The Experimental Study Of The Relationship Between Protein Kinase B And Salivary Adenoid Cystic Carcinoma

ObjectiveProtein kinase B(PKB) is serine -threonine kinase. PKB was discovered in 1991. There are three study groups discover the PKB at the same time. Two groups discovered which its catalyse field was the same as protein kinase C (PKC,73% ) and protein kinase A(PKA,68%) ,so called it as protein kinase B (PKB) and related the A and C kinase ( RAC - PK). The other group discovered that PKB is the production of T cell lymphangioma reverse transcription the viral oncogene v - akt, called Akt. This 58 kDa protein plays an important role in anti - apoptosis, which is relation with tumor. Salivary adenoid cystic carcinoma (SACC) is one of the most common salivary malignant tumors. It is the first or the second of malignant salivary tumor. The typically characterizion of SACC is slower in growth, stronger in invasion, growth along nerves and blood vessel, ea-ser recrudescence and metastasis.The mechanism of SACC proliferative, differentiation, invasion and metastasis is still unknow now. In clinic, there are few potent drugs to treat it. We study the expression of PKB (Ser473) and PTEN in tissue with SP method, analysing the relationship of PKB ( Ser473) and PTEN. Using Western - blot and RT -PCR detected the protein and mRNA level of PKB both in SACC - 83 and SACC -LM cell line, absent and present LY294002. In vitro, we used TEM, MTT method and Flow cytometry to investigate the influences of LY294002 to cell proliferative , cell cycle and cell apoptosis, in order to study mechanism of SACC carcinogenesis and treatment in clinic.Methods1. Immumohistochemical methodThere are 63 tissue samples of SACC , include of 38 cases both non - cancer tissue near by cancer. 4μm sections were deparaffinized . The sections were stained by Streptavivdin -Peroxidase method(SP).2. Cell cultureSACC - 83 and SACC - LM cells were cultured with RPMI -1640 medium containing 15% fetal bovine serum ,100u/ml penicillin and 100u/ml streptomycin. Cells were incubated in a 37℃. and a humidified 5% CO2 atmosphere incubator.3. Protein quantity determinationCoomessie method was used to detect the concentration of protein solution.4. Western blot AnalysisLY294002(50μmol/L) was added 2h before lysing the cells. 20μg Protein were subjected to SDS - PAGE( 10% ) , 150v,1. 5h. The separated protein were transferred to nitrocellulose membrane(50v,2h). Membranes were blocked with 5% BSA for 1h. Membranes were incubated with the primary antibody (1 :500) overnight at 4℃. Membranes were then incubated with the secondary antibody (1:2000) for 2h. Immunocomplex was visualized with the staining agents.5. RT-PCRLY294002(50μmol/L) was added 2h before lysing the cells. The quality and quantity of the RNA was determined by measuring the absorbance of the total RNA at 260nm and 280nm. According to the manufacture' s instructions to reverse transcribe and amplify PKB sequence, after 94℃ for 2min, PCR amplification were 30 cycles of 94℃ for 30S, 60℃ for 30s, and 72℃ for 1. 5min,in the end 72℃ for 10min. The PCR products were electrophoresed on 2% agarose gel and visualized with EB(ethidium bromide).6. Cell morphological changesSACC -83 cells were incubated in RPMI - 1640 medium containing 15% fetal bovine serum ,100u/ml penicillin and 100u/ml streptomycin, in the ab-sence or presence of LY294002(50μmol/L) for 72h. Microscope was used to observe the cells morphological changes. The cells were centrifugated at 4℃ for 6min at 1000rpm,2.5% amyldialdehyde fixation, abserved by TEM.7. MTT assayThe cells were seeded at a density of 5000/well in a 96 well culture dish. SACC -83 cells were incubated in RPMI - 1640 medium containing 10% fetal bovine serum ,100u/ml penicillin and 100u/ml streptomycin, in the absence or presence of LY294002(50μmol/L) . After cells were treated for 4 days, MTT was added into the dish with 20 μl/well, the cells were incubated for 4 hours with MTT followed by DMSO added,then the OD values were detected at 490nm.8. Flow cytometryCells were serum - starved for 24 hours . SACC - 83 cells were incubated in RPMI - 1640 medium containing 15% fetal bovine serum ,100u/ml penicillin and 100u/ml streptomycin, in the absence or presence of LY294002(50μmol/ L) for 24,48 and 72 hours. Cells were harvested,washed twice with PBS,stained for 30 min at 4℃ with PI while protected from light. The samples were analysed on a FACscan.Results1. The expression of PKB ( Ser473) in SACC tissuesThe expression of PKB ( Ser473) was higher in cancer tissue than in non -cancer tissue near by cancer ( P = 0. 008, P < 0. 01 ). PKB expression were no significant difference between the different sex, age and sorts of pathology(P > 0.05).2. The expression of PTEN in SACC tissuesThe expression of PTEN was lower in cancer tissue than in non - cancer tissue near by cancer ( P=0.013,P<0.05). PTEN expression were no significant difference between the different sex, age and sorts of pathology(P >0.05).3. Relationship between expression of PKB (Ser473) and PTENThe expression of PKB ( Ser473) and PTEN were negatively correlated in SACC tissues(r3= -0. 336, P =0.006, P <0.01).4. The expression of PKB(Ser473) in SACC -83 and SACC - LM cell lines The expression of PKB( Ser473) in SACC - 83 is lower than SACC - LM( t =2. 683, P = 0. 025, P < 0. 05 ). The expression of PKB ( Ser473 ) in SACC cell lines with LY294002 is lower than in SACC cell lines without LY294002 (tSACC-83 =5. 075 ,tSACC-LM =4. 83 ,P < 0.01). PKB expression were no significant difference in SACC -83 with LY294002 and in SACC - LM with LY294002 (t = 1.887,P > 0.05).5. The transcription level of PKB in SACC -83 and SACC - LM cell lines The transcription level of PKB is lower in SACC - 83 than in SACC - LM( t=2. 827, P < 0.05). The transcription level of PKB is on difference in SACC cell lines with and without LY294002(P > 0.05).6. The morphology of SACC -83 cell with LY294002In light microscope, cells treated with LY294002 showed changes in morphology. The volume of cells was decreased and suspended cells increased.In TEM, SACC - 83 cells showd chromatin condensation into dense granula caps under the nuclear membrane. The microvilli on the cell surface disappeared. The nuclear membrane was sunken to inside. The severity occurs rupture.7. MTT assayCells with LY294002 growths were significantly inhibited, with elongation of action time the inhibitory effects increase(F = 111.51,P < 0.01).8. Flow cytometry for detecting cell cycleAfter LY294002 treated, with elongation of action time, the G0/G1 phase of cells were increased ( F(药物作用) = 681. 58,F(时间作用) =1660.87, P < 0.01) ,the S phase of cells were decrease(F(ffftfMi) = 508.98,F(BtHffua) =173. 56,P < 0. 01) , and G2/M phase of cells were decrease (F(药物作用) = 278.43,F(时间作用) = 637.33,P < 0.01).9. Flow cytometry for detecting apoptosisAfter LY294002 treated, with elongation of action time, the apoptosis cells was increased(F(药物作用) = 134.69,F(时间作用) =43.46,P < 0.01).