Hsp90Alpha Inhibition Coordinated With Cyclin B1 Accumulation Were Involved In G2/M Cell Cycle Arrest Induced By Oxidative Stress

BackgroundHeat shock protein 90(Hsp90) is a highly conserved molecular chaperone that is critical to maintaining the maturity and stability of its client proteins which including hundreds of pivotal molecules in the signal transduction and cell cycle regulation. It is well known that human Hsp90 includes four subtypes:Hsp90a, Hsp90β\TRAP1 and Grp94. Hsp90a, which is easily induced by thermal, plays an important part in biological behavior, tumor cells proliferation, tumor development and prognosis. So Hsp90a is the focus of this study. Hsp90 plays a key role in G2/M checkpoint regulation by interacting with its client proteins including CDK1, Weel, Myt1 and cyclin B1 through regulation of their stability. It is clear that cyclin B1-CDK1 is a primary regulator of G2/M transition, and levels of the cyclin B1 is tightly related to cell cycle arrest during the DNA damage response (DDR). Interestingly, it has been suggested that Hsp90 is vital for the proper folding or function of a specific domain of cyclin B that is necessary for the localisation of cyclin B on centrosomes and spindles. In addition, Hsp90 inhibitor,17-AAG arrested cells in mitosis concomitant with up regulation of cyclin B1. Nevertheless, how Hsp90 affects the level of cyclin B1, and the following influence on cell cycle progression remain unclear.Our previous studies have shown that in the keratin pearls of well-differentiated esophageal squamous cell carcinoma (ESCC), Hsp90a expression was negatively correlated to cyclin B1 expression,ie. Hsp90a expression level is low in the center of keratin pearl, whereas cyclin B1 is significantly high. Interestingly, a recent report suggests that immature and mature keratin pearls in oral carcinoma in situ and squamous cell carcinoma were generated by oxidative stress derived from erythro-hemophagocytosis. Studies have demonstrated that cancer cells are known to be under increased oxidative stress and metabolically active, probably connected with unbounded cell proliferation and dysfunction of metabolic regulation. Furthermore, the oxidative damage conduces to pathogeneses of several human diseases, including Alzheimer’s disease and pulmonary fibrosis. In addition, oxidative stress can directly provoke cell cycle checkpoint responses via the induced damage to DNA. Therefore, we hypothesize that the formation of keratin pearls in ESCC may be related to oxidative stress and Hsp90a might be involved in the regulation of cyclin B1 related cell cycle arrest in keratin peal formation of ESCC.It is well believed that ubiquitin-proteasome-mediated proteolysis is a pivotal pathway for the degradation of cell cycle regulatory proteins. For example, dropping out of mitosis needs the proteasome-dependent separation of the regulatory component cyclin B1 from the mitosis promoting factor (MPF) complex. In addition, ubiquitin-proteasome pathway (UPP) plays an essential role in protecting cells from oxidative damage and maintaining cell homeostasis. Although proteasomal degradation increases owing to mild oxidation, it decreases at higher levels of oxidative damage due to proteasome inhibition by protein aggregates. Intriguingly, hydrogen peroxide treatment can inhibit the ubiquitin transfer to anaphase-promoting complex/cyclosome (APC/C) substrates, which deters the degradation of cyclin B1 and conduces to the growth arrest induced by oxidative stress. As a molecular chaperone, Hsp90 has been reported to be involved in maintaining the activity of ubquitin-proteasome system (UPS) and maintaining the steady-state level of the 26S proteasome, on the other side, Hsp90a itself also could be cleaved and deactivated by oxidative stress. Thus, we speculate that inhibition of Hsp90a may prevent the degradation of cyclin B1 in response to oxidative stress which may be related to the formation of keratin pearls in ESCC.The aim of this study was to confirm whether Hsp90 can alter cyclin B1 expression in the formation of keratin pearls in ESCC and to explore possible mechanisms that cause the alteration in cyclin B1 expression. This study may provide another possible therapeutic avenue for ESCC and other solid tumors.Objectives1. To establish the TE-1 cell model of oxidative stress and detect whether hydrogen peroxide (H2O2) treatment can lead to DNA double strand breaks;2. To study whether oxidatie stress treatment can affect the TE-1 cell cycle;3.To explore the change of cyclin B1 and Hsp90a in TE-1 cells under oxidatie stress condition;4. To explore the change of proteasomal activity in TE-1 cells after oxidative stress by H2O2;5. To demonstrate that inhibition of Hsp90 and protesome would subsequently affect cell cycle and the amount of cyclin B1 in TE-1 cells by using the Hsp90 inhibitor 17-DMAG and proteasome inhibitor MG132 as positive controls.6. On the basis of the previous steps, to elucidate whether Hsp90a can affect cyclin B1 protein level and cell cycle arrest. And to explore possible mechanisms that cause the cell cycle arrest and the role of Hsp90a in the formation of the keratin pearls in well-differentiated esophageal squamous cell carcinoma (ESCC).Methods1. After treated with different concentrations of H2O2 in esophageal cancer TE-1 cells, measure CCK8 values and select the appropriate concentration to establish the TE-1 cell model of oxidative stress;2. Flow cytometry was used to detect cell cycle distribution at various time-points in oxidative stress;3. Western-blotting was used to detect the change of DNA double strand breaks in esophageal cancer cells;4. Western-blotting was used to detect the expression of cyclin B1 and other cell cycle regulatory proteins in oxidative stress. And Quantitative Real-time PCR was used to detect cyclin B1mRNA level. After pre-incubating with 100 nM cycloheximide (CHX) for one hour and subsequently treated with H2O2, the expression of cyclin B1 was detected by Western-blotting;5. Western-blotting was used to detect the expression of Hsp90a;6. The change of proteasomal activity, proteasome and proteasome-dependent cyclin degradation pathway related protein were detected in oxidative stress;7. The amount of cyclin B1 was detected in TE-1 cells by using the Hsp90 inhibitor 17-DMAG and proteasome inhibitor MG132 as positive controls;8. Cyclin B1 siRNA was used to determine whether cyclin B1 accumulation plays a role in the cell cycle arrest induced by oxidative stress;9. Antioxidant N-acetylcysteine (NAC) was pretreated in TE-1 cells to determine whether elevated production of ROS may have a role in cell cycle arrest after oxidative stress;10. Immunofluorescence and Co-IP were used to detect the co-localization and interactions of Hsp90a and the cyclin B1. And above experimental methods were also used to detect the ubiquitinated cyclin B1;11. Immunohistochemistry was used to demonstrate the change of Hsp90a and cyclin B1 in the central carcinoma nests or keratin pearls in ESCC. And heme oxygenase-1 (HO-1) and phosphorylation of H2A.X at Ser139 were also detected.Results1. Oxidative stress by H2O2 can inhibit the growth of human esophageal cancer TE-1 cells. The expression of phosphorylation of histone H2A.X and Thr5/7-phosphorylated Hsp90a increased from 2 h to 24 h in the H2O2-treated cells as demonstrated by the Western blot;2. Oxidative stress by H2O2 caused G2/M arrest at 24h and the inactivation of CDK1 in TE-1 cells;3. Cyclin B1 was markedly increased within 24 h after exposuring to oxidative stress by H2O2, but a general decrease in the amount of cyclin B1 mRNA transcript was shown, and the amount of cyclin B1 in CHX/H2O2 co-treated cells did not reduce to the baseline level of the negative control at 24h. Oxidative stress by H2O2 also induced the cleavage and deactivation of Hsp90α. In addition, oxidative stress by H2O2 reduces the proteasomal activity in TE-1 cells;4. Targeting Hsp90 with 17-DMAG and inhibiting proteasome with MG132 induce G2/M arrest and the accumulation of cyclin B1 in TE-1 cells.17-DMAG also reduced the proteasomal activity in TE-1 cells;5. Though mRNA level cyclin B1 was lower after SiRNA of cyclin B, the protein level showed obviously increase after H2O2 and 17-DMAG treatment and so as the G2/M arrest;6. The induction of the G2/M phase arrest by H2O2,17-DMAG and MG132 did not involve ROS after pretreatment with NAC.The G2/M phase arrest in TE-1 cells might be due to the inhibition of Hsp90α, rather than the accumulation of ROS;7. Immunofluorescence and Co-IP results demonstrated that oxidative stress by H2O2 reduced cyclin B1 binding to Hsp90a and suppressed cyclin B1 ubiquitination; 8. Our immunohistochemical study showed that Hsp90a expression was negatively correlated to cyclin B1 expression in the developing process of keratin pearls (Hsp90a expression level is low in the central keratin pearl, whereas cyclin B1 is significantly high) in well-differentiated ESCC. In addition, the central carcinoma nests were strongly positive for heme oxygenase-1 (HO-1) and phosphorylation of H2A.X, which indicated oxidative damage came into being.Conclusions1. Oxidative Stress by H2O2-induced G2/M arrest in TE-1 cells is accompanied with the accumulation of cyclin B1 due to the Hsp90 inhibition caused 26S proteasome impairment.2. The results of vitro experiments combined with Immunohistochemistry demonstrated that the formation of keratin pearls in ESCC might be related to oxidative stress, and Hsp90a involves in cyclin B1 expression and cell cycle regulation in keratin pearl formation of ESCC.