3,4-dimethoxybenzyl isothiocyanate enhances doxorubicin efficacy in LoVoDX doxorubicin-resistant colon cancer and attenuates its toxicity in vivo

Abstract

Aims

The aim of the study was to evaluate the potential of naturally occurring isothiocyanates and doxorubicin in combined treatment of doxorubicin-resistant colon cancer. Doxorubicin is a cytostatic commonly used to treat many different types of cancer but its usage is often abrogated by severe side-effects and drug-induced resistance.

Main methods

The antiproliferative potential of the combined treatment was analyzed in vitro by the SRB method (sulforhodamine B) and further evaluated for the mechanisms that determine the treatment outcome using a series of assays which included oxidative stress, apoptosis and compounds accumulation assessment. Ultimately, a combined treatment potential was assessed in vivo utilizing doxorubicin-resistant colon cancer model.

Key finding

The results indicate that naturally occurring isothiocyanates, represented by 3,4-dimethoxybenzyl isothiocyanate (dMBITC) increase doxorubicin the efficacy in doxorubicin-resistant human colon adenocarcinoma model by attenuated drug efflux, an increased reactive oxygen species production and an increased rate of apoptosis. In in vitro studies, over a 3-fold decrease in doxorubicin IC₅₀ value was observed on the LoVoDX cell line when used in combination with suboptimal concentrations of dMBITC. The combined therapy exhibited a significantly higher efficacy than doxorubicin-alone treatment (c.a. 50% tumor growth inhibition in comparison to c.a. 25% for doxorubicin-alone treatment) in vivo. At the same time, the combined treatment attenuates doxorubicin toxicity as evidenced by improved animals body mass, main organs weight and biochemical markers of toxicity.

Significance

The adopted approach provides evidence that isothiocyanates can be successfully applied in the treatment of doxorubicin-resistant colon cancer, which warrants further studies.

Introduction

The brassicaceae family, which encompasses many frequently consumed vegetables (like cauliflower, horseradish, Brussels sprouts, broccoli etc.), is a rich source of chemically diverse glucosinolates, biologically inert precursors of isothiocyanates. These naturally occurring compounds for over 30 years receive sustained attention as potential anticancer agents [1]. The high reactivity of isothiocyanates arises from the electrophilic character of the central carbon atom in the isothiocyanato moiety and is responsible for its rapid addition to sulfhydryl groups of which the most abundant intracellular source is glutathione. The adduct and the products of its metabolism (including the N-acetylcysteine derivative) can act as an intracellular depot of isothiocyanates, which allows for the protein's sulfhydryl groups subsequent modifications via transthiocarbamoylation [2]. This unique feature enables rapid isothiocyanates intracellular accumulation and the modulation of multiple signaling and metabolic pathways, which is a result of the covalent modification of over 30 proteins, including cytochromes P450, Kelch-like ECH-associated protein 1 (Keap1), α- and β-tubulin, and many more [3]. In numerous studies isothiocyanates and their mercapturic derivatives exhibited a high anticancer activity at multiple diverse cancer models (both in vitro and in vivo) with but negligible toxicity [4].

Doxorubicin (Doxo) belongs to anthracyclines – antineoplastic antibiotics – and is one of the most potent and widely used chemotherapeutics, recognized by the World Health Organization (WHO) as the essential medicine. Its usage covers, but is not limited to, several types of sarcomas, lymphomas, breast, colon and bladder cancer. Unfortunately, apart from the activity as an intercalating agent and topoisomerase II inhibitor, doxorubicin causes severe toxic effects because it affects negatively mitochondrial respiration and elevated reactive oxygen species (ROS) production [5]. The most dangerous side effect is cardiomyopathy whose rate strongly correlates with doxorubicin cumulative dose (600 mg/m² body surface is estimated as the highest dose that can be received in a lifetime). Prolonged anthracycline-based treatment is often associated with the occurrence of chemoresistance, mainly caused by the elevated expression and activity of ABC multidrug-resistance pumps, which further decreases the compound's usability [6].

Recently, isothiocyanates and their metabolites – mercapturic acids – have been sparking a keen interest as a common diet constituent whose regular consumption substantially reduces tumor incidence [7]. In several studies, isothiocyanates have been revealed to exhibit considerable capabilities as adjuvants for classical chemotherapeutics with markedly increased treatment outcomes [8]. Herein, we report that naturally occurring 3,4-dimetoksybenzyl isothiocyanate not only increases doxorubicin antitumor potential in the doxorubicin-resistant colon adenocarcinoma model, but also significantly reduces its toxicity.