Study On Biological Functions Of BTG1 And Relevant Mechanisms In Breast Cancer

ObjectiveThe current study was designed to investigate the effect of BTG1 overexpression by transfection with a pcDNA3-BTG1 expression vector on cell growth,proliferation, apoptosis, migration and invasion as well as ridiosensitivity in human breast cancer cells via either in vitro cell model and in vivo animal model. These findings will provide experimental evidence for BTG1 as a new target in therapy of breast cancer.Methods1. The recombinant plasmid pcDNA3-BTG1 was transfected into the breast cancer cell lines, MDA-MB-231 and MCF-7, via Lipofectamine transfection assay. G418 resistant clones were developed and collected by cultured in G418-containing medium. The expression of BTG1 mRNA and protein were monitored by RT- PCR and Western blot assay, respectively.2. Cell proliferation was assessed by MTT assay; Flow cytometric analysis was used to detect the alteration of cell cycle distribution and apoptosis; Apoptosis was also detected by assay of phosphatidylserine valgus. Western blot assay was used to detect the expression of cell cycle and apoptosis-relevent proteins,3. The allograft tumor model was established in BALB/c nude mice, which were subcutaneously inoculated with un-trasfected and transfected cells. The effect of BTG1 on tumor growth was examined. The Bcl-2 protein in tumors was measured by immunohistochemistry. Apoptosis induction was detected by TUNLE.4. Wound healing assay and Transwell chamber assay were performed to assess cell migration and invasion. Western blot assay was used to measure change of proteins related to invasion and migration. 5. The effects of BTG1 on tumor invasion and metastasis were examined in allograft tumor model established in BALB/c nude mice. The amount of microvessels and the expression of VEGF in tumors was measured by immunohistochemistry.6. After irradiated with X-ray, colony formation assay was used to evaluate survival, flow cytometric analysis was performed to assess the alteration of cell cycle distribution and apoptosis caused by radiation. Western blot assay was used to detect the change of DNA damage repair and NEJH related proteins.7. After irradiated, qRT-PCR was used to quantitative mRNA level changes. Comet assay was used to analyze DNA damage and repair. Immune fluorescence in situ hybridization was employed to detectγH2AX foci. Fluorescent probe was performed to assessγH2AX and ROS.8. Analysis of chromosome aberrations was used to monitor the genome stability in cells with X-ray irradiation.9. The amount of microvessels and the expression of VEGF and Bcl-2 in tumors were measured by immunohistochemistry, while apoptosis induction was detected by TUNLE.Results1.A pcDNA3-BTG1 expression vector carrying a full-length of BTG1 cDNA was successfully constructed and transfected into breast cancer cell lines, MDA-MB-231 and MCF-7. A higher expression of BTG1 at both mRNA and protein levels was observed in the G418-resistant clonies transfected with a pcDNA3-BTG1 vector.2.Compared with parental cells and Neo cells transfected with a“empty”pcDNA3 vector, a significant decrease of cell proliferation was observed in pcDNA3-BTG1 transfected cells, accompanied with a G2/M phase arrest, a decrease of cyclin D1 and an increase in cyclin B1 protein expression.3.Overexpression of BTG1 caused a decreased Bcl-2 protein expression and an up-regulated expression of Bax.4 . A slower growth, average weight and volume were observed in BTG1-overexpressed tumors, accompanying with a decreased expression of Bcl-2 and a large number of pyknotic nuclei, debris and apoptotic cells.5. Overexpression of BTG1 inhibited invasion and migration of MDA-MB-231 and MCF-7 cells, along with a decreased expression of NF-κB, MMP-2 and MMP-9 protein.6. Metastasis of tumors was lower in tumors with BTG1 overexpression, in which the amount of microvessels and VEGF expression in tumors was also significantly less.7. After irradiated with X-rays, overexpression of BTG1 increased the radiosensitivity in both of MDA-MB-231 and MCF-7 cells, along with a G2/M arrest, a decreased expression of cyclin B1 and cyclin D1 protein and increased expression of cyclin E1. Enhanced expression of BTG1 promote apoptosis induction with a reduced expression of Bcl-2, an increased expression of Bax, caspase-3, Bad and phosphorylated Akt proteins. NF-κB was not altered. Moreover, the alterations of a series of DNA damage repair protein levels were observed, including an increase of phosphorylated P53, ATM and CHK2 expression, a decrease of FEN-1 expression and no change of BRCA1 and RAD51. Finally, the expression levels of Ku-70, Ku-80 and XRCC4 proteins significantly reduced with an unchanged expression of DNA-PKcs.8. After irradiated with X-rays, BTG1 mRNA was increased along a decreased expression of DNA damage repair related gene ligaseⅣmRNA.9. After irradiated with X-rays, the "comet" tail length and tail moment significantly increased in BTG1-overexpressed cells. Moreover, the tailed DNA content increased with an increase of doses, to maximum at 2 hr.10. After irradiated with X-rays, the number ofγH2AX foci increased in BTG1-overexpressed cells. The fluorescence intensity ofγH2AX was also changed with times, reaching maximum at 2 h.11. In BTG1-overexpressed cells, ROS increased linearly with doses of X-rays.12. After irradiated with X-rays, the vascular density of tumor was less in BTG1-overexpressed cells, with decreased expression of VEGF and Bcl-2.Conclusion1. An overexpression of BTG1 has been obtained in breast cancer cell lines, MDA-MB-231 and MCF-7, trasfected with a pcDNA3-BTG1 mammalian expression plasmid.2. Overexpression of BTG1 inhibits growth of breast cancer cells in vitro and in vivo, which is related to its regulation of cell cycle distribution and apoptosis induction.3. Significant suppression of invasion, metastasis and angiogenesis is observed in breast cancer cells in vitro and in vivo, with enforced expression of BTG1, along with in an alteration of NF-κB, MMP-2, MMP-9 and VEGF.4. Overexpression of BTG1 increases cell sensitivity to X-rays in vitro and in vivo, which may be related with a change of cell cycle progression and apoptosis induction. BTG1 may participate in the regulation ATM pathway, DNA damage repair, ROS generation and scavenging free radicals.