The Actions Of APAF-1 And S100A8 Gene In The Pathogenesis Of Laryngeal Squamous Cell Carcinoma

Objective Part IThe Study of Relationship Between APAF - 1 Gene and Laryngeal Squamous Cell CarcinomaUp - regulation of oncogenes, loss of tumor suppressor genes and apoptosis - associated genes are common events in laryngeal squamous cell carcinoma (LSCC) , which is resistant to chemotherapy and radiotherapy. Apoptosis plays a role in drug - resistance and side - effect of chemotherapy. Apaf - 1 ( apoptotic protease activating factor - 1) is a key factor in cytochrome C - dependent apoptotic pathway. Previous studies showed APAF - 1 gene was less likely to mutate , however, LOH and methylation were the main mechanisms in downregula-tion of APAF - 1 gene. To investigate the effect of APAF - 1 gene in progression of LSCC, the expression, LOH and methylation of APAF - 1 were analyzed in this paper.Materials and methodsMaterials1. Laryngeal squamous carcinomas (LSCCs), benign lesion tissues and their paired adjacent normal laryngeal tissues (PANLs) were from the patients admitted to the First Affiliated Hospital of China Medical University, the 463Hospital of PLA and the General Hospital of Shenyang Military Region. All human tissues were collected following signed patient consent.2. TRIZOI reagent and Taq DNA polymerase, transcription kit and DNA pu-rifaction kit were purchased from Promega company. Apaf - 1 - IgG was purchased from Santa Cruz Company and other reagents from sigma company.MethodsExpression of APAF - 1 mRNATotal RNAs were extracted by TRIZOL reagents, and the first strands of cDNA were synthesized using reverse transcriptase system. PCR primers were designed according to APAF - 1 mRNA sequence by Primer3 and consisted of the following sequences: forward: 5'- TTG CTG CCC TTC TCC ATG AT - 3', reverse:? 5'- TCC CAA CTG AAA CCC AAT GC -3';PCR was performated under the following conditions: first, 94℃ 5min, then 35 cycles of 94℃ 30s, 61℃ 45s,72℃ 60s, and finally 72℃ 10min. RT - PCR pro - duction is separated by 1 1.5% agarose gel and images were taken with AlphaImage 2000 auto - imaging apparatus and analyzed by Fluorchem v2.0 Stand Alone software.2. Expression of Apaf - 1 proteinFor immunohistochemical analysis, 5 mm thick sections were cut from for -malin - fixed paraffin - embedded LSCCs and lryngeal benign tissues specimens. The sections were deparaffinized, rehydrated in a series of 100% , 90% and 70% ethanol, then dripped in 3% hydrogen peroxide solution for 15 min. Then proteins were blocked using normal rabbit serum for 30 min at room temperature. Goat anti - Apaf - 1 polyclonal antibody was applied to the sections at a 1: 150 dilution, and sections were then incubated at 4℃ overnight.3. LOH analysis of APAF -1 geneFive polymorphic loci of D12S327, D12S1657, D12S393, D12S1706 and D12S346 on 12q22 - 23 were amplification by PCR, the productions were elec-trophoresed using a 10% denature PAGE gel, subsequently stained by AgNO3.4. Methylation - specific PCR (MSP) analyses of the APAF - 1 promoterMethylation and unmethylation specific primers were selected based upon the sequences identified by the computer software CpG Island Searcher. Totally1 g DNA was denatured by NaOH and treated with 3M sodium bisulfite, 10 mM hydroquinone for 16 hours at 50℃. The modified DNA,which was purified using Wizard DNA purification system ,was used for MSP assays. The expected PCR products were visualized by 2.0% agarose gel with ethidium bromide staining.Results1. Expression of APAF -1 mRNA in LSCC. Semi - quantitative RT -PCRanalysis of APAF - 1 mRNA showed that 38. 1% of LSCCs(16/42) were down - regulation compared with PANLs.2. Expression of Apaf - 1 protein in LSCC:The results of Immunohisto -chemistry staining showed Apaf - 1 protein decreased in 45. 1% of LSCCs (14/ 31).3. LOH analyses of APAF -1 gene in LSCCs:The LOH frequencies of D12S327, D12S1657, D12S393, D12S1706, D12S346 on 12q22 -23 were 18. 2% ,3.6% ,8. 2% ,22. 2% and 16. 6% in 72 matched samples of LSCCs, respectively,4. Methylation analysis of APAF - 1 gene promoter: MSP analysis displayed that all of 11 LSCCs, with down - regulation of APAF - 1 mRNA, were methylated in promoter regions. In contrast, only 1 of 16 LSCCs without alteration of APAF - 1 mRNA was also methylated, which showed significant difference (P =0.0001).Conclusion1. APAF - 1 gene participated in the pathogenesis of LSCCs.2. Methylation of promoter regions plays an important role in down - regulation of APAF -1 gene in LSCCs.Laryngeal squamous cell carcinoma (LSCCs) is one of the prevalent tumor of head and neck. The incidence of LSCCs is increased in China over past decades. Chronic inflammation, harmful chemical substances and virus involve in the pa - thogenesis of LSCCs. It has reported that oncogenes RAS , c - MYC , EFGR , ERBB -2 and tumor - suppress genes P53 , RB , P16 , FHIT , P21 , cyclin Dl are abnormal expression in LSCCs. In order to explore the profile of genes expre — ssion, cDNA microarray with 506 genes designed by ourselves is performed in different stages of LSCCs. 29 genes up - regulated and 211 genes down - regulated are iden - tified,in which S100A8 ( MRP8 ) is down - regulated in LSCCs and met - astatic lymphocytes compared with their paired adjacent normal laryngeal tissues (PANLs). S100A8 is also known as calgranulin A which is a sub - family of proteins with Ca²⁺ - binding motif, EF - hand in each molecule. Whole length of S100A8 gene is 4195bp with 2 exons, located on 1q21. Studies show it involved in inflamm — ation. It is also suggested that high level of S100A8 protein present in the inflammatory sites , which might lead to delay in tissue repairment and a deleterious effect on the inflamed tissue. However, it is deregulated in many cancers.Recent data have suggested that inflammation is a critical component of tumor progression. Many cancers originate from sites of infection, chronic irritation and inflammation. The incidence of laryngeal tumor is common event resul-ting from chronical inflammatory lesions. The present study is to explore the effect of S100A8 gene on the pathogenesis of LSCCs.Material and methodsMaterialsTumor samples have been described in part I. Hep2 , BGC823 and HEC cells. Anti - MRP8 polyclonal antibody was brought from Santa Cruz, apoptosis detection kit: Anexin V from BaoShai, DeadEnd^TM Fluometric TUNEL System from Promega, Transwell from Coster, Silencer^TM siRNA Construction Kit from Ambion, and Trans - Messenger^TM Transfection Reagent from Qiagen Company.Cell cultureHep2 ,BGC823 and Hec cells were cultured in RPMI 1640 medium contai - ning 10% fetal bovine serum (FBS) , 100 U/ml penicillin, and streptomycin in a humidified atmosphere of 5% CO2 at 37℃.Semiquantitative PCR analysesThe total RNA was isolated from LSCCs, PANLs and Hep2 cells using Tr-iZol reagent. Reverse transcription and PCR analyses were performed by the First Stranded cDNA synthesis kit from Promega. PCR were carried out according to the primers (See Materials). The relative expression level of S100A8 mR-NA was normalized with the β - actin within the same samples.Immunohistochemical analysis (same as part â… )Western blot analysisLSCC tissues and harvested Hep2 Cells were lysed in 1 x SDS sample buffer. Twenty micrograms of cell protein/lane was electrophoresed on a 10% SDS PAGE gel and then transferred to a PVDF membrane. After blocked at room temperature for overnight , the membrane was washed three times and then incubated in blocking buffer containing 1 : 500 dilution of anti - S100A8 antibody at room temperature for 3h. Subsequently, the membrane was incubated with a donkey peroxidase linked antibody against goat IgG (1 :5000) for 1h. .RNA Interference (RNAi) analysisCells were plated in 6 - well tissue culture dishes with RPMI 1640 mediumand incubated for 24 to 48 hours prior to transfection. On the day of transfec-tion, media was replaced with 900μl serum - free and antibiotic - free RPMI 1640. Transient transfections were performed using 90μl of complex with 3ng S100A8 dsRNA at 37℃ for 4 hours. Meantime, cells transfected with liposome served as a control for specificity. Following transfection, the media was removed and replaced with the respective serum containing maintenance media for each cell line. All experiments were repeated at least three times.MTT assays for cell proliferationProliferation of Hep2 cells was measured using the methyl thiazolyl blue tet-razo - lium bromide (MTT) colorimetric dye assay. At each time point, 0. 1ml Hep2 cells suspend (2 × 10⁶cells/ml) were transferred to a 96 - well plate, cultured for 24 hours, then incubated with 0. 3ml MTT dye (1mg/ml in serum -free RPMI1640 media) at 37℃ for 2 hours. Color was developed by the addition of 200μl 0. 04N HCL in isopropyl alcohol monitored at 570 nm.Apoptosis analysisTUNEL assayAs FITC staining, Hep2 cells were cultured in glass slides for 24 hour, then taken out of the culture, washed with PBS once, fixed with 4% paraformal-dehyde (free ethanol) for 25 min at 4℃ , foll - owed by washed with PBS for 3 times and stained with FITC for 10 min at room temperature . Cells were prepared on glass slides and ob — served with a fluorescent microscope. Apoptotic cells were identified on the basis of the presence of highly condensed or fragmented nuclei.Flow cytometer assayTo calculate the percentage of apoptosis, Flow cyto - meter assay were performed according to the recommend of Anexin V kit. Briefly, Hep2 cells, which were cultured in different day after RNAi of S100A8 gene, were diluted to 1 10⁶ cells/ml with PBS, then cells were washed with PBS and centrifugated in 1, 000rpm at 4℃ for 10 min. Subsequence, cells were stained by Anexin V and PI for 15 min and detected by Flow cytometer. The data of apoptotic cells were averaged from three times of experiments use for further analysis.Results1. Expression of S100A8 in LSCCsSemi - quantitative RT - PCR analysis showed that 47. 2% of LSCCs (17/ 36) were up - regulation compared to the PANLs, but only 28. 5% of benign tumors up - regulate (2/7). The results of Western blot analysis displayed that S100A8 protein were up - regulation in 7 of 13 LSCCs. Immunohistochemistry staining results indicated 5100/18 proteins were positively stained in 54.3% of LSCCs, The positive frequencies were 71.4% in well - differentiated, 53. 8% in moderately - differentiated and 12. 5% in poorly - differentiated LSCCs respectively ( P =0.023 ,P =0.14) , which suggested S100A8 involved in the differentiation of LSCCs.2. The effects of S100A8 RNAi on Hep2 cells5100/18 RNAi induced Hep2 cells apoptosis. After RNAi, the rates of ap-optosis in Hep2 cells reached 17. 32% , which enhanced nearly 9 - fold. S100A8 RNAi gene also showed inhibition the invasion of hep2 cells, but has no effect on colony formation of Hep2 and less effects on on Hep2 cells proliferation.3. The mechanism of S100A8 gene involved in LSCCsS100A8 RNAi affected the expression of BCL -2 genes but not APAF -1. Expression of P65 mRNA were up - regulation in 12 of 17 LSCCs whose S100A8 mRNA increased, meantime, expression of S100A8 proteins increase in 6 of 8 LSCCs with also enhancement of p65 protein. Up - regulation of P65 gene coincided with increase of S100A8 gene in LSCCs which indicated 5100/18 gene related to P65 . P65 gene by RNAi showed enhanced cells apoptosis up to 15. 1% , increased nearly 7 - fold. S100A8 RNAi showed that it had no effects on the expression of P65 , RELB , IKKα. and IKKβ genes, but P65 RNAi showed down -regulation of S100A8 mRNA.Conclution1. We represent the first report that S100A8 gene involves in the pathogene-...