| Objectives: Esophageal carcinoma(EC) is one of the most common malignant digestive tumors in the world. The incidence of EC is high in China, which lines the second in digestive gastrointestinal cancers, and it is a great threaten to human health. Therefore, it is extremely important to investigate pathogenesis and search new target and effective drugs for EC therapy to decrease mortality and prolong life span of EC patients.Dysregulation of cell cycle progression is believed to play an important role in the tumorigenesis. There is increasing interest in the relationship between CDC25, an essential positive regulator of cell cycle transitions, and human tumors. Three CDC25-related genes, CDC25A, CDC25B, and CDC25C, which share approximately 40%~50% amino acid identity, have been identified in human. Among them, CDC25A activates cyclin-dependent kinase (CDK) by removal of the inhibitory phosphates of threonine and tyrosine residues in the APT-binding sites of CDK and activated CDK interacts with a specific subset of cyclins, which is essential for progression from G0/G1 to S and G2 to M phase. Overexpression of CDC25A has been observed in a variety of cancers(i.e., breast, colon, ovary, and gastric cancer). These findings provided an explanation for dysfunction of cell cycle, exorbitant proliferation and expansion of tumors. These data suggested that CDC25A might be a promising novel target for cell-cycle-regulating anticancer dugs.Artesunate (ART) is a semisynthetic derivative of artemisinin, the active principle initially isolated from Artemisia annua L by Chinese researchers in 1972. Recent studies showed that in addition to the well known antimalarial activity, ART has also been confirmed to possess a profound antitumor action against various cancer cell lines(i.e., lymphocytic leukemia, breast, ovarian, and hepatocellular carcinoma) by regulating cell cycle transition. However, information regarding the functions and mechanisms of ART in esophageal carcinoma is limited.CDC25A expression is mainly modulated by ubiquitin-dependent proteasomal degradation in normal cells. Previous reports demonstrated that signal transduction elicited by transforming growth factor-β(TGF-β) facilitated ubiquitination of target proteins and Smad3 was a rate-limiting factor for this process. At present, only limited evidence, However, is provided in relation to CDC25A expression.Recently it was shown that CDC25A overexpression in several cancer cell lines resulted from impaired protein degradation, which was usually accompanied by TGF-β/Smad3 signal dysfunction. These data again highlighted the relevance of TGF-β/Smad3 signal transduction to CDC25A level modulated by ubiquitin-dependent proteasomal degradation, but the detailed mechanisms remain unclear.Therefore, in the present study we first examined the expression of CDC25A, TGF-βand Smad3 in the process of carcinogenesis of esophageal epithelium and determined the correlation between the levels of these proteins and various clinical and pathological features; Next, we studied the effects of ART on proliferation, cell cycle distribution, and expression of CDC25A, TGF-βand Smad3 in human esophageal Eca-109 cells and the role of ART on proliferation of human peripheral blood mononuclear cells (hPBMC); Furthermore, we investigated the significance of TGF-β/Smad3 signal transduction in CDC25A stability and in cell cycle arrest effect of ART by application of Smad3 RNAi(RNA interference), which might contribute to forward understanding of intra-cellular signal transduction that was associated with cell cycle arrest induced by ART; Finally, we studied the effects of ART on proliferation, cell cycle distribution, and expression of CDC25A, Smad3 in human esophageal xenografts. It might provide theoretical and experimental evidence for utilization of ART in esophageal carcinoma therapy. Methods1 Expression of CDC25A, TGF-βand Smad3 in esophageal tissues and the correlation between the levels of these proteins and clinical-pathological featuresEsophageal carcinoma tissues, paired adjacent mucosa (2~5cm from margin of esophageal carcinoma), and paired normal mucosa (at least 5cm from margin of esophageal carcinoma) were obtained from 52 resected surgical specimens of esophageal squamous cell carcinoma (SCC). All the specimens were verified by pathologic diagnosis. CDC25A, TGF-βand Smad3 protein expression in normal squamous epithelium (n=24), squmous dysplasia [mild dysplasia(n=23), moderate and severe dysplasia(n=2)] and advanced SCC [well differentiated SCC(n=21), moderately and poorly differentiated SCC(n=31)] were examined by immunohistochemistry(IHC). CDC25A mRNA expression was detected in 40 resected surgical specimens of fresh esophageal carcinoma, paired adjacent esophageal mucosa, and normal esophageal mucosa by reverse transcription-polymerase chain reaction (RT-PCR). Relationship between their expressions and clinical pathological features was analyzed.2 Effect of ART on proliferation and cell cycle distribution of human esophageal carcinoma Eca-109 cells and hPBMCFirst, human esophageal carcinoma Eca-109 cells were conventionally cultured. ART was administered with a final concentration of 1, 10 and 100μmol/L. The inhibition effect of ART on proliferation of Eca-109 cells at 24, 48 and 72h was determined by MTT method; Next, hPBMC were conventionally isolated and cultured, ART was added with a final concentration of 1, 10, and 100μmol/L, and 10μg/ml ConA was added at the same time. The inhibition effect of ART on proliferation of hPBMC at 24, 48 and 72h was determined by MTT method; Then, cell cycle distribution was assayed by flow cytometry (FCM); CDC25A, TGF-βand Smad3 mRNA expression were measured by RT-PCR; CDC25A, TGF-βand Smad3 protein expression were detected by Western blot; Finally, Smad3 expression was suppressed in Eca-109 cells by RNAi. Two plasmid vectors (S1, S2) encoding shRNA(small hairpin RNA) were constructed by WuHan JingSai company according to principle of small interference RNA(siRNA) and transcription RNA site of human Smad3 gene sequence, a negative control plasmid vector HK was constructed at the same time. These plasmid vectors expressed green fluorescence protein (GFP) and resisted to neomycin (neo). They were transfected into human esophageal carcinoma Eca-109 cells by cations liposome vector. The transfection efficiency of plasmid vector was evaluated by FCM to determine the most optimal ratio of cations liposome vector to plasmid vector; Smad3 mRNA and protein of Eca-109 cells were detected by RT-PCR and Western blot 48h after transfection to determine the most effective Smad3 RNAi which was used in the subsequencial experiments. The inhibition effect of ART on proliferation of Eca-109 cells after Smad3 RNAi transfection was assayed by MTT method; Expression of CDC25A at different times were measured by RT-PCR and FCM; Cell cycle distribution was assayed by FCM.3 Effect of ART on growth of human esophageal xenograft in nude mice and its mechanismsEach of 30 (15 of male and 15 of female) nude mice (BALB/c, nu/nu, 4 wk old, 18~20g) was inoculated with 200μl Eca-109 cells(6×106 each mouse) subcutaneously on the left axillary fossa. The mice were randomly divided into experiment and control groups (6 in each group). On the seventh day after inoculation, the experimental groups received ART (100, 200, 300mg/kg/d, respectively) by peritomeal injection for 2 courses of treatment, each course contained 7 days and there was a seven-day interval between two courses. The positive control group received diamminedichloroplatinum (DDP, 3mg/kg/d) by peritomeal injection for 7 days. The negative control group received saline instead of ART. The shortest and the longest diameters of the tumors were measured with vernier caliper each 3 days, and the growth curve of tumors was drawn according to the tumor volume, the tumor volume (mm3) was calculated using the following standard formula: (the longest diameter)×(the shortest diameter)2/2. Mice were sacrificed on day 30 and all tumor tissure samples were weighed. FCM was used to examine cell cycle distribution. Expression of CDC25A and Smad3 were determined by RT-PCR, IHC and FCM.Results1 Expression of CDC25A, TGF-βand Smad3 in esophageal tissues and the correlation between the levels of these proteins and clinical-pathological features①CDC25A mRNA expression corresponded with its protein expression in normal squamous epithelium, squamous dysplasia and advanced SCC. The positive rates of CDC25A mRNA and protein were significantly higher in advanced SCC (53.8% and 50.0%) than those in squamous dysplasia (20.0% and 16.0%) and normal squamous epithelium (4.2% and 0.0%) (all the same, P﹤0.01), there was no significance between squamous dysplasia and normal squamous epithelium (P>0.05). The positive rate of CDC25A protein expression was significantly lower in esophageal carcinoma with well differentiation, fibrous membrane invasion and lymph node metastasis than those in esophageal carcinoma with moderately and poorly differentiation and without fibrous membrane invasion and lymph node metastasis (all the same, P<0.05).②The positive rate of TGF-βprotein was significantly higher in advanced SCC (67.3%) than in squamous dysplasia (36.0%) and normal squamous epithelium (33.3%) (all the same, P﹤0.01), there was no significance between squamous dysplasia and normal squamous epithelium (P>0.05). The positive rate of TGF-βprotein expression was significantly higher in esophageal carcinoma with lymph node metastasis than in esophageal carcinoma without lymph node metastasis (P<0.05).③The positive rate of Smad3 protein was significantly lower in advanced SCC (28.8%) than those in squamous dysplasia (56.0%) and normal squamous epithelium (58.3%) (all the same, P﹤0.01), there was no significance between squamous dysplasia and normal squamous epithelium (P>0.05). There was no correlation between the positive rate of Smad3 protein expression and clinical-pathological features.④Expression of CDC25A protein was negatively associated with Smad3 protein expression (rs=-0.473, P=0.000). There was no significant association between TGF-βprotein expression and CDC25A, Smad3 protein expression (rs =0.117,P=0.182; rs =0.133,P=0.121, respectively).2 Effect of ART on proliferation and cell cycle distribution of human esophageal carcinoma Eca-109 cells and hPBMC①1, 10, 100μmol/L ART inhibited growth of Eca-109 cells in a time and dose dependent manner. The highest inhibition rate was (78.31±1.48)%, the IC50 of ART was (68.80±0.76)μmol/L.②Proliferation of hPBMC stimulated by ConA was inhibited slightly by ART, proliferative inhibition rate was about 2%~8%.③Most of cells were arrested at G0/G1 phases when 1, 10μmol/L ART were used, whereas the majority of cells were arrested at G2/M phase when 100μmol/L ART was applied. The percentage of S phase cells decreased in all of ART groups.④CDC25A mRNA and protein expression significantly decreased while Smad3 mRNA and protein expression increased compared to the control group when ART were administered (P<0.05). There was no significant difference in the levels of TGF-β(P>0.05).⑤When the ratio of cations liposome vector to plasmid vector was 6μl to 1.0μg, the transfection efficiency was 72.6%. Expression of Smad3 mRNA and protein in Eca-109 cells were suppressed most effectively by S2 plasmid vector 48 h after transfection.⑥Smad3 mRNA and protein expression of Eca-109 cells transfected with S2 began to decrease 24h after transfection and couldn't be detected 48h after transfection and could be faintly detected at 72h after transfection.⑦Proliferation of Eca-109 cells which were transfected with S2 plasmid vector was not significantly inhibited by ART.⑧Cell cycle distribution of Eca-109 cells transfected with S2 was not changed by ART detected by FCM.⑨CDC25A expression of Eca-109 cells transfected with S2 was not significantly changed by ART assayed by RT-PCR and FCM.3 Effect of ART on growth of human esophageal xenograft in nude mice and its mechanisms ①The model of esophageal cancer xenograft in nude mice was successfully established, the tumorigenic rate was 100%.②The tumor volume and the tumor weight in experiment groups decreased significantly compared with control group(P<0.01). The volume inhibition rate of ART groups were 32.6%, 76.4% and 18.0%, respectively. The weight inhibition rate of ART groups were 11.4%, 63.2% and 21.6%, respectively. The volume inhibition rate of DDP group was 78.3% and the weight inhibition rate of DDP group was 64.7%.③The proportion of cells in the G0/G1 phase increased while the proportion in the S phase decreased in ART-treated groups. Furthermore, the expression of CDC25A was decreased, whereas the expression of Smad3 were increased in ART-treated groups.Conclusions1 In the process of carcinogenesis of the esophagus, the expression of CDC25A protein in advanced SCC was significantly higher than that in normal squamous epithelium and squamous dysplasia. In advanced SCC, higher expression of CDC25A might be correlated with tumor progression and metastasis. Our results indicated that CDC25A is an important factor in tumorigenesis and tumor progression. CDC25A specific inhibition may serve as a potential strategy for the prevention and treatment of esophageal carcinoma.2 For the first time, we analyzed expression of Smad3, an important member in TGF-βsignal transduction pathway, in squamous carcinogenesis of the esophagus. From normal squamous epithelium to dysplasia and carcinoma, the expression of Smad3 decreased gradually. In addition, Smad3 expression was negatively associated with CDC25A expression in advanced SCC. It is reasonable to speculate that abnormal expression of Smad3 and CDC25A might participate in tumorigenesis and tumor progression. It may be a promising approach to inhibit the expression of CDC25A by increasing the expression of Smad3.3 ART could inhibit Eca109 cells proliferation in vitro, but had a weaker effect on hPBMC. G0/G1 and G2/M phase arrest might be involved in the antitumor mechanism of ART.4 For the first time, it was reported that ART could significantly suppress the growth of EC xenograft in vivo without causing obvious side effects in treated mice, which may provide a promising strategy for cancer therapy in EC.5 ART could inhibit the expression of CDC25A which might be a major reason for ART's antitumor effect. The augment of the expression of TGF-βand Smad3 caused by ART administration might be an important pathway for CDC25A degradation. It was shown for the first time that RNAi could effectively silence Smad3 expression in Eca-109 cells. Such silence inhibited the degradation of CDC25A caused by ART in Eca-109 cells. These suggested that Smad3 might play a key role in the process of CDC25A degradation induced by ART.
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