Study on the enhancement of malignant biological behavior and cisplatin resistance in laryngeal squamous cell carcinoma by GAPDH-mediated inhibition of ferroptosis under hypoxic conditions
10.16066/j.1672-7002.2024.10.001
- VernacularTitle:缺氧环境下GAPDH通过抑制铁死亡增强喉鳞状细胞癌恶性生物学行为和顺铂耐药性研究
- Author:
Ruixiang CEN
1
;
Dan LIU
;
Cong PENG
;
Lang WAN
Author Information
1. 黄石市中心医院(湖北理工学院附属医院)耳鼻咽喉科,湖北 黄石 435000
- Keywords:
Hypoxia;
Laryngeal Neoplasms;
Carcinoma,Squamous Cell;
Cisplatin;
glyceraldehyde-3-phosphate dehydrogenase;
ferroptosis
- From:
Chinese Archives of Otolaryngology-Head and Neck Surgery
2024;31(10):613-619
- CountryChina
- Language:Chinese
-
Abstract:
OBJECTIVE To investigate the role of glyceraldehyde-3-phosphate dehydrogenase(GAPDH)in regulating ferroptosis,malignant biological behavior,and cisplatin resistance in laryngeal squamous cell carcinoma(LSCC)under hypoxic conditions.METHODS Human laryngeal cancer Hep-2 and TU212 cell lines were cultured in vitro under hypoxic(1%O2)and normoxic(21%O2)conditions without serum.Hep-2 cells were divided into five experimental groups:DMSO control group,Erastin treatment group,Hypoxia+Erastin group,Hypoxia+Erastin+NC-GAPDH group,and Hypoxia+Erastin+Silenced GAPDH group,to study the cellular response under various experimental conditions.Quantitative polymerase chain reaction(qPCR)and Western blot(WB)techniques were employed to analyze GAPDH mRNA and protein expression levels in each cell line.Cell Counting Kit-8(CCK-8),colony formation,and Transwell invasion assays were used to assess the vitality,proliferation,and invasion capabilities of each group of laryngeal cancer cells.Additionally,CCK-8 assays were used to determine cell resistance to various concentrations of cisplatin(5,15,20 μg/ml).Kit methods were used to evaluate intracellular malondialdehyde(MDA)and glutathione(GSH)levels.WB was employed to detect the expression levels of glutathione peroxidase 4(GPX4)and 4-hydroxynonenal(4-HNE)proteins.Fe2+probes and reactive oxygen species(ROS)fluorescence probes were used to measure intracellular Fe2+content and ROS levels.Transmission electron microscopy was utilized to observe mitochondrial morphology changes to assess ferroptosis occurrence.RESULTS Compared to normoxic conditions,hypoxia significantly increased the expression of GAPDH mRNA and protein in Hep-2 and Tu212 cells(P<0.05).Under hypoxic conditions,the number of cell colonies,migration capacity,and cell viability at 15 and 20 μg/ml cisplatin treatment were significantly higher than those under normoxic conditions(P<0.05).When GAPDH was silenced,these hypoxia-induced enhancements were reversed.Under normoxic conditions,compared to the DMSO control group,Erastin treatment significantly reduced the number of colonies,migration capacity,and cell viability at 15 and 20 μg/ml cisplatin treatment(P<0.05).However,under hypoxic conditions,the inhibitory effect of Erastin was weakened.Additionally,in the hypoxia+Erastin+si-GAPDH group,the number of colonies,migration capacity,and cell viability at 15 and 20 μg/ml cisplatin treatment were significantly lower than those in the hypoxia+Erastin+NC-GAPDH group(P<0.05).CONCLUSION This study reveals that silencing GAPDH under hypoxic conditions can reverse the inhibition of ferroptosis in LSCC cells,thereby suppressing their malignant biological behavior and cisplatin resistance.
