Combination treatments in in vitro and in vivo models between molecules reverting epigenetic gene silencing and DNA-interacting anticancer drugs

Sabatino, Maria Antonietta (2008). Combination treatments in in vitro and in vivo models between molecules reverting epigenetic gene silencing and DNA-interacting anticancer drugs. PhD thesis The Open University.

DOI: https://doi.org/10.21954/ou.ro.0000eb08

Abstract

Epigenetic transcriptional gene silencing plays a fundamental role in cancer development and has been considered as a target for cancer therapy in the last few years, mainly due to its reversibility by small molecules. Among the several methylated genes investigated, glutathione-S-transferase (GST) P1, a protein belonging to cellular detoxification systems, has been shown to be extensively promoter-methylated in prostate cancer. My study therefore describes a new therapeutic approach against prostate cancer, based on the combination of demethylating agents and brostallicin, a DNA minor groove binding drug, which is activated in the cell by binding to glutathione, a reaction catalyzed by GST.

Among the demethylating molecules tested on the prostatic cancer cell line LNCaP in in vitro combinations with brostallicin, zebularine was able to increase brostallicin activity with little toxicity compared to the other tested demethylating drugs.

These in vitro results prompted the in vivo testing of zebularine with brostallicin on LNCaP cells transplanted in mice. Prolonged treatment with zebularine was able to significantly improve brostallicin antitumour activity compared to both drugs administrated as single agents. When GSTP1 expression was investigated in treated samples versus untreated controls, no protein re-expression was found and this was related to the unchanged levels of GSTP1 promoter methylation. In contrast, the demethylating effect of zebularine was clearly evident in the promoter of GSTM1 gene, which is also silenced by methylation in LNCaP cells. GSTM1 codes for a class of GST enzymes that has recently been found to be more active on brostallicin than GSTP1.

This indicates that the activation of brostallicin cytotoxicity in LNCaP cells by zebularine likely depends on enzymatic activation by GSTM1 rather than GSTP1 and strengthens the feasibility of this combination as a treatment for prostate cancer in the clinic, and as model for the therapy of other solid tumours.