Abstract A175: Novel natural withanolides potently inhibit adrenocortical carcinomas in vivo with superior safety over standard cytotoxic therapeutics.

Background: Current adjuvant strategies for an adrenocortical carcinoma (ACC) using mitotane or multidrug cytotoxics as in the Italian protocol have limited efficacy yet carry significant morbidity and toxicity. We recently demonstrated that novel natural withanolides from the North American Physalis plant potently and selectively inhibit several solid tumors including ACCs in vitro. These anticancer mechanisms through a combination of oxidative stress and heat shock protein client modulation in ACCs specifically target key tumorigenic proteins and pathways in the disease. Therefore, we hypothesize that these novel withanolides will potently inhibit ACC tumor growth in xenograft murine models without the toxicity associated with standard therapies for this disease. Methods: Tumor xenografts were established by subcutaneous injection of 5x106mouse Y1 cells into the flank region of 6-week old female, athymic, Nu/Nu mice. Drug treatment with 5mg/kg/day of withanolides (WA or WGA-TA or WGB-DB), mitotane (300mg.kg/day), or cisplatin (5-7.5 mg/Kg once weekly for 3 weeks) or control was administered ip daily for 21 days once tumors were at least 4mm in diameter. Tumor size, weight and body score as an estimate of gross toxicity were measured thrice weekly. Assessment of apoptosis and modulation of oncogenic pathway proteins was by done by immunoblot analysis. Significance was set for p<0.05 with differences between two or more means determined by two-way ANOVA t test. Results: Daily treatment of Y1 tumors with the novel acetyleated withanolide, WGA-TA, resulted in a two-fold decrease in tumor volume compared to controls (p<0.01) while WA and WGB-DB showed only slight decrease in tumor volume. All the withanolides were well tolerated in vivo without any weight loss or adverse side effects noted during treatment. While response rates for mitotane and cisplatin were similar to WGA-TA (∼2-fold decrease) compared to controls, both of these drugs carried mild to moderate toxicity that was not observed with any of the withanolides, suggesting their superior safety and potent efficacy in this disease. Induction of apoptosis, including PARP cleavage and Caspase 3 activation was measured on Western blot analysis of tumor samples. Evaluation of several key regulatory proteins of ACC pathogenesis, including Akt, mTOR, HEDGEHOG, and heat shock protein 90 client proteins after treatment with withanolides indicated significant down regulation of HSP90 client proteins (phospho-Akt, jagged 1, HSF1 and mTOR downstream regulators p70S6K and 4EBP1) compared to untreated controls, indicating a putative role of HSP90 modulation in the therapeutic efficacy of withanolides in ACC. Conclusion: Natural withanolides are potent novel therapeutics against adrenocortical carcinomas in vivo with improved toxicity compared to standard agents like mitotane and cisplatin. These data support further translational evaluation of WGA-TA toward clinical evaluation in this difficult malignancy. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):A175. Citation Format: Chitra Subramanian, Patrick T. Grogan, Joseph Bazzill, Rob Gallagher, Hauping Zhang, Barbara Timmermann, Mark S. Cohen. Novel natural withanolides potently inhibit adrenocortical carcinomas in vivo with superior safety over standard cytotoxic therapeutics. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr A175.