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Realgar‑induced KRAS mutation lung cancer cell death via KRAS/Raf/MAPK mediates ferroptosis
Xiaofeng Liu1, Yang Hai2, Jinqu Dong1
1School of Pharmacy, Gansu University of Chinese Medicine, Lanzhou, Gansu 730000, P.R. China.
Abstract:
KRAS is a biomarker for non‑small cell lung cancer‑targeted therapy, but there is currently no effective KRAS‑targeting medication. Realgar is an impelling anticancer drug, however its significance in KRAS mutant lung cancer is uncertain. According to our findings, the IC50 of H23 (KRAS mutant) cells is 2.99 times lower than that of H1650 (non‑KRAS mutant) cells. Flow cytometry and the Hoechst 33258 staining assay revealed that H1650 cells treated with 4 µg/ml realgar had an apoptotic rate of 8.2%, while H23 cells had a rate of 21.46%. Accordingly, realgar was more sensitive to KRAS mutant cells. Transcriptome sequencing test indicated that there were 481 different expression genes in H23 cells treated with realgar. In H23 cells treated with realgar, mitochondria shrank, inner membrane folding was disturbed, and mitochondrial membrane potential crushed. Realgar boosted intracellular Fe2+, reactive oxygen species, malondialdehyde and glutathione levels, which were all reversed by ferroptosis inhibitor Fer‑1. Realgar decreased phosphorylated p‑Raf, p‑ERK1/2 and increased p‑p38 and p‑JNK, whereas only p‑Raf was abolished by Fer‑1. Raf inhibitor Sorafenib accelerated the realgar‑induced ferroptosis. On H23 cells treated with realgar, the expression of GPX4, SCL7A11 decreased while ACSL4 expression increased; this effect could also be amplified by Sorafenib. In conclusion, the present study indicated that realgar may induce ferroptosis by regulating the Raf, and hence plays a role in anti‑KRAS mutant lung cancer.
Insights
Realgar demonstrates increased sensitivity towards KRAS-mutant non-small cell lung cancer cells. This traditional Chinese medicine induces ferroptosis by regulating the Raf pathway, offering a potential therapeutic strategy for KRAS-mutant lung cancer.
Area of Science:
- Oncology
- Molecular Biology
- Pharmacology
Background:
- KRAS mutations are key biomarkers in non-small cell lung cancer (NSCLC) targeted therapy.
- Currently, effective KRAS-targeting medications are lacking.
- The anti-cancer potential of Realgar, particularly in KRAS-mutant NSCLC, remains uncertain.
Purpose of the Study:
- To investigate the efficacy and mechanism of Realgar in KRAS-mutant NSCLC.
- To determine if Realgar exhibits differential sensitivity towards KRAS-mutant versus non-mutant NSCLC cells.
Main Methods:
- Cell viability assays (IC50 determination) on KRAS-mutant (H23) and non-mutant (H1650) NSCLC cell lines.
- Flow cytometry and Hoechst 33258 staining to assess apoptosis rates.
- Transcriptome sequencing to identify gene expression changes.
- Mitochondrial function assays and measurement of reactive oxygen species (ROS), iron, and lipid peroxidation markers.
- Western blotting to analyze key protein expressions in signaling pathways (Raf, ERK, p38, JNK, GPX4, ACSL4).
- Utilized ferroptosis inhibitor (Fer-1) and Raf inhibitor (Sorafenib) to elucidate mechanisms.
Main Results:
- Realgar showed significantly higher cytotoxicity against KRAS-mutant H23 cells (lower IC50) compared to non-mutant H1650 cells.
- Realgar treatment led to a higher apoptotic rate in H23 cells (21.46%) than in H1650 cells (8.2%).
- Transcriptome analysis revealed 481 differentially expressed genes in Realgar-treated H23 cells.
- Realgar induced mitochondrial dysfunction, increased intracellular Fe2+, ROS, malondialdehyde, and glutathione levels, characteristic of ferroptosis, all reversed by Fer-1.
- Realgar modulated the Raf/ERK pathway (decreased p-Raf, p-ERK1/2; increased p-p38, p-JNK), with p-Raf reduction abolished by Fer-1.
- Realgar decreased GPX4 and increased ACSL4 expression in H23 cells, effects amplified by Sorafenib.
- Sorafenib accelerated Realgar-induced ferroptosis.
Conclusions:
- Realgar exhibits selective anti-cancer activity against KRAS-mutant NSCLC cells.
- Realgar induces cell death primarily through ferroptosis, involving mitochondrial damage and oxidative stress.
- The mechanism involves the regulation of the Raf signaling pathway and ferroptosis-related genes (GPX4, ACSL4).
- Realgar represents a potential therapeutic agent for KRAS-mutant NSCLC.
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