Down-regulates The Expression Of STAT3 In JAK-STAT Signaling Pathway To The Occurrence And Treatment Of Esophageal Squamous Cell Carcinomas

Objective Esophageal cancer is one of the most common malignant gastrointestinal tumors. In China, esophageal cancer was regarded as the fourth leading death cause of cancer, and it is serious threat to human health of body and mind. The pathological type of esophageal cancer is mainly divided into esophagus squamous cell carcinoma(ESCC) and esophageal adenocarcinoma. In our country, ESCC is the main type of esophageal cancer, accounting for more than 90% of all types of esophageal cancer. The occurrence and development of ESCC is a complex and long time process. Like other malignant tumors, the occurrence of esophageal cancer is results of the interaction triggered by a variety of factors and multiple genes, and its pathogenesis is a multi-stage development process, but its molecular mechanisms remain elusive, it also lack effective strategies in the diagnosis and treatment. With the progress of tumor targeted therapy, it is dramatically imperative to investigate the pathogenesis of ESCC, and seek for the new tumor markers and therapeutic targets. The Signal transduction and activation of transcription factor(STAT) protein family was first identified by investigating the interferon induced gene transcription by Fu et al. in 1992. STAT members of the protein family paticipate in the occurrence and development of a wide variety of tumor cells, including cell cycle progression, angiogenesis, apoptosis, invasion, metastasis, and immune evasion. Currently, there are seven members in the STAT family, including STATl, STAT2, STAT3, STAT4, STAT5 a, STAT5 b, and STAT6. The human STAT3 gene, one of the STAT family members, is located in chromosome 12, and it was purified as the acute phase response factor in the IL- 6 signal transduction in 1994. STAT3 is widely distributed in different types of tissues and cells. The association of STAT3 with tumor, was initially found in the activation of STAT3 signaling pathways in the v-Ser transfection cell lines, activated STAT3 protein transformed the cultured fibroblasts, and led to cancer in mice; however, fibroblasts ttransformation was suppressd after blocking STAT3 signal. Activated STAT3 signal was also found in the process of multiple myeloma cells and head and neck cancer cells growth, which firstly revealed the tight correlation between the STAT3 and the occurrence of human malignant tumors. Mounting evidence has demonstrated that STAT3 is abnormally expressed in a large number of tumors, such as head and neck, breast, lung, prostate, ovarian, colorecal, gastric tumors, etc, and participates in the occurrence and development of these tumors. The abnormal expression of STAT3 was detected in a wide variety of tumors, and blocking STAT3 signaling pathway suppressed the growth and induced apoptosis in tumor cells. With the increasing understand of STAT3 signaling pathway, the roles of STAT3 in the process of tumor occurrence and development gain wide attention.Targeting STAT3 may be a novel therapeutic strategy for some malignant tumor. In the current study, the underlying biological role of STAT3 in the occurrence and development of ESCC were investigated based on small molecule inhibitor stattic to inhibit the activation of STAT3 signaling pathway.PART 1: Effects of blocking STAT3 signaling on esophageal squamous cell lines in vitroMethods: 1. Western blotting was utilized to detect the activation of STAT3 in a variety ofESCC cell lines by Western blot. 2. The toxic effects of different concentrations(DMSO, 1, 3, 10, 30μM) of stattic on ESCC cell lines were investigated by cell toxicity experiment, and the appropriate concentration of stattic for cell proliferation experiment was selected. 3. The maximal nontoxic concentration for cell proliferation experiment was selected according to the results of cell toxicity experiment, and the effects of different cocentration stattic(DMSO, 1, 2, 3, 4μM) on ESCC cells were detected at different time points(0, 24, 48, 72, 96h), and the relationship between the inhibitory effect of stattic and the STAT3 expression was observed. 4. Soft-agar assay was employed to examine the effects of different concentration(DMSO, 1, 2, 3, 4μM) of stattic on clone formation ability of ECSS cells. 5. Western blotting was utilized to test the effects of different concentration(DMSO, 5, 10, 20μM) of stattic on STAT3 activation and related gene expressions in ESCC cells. 6. Western blotting was utilized to analyze the effects of different concentration(DMSO, 5, 10, 20μM) of stattic plus IL-6(30ng/ml) on STAT3 activation and blockage in ESCC cells. 7. Annexin V, PI and Hochest were utilized to test the effects of different concentration(DMSO, 1, 2, 3, 4μM) of stattic on apoptosis and cell cycle analysis in ESCC cells.Results: 1. The abnormal activation of STAT3 was found in many ESCC cell lines, but theirs phosphorylation levels were different, in which Eca109, EC9706, TE1, TE13 and KYSE450 displayed aberrant STAT3 activation, whereas there was no STAT3 phosphorylation in EC1, KYSE70 and KYSE140 cells. 2. Stattic presented the obviously inhibitory effect on ESCC cells, stattic suppressed the proliferation of ESCC cells harboring the constitutive activation of STAT3, and this inhibition ability was positively coorelated with the concentration of stattic. 3. Stattic didn’t supress the proliferation of ESCC cells with non-STAT3 activation.4. Soft-agar assay showed that the higher concentration of stattic resulted in much less cell clone numbers in ESCC cells with constitutive activation of STAT3. 5. The results of Western blotting revealed that stattic inhibited the phosphorylation of STAT3 and the expression of Cox2, but increased the expressions of i NOS and Pentraxin-3, which displayed in a concentration-dependent manner. 6. IL-6 activated STAT3 in ESCC cells, whereas stattic blocked the activation of STAT3, and this regulatory effect was dependent the concentration of stattic. 7. Stattic promoted the apoptosis of EC9706 cells and blocked the cell cycle in S phase.PART 2: The therapeutic value of blocking STAT3 signaling in ESCC PDX mice model in vivoMethods: 1. Western blotting was employed to detect the activation of STAT3 in some cases of ESCC tissues. 2. ESCC tissue with high STAT3 activation(EG30) was selected and antitumor efficacy of inhibitor stattic and 5-FU alone or combined treatment was investigated using the PDX model in SCID mice. 3. The effects of stattic and 5-FU alone or combine treatment on the activation of STAT3 signaling pathway and the expression of i NOS/Cox2/Pentraxin-3 in the tumor tissues were detected by Western blotting. 4. The effects of stattic and 5-FU alone or combined treatment on the targeted protein expressions(p-STAT3, Cox2, Pentraxin-3 and caspase-3) in tumor tissues were investigated by immunohistochemistry. 5. TUNEL was uesd to detect cell apoptosis in tumor tissues after stattic and 5-FU alone or combined treatment.Results: 1. In the ESCC PDX mice model with constitutive activated STAT3, stattic showed a significantly tumor growth inhibition, but those tumors with 5-FU treatment presented a drug resistance, lack of therapy efficacy; but compared with stattic alone, 5-FU combined with stattic displayed a stronger ability to inhibit the tumor growth. 2. Stattic blocked the activation of STAT3, and lowered the expression of Cox2, but upregulated the expression of i NOS and Pentraxin-3 in tumor tissues. 3. The results of immunohistochemistry revealed that, stattic downregulated the phosphorylation of STAT3 and the expression of Cox2, but promoted the expressions of i NOS and Pentraxin-3. 4. The investigation from TUNEL assay demonstrated that, stattic markedly induced the apoptosis of tumor cells, and 5-FU with stattic evoked more significant apoptosis in the ESCC with constitutive activation of STAT3.Conclusion: 1. In ESCC cell lines and tissues, STAT3 is commonly abnormally activated, but their phosphorylation levels are different. 2. The small molecular inhibitor stattic inhibits the constitutive and IL-6-induced STAT3 activation, leading to the surpressions of cell proliferation and clone formation ability, as well as cell apoptosis, which may be tightly associated with the decrease of Cox2 level as well as the increase of i NOS and Pentraxin 3 levels. 3. Stattic effectively restrains the tumor growth in PDX model of SCID mice harboring the constitutive activation of STAT3 increases the sensitivity of 5-FU, and promotes the apoptosis of ESCC cells in vivo.