| IntroductionApoptosis is a genetically programmed cell death which is regulated by apoptosis correlated genes. It plays an important role to maintain economy homeo-stasis. Recent studies have demonstrated that the process of tumorigenesis at cellular level is related to inhibition of celluar apoptosis and disorder of cell proliferation. With respect to apoptosis , tumorigenesis is correlated with inhibition of apoptosis mechanism, absence of cell death and disability of eliminating aberrant cell . Elevation of tumor cell survival contributes to tumorigenesis. Studies on molecular mechanism of apoptosis suggested that caspase family is a protease which directly results in cellular apoptosis and it is essential for cellular apoptosis mechanism network. Caspase - 3 is a member of caspase family and it is crucial for protease responses . Caspase - 3 is activated by its upstream proteases and directly hydrolyzes several substrates responsible for alteration of cellular biochemistry and morphology . The abnormal expression of caspase - 3 has been well documented in a wide variety of malignancies including breast, lung, liver, prostate, stomach and large intestine tumors. However, there is few investigations reported about the expression of caspase - 3 in esophageal carcinoma and precancerous lesion. To investigate the relationship between caspase - 3 and cellular apoptosis, immunohistochemistry S - P and TUNEL methods were used to detect the expression of caspase - 3 and cellular apoptosis in normal esophageal epithelium, dysplasia, esophageal carcinoma and metastasis lymph node. The purpose is to figure out the role of caspase - 3 in biology significance and relatedmolecular pathology mechanism during the process of esophageal tumorigenesis.Materials and Methods127 tissue samples were collected from the first clinical academy of China Medical University between 1995 and 2004. Specimens were fixed in 10% neutrally buffered formalin and 95% alcohol separately and were embedded in paraffin. Four - micrometer sections prepared from each block. All the specimens were diagnosed by pathologists and stained by hematoxylin - eosin, including 30 cases of normal esophageal epithelium: male 23, female 7; 18 cases of dysplasi-a;male 18, female 0; 79 cases of esophageal carcinoma (41 lymph node - positive and 38 lymph node - negative) : male 66, female 13. The pathological grades were classified as high differentiation in 24, moderate differentiation in 34 and low differentiation in 21. The pathological stages were classified as stageI in 20 patients, II a + IIb in 38, III in 21 and IV in 0 according to the P -TNM classification of UICC. Polyclonal antibody to caspase -3 was purchased from Santa Cruz . UltraSensitiveTM S - P kit and DAB were purchased from Beking zhongshan . In situ cell death detection kit was purchased from Boehring-er Mannheim (German). For immunostainy, streptavidin peroxidase method was used to detect the expression of caspase - 3 ( diluted 1:100) in normal esophageal epithelium , dysplasia , esophageal carcinoma and metastasis lymph node. TUNEL method was used to detect cellular apoptosis. Caspase - 3 staining was brown -yellow particles (DAB) and located in cytoplasm and (or) nucleus. We used an intensity - adjusted scoring system to evaluate immunostaining indices. With respect to the staining cell count ( b) , no positive cells were scored as 0,< 25% were scored as 1,25 -50% were scored as 2, >50% were scored as 3. The staining intensity (a)was graded 0 -3,corresponding to no,weak ,moderately strong, and intense staining, respectively. By multiplying these two factors , an immunoreactive score was obstained. Positive indices was graded 0-4,corresponding to a + b. Integrating grades <2 is negative, ≥ 2 is positive. The cells that contain brown - yellow particles were positive cellular apoptosis was assessed by Apoptotic Indices (AI) , which was obtained through containingpositive cells per 100 tumor cells . The sections were scanned at high — power magnifications covering 10 fields, 100 cells in each field were counted irrespective of immunoreactive status. The statistical methods were according to x test , t - test and correlation analysis.For immunostainy, streptavidin peroxidase method was used. The sections were dewaxed, water processed and then treated by buffer A for 20 minutes to quench the endogenous peroxidase activity. The sections were incubated for 20 minutes with normal non - immuneserum to eliminate nonspecific staining. They were incubated at 4X1 with primary antibody. This was followed by incubation with biotin - labelled condary antibody for 20 minutes and streptavition - peroxidase coplx for 20 minutes. The color was developed with diaminobenzidine and the sections were counterstained with hematoxylin. The blank control were carried out by replacing the primary antibody with PBS. Cellular apoptosis was detected by terminal deoxyribonucleotidyl transferase - mediated dUTP - biotin nick end labeling ( TUNEL ). Briefly, 4 - μm - thick sections were collected from specimens embedded by paraffin, affixed, deparaffinized, and rehydrated. After digestion with 0.25% pepsin in PBS at 37℃ for 30 minutes and extensively washed in PBS, the sections were incubated in 0.3% hydrogen peroxide for 10 minutes. They were incubated in 37℃. with Tunel liquid for 60 minutes in methanol. Then the sections were incubated with peroxidase - labelled condary antibody for 30 minutes in 37 ℃. The color was developed with DAB and the sections were counterstained with hematoxylin.Results1. The expression of caspase — 3 by irrimunohistochemistry The expression of caspase -3 was localized in cytoplasm and (or) nucleo-lus, and brown - yellow granule could be observed. The positive expression rate of caspase - 3 in normal esophageal epithelium, dysplasia, esophageal carcinoma and metastasis lymph node was 93. 33% ,72. 22% ,48. 10% ,46. 34% respectively. The positive expression rate of caspase - 3 in normal esophageal epithelium and dyspalsia were significantly different in statistics ( p < 0. 05 ). Nosignificant differences between esophageal carcinoma and metastasis lymph node ( p > 0.05 ). The expression of caspase - 3 is related to carcinoma differentiation (p <0. 05). The expression of caspase -3 is no correlation with pathological staging , lymph node metastasis, positions and sizes of tumors, as well as ages and gender2. Cellular apoptosis were detected in all tissue samples. The rate of apoptosis in normal esophageal epithelium is significantly lower than dysplasia, e-sophageal carcinoma and metastasis lymph node ( p < 0. 01). No significant differences were observed between dysplasia and esophageal carcinoma(p >0. 05 ). Esophageal carcinoma and metastasis lymph node were significantly different in statistics ( p < 0.05 ). Levels of cellular apoptosis in esophageal carcinoma is related to lymph node metastasis and cell differentiation ( p <0.05 ). However , it is no correlation with staging , positions and sizes of tumors, as well as a-ges and gender( p >0.05 ).3. The relationship analysis between caspase - 3 and esophageal carcinoma cellular apoptosis: Beeline correlation analysis was used to detect caspase - 3 integrating scores, r = 0. 177, p =0. 468. No statistical significance between caspase - 3 and cellular apoptosis of esophageal carcinoma.DiscussionOur studies suggested that the expression of caspase - 3 is down - regulated ,but apoptosis indices have a trend of up — regulation during the malignant process from normal esophageal epithelium , dysplasia to esophageal carcinoma. The results show that there are other factors inducing apoptosis except for caspase —3 during the process of tumorigenesis. Our studies also find that the expression of caspase - 3 has no significant correlation with esophageal carcinoma cellular apoptosis indices, which may result from apoptotic cells being eliminated and result in the limitation of apoptosis indices score. TUNEL method could detect apoptotic cells in advanced stage, but could not value forepart e-vents. Moreover, the results of our research indicates although caspase plays a key role in cellular apoptosis , caspase family is not an exclusive pathway during...
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