Research On Resistance To Paclitaxel In Cervical Carcinoma Cells And Effects Of Ginsenoside RG3on The Promoter Activity Of The Nep Gene

Cervical carcinoma is a common gynecologic tumor. Its incidence rate and mortality rate rank first in female tumors. At present, operation treatment is the main method for the treatment of early stage cervical carcinoma, and chemotherapy and radiotherapy are often used to treat advanced stage of cervical carcinoma. As a first-line drug, paclitaxel (PTX)-based combined chemotherapy is the main therapy for patients with advanced cervical carcinoma However, the majority of cervical carcinoma cells have gradually emerged resistance to PTX. The PTX resistance has become a thorny issue. Therefore, is very important to develop methods to ascende the PTX sensitivity of cervical carcinoma.The levels of reactive oxygen species (ROS) level and antioxidant system are related tothe occurrence and development of cervical carcinoma and resistance of it to chemotherapeutic drugs. The PTX anti-cancer effects are also related to the cellular redox balance.Taxol resistance gene1(Txrl) is associated with PTX resistant, which encodes an18-kDa proline-and serine-rich nuclear protein and whose altered expression confers resistance to PTX.N-acetylcysteine (NAC) is a thiol antioxidant, which can neutralize ROS and significantly affect the intracellular redox state. NAC can clear intracellular free radicals, increase intracellular GSH level, and enhance the anti-tumor effects of multiple drugsObjective:To construct a PTX-resistant human cervical carcinoma cell line and observe effects of NAC on resistance to PTX in the cells.Methods:1. HeLa cells were treated with PTX in order to construct the PTX-resistant HeLa/PTX cells.2. The cell shape of HeLa and HeLa/PTX cells were observed by inverted microscope. The cell growth curves and drug resistance index were drawn or determined respectively.3. Flow cytometry was used to detect the ROS levels of two kinds of cells. The cellular glutathione (GSH) and oxidized glutathione (GSSG) levels and the superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GPx) activities were determined as well. RT-PCR assay was adopted to detect the mRNA expression of Txrl.4. HeLa/PTX cells were treated with NAC for observing the effects of NAC on PTX resistance.Results:1. After HeLa cells were treated with PTX for10months, the PTX-resistant cervical carcinoma HeLa/PTX cells could be obtained, which grew well in500μg/L of PTX.2. The size of HeLa/PTX cells was larger, and there were more cytoplasmic granules in them. The population doubling time of HeLa/PTX cells was1.32times of that of HeLa cells. HeLa/PTX cells manifested the stronger resistance to PTX than HeLa cells with resistance index122.69.3. HeLa/PTX cells had higher levels of ROS and Txrl mRNA, a lower level of reduced GSH, and lower activities of superoxide dismutase, catalase and glutathione peroxidase than Hela cells. There was no significant difference between the levels of GSSG or the GSH to GSSG ratios of HeLa/PTX and Hela cells.4. After HeLa/PTX cells were treated with NAC, the levels of ROS and Txrl mRNA in them decreased, and they were more sensitive to PTX.Conclusion:Redox imbalance and the Txrl transcripts increased were found in HeLa/PTX cells. NAC could reduce the levels of ROS and Txrl mRNA in Hela/PTX cells and reversed the resistance of them to PTX. Alzheimer’s disease (AD) is a kind of degenerative disease in nervous system with the characteristics of concealment and progressive development. The pathogenesis and the progress of etiopathogenesis have not been fully understood. There are no effective treatment measures to prevent or reverse disease presently yet. Researches have shown that the high expression of beta amyloid peptide (Aβ) could be caused by mutations of genes coding amyloid precursor protein (APP), presenilins1and2. The deposition and aggregation of AP is a major pathological feature and one of the key factors of AD.Neprilysin (NEP), a kind of type II membrane metallo-endopeptidases, is thought to be a key enzyme for Aβ-degradation, and can maintain the balance of synthesis and degradation of Aβ in the brain. Therefore, improving enzyme activity or increasing the expression of NEP, which contribute to the degradation of Aβ, is potential targets for treatment of AD.Ginsenoside Rg3, one of the active substances extracted from ginseng, can exert many beneficial effects including enhancement of memory and cognitive functions. It was also observed that Rg3could reduce the accumulation of Aβ in a cell-based model system, as well as in a mouse model of AD, indicating the potentiality of Rg3in the treatment of AD.Objective:To investigate sites in the NEP promoter region and relevant transcription factors related to the regulation of the NEP promoter activity by Rg3.Methods:1. SH-SY5Y cells were transiently transfected with pGL3-nep2.4and treated with Rg3,and then the dual luciferase activity in them was detected. 2. Dual-luciferase reporter assay was conducted after SH-SY5Y cells were transfected with the mutants of Region I, II, III, IV and treated with Rg3.3. Transcription factor analysis software TFSearch and TFBIND were applied to analyze the sequence, which was related to the regulation of the NEP promoter activity by Rg3.4. RT-PCR and Western blot assay were adopted to detect the expression of relevant transcription factors in SH-SY5Y cells treated in Rg3.Results:1. The dual-luciferase assay showed that the luciferase activity in SH-SY5Y cells transfected with pGL3-nep2.4was13.1times of that with pGL3-basic. After treatment with Rg3, the luciferase activity in SH-SY5Y cells transfected with pGL3-nep2.4was1.98times that of the control cells.2. After treatment with Rg3, the luciferase activity in SH-SY5Y cells transfected with the mutant deleting Region Ⅰ, Ⅲ or Ⅳ was no different from that in the control cells, and the luciferase activity in SH-SY5Y cells transfected with the mutant containing Region Ⅱ was3.2times of that in the control cells without Region II was no different from that in the control cells.3. There were sites recognized and bound by transcription factor HSF1, SRY, C/EBPβ, CP2, and GATA3in the region Ⅱ (-559～-534bp).4. RT-PCR analysis showed that Rg3could increase the C/EBPβ mRNA levels and decreased the HSF1mRNA level. Western Blot analysis showed that the expression of CEBP/β protein could be significantly increased by Rg3.Conclusion:1. The recombinant plasmid pGL3-nep2.4had the strong promoter activity in SH-SY5Y cells that could be increased by Rg3.2. There are four regulatory regions (Region Ⅰ, Ⅱ, Ⅲ and Ⅳ) in the NEP promoter, and the change of the NEP promoter activity caused by Rg3was related to Region Ⅱ.3. Rg3could significantly increase the expression of transcription factor C/EBPβ, indicating that Rg3may influence the expression of the NEP gene by transcription factor C/EBPβ.