Antiapoptosis Gene,Survivin Expression In Laryngeal Squamous Cell Carcinoma And Its Correlation With Bcl-2 And Angiogenesis

IntroductionThe laryngeal squamous cell carcinoma ( LSCC) is one of the familiar malignant tumors in Northern China. Along with the continuous developing of diagnosis and treatment technic, there is obviously progress in the reservation of laryngeal function , the post - treatment living time of the patient has been longer than before. But the therapeutic efficacy of advanced stage cancer is still not as good as what we hoped. In order to improve the therapeutic efficacy, the research a-bout the correlation factors of this disease has become one of the major tasks of early discovery and early treatment. The genesis of malignant tumor is a multiple stage involving many kinds of oncogenes and anti - oncogenes. The reasons had been formerly considered that the cell proliferation was out of control due to the disequilibrium between the cell multiplication and the cell decease. For the past few years, in the wake of developed investigation, a lot of studies show that apoptosis is a equally reason of the tumor pathogenesis with the malignant proliferation and obstructive differentiation. Apoptosis have a significant contribution to the inhibition of cell proliferation, the genesis and growth of tumor. Disequilibrium between cell proliferation and apoptosis induce the tumorigenesis and affect the biological behaviour of tumor. It is considered recently that the" activation of apoptosis is on account of series gene expression and there are many genes take part in this process.Inhibitor of apoptosis proteins (IAPs) is a important family of apoptosis in-hibition. Survivin found recently was a novel inhibitor of apoptosis family members. The distribution of Survivin had a high selectivity. There was no Survivin expression in normal tissues but in a lot of tumors,which was different from another antiapoptosis gene Bel - 2. Survivin expression had correlation with the biological behaviour of tumor. The studies of recent years discovered that the expression of Survivin had a relationship with the tumor angiogenesis.At present, the reports about Survivin expression in laryngeal squamous cell carcinoma were absence and the correlation between its expression and clinical pathology was not clear. There were no reports about the relationship between Survivin expression and angiogenesis of LSCC. RT — PCR method (reverse transcription polymerase chain reaction) was used to detect Survivin mRNA in 64 cases of laryngeal squamous cell carcinoma, 20 cases of benign tumors and 28 cases of laryngeal normal tissues. The expression of Survivin and Bel -2 protein in these tissues was detected by the immunohistochemistry method in order to find the relationship between them. The vascular endothelial cells were marked by CD^to investigate whether there were dependability between Survivin and LSCC angiogenesis base on the microvascular density ( MVD).Materials and methodsMaterials64 samples of LSCC were collected from the department of otolaryngology during March 2003 to May 2004, First Affiliated Hospital China Medical University. The pathologic diagnosis of each disease was LSCC. According to the tumor position, 22 cases of glottic carcinoma and 42 cases of supraglottic carcinoma; According to the differentiating degree,the cases were classified as high - differentiated (19 cases) , moderately differentiated (32 cases) and poorly differentiated(13 cases); According to 1997 UICC stating criteria,7 cases were classified as stage 1,14 cases II ,34 cases III and 9 cases IV; The cervical lymph node metastasis happed in 37 cases and 27 cases were free. None of the patients was received radiotherapy or chemiotherapy before operation. 28 cases of normal tissues beside the malignant tumors and 20 cases of laryngeal benign diseases (2cases of laryngeal papilloma and 18 cases of polyp of vocal cord ) were obtained in the same time.Methods:1 . RT - PCR for Survivin geneTotal RNA firom each sample (contain 50 - lOOmg tissue) were isolated by using Tripure Solution, then the concentration and purity of RNA were detected by DNA/RNA detector. The solution contained 25mM MgCl^yi, 10 x RNA PCR Buffer ljxl, RNase Free dH2O 3.75jxl, lOmM dNTP lui, RNase inhibitor 0.25|xl, AMV reverse tanscriptase 0. 5|xl, Oligo dT - Adaptor Primer 0. 5uJ, RNA 10jxg. Followed by the condition; lOmin at 30t ,30min at 42^ , 5min at 99T:, 5minat5T:.PCR amplification was performed on a PCR thermal cycler. 10 |xl of cDNA mixture was subject to amplification in 40yj solution containing the following; 5 xPCR buffer lOui, lOmM dNTP ljjd, sterile purified water 27. 75 ui, Taq DNA polymerase 0. 25(jd , each of 5 ' and 3 ' primers 0. 5jxl. PCR conditions were initial denaturation for 2 min at 94^ , followed by 30 cycles of denaturation of 94X: for 30sec and annealed at 55^ for 30sec,then extended at 72V for 4min and 4°C for 5min. The efficiency were detected by GAPDH. PCR products were resolved on a 2% agarose gel and the bands were visualized by ethidium bromide staining,then ascertained with the comparison to DL2000 DNA Markers (TaKaRa Co. ). Band Leader Ver. 3.0 software was used to analysis electropho-resis strip, the relative values were obtained by calculating the values between Survivin and GAPDH.2. Immunohistochemistry for Survivin and Bel -2 protein and VECs labled by CD*Immunohistochemical tests were performed by SP method. The SP Kit was produced by Fuzhou Maixin Biological Corp. China. The primary antibodies were polyclonal rabbit anti - human against Survivin, mouse monoclonal and -human against Bel -2,mouse monoclonal anti - human against CD^and all the procedures were followed by the instruction. The negative control was prepared in the PBS, the positive control for staining was obtained from the company. The positive were defined as brown to yellow staining in cytoplasm and cell mem-brane. Semiquantitative integration was adopted to describe the results of Sur-vivin and Bel - 2 proteins. The MVD values were calculated by Weidners method.3. Statistical analysisChi - squared test was used in data of quantity. T - rest or ONE - WAY ANOVA method was used in data of quality. Spearman method was applied for the correlation analysis. The data were analyzed by the SPSS software vl2.0.Results1. The expression of Survivin mRNA: (0. 31 ±0.10) in LSCC, (0.12 ± 0 . 0 5 ) in laryngeal normal tissues and (0. 17 ±0.07) in laryngeal ( P < 0.05).2. The expression of Survivin mRNA: (0. 33 ± 0. 08) in supraglottic carcinoma and(0.27 ±0.12) in glottic carcinoma( P <0.05) ; (0.33 ±0.08) in IH and IV stages and(0.25 ±0.11) in I and II stages( P <0.01) ; (0.34 ±0.07) in the cases with cervical lymph node metastasis and(0.25 ±0.11) in the cases without cervical lymph node metastasis (P =0.001).3. The expression of Survivin mRNA in moderately differentiated LSCC was (0.32 ±0.09) , in poorly differentiated LSCC was (0.33 ±0.10) and in well -differentiated LSCC was (0.26 ±0.11) (P <0.05).4. The expression of Survivin protein; 67. 4% (43/64) in LSCC, 3. 6% ( 1/28 ) in the normal tissues and 1 0 % ( 2/20 ) in the benign diseases ( P < 0.01).5. The expression of Survivin protein: 76.2% (32/42) in supraglottic carcinoma and 50.0% (11/22) in glottic carcinoma (P < 0.05) ; 76.1% (33/43) in I and IV stages and 47.6% (10/21)in I andEstages(P<0.05) ; 81.1% (30/37) in the cases with cervical lymph node metastasis and 48.1% ( 13/27) in the cases without cervical lymph node metastasis (P < 0. 01). 75. 6% (34/ 45) in the moderate and poorly differentiated LSCC and 47.4% (9/19) in well -differentiated LSCC (P < 0.05).6. The expression of Bel -2 protein; 55. 6% (30/64) in LSCC, 7.1%( 2/28 ) in the normal tissues and 15% (3/20) in the benign diseases ( P < 0.01).7. The expression of Bel -2 protein: 67. 6% (25/37) in the cases with cervical lymph node metastasis and 18.5% (5/27) in the cases without cervical lymph node metastasis. (P < 0.01).8. The expression of Survivin protein in LSCC had close relationship with the expression of Bel - 2 protein. Among the Bel - 2 protein expression, the ratio of positive expression of Survivin protein was 80% (24/30) and it was 55. 9% ( 19/34 ) among the negative expression of Bel - 2 protein. ( P < 0. 05 , r = 0.256).9. The positive expression of Survivin protein were 43 cases and 21 cases were negative expression detected by immunohistochemistry technic, The relative value of Survivin mRNA examined by RT - PCR was (0. 36 ± 0. 03 ) and (0.20 ±0.10) respectively in the same time. The positive correlation between theresults( r =0.279).10. The value of MVD in LSCC was (47.31 ±21.73) and it was(4.25 ± 2.80) in normal tissues and(6.80 ±4.54) in benign diseases(P <0.05).11. There was statistical significance between the value of MVD in well -differentiated LSCC (38.47 ± 20. 82) and poorly differentiated LSCC (57. 31 ± 18.10)(P<0.05).1 2. The values of MVD : (56.74 ±17.42)in supraglottic LSCC and (29.32 ±17.52) in glottic LSCC(P <0.01) ; (56. 35 ± 18.09) in III and IV stages and (28.81 ±16.28) in I and II stages(P<0.01) ; (59.54 ±16.13) in the cases with cervical lymph node metastasis and (30.56 ± 16.71) without metastasis (P<0.01).13. The MVD value in the LSCC cases with positive expression of Survivin protein was (57.35 ± 17.59) and it was (26.76 ±13.31) in the negative cases. There was correlation between MVD value and Survivin protein(P <0.01, r = 0.658).