[Expression and mutation of Fas-associated death domain (FADD) gene in non-small cell lung cancer].

BACKGROUND & OBJECTIVE: Abnormal expression of Fas-associated death domain (FADD) protein, an important adapter in cell apoptosis signal conduction, may closely relate with tumorigenesis. This study was to detect expression and mutation of FADD gene in non-small cell lung cancer (NSCLC), evaluate its effect on development of NSCLC, and explore the mechanism. METHODS: Polymerase chain reaction and single-strand conformation polymorphism (PCR-SSCP) was used to detect FADD gene mutation in 62 specimens of NSCLC tissues and 13 specimens of adjacent non-cancerous lung tissues. Immunohistochemistry was used to detect its protein expression. In situ hybridization (ISH) was used to detect FADD mRNA expression in 30 of the 62 specimens of NSCLC tissues. Terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick labeling (TUNEL) was used to detect apoptotic cells in NSCLC tissues. RESULTS: Of the 62 specimens of NSCLC tissues, 4 cases of stage N2 showed FADD gene mutation. Positive rate of FADD protein in NSCLC tissues was 80.6% (50/62), its protein level positively correlated with differentiation of NSCLC (rs=0.411, P<0.01). Protein level of FADD in NSCLC tissue was significantly higher than that in non-cancerous tissue (P<0.05). Positive rate of FADD mRNA in NSCLC tissue was 80.0%, its concordant rate with positive rate of FADD protein was 88.6% (P>0.05). Apoptotic cells were observed in all specimens of NSCLC, apoptosis indexes of the 4 cases with FADD gene mutation were lower than the mean level, although they showed positive expression of FADD protein. Protein level of FADD was positively related with cell apoptosis of NSCLC (rs=0.599, P<0.001). CONCLUSIONS: FADD gene mutation exists in NSCLC, its mutation and abnormal expression might play a crucial role in carcinogenesis of NSCLC. Protein level of FADD closely correlates with cell apoptosis of NSCLC.

Expression levels and significance of hypoxia inducible factor-1 alpha and vascular endothelial growth factor in human colorectal adenocarcinoma.

BACKGROUND: Hypoxia-inducible factor 1 (HIF-1), a transcription factor, is overexpressed in common human cancers and their metastases. This study aimed at determining the expression levels of HIF-1alpha and vascular endothelial growth factor (VEGF) in SW480 cells and in colorectal adenocarcinoma tissue and ascertaining whether HIF-1alpha and VEGF play important roles in tumor angiogenesis. METHODS: HIF-1alpha mRNA expression was analyzed using in situ hybridization and RT-PCR. HIF-1alpha and VEGF protein were detected in SW480 cells and colorectal adenomas and adenocarcinomas by immunohistochemistry using streptavidin/peroxidase (SP). Western blot was used to detect HIF-1alpha protein extracted from SW480 cells. Microvessel density (MVD) in colorectal carcinomas was determined by anti-CD34 immunostaining in colorectal carcinomas. RESULTS: Optical density values representing HIF-1alpha mRNA expression levels were found to be significantly higher in SW480 cells in hypoxic conditions than in cells under normoxic conditions (P < 0.05) or in hypoxic conditions but treated with genistein (P < 0.05). The levels of HIF-1alpha and VEGF protein expression in SW480 cells were significantly higher in the hypoxia group than in the normoxia group (P < 0.01, P < 0.05, respectively) and hypoxia/genistein group (P < 0.01, P < 0.05, respectively). The positive expression rates of HIF-1alpha mRNA changed dramatically when comparing colorectal adenomas with adenocarcinomas of different Dukes' stages (P < 0.05). HIF-1alpha mRNA was also expressed at higher levels in adenocarcinomas than that in adenomas (P < 0.01). HIF-1alpha protein expression correlated significantly with VEGF protein expression and MVD. CONCLUSIONS: Hypoxia induces the expression of HIF-1alpha and VEGF in colorectal adenocarcinomas. HIF-1alpha may play an important role in angiogenesis and tumor progression by regulating the expression of VEGF in human colorectal carcinomas.

[Expression and pathobiological implication of hypoxia-inducible factor-1alpha in human colorectal carcinoma].

OBJECTIVE: To investigate the transcription level and protein expression of HIF-1alpha and VEGF in SW480 cell line and colorectal adenocarcinoma, and to determine whether HIF-1alpha plays a role in angiogenesis through its regulation of VEGF. METHODS: HIF-1alpha mRNA expression was analyzed by in situ hybridization. HIF-1alpha and VEGF protein expressions were determined by immunochemical streptavidin/peroxidase (SP) in SW480 cells and colorectal carcinoma tissue samples and Western blot, using proteins extracted from SW480 cells. Tumor tissue microvessel density (MVD) was determined by CD34 immunostaining of colorectal carcinomas. RESULTS: The levels of HIF-1alpha mRNA changed significantly in response to different oxygen concentrations and an addition of genistein in SW480 cells. Immunocytochemistry revealed that the levels of HIF-1alpha, VEGF protein expression in SW480 cells were significantly higher under hypoxia than those in nomoxia (P < 0.01, P < 0.05 respectively). However, addition of genistein, an inhibitor of HIF-1alpha, suppressed such responses to hypoxia. Western blot analysis showed that SW480 cells exposed to hypoxia expressed a high level of HIF-1alpha protein, compared to a weak expression in nomoxia. The addition of genistein in hypoxia suppressed the over-expression of HIF-1alpha. The positive rates of HIF-1alpha mRNA by in situ hybridization in colorectal adenomas and adenocarcinomas were 38.9% (7/18) and 67.7% (42/62), respectively. The percentage of HIF-1alpha mRNA positive cells varied significantly from colorectal adenomas to adenocarcinomas at different Duke stages (P < 0.05), and HIF-1alpha mRNA was higher in adenocarcinomas than in adenomas (P < 0.01). The positive rates of HIF-1alpha and VEGF protein expression in adenocarcinomas were 43.5% (27/62) and 37.1% (23/62), respectively. The expression of VEGF elevated as the Duke tumor staging increased. The conformation rate of HIF-1alpha and VEGF was 74.2% (46/62). MVD was significantly higher in HIF-1alpha and/or VEGF positive tumors than those without (P < 0.01 and P < 0.05 respectively). Among the four groups, i.e. HIF-1alpha+/VEGF+, HIF-1alpha+/VEGF-, HIF-1alpha+/VEGF- and HIF-1alpha-/VEGF-, the difference of MVD was highly significant (P < 0.01). HIF-1alpha expression was correlated significantly with VEGF expression and microvessel density. CONCLUSIONS: These findings suggest hypoxia induces the expression of HIF-1alpha and VEGF in colorectal adenocarcinoma. HIF-1alpha may play an important role in angiogenesis and tumor progression by regulating the expression of VEGF in human colorectal carcinoma.

[Relationship of MAGE-A1 expression with Ki-67 and tumor-infiltrating lymphocyte response in non-small cell lung carcinoma].

BACKGROUND & OBJECTIVE: Recent studies revealed a possible close association between the expression of some members of tumor-specific antigen MAGE (melanoma antigen) family and actively proliferated infantile cells. But the correlation of MAGE-A1 expression with proliferation of tumor cells and immune response at host local site has not been reported to date. Our study was to investigate the expression of MAGE-A1 in non-small cell lung carcinoma (NSCLC), and its relationship with Ki-67 expression, tumor-infiltrating lymphocyte (TIL) response, histologic grade, and pathological type. METHODS: Thirty NSCLC samples in formalin-fixed, paraffin-embedded sections were examined for MAGE-A1, Ki-67 and TIL response using SP immunohistochemical technique. RESULTS: The positive expression rate of MAGE-A1 was 80.00%(24/30) with high expression rate of 58.33%(14/24) and low expression rate of 41.67%(10/24). The positive expression rate of Ki-67 was 93.33%(28/30) with high expression rate of 57.14%(16/28) and low expression rate of 42.86% (12/28). TIL response was observed in 22 patients. There was a significant relationship between MAGE-A1 positive expression and Ki-67 positive expression (rs=0.578, P< 0.005), as well as between MAGE-A1 positive expression and TIL response (rs=0.505, P< 0.005). However, MAGE-A1 expression was not significantly correlated with histologic grade and pathological type (P >0.05). CONCLUSION: The NSCLC cells with MAGE-A1 positive expression possess high proliferation activity; meanwhile, the up-regulation of MAGE-A1 indicates the increase of antigen in tumor cells and the increase of local TIL response, indicating that MAGE-A1 may have potential to be used as a target for immunotherapy in NSCLC patient.

[Relationship between hMSH2 and FHIT gene expression in non-small cell lung cancer].

BACKGROUND & OBJECTIVE: Abnormality of FHIT gene has been proved to be frequent in certain malignant tumors closely related to environmental oncogenic factors, such as lung cancer. Foreign scholars have begun to explore the relationship between FHIT gene and other tumor suppressor genes, which are implicated in the pathogenesis of lung cancer. This study was designed to investigate the relationship between hMSH(2) and FHIT protein expression and to explore the correlation of hMSH(2) and FHIT protein expression with clinicopathologic features of lung cancer. METHODS: Immunohistochemical analysis of hMSH(2) and FHIT protein expression in 40 lung cancer cases and 15 adjacent non-cancer lung tissues was performed; the positive rates of FHIT and hMSH(2) proteins were measured by image analysis system. RESULTS: (1)The positive rates of FHIT and hMSH(2) proteins were 58.2% and 45.8% respectively in lung cancer tissues compared with 89.1% and 65.3% in non-cancer lung tissues. The expression levels of FHIT and hMSH(2) proteins were significantly lower in lung cancer tissues than that in non-cancer lung tissues (P< 0.01). (2)Reduced expression levels of both proteins were significantly related to tumor histology. The positive rate of the FHIT protein was 52.2% in squamous cell carcinoma compared with 63.4% in adenocarcinomas(P< 0.01), whereas the positive rate of the hMSH(2) protein was 35.6% in adenocarcinomas compared with 53.2% in squamous cell carcinoma(P< 0.01). (3)A correlation between FHIT reduced expression and lymph node metastasis was observed(P< 0.01). The positive rate of the FHIT protein was 54.1% in lung cancer tissues with metastasis compared with 60.5% in lung cancer tissues without metastasis. No association was found between hMSH(2) reduced expression and nodal metastasis(P >0.05). (4)Loss of FHIT protein correlated significantly with lasting and heavy smoking(P< 0.01). The positive rate of the FHIT protein was 53.1% in smoking group compared with 66.1% in non-smoking group. The reduction of hMSH(2) expression was not associated with smoking(P >0.05). (5)An inverse correlation was found between hMSH(2) reduced expression and FHIT protein loss (P< 0.01, RR=-0.54). CONCLUSION: FHIT gene may be a negative regulatory gene of hMSH(2) gene, and play an important role in the inactivation mechanism of hMSH(2) gene.

[Expression of apoptosis and proliferation related genes in carcinogenesis of lung squamous cell carcinoma in rats].

BACKGROUND & OBJECTIVE: The activation of caspase-3 protein, located the downstream of apoptosis, is the key of cell apoptosis signal conduction. Caspase-3 is the most important performer in accelerating apoptosis in the caspase family. Much progress has been achieved in the study of caspase-3 and human non-small cell lung cancer. This study was designed to investigate the effect of cellular proliferation and apoptosis related genes caspase-3 and proliferating cell nuclear antigen (PCNA), and to seek whether they could be chosen as molecular biology markers for lung cancer. METHODS: 3-Methylcholanthrene (MCA) and diethyinitrosamine (DEN) were used to induce lung squamous cell carcinoma by intra-left lobar-bronchial instillation in 50 Wistar rats. The other 10 rats instilled with iodized oil were regarded as control group. The expression of caspase-3 and PCNA were evaluated by SP immunohistochemistry during carcinogenesis. TUNEL(TDT-mediated dUTP nick end labeling) method was used to examine apoptotic cells. RESULTS: For the rat bronchial epithelium cells in control group, precancerous lesions, and lung squamous cell carcinoma, positive coefficient values of caspase-3 protein were 3.10+/-0.99, 2.25+/-1.13, and 1.38+/-0.95 on average, respectively; the means of PCNA-labeling index (PCNA-LI)were 14.10+/-5.02, 28.13+/-8.72, and 41.88+/-14.24, respectively; the means of apoptotic index(AI) were 0.60+/-0.52, 2.06+/-0.85, and 2.26+/-1.14, respectively.Significant differences in caspase-3 protein expression were observed between bronchial epithelium in control group and lung squamous cell carcinoma (P< 0.01). Caspase-3 expression was showed stronger in precancerous lesions than that in lung cancer (P< 0.05). Low proliferation index and low AI were detected in rat bronchial epithelium region in control group,which were significant differences in precancerous lesions and lung cancer, respectively (P< 0.01). In 34 rats with lung squamous cell carcinoma, there was negative relationship between caspase-3 and PCNA-LI (r=-0.7306, P< 0.01), so did it in AI and PCNA-LI(r=-0.8127,P< 0.01), but there was no relationship between caspase-3 expression and AI(P >0.05). CONCLUSION: Loss of caspase-3 expression may be associated with the development of lung squamous cell carcinoma in Wistar rats, but it is not associated with AI. PCNA-LI is an important marker for malignant progression of lung cancer.

[Expression of HIF-1 alpha and its relationship to apoptosis and proliferation in lung cancer].

BACKGROUND & OBJECTIVES: Hipoxia-inducible factor-1 is a transcriptive factor that regulates genes involved in metabolism, angiogenesis, proliferation, and apoptosis. This study was designed to investigate the expression of hypoxia inducible facter-1 alpha(HIF-1 alpha) and its relationship to bcl-2, Bax, PCNA in lung cancer. METHOD: Immunohistochemical streptavidin/peroxidase(SP) was used to examine the expression of HIF-1a, bcl-2, Bax, and PCNA in 60 cases of lung cancer. RESULTS: In 60 cases of lung cancer, positive rate for HIF-1a was 28.3% (17/60), specially the positive rate of small cell lung cancer(66.7%) was significantly higher than non-small cell lung cancer (21.6%). HIF-1a expression increased as clinical stage and metastasis increased(P < 0.01). The positive rate of bcl-2, Bax, and PCNA were 31.7% (19/60), 40.0% (24/60), 76.7% (46/60), respectively. Inverse relationship was found between the expression of HIF-1 alpha and bcl-2; while the correlation of HIF-1 alpha and Bax was positive(P < 0.01). The relationship between HIF-1 alpha and Bax was positive(P < 0.01). The relationship between HIF-1 alpha and PCNA was not observed(P > 0.05). CONCLUSION: HIF-1 alpha is correlated with apopotosis, but has no relationship with proliferation.