Effect of Gefitinib inhibiting epidermal growth factor receptor signaling pathway on proliferation and osteogenic differentiation of endosteum-derived stem cells in rats

Abstract

Objective To investigate the effect of gefitinib, an inhibitor of epidermal growth factor receptor (EGFR), on the proliferation and osteogenic differentiation of endosteum-derived stem cells (EDSCs) in rats. Methods Femoral fracture models were established in healthy male 4-week old SD rats. They were randomly divided into 2 groups. The experimental group was subjected to intragastric lavage with gefitinib, an EGFR signaling inhibitor (100 mg/kg·d) while the control group to intragastric lavage with an isodose of methyl cellulose. Bilateral femurs and tibias were harvested one week after lavage for separation of EDSCs and bone marrow mesenchymal stem cells (BMSCs) respectively using density gradient centrifugation. After proliferative cloning in vitro, expression of the cell surface antigens (CD29, CD34, CD44 and CD45) of the third passage cells was detected by flow cytometry (FCM). Proliferation of the cells was detected by BrdU, cell cycle was measured by FCM, and expression of the genes related to cell cycle inhibitory factors (p15, p16, p21 and p27) was determined by PCR. ALP staining was performed 14 days after osteogenesis induction. After 21 days of chondrogenic induction, von Kossa staining was conducted. qRT-PCR of the mRNA obtained was used to detect expression of osteogenic differentiation of related genes (osteocalcin, bsp, runx2 and osterix). Results CD29 and CD44 were positively expressed while CD34 and CD45 negatively expressed in EDSCs and BMSCs. After the EGFR signaling pathway was blocked by gefitinib, BrdU detection found that gefitinib inhibited BMSCs (11.15%) much more than EDSCs (0.25%). Cell cycle detection showed that the volume of EDSCs was increased in phases G0/G1 and S but decreased significantly in phase G2-M. ALP staining showed that the increase of EDSCs ALP+cells (53.31%) was significantly higher than that of BMSCs (25.04%). The increased expression percentages of the genes related to cell cycle inhibitors in EDSCs (103.9%, 58.0%, 117.3% and 105.1%, respectively) were significantly higher than those in BMSCs (39.3%, 38.4%, 24.5% and 83.4%, respectively)(P< 0.05). The increased expression percentages of the genes related to osteogenic differentiation in EDSCs (247.0%, 289.9%, 66.1% and 233.2%, respectively) were significantly higher than those in BMSCs (106.5%, 186.4%, 41.7% and 190.8%, respectively). All the above differences were statistically significant (P< 0.05). Conclusions Gefitinib, an EGFR inhibitor, can inhibit proliferation of EDSCs and BMSCs but promote their osteogenic differentiation. It inhibits proliferation of BMSCs more significantly as it promotes osteogenic differentiation of EDSCs. Key words: Receptor, epidermal growth factor; Bone marrow cells; Stem cells; Osteogenic differentiation