Experimental Study Of Inhibitory Effects Of Triethyltin Chloride On Rat C6 Glioma Cells In Vitro And In Vivo

Glioma is one of the commonest malignant tumors among brain tumors.Surgery operation is the primary therapy to glioma. Because of its infiltratinggrowth characterization,glioma is diffcult to be eradicated by surgery operation.Seventy-seven percent of glioma patients will die one year after the surgeryoperation because of the recurrence of glioma. Chemotherapy is the importantsupplement to treat glioma. Drug-resistance of tumor cells and poor drugdelivery through the blood-brain barrier play a relevant role in the lowresponsiveness of glioma to antitumor agents, therefore, the efficacy ofantitumor agents is not satisfied. Searching effective antitumor agents whichcan deliver through the blood-brain barrier easily is the task for our medicalworkers. Triethyltin chloride,which can deliver through blood-brain barriereasily,is neurotoxic drugs, and brain is its target organ.We guess it would bepossible for triethyltin chloride to cure glioma. This study discussed theinhibitory effects of triethyltin chloride on rat C6 glioma cells in vitro and invivo and its mechanisms.1. Experimental study of inhibitory effect of triethyltin chloride on rat C6glioma cells in vitro1.1 Inhibitory effect of triethyltin chloride on the proliferation of culturedrat C6 glioma cellsMTT assay and inverted light microscope were used to detect theinhibitory effect of triethyltin chloride of different concentrations on theproliferation of cultured C6 glioma cells.The results showed the inhibitoryeffect of triethyltin chloride on the proliferation of C6 glioma cells after C6exposed to 0.5μM,1.0μM,2.0μM triethyltin chloride for 24h and the inhibitoryrate increased in a dose-dependent manner.With longer time exposure totriethyltin chloride for 48h, the inhibitory rate increased further and theinhibitory rate were 15.66%, 36.17% and 41.96%,respectively. A570 value wassignificant difference compared with control(p<0.05) and compared with eachother among every two adjacent–dose groups(p<0.01).Cell morphology ofcultures exposed to 0μM(control), 0.5μM, 1.0μM, 2.0μM and 4.0μM triethyltinchloride for 48h was observed under inverted light microscope. C6 glioma cellsin control group showed adherent cells, cells grew densely and exhibitedfusiform or polygon shape.C6 cells sticked to the walls of culture plate firmly.2.0μM triethyltin chloride treated group showed that the shapes of C6 cells wasirregular and cell volume began to reduce.The number of the treated cells wasless than that of control;some round cells were found under light microscope.The number of C6 glioma cells in 0.5μM or 1.0μM triethyltin chloride treatedgroups was between that in control group and 2.0μM triethyltin chloride treatedgroup.The cell volume of C6 treated by 0.5μM or 1.0μM triethyltin chloridebegan to reduce in different degree and some round cells were also seen underlight microscope. The cell number of 4.0μM triethyltin chloride treated groupwas significant lesser than that of control , and C6 cells could not stick to thewalls of culture plate firmly.Most cells died and broke down, and the left cellsbecame round cells which were easily detached from the walls of culture plateand floated in the culture medium.The inhibitory effect of triethyltin chloride onthe proliferation of C6 cells observed under inverted light microscope wascoincidence with that measured by MTT assay. We noticed that the cell volumesof all C6 cells began to reduce as soon as C6 cells exposed to 4.0μM triethyltinchloride , and most cells died and broke down and all the left cells becameround cells just after treatment by 4.0μM triethyltin chloride for 4h.This resultindicated that the dose of 4.0μM triethyltin chloride was so high that it exertedcytotoxic effects on C6 cells and most C6 cells died of necrosis. We couldconclude that the inhibitory rate of 4.0μM triethyltin chloride on theproliferation of C6 cells would be higher than that of 2.0μM triethyltin chloridetreated group from the cell morphological changes.Because of that,we did notmeasure the inhititory rate of 4.0μM triethyltin chloride on the proliferation ofC6 cells.It indicated from the inhibitory rates of triethyltin chloride on the growthof C6 glioam cells measured by MTT assay and cell morphological changesobserved under inverted light microscope that triethyltin chloride could inhibitethe proliferation of cultured C6 glioma cells and inhibitory rate increased in adose-dependent and time-dependent manner.During investigating the inhibitoryeffect of some drugs on the growth of some cells and choosing drug dose,weshould combine the MTT assay and cell morphology observation method .1.2 Mechanisms of inhibitory effect of triethyltin chlorid on theproliferation of cultured rat C6 glioma cells1.2.1 Effect of triethyltin chloride on cell cycle of C6 glioma cellsThree concentrations (0.5μM,1.0μM,2.0μM) of triethyltin chloride wereused to study the changes in cell cycle of C6 glioma cells by FCM.Resultsshowed that the pencentage of C6 glioma cells in G0/G1 phase was increasedsignificantly 48h after exposure to 1.0μM ,2.0μM triethyltin chloride comparedwith control(p<0.05). It indicated that G0/G1 phase arrest could be induced bytriethyltin chloride in C6 glioma cells.1.2.2 Effect of triethyltin chloride on apoptosis of C6 glioma cellsHE staining,electron microscope,fluorescent microscope and DNA agarosegel electrophoresis were used to detect the apoptosis of C6 glioma cells treatedby triethyltin chloride for 48h.Fluorescent TUNEL assay and FCM assay wereused to detect the percentage of apoptosis in C6 glioma cells.Apoptotic changesrevealed cell shrinkage,nuclear pyknosis, chromatin condensation, nuclearfragment and chromatin margination.These changes were seen in part of C6glioma cells stained with HE under light microscope or in part of C6 gliomacells observed by electron microscope.Apoptotic changes,such as chromatincondensation and nuclear fragment, were also seen under fluorescentmicroscope. " DNA ladder " was shown by DNA agarose gel electro-phoresis.These results indicated that triethyltin chloride induced the apoptosisof C6 glioma cells in vitro.Fluorescent TUNEL assay(using ApoAlertTM DNA Fragmentation AssayKit) and FCM assay were used to measure the percentage of apoptosis in C6glioma cells treated by 0.5μM, 1.0μM and 2.0μM triethyltin chloride for48h.The results measured by Fluorescent TUNEL assay showed that thepercentage of apoptosis in various experimental groups were significantlyhigher than that of control group(P<0.01). The same results were also shown byFCM assay.These results indicated that triethyltin chloride could inhibit theproliferation of C6 glioma cells by inducing apoptosis and the percentage ofapoptosis increased in a does-dependent manner.1.2.3 Changes in mRNA level and protein expression of apoptosis-relatedgenesFour genes were studied, including bcl-2, bax, caspase-3 and survivin.RT-PCR assay was employed for the measurement of bcl-2 mRNA and baxmRNA level. Western blot analysis was employed for the measurement of bcl-2and bax protein expression.Immunocytochemistry analysis and Western blotanalysis were employed for the measurement of caspase-3 and survivin proteinexpression.1.2.3.1 Changes in bcl-2 mRNA level and protein expressionResults showed that bcl-2 mRNA level tended to decrease at 3-12h in C6cells treated by triethyltin chloride at 2.0μM.The changes of bcl-2 proteinexpression were the same with that of bcl-2 mRNA expression,that is bcl-2protein expression decreased at 3-12h in C6 cells after treatment by 2.0μMtriethyltin chloride.1.2.3.2 Changes in bax mRNA level and protein expressionResults showed that bax mRNA level tended to increase at 3-12h in C6cells treated by triethyltin chloride at 2.0μM.The changes of bax proteinexpression were the same with that of bax mRNA expression,that is bax proteinexpression increased at 3-12h in C6 cells after treatment by 2.0μM triethyltinchloride.1.2.3.3 Changes in caspase-3 protein expressionThe expression of caspase-3 protein level detected by Immunocyto-chemistry and Western blot analysis showed the same results that the caspase-3protein increased at 3-12h in C6 cells after treatment by 2.0μM triethyltinchloride.1.2.3.4 Changes in survivin protein expressionThe expression of survivin protein level detected by Immunocyto-chemistry and Western blot analysis showed the same results that the survivinprotein decreased at 3-12h in C6 cells after treatment by 2.0μM triethyltinchloride.It is concluded from the experiments in vitro,therefore, triethyltin chloridecould inhibit the proliferation of cultured C6 glioma cells and induceapoptosis .The mechanism of inducing apoptosis was possibly correlative withpromoting the expression of bax mRNA, bax protein and caspase-3 protein andinhibiting the expression of bcl-2 mRNA, bcl-2 protein and survivin protein.2. Experimental study of inhibitory effect of triethyltin chloride on rat C6glioma cells in vivoAfter autotransplanted of C6 cells into Wistar rats,we injected triethyltinchloride into each rat through intraperitoneal route at the dose of 1mg/kg/dayand the injection lasting 4 days. Fourteen days after the C6 cells auto-transplanted into rats, the weight of C6 glioma was measured and theproliferation rate of the tumor was calculated. Results showed that theproliferation rate of treatment group was 33.8% and the inhibitory rate was66.2%. Tumor weight of treatment group was significant lower than that ofcontrol group(P<0.01).Morphological changes of C6 glioma were investigated by HE stainingand electron microscope.The number of C6 cells in treatment group was lesserthan that in control group.Apoptotic changes,such as nuclear pyknosis andnuclear margination,were observed in triethyltin chloride treated group.It is concluded from the experiments in vivo that triethyltin chloride couldinhibite the proliferation of C6 glioma cells in rats.The inhibitory effect oftriethyltin chloride on the proliferation of C6 glioma cells in rats was possiblyinvolved in apoptosis inducement. Whether triethyltin chloride could induceapoptosis of C6 glioma cells in rats needs further testified.This experimental study proved that triethyltin chloride could inhibit theproliferation of cultured C6 glioma cells and induce apoptosis .The mechanismwas possibly correlative with cell cycle arrest and apoptosis inducement byregulating the expression of pro-apoptotic gene and promo-oncogene.Inhibitingthe proliferation of C6 glioma cells in rats by triethyltin chloride was alsoproved in this study.The results of this study will provide us a new idea forfinding anti-glioma drugs and experimental basis for clinical application oftriethyltin chloride.