| Deubiquitinases(DUBs) act on ubiquitinated substratesto catalyze the removal of ubiquitin moieties. DUBs can reverse the process of protein degradation, and further affect or regulater cell metabolism, proliferation and differentiation. Recent studies have demonstrated that aberrant expression or fysfunction of DUBs are closely related with tumor oncogrnrsis and development. As a noval small molecular inhibitor, b-AP15 can specifically inhibit the acivity of UCHL5(ubiquitin C-terminal hydrolase 5) and USP14(ubiquitin-specific peptidase 14) by inhibiting the deubiquitinating activity of 19 S regulatory subnit of proteinase in cancer cells. Researches have shown that b-AP15 can suppress tumor growth in multiple myeloma, colon cancer and acute myeloid leukemia models. Previous studies have shown that UCH37, b-AP15 targeted substrate, was overexpressed in tumor tissues and was significantly correlated with TNM stage and lymph nodes metastasis. This suggest that b-AP15 might be a good candidate for targeted therapies in ESCC. However, the machanisms remain largely unknown and need to be further explored.In this study, we aimed to check whether b-AP15 have anti-tumor activity in esophageal cancer cells in vitro and try to explor the underling mechanisms, and thus will lay the foundation to the diagnosis and treatment of esophageal cancer.Materials and Methods1. CCK-8 assay, colony formation assay and morphological observation were used to check whether b-AP15 have anti-tumor activity in EC1 and Kyse 450 esophageal cance cell lines.2. Flow cytometry was used to check the change of cell cycle and apoptosis in EC1 and Kyse 450 cell lines after being treated with b-AP15 at different concentrations(DMSO, 0.4μM、0.6 μM、0.8 μM), at the same time, Western Blot was used to estimate the variation of cell-cycle proteins, pro-apoptotic prteins and anti-apoptotic proteins in the two EC cell lines after being treated with b-AP 15.3. Mitochondrial membrane potential assay kit with JC-1 was used to check the change of mitochondrial membrane potential, FITC-DEVD-FMK was used to check the activation of total apoptosis proteinase caspase 3 in two EC cell lines treated with b-AP 15 at different concentrations.4. Real –time q PCR was used to check the change of Noxa m RNA in two EC cell lines after being treated with b-AP15, and we fureher check the apoptosis rate after treated with b-AP15 in these two cell lines with Noxa knockdown by flow cytometry.5. Flow cytometry was used to estimate and compare the effect of b-AP15 treatment in both cell lines after Noxa being knockdown and Noxa upsteam regulator Myc being knockdown respectively. Western Blot was used to evaluate changes of Noxa after Nyc being knockdown.Result1. Our results indicate that b-AP15 can significantly inhibit the prolifriation activity of cell lines in a concentration-dependent manner in vitro studies.2. Flow cytometry results show EC1 and Kyse 450 cells were arrested in G2/M phase after being treated with b-AP15. In accordance with these findings, western blot results show that the expression of G2/M phase related proteins p21, p27 and p Wee1 significantly increased while the expression of the G1/S phase marker proteins including cyclin A, cyclin D show no significant change after being treated with b-AP15.3. FITC annexin V Apoptosis detection kit with PI and Caspase 3 Assay kit were used to evaluate apotosis in two cell lines after treatment. The results show that b-AP15 treatment can significantly induce apoptosis, and total apoptosis rate, including early phase and late phase apotosis rate, significantly increased in a dose-dependment manner in two EC cell lines. The results from western blot show that the expression of apoptotic protein c-PARP and c-caspase 3 significantly increased and was dose-depedent. JC-1 flow cytometry analysis show that b-AP15 treatment can significantly derease mitochondrial membrane potential. All these results suggest that b-AP15 can induce EC cell apotosis and may play certain role in mitochondrial control of apoptosis signaling pathway.4. Results from Q-PCR show that the expression of Noxa m RNA significantly increased and this further confirmed the results from western blot that the expression of this apoptotic protein also increased after being treated with b-AP15.5. Apoptosis rate slightly decreased in Noxa or Myc knockdown EC cell lines after b-AP15 treatment. Western blot showed similar results in protein level. This suggest that Noxa might be a key factor in b-AP15 induced cell apoptosis.Conclusion1. b-AP15 can inhibit proliferation of EC1 and Kyse 450 cells by inducing cell-cycle arrest and promoting cell apoptosis.2. b-AP15 can upregulate the expression of Myc, and as a transcriptional factor, Myc can upregulate the expression of apoptotic protein Noxa and further trigger the activation of mitochondrial control apoptosis signaling pathway. |
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