Expression Of Hypoxia-inducible Factor 1α In Pediatrics Non-Hodgkin's Lymphoma And A Study On Effect Of Sirolimus On Growth And Apoptosis Of Cell Raji

Objective: Lymphoma is one of the most dangerous cancers in pediatrics with an increasing incidence. Non- Hodgkin's lymphomas(NHL)represent the most frequent type of lymphoma一80% to 90% of lymphoma.Conventional treatment for patients with NHL is chemotherapy. Although these tumors can be treated successfully with chemotherapy, primary. Chemo resistance and relapse often occur and are the major causes of death in these patients.So it is important for longer survival period to find available therapeutic approaches. Hypoxia- inducible factor-1α(HIF-1α) is an important transcription factor under hypoxia condition. It plays the role of dominating the expressions of correlative genes. It also promotes tumor deterioration, tumor metastasis and tumor invasion. The molecular role of HIF-1αhas been intensively studied in cancer basic research. Presently, hypoxia inducible factor 1alpha is believed to play a role in on cogenesis. Tumor growth and angiogenesis are correlated with the expression of HIF-1α, therefore, inhibiting the activity of HIF-1αmay be a strategy for cancer therapy. In in-vitro studies, HIF-1αtargeting with anti sense reduces tumor growth potential, and sensitizes cells to chemotherapeutic drugs. Sirolimus is a kind of macrolide antibiotics as well as immunosuppressant. Newly find sirolimus also have resist tumor action, the mechanisms maybe sirolimus inhibited protein expression of HIF-1α, but specific mechanisms is not clarity. To identify the role of HIF-1αin pediatrics NHL tumorigenesis, we investigated tissue expression of HIF-1αand vascular endothelia cell growth factor (VEGF) in pediatrics NHL. In addition, we investigated the effect of Sirolimus on the proliferation, apoptosis, cell cycle distribution and HIF-1αexpression in human malignant lymphoma cell line Raji and the possible anti tumor mechanism. And study the combinational effects of Sirolimus and Adriamycin (ADM) on proliferation and apoptosis of Raji cells in vitro and to explore the possible mechanisms. Thus, theory evidence of sirolimus in clinical application will be provided.Methods: 1 Choose 30 tissues of Pediatrics non-Hodgkin's lymphoma in the fourth hospital of He Bei Medical University from March, 2004 to September, 2008. Among them the T cell type is 13 and the B cell type is 17.According to the St.Jude non-Hodgkin's lymphoma staging system, is divided into the above case low degree dangerous group 11 is aⅠ,Ⅱperiod; height degree dangerous group 19 isⅢ,Ⅳperiod. Take another 10 Reactive hyperplasia lymph node as a control group. Using immunohistochemical method(S-P) detection the expression of the HIF-1αand the VEGF in each one, and analyze the relativity of their expression.2 Human Burkitt's lymphoma cells Raji were cultivated in vitro and treated with various concentrations of Sirolimus and ADM.Cell inhibitive rate was investigated by MTT assay.Also compute half growth depressant quantity (IC50).Choose function density and function of medicine according to the IC50 for time.3 Is divided into the matched control to the Raji cell:Join in the cell culture medium with DMSO of experiment set etc. physical volume;Sirolimus set: Join in the cell culture medium with Sirolimus which solution of DMSO eventually the concentration to 5,10,15,20ng·mL-1; ADM set: Join in the cell culture medium with ADM which solution of PBS eventually the concentration to 10mg·L-1; Sirolimus and ADM set: Join in the cell culture medium with Sirolimus which solution of DMSO eventually the concentration to 10ng·mL-1 and ADM which solution of PBS eventually the concentration to 10mg·L-1. Cell inhibitive rate was investigated by MTT assay. Apoptosis rate, cell cycle distribution and expression of HIF-1αprotein in Raji cells were detected by FCM and immunocytochemistry method (S-P).Results:1 The expression rate of HIF-1αin the control group, low degree dangerous group and height degree dangerous group discern to 0.00%, 54.55%, 73.68%. Three sets of comparison differences have covariance to learn meaning (P<0.05). The expression rate of VEGF in the three sets discern to 0.00%,63.64%,84.21%.Three sets of comparison differences have covariance to learn meaning (P<0.05).2The expression rate of HIF-1αin the T,B cell type of Pediatrics NHL tissue discern to 61.54%,70.59%.Two sets of comparison differences have covariance to no learn meaning (P>0.05). The expression rate of VEGF in the two sets discern to 69.23%,82.35%.Two sets of comparison differences have covariance to learn meaning (P<0.05).3 Spearman ranking correlation analysis display: the expression of HIF-1αand VEGF in the Pediatrics NHL just related (r=0.671, P<0.05).4 MTT assay demonstrated that: PBS and DMSO which eventually concentration is 0.1% have no obvious influence to the Raji cell's growth; Sirolimus and ADM repress Raji cell a function to present the quantity dependence of the time in the certain scope; after 48h the IC50 of ADM is 11.47mg·L-1, the IC50 of Sirolimus is 17.19ng·mL-1. 5 MTT assay demonstrated that: Cell inhibitive rate of Raji after treated with various concentrations of Sirolimus for 48 hours were 25.99%,34.28%,53.08%,6.40%;Cell inhibitive rate of Raji after treated with ADM for 48 hours were 47.47%; Cell inhibitive rate of Raji after treated with Sirolimus and ADM for 48 hours were 65.98%. Absorbance light degree in experiment groups after treated Raji with various concentrations of Sirolimus for 48h were significantly different compared with control group (P<0.05);the concentrations group of 15,20ng·mL-1 were no significantly different (P>0.05), the other group were significantly different (P<0.05). Absorbance light degree in experiment groups after treated Raji with Sirolimus and ADM for 48h (0.431±0.047) were significantly different compared with just treated the same concentrations of Sirolimus (0.832±0.071) or ADM (0.601±0.041) (P<0.05).6 FCM showed: Apoptosis percentage (AP) of Raji after treated with various concentrations of Sirolimus for 48 hours were (7.60±1.02)%, (11.32±0.95)%, (23.17±1.95)%, (32.20±1.80)%; AP in control was (2.29±0.31)%. There was remarkable difference between experiment groups and control group by least significant difference test (P<0.05). By one way ANOVA, AP were significantly different among concentration groups (P<0.05). AP in ADM group was (0.93±0.17)%, There was no remarkable difference between ADM group and control group(P>0.05). AP in Sirolimus and ADM was(10.63±0.94)%; There was remarkable difference between this group and control group or ADM group (P<0.05), There was no remarkable difference between this group and Sirolimus of the same concentration group(P>0.05).7 FCM showed: After Raji cells treated with various concentrations of Sirolimus for 48 hours, the cell percentage in G0/G1 phase of test groups were (57.27±1.16)%, (62.07±1.79)%, (68.67±0.06)%, (72.57±0.85)% respectively; the cell percentage in S phase were (29.67±1.00)%, (27.33±0.87)%,(21.87±0.42)%,(18.87±1.56)% respectively; the cell percentage in G2/M phase were (13.07±0.25)%, (10.63±0.88)%, (9.48±0.38)%, (8.56±0.75)% respectively. The cell percentage in G0/G1, S, G2/M phase of control group were (39.97±0.74)%, (45.03±0.93)%, (15.00±0.92)% respectively. Compared with control group, cell cycle distribution in test groups of Sirolimus existed a significant difference (P<0.05). By one way ANOVA, cell cycle distribution were significantly different among concentration groups (P<0.05). After Raji cells treated with ADM for 48 hours, the cell percentage in G0/G1,S,G2/M phase of control group were (32.13±0.76)%, (46.40±0.72)%, (21.50±1.32)%. Compared with control group, cell cycle distribution existed no significant difference (P>0.05). After Raji cells treated with Sirolimus and ADM for 48 hours, the cell percentage in G0/G1,S,G2/M phase of control group were (53.57±1.10)%, (32.67±1.17)%, (13.77±0.15)%.Compared with control group or ADM group, cell cycle distribution existed significant difference (P<0.05). Compared with the same concentration of Sirolimus, cell cycle distribution existed no significant difference (P>0.05).8 FCM showed: The FI of HIF-1αprotein expression in Raji cells after treated with various concentrations of Sirolimus for 48 hours were 0.91±0.015, 0.78±0.021, 0.64±0.025, 0.57±0.026 respectively. The FI of HIF-1αprotein expression in control group was regarded as 1, there were significantly different among every group compared with control group (P<0.05). 9 Immun- ocytochemistry method showed: HIF-1αwas stained in nucleolus and cytoplasm of the Raji cells, to assume stramineous to Buffy granula. After treated with Sirolimus, the color in nucleolus and cytoplasm of the Raji cells was light than the control group, and the granula reduce. These state that after the Raji cells treated with Sirolimus, the expression of the HIF-1αwas decreased.Conclusions: 1There are high expression of HIF-1αand VEGF in Pediatrics NHL, and the expression between HIF-1αand VEGF in NHL was significantly correlated, so the pathway HIF-1α→VEGF may play a role in the pathogenesis of Pediatrics NHL. 2 The survival of Raji cells was inhibited by Sirolimus in vitro with a concentration and time dependent manners in a circumscription. 3 The number of Raji cells in G1 phase was increased, while those in S and G2 phase was decreased by the effect of Sirolimus. Sirolimus induced cell cycle arrest in G1 phase and inhibited the growth of Raji cells, which maybe the mechanism that sirolimus have resist tumor action. 4 The expression of HIF-1αcan be decreased by Sirolimus in vitro with a concentration dependent manner, and apoptosis in Raji cells can be induced by Sirolimus through the downregulation of HIF-1αexpression, which maybe another possible anticancer mechanism of Sirolimus. 5 Raji cells treated with Sirolimus and ADM was conspicuous repressed compared with Raji cells treated with Sirolimus or ADM. But the AP and cell cycle distribution after Raji cells treated with Sirolimus and ADM was no significantly different compared with Sirolimus. This maybe ADM is the non- particularity of the cell cycle medicine, the cell poisonous function can be take place in each period.