The Mechanism Of ERK Expression And Activation In Esophageal Cancer Progression

Esophageal cancer is the one of the most common malignant tumor in China, the WHO data show that patients with esophageal cancer in China accounted for 46.6% of the world. The 90% of esophageal cancer is a squamous cell carcinoma, and the adenocarcinoma accounts for about 5% ~ 10% in 1999. The rate of deaths dues to esophageal cancer is the fourth of malignant tumors each year. Although pathological diagnosis of precancerous lesions of esophageal cancer technology has greatly improved, new preventive measures and more effective method of improving the combination therapy still can not stop the increase in incidence of esophageal cancer. Therefore, further study of the role of the clinical anticancer drug target and esophageal cancer-related change of signal transduction pathways activation, is still the goal in the treatment of esophageal cancer.Hydroxycamptothecin (HCPT) is the camptothecin derivative extracted from Nyssaceae plant in china in recent years. It is the clinical first-line chemotherapy drugs, and widely used in gastrointestinal malignancies, liver cancer, leukemia, bladder cancer, lung cancer and other cancer chemotherapy. But its anti-cancer mechanism is not yet very clear. Recent studies have found that HCPT can prevent tumor cells from S phase into G2 / M phase, and can induce their apoptosis.Extracellular signal-regulated kinase (ERKs) is a sub-tribe of mitogen -activated protein kinase (MAPK) family, and widely exists in a variety of organisms, plays an important role in the process of carcinogenesis in the cell. ERK can also be activated into the state of phosphorylation (P-ERK) by variety of mitogen-activated signaling and oncogene products. P-ERK transducts signal to the nucleus, involves in regulating cell proliferation, differentiation, apoptosis and other physiological processes, and plays an important role in malignant transformation of cell.This study investigated the significance of ERK pathway in the development of esophageal cancer, to provide experimental evidence of the comprehensive treatment for esophageal cancer, through researches the expression of ERK and phosphorylation level (P-ERK) in esophageal squamous cell carcinoma and the relationship between clinicopathological features of esophageal squamous cell carcinoma, and the related effects of HCPT on P-ERK and esophageal cancer cell lines Eca109.PartⅠ: The role and significance of ERK in esophageal carcinomaObjective: To investigate human esophageal squamous cell carcinoma and normal tissue in the ERK mRNA and protein expression and the level of phosphorylation,and to explore the role and significance of ERK in development of the human esophageal squamous cell carcinoma.Methods: Collected 60 cases of fresh esophageal cancer tissues and 25 cases of normal tissue. RT-PCR and Western blot assay were used to detect the mRNA expression level of ERK, protein expression level of ERK and P-ERK respectively, and to discuss the relation of pathological features of esophageal cancer in clinical.Result:1. RT-PCR results showed that: the expression of ERK gene in esophageal cancer tissue is significantly higher than in normal mucosa. It can be seen that ERK mRNA gene expression in esophageal cancer tissues was increased significantly, while the one in normal tissues showed lower expression from the table. The relative expression levels of ERK mRNA in esophageal carcinoma is (0.656±0.070), the one in normal tissues is (0.450±0.055). The difference was statistically significant (P <0.01).2. It can be seen the expression of ERK and its activation (P-ERK) in esophageal cancer tissues was markedly enhanced, while its one in normal esophageal mucosa was in the lower expression by the Western blotting results. The relative expression levels of ERK and P-ERK in esophageal normal tissues and cancer tissues are respectively (0.581±0.053 vs 0.702±0.703;0.542±0.053 vs 0.659±0.072). Differences were statistically significant (P <0.01).3. The relationship between the ERK expression and features of clinical pathologicalRT-PCR results showed that: the expression of ERK among the patient's age (≥60 and <60), gender (male and female) and the degree of differentiation (high division and poorly differentiated) has not significant difference (P> 0.05), while there was significant difference among the clinical stage, lymph node metastasis(P<0.05), and T3-4 gene in patients with ERK mRNA expression level was significantly higher than T1-2 in patients (0.717±0.049 vs 0.643±0.067,P<0.01); the lymph node metastasis in esophageal carcinoma with ERK mRNA gene expression level was significantly higher than those without lymph node metastasis (0.705±0.071 vs 0.645±0.066,P<0.05). Western blotting results showed consistent with the RT-PCR results. T3-4 period with T1-2 expression intensity of P-ERK and ERK protein are respectively (0.700±0.068 vs 0.648±0.069 , P<0.05 ; 0.743±0.056 vs 0.691±0.070,P<0.05); lymph node metastasis of P-ERK and ERK protein expression intensity and non-lymph node metastasis are respectively (0.695±0.054 vs 0.648±0.072,P<0.05;0.736±0.072 vs 0.690±0.066,P<0.05).Conclusion:1. ERK and its phosphorylation level increased in ESCC tissues, suggesting that activation of ERK plays an integral role in the ESCC;2. ERK expression and phosphorylation levels have a certain correlation on ESCC clinical stage, lymph node metastasis. It indicates that the expression of ERK and phosphorylation level plays an important role in the development of ESCC;3. We may feel that further study of anticancer drugs inhibiting ERK pathway can be as a target for ESCC drug therapy.PartⅡ:The mechanism of ERK activation in ESCCObjective: To observe the effects of the hydroxyeamtothecin on P-ERK activity and cell growth and apoptosis of the human esophageal cancer cell line Eca109,to clarify the mechanism of ERK to promote the progress of ESCC, and to offer reasonable experiment evidence in the clinic theapy.Method: MTT is used to measure the suppress rate of tumor cell, FCM is used to analyze the apoptosis of esophageal cell lines , and the Immunocytochemistry method is used to detect the amount of P-ERK of the human esophageal cancer cell line Eca109.Result:1. MTT method showed that HCPT inhibited the growth of esophageal cancer cell lines Eca109 significantly. Different concentrations of HCPT treated Eca109 cell lines, OD values were reduced to varying degrees, the difference was statistically significant (P<0.01). The level of cell proliferation decreased with the rise in the concentration of HCPT(0.989±0.047 ;0.860±0.095;0.748±0.036;0.541±0.045), and reached the peak at theconcentration of 20um/l. 2. FCM also showed that Eca109 cells growth were inhibited significantly by the HCPT for 48 hours, and with increasing the concentration of HCPT, the apoptosis rate increased gradually (0.80%;3.52%;3.91%;12.72%;P<0.01). The results are consistent with the previous experiment. It shows that HCPT promotes apoptosis of esophageal cancer Eca109 cell lines, and inhibition were dose-dependent.3. The Immunocytochemistry method showed that the hydroxycam- ptotheich has reduced the amount of P-ERK of the human esophageal cancer cell line Eca109. With the concentration of HCPT increased, the number of positive cells gradually reduced, brown-yellow particles became smaller, the density gradually reduced (P<0.01).Conclusion:1. HCPT inhibited the proliferation of esophageal cancer cell lines Eca109, and showing dose-dependent at certain concentration range;2. HCPT induced apoptosis of esophageal cancer cell lines Eca109, and has a certain effect on arresting cell cycle S phase;3. P-ERK expression in cancer cells can be effectively inhibited by HCPT. The mechanism of ERK promoting the progress of ESCC may be associated with cell cycle regulation.