Inhibition Of PFGFR By CP-673451 Induces Cytotoxicity In NSCLC And Increases The Effect Of Cisplatin

Object : The platelet-derived growth factor receptor(PDGFR)belongs to the receptor tyrosine kinase.Abnormalities such as overexpression and mutation activationof PDGFR play an important role in the occurrence,development and metastasis of certain tumors.PDGFR is expressed by stromal cells in the tumor microenvironment,and in a subset of cancer cells,usually with overexpression and/or mutation of PDGFRs.In NSCLC,overexpression of PDGF/PDGFR is often associated with poor prognosis of cancer.The aim of this study was to investigate the cytotoxic effect of PDGFRβ inhibitor CP-673451 on NSCLC cells and its potential mechanism.Furthermore,explore the cytotoxicity of CP-673451 sensitizing cisplatin in NSCLC,in order to provide experimental basis for clinical application of PDGFR targeted therapy for NSCLC.Methods:(1)The expression of PDGFRβ in A549 and H358 cells was detected by immunofluorescence staining.The inhibition of PDGFRβ in A549 and H358 cells by CP-673451 was detected by enzyme-linked immunosorbent assay(ELISA).The effect of CP-673451 on the proliferation of A549,H358 and BEAS-2B cells was measured by MTT assay.(2)Flow cytometry and Caspase-Glo 3/7 activity assay were used to detect the effect of CP-673451 on apoptosis of A549 and BEAS-2B cells.(3)The effect of CP-673451 on intracellular ROS levels was determined by DCFH-DA Reactive Oxygen Assay Kit.Flow cytometry and Caspase-Glo 3/7 were used to determine apoptosis of A549 cells after addition of antioxidant NAC.(4)Western blotting and Real-time quantitative gene amplification fluorescence(RT-qPCR)were used to detect the CP-673451 on the protein and mRNA expression of Nrf2 in A549 cells and also to detect the mRNA expression of Nrf2 downstream target genes HO-1 and NQO-1.GSH kit was used to detect the total GSH level of A549 cell after given CP-673451.(5)After addition of Nrf2 activator tBHQ,flow cytometry,DCFH-DA and GSH kit were used to measuer the effect caused by CP-673451 in apoptosis,ROS level and GSH content of A549 cells.(6)A549 cellswith stably low-express of PDGFRβ was constructed,and then techniques such as Western blotting,MTT,flow cytometry,Caspase-Glo 3/7,DCF-DA fluorescence analysis,and GSH kits are used to detect the effect of CP-673451 on A549 cells with low-express of PDGFRβ.(7)MTT assay,flow cytometry,Caspase-Glo 3/7,DCFH-DA assay and GSH kit were used to detect the effect of CP-673451 combined with cisplatin on the toxicity of A549 cells.Results:(1)PDGFRβ is highly expressed in both A549 and H358 cells,and CP-673451 can inhibit the phosphorylation of PDGFRβ in A549 and H358 cells.CP-673451 significantly inhibited the proliferation of NSCLC cell lines(A549 and H358 cells),but in normal cells(BEAS-2B cells)the effect was small.(2)CP-673451 could induce apoptosis in A549 cells.However,there was no significant difference in the effect of apoptosis on BEAS-2B cells in the same dose range.CP-673451 significantly increased the ROS level in A549 cells in a dose-dependent manner.The anti-oxidant NAC can antagonize the apoptosis of A549 cells induced by CP-673451.(3)CP-673451 can reduce the expression of Nrf2 and its downstream target genes HO-1 and NQO-1 in A549 cells.And it can reduced the content of GSH in A549 cells.After the addition of Nrf2 activator tBHQ,it can reverse the increase of ROS level,cell apoptosis increase and GSH content decrease in A549 cells induced by CP-673451.(4)Application database analysis and experimental techniques confirmed that CP-673451 inhibits the expression of Nrf2 in A549 cells through the PI3K/Akt pathway.(5)By constructing silences expression of PDGFRβ in A549 cells,the experiments confirmed that the effect of CP-673451 on A549 cells was achieved by inhibiting PDGFRβ,thereby eliminating the effect of“off-target effect”.(6)Finally,our experiments confirmed that CP-673451 can enhance the cytotoxic effect of cisplatin on A549 cells.Conclusion:(1)The PDGFRβ inhibitor CP-673451 caused cytotoxicity by increasing ROS levels in A549 cells.(2)This effect is achieved by inhibiting the PI3K/Akt/Nrf2 pathway.(3)CP-673451 can enhance the cytotoxic effect of cisplatin on A549 cells.