Hsp90α Is Involved In Stress Response Of HepG2Liver Carcinoma Cells And L02Hepatocytes To Hydrogen Peroxide Induced Oxidative Stress

BackgroudOxidative stress, resulting from exposure to reactive oxygen species (ROS) and reactive nitrogen species (RNS), which can damage the balance between the oxidant system and antioxidant system and cause the stress injury.Proteins are known to be damaged by reactive oxygen species directly and to be targets of secondary modifications by aldehydic products of lipid peroxidation or glycoxidation(eg:carbonyl-groups in the protein). In many diseases and environmental damage, the imbalance of ROS generation and elimination can cause aging, tumorigenes, cardiovascular disease, neurodegenerative diseases, rheumatoid arthritis、lung inflammation lesions and other diseases.Heat shock proteins (Hsps) are important chaperone proteins produced endogenously and Hsps are a highly conserved family of molecular chaperones. The name "heat shock" was used because some members (e.g., Hsp90and Hsp70) can be induced by various stresses, such as temperature elevation, hypoxia, oxidative damage or cancer. Hsps are classified according to their molecular weight including Hsp100, Hsp90, Hsp70, Hsp60、Hsp40and the small Hsps(<30kDa).The90-kDa heatshock protein, Hsp90, is an abundant molecular chaperone participating in the cytoprotection of eukaryotic cells. It accounts for1-2%of total proteins under basal, nonstressed conditions and is upregulated in cells in response to stressors, during which its abundance increases up to4-6%of cellular proteins. Hsp90has emerged as an important target in cancer therapeutics and has subsequently become the focus of several drug discovery and development efforts.Hsp90is often referred to as the "cancer chaperone". Multiple isoforms of HSP90exist and these include Hsp90a and Hsp90β in the cytoplasm and nucleus, GRP94in the endoplasmic reticulum, and TRAP1in the mitochondria. Hsp90a is inducible and its functions include stress-induced cytoprotection and cell-cycle regulation, whereas Hsp90β is constitutively expressed and is involved in early embryonic development, signal transduction, and long-term cell adaptation. Because of this, Hsp90α is preferred to be selected in the present study.Hydrogen peroxide (H2O2) is a representative membrane-permeable oxidant and is the most abundant ROS in cells and regulates metabolism, aging, apoptosis and the intensity of growth factor signaling. In addition, it acts as a negative or positive regulator of cell signaling, and is very diffusible within and between cells and so any H2O2might presumably diffuse into the cells for removal by catalase, glutathione peroxidases and other H2O2-removing systems.An environment of oxidative stress mediated by H2O2markedly up-regulates the expression of oxidation-sensing regulator. However, the final working effect of H2O2and its relationship with Hsps during tumor and healthy cells under oxidative stress are not clear. In addition, under normal physiological conditions, the body’s antioxidant defense system can timely remove free radicals (as of ROS, RNS), to maintain the body’s redox balance; while under pathological conditions, once free radicals generated beyond the antioxidant defense capacity of the body, they will accumulate in the body, and lead to oxidative stress. Studies have shown that oxidative DNA damage caused by oxidative stress or lipid peroxidation is an important pathogenic mechanism of tumor, the sensitivity to oxidative stress in the tumor cells is significantly higher than that in normal cells. However, the specific mechanism remains to be elucidated.Therefore, determining the H2O2concentration in the culture medium of the oxidative stress model is crucial to understanding the interactions among the H2O2, the cell lines, the culture medium and the Hsps’ micro-environment. The mechanism will provides a new way to explore the mechanism of the various stress disorders,and achieve preventionand therapy of such disease.Objectives1. Under different conditions of oxidative stress, to research the subduction of H2O2in two different culture media(DMEM、RPMI1640) cultered with two different liver cell lines(HepG2, L02cells);2. To establish models of oxidative stress in HepG2and L02cells;3. To make clear the changes of oxidative stress state in HepG2and L02cells under same oxidative stress conditions;4. To study the change of Hsps in HepG2and L02cells, especially Hsp90a under oxidatie stress condition;5. To detect the expression of Hsp90a in human liver tissues, and to further investigate the molecular mechanism of the protective function of heat shock proteins in cells under oxidative stress.Methods 1. Established model of oxidative stress in HepG2and L02cell lines:The cells were divided into control group and oxidative stress group,. Oxidative stress factors: initial concertration of100μM、200μM、500μM、1000μM H2O2to stimulate oxidative stress for the appropriate durations (2h,4h,6h,8h,10h,12h,24h,48h). Use CCK-8to detect cell survival;2. Detect the the final working concertration of H2O2:The cells were divided into groups of oxidative stress included HepG2and L02cells, group of stimulated by H2O2not included HepG2and L02cells. Oxidative stress factors:100μM、200μM、500μM、1000μM H2O2to stimulate oxidative stress for the appropriate durations. Use FOX (Ferrous Oxidation of Xylenol Orange) to detect the final working concertration of H2O2in different culture medium;3. A range of H2O2concentrations (100,200,500,1000μmol/L) at different time points (0,2,4,6,8,10,12,24, and48h) in human liver cancer cell line HepG2and human normal liver cell line L02, the cell viabilty of H2O2treatment was assayed in HepG2and L02cell lines by CCK-8kit, which confirmed the appropriate time course and concentration of HepG2and L02cell lines under the condition of oxidative stress.4. Under100μM H2O2concentration, the HepG2and L02cells were divided into control group and group of oxidative stress to stimulate oxidative stress for the appropriate duration (20min,40min,60min,80min, lOOmin,120min). Use the ROS probe to observe the content of ROS in cells under different conditions. The levels of GSH in intracellular was observed by colorimetric method;5. Use Western blot to detect the protein expression of Hsp90a, Hsp70、Hsp40at different time points of cells stimulated by H2O2;6. Immunohistochemistry assay were used to detect the protein expression of Hsp90a, Hsp70、Hsp40in human liver tissues. Results1. Depending on its concentration, H2O2can have a dual effect on cell viability: Time course of extracellular H2O2concentration following its administration to cell lines (HepG2and L02) and culture medium (DMEM and RPMI1640):After treatment with a range of H2O2concentrations (100,200,500,1000μM) at different time points (0,2,4,6,8,10,12,24, and48h), the activity of H2O2was assayed in HepG2and L02cell lines by CCK-8kit. H2O2induced cell growth inhibition in high concentrations (500-1000μM). Low concentration (100-200μM) stimulated the cell growth on HepG2in DMEM, and had concentration-dependent manner on L02in RPMI1640expect under the H2O2concertration of100μM.2. Different culture medium have different subduction to H2O2:The result of FOX (ferrous ion oxidation-xylenol orange) colorimetric assay showed:in the culture medium, the H2O2concentrations (100,200,500μM) returned to basal level within2hours and higher H2O2concentration could last for almost48h after it had been added to DMEM. By contrast, When used the same H2O2concentration used in RPMI1640, it showed that H2O2lasting longer with slowly reducing no matter what concentrations they are. In DMEM with HepG2, the H2O2reducing phenomenon was parallel to what happened in DMEM culture medium. The lower concentrations of H2O2(100,200μM) reduced quickly to basal level within40min, and the500μM H2O2concentrations returned to basal level within2hours, though at H2O2concentrations of1000μM could last for about10hours after it was added to HepG2cells. In RPMI1640of H2O2’s treatment to L02, it can last on slowly reducing among different H2O2concentrations. They returned to basal level after4hours or longer. Furthermore, H2O2subducted more quickly in cell lines than culture medium only with the prolonging of time;3. HepG2cells are more sensitive than L02cells in terms of hydrogen peroxide induced oxidative stress:Under100μM H2O2concentration in HepG2and L02cells cultured in RPMI1640, the inhibition of cell proliferation was time dependent.With the time prolongation, the cells viability was39%(39.82±2.84) and80%(80.80±3.69) in HepG2and L02cells, respectively. Thus, the concentration of100μM and the time points within2hours were chosen for the following experiments;4. The decreasing of antioxidant capacity of HepG2is more serious than that of L02cells to hydrogen peroxide induced oxidative stress:100μM H2O2treatment induced ROS generation in HepG2was higher than that of L02cells. In contrast, the levels of GSH in intracellular was higher in L02than in HepG2cells with the prolongation of H2O2treatment;5. The western-blotting results demonstrating the different change of Hsp90α、 Hsp70and Hsp40under hydrogen peroxide induced oxidative stress:the results showed that H2O2treatment increased Hsp90a accumulation after20min,40min and60min H2O2treatment, then decreased after80min,100min and120min H2O2treatment in HepG2. Changes of Hsp90a level in L02was parallel to HepG2cells, which increased at20min to40min, and then decreased after60min,80min,100min and120min H2O2treatment. Furthermore, as to Hsp90a level, there was an obvious less of Hsp90a expression in L02than that in HepG2cells. In addition, Western blotting analysis showed the appearance of an additional Hsp90a protein cleavage band of approximately70kDa in both cell lines, which suggested that Hsp90a cleavage and oxidative cleavage of Hsp90a was preferentially observed in cancer cells. As to Hsp70and Hsp40, the protein expression was parallel to Hsp90a, however, did not change so obviously like Hsp90a after oxidative stress;6. The results of immunohistochemical detecting the expression of Hsp90a in human liver cancer tissues:Immunohistochemical analysis showed the parallel tendency to western blot, which was that Hsp90a overexpressed and Hsp70and Hsp40expressed weakly in liver cancer tissues.Conclusions1. Under the conditions of oxidative stress, the experimental results may have significance difference due to the type of culture medium and cell lines;2. Oxidative stress has a dual effect both in liver cancer cell and liver normal cells, because the actual H2O2concentrations decreased either instantly or gradually. in different culture medium Cancer cells and normal cells’reactivity to hydrogen peroxide induced oxidative stress is different.3. The basal level of oxidative stress in liver cancer cells were significantly higeer than the normal liver cells and went higher after hydrogen peroxide induced oxidative stress comparing to normal liver cells with low cell viability these indicated that the liver tumor cells were more vulnerable to oxidative stress to normal liver cells;4. Under the conditions of oxidative stress, the protein level of Hsp90a was different among Hsp70and Hsp40between liver canner cells and liver normal cells, and the oxidative stress caused a cleavage of Hsp90a and, it appears to preferentially affect cancer cells (40min) earlier than normal cells (80min), indicating that Hsp90a was obvious sensitive than Hsp70and Hsp40and liver tumor cells of Hsp90a’ oxidative stress sensitivity is higher than normal cells.