Study On Survivin Antisense Oligonucleotide Combined With Chemotherapeutic Drugs On Induced Apoptosis In Laryngeal Carcinoma Cells

Introduction Laryngeal squamous carcinoma is one of the most common malignantneoplasms in our country. The mechanism of incidence and development oflaryngeal squamous carcinoma is still unclear. At present, there are manytraditional ways of curing laryngeal cancer, such as radiotherapy, chemotherapy,surgical operation, laser therapy and so on. Unfortunately, none of them is idealand all of them have their own limitation .The useness of them is not widelyenough. How to find and chose the efficient anticancer drugs for medication stillis a difficult problem to E.N.T expert. Survivin is a new member in inhibitor ofapoptosis family of proteins (IAPs). This new anti-apoptosis protein has doublefunction of modulate the cell apoptosis and cell cycle. The relationship betweeninhibition of cell apoptosis and incidence of the malignant neoplasms,unsensitivity of radiotherapy and chemotherapy is closely. On the other hand, ithas special expression character that is selectively expressed in the carcinomatissue rather in the terminate-undifferentiated tissue of the adults, which make itthe new target point in the research of the diagnosis and the treatment of themalignant neoplasms. Antisense oligodeoxynucleotide (ASODN) is an artificialsynthesized and decorated sequence that can combine with the target mRNAthrough self-designed special sequence. It also can interfere with the synthesis ofthe protein in the gene level and then, it can inhibit the biological function of thetarget gene. Since ASODN has higher selectivity and less by-product, these drugshave been the hotpoint in the study and the development of new drugs recently. Inour study, we investigated the follow question: 1.the relationship between theexpression of a novel apoptosis inhibitor-survivin in laryngeal squamouscarcinoma and the apoptosis, the parameter of the clinical pathology. 2. the role ofsurvivin on the apoptosis of human laryngeal carcinoma cell line Hep2 induced byDDP. 3. the apoptosis of Hep2 induced by ASODN targeting survivin and theeffect of the survivin on the chemotherapy sensitivity of the laryngeal carcinoma,So as to get information of laryngeal carcinoma and to provide the reference andtheory basis of the investigation on etiology and clinical treatment of laryngealcarcinoma.Material and Methods1. Paraffin-embedded tissue specimens used for this study were obtainedfrom 40 patients of laryngeal squamous carcinomas, who were resectedsurgically at Bethune International Peaceful Hospital from December 1999 toMarch 2004.The patients had received neither chemotherapy nor radiationtherapy before tumor resection. The expression of survivin protein in 40laryngeal carcinoma and 8 normal laryngeal tissue were detected byimmunohistochemical staining. The expression of survivin mRNA was detectedby RT-PCR. In laryngeal carcinoma tissue, we detected apoptosis index (AI)through the situ TUNEL technique.2. Hep2 cells were treated with 2,3,4 or 5μg /ml DDP for 24 hours and 3μg /ml DDP for 12,24,36 or 48 hours Using crystal violet, DNA agarose gelelectrophoresis, RT-PCR and FCM, we investigated the rate of the cellinhibitition, apoptosis and the expressions of survivin protein and mRNA atdifferent times and concentrations. Moreover we observed cell apoptosisconformation by acridine orange staining.3. Hep2 cells were transfected mediated by LipofectamineTM2000 reagent,100, 200, 400 or 600 nmol/L survivin ASODN for 24 hours and 200 nmol/Lsurvivin ASODN for 12,24,36,48 or 60 hours. Using crystal violet, RT-PCR andFCM, we investigated the rate of the cell inhibitition, apoptosis and theexpressions of survivin protein, mRNA and capspase-3 protein at differentconcentrations and times. Cell ultrastructure was observed by electronmicroscope. After 200 nmol/L survivin ASODN transfected into Hep2 cells for24 h, treated the Hep2 cells with 2,3,4 or 5μg /ml DDP or 10,20,30 or 40μg/ml 5-Fu for another 24 h. Using crystal violet FCM we investigated the rateof the cell inhibitition and apoptosis.Results1. The products of RT-PCR were analyzed on the 1.2% agarose gel. Thesurvivin cDNA was detected in the position of 439bp and survivin mRNAexpression in 23 of 40 (57.5%) cases of laryngeal carcinomas. In contrast, itdidn't express in normal laryngeal tissue.The result of survivin proteinexpression was equal to that of mRNA expression by RT-PCR. The statisticalanalysis was performed for survivin protein expression with clinicalparameters.It revealed in laryngeal squamous carcinomas that survivin proteinexpression had no relationship with the elements such as age (P=0.491), sex( P=0.068), smoking history (P=0.3725). But there was significant correlationbetween survivin expression and tumor clinical stage (P <0.05), pathologicalstage (P<0.05) and lymph node metastases. AI of survivin protein positive cases(2.93±1.20) was lower than that of negative cases (4.05±1.42, P<0.05). AI oflaryngeal carcinoma (3.41±1.40) was lower than that of normal laryngeal tissue(23.62±1.22, P<0.01).2. The expressions of survivin protein and mRNA in Hep2 cells waspositive and negative in periodontal ligament cell (PDLC), respectively. Hep2cells were adherent in normal survival condition. After DDP treatment, thevolume of adherent cells increased, and the suspended cells increasedovertimes. The rate of inhibition was remarkably different between 2,3,4 or5μg /ml DDP concentration (F=203.78,P<0.01). The rate of inhibition wasalso remarkably different after treating with 3μg/ml DDP for 12, 24, 36 or 48hours (F=203.78,P <0.01). DDP obviously inhibited laryngeal carcinoma cellline growth with dose-and time-dependent.DDP had inhibition function on the Hep2 cells in all concentration. Thisinhibition function was dependent on the concentration and the time of DDPadministration. Acridine orange staining showed apoptotic changes in addingDDP groups: kelly granule, even the kelly fragment was found in thenucleuous and the cytoplasm, and the morphology of cell was normal incontrol groups. On 24 h and 48 h after adding 3μg/ml DDP, DNA"ladder"was observed for cells by agarose gel electrophoresis, the expressionof survivin mRNA and protein decreased with the increase of DDPconcentration and time elapsing. (respectively, P<0.01). The expression ofsurvivin mRNA and protein was significantly different among the effect of0,2,3,4 or 5 μ g/ml concenration DDP (F=12.31, F=37.96, respectivelyP<0.01). And there was remarkable difference before and after the effect ofthe 3μg/ml DDP for 12,24,36 or 48 hours (F=19.33, F=16.05, respectivelyP<0.01). The expression of survivin protein and mRNA were downregulatedwith the DDP concentration increased and the time of the effect prolonged.The results showed the levels of survivin mRNA and protein were negativelydependent on the the concentration and the time of the DDP administration.3. Under the microscope, we observed that the ASODN transfectedgroups in all concentration had green fluorescence. lipid-ASODN entered thecell 30 min after the transfection. About 80-90% cells were detected greenfluorescence 1 h after tansfection and about more than 90% cells had greenfluorescence 6 h. Survivin ASODN could not inhibite PDLC growth. Wedetected that the transfection rate was up to 94-99%.Aryngeal carcinoma Hep2 cell line growth were obviously inhibitedafter transfected with100, 200, 400 or 600 nmol/L survivin ASODN and therate of inhibition of A-or B-chain was gradually increased with increasingconcentrations and the time of the effect prolonged (F=93.93, F=58.36, P<0.01). Rate of inhibition of ASODN survivin in 600 nml/L was the highest.There was no obvious difference in all kinds of the contral groups. Theinhibition in B-chain was bigger than A-chain (F=4.71, P<0.05). The rate ofcells inhibition chemotherapeutic drugs in ASODN groups (ASODN+5-Fu,ASODN+DDP) were significantly higher than control groups(5-Fu, DDP,respectively, P<0.01).The rate of cells apoptosis increased from 0.70% to 17.98% aftertransfection. The expression of survivin mRNA and protein weredownregulated to 34.33% and 30.67% compare with control groups,respectively. There was no significant change in capspase-3 protein beforeand after transfection. The rate of the cells apoptosis induced by ASODNcombined with DDPgroups was significantly higher than DDP groups.Conclusions1. Survivin is expressed highly in laryngeal squamous carcinomas. Theapoptosis inhibition caused by survivin protein may play a important role inthe origin and progression of laryngeal carcinoma.2. The inhibition of survivin gene expression may play a critical role onthe laryngeal carcinoma cell apoptosis induced by DDP.3. Survivin ASODN can downregulate the expression of the survivingene and this can induce the apotosis of the laryngeal carcinoma cells, then itcan increase the chemotherapy sensitivity of laryngeal carcinoma cells.