# Synergistic Induction of Oxidative and Endoplasmic Reticulum Stress by Tempol and ML210 Combination Therapy in B16F10 Melanoma Cells

## TL;DR

Combining Tempol and ML210 may offer a new treatment for melanoma by causing cell death through stress and antioxidant disruption.

## Contribution

The study reveals a novel synergistic mechanism of Tempol and ML210 in inducing apoptosis in melanoma cells.

## Key findings

- Combination treatment significantly inhibited B16F10 cell proliferation compared to monotherapies.
- The treatment increased oxidative stress and ER stress markers while reducing antioxidant levels.
- The combination upregulated pro-apoptotic proteins and suppressed anti-apoptotic Bcl-2 expression.

## Abstract

Given the challenges in treating metastatic melanomas, there is a growing need for novel and effective therapeutic strategies. This study aimed to understand molecular mechanisms underlying synergistic effects of a Tempol and ML210 combination in B16F10 murine melanoma cells and to evaluate its therapeutic potential. We hypothesized that this combination would synergistically induce cell death by increasing oxidative stress and triggering ER stress. B16F10 melanoma cells were treated with Tempol and ML210 alone or in combination for 48 h. Cell viability was determined using MTT assay. Oxidative stress was evaluated by measuring Total Antioxidant Status (TAS), Total Oxidant Status (TOS), and intracellular H2O2 levels. Apoptotic markers (caspase-3, Bax, Bcl-2) and ER stress proteins (GRP78, GADD153, IRE1α, ATF6) were quantified by ELISA. Combination treatment significantly inhibited cell proliferation compared to monotherapies. Molecular analyses revealed that combination caused depletion of TAS and increase in TOS and intracellular H2O2 levels. Furthermore, combination treatment synergistically upregulated ER stress markers and pro-apoptotic proteins while significantly suppressing anti-apoptotic Bcl-2 expression. In conclusion, the combination of Tempol and ML210 synergistically induces cell death in B16F10 melanoma cells by disrupting redox balance and activating ER stress-mediated apoptosis. These findings suggest a potential strategy for melanoma treatment that warrants further in vivo investigation.