
Methods: In vitro: Withaferin A (WA), withalongolide A (WGA) and acetylated derivatives of each were first tested for anticancer effect on tumor cell viability on 5 different colon cancer lines (SW48, SW480, HT-29, SW-620, HCT-116) by MTS assay, then for apoptosis by flow cytometry and confirmed by western analysis for caspase 3 activation and PARP cleavage. PI3K-mTOR inhibition was confirmed by western blot analysis for total and phosphorylated levels of: mTOR, Akt, 4EBP1, p70-S6K; as well as for specific effects on colon cancer regulatory pathways including: GSK3β, β-catenin, Notch1 cleavage, and cyclin D1. In vivo: Athymic nude mice were injected with 10^7 HT-29 human colon cancer-cells in the left posterior flank and treated x 21days once tumors were &gt;100mm3(N=10/drug) and followed for endpoints of toxicity (decreased body weight or body score),tumor growth&gt;1500mm3 and survival.
Results: IC50 levels of the parent compound WA averaged 640nM whereas the acetylated derivatives were as low as 30-100nM in the 5 cell lines compared to 4500nM in normal fibroblast cells. The withanolides potently induced apoptosis (starting at 250-500nM drug) on flow cytometry and this was confirmed by caspase 3 activation and PARP cleavage on Western analysis. These drugs also selectively inhibited phosphorylation of mTOR, Akt, 4EBP1, and p70S6K in a dose-dependent manner in the colon cancer lines starting at only 500nM of drug. Specific to colon cancers, the withanolides modulated GSK3β, β-catenin, Notch1 cleavage, and cyclin D1 levels in a dose-dependent manner all more potently than targeted tyrosine kinase inhibitors like ventadenib and cabozantenib which did not inhibit the PI3K-mTOR pathway. In vivo, all control animals progressed to metastatic disease to the liver and a terminal study endpoint within 6 weeks. All treatment arms inhibited HT-29 colon cancer growth at varying rates during the treatment period with only the WA treatment arm at 8mg/kg/day demonstrating a complete pathologic response to treatment in 80% of mice (p&lt;0.001 vs controls with a tumor increase of 363±31% by day 21); (p&lt;0.001 vs. vandetanib or cabozantenib 31-282% increase by day 21 respectively). WA-treated mice also had 100% survival after 8wks vs. all other arms(0-71%survival;p&lt;0.01). Body weight loss during treatment was worst with vandetanib (18.5±1.3%) and cabozantinib (6.85±1.8%) compared to the withanolide treatment arms(p&lt;0.01) with return to baseline weight by week 5.
Conclusions: Natural withanolides are a novel class of chemotherapy drugs that selectively target PI3K-mTOR in colon cancers and demonstrate potent efficacy in vitro and in vivo compared to TKIs being evaluated in clinical trials with improved tolerability and less toxicity in vivo. These compounds warrant further evaluation to determine their clinical potential in this disease.
Citation Format: Chitra Subramanian, Peter White, Alicia Gingrich, Barbara N. Timmermann, Mark Steven Cohen. Natural withanolides: A new group of anticancer drugs that selectively target the PI3K-mTOR pathway as novel potent therapeutics against colon cancers in vitro and in vivo. [abstract]. In: Proceedings of the AACR Special Conference: Targeting the PI3K-mTOR Network in Cancer; Sep 14-17, 2014; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(7 Suppl):Abstract nr B24.