Effect Of Stattic On The Biological Activity Of Epidermal Growth Factor Receptor Inhibition-mediated Osteosarcoma

Part I Effect of EGFR tyrosine kinase inhibitor on the biological activities of OS Objective: Osteosarcoma(OS),the most common malignant bone tumor,often occurs in children and adolescents with a high metastatic potential.Epidermal growth factor receptor(EGFR)is the driving factor of various tumors,and tyrosine kinase inhibitors targeting EGFR have shown encouraging antitumor activity in patients with various types of solid tumors.However,there remains controversial regarding the effect of EGFR inhibitors on osteosarcoma.This part of the study aimed to determine the antitumor activity of gefitinib,a tyrosine kinase inhibitor of EGFR,against OS and to explore its mechanism.Methods: The expression of EGFR in OS cells and normal osteoblasts was firstly detected by Western blot and real-time quantitative polymerase chain reaction(RT-q PCR).The effect of gefitinib on OS cell proliferation and tumor growth was detected by CCK-8 assay,clone formation assay and subcutaneous tumor transplantation model in nude mice.Transwell assay and Western blot were used to detect the effect of gefitinib on OS cell migration and invasion and its mechanism.Flow cytometry and Western blot were used to detect the effect of gefitinib on the apoptosis and cycle of OS cells and its mechanism.To investigate the mechanism of gefitinib affecting OS cells,we used Western blot to detect the protein expressions of EGFR,protein kinase B(Akt),extracellular signal regulatory enzyme(ERK),and signal transducer and activator of transcription 3(STAT3).Results: Western blot and RT-q PCR assays showed that the expression of EGFR protein and gene in MG63,143 B and MNNG-HOS cells with fibroblast or mixed characteristics was increased,with the difference of gene expression statistically significant compared with osteoblasts(P<0.01).U2 OS and Saos-2 cells with epithelial characteristics showed low expression of EGFR protein and gene.Gefitinib inhibited OS cell proliferation and tumor growth in a dose-dependent manner.Transwell assay showed that gefitinib inhibited the migration and invasion of OS cells by increasing E-cad and decreasing N-cad expressions.In addition,after gefitinib treatment,the apoptosis rate and the proportion of G1 phase of OS cells increased,with a statistical significance compared with their respective control groups(P<0.05).Western blot assay showed that the Bax/Bcl-2 ratio and P27 increased,and cyclin D1 and cyclin-dependent kinase 4(CDK4)expressions decreased.Further mechanism exploration showed that gefitinib inhibited the phosphorylation of EGFR,Akt,and ERK,while increased the phosphorylation of STAT3.Gefitinib had no effect on the total protein levels of EGFR,Akt,ERK and STAT3.Conclusion: Gefitinib could inhibit the tumorigenesis and metastasis,and promote the G1 phase arrest and apoptosis of OS cells by blocking the activation of EGFR,Akt and ERK signaling pathways.The feedback activation of STAT3 protein mediated by gefitinib may be involved in the resistance mechanism of OS cells to EGFR inhibitors.Part Ⅱ Effect of EGFR knockdown on the biological activities of OS Objective: The results of the first part of this study showed that gefitinib,an EGFR tyrosinase inhibitor,could inhibit the biological activities of OS cells.However,the effect of EGFR inhibition on the occurrence and progression of OS requires to be further explored.This part of the study aimed to elucidate the anti-tumor effect of EGFR protein expression changes on OS.Methods: EGFR knockdown(sh-EGFR group)and control(sh-NC group)lentiviral particles were used to transfect OS cells,with the m RNA and protein expression of EGFR after transfection detected by RT-q PCR and Western blot.The effect of EGFR knockdown on OS cell proliferation and tumor growth was detected by CCK-8 assay,clone formation assay and subcutaneous xenograft model in nude mice.The effect of EGFR knockdown on OS cell migration and invasion abilities was detected by Transwell assay.The effect of EGFR knockdown on OS cell apoptosis and cycle distribution was detected by flow cytometry.Results: RT-q PCR and Western blot results showed that the m RNA and protein expressions of EGFR in 143 B and U2 OS cells in their sh-EGFR groups were significantly lower than those in their respective sh-NC groups,with the differences statistically significant(P<0.01).The cell proliferation,number of colony formation and tumor growth of 143 B and U2 OS cells in their sh-EGFR groups were significantly lower than those in their respective sh-NC groups,with the differences statistically significant(P<0.01).Transwell assay results showed that the number of migrated and invasive cells of 143 B and U2 OS in their sh-EGFR group was significantly lower than that in their respective sh-NC groups(P<0.01).Flow cytometry results showed that the apoptosis rate and cell cycle arrest in G1 phase of143 B and U2 OS cells increased after EGFR knockdown,while the S phase number of tumor cells decreased,all with statistically significant differences compared with their respective control groups(P<0.05).Conclusion: Lentivirus-induced EGFR knockdown could inhibit the proliferation,tumor growth and metastasis of OS cells,and promote the G1 phase arrest and apoptosis of OS cells.Part Ⅲ Effect of Stattic on gefitinib-mediated biological activities of OSObjective: In the first part of the study,we found that gefitinib-mediated inhibition of EGFR phosphorylation resulted in the feedback activation of STAT3 protein phosphorylation.STAT3 phosphorylation in soft tissue sarcomas has been reported to be associated with tumor resistance to gefitinib.However,the interaction between EGFR and STAT3 in OS has not yet been reported.This part of the study aimed to explore the role of STAT3 in gefitinib mediated anti-tumor activity in OS and its related mechanisms.Methods: Western blot was firstly used to detect the changes of EGFR protein in OS cells treated with Stattic,a STAT3 tyrosine kinase inhibitor.The effects of gefitinib(G),Stattic(S)and their combination(G+S)on OS cell proliferation and tumor growth were compared by the CCK-8 assay,clone formation assay and subcutaneous xenograft model construction in nude mice.Transwell method and Western blot were used to compare the effects of G,S and G+S treatments on OS cell migration and invasion,and the changes of E-cad and N-cad,respectively.Flow cytometry was used to compare the effects of G,S and G+S treatments on the apoptosis and cell cycle distribution of OS cells,with Western blot applied to detect the expression changes of cycle and apoptosis-related proteins.Finally,the expression changes of EGFR,Akt,ERK and STAT3 proteins in OS cells treated with G,S and G+S were detected by Western blot.Results: The results of Western blot showed that S decreased the expression of p-STAT3,but increased the expression of p-EGFR.CCK-8 results showed that the half-inhibitory concentrations of 143 B and U2 OS cells in their G+S groups were lower than those in their respective G or S groups.In addition,the colony formation number of 143 B and U2 OS cells,and the xenografted tumor volume and weight in their G+S groups were significantly lower than those in their respective G and S groups,all with statistically significant differences(P<0.05).The number of 143 B and U2 OS cells migrated or passed through the Matrigel in their G+S groups was significantly lower than that in their G and S groups,with the differences statistically significant(P<0.001).Western blot results showed that,compared with their respective G and S groups,the expression of E-cad in OS cells in G+S group was the highest,while the expression of N-cad was the lowest.In addition,the proportions of apoptosis and G1 phase of OS cells in their G+S groups were significantly higher than those in their respective G and S groups,all with statistically significant differences(P<0.01).Western blot results showed that the Bax/Bcl-2 ratio and P27 protein expression in OS cells in their G+S groups increased most obviously,while the expression of cyclin D1 and CDK4 proteins decreased most obviously.Further mechanism exploration showed that G+S treatment inhibited both the phosphorylation of EGFR and STAT3,and the levels of p-Akt and p-ERK were lower than those in the G and S groups.G,S and G+S treatments had no effect on the total protein levels of EGFR,Akt,ERK and STAT3.Conclusion: Stattic could block the negative feedback activation pathway between STAT3 and EGFR via inhibiting STAT3 phosphorylation,thereby increasing the inhibitory effect of gefitinib on OS cell proliferation and metastasis,as well as promoting OS tumor cell apoptosis and cell cycle arrest.Combined treatment targeting both EGFR and STAT3 may serve as a potential therapy for OS.Part Ⅳ Effect of Stattic on EGFR knockdown-induced biological activities of OS Objective: In the third part of this study,it was found that Stattic increased gefitinib-mediated inhibition of OS cell proliferation,tumor growth and metastasis,as well as increased gefitinib-mediated blocking of cell cycle progression and promotion of apoptosis.However,the synergistic effect of Stattic on EGFR knockdown-induced biological alterations of OS cells required to be further verified.This part of the study aimed to explore the contribution of Stattic to EGFR knockdown-mediated anti-OS activity.Methods: The number of proliferation and colony formation of 143 B and U2 OS cells in the EGFR-knockdown(sh-EGFR)group and the control group(sh-NC)were compared by the CCK-8 assay and clony formation assay,respectively.Transwell method was used to compare the difference in the number of migrated and invasive OS cells between sh-EGFR and sh-NC groups after treatment with S.Flow cytometry was used to compare the apoptotic proportion and cell cycle distribution of OS cells in sh-EGFR and sh-NC groups after treatment with S.Results: Under the condition of the same S concentrations,the half-inhibitory concentrations of 143 B and U2 OS cells in their sh-EGFR groups were lower than those in their respective sh-NC groups.The number of colony formation of 143 B and U2 OS cells in their sh-EGFR + S groups was significantly lower than that in their respective sh-EGFR and S groups,all with statistically significant differences(P<0.001).The number of 143 B and U2 OS cells migrated and passed through the Matrigel in their sh-EGFR + S groups was significantly lower than that in their respective sh-EGFR and S groups,all with statistically significant differences(P<0.01).In addition,the proportions of apoptosis and G1 phase of OS cells in sh-EGFR + S group were significantly higher than those in sh-EGFR and S groups,the differences of which were statistically significant(P<0.001).Conclusion: Stattic could increase the inhibition effect of EGFR knockdown on OS cell proliferation,migration and invasion,as well as increase the promotion effect on tumor cell apoptosis and G1 phase arrest.Combined targeting of EGFR and STAT3 should be considered in future OS therapy.