| Objective: Esophageal Carcinoma is highly prevalent in China, the mortality due to esophageal cancer are approximately 150,000 people per year, accounting for nearly a quarter of mortality caused by cancer. The mortality rate of esophageal cancer is 31 fold higher in Henan Province than Yunnan Province(1,050,000/100,000), It indicates the incidence of esophageal cancer varies greatly with location. In recent years environmental carcinogene(such as mycotoxins and viruses, etc.) has become one of the hot theses in the development of various types of cancer. Relationship between human papillomavirus(HPV) and esophageal cancer was first proposed by Syrjanen in 1982, his research found that the histological changes of 40%(24/60) esophageal cancer is very similar to the change of genital tract warts, and then he and his colleagues discovered the structure protein of HPV in esophageal squamous cell carcinomas(ESCC) by immunohistochemistry. The role of HPV as an important factor in the development of cervical cancer has been confirmed. Some scholars have found oral squamous cell carcinomas(OSCC) and ESCC also have relationship with the infection of HPV. In 2008, WHO identified there is a direct relationship between HPV status and OSCC. However, the relationship between HPV and ESCC remain to be elucidated. Fas belongs to the tumor necrosis factor and nerve growth factor receptor family, after Fas binding to its corresponding ligand Fas L(CD95L), Fas receptor are trimerization and activation, activated receptor bind with FADD, it interacts with caspase-8 and activates the latter, form the death inducing signaling complex(DISC),that activates a series of Caspase-1, 3, 7, etc, Wherein Caspase-3 cleaves a substrate for PARP, the final step in the regulation of apoptosis execution. FADD as a crucial protein in Fas pathway can trigger apoptosis cascade. Recent studies show that human colon adenocarcinoma cell line HCT116 has high expression of HPV16 E6 protein, can influence apoptosis pathway mediated by TRAIL, causes cells to escape apoptosis. The study also found that E6 can induce the ubiquitination of FADD and caspase-8, to inhibit apoptosis by inhibiting the activation of the Fas signaling pathway. In preliminary experiments we have mastered the infection of HPV can decrease the expression of Fas protein in ESCC. In order to further clarify the role of HPV in the development and progression of ESCC, we first observe the expression of caspase-8 and FADD protein of ESCC in Hebei Province, and then analyze the relationship between HPV status and the expression of FADD and caspase-8 protein. Secondly, we transfect normal esophageal epithelium and well-differentiated ESCC cell lines with HPV16 E6 to investigate whether overexpression of E6 has an effect on Fas/Fas L apoptosis pathway(Fas/Fas L, FADD, caspase-8, et al.). To investigate the effect of HPV status on associated protein of Fas-mediated apoptosis pathway from the overall and cellular level, help us further clarify the relationship between the infection of HPV and tumorigenesis in ESCC, then clarify the mechanism from the perspective of apoptosis pathway. That provides new ideas and perspectives for the study of the mechanism of esophageal cancer, also provides a scientific basis for the clinical treatment, prognosis and prevention of esophageal cancer.Methods: 1 Specimen Collection This study included 159 patients with ESCC, which were consecutive cases seen at NO.2 Hospital of Hebei Medical University, China between 2009.01 and 2015.12 who had histopathologically confirmed primary ESCC. Patients who had primary tumors outside of the esophagus, tumors of unknown origin, or any histopathologic diagnosis other than ESCC were excluded. Clinicopathological Features of 159 ESCCs include: age, gender, lymph node metastasis and histological differentiation.2 DNA isolation and direct sequencing DNA was extracted from formalin fixed paraffin embedded(FFPE) samples. HPV was analyzed by PCR, which was used for the amplification of the HPV L1(MY09/MY11) gene in all samples, with proper negative and positive PCR controls in both amplifications. PCR was used to amplify the housekeeping gene β-actin(200 bp) to assess the presence of PCR inhibitors in the samples used in this study. HPV positive controls were obtained from cervix tumor FFPE samples, which had been ensured HPV infection. HPV negative controls were dd H2 O instead of HPV L1 primers in PCR. 3 Observe the expression of FADD and caspase-8 in ESCC by immunohistochemistry. The expression of FADD and caspase-8 protein was investigated by SP immunohistochemical staining method. FADD and caspase-8 protein positive reaction mainly located in cell membrane。 4 EX-GS3066-M90(HPV16 E6), EX-NEG-M90(negative control) plasmid purification and concentration detection. 5 Vitro Het-1A(human normal esophageal epithelial cells) and Eca-109(human well-differentiated squamous cell carcinoma) cells were transfected with HPV16 E6 plasmid. Het-1A and Eca-109 cells were transfected with HPV16 E6 plasmid. RT-PCR and Western blot method were used to detect the proteins associated with Fas/Fas L apoptotic pathway in Het-1A and Eca-109 cells. Analyze the mechanisms of Fas/Fas L apoptotic pathway affected by HPV16 E6. In order to further clarify the relationship between the role of HPV16 E6 protein and proteins associated with Fas-mediated apoptosis pathway in Het-1A cells, we collected cells at 12 h, 24 h and 48 h after transfection. Observe the changes of Fas/Fas L, FADD and caspase-8 expression. 6 Statistics All statistical analyses were performed using SPSS19.0. Count data using χ2 test analysis, P<0.05 was considered statistically significance. Measurement data were presented as mean ± standard deviation( (?)±s), P<0.05 was considered statistically significance in one-way ANOVA.Results: 1.1 Results of clinical pathological morphology in ESCC The patients of ESCC was 159 cases, the median age of the patients was 62 years(range, 27-87 years). Male patients were 124 cases, female patients were 35 cases, and the male: female ratio was 3.54:1. Pathological morphology: 47 cases were lymph node metastasis. Well-differentiated ESCC of the patients were 39 cases, moderate-differentiated ESCC of the patients were 72 cases, and poor-differentiated ESCC of the patients were 48 cases. There was no statistically significant difference between the lymph node metastasis and differentiated in ESCC(P>0.05). 1.2 Relationship between the HPV status with clinical pathological features in ESCC In 159 cases ESCC tissues, the HPV detection rate was 32.1%. Patients were divided into two groups according to the tumor HPV status. In the HPV-positive group, moderate/well-differentiated ESCC accounted for 56.9%, significantly lower than that in HPV-negative group was 75.9%, poor-differentiated ESCC accounted for 43.1%, significantly higher than that in HPV-negative group was 24.1%(P<0.05). There were no statistically significant differences between the HPV status and respect to age, gender and lymph node metastasis(P>0.05). 2 Immunohistochemistry 2.1 The expression of FADD and caspase-8 in ESCC In 159 cases ESCC tissues, the positive expression of FADD and caspase-8 were 81 cases and 93 cases, the negative expression were 78 cases and 66 cases. In 159 cases adjacent tissues, the positive expression of FADD and caspase-8 were 150 cases and 131 cases, the negative expression were 9 cases and 28 cases. The positive expression rate of FADD and caspase-8 respectively were 50.9% and 58.5% in ESCC tissues, significantly lower than those in adjacent tissues were 94.3% and 82.4%(P<0.05). 2.2 Relationship between expression of FADD, caspase-8 proteins and HPV in ESCC In the HPV-positive group, the positive expression of FADD and caspase-8 were 19 cases and 24 cases, the negative expression were 32 cases and 27 cases. In the HPV-negative group, the positive expression of FADD and caspase-8 were 62 cases and 69 cases, the negative expression were 46 cases and 39 cases. The positive expression rate of FADD and caspase-8 in HPV- positive group respectively were 37.3% and 47.1%, significantly lower than those in HPV-negative group were 57.4% and 63.9%(P<0.05). 2.3 Relationship between the expression of FADD, caspase-8 proteins and clinical pathological features in ESCC In male patients, the positive expression rate of FADD was 59.7%, significantly higher than that in female patients was 20.0%(P<0.05). There were no statistically significant differences between the expression of FADD and respect to age, lymph node metastasis and differentiation(P>0.05). In male patients, the positive expression rate of caspase-8 was 63.7%, significantly higher than that in female patients was 40.0%(P<0.05). There were no statistically significant differences between the expression of caspase-8 and respect to age, lymph node metastasis and differentiation(P>0.05). 2.4 Relationship between the expression of FADD, caspase-8 proteins and clinical pathological features in HPV-positive group in ESCC HPV-positive group was 51 cases in ESCC. The positive expression rate of FADD in more than 62-year-old(including 62 years) patients was 24.1%, significantly lower than that less than 62-year-old patients was 54.5%(P<0.05). The positive expression rate of FADD in poor-differentiated group was 13.6%, significantly lower than that in Well/moderate-differentiated group was 55.2%(P<0.05). There were no statistically significant differences between the expression of FADD and respect to gender and lymph node metastasis(P>0.05). The positive expression rate of caspase-8 in poor-differentiated group was 27.3%, significantly lower than that in Well/moderate-differentiated group was 62.1%(P<0.05). There were no statistically significant differences between the expression of caspase-8 and respect to age, gender and lymph node metastasis(P>0.05). 3 Cell Experiment The expression of HPV16E6, Fas, Fas L, FADD, caspase-8 after transfection 3.1 The results of RT-PCR show After Het-1A cells were transfected with E6 gene plasmid, the m RNA level of E6 increased significantly compared with the empty plasmid group(P<0.05), the m RNA levels of FADD and caspase-8 were significantly decreased compared with the empty plasmid group(P<0.05), the m RNA level of Fas increased significantly compared with the empty plasmid group(P<0.05), the m RNA level of Fas L had no significantly change(P>0.05). After Eca-109 cells were transfected with E6 gene plasmid, the m RNA level of E6 increased significantly compared with the empty plasmid group(P<0.05), the m RNA levels of FADD, caspase-8 and Fas significantly increased compared with the empty plasmid group(P<0.05), the m RNA level of Fas L had no significantly change(P>0.05). After Het-1A cells were transfected with E6 gene plasmid, the m RNA levels of E6 increased significantly at 12 h, 24 h and 48 h compared with the empty plasmid group(P<0.05). The m RNA level of FADD and caspase-8 decreased significantly at 12 h, 24 h and 48 h compared with the empty plasmid group(P<0.05). The m RNA levels of Fas increased significantly at 12 h, 24 h and 48 h compared with the empty plasmid group(P<0.05). The expression of Fas L had no significantly change(P>0.05). 3.2 The results of Western blot show After Het-1A cells were transfected with E6 gene plasmid, the expression of E6 protein increased significantly compared with the empty plasmid group(P<0.05), the expression of FADD, caspase-8 and Fas proteins significantly decreased compared with the empty plasmid group(P<0.05), the expression of Fas L had no significantly change(P>0.05). After Eca-109 cells were transfected with E6 gene plasmid, the expression of E6 protein increased significantly compared with the empty plasmid group(P<0.05), the expression of FADD, caspase-8 and Fas proteins significantly decreased compared with the empty plasmid group(P<0.05), the expression of Fas L had no significantly change(P>0.05). After Het-1A cells were transfected with E6 gene plasmid, the expression of E6 proteins increased significantly at 12 h, 24 h and 48 h compared with the empty plasmid group(P<0.05). The expression of FADD and caspase-8 proteins increased significantly at 12 h, 24 h and 48 h compared with the empty plasmid group(P<0.05). The expression of Fas increased significantly at 12 h and 24h(P<0.05), and decreased significantly at 48 h compared with the empty plasmid group(P<0.05). The expression of Fas L had no significantly change(P>0.05).Conclusions: 1 In the study, the HPV detection rate was 32.1%. In HPV-positive group, poorly differentiated esophageal squamous cell carcinoma has the higher proportion than HPV-negative group. It suggested that there be a close correlation between HPV and the differentiation of ESCC. 2 The expression of FADD and caspase-8 were decreased in ESCC, moreover, they have a more significant decrease in HPV-positive group. 3 HPV16 E6 gene can reduce the protein expression of FADD, caspase-8, and Fas in Het-1A and Eca-109 cells. 4 The results of study suggested that HPV may inhibit FAS-mediated apoptosis pathway and play a role in carcinogenesis of ESCC. |
PDF Full Text Request