Tissue Expression Of XIAP In Laryngeal Squamous Carcinomas And The Effect Of XIAP In Cisplatin Induced Apoptosis Of Hep2 Cell

Objective:Laryngeal squamous carcinoma is one of the most common malignant neoplasms in the head and neck region,accounting for 13.9% of all malignant neoplasms in the head and neck region.How to control the carcinogenesis and development of laryngeal squamous carcinoma and chose the efficient drugs for medicateon is still a difficult problem to E.N.T expert.XIAP( X chromosome-linked inhibitor of apoptosis)is important to the carcinogenesis and the development of the malignant neoplasms.But there are few reports obout expression of XIAP in laryngeal squamous carcinomas.Therefore,we investingated tissue expression of XIAP in laryngeal squamous carcinomas and the effect of XIAP in cisplatin induced apoptosis of Hep2 cell by immuneohistochemistry, FCM and RT-PCR.Methods:Paraffin-embedded tissue specimens used for this study were obtained from 50 patients of laryngeal squamous carcinomas,which were resected surgically at Bethune International Peace Hospital and the Fourth Hospital of Hebei Medical University from December 1999 to December 2004.The patients had received neither chemotherapy norradiation therapy before tumor resection.Using immuneohistochemical staining for the paraffin sections(S-P methods),we detected the expression of XIAP protein in 50 laryngeal squamous carcinomas tissues and 8 normal laryngeal tissues,investgate the connection of the XIAP expression with the clinical parameters and the pathological parameters.Hep2 cells were treated with 2,3,4,5ug/ml cisplatin for 24 hours and 3ug/ml cisplatin for 12,24,36,48 hours.Using crystal violet,RT-PCR and FCM,we investingated the rate of the cell apoptosis and the expression of XIAP protein,mRNA at different times and concentrations.Results:The expression of XIAP protein was observed mainly in the cytoplasm and nucleus.The staining color was dark brown.XIAP was expressed masculine in 50 patients of laryngeal squamous carcinomas,about:+16%(8/50),++ 18%(9/50),+++ 40%(20/50), +++ 26%(13/50) in 50 cases of this laryngeal squamous carcinomas.But XIAP expression was about:negative37.5%(3/8),+62.5%(5/8) in normal laryngeal tissue specimens.There is very remarkable difference between thetwogroups(P<0.01).The statistical analysis was performed for the XIAP expression with clinical parameters.lt revealed in laryngeal squamous carcinomas that XIAP expression had no relation with the elements such as age(p = 0.510),sex(p = 0.664),smoking history(p=0.113),tumor site(p=0.276) and lymph node metastases(p=0.106).But there is significant correlation betweenXIAP expression and tumor clinical stage(p<0.05),T stage(p<0.05) and pathological stage(p<0.05).Hep2 was adherent in normal survival condition.After cisplatin treatment,the volume of adherent cells increased,and the suspended cells increased overtimes. The rate of inhibition was remarkably different between 2^ 3^ 4> 5ug/ml cisplatin concentration F=1336.95, P<0.05).Correlation analysis showed r=0.998, P<0.01. The rate of inhibition was also remarkably different too after treating with 3ug/ml cisplatin for 12,24,36,48h ( F=775.22, P<0.05). Correlation analysis show r=0.955, P<0.01.The results showed that the dead cells were gradually increased with increasing concentrations and the times of the effect prolonged.The products of RT-PCR were analyzed on the 1.2% agarose gel.The XIAP cDNA was detected in the position of 802bp and down-regulation with the concentration increased and the time of the effect prolonged. The results showed the levels of XIAP mRNA were dependent on the the concentration and the time of the cisplatin administration.The expression of XIAP protein was significantly different among the effect of 0,2,3,4,5ug/ml concenration cisplatin(F=18.12,P<0.05).Correlation analysis show r=-0.894, P<0.01.And there was remarkable difference before and after the effect of the 3ug/ml cisplatin for 12 , 24 > 36 > 48 hours(F=19.89,P<0.05). Correlation analysis showed r=-0.936, P<0.01.The apoptosis rate measured by FCM:Apoptosis happened in each sample.The rates of apoptosis were remarkably different between the cisplatin concentration of 2,3,4,5ug/ml we selected (F=322.20,P<0.05). Correlation analysis showed r=0.920, P<0.01.And between different effected times,the rates of apoptosis differ significantly too(3ug/ml cisplatin effect for 12,24,36,48 hours). Correlation analysis showed r=0.972, P<0.01.The results demonstrated that the apoptosis was gradually increased with increased the drug concentration increased and the effected time prolonged.Conclusion: 1 .XIAP is expressed highly in laryngeal squamous carcinomas,but lowly in normal laryngeal tissues.The level of XIAP expression is associated with tumor clinicopathologic characteristics in laryngeal squamous carcinomas.With tumor growth and malignancy increasing,the expression of XIAP is up-regulated in laryngeal squamous carcinomas.lt may play an anti-apoptosis role in the pathway of carcinogenesis and development in laryngeal squamous carcinomas.2.Cisplatin can inhibit the growth of Hep2 cells.The inhibition of the growth inceases gradually with the increased drug concentrations and the effected times prolongs.The most important pathway that cisplatin induces cell death is apoptosis.The rate of apoptosis is time- and concentration-dependent. 3.The expression of XIAP protein and mRNA in Hep2 cells is time- and concentration- dependent with cisplatin.The expression of XIAP protein is negative to the rates...