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

Sep 22, 2026

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Although combination therapies using mitogen-activated protein kinase inhibitors can treat malignant melanoma effectively, benefits vary across subtypes and decline in the presence of distant metastases. Drug resistance and treatment-related adverse effects often reduce patient survival, with elevated tumor autophagy frequently contributing. This study evaluated the cytotoxicity of chloroquine as a late-stage autophagy blocker combined with thymoquinone in metastatic melanoma cell lines, focusing on autophagy-dependent and -independent mechanisms.

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CategoryHealthcare

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1. Introduction

Article The Combination of Thymoquinone and Chloroquine Dose-Dependently Regulates Autophagy and Potentiates Metastatic Melanoma Cell Death via Autophagy-Dependent and-Independent Mechanisms Patrycja Kłos 1, *, Krzysztof Safranow 1, Magdalena Peruy ´nska 2, Radosław Birger 2, Agata St˛epniewska 1, Violetta Dziedziejko 1, Marek Dro´zdzik 2 and Dariusz Chlubek 1 Academic Editors: Simona Serini and Gabriella Calviello Received: 29 December 2025 Revised: 31 January 2026 Accepted: 9 February 2026 Published: 11 February 2026 Copyright: © 2026 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license. 1 Department of Biochemistry and Medical Chemistry, Pomeranian Medical University in Szczecin, Powsta ´nców Wlkp. 72, 70-111 Szczecin, Poland; [krzysztof.safranow@pum.edu.pl](krzysztof.safranow@pum.edu.pl) (K.S.); [58644@student.pum.edu.pl](58644@student.pum.edu.pl) (A.S.); [violetta.dziedziejko@pum.edu.pl](violetta.dziedziejko@pum.edu.pl) (V.D.); [dariusz.chlubek@pum.edu.pl](dariusz.chlubek@pum.edu.pl) (D.C.) 2 Department of Clinical and Experimental Pharmacology, Pomeranian Medical University in Szczecin, Powsta ´nców Wlkp. 72, 70-111 Szczecin, Poland; [magdalena.peruzynska@pum.edu.pl](magdalena.peruzynska@pum.edu.pl) (M.P.); [radoslaw.birger@pum.edu.pl](radoslaw.birger@pum.edu.pl) (R.B.); [marek.drozdzik@pum.edu.pl](marek.drozdzik@pum.edu.pl) (M.D.) * [Correspondence: patrycja.klos@pum.edu.pl](Correspondence: patrycja.klos@pum.edu.pl) 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. Keywords: melanoma; metastasis; thymoquinone; chloroquine; cytotoxicity; apoptosis; autophagy; additivity 1. Introduction Melanoma is a malignant tumor resulting from a mutation in melanocytes [1] . Although it is not the most frequently diagnosed skin cancer, it is responsible for the largest number of deaths caused by skin cancers [2], which constitutes 0.6% of cancer-related deaths worldwide [3] . Melanoma occurs mainly in the skin, but can also appear in other tissues where melanocytes are present, such as the eyes, digestive and genitourinary systems, and the nasopharynx [1] . Genetic mutations associated with melanoma affect signaling pathways that regulate key cellular processes such as growth, differentiation, migration, division, and apoptosis. Dysfunctions in t

FAQs

What was the main purpose of combining chloroquine with thymoquinone in metastatic melanoma cells?

To assess the cytotoxicity of the combination in metastatic melanoma cell lines, with attention to mechanisms that involve both autophagy-dependent and autophagy-independent pathways.

How was cytotoxicity measured and which cell lines were tested?

Cytotoxicity of chloroquine and thymoquinone, alone and in combination, was examined using the WST-1 assay in WM9 and WM852 melanoma cell lines.

What autophagy-related effects were observed when thymoquinone was combined with chloroquine?

In WM9 cells, thymoquinone plus chloroquine increased autophagosome accumulation while attenuating chloroquine’s anti-autophagic effect.

Sep 22, 2026

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