# The Combination of Thymoquinone and Chloroquine Dose-Dependently Regulates Autophagy and Potentiates Metastatic Melanoma Cell Death via Autophagy-Dependent and -Independent Mechanisms

## TL;DR

Combining thymoquinone and chloroquine increases cancer cell death in melanoma by affecting autophagy, a process cells use to survive stress.

## Contribution

This study reveals how thymoquinone and chloroquine interact to enhance melanoma cell death through both autophagy-dependent and -independent mechanisms.

## Key findings

- The combination of thymoquinone and chloroquine showed additive or sub-additive cytotoxic effects in melanoma cell lines.
- Thymoquinone increased autophagosome accumulation in WM9 cells while reducing chloroquine's anti-autophagic effect.
- The drug combination altered cell morphology and autophagy dynamics in metastatic melanoma cells.

## Abstract

Although combination therapies with mitogen activated protein kinase inhibitors remain among the most effective treatments for malignant melanoma, they are not universally applicable to all subtypes of this cancer, and their efficacy decreases in the presence of distant metastases. Drug resistance, often associated with elevated autophagy in tumor cells, and adverse effects of the treatment also reduce the survival time of melanoma patients. Therefore, the aim of our research was to assess the cytotoxicity of the combination of a late-stage autophagy blocker chloroquine with thymoquinone, a natural substance with anticancer potential and low toxicity towards healthy cells, in metastatic melanoma cell lines. Using the WST-1 assay, we examined the cytotoxicity of the combination of chloroquine and thymoquinone in melanoma WM9 and WM852 cell lines and assessed the type of their interactions. Additionally, using time-lapse bright field and fluorescent microscopy, we assessed changes in cell morphology during 48 h incubation with the tested compounds and their combinations, as well as their effect on autophagy. We identified an additive and sub-additive cytotoxic effect of thymoquinone/chloroquine combinations against WM9 and WM852 cells. Moreover, we found that thymoquinone combined with chloroquine caused an increase in autophagosome accumulation in WM9 cells, while attenuating the chloroquine’s anti-autophagic effect. A thorough understanding of the mechanism of drug interactions with natural substances is crucial for the development of new effective anticancer therapies.