Hsp90α Inhibition Affects The Redox Status And Induces CyclinB1 Accumulation Related G2/M Cell Cycle Arrest In HepG2 Cells

BackgroundHeat shock proteins (heat shock proteins, HSPs) is a group of highly conservative proteins which synthetize in the stimulation such as hyperthermia, radiation and chemical poison. HSP is widespread in prokaryote and eucaryon. It plays a role as molecular chaperone. On the one hand, HSP participate in the substrate protein synthesis, folding and assembly. On the other hand, HSP participate in immune response as foundation of Anti-infection immune and autoimmune reaction. Hsp90 (heat shock protein 90) is largely exist in cytoplasm as an important member of HSP, which participate in various cell progress contain signal transduction, protein folding and degradation. In addition, Hsp90 is known as molecular chaperone for regulating substrates protein without changing target protein structure. It promotes substrate proteins folding and contacts with other co-molecular chaperone to accelerate mistake folding proteins degradation by proteasome system. Until now, there are more than 200 proteins found to be related with Hsp90, most of which are kinase. In addition, the expression of Hsp90 in tumor is 2 to 10 times higher than normal cells. In recent years, Hsp90 becomes the target of cancer treatment for its important role in the tumor cell growth, proliferatio.Antioxidant defense system will play a role in maintaining the balance of redox status when the body is under attack by endogenous or exogenous ROS. However, if the balance is broken, body will be damaged and then result in diseases. The redox status direction is depended on the activity or proportion of intracellular ROS and GSH. ROS can be directly applied to protein, leading S-S formation, carbon chain fracture and degradation, and then destroy cell homeostasis and vitality. The broken balance of ROS generation and eliminate can induce oxidative stress, which is associate with aging and many diseases. Redox balance disorders induce damage of lipid, protein, nucleic acid function, and result in a variety of pathological processes, tumor, ischemia-reperfusion injury, diabetes and neural degenerative diseases.Proteasome is a protease complex, which decompose proteins by interrupting the peptide bond. Ubiquitin—Proteasome pathway (UPP) is the most important degradation pathway of lysosome proteins, including proteasome and ubiquitin. UPP plays an important role in maintaining cell homeostasis, protecting cells from oxidative damage.26S proteasome contains a 20S core granule and two 19S regulatory particles. When a protein tagged with a single ubiquitin molecule, the additional ubiquitin molecules will attach to this protein subsequently. The polyubiquitin chain is bound to the proteasome, and the tagged protein degraded. Proteosome plays an important role in gene transcription, cell cycle regulation, cell swallowed and a variety of cellular physiological processes. For these, it involves a variety of diseases, including cancer, nervous system diseases, autoimmune diseases and other diseases.The basic composition of cell cycle control system is highly conservative and the core component s is cyclin-dependent protein kinases (CDKs). The activity of CDK depends on cyclins and the ordered degradation of cyclins is critical to cell cycle progress. CyclinB1 is a crucial regulator in G2/M phase, which first appeared in late Gl phase and degraded in middle mitosis phase. The expression of cyclinB1 is closely related with G2/M phase. Only cyclinB1 bounded to its cyclin-dependent kinase and the CDK-cyclin complex activated, cell cycle progression will go with a swing. If the degradation is inhibited, cyclinB1 will accumulate and cell will be blocked in mitosis phase. For these, cyclinBl is closely associated with tumors and has been confirmed as an oncogene. CyclinB1 is a crucial regulator in cell cycle arrest and it is one of client proteins of Hsp90. So it can be the effect index of cell cycle, which clarifies the relationship among Hsp90, the oxidative stress induced by Hsp90 and proteasome function.Hsp90 is closely related to important substrate proteins in various cancer cells. Oncogene proteins are usually expressed in mutation types and need to rely on Hsp90 in order to maintain itself requirement. Nowadays, increasing research focus on Hsp90 as molecular targets for cancer treatment, so clearing Hsp90’s important role in the growth of liver cancer cells is benefit to drug compatibility application development of oxidative stress injury and adaptation.Objectives1. To study whether Hsp90 inhibition influence the HepG2 cell cycle;2. To explore the effect of Hsp90 inhibition in redox status in HepG2 cells;3. To investigate the effect of Hsp90 inhibition in proteasome activity and oxidative damaged proteins in HepG2 cells;4. Treament with exogenous inhibitor 17-DMAG and then analysis the cyclinB1 mRNA synthesis and the expression of cyclinB1, p-cyclinB1 and p-Hsp90;5. To study the co-localization and mutually combination of ubiquitinlate proteins and cyclinB1;6. Pretreament with exogenous antioxygen NAC to promote redox status and then research the changes of proteasome activity and cell cycle;7. In the previous step analysis the effect of Hsp90 inhibition in cell cycle, and in order to further investigate the relationship among oxidative stress induced by Hsp90 inhibition, proteasome activity and cell cycle. In addition, investigate the mechanism of cell cycle arrest which induced by 17-DMAG and regulating effect mechanism of Hsp90a.Methods1. Inhibiting Hsp90 by three methods:exogenous inhibitor 17-DMAG, Hsp90a SiRNA HepG2 cells and Hsp90a ATPase HepG2 mutant. Cell survival of 17-DMAG treatment cells is detected by CCK-8. And then determine the corresponding treatment concentration and time. Hsp90a SiRNA HepG2 cells and Hsp90a ATPase HepG2 mutant are built formerly by our research group.2. Flow cytometry is used to detect cell cycle of cells;3. ROS fluorescent probe reagents and Reduced Glutathione Detection Kit are used to detect redox status indicator-intracellular ROS and GSH;4. Poteasomal Peptidase Activity Assay Kit is used to assay the Chemotrypsin-like activity;5. Western blot is used to detect content of protein carbonyl, Hsp90a, P-Hsp90a, cyclinB1 and P-cyclinB1;6. Quantitative Real-time PCR is used to detect cyclinB1 mRNA level;7. Immunofluorescence is used to detect the intracellular co-localization of cyclinB1 and ubiquitin protein;8. CO-IP is used to detect the combination of cyclinB 1 and ubiquitin protein.Results1. HepG2 cells were treated with 17-DMAG and then cell proliferation was inhibited at all concentration-dependent and time-dependent manner;2. Inhibition of Hsp90 induced G2/M cell cycle arrest in HepG2 cells. In addition 17-DMAG and Hsp90a ATPase mutant HepG2 cells presented more obvious cell cycle G2/M arrest;3. Inhibition of Hsp90a changed redox status in HepG2 cells:significant increase of ROS and reduce of GSH;4. Hsp90 inhibition decreased proteasome activity and the effect of 17-DMAG treament is with concentration gradients;5. After cells pretreated with NAC and then incubated with 17-DMAG, intracellular redox status and proteasome activity were promoted but cell cycle not.6. Hsp90 inhibitor had a promotive effect on cyclinB 1 mRNA synthesis;7.17-DMAG treament induced increasing of carbonyl-proteihs, cyclinB 1, P-Hsp90a and decreasing of P-cyclinBl in HepG2 cells;8. The co-localization of ubiquitin cyclinB 1 after 17-DMAG treatment was decreased at concentration-dependent manner;9. Ubiquitination of cyclinB 1 protein was decreased by 17-DMAG;Conclusions1. Hsp90 is important to cell growth and Hsp90 inhibitor induces cell survival inhibition and G2/M cell cycle arrest;2. Hsp90a ATPase inhibition may induce more significant biological effect than Hsp90a expression inhibition;3. Hsp90 plays an important role in redox status. Both Hsp90a expression inhibition and Hsp90a ATPase inhibition may break the balance of intracellular redox status and lead to oxidative damage;4. Both Hsp90a expression inhibition and Hsp90a ATPase inhibition may decrease intracellular proteasome activity, suggesting that Hsp90 is important to maintainance of proteasome function;5. NAC protects pancreas from oxidative damage and proteasome function, but ineffectively to decrease cell cycle arrest induced by 17-DMAG6. G2/M cell cycle arrest induced by Hsp90a ATPase inhibition may due to cyclinBl accumulation:increase of cyclinBl mRNA level, decreased ubiquitinated cyclinB1 and decreased proteasome activity. All these are different from H2O2 induced oxidative stress;7. Hsp90α ATPase inhibition induced decresed P-cyclinBl, suggesting that mitosis initiation was blocked. This is associated with G2/M phase arrest induced by 17-DMAG.