Expression Of CDC25A, CDC25B, TGF-beta, Smad3 In Esophageal Squamous Cell Carcinoma And Their Role In Antitumor Effect Of Artesunate

Objective:This study is to investigate:①The relationship of CDC25A, CDC25B,Smad3, TGF-beta expression with carcinogenesis and progression of esophageal squamous cell carcinoma(ESCC).②The relationship between CDC25A, CDC25B expression and Smad3, TGF-beta expression.③The relationship of CDC25A, CDC25B protein content with cell apoptosis and proliferation index (PI).④Observe the growth inhibitory effect and side effect of artesunate on human esophageal tumor xenografts, and to elucidate the possible mechanism.Methods:1 Expressions of CDC25A, CDC25B, TGF-beta were examined by s-p immunohistochemical stain (IHC) in 52 cases of ESCC and 25 cases of dysplasia to carcinoma and 24 cases of normal mucosa.2 Expressions of CDC25A, CDC25B, Smad3 and cell apoptosis, PI in 30 cases of ESCC and 5 cases of normal mucosa were quantitatively examined by FCM.3 The model of esophageal cancer xenograft in nude mice was established and the the inhibitory effects of Art on xenograft of nude mice was observed.Results:1 The expression of CDC25A in different esophageal tissues (IHC and FCM)Expressions of CDC25A were detected in 52 cases of ESCC and 25 cases of dysplasia and 24 cases of normal mucosa by IHC, the results showed that the positive expression rate of CDC25A protein (50%) in carcinoma was higher than those in dysplasia (16%) and normal mucosa (0%) (P<0.01), but the difference between dysplasia and normal mucosa was not significant (P>0.05). Expression of CDC25A wasn't correlated with age, sex, but with cell differential degree (P<0.01), fibromembranous invasion (P<0.01), lymph node metastasis (P<0.05).Expressions of CDC25A were detected in 30 cases of ESCC and 5 normal mucosa by FCM, the results showed that the CDC25A protein content (Fluscence Index, FI) in esophageal carcinoma (1.15±0.09) was higher than that in normal mucosa(1.00±0.06) (P<0.01); and the CDC25A protein content (FI) correlated with lymph node metastasis (P<0.01) and fibromembranous infiltration (P<0.05); but not with age, sex, cell differential degree(P>0.05). This result was consistent with those of IHC approximately.2 The expression of CDC25B in different esophageal tissues (IHC and FCM)Expressions of CDC25B were detected in 52 cases of esophageal carcinoma and 25 cases of dysplasia and 24 cases of normal mucosa by IHC, the results show that the positive expression rate of CDC25B(48.1%) in carcinoma was higher than that in dysplasia (16%) and normal mucosa (0%) (P<0.01), but the difference between dysplasia and normal mucosa was not significant (P>0.05). Expression of CDC25B wasn't correlated with age, sex, lymph node metastasis, but with cell differential degree, fibromembranous invasion (P<0.01).Expressions of CDC25B protein were detected in 30 cases of esophageal carcinoma and 5 cases of normal mucosa by FCM, the results showed that the CDC25B protein content(FI) in esophageal carcinoma (1.11±0.11) was higher than that in normal mucosa (1.00±0.06) (P<0.05); the CDC25B protein content (FI) was correlated with fibromembranous infiltration only (P<0.05); but not with age, sex, cell differential degree, lymph node metastasis (P>0.05). This result was consistent with those of IHC approximately.3 The expression of TGF-beta in different esophageal tissues (IHC).Expressions of TGF-beta were detected in 52 cases of esophageal carcinoma, 25 cases of dysplasia and 24 cases of normal mucosa by IHC, the results show that the positive expression rate of TGF-beta(67.3%) in carcinoma was higher than that in dysplasia(36.0%) and normal mucosa(33.3%)(P<0.01), but the difference between dysplasia and normal mucosa was not significant (P>0.05). Expression of TGF-beta wasn't correlated with age, sex, cell differential degree, fibromembranous invasion, but with lymph node metastasis (P<0.05).4 The expression of Smad3 in different esophageal tissues (FCM)Expressions of Smad3 protein were detected in 30 esophageal carcinoma and 5 normal mucosa by FCM, the results showed that the Smad3 protein content (FI) in esophageal carcinoma (0.92±0.06) is lower than that in normal mucosa(1.00±0.07) (P<0.05); The Smad3 protein content (FI) wasn't correlated with age, sex, cell differential degree, fibromembranous invasion and lymph node metastasis (P>0.05)5 There was no correlation between the expression of CDC25A and CDC25B protein in the esophageal carcinoma tissues (x2=3.78, P>0.05) .6 There was no correlation between the expression of CDC25A and TGF-beta protein in the esophageal carcinoma tissues (x2=2.19, P>0.05).7 There was no correlation between the expression of CDC25B and TGF-beta protein in the esophageal carcinoma tissues (x2=3.53, P>0.05).8 There was a negative correlation between the CDC25A protein content (FI) and Smad3 protein content (FI) (r=-0.482, P<0.01).9 The relationship between CDC25A protein content (FI) and cell apoptosis, proliferation index (PI) (FCM). There was no correlation between CDC25A protein content (FI) and cell apoptosis rate (P>0.05), but the cell apoptosis rate (8.53±4.32) in group without the cytoplasmic expression of CDC25A by IHC result was higher than that (6.62±2.01) with positive cytoplasmic expression, but without statistical significance (P>0.05); the PI (30.71±12.04) with the positive nuclear membrane expression was higher than that (21.78±8.22) without expression (p<0.01).10 There was no correlation between CDC25B protein content (FI) and cell apoptosis rate (P>0.05) (FCM)11 There was no correlation between CDC25B protein content (FI) and Smad3 protein (P>0.05) (FCM).12 Growth inhibition of Art on human esophageal tumor xenografts in nude mice①The model of esophageal cancer xenograft in nude mice was successfully established, the tumorigenic rate in nude mice injected with Eca-109 cells was 100%.②The inhibitory rates of volume of 100mg/kg, 200mg/kg, 300mg/kgArt and DDP were 30.30%, 74.76%, 36.36%, 74.75% respectively, the inhibitory rates of weight were 29.35%, 65.22%, 32.61%, 64.13% respectively.③The positive labling frequency of CDC25A in Art group was lower than that in the control group significantly. The effect of 200mg/kg Art was best.④There was no significant difference between the positive labling frequency of CDC25B in five groups.Conclusion: 1 The reduction of Smad3 protein probably resulted in abundance of CDC25A protein in esophageal squamous cell carcinoma; CDC25A could be involved in carcinogenesis and progression of esophageal squamous cell carcinoma; Overexpression of CDC25A protein in nuclear membrane may contribute to cell proliferation.2 CDC25B could be involved in carcinogenesis but might play a role in early phase of esophageal squamous cell carcinoma only.3 The mechanism of CDC25A and CDC25B overexpression might be different and their functions might be independent in carcinogenesis of esophageal squamous cell carcinoma.4 Art could inhibit the transplantation tumor of nude mice with no apparent side effects, inhibition of CDC25A might be the important mechanism of antitumor effects of it.