Exploration of the regulatory mechanism of PD-L1 expression in pMMR AGS gastric cancer cell by cytotoxic drugs and anti-angiogenic drugs

Abstract

Immune checkpoint inhibitors (ICIs) play a key role in treating advanced gastric cancer; however, their efficacy is limited in patients with proficient mismatch repair (pMMR) tumors. Therefore, enhancing ICIs sensitivity in this subgroup is crucial. Preclinical studies have indicated that cytotoxic and anti-angiogenic drugs may boost ICIs efficacy by modulating programmed death-ligand 1(PD-L1) expression via pathways such as PI3K/AKT and MAPK in other cancers. However, how these agents regulate PD-L1 specifically in pMMR gastric cancer cells remains unclear, hindering the development of effective combination therapies. The impact of various drug concentrations on AGS cell proliferation was assessed using the CCK-8 assay to select the appropriate concentrations for subsequent experiments. Real-time quantitative PCR (qPCR) and western blotting were used to evaluate the effects of 72-h drug treatment on PD-L1 mRNA and protein expression levels, respectively. Furthermore, Western blot analysis was used to examine drug-induced changes in the expression of key proteins (PI3K, Akt, MEK1, and ERK) within the PI3K/Akt/mTOR and RAS/RAF/MEK/ERK signaling pathways, which are potentially involved in PD-L1 regulation. We investigated the effects of cytotoxic (5-fluorouracil and cisplatin) and anti-angiogenic (apatinib) drugs on PD-L1 expression and related signaling pathways in proficient mismatch repair (pMMR) AGS gastric cancer cells. Initial characterization confirmed the pMMR status of the cell line via detection of key MMR proteins (MSH6, MSH2, MLH1, and PMS2) and established constitutive baseline expression of both PD-L1 mRNA and protein. Cell viability assays (CCK-8) demonstrated that all three drugs significantly inhibited AGS cell proliferation in time- and concentration-dependent manner over 72–96 h. Subsequent gene expression analysis (qPCR) revealed that specific drug concentrations (5-FU 64 µM, cisplatin 16 µM, apatinib 320 nM and 640 nM) significantly upregulated PD-L1 mRNA levels after 72-h treatment. Western blot analysis confirmed that the same concentrations (5-FU 64 µM, cisplatin 16 µM, apatinib 320 nM) also significantly increased PD-L1 protein expression. To explore the underlying mechanisms, key proteins in the PI3K/Akt/mTOR and RAS/RAF/MEK/ERK pathways, which are known regulators of PD-L1, were analyzed. Western blotting results showed that all three drugs significantly upregulated PI3K protein expression. However, their effects on downstream effectors varied: apatinib increased Akt phosphorylation, whereas 5-FU and cisplatin decreased Akt phosphorylation. Furthermore, 5-FU treatment upregulated MEK1 and ERK protein levels, whereas apatinib and cisplatin downregulated them. This study demonstrated that cytotoxic drugs (5-fluorouracil and cisplatin) and the anti-angiogenic drug(apatinib) effectively inhibited the proliferation of pMMR-type AGS gastric cancer cells and may also have the potential to upregulate PD-L1 mRNA and protein expression in pMMR-type AGS gastric cancer cells. Further experimental results suggested that this upregulation of PD-L1 mRNA and protein may be correlated with the PI3K/Akt/mTOR and RAS/RAF/MEK/ERK signaling pathways. These findings provide a potential theoretical basis for combining cytotoxic and anti-angiogenic drugs with PD-L1 inhibitors to further enhance therapeutic efficacy against pMMR-type gastric cancer.