| PART ⅠEXPRESSION OF MCTS IN HUMANASTROCYTIC TUMORSObjectivesTo investigate the expression patterns of monocarboxlate transporterMCTs in human astrcytoma with different pathologic grades, so as toexplore the molecular mechanism underlying the growth and proliferationof astrocytoma.Materials and Methods1. Specimen collection：Fifty-one astrocytoma samples werecollected from the first hospital affiliated to Chongqing MedicalUniversity and the Daping hostpital of the Third Military MedicalUniversity. All patients were not received any treatments were determinedby pathological examinations and detail historys. 2. The pathological grade appraisal：To identify the pathologicaldiagnosis through the techniques of HE staining.3. Each specimen was divided into two parts equally and conducted bythe Immunohistochemistry and Western Blot.(1). The immunohistochemistry technology was used to detect theexpression of MCTs in different pathologic grade of human astrocytomasand the normal brain tissues.(2). Western Blot technology was applied to detect the expressionalterations of MCTs in different grade of astrocytic tumors and the normalbrain specimens.4. statistical analysis：All of the experimental results were analyzedby graphic analysis software and SPSS17.0.Results1. The IHC results showed that MCT1was distributed in endothelialcells and astrocyte in normal brain tissues and in the nucleus and cytoplasmin different grade of astrocytoma tissues. Compared with normal braintissues, Western Blot showed that MCT1had strong expression in humanastrocytoma, the expression of MCT1in high grade astrocytoma was muchhigher than that in low grade astrocytoma. there was a significantdifference in statistical analysis.(P<0.05)2. The IHC results showed that MCT2was mainly distributed in thethe neuron and astrocyte of normal brain tissue and in the plasmalemma and cytoplasm of different pathological grade of astrocytoma. Theexpression of MCT2were increased with the ascending pathologic grade.(P<0.05)3. MCT4was detected in the astrocyte of normal brain tissue, and inthe cytoplasm of the low grade（ⅠandⅡ）of astrocytoma tissue, and in theplasmalemma and cytoplasm of anaplastic astrocytoma and glioblastomamultiforme. Western Blot showed that the expression of MCT4wereincreased in the low grade（ⅠandⅡ）of astrocytoma tissue and statisticalanalysis showed a significant difference （P＜0.05）,the expression ofMCT4were progressively increased in the high grade （Ⅲ and Ⅳ）ofastrocytoma and there was a significant difference in statistical analysis （P＜0.05）. There was no significant difference in statistical analysis in thecomparison between the high grade （Ⅲ and Ⅳ）and the low grade of（ⅠandⅡ）astrocytic tumors.（P＞0.05）Conclusions1.The expression of MCT1and MCT2in different grade of humanastrocytoma were increased compared to the normal brain tissue（P＜0.05），and enhanced with the increasing of pathologic grades. Theexpression of MCT1and MCT2in high grade astrocytoma was muchhigher than that in low grade astrocytoma (P<0.05). Protein expressions ofMCT1and MCT2are increased in human astrocytoma compared tonormal controls, and have a positive correlation with pathologic grades. 2. MCT4was mainly detected in the plasmalemma and cytoplasm ofdifferent pathological grade of astrocytoma. MCT4were increased in thelow grade of astrocytoma tissue, and were progressively increased in thehigh grade of astrocytoma. This suggested MCT1might play an importantrole in the proliferation and migration of astrocytoma which could be usedas a new treatment target for astrocytoma. PARTⅡ IMPACT OF MCT INHIBITOR CHCINTERFERENCE IN THE PROLIFERATION OF HUMANASTROCYTOMA CELL LINE U-251ObjectivesTo study the antitumour impact of a-Cyano-4-hydroxycinnamic Acid(CHC, MCT inhibitor) in the treat of human astrcytoma cell line U251.Methods1. U-251cell line was cultured.2. Group of experiments：U251cells were divided into control group,low dose group (CHC：5mmol/L) and high dose group (CHC：10mmol/L).3. To detection the capacities of proliferation of U251cells afterinterfered by CHC： MTT was used to detect the cell proliferation abilityof control group, low dose group and high dose groupat24,48,72h. 3. To investigate the expression of MCTs protein in U251cells afterinterfered by CHC: Immunocytochemical technique and Western Blottechnology were used to investigate the expression of MCT1,2,4proteinin control group, low dose group, high dose group at48h.4. To measure the intracellular lactate contect after interfered byCHC: Spectrophotometer was used to measure the intracellular lactatecontect of control group, low dose group and high dose group at48h.5. To observe the U251cell cycle change after interfered by CHC:Flow cytometry screening was used to observe the cell cycle change(G1phase、S phase and G2phase) in control group, low dose group and highdose group at48h.Results1. MTT results showed that the proliferation of the low and high dosegroups significantly were suppressed compared with the control groupwithin24h,48h and72h, the inhibitory effect of the high dose group werestronger than the low dose group in the above three time point, and thestrongest effect at48h.2. The ICC results showed that MCT1was linearly expressed in cellmembrane of the control group, interfered by CHC, the expression ofMCT1was translocated from cell membrane to cell cytoplasma. WesternBlot showed that the expression of MCT1were reduced in low and highdose groups compared with the control group, MCT1expression gradually reduced with the increase of drug concentration, there was a significantdifference in statistical analysis (P<0.05).3. MCT2was mainly distributed in U251cell cytoplasm, comparedwith the control group, the expression of MCT2was decreased in low andhigh dose groups. Western Blot results showed that the expression ofMCT2was reduced in low and high dose groups compared with thecontrol group and statistical analysis showed a significantdifference(P<0.05), there was no significant difference in statisticalanalysis in the comparison between low dose group and high dosegroup(P>0.05).4. MCT4was expression in cell membrane of all groups. WesternBlot results showed that the expression of MCT4was no obvious changein low and high dose groups compared with the control group, there wasno significant difference in statistical analysis(P>0.05).5. Compared with the control group, the expression of intracellularlactate content were significantly enhanced in low and high dose groups,control group (0.189±0.012mmol/L), low dose group (0.412±0.015mmol/L), high dose group (0.895±0.010mmol/L). The variation ofintracellular lactate content had significant statistical difference betweenthree groups (P<0.05).6. Flow cytometry screening results proved that the cells number oflow and high dose group are49.08%,56.21%in G0/G1phase, higher than the control group(35.10%), meanwhile, the cells of low and high dosegroup are41.63%and31.27%, lower than the control group(55.38%).Conclusions1. After application of MCT inhibitor CHC interference U251cells,the expression of MCT1significantly suppressed, and the inhibition effecthas a positive correlation with the drug concentration, the inhibitory effectof MCT2is much less than MCT1, and the expression of MCT4in U251cell could not affected by CHC.2. CHC could increase the production of intracellular lactate, thusaffect the proliferation and growth of astrocytomas.3. CHC could prevent or inhibit mitosis of U251cells, decrease thecells number of S phase, increase the cells of G0/G1phase. U251cellsstopped at G1phase, which can inhibit of the cells proliferation. |
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