The Effect And Mechanism Of Adipocytes Modulated By Metformin On The Chemoprotection Of Leukemia Cells

Objective:The high malignancy and poor prognosis of acute myeloid leukemia(AML) are closely associated with bone marrow microenvironment. As main components of bone marrow microenvironment, adipocytes play a key role in the development of leukemia, but researches on which have many blind spots.Metformin is a widely-used oral antidiabetic drug,which has been demonstrated to suppress the proliferation and metastasis of solid tumor cells by inhibiting adipogenesis. However, the effect of metformin on bone marrow adipocytes in leukemia hasn't been reported. In the present study, we retrospectively analyzed the bone marrow biopsies of primary AML patients and normal controls and characterized the bone marrow adipocytes of which quantitatively by using a self-developed grid-computing software to explore the correlation between bone marrow adipocytes and the refractoriness of AML. Adipogenic differentiation of bone marrow mesenchymal stem cells(MSC) was induced in vitro to investigate the effects of adipocytes on chemoprotection of leukemic cells and the underlying mechanism. Meanwhile, we explored the effect of adipocytes modulated by metformin on the chemoresistance of leukemic cells, which may provide a novel insight into the treatment of AML.Methods:(1) Characteristics of bone marrow adipocytes(the volume and size) were detected in 61 primary AML patients and 71 normal controls with a self-developed grid-computing software,Compare the differences between them;Primary AML patients were divided into remission and refractory group according to their prognosis to analyze the correlation between the volume and size of bone marrow adipocytes and the refractoriness of primary AML.(2)Adipogenic differentiation of bone marrow MSC of primary AML patients and normal controls were induced in vitro. Observation of adipocytes morphology by oil red staining and detection of adipogenic genes(PPAR? ?C/EBP? ?FABP4) were carried out to verify the adipogenesis of MSC.(3)Screening the optimal chemotherapeutic concentration of cytarabine(Ara-c), and co-culture of adipocytes and THP-1 cells was established to study the difference between the effects of adipocytes derived from AML and normal bone marrow MSC on the chemosensitivity of leukemia cells;Adipokines in bone marrow serum of primary AML were detected by protein microarray to screen the proteins with significant changes in expression level and the target adipokines were selected according to references. The result was confirmed by Western Blot and q RT-PCR;(4)At day 19 of adipogenic differentiation of bone marrow MSC of primary AML, 5mmol/L metformin was added for 48 hours.and set up the controls.Adipogenic rate was tested by oil red staining and the expression level of adipogenic genes PPAR?, C/EBP? and FABP4 was detected by q RT-PCR.Co-culture of adipocytes and THP-1 cells was established. The difference in chemosensitivity of THP-1 to Ara-c was compared by counting viable cells and the differences in apoptosis of THP-1 was determined by flow cytometry.(5) Conditioned medium was collected from adipocytes modulated by metformin and control. THP-1 cells were cultured in mixture of conventional culture medium and conditioned medium with different ratios for 48 hours in the presence of Ara-c and viable cells were counted to compare the effects of adipocytes of both groups on the chemosensitivity of leukemia cells;(6)The detection of expression level of Leptin in adipocytes modulated by metformin or not was carried out by Western Blot and q RT-PCR to explicit the role of Leptin in chemoprotection of adipocytes modulated by metformin in AML.Result:(1)The bone marrow adipocytes of primary AML were significantly decreased in volume and size compared to normal controls(P<0.0001); the adipocytes of refractory group had remarkably lower volume than non-refractory group(P<0.05). In addition, the volume of adipocytes was found to be positively correlated with age except for other factors as primary bone marrow leukemic cell number, sex and BMI;(2)With treatment of Ara-c,Adipocytes derived from MSC of primary AML and THP-1 cells co-culture,compared with the normal group,THP-1 cells Chemosen-sitivity significantly decreased and viable cell number remarkably increased(P<0.001);(3) Protein microarray revealed that bone marrow serum of primary AML overexpressed Leptin, Resistin and Growth Hormone. Adipocytes derived from MSC of primary AML overexpressed Leptin compared to normal controls(P<0.001);(4) Compared to controls,MSC of AML derived adipocytes regulated by metformin adipogenic rate and adipogenic genes have no significant change(P>0.05);However, the chemo-resistance of THP-1 was attenuated with promoted apoptosis when co-cultured directly or indirectly with adipocytes modulated by metformin.(P<0.05);(5)Protein and m RNA expression level of Leptin was significantly reduced with the treatment of metformin(P<0.01).Conclusion: 1. Bone marrow adipocytes of primary AML were significantly decreased in volume and size,Involved in the chemoresistance of AML,which is closely associated with the refractoriness of AML. Abnormally high secretion of Leptin may be responsible for regulating bone marrow adipocytes to protect leukemic cells from chemotherapy.It is suggested that bone marrow adipocytes of primary AML may act as an important prognosis factor and further study of the mechanism may provide guidance for early interventing treatment of AML. 2.The protection of adipocytes for leukemia cells is attenuated by the treatment of metformin. The possible mechanism, on one hand, is that metformin downregulates the expression of Leptin in adipocytes;on the other hand it attenuates the anti-apoptotic effection of adipocyte to leukemia cells.