Growth Inhibitory And Antimetastatic Effect Of Metformin On Mammary Cancer 4T1 In Vitro And In Vivo

ObjectivesTo confirm the antitumoral action of metformin, this part was gone through cell experiments to evaluate the effects of metformin on the proliferation, apoptosis and invasion of mammary cancer 4T1, as well as to explore the possible signaling mechanisms involved by which metformin exerts its effects.MethodsMurine mammary carcinoma 4T1 cell is cultured to test the effects of different concentrations of metformin on cell proliferation, apoptosis and invasion. To detect the effect of metformin on apoptosis-associated protein, cell-cycle regulatory protein and invasion-associated protein of mammary cancer 4T1 and to investigate whether the AMPK/mTOR pathway was involved in the antitumor effects of metformin in 4T1 we used Western Blot.Results1. Metformin inhibits mammary cancer 4T1 proliferation.MTT analysis shows a result of that metformin is able to inhibit mammary cancer 4T1 proliferation in a dose-and time-dependent manner (P<0.05) and Compound C can antagonize the effect of metformin.2. Metformin can activate the AMPK/mTOR signaling pathways in mammary cancer 4T1, whereas Compound C weakens the effect of metformin.4T1 cells were treated with metformin for 48 hours. Western Blot founds Metformin treatment increased AMPK protein phosphorylation levels and reduced p70S6K protein phosphorylation levels in a dose-dependent manner. Compound C can antagonize the effect of metformin on 4T1.3. Metformin inhibits 4T1 cell colony formation.Metformin prevented the colony formation of 4T1 cells in a dose-dependent manner (P<0.05). When used at 5mM, metformin inhibited cell colony formation by 4.11% compared to control.4. Metformin induces cell cycle arrest in 4T1 cell.Treatment of proliferating 4T1 cells with metformin at varying concentrations for 24h caused delayed entry into S phase and induced G0/G1 arrest. Western Blot indicates cyclin Dl, a key protein implicated in the transition of the G0/G1 phase, was significantly reduced in a dose-dependent manner in metformin-treated cells.5. Metformin induces apoptosis of 4T1 cell.4T1 cells were incubated with various concentrations of metformin. Apoptosis (annexin V fluorescein isothiocyanate and PI staining), nuclear morphology (DAPI staining), and apoptosis biomarker (cleaved caspase-3) were assessed. The results indicate metformin induces 4T1 cell apoptosis in a dose-dependent manner.6. Metformin suppresses 4T1 cell migration and invasion in vitro.Wound-healing migration is performed, and metformin-treated group shows reduced migrated cell numbers in mammary cancer 4T1. Similar results were obtained in Matrigel-coated transwell invasion assay. Western blot results show that after metformin treatment the invasion related proteins (MPP-2 and MPP-9) in 4T1 cell expression and activity decreased.Conclusion1. Metformin can inhibit 4T1 cell proliferation which induces G0/G1 phase cell cycle arrest and cell apoptosis.2.4T1 cells proliferation can be inhibited by metformin through activating the AMPK/mTOR signaling pathways. Compound C can antagonize the effect of metformin on 4T1 proliferation.3. Metformin suppresses 4T1 cell migration and invasion in vitro.ObjectivesTo investigate the potential effects of metformin on the growth and metastasis of mammary cancer,4T1 xenograft tumors were established in female BALB/c mice, and tumor growth was monitored during metformin treatment. We hope the results provide the new evidence for the antineoplastic effect of metformin and new methods and options of advanced breast cancer clinical treatment.MethodsObserve the effect of metformin on xenograft tumor growing and lung metastasis through building up the BALB/C model of advanced breast cancer. Detect the effect of metformin on transplanted tumor of ki-67 and p-mTOR by IHC-F. The tumor cell apoptosis can be detected by fluorescence TUNEL. Western Blot is used to detect the change of invasion related protein in xenograft tumor after metformin treatment. The serum insulin and IGF-1 levels in tumor-bearing mice were detected by ELISA.Results1. Metformin inhibits the growth of 4T1 xenograft tumor in vivo.After metformin treatment, it is found by the measurement of vernier caliper and precision scales, the size of 4T1 xenograft tumor was significantly reduced. It is found by IHC-F, in the xenograft tumor tissues, metformin treatment significantly decreased ki-67 index. In addition, it is found by fluorescence TUNEL, after metformin treatment the apoptosis index in xenograft tumor tissues was prominently increased.2. Metformin suppresses mammary cancer metastasis in vivo.It is showed by Western Blot, after metformin treatment, the level of invasion related proteins (MPP-2 and MPP-9) in xenograft tumor tissues were reduced. It is found the number of metastases to the lungs in BALB/C mice 4T1 xenograft model was prominently decreased after taken oral metformin by counting its number and together with the routine pathologic examination.Conclusion1. Metformin can inhibit the growth of 4T1 xenograft tumor through inducing G0/G1 phase cell cycle arrest and cell apoptosis and activating the signal pathways of AMPK/mTOR.2. Metformin can suppress BALB/C mice model of advanced breast cancer metastasis through decreasing the level of invasion related proteins (MPP-2 and MPP-9) in xenograft tumor tissues.