Melanogenesis inhibition by (2-Methylbutyryl) Shikonin, a naturally occurring naphthoquinone potentiates dacarbazine anti-melanoma efficacy via ROS mediated apoptotic pathway

Abstract

Introduction

Melanoma is a highly aggressive skin cancer with strong metastatic potential and resistance to conventional chemotherapy. Dacarbazine (DTIC) is widely used for melanoma treatment, but its effectiveness is often compromised by acquired resistance, resulting in relapse and poor prognosis. (2-Methylbutyryl) Shikonin (BSHK), a naturally derived naphthoquinone pigment isolated from Lithospermum erythrorhizon, is a bioactive derivative of shikonin—an established compound in Traditional Chinese Medicine (TCM). In TCM, shikonin and its derivatives are key constituents of formulations such as Zicao (purple gromwell root), traditionally used for detoxification, reducing inflammation, and treating skin disorders, including eczema, burns, and malignancies. BSHK has demonstrated broad anti-cancer activity by inhibiting cancer cell proliferation and possesses the ability to suppress tyrosinase enzyme activity, a key enzyme in melanogenesis. This study investigates whether BSHK can enhance the therapeutic efficacy of dacarbazine in melanoma cells and explores the molecular mechanisms underlying their combined effect, while also highlighting its traditional use and therapeutic relevance in Chinese herbal medicine.

Methods

Melanoma cells were treated with BSHK, dacarbazine, or their combination. Cell viability was assessed by MTT assay. Apoptosis was analyzed using Annexin-V/FITC staining and flow cytometry. Protein expression levels of key Oxidative stress, apoptotic and melanogenic markers such as SOD, Catalase, Nrf-2 Bcl-2, BAX, Caspase-3, and MITF was evaluated by immunoblotting.

Results

BSHK significantly enhanced the anti-proliferative effect of dacarbazine on melanoma cells. Co-treatment increased apoptotic cell populations and ROS levels compared to single treatments. Western blot analysis revealed that combination therapy downregulated MITF and Bcl-2, while upregulating BAX and cleaved Caspase-3, indicating enhanced apoptotic signaling and melanogenesis inhibition.

Discussion

In conclusion, (2-Methylbutyryl) Shikonin sensitizes melanoma cells to dacarbazine by inhibiting melanogensis thereby increasing their susceptibility to apoptosis. The synergistic anticancer effects of (2-Methylbutyryl) Shikonin and dacarbazine suggest that their combination could be a promising therapeutic strategy for treating melanoma