Effect Of Hypoxia On Invasion And Metastasis Of Leukemia Cells

Backgroud:Invasion and metastasis is one features of tumor, and is also caused death of most tumor patients. Leukemia is the malignant tumor from hematopoietic system. Recent years, with the improvement of combined chemotherapy, the complete remission rate of leukemia has been heightened. But invasion and metastasis is main reason of lead to the recurrence and treatment failure for complete remission leukemia patients in clinic, and is also one of the most difficult problems for clinician. Recently research found that hypoxia was widely exists in malignant tumors, which make solid tumors invasion and metastasis to distant tissues more easy. The hypoxia-inducible factor 1 alpha (HIF-1α) is the key nuclear transcription factor in tumor cells by hypoxia, which is most important in proliferation, invasion and metastasis in tumor. Recent reports indicate HIF-1αcould promote tumor cell proliferation and angiogenesis by control the transcription of vascular endothelial growth factor (VEGF), and degradation of extracellular matrix (ECM) by control the transcription of metalloproteinases (MMPs). However, we known few about the effects of hypoxia on invasion and metastasis in leukemia.Objective:To study the potential effect of hypoxia on invasion and metastasis of leukemia cells, and investigate their possible mechanism of action.Methods: K562 cells and Raji cells were cultured with normal method in vitro and treated with 1%, 3%, 5% oxygen for 24h, the normoxic cultured group was used as control. Then cell adhesion assay,cell migration assay and cell invasion assay were used to observe the effects of oxygen on adhesion, migration and invasion ability of Raji cells, and RT-PCR was used to evaluate the mRNA expression levels of VEGF, MMP-2 and MMP-9. The protein level of HIF-1αwas observed by Western Blot.Results :1. Treat by hypoxia in normoxic, 1%, 3%, 5% oxygen concentration for 24 hours, cells adhesion rates of K562 were 100%, (101.20±0.17)%, (191.60±0.27)%, (160.00±0.24) respectively; cells adhesion rates of Raji were 100%, (101.15±0.14)%, (179.08±0.17)% , (138.98±0.25) % respectively.2. Treat by hypoxia in normoxic, 1%, 3%, 5% oxygen concentration for 24 hours, cells migration rates of K562 were 100%, (110.09±0.20)%, (214.71±0.30)%, (154.60±0.29) respectively; cells migration rates of Raji were 100%, (99.81±0.10)%, (174.16±0.12)%, (138.51±0.22)% respectively.3. Treat by hypoxia in normoxic, 1%, 3%, 5% oxygen concentration for 24 hours, cells invasion rates of K562 were 100%, (100.65±0.09)%, (146.34±0.07)%, (127.47±0.09)% respectively; cells invasion rates of Raji were 100%, (102.41±0.11)%, (167.50±0.19)% , (134.46±0.13)% respectively.4. Treat by hypoxia in normoxic, 1%, 3%, 5% oxygen concentration for 24 hours, the absorbance of HIF-1αprotein of K562 were 0.04±0.01, 0.03±0.01, 1.04±0.05, 0.88±0.23 respectively; the absorbance of HIF-1αprotein of Raji were 0.03±0.02, 0.02±0.03, 1.81±0.05, 1.61±0.10 respectively.5. Treat by hypoxia in normoxic, 1%, 3%, 5% oxygen concentration for 24 hours, the absorbance of HIF-1αmRNA of K562 were 0.08±0.02, 0.08±0.02, 0.91±0.06, 0.66±0.04 respectively; the absorbance of HIF-1αmRNA of Raji were 0.10±0.04, 0.11±0.07, 0.81±0.05, 0.65±0.05 respectively.6. Treat by hypoxia in normoxic, 1%, 3%, 5% oxygen concentration for 24 hours, the absorbance of VEGF mRNA of K562 were 0.41±0.03, 0.42±0.04, 0.72±0.04, 0.65±0.03 respectively; the absorbance of VEGF mRNA of Raji were 0.38±0.05, 0.37±0.06, 0.86±0.04, 0.76±0.08 respectively.7. Treat by hypoxia in normoxic, 1%, 3%, 5% oxygen concentration for 24 hours, the absorbance of MMP-9 mRNA of K562 were 0.52±0.06, 0.50±0.06, 0.98±0.03, 0.87±0.04 respectively; the absorbance of MMP-9 mRNA of Raji were 0.30±0.04, 0.29±0.04, 0.80±0.06, 0.65±0.05 respectively.8. Treat by hypoxia in normoxic, 1%, 3%, 5% oxygen concentration for 24 hours, the absorbance of MMP-2 mRNA of K562 were 0.52±0.06, 0.50±0.06, 0.98±0.03, 0.87±0.04 respectively; the absorbance of MMP-9 mRNA of Raji were 0.30±0.04, 0.29±0.04, 0.80±0.06, 0.65±0.05. MMP-2 mRNA was not obvious expression in Raji cells.Conclusion:1. Moderate hypoxia could enhance the ability of adhesion, metastasis and invasion of K562 cells and Raji cells.2. Moderate hypoxia could up-regulating the expressions of HIF-1αprotein and HIF-1αmRNA in K562 cells and Raji cells.3. Moderate hypoxia could up-regulating the expressions of VEGF mRNA and MMP-9 mRNA in K562 cells and Raji cells.4. Moderate hypoxia could up-regulating the expressions of MMP-2 mRNA and MMP-9 mRNA in K562 cells. But the mRNA level of MMP-2 was not obviously expression in Raji cells.5. The possible mechanism that Moderate hypoxia could enhance the ability of invasion and metastasis of leukemia cells, is HIF-1αup-regulate the expression levels of VEGF, MMP-9 and /or MMP-2.