Abstract 1530: Derivatives of betulinic acid act as modulators of the androgen receptor and report cytotoxicity towards cancer cell lines
Authors: Srovnalova Alzbeta , Gurska Sona , Urban Milan , Sarek Jan , Rehulka Jiri , Dzubak Petr , Hajduch Marian 
Published: June 2022

Abstract
INTRODUCTION Triterpenoids are naturally occurring substances with a broad spectrum of beneficial effects for human health. Betulinic acid belongs to the group of pentacyclic triterpenoids and is well known for its antitumor activities. The library of about 1000 betulinic acid-derived triterpenes was collected and analyzed for cytotoxic activity on cancer and non-cancer cell lines (A549, CCRF-CEM, CEM-DNR, HCT116, K562, K562-TAX, U2OS, BJ and MRC-5) in our Institute over the last 20 years. Steroidal triterpenes are precursors of cholesterol, the precursor of testosterone, the fundamental ligand of the androgen receptor that is the key regulator in several physiological and pathological processes. Finding new modulators of androgen receptor is critical for the treatment of various androgen-dependent diseases, including prostate cancer or anabolic deficiencies. Betulinic acid and its derivatives showed structural similarity with steroids and therefore were selected to be tested for androgen receptor activity modulation.
METHODS The stably transfected cell line HEK 293T GFP-AR was used to determine the translocation activity of the androgen receptor from the cytoplasm to the nucleus after the treatment with the derivatives. The high throughput fluorescent microscopy device and image analysis comprised the data, and compounds with the proved activity were selected for subsequent analysis. The luciferase reporter gene assay was further used to evaluate the effect of compounds on the transcriptional activity of the androgen receptor. The MTS assay was performed to assess the cytotoxicity.
RESULTS Betulinic acid and its eight derivatives were identified to have 5-6 - fold higher translocation activity of AR (ratio cytoplasm GFP intensity/nuclear GFP intensity) than the negative control. Validation on luminescence luciferase reporter assay confirmed 4-5 - fold induction for five derivatives, whereas three derivatives were not active. Interestingly only two of them showed substantial cytotoxicities (IC50&lt; 50 µM) as they possess significant inhibitory activity against all tested cancer cell lines with favorable therapeutic index against the non-cancer cell lines.
CONCLUSION Derivatives of betulinic acid were proven to have modulatory activity on the androgen receptor pathway. The two derivatives of betulinic acid also displayed cytotoxicity towards almost all of the tested cancer cell lines. There could be not only AR modulation responsible for anticancer activity as the derivatives of betulinic acid are well know disruptors of mitochondrion functions as well. The synergy of these two mechanisms of action could potentiate the antitumor activity of the selected compounds.
This work was supported by ENOCH CZ.02.1.01/0.0/0.0/16_019/0000868, CZ-OPENSCREEN - LM2018130, EATRIS-CZ - LM2018133.
Citation Format: Alzbeta Srovnalova, Sona Gurska, Milan Urban, Jan Sarek, Jiri Rehulka, Petr Dzubak, Marian Hajduch. Derivatives of betulinic acid act as modulators of the androgen receptor and report cytotoxicity towards cancer cell lines [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1530.