Inhibitory Effects Of Matrine And Ligustrazine On Invasion And Metastasis Of Leukemia Cells Treated With Hypoxia Culture

Background: Leukemia was the most common nausea tumors of hematopoietic system, and one of the most common nausea tumors in children. In recent years, with the improvement of combined chemotherapy, the complete remission rate of leukemia has been increased. But invasion and metastasis is the main reason that leads to the recurrence and treatment failure for complete remission leukemia patients in clinic. So it is very important to study the pathogenesis of invasion and metastasis in leukemia, and looking for new drugs for effectively treating and preventing leukemia. Matrine and Ligustrazine, as two kinds of traditional Chinese medicine, have many antitumor effects. Our early results indicated that Matrine and Ligustrazine could effectively restrain the proliferation, invasion and metastasis, and reduce the expression of MMP-2,MMP-9 in leukemia cells. In addition, researches showed that hypoxia was one of the essential characteristic of malignant tumor microenvironment, and hypoxia-inducible factor-1 alpha (HIF-1α) was one of the main transcription factor that directly controlled by hypoxia. In solid tumor, HIF-1αcould promote the formation of blood vessels and the degradation of extracellular matrix by controlling the transcription of target genes, such as VEGF,MMPs, etc, to facilitate invasion and metastasis of the occurrence tumor cells. Our early results suggested that hypoxia(3%O₂) could increase the invasion and metastasis of leukemia cells, meanwhile increase the expression of HIF1-α,VEGF,MMP-2 and MMP-9. But the inhibitory effects of Matrine and Ligustrazine on invasion and metastasis in leukemia cells concerned with reducing expression of HIF1-α,VEGF,MMP-2 and MMP-9 that has not been seen on the domestic and international reports. So this research will continue to study the interaction mechanisms of Matrine and Ligustrazine in leukemia cells.Objective:To study the inhibitory effects and mechanism of Matrine and Ligustrazine in different dose on invasion and metastasis of leukemia cells which was treated with hypoxia.Methods:Raji and K562 cells were culture with normal method in vitro and treated with 3% oxygen for 24 hours, and then treated by 0g/L,0.15g/L,0.20g/L,0.25g/L Matrine or 0g/L,0.10/L,0.15L,0.20/L Ligutrazine. The nomoxic cultured group was used as the control group. Then cell adhesion assay﹑cell migration assay and cell invasion assay were used to observe the effect of matrine or ligutrazine on adhesion﹑migration and invasion of Raji and K562 cells which were treated with hypoxia, and RT-PCR was used to evaluate the mRNA expression levels of HIF-1α﹑VEGF﹑MMP-2 and MMP-9.Results:1. In the normoxic cultured group, the cells adhesion,migration and invasion rates of Raji cells and K562 cells were 100%;2. The group treated with hypoxia(3%O₂) but no drugs , cells adhesion,migration and invasion rates of Raji cells component were (172.216±13.44)%,(164.47±4.73)%,(142.817±2.665)%,and of K562 cells component were (182.49±8.33)%,(192.31±2.77)%, (139.607±7.039)%;3. The groups treated with hypoxia(3%O₂) and then treated by 0.15g/L,0.20g/L,0.25g/L Matrine, cells adhesion rates of Raji cells component were (160.774±11.03)%,(143.877±7.813)%,(127.419±2.581)%,cells migration rates component were ( 146.68±5.30 ) %,(132.11±1.50)%,(117.21±5.49)%,and the invasion rates component were (122.533±2.520)%,(109.203±6.576)%,(94.636±2.000)%;4. The groups treated with hypoxia(3%O₂) and then treated by 0.15g/L,0.20g/L,0.25g/L Matrine, cells adhesion rates of K562 cells component were (172.3±3.60)%,(139.8±10.4)%,(133.9±4.8)% ,cells migration rates component were ( 175.13±7.85 ) %,( 139.74±14.76 ) %,( 117.63±6.87 ) %,and the invasion rates component were (121.164±7.876)%,(83.539±15.741)%,(63.630±7.331)%;5. The groups treated with hypoxia(3%O₂) and then treated by 0.10g/L,0.15g/L,0.20g/L Ligutrazine, cells adhesion rates of Raji cells component were (154.56±9.75)%,(138.50±9.76)%,(128.63±10.88)%%,cells migration rates component were ( 150.80±1.90 ) %,(137.15±3.54)%,(124.66±4.57)%,and the invasion rates component were (116.549±10.071)%,(106.06±5.017)%,(95.964±6.398)%;6. The groups treated with hypoxia(3%O₂) and then treated by 0.10g/L,0.15g/L,0.20g/L Ligutrazine, cells adhesion rates of K562 cells component were (175.13±7.85)%,(139.74±14.76)%,(117.63±6.87)%,cells migration rates component were ( 150.80±1.90 ) %,(137.15±3.54)%,(124.66±4.57)%,and the invasion rates component were (127.180±13.846)%,(104.650±6.999)%,(79.813±11.337)%;7. The group cultured with normoxic,group treated with hypoxia(3%O₂) but no drugs and groups treated with hypoxia(3%O₂) and then treated by 0.15g/L,0.20g/L,0.25g/L Matrine , the expression of Raji cells of HIF1-αmRNA component were 0.70±0.06,2.41±0.01,1.99±0.07,1.61±0.09,1.27±0.08, and of VEGF component were 0.02±0.00,0.47±0.00,0.24±0.08,0.16±0.03,0.09±0.07;the expression of k562 cells of HIF1-αmRNA component were 0.36±0.09,3.32±0.02,3.27±0.05,2.51±0.04,1.23±0.01, and of VEGF component were 0.05±0.02,0.34±0.00,0.32±0.08,0.22±0.08,0.15±0.06;8. The group cultured with normoxic,group treated with hypoxia(3%O₂) but no drugs and groups treated with hypoxia(3%O₂) and then treated by 0.10g/L,0.15g/L,0.20g/L Ligutrazine , the expression of Raji cells of HIF1-αmRNA component were 0.01±0.00,1.52±0.00,1.28±0.01,0.90±0.09,0.57±0.01, and of VEGF component were 0.02±0.00,0.51±0.00,0.24±0.04,0.14±0.02,0.09±0.02;the expression of k562 cells of HIF1-αmRNA component were 0.32±0.03,2.51±0.09,1.57±0.08,1.17±0.00,0.79±0.02, and of VEGF component were 0.01±0.00,0.98±0.09,0.57±0.07,0.23±0.02,0.08±0.00.Conclusion:1. Contrasting to the control group, treated with hypoxia but without any drugs groups could significantly enhanced the adhesion, migration and invasion of Raji cells and K562 cells (P<0.01);2. 0.15g/L,0.20g/L,0.25g/L Matrine could significantly inhibit the adhesion, migration and invasion of Raji cells which were treated with hypoxia(P<0.05 , P<0.01);3. 0.20g/L,0.25g/L Matrine could significantly inhibit the adhesion, migration and invasion of K562 cells which were treated with hypoxia(P<0.05 , P<0.01);4. 0.10g/L,0.15g/L,0.20g/L Lingutrazine could significantly inhibit the adhesion, migration and invasion of Raji cells which were treated with hypoxia(P<0.05 , P<0.01);5. 0.10g/L,0.15g/L,0.20g/L Lingutrazine could significantly inhibit the adhesion, migration and invasion of K562 cells which were treated with hypoxia(P<0.05 , P<0.01);6. 0.15g/L,0.20g/L,0.25g/L Matrine could significantly inhibit the expression of HIF-1α﹑VEGF mRNA of Raji cells which were treated with hypoxia, 0.20g/L,0.25g/L Matrine could significantly inhibit the expression of HIF-1α﹑VEGF mRNA of K562 cells which were treated with hypoxia;7. 0.10g/L,0.15g/L,0.20g/L Lingutrazine could significantly inhibit the expression of HIF-1α﹑VEGF mRNA of Raji cells or K562 cells which were treated with hypoxia.