The Study Of Apoptosis Induced By All-Trans Retinoic Acid In Rat C6 Glioma.

Objective and backgrounds: Glioma is the most common malignant braintumor and the outcome is very bad. The median survival for patients doesn'timprove significently in twenty years,despite treatments that include surgicalresection, radiotherapy and cytotoxic chemotherapy. The serious toxity oftraditional radiation and chemprevention restricts the dosage and therapy time.Thus it is the most challenging and emergengcy question to explore effective andsafe drugs in order to length tumor relapse and improve prognosis.One of the mostimportant anti-cancer mechanisms of all-trans retinoic acid is believed to be theinduction of tumor cell's apoptosis. Apoptosis is the cell programmed death,which is regulated by multiple genes. It has been reported that all-trans retinoicacid can induce many tumors apoptosis in vitro. It is not reported that retinoic acidcan induce rat C6 glioma apoptosis.Although so much research work has beendone,ATRA's antineoplastic pathway, target point and its influence on genes arestill remained unknown,especially its molecule mechanism. Those are what wewant to emphasize in our study. Methods: MTT assay was used to observe the effect of ATRA on the growthof the cell line C6 with 1,2,4,8,16 μmol/L concentrations for 24h,48h,72hin vitro and the inhibition rate was examined. Flow cytometry was used todetermine the cell cycle distribution and the rate of apoptosis. Observation withtrasmission electron microscope showed the variation of ultramicroscopicstructure of C6 cells when given by 4 μmol/L ATRA at different time duration(24h,48h,72h). RT-PCR was used to detect expression of cyclin E mRNA.RT-PCR and Western-blot analysis were performed to analyze the expressions ofcaspase-3 mRNA and proteins in rat C6 glioma cells respectively.C6 glioma cellsfor each male rat were stereotactically implanted into the right caudate nuclei ofWistar rats. ATRA(2 and 4 mg/kg bodyweight) was used in the treatment of theimplanted tumors formed by C6 cells. For evaluation of the effects of ATRA onthe tumors, The size of tumor was measured and morphology was observed byTEM. RT-PCR for cyclin E,caspase-3 mRNA was performed on the tumortissues. Western-blot was used to detect the expression of caspase-3 proteins.Results: MTT showed that ATRA inhibited C6 cells with dose-dependenceand time-dependence. The OD value and the inhibition rate had a remarkabledifference between ATRA experimental groups and control group (P<0.01). IC50was 9.32±0.13 μmol/L,5.23±0.15 μmol/L,2.43±0.03 μmol/L in 24h,48h,72h respectively. For 48h, the growth of cells was inhibited remarkably. Its ODvalue was 0.602±0.009, while its inhibition rate was 40.44±1.51%. If the timewas prolonged to 72h, the two value would be 0.339±0.011 and 66.8±2.91%.The normal figure of C6 cell under microscope was a fusiform one, whichmultiplicated rapidly. Their multiplicate rate will be lowered after given by ATRA,Their density reduced, some of the cells were round, died from cytoderms. Theproportion increased as the effection time of ATRA grew. In flow cytometryanalysis C6 cells arrested in G1 phase from 42.18±2.52% to 65.44±4.05%(P<0.01);The rate of S,G2 phase was declined from 43.12±3.01% to 20.75±1.51% (P<0.01), 18.59±2.02% to 11.2±2.50% (P<0.05) respectively;The rate ofapoptosis was increased from 3.55±1.60% to 45.49±2.03% (P<0.01). a typicalsubdiploid peak was detected in DNA frequency distribution histograms.TEMshowed that untreated cells generally had abnormal nucleus, whose endoplasmwas not so rich, scant rough endoplasmic reticulum , numerous polyribosomes,and had a great deal of microrill. By contrast, the C6 cells apoptosised and we cansee phenomenon of this, such as condensation of nuclei and margination ofnuclear chromatin in cells of the ATRA experimental group in 72h. RT-PCRshowed ATRA can obviously down-regulate the expression of cyclin E mRNAand up-regulate the expression of caspase-3 mRNA: The expression of cyclin EmRNA was 0.815±0.062,0.534±0.050,0.280±0.032,0.159±0.015 in controlgroup,ATRA experimental groups of 24h,48h,72h respectively and there wasa statistically significant difference among different groups (P<0.01). Theexpression of caspase-3 mRNA was 0.151±0.013,0.432±0.057,0.698±0.025,0.815±0.064 in control group, ATRA experimental groups of 24h, 48h, 72hrespectively and there was a statistically significant difference between controlgroup and experimental groups(P<0.01). Westorn blot results indicatedexperimental groups increased the expression of caspase-3 active proteins (17KD,12KD) in rat C6 glioma cells in contrast to control group. The expression ofcaspase-3 proenzyme was 547494±131351,3057878±145420,3431024±109280 , 2984956 ± 103658 respectively. There was statistically significantbetween control group and experimental groups (P<0.05) . There was nostatistically significant difference between 24h and 48h ,72h (P>0.05)and therewas statistically significant difference between 48h and 72h (P<0.05).Theexpression of 17KD active-caspase-3 was 3047924±137831,3838953±819573,4836716 ±135564 in 24h , 48h, 72h respectively. There was statisticallysignificant difference between 24h and 72h (P<0.05) and there was no statisticallysignificant difference between 48h and 24h,72h (P>0.05).The expression of12KD active-caspase-3 was 2583224±476516,3251944±121801,4177306±383389 in 24h,48h,72h respectively. There was statistically significant differencebetween 24h and 72h (P<0.05) and there was no statistically significant differencebetween 48h and 24h,72h (P>0.05).C6 glioma mode was constructed in Wistarrats successfully. Most of their food intake and water intake were at some degreereduced,their reactive ability was inhibited, convulsive seizure,and then walkedunsteadily, their hair lost brightness.The rate of success of C6 cells gliomainoculatiny to 30 rats was 96.7%. At 6th day after inoculation, we can see tumorsunder the help of MRI. The implanted tumors were smaller in sizes inATRA-treated groups than in control group. The inhibition rates of ATRA-treatedgroup(1) and ATRA-treated group(2) were 89.2% and 77.2% respectively. TEMshowed that control group had unregular nucleus, nucleoles apparent.The structureof neurofilament could be seen in endoplasm, also there existed glycogen grannles.The variation of apoptosis could be seen easily in ATRA treated groups such askaryopyknosis and chromatin margination. Apoptosis number in ATRA-treatedgroup(2) was more than that in ATRA-treated group(1). The expression of cyclinE mRNA was down-regulated and that of caspase-3 mRNA was up-regulated: Theexpression of cyclin E mRNA was 0.816±0.141,0.221±0.036,0.144±0.021(n=9) in control group,ATRA-treated group (1), ATRA-treated group(2)respectively and there was a remarkable difference in different groups (P<0.01).The expression of caspase-3 mRNA was 0.155±0.032,0.676±0.074,0.816±0.121 (n=9) in control group,ATRA-treated group (1),ATRA-treated group(2)respectively and there was a remarkable difference in different groups (P<0.01).Westorn blot results indicated there appeared active fragment expression ofcaspase-3 proteins in ATRA-treated groups in contrast to control group. Theexpression of caspase-3 proenzyme was 779038±66580,3513553±100974,3112862±102607(n=9)respectively. There was statistically significant differenceamong different groups (P<0.05). The expression of 17KD active-caspase-3 was1222437±85875,2019649±133810(n=9)between ATRA-treated group(1) andATRA-treated group(2) respectively. There was statistically significant differencein two groups (P<0.05). The expression of 12KD active-caspase-3 was 1555585±100521,1942463±121822(n=9)in ATRA-treated group(1) and ATRA-treatedgroup(2) respectively. There was statistically significant difference in two groups(P<0.05).Conclusions: ATRA inhibits the growth of C6 cells with time-dependenceand dose-dependence.ATRA can induce apoptosis of C6 glioma cells.C6 cellsarrest in G1 phase and the cells of S,G2 phase decline.The rate of apoptosisincreases and cells appear typical subdiploid peak with time-dependence. TEMshows C6 cells the morphology of apoptosis. The mechanism that ATRA inhibitsC6 glioma may be to induce apoptosis,to alter relevant cell cycle protein andapoptosis relevant gene expression.ATRA may remarkably increase theexpression of caspase-3 mRNA and active protein fragment,decrease theexpression of cyclin E mRNA.We treat the Wistar rats with different dosage ofATRA and find that the implanted tumors are smaller in treated groups than incontrol group with dose-dependence.