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Hyperglycaemia and oxidative stress upregulate HSP60 & HSP70 expression in HeLa cells

Hyperglycaemia and oxidative stress upregulate HSP60 & HSP70 expression in HeLa cells Heat Shock Proteins 60 & 70 (HSP60 & HSP70) are intracellular protein that has been shown to be present at elevated levels in systemic circulation in Type 2 Diabetes mellitus (T2DM) patients. Conditions that lead to its secretion, and the mechanism of its translocation from cells, have not yet been defined. The aim of this study was to determine if specific cell stressors associated with T2DM, namely hyperglycaemia and oxidative stress, result in the upregulation of HSP60 in human cells in vitro. Human HeLa cells were grown in media supplemented with 100 mM glucose, 200 μM hydrogen peroxide (H O ), and 50 μM sodium azide. Initially, the effect of these 2 2 treatments on cell growth rate was examined, with each treatment significantly inhibiting growth rate. LDH and MTT assays were also used to successfully demonstrate that these treatments do not significantly increase cell lysis, but do significantly impair mitochondrial dehydrogenase activity. To confirm this mitochondria specific form of inhibition, DCFDA assay were used to investigate any increases in intracellular reactive oxygen species (ROS) generation. All three treatments resulted in significantly increased ROS generation, with greater ROS production occurring with a greater exposure time. Interestingly, when the protein levels of HSP60 and HSP70 were measured after 3 and 7 days of exposure of the HeLa cells to 100 mM glucose, 200 μMH O , and 50 μM sodium azide 2 2 significant induction of these two molecular stress proteins were observed ranging from 2.43-5.08 fold compared to untreated control cells. Keywords: HSP60; HSP70; Mitochondria; Diabetes mellitus; Hyperglycaemia; Oxidative stress Introduction tionally related proteins found in all living organisms. T2DM is the most common metabolic disease in the Their expression is increased in response to various world, and the World Health Organisation (WHO) has cellular stressors in what is referred to as the heat predicted that T2DM related deaths will double between shock response (Ritossa, 1996). The induction of one 2005 and 2030 (Danaei et al. 2011). Patients with T2DM particular HSP, HSP60, has been found to be correlated have an increased risk of cardiovascular disease (CVD), with mitochondria specific cell stress (Martinus et al., which is responsible for 50-80% of deaths in people with 1996). Recently HSP60 has been found to be secreted diabetes (Danaei et al. 2011). and expressed extracellularly, after first being thought T2D has been found to be associated with mitochon- to be strictly intracellular where it performs roles as a drial specific cell stress (Giacco and Brownlee, 2010). chaperone and in protein folding (Yuan et al., 2011). The primary stressors that elicit this response in T2D However, these mechanisms of translocation and secretion are hyperglycaemia and oxidative stress (Mulder and have not been clearly identified. Extracellularly expressed Ling, 2009; Kim et al., 2008; Lowell and Shulman, 2005). HSP60 is believed to contribute to atherosclerotic de- T2D has also been found to be associated with elevated velopment (Ellins et al., 2008; Pockley et al., 2003). levels of heat shock protein 60 (HSP60) (Aguilar-Zavala It is therefore plausible that the stressors associated et al., 2008; Nakhjavani et al., 2010; Yuan et al., 2011). with T2D are responsible for the elevated levels of extra- HSPs are a class of ubiquitously expressed and func- cellular HSP60 seen in T2D (Nakhjavani et al., 2010; Yuan et al., 2011). To determine if hyperglycaemia and oxidative stress results in the induction of HSP60 * Correspondence: martinus@waikato.ac.nz Department of Biological Sciences, Faculty of Science & Engineering, The expression, HeLa cells were grown in media supplemented University of Waikato, Private Bag 3105, Hamilton, New Zealand © 2013 Hall and Martinus; licensee Springer. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Hall and Martinus SpringerPlus 2013, 2:431 Page 2 of 10 http://www.springerplus.com/content/2/1/431 with glucose and hydrogen peroxide to simulate hypergly- 100 μM). The plate was then returned to incubate in caemia and oxidative stress respectively. Sodium azide standard incubation conditions for 3 days, when the cells was used as a third treatment as it is a known mitochon- would be in the exponential growth phase. After 72 hours, drial stressor, irreversibly binding to the heme component the media was removed and replaced with 10 μL of complex IV in the electron transport chain. LDH reconstituted MTT in DMEM. The plate was then and MTT assays were also implemented to show that incubated at standard incubation conditions for 2 hours. the selected concentrations of glucose, hydrogen peroxide, After the incubation period, the culture fluid was removed. and sodium azide did not lead to cell lysis, but did inhibit This was followed by the addition of 100 μLMTT mitochondrial bioenergetics functions. ROS generation Solubilisation Solution. The plate was then placed on a was also measured using a DCFDA assay, as evidence minishaker for 10 minutes to assist in dissolving the in the literature suggests that ROS activity mediates the crystals, and the absorbance was read at 570 nm and mechanisms of HSP induction (Ahn and Thiele, 2002). thebackgroundreadat655 nm. HeLa cells were used as a representation of a peripheral tissue. DCFDA assay Near confluent HeLa cells were seeded onto a 96-well Methods black culture plate in 100 μL of DMEM supplemented Cell culture and dose response with glucose (25-125 mM), H O (0-250 μM), and sodium 2 2 HeLa cells were grown in complete DMEM media at azide (0-100 μM). The plate was then returned to incubate 37°C, 5% CO , in a humidified incubator (standard in standard incubation conditions for time periods of incubation conditions). Cells were passaged every 7 days, 24 hours, 3 days, and 7 days. At the end of the incubation and media was changed 3 days after passage. For the period, the DMEM media was removed and the cells dose response experiments, cells were seeded at a density were washed with 100 μL pre-warmed PBS. The cells of approximately 60,000 cells/mL onto 24 well plates were then incubated for a further hour in 100 μL Opti- containing a range of glucose (25-125 mM), H O (0- MEM containing only 2% FBS and further supplemented 2 2 250 μM), and sodium azide (0-100 μM) concentrations. with 2.5 μLof400 μM DCFDA. After the one hour incu- Cell density was estimated by the trypan blue exclusion bation period, the media was again removed and the method every 24 hours over a 7 day period. cells washed twice with 100 μL PBS. 200 μL Opti-MEM was then added, containing 2% FBS, and the fluorescence LDH assay intensity over a 30 minute period at excitation and Near confluent HeLa cells were seeded onto a 96-well emission wavelengths of 485 nm and 520 nm respectively plate in 100 μL of DMEM supplemented with glucose were determined. Data was standardised to the control (25-125 mM), H O (0-250 μM), and sodium azide (0- analysis done in the absence of H O (at the three dif- 2 2 2 2 100 μM). The plate was then returned to incubate in ferent incubation times (24 h, 3 & 7 days)) and then standard incubation conditions for 3 days, when the cells expressed as relative fluorescence units to the controls. would be in the exponential growth phase. After which, one set of control cells (maximum control) was treated Heat shock control cells with 100 μL 10× lysis solution and the plate was returned As a means of obtaining a positive control a heat shock to standard incubating conditions for 45 minutes. After treatment was utilised (Martinus et al., 1996). HeLa cells this time period, the plate was centrifuged at 250×g for were grown in a culture flask for 3 days, so as to reach 5 minutes at room temperature (RT) to pellet cell debris. the exponential growth stage. The media was then 50 μL of the supernatant from each well of the culture replaced with DMEM pre-warmed to 45°C, and the plate was transferred to the corresponding well of a flask was placed into a 45°C incubator for 20 minutes. 96-well enzymatic assay plate. 50 μLofreconstituted Following heat treatment, the media was again replaced, substrate was then added to the supernatant in each this time with DMEM pre-warmed to 37°C. The culture well of the assay plate. The plate was then covered was returned to standard incubating conditions for from light and incubated at RT for 30 minutes. The 6 hours. After this time, the cells were harvested for assay was terminated by the addition of 50 μLofStop protein extraction. Solution to each well of the plate and the absorbance was read at 490 nm. Protein extraction Total protein was isolated using TENT buffer and freshly MTT assay added 0.4 mM phenylmethylsulfonyl fluoride (PMSF) Near confluent HeLa cells were seeded onto a 96-well as a protease inhibitor. Samples were ruptured with plate in 100 μL of DMEM supplemented with glucose 100 μL TENT buffer by vortexing, and were subsequently (25-125 mM), H O (0-250 μM), and sodium azide (0- incubated at 4°C for 30 minutes. After this incubation 2 2 Hall and Martinus SpringerPlus 2013, 2:431 Page 3 of 10 http://www.springerplus.com/content/2/1/431 period, samples were spun at 10,000 rcf for 5 minutes at to determine the significance of the data, and the accepted 4°C to pellet cell debris. The supernatant containing whole level of significance was p < 0.05, which was denoted as ’*’. protein was transferred to a new tube and stored at −20°C. Results and discussion Cell culture and dose response curves Protein separation and transfer Cells treated with 50, 100 and 150 μMH O and 1, 10 and 2 2 Total protein was separated on a 10% polyacrylamide 25 μM sodium azide showed growth rates comparable, discontinuous gel (0.76 mm) and run in a Mini-Protean if not greater than to that of untreated control cultures. 3 Cell gel tank. Cells treated with 50 and 75 mM glucose showed a slightly Each sample containing 20 μg total protein was made reduced growth rate compared with the control. Finally up to 20 μL with protein loading buffer. The samples the 100 and 125 mM glucose groups, the 200 and 250 μM were denatured in boiling water for approximately 5 H O , and the 50 and 100 μM sodium azide groups had 2 2 minutes before loading into the wells. The gel was considerably reduced growth rate through to the end of electrophoresed at a constant 20 mA as the bands ran the culture period. through the stacking gel, and 30 mA through the To compare cell growth across treatments, the num- resolving gel. The end point was determined as when ber of cells at each time point was normalised to the theloading dyeran offthe gel. number of control cells. The rate of cell growth was Semi-dry transfer was completed using a PVDF mem- determined during the exponential growth phase be- braneand a3-buffersystemcontainingcathode,anode tween days 3 and 5 of the culture period, and is exhibited I and anode II buffers. The transfer cell was run at in Figure 1a, b, and c. 15 V for 35 minutes. After the transfer was complete, As was to be expected, increasing the concentration themembranewas brieflyrinsedindistilled water of the treatment resulted in increased inhibition of cell before being stained with Ponceau S for 15 seconds to growth. Slight growth inhibition resulted from treatments ascertain whether efficient transfer had occurred. The of 50 and 75 mM glucose (11% and 30% inhibition stain was then washed out with distilled water. respectively), 150 μMH O (4%), and 10 and 25 μM 2 2 sodium azide (11% and 9% respectively). Strong growth Western blotting inhibition resulted from treatments of 100 and 125 mM The membrane was blocked in 10% skim milk in TBST glucose (36% and 84% respectively), 200 and 250 μMH O 2 2 overnight at 4°C. The following day, the membrane was (54% and 91% respectively), and 50 and 100 μMsodium washed in TBST. The membrane was then incubated in azide (47% and 78% respectively). the primary polyclonal rabbit HSP60 (Sapphire Bioscience) It is interesting that a degree of hormesis was present 1: 250 in 5% skim milk in TBST overnight at 4° in a in all treatments. Hormesis is the name given to the humidified chamber. After another set of washes in TBST, stimulatory effects caused by low levels of potentially the membrane was incubated in peroxidase conjugated toxic agents (Calabrese et al., 2012). The 25 mM glucose, goat anti-rabbit IgG (Sigma) 1:1000 in 5% skim milk in and 50 and 100 μMH O treatments all resulted in 2 2 TBST for 5 hours in a humidified chamber on a gyro increased growth rates compared to the control over the rocker. After another set of washes in TBST on a gyro exponential growth phase. The 1 μM sodium azide was rocker, the membrane was transferred onto a glass plate found to have a comparable growth rate to the control. and incubated for 1 minute with SuperSignal West Pico While it may not be surprising that the comparatively Chemiluminescent Substrate (Pierce). The membrane was low glucose levels result in increased growth, as it would visualised using the LAS-100 Plus Gel Documentation convey a greater energy source to the cell culture, it is of System (Fujifilm), and the intensity of the bands was interest that H O does increase growth, which agrees 2 2 measured using Gel Quant software. After stripping, the with results of previous studies (Burdon, 1995). H O 2 2 same blots were then probed with polyclonal rabbit and its stimulatory effect on cell proliferation have been HSP70 (Sapphire Bioscience). The Quant values generated of particular interest as it has been identified that the for the proteins of interest were normalised to the house elevated levels of H O that result from regular exercise 2 2 keeper gene actin after probing the same membrane have a hormetic effect, indicating that minor oxidative with the anti-actin antibody. stress may have a beneficial effect (Radak et al., 2008). To confirm that the impaired cell growth noted above Statistical analysis was indeed a result of mitochondrial inhibition, and not All statistical analysis in this study was carried out using necrotic cell death, the release of lactate dehydrogenase Microsoft Excel. Data was averaged where appropriate, (LDH) into the growth media and formazan formation and the standard error of the mean (S.E.M.) was calculated via the MTT assays as an indicator of mitochondrial de- using Excel. A two-tailed student’s t-test was carried out hydrogenase activity were carried out. Hall and Martinus SpringerPlus 2013, 2:431 Page 4 of 10 http://www.springerplus.com/content/2/1/431 Figure 1 Growth rates of HeLa cells supplemented with glucose, H2O2 and sodium azide. a- Growth rate of HeLa cells supplemented with glucose (25-125 mM). Error bars showing S.E.M. The growth rate between days 3 and 5 has been taken as the growth rate during the exponential phase of growth. b- Growth rate of HeLa cells supplemented with H O (0-250 μM). Error bars showing S.E.M. The growth 2 2 rate between days 3 and 5 has been taken as the growth rate during the exponential phase of growth. c- Growth rate of HeLa cells supplemented with sodium azide (0-100 μM). Error bars showing S.E.M. The growth rate between days 3 and 5 has been taken as the growth rate during the exponential phase of growth. Hall and Martinus SpringerPlus 2013, 2:431 Page 5 of 10 http://www.springerplus.com/content/2/1/431 LDH assay lead to an increase in cell lysis, indicative of a potential After 3 days in culture, the percentage of LDH content state of cellular stress. To determine if this stress is relative to corresponding maximum control samples was targeted at the level of the mitochondria MTT assays calculated. It was assumed that every dead cell released were implemented. an equal amount of LDH. Therefore, the percentage of LDH content was interpreted as the percentage of lysed MTT assay cells, as shown in Figure 2. After 3 days in culture, cells were incubated with MTT for The glucose control sample had 25.50 ± 1.31% lysed 2 hours. After which the formazan crystals were solubilised, cells, and was not significantly different from the 75 the absorbance read, and results were interpreted as a and 100 mM glucose groups, which had 22.76 ± 1.29% percentage of mitochondrial activity relative to the control and 26.46 ± 1.00% of lysed cells, respectively. However, group, as seen in Figure 3. treatment with 125 mM glucose resulted in a significantly All glucose samples were found to have significantly increased (p < 0.05) percentage of lysed cells compared decreased (p < 0.05) mitochondrial dehydrogenase activity to the control; resulting in 31.46 ± 1.37% lysed cells. compared to control cells. 75 mM glucose resulted in The H O control sample had 27.84 ± 3.04% lysed cells, 84.29 ± 1.20%, 100 mM resulted in 78.21 ± 1.13%, and 2 2 and was not significantly different from the 150 and 125 mM glucose resulted in 73.96 ± 1.12% mitochon- 200 μMH O groups, which had 27.61 ± 2.33% and drial dehydrogenase activity. 2 2 30.69 ± 2.19% of lysed cells, respectively. However, samples All H O samples were found to have significantly de- 2 2 treated with 250 μMH O resulted in a significantly in- creased (p < 0.05) mitochondrial dehydrogenase activity 2 2 creased (p < 0.05) percentage of lysed cells compared to relative to control cells. 150 μMH O resulted in 87.70 ± 2 2 the control; resulting in 37.71 ± 3.09% lysed cells. 3.07%, 200 μM resulted in 81.97 ± 1.41%, and 250 μM The sodium azide control sample had 24.42 ± 1.13% resulted in 59.61 ± 4.21% mitochondrial dehydrogenase lysed cells, and was not significantly different from the activity. 25 and 50 μM sodium azide groups, which had 26.80 ± All sodium azide samples were found to have signifi- 1.18% and 27.57 ± 1.51% of lysed cells, respectively. cantly decreased (p < 0.05) mitochondrial dehydrogenase However, treatment with 100 μM sodium azide resulted activity relative to control cells. 25 μMsodiumazide in a significantly increased (p < 0.05) percentage of lysed resulted in 91.76 ± 2.27%, 50 μM resulted in 73.61 ± 3.53%, cells compared to the control; resulting in 32.08 ± 2.85% and 100 μM resulted in 75.33 ± 1.20% mitochondrial de- lysed cells. hydrogenase activity. From the LDH assay results it can be concluded that From the MTT assay results it can be concluded that 100 mM glucose, 200 μM hydrogen peroxide and 50 μM 100 mM glucose, 200 μM hydrogen peroxide and 50 μM sodium azide do not result in significant cell lysis com- sodium azide do result in significant inhibition of mito- pared to control cells. These concentrations are of par- chondrial bioenergetics compared to control cells. In ticular interest as they reduced growth rate but do not conjunction with the dose response and LDH results, Figure 2 Percentage of lysed cells in the presence of glucose, H O , and sodium azide. The only concentrations that gave significantly 2 2 elevated degrees of cell lysis compared to the control group were the maximum treatment concentrations. All other treatment concentrations had no significant difference in the degree of cell lysis. (* represents statistically significant values, p < 0.05). Hall and Martinus SpringerPlus 2013, 2:431 Page 6 of 10 http://www.springerplus.com/content/2/1/431 Figure 3 Mitochondrial dehydrogenase activity in the presence of glucose, H O , and sodium azide. All concentrations significantly 2 2 inhibited mitochondrial bioenergetic function compared to respective control. (* represents statistically significant values, p < 0.05). the MTT results indicate that 100 mM glucose, 200 μM resulted in 112.93 ± 2.71%, and the sodium azide treat- hydrogen peroxide and 50 μM sodium azide result in ment resulted in 110.77 ± 2.24% ROS levels. mitochondrial specific cell stress. To further support this After 3 days, all samples were found to have signifi- conclusion, DCFDA assays were used to investigate any cantly increased (p < 0.05) ROS levels. The glucose treat- ROS generation, a common association with mitochon- ment resulted in 123.63 ± 6.24%, the H O treatment 2 2 drial impairment. resulted in 135.36 ± 7.33%, and the sodium azide treat- ment resulted in 134.77 ± 7.15% ROS levels. DCFDA assay After 7 days, all samples were found to have signifi- After 1, 3, and 7 days in culture, cells were incubated cantly increased (p < 0.05) ROS levels. The glucose treat- with DCFDA for 1 hour. After which the fluorescence ment resulted in 179.95 ± 23.78%, the H O treatment 2 2 was read, and results were normalised to the H O absent resulted in 304.56 ± 29.02%, and the sodium azide treat- 2 2 control. Results were interpreted as a percentage of ROS ment resulted in 279.13 ± 43.82% ROS levels. levels relative to the control group, as seen in Figure 4. The DCFDA assay results show that increasing the time After 24 hours, all samples were found to have signifi- of exposure to each of the treatments increases the degree cantly increased (p < 0.05) ROS levels. The glucose treat- of intracellular ROS generation, supporting the conclusion ment resulted in 109.06 ± 2.27%, the H O treatment that 100 mM glucose, 200 μM hydrogen peroxide, and 2 2 Figure 4 ROS levels in the presence of glucose, H O , and sodium azide over 24 hours, 3, and 7 days. Each sample contained significantly 2 2 elevated ROS activity after 24 hours, which continued to rise over 3 and 7 days. A dramatic increase in ROS activity is observed between days 3 and 7. (* represents statistically significant values, p < 0.05). Hall and Martinus SpringerPlus 2013, 2:431 Page 7 of 10 http://www.springerplus.com/content/2/1/431 50 μM sodium azide result in mitochondrial specific HSP60 protein expression was found to be upregulated cell stress. Increased levels of intracellular ROS activity in all treatments over 3 days, as demonstrated in Figures 5a have been shown to induce a concentration-dependent and 6. The heat shocked cells had a 2.23 fold increase in transactivation and DNA-binding activity of heat shock HSP60 expression, while the 100 mM glucose, 200 μM factor-1 (HSF-1), the principal transcription factor of H O , and 50 μM sodium azide had 1.61, 1.54 and 2.12 2 2 HSP60 (Jacquier-Sarlin and Polla, 1996). Therefore it fold increases respectively. All were significantly different can be expected that if these treatments do induce a heat to the control (p < 0.05). shock response, a longer treatment period will result in Additionally, HSP60 protein expression was found to a more pronounced induction of HSP60. Western blots be further upregulated in all treatments over a 7 day were implemented to investigate the effect of 100 mM treatment period, as shown in Figures 5b and 6. The glucose, 200 μM hydrogen peroxide, and 50 μMsodium heat shocked cells had a 2.38 fold increase in HSP60 ex- azide on HSP60 & HSP70 expression. pression according to these membranes. The 100 mM glucose, 200 μMH O , and 50 μM sodium azide had 2 2 Western blotting 2.43, 3.58 and 4.74 fold increases respectively, and each After total protein was separated on a polyacrylamide was significantly different from the control (p < 0.05). gel and transferred to a PVDF membrane, Ponceau S The western blots for HSP60 show that treatment with staining was used to show the standard HSP60 band to glucose (100 mM), H O (200 μM), and sodium azide 2 2 be at 60 kDa and that protein loading was even. After (50 μM) all result in a significant increase in expression western blot, a single band for each sample was seen of HSP60. Length of exposure appears to have a pro- and the bands appeared to be the same size as the stand- nounced effect on HSP60 induction, with the 7 day ard HSP60 protein band. The bands were then quantitated treatment having a marked increase in HSP60 expres- using the Gel Quant software, and the relative expression sion. This drastic rise appears to mirror that seen in to the control was plotted. ROS activity over the same time period. HS Control Glucose H O NaN 2 2 3 HSP60 Actin HS Control Glucose H O NaN 2 2 3 HSP60 Actin Figure 5 Western blots of HSP60 expression in HeLa cells treated with heat shock, glucose, H2O2 and sodium azide over 3 and 7 days. a- Western blots of HSP60 and actin in cells treated with heat shock, glucose (100 mM), H O (200 μM), and sodium azide (50 μM), respectively, 2 2 over 3 days. The greatest induction of HSP60 is observed in the sodium azide treated sample, with similar levels of HSP60 observed in the glucose and hydrogen peroxide treatments. HSP60 was tested for a minimum of three times on different blots to provide an accurate representation of its induction. b- Western blots of HSP60 and actin in cells treated with heat shock, glucose (100 mM), H O (200 μM), and 2 2 sodium azide (50 μM) over 7 days. The greatest induction of HSP60 is observed in the sodium azide treated sample, followed by the hydrogen peroxide treatment, with samples treated with glucose having a lesser degree of induction. HSP60 was tested for a minimum of three times on different blots to provide an accurate representation of its induction. Hall and Martinus SpringerPlus 2013, 2:431 Page 8 of 10 http://www.springerplus.com/content/2/1/431 Figure 6 Relative HSP60 expression in cells treated with heat shock, glucose (100 mM), H2O2 (200 μM), and sodium azide (50 μM) over both 3 and 7 days. All treatments and time periods give a significantly increased level of expression of HSP60 compared to control samples. (* represents statistically significant values, p < 0.05). The fold change in HSP60 expression is even across all treatments after 3 days followed by a dramatic rise in expression levels after 7 days, a similar trend to that seen in the DCFDA assay. Even though the Gel Quant results for HSP60 induc- the western blots were re-probed with antibodies against tion are an average of three different blots, the band in- HSP70. Figures 7a,b, and 8 clearly provide additional tensities seen in the images in Figure 5a and b are not evidence that a general cellular stress response is being entirely convincing. To further support the conclusion evoked, resulting in a heat shock response from at least that the cellular stressors being investigated was indeed HSP60 and HSP70. The degree of induction of HSP70 resulting in the induction of molecular stress proteins, closely mirrors that of HSP60. HS Control Glucose H O NaN 2 2 3 HSP70 Actin HS Control Glucose H O NaN 2 2 3 HSP70 Actin Figure 7 Western blots of HSP70 expression in HeLa cells treated with heat shock, glucose, H2O2 and sodium azide over 3 and 7 days. a- Western blots of HSP70 and actin in cells treated with heat shock, glucose (100 mM), H O (200 μM), and sodium azide (50 μM) over 3 days. 2 2 The greatest induction of HSP70 is observed in the sodium azide treated sample, followed by the hydrogen peroxide and glucose treated samples. HSP70 was tested for a minimum of three times on different blots to provide an accurate representation of its induction. b- Western blots of HSP70 and actin in cells treated with heat shock, glucose (100 mM), H O (200 μM), and sodium azide (50 μM) over 7 days. The greatest 2 2 induction of HSP70 is observed in the sodium azide treated sample, followed by the hydrogen peroxide treatment, with samples treated with glucose having a lesser degree of induction. HSP70 was tested for a minimum of three times on different blots to provide an accurate representation of its induction. Hall and Martinus SpringerPlus 2013, 2:431 Page 9 of 10 http://www.springerplus.com/content/2/1/431 Figure 8 Relative HSP70 expression in cells treated with heat shock, glucose (100 mM), H O (200 μM), and sodium azide (50 μM) over 2 2 both 3 and 7 days. All treatments and time periods give a significantly increased level of expression of HSP70 compared to control samples. (* represents statistically significant values, p < 0.05). The fold change in HSP70 expression is even across all treatments after 3 days followed by a dramatic rise in expression levels after 7 days, a similar trend to that seen in the DCFDA assay. HSP60 plays a critical role in the molecular cellular and Polla, 1996). Sensing of the oxidative stress requires stress response targeted at the level of the mitochondrion. two cysteine residues within the HSF-1 DNA-binding do- The primary role of HSPs is that of a molecular chap- main that are engaged in redox-sensitive disulfide bonds. erone, where they act to mediate the folding, assembly or HSF-1 derivatives in which either or both of these cysteine translocation across the intracellular membranes of other residues are mutated have been found to be defective polypeptides; and a role in protein degradation, making in stress-inducible trimerization and DNA binding, stress- up some of the essential components of the cytoplasmic inducible nuclear translocation and HSP gene trans- ubiquitin-dependent degradative pathway (Burel et al., activation, and in the protection of mouse cells from 1992). Additionally, when exposed to a various proteotoxic stress-induced apoptosis (Ahn and Thiele, 2002). In stressors, the expression of HSPs is induced in order to this way, H O is thoughttoexertstwo effectson the 2 2 minimise cellular damage, as well as to stave off apoptosis, activation and the DNA-binding activity of HSF; H O 2 2 by stabilising compromised proteins (Santoro, 2000). favours the nuclear translocation of HSF, while also alter- The inducible HSP component of a cell’s total HSP ing the HSFs DNA-binding activity, which is achieved by pool is regulated by HSFs, of which HSF-1 is the major oxidizing the two critical cysteine residues within the regulator. In the absence of cellular stress, HSF-1 is DNA-binding domain (Jacquier-Sarlin and Polla, 1996). inhibited due to its association with HSPs and is therefore Hyperglycaemia has been reported to increase oxidative maintained in an inactive state. If and when a cellular stress by increasing the rate of glycolysis while inhibiting stress does occur, the HSPs bind to any misfolded pro- oxidative phosphorylation (Crabtree, 1928). The build-up, teins, and subsequently dissociate from HSF-1. This allows and subsequent auto-oxidation, of glyceraldehyde-6-phos- the HSF-1 monomers to oligomerise and form active phate which ensues from increased glycolysis results in trimers, regaining their DNA binding activity. The trimers the generation of H O . As a result, the mitochondrion 2 2 undergo stress-induced serine phosphorylation and are experiences an increase in ROS generation. As mentioned translocated to the nucleus (Prahlad and Morimoto, earlier, activation of HSF-1, the major regulator of HSPs, 2008). Upon nuclear localisation, HSF-1 binds to the HSE is redox dependent. situated upstream of heat shock responsive genes, which results in HSP gene transcription. However, the mecha- Conclusion nisms for stress sensing and signalling to activate HSF-1 This study demonstrated that hyperglycaemic conditions have not been fully elucidated. However, there is growing and oxidative stress can lead to the induction of HSP60 evidence in the literature that this mechanism is mediated and HSP70expression, suggesting that the increased serum by ROS, and in particular H O (Ahn and Thiele, 2002). levels of these molecular stress proteins observed in 2 2 H O has been previously documented to induce a T2DM patients could also be due to uncontrolled hyper- 2 2 concentration-dependent transactivation and DNA-bind- glycaemia and oxidative stress. Interestingly there was also ing activity of HSF-1, although to a lesser extent than a significant and concomitant increase in the intracellular that of the classical heat shock treatment (Jacquier-Sarlin levels of ROS generated during the exposure of the HeLa Hall and Martinus SpringerPlus 2013, 2:431 Page 10 of 10 http://www.springerplus.com/content/2/1/431 cells to the stressors being investigated; suggesting that Ritossa FM (1996) Discovery of the heat shock response. Cell Stress Chaperones 1(2):97–98 the induction of HSP70 & HSP60 was related to ROS Santoro MG (2000) Heat shock factors and the control of the stress response. mediated processes. Biochem Pharmacol 59(1):55–63 Yuan J, Dunn P, Martinus RD (2011) Detection of HSP60 in saliva and serum from type 2 diabetic and Non-diabetic control subjects. Cell Stress Competing interests Chaperones 16(6):689–693 The authors declare that they have no competing interests. doi:10.1186/2193-1801-2-431 Authors’ contribution Cite this article as: Hall and Martinus: Hyperglycaemia and oxidative LH carried out the experiments described in the study as part of his MSc stress upregulate HSP60 & HSP70 expression in HeLa cells. SpringerPlus 2013 2:431. research program at The University of Waikato under the supervision of RDM. All authors have read and approved the final manuscript. Received: 26 August 2013 Accepted: 30 August 2013 Published: 3 September 2013 References Aguilar-Zavala H, Garay-Sevilla ME, Malacara JM, Perez-Luque EL (2008) Stress, inflammatory markers and factors associated in patients with type 2 diabetes mellitus. Stress Health 24(1):49–54 Ahn SG, Thiele DJ (2002) Redox regulation of mammalian heat shock factor 1 is essential for Hsp gene activation and protection from stress. 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Eur J Biochem 240:98–103 Mulder H, Ling C (2009) Mitochondrial dysfunction in pancreatic β-cells in type 2 diabetes. Mol Cell Endocrinol 297:34–40 Nakhjavani M, Morteza A, Khajeali L, Esteghamati A, Khalilzadeh O, Asgarani F, Submit your manuscript to a Outeiro TF (2010) Increased serum HSP70 levels are associated with the journal and benefi t from: duration of diabetes. Cell Stress Chaperones 15:959–964 Pockley AG, Georgiades A, Thulin A, de Faire U, Frostegard J (2003) Serum heat 7 Convenient online submission shock protein 70 levels predict the development of atherosclerosis in 7 Rigorous peer review subjects with established hypertension. J Hypertens 42(3):235–238 Prahlad V, Morimoto RI (2008) Integrating the stress response: lessons for 7 Immediate publication on acceptance neurodegenerative diseases from C. Elegans. Trends Cell Biol 19(2):52–61 7 Open access: articles freely available online Radak Z, Chung HY, Koltai E, Taylor AW, Goto S (2008) Exercise, oxidative stress 7 High visibility within the fi eld and hormesis. Ageing Res Rev 7:34–42 7 Retaining the copyright to your article Submit your next manuscript at 7 springeropen.com http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png SpringerPlus Springer Journals

Hyperglycaemia and oxidative stress upregulate HSP60 & HSP70 expression in HeLa cells

SpringerPlus , Volume 2 (1) – Sep 3, 2013

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Copyright © 2013 by Hall and Martinus; licensee Springer.
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Science; Science, general
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2193-1801
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10.1186/2193-1801-2-431
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Abstract

Heat Shock Proteins 60 & 70 (HSP60 & HSP70) are intracellular protein that has been shown to be present at elevated levels in systemic circulation in Type 2 Diabetes mellitus (T2DM) patients. Conditions that lead to its secretion, and the mechanism of its translocation from cells, have not yet been defined. The aim of this study was to determine if specific cell stressors associated with T2DM, namely hyperglycaemia and oxidative stress, result in the upregulation of HSP60 in human cells in vitro. Human HeLa cells were grown in media supplemented with 100 mM glucose, 200 μM hydrogen peroxide (H O ), and 50 μM sodium azide. Initially, the effect of these 2 2 treatments on cell growth rate was examined, with each treatment significantly inhibiting growth rate. LDH and MTT assays were also used to successfully demonstrate that these treatments do not significantly increase cell lysis, but do significantly impair mitochondrial dehydrogenase activity. To confirm this mitochondria specific form of inhibition, DCFDA assay were used to investigate any increases in intracellular reactive oxygen species (ROS) generation. All three treatments resulted in significantly increased ROS generation, with greater ROS production occurring with a greater exposure time. Interestingly, when the protein levels of HSP60 and HSP70 were measured after 3 and 7 days of exposure of the HeLa cells to 100 mM glucose, 200 μMH O , and 50 μM sodium azide 2 2 significant induction of these two molecular stress proteins were observed ranging from 2.43-5.08 fold compared to untreated control cells. Keywords: HSP60; HSP70; Mitochondria; Diabetes mellitus; Hyperglycaemia; Oxidative stress Introduction tionally related proteins found in all living organisms. T2DM is the most common metabolic disease in the Their expression is increased in response to various world, and the World Health Organisation (WHO) has cellular stressors in what is referred to as the heat predicted that T2DM related deaths will double between shock response (Ritossa, 1996). The induction of one 2005 and 2030 (Danaei et al. 2011). Patients with T2DM particular HSP, HSP60, has been found to be correlated have an increased risk of cardiovascular disease (CVD), with mitochondria specific cell stress (Martinus et al., which is responsible for 50-80% of deaths in people with 1996). Recently HSP60 has been found to be secreted diabetes (Danaei et al. 2011). and expressed extracellularly, after first being thought T2D has been found to be associated with mitochon- to be strictly intracellular where it performs roles as a drial specific cell stress (Giacco and Brownlee, 2010). chaperone and in protein folding (Yuan et al., 2011). The primary stressors that elicit this response in T2D However, these mechanisms of translocation and secretion are hyperglycaemia and oxidative stress (Mulder and have not been clearly identified. Extracellularly expressed Ling, 2009; Kim et al., 2008; Lowell and Shulman, 2005). HSP60 is believed to contribute to atherosclerotic de- T2D has also been found to be associated with elevated velopment (Ellins et al., 2008; Pockley et al., 2003). levels of heat shock protein 60 (HSP60) (Aguilar-Zavala It is therefore plausible that the stressors associated et al., 2008; Nakhjavani et al., 2010; Yuan et al., 2011). with T2D are responsible for the elevated levels of extra- HSPs are a class of ubiquitously expressed and func- cellular HSP60 seen in T2D (Nakhjavani et al., 2010; Yuan et al., 2011). To determine if hyperglycaemia and oxidative stress results in the induction of HSP60 * Correspondence: martinus@waikato.ac.nz Department of Biological Sciences, Faculty of Science & Engineering, The expression, HeLa cells were grown in media supplemented University of Waikato, Private Bag 3105, Hamilton, New Zealand © 2013 Hall and Martinus; licensee Springer. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Hall and Martinus SpringerPlus 2013, 2:431 Page 2 of 10 http://www.springerplus.com/content/2/1/431 with glucose and hydrogen peroxide to simulate hypergly- 100 μM). The plate was then returned to incubate in caemia and oxidative stress respectively. Sodium azide standard incubation conditions for 3 days, when the cells was used as a third treatment as it is a known mitochon- would be in the exponential growth phase. After 72 hours, drial stressor, irreversibly binding to the heme component the media was removed and replaced with 10 μL of complex IV in the electron transport chain. LDH reconstituted MTT in DMEM. The plate was then and MTT assays were also implemented to show that incubated at standard incubation conditions for 2 hours. the selected concentrations of glucose, hydrogen peroxide, After the incubation period, the culture fluid was removed. and sodium azide did not lead to cell lysis, but did inhibit This was followed by the addition of 100 μLMTT mitochondrial bioenergetics functions. ROS generation Solubilisation Solution. The plate was then placed on a was also measured using a DCFDA assay, as evidence minishaker for 10 minutes to assist in dissolving the in the literature suggests that ROS activity mediates the crystals, and the absorbance was read at 570 nm and mechanisms of HSP induction (Ahn and Thiele, 2002). thebackgroundreadat655 nm. HeLa cells were used as a representation of a peripheral tissue. DCFDA assay Near confluent HeLa cells were seeded onto a 96-well Methods black culture plate in 100 μL of DMEM supplemented Cell culture and dose response with glucose (25-125 mM), H O (0-250 μM), and sodium 2 2 HeLa cells were grown in complete DMEM media at azide (0-100 μM). The plate was then returned to incubate 37°C, 5% CO , in a humidified incubator (standard in standard incubation conditions for time periods of incubation conditions). Cells were passaged every 7 days, 24 hours, 3 days, and 7 days. At the end of the incubation and media was changed 3 days after passage. For the period, the DMEM media was removed and the cells dose response experiments, cells were seeded at a density were washed with 100 μL pre-warmed PBS. The cells of approximately 60,000 cells/mL onto 24 well plates were then incubated for a further hour in 100 μL Opti- containing a range of glucose (25-125 mM), H O (0- MEM containing only 2% FBS and further supplemented 2 2 250 μM), and sodium azide (0-100 μM) concentrations. with 2.5 μLof400 μM DCFDA. After the one hour incu- Cell density was estimated by the trypan blue exclusion bation period, the media was again removed and the method every 24 hours over a 7 day period. cells washed twice with 100 μL PBS. 200 μL Opti-MEM was then added, containing 2% FBS, and the fluorescence LDH assay intensity over a 30 minute period at excitation and Near confluent HeLa cells were seeded onto a 96-well emission wavelengths of 485 nm and 520 nm respectively plate in 100 μL of DMEM supplemented with glucose were determined. Data was standardised to the control (25-125 mM), H O (0-250 μM), and sodium azide (0- analysis done in the absence of H O (at the three dif- 2 2 2 2 100 μM). The plate was then returned to incubate in ferent incubation times (24 h, 3 & 7 days)) and then standard incubation conditions for 3 days, when the cells expressed as relative fluorescence units to the controls. would be in the exponential growth phase. After which, one set of control cells (maximum control) was treated Heat shock control cells with 100 μL 10× lysis solution and the plate was returned As a means of obtaining a positive control a heat shock to standard incubating conditions for 45 minutes. After treatment was utilised (Martinus et al., 1996). HeLa cells this time period, the plate was centrifuged at 250×g for were grown in a culture flask for 3 days, so as to reach 5 minutes at room temperature (RT) to pellet cell debris. the exponential growth stage. The media was then 50 μL of the supernatant from each well of the culture replaced with DMEM pre-warmed to 45°C, and the plate was transferred to the corresponding well of a flask was placed into a 45°C incubator for 20 minutes. 96-well enzymatic assay plate. 50 μLofreconstituted Following heat treatment, the media was again replaced, substrate was then added to the supernatant in each this time with DMEM pre-warmed to 37°C. The culture well of the assay plate. The plate was then covered was returned to standard incubating conditions for from light and incubated at RT for 30 minutes. The 6 hours. After this time, the cells were harvested for assay was terminated by the addition of 50 μLofStop protein extraction. Solution to each well of the plate and the absorbance was read at 490 nm. Protein extraction Total protein was isolated using TENT buffer and freshly MTT assay added 0.4 mM phenylmethylsulfonyl fluoride (PMSF) Near confluent HeLa cells were seeded onto a 96-well as a protease inhibitor. Samples were ruptured with plate in 100 μL of DMEM supplemented with glucose 100 μL TENT buffer by vortexing, and were subsequently (25-125 mM), H O (0-250 μM), and sodium azide (0- incubated at 4°C for 30 minutes. After this incubation 2 2 Hall and Martinus SpringerPlus 2013, 2:431 Page 3 of 10 http://www.springerplus.com/content/2/1/431 period, samples were spun at 10,000 rcf for 5 minutes at to determine the significance of the data, and the accepted 4°C to pellet cell debris. The supernatant containing whole level of significance was p < 0.05, which was denoted as ’*’. protein was transferred to a new tube and stored at −20°C. Results and discussion Cell culture and dose response curves Protein separation and transfer Cells treated with 50, 100 and 150 μMH O and 1, 10 and 2 2 Total protein was separated on a 10% polyacrylamide 25 μM sodium azide showed growth rates comparable, discontinuous gel (0.76 mm) and run in a Mini-Protean if not greater than to that of untreated control cultures. 3 Cell gel tank. Cells treated with 50 and 75 mM glucose showed a slightly Each sample containing 20 μg total protein was made reduced growth rate compared with the control. Finally up to 20 μL with protein loading buffer. The samples the 100 and 125 mM glucose groups, the 200 and 250 μM were denatured in boiling water for approximately 5 H O , and the 50 and 100 μM sodium azide groups had 2 2 minutes before loading into the wells. The gel was considerably reduced growth rate through to the end of electrophoresed at a constant 20 mA as the bands ran the culture period. through the stacking gel, and 30 mA through the To compare cell growth across treatments, the num- resolving gel. The end point was determined as when ber of cells at each time point was normalised to the theloading dyeran offthe gel. number of control cells. The rate of cell growth was Semi-dry transfer was completed using a PVDF mem- determined during the exponential growth phase be- braneand a3-buffersystemcontainingcathode,anode tween days 3 and 5 of the culture period, and is exhibited I and anode II buffers. The transfer cell was run at in Figure 1a, b, and c. 15 V for 35 minutes. After the transfer was complete, As was to be expected, increasing the concentration themembranewas brieflyrinsedindistilled water of the treatment resulted in increased inhibition of cell before being stained with Ponceau S for 15 seconds to growth. Slight growth inhibition resulted from treatments ascertain whether efficient transfer had occurred. The of 50 and 75 mM glucose (11% and 30% inhibition stain was then washed out with distilled water. respectively), 150 μMH O (4%), and 10 and 25 μM 2 2 sodium azide (11% and 9% respectively). Strong growth Western blotting inhibition resulted from treatments of 100 and 125 mM The membrane was blocked in 10% skim milk in TBST glucose (36% and 84% respectively), 200 and 250 μMH O 2 2 overnight at 4°C. The following day, the membrane was (54% and 91% respectively), and 50 and 100 μMsodium washed in TBST. The membrane was then incubated in azide (47% and 78% respectively). the primary polyclonal rabbit HSP60 (Sapphire Bioscience) It is interesting that a degree of hormesis was present 1: 250 in 5% skim milk in TBST overnight at 4° in a in all treatments. Hormesis is the name given to the humidified chamber. After another set of washes in TBST, stimulatory effects caused by low levels of potentially the membrane was incubated in peroxidase conjugated toxic agents (Calabrese et al., 2012). The 25 mM glucose, goat anti-rabbit IgG (Sigma) 1:1000 in 5% skim milk in and 50 and 100 μMH O treatments all resulted in 2 2 TBST for 5 hours in a humidified chamber on a gyro increased growth rates compared to the control over the rocker. After another set of washes in TBST on a gyro exponential growth phase. The 1 μM sodium azide was rocker, the membrane was transferred onto a glass plate found to have a comparable growth rate to the control. and incubated for 1 minute with SuperSignal West Pico While it may not be surprising that the comparatively Chemiluminescent Substrate (Pierce). The membrane was low glucose levels result in increased growth, as it would visualised using the LAS-100 Plus Gel Documentation convey a greater energy source to the cell culture, it is of System (Fujifilm), and the intensity of the bands was interest that H O does increase growth, which agrees 2 2 measured using Gel Quant software. After stripping, the with results of previous studies (Burdon, 1995). H O 2 2 same blots were then probed with polyclonal rabbit and its stimulatory effect on cell proliferation have been HSP70 (Sapphire Bioscience). The Quant values generated of particular interest as it has been identified that the for the proteins of interest were normalised to the house elevated levels of H O that result from regular exercise 2 2 keeper gene actin after probing the same membrane have a hormetic effect, indicating that minor oxidative with the anti-actin antibody. stress may have a beneficial effect (Radak et al., 2008). To confirm that the impaired cell growth noted above Statistical analysis was indeed a result of mitochondrial inhibition, and not All statistical analysis in this study was carried out using necrotic cell death, the release of lactate dehydrogenase Microsoft Excel. Data was averaged where appropriate, (LDH) into the growth media and formazan formation and the standard error of the mean (S.E.M.) was calculated via the MTT assays as an indicator of mitochondrial de- using Excel. A two-tailed student’s t-test was carried out hydrogenase activity were carried out. Hall and Martinus SpringerPlus 2013, 2:431 Page 4 of 10 http://www.springerplus.com/content/2/1/431 Figure 1 Growth rates of HeLa cells supplemented with glucose, H2O2 and sodium azide. a- Growth rate of HeLa cells supplemented with glucose (25-125 mM). Error bars showing S.E.M. The growth rate between days 3 and 5 has been taken as the growth rate during the exponential phase of growth. b- Growth rate of HeLa cells supplemented with H O (0-250 μM). Error bars showing S.E.M. The growth 2 2 rate between days 3 and 5 has been taken as the growth rate during the exponential phase of growth. c- Growth rate of HeLa cells supplemented with sodium azide (0-100 μM). Error bars showing S.E.M. The growth rate between days 3 and 5 has been taken as the growth rate during the exponential phase of growth. Hall and Martinus SpringerPlus 2013, 2:431 Page 5 of 10 http://www.springerplus.com/content/2/1/431 LDH assay lead to an increase in cell lysis, indicative of a potential After 3 days in culture, the percentage of LDH content state of cellular stress. To determine if this stress is relative to corresponding maximum control samples was targeted at the level of the mitochondria MTT assays calculated. It was assumed that every dead cell released were implemented. an equal amount of LDH. Therefore, the percentage of LDH content was interpreted as the percentage of lysed MTT assay cells, as shown in Figure 2. After 3 days in culture, cells were incubated with MTT for The glucose control sample had 25.50 ± 1.31% lysed 2 hours. After which the formazan crystals were solubilised, cells, and was not significantly different from the 75 the absorbance read, and results were interpreted as a and 100 mM glucose groups, which had 22.76 ± 1.29% percentage of mitochondrial activity relative to the control and 26.46 ± 1.00% of lysed cells, respectively. However, group, as seen in Figure 3. treatment with 125 mM glucose resulted in a significantly All glucose samples were found to have significantly increased (p < 0.05) percentage of lysed cells compared decreased (p < 0.05) mitochondrial dehydrogenase activity to the control; resulting in 31.46 ± 1.37% lysed cells. compared to control cells. 75 mM glucose resulted in The H O control sample had 27.84 ± 3.04% lysed cells, 84.29 ± 1.20%, 100 mM resulted in 78.21 ± 1.13%, and 2 2 and was not significantly different from the 150 and 125 mM glucose resulted in 73.96 ± 1.12% mitochon- 200 μMH O groups, which had 27.61 ± 2.33% and drial dehydrogenase activity. 2 2 30.69 ± 2.19% of lysed cells, respectively. However, samples All H O samples were found to have significantly de- 2 2 treated with 250 μMH O resulted in a significantly in- creased (p < 0.05) mitochondrial dehydrogenase activity 2 2 creased (p < 0.05) percentage of lysed cells compared to relative to control cells. 150 μMH O resulted in 87.70 ± 2 2 the control; resulting in 37.71 ± 3.09% lysed cells. 3.07%, 200 μM resulted in 81.97 ± 1.41%, and 250 μM The sodium azide control sample had 24.42 ± 1.13% resulted in 59.61 ± 4.21% mitochondrial dehydrogenase lysed cells, and was not significantly different from the activity. 25 and 50 μM sodium azide groups, which had 26.80 ± All sodium azide samples were found to have signifi- 1.18% and 27.57 ± 1.51% of lysed cells, respectively. cantly decreased (p < 0.05) mitochondrial dehydrogenase However, treatment with 100 μM sodium azide resulted activity relative to control cells. 25 μMsodiumazide in a significantly increased (p < 0.05) percentage of lysed resulted in 91.76 ± 2.27%, 50 μM resulted in 73.61 ± 3.53%, cells compared to the control; resulting in 32.08 ± 2.85% and 100 μM resulted in 75.33 ± 1.20% mitochondrial de- lysed cells. hydrogenase activity. From the LDH assay results it can be concluded that From the MTT assay results it can be concluded that 100 mM glucose, 200 μM hydrogen peroxide and 50 μM 100 mM glucose, 200 μM hydrogen peroxide and 50 μM sodium azide do not result in significant cell lysis com- sodium azide do result in significant inhibition of mito- pared to control cells. These concentrations are of par- chondrial bioenergetics compared to control cells. In ticular interest as they reduced growth rate but do not conjunction with the dose response and LDH results, Figure 2 Percentage of lysed cells in the presence of glucose, H O , and sodium azide. The only concentrations that gave significantly 2 2 elevated degrees of cell lysis compared to the control group were the maximum treatment concentrations. All other treatment concentrations had no significant difference in the degree of cell lysis. (* represents statistically significant values, p < 0.05). Hall and Martinus SpringerPlus 2013, 2:431 Page 6 of 10 http://www.springerplus.com/content/2/1/431 Figure 3 Mitochondrial dehydrogenase activity in the presence of glucose, H O , and sodium azide. All concentrations significantly 2 2 inhibited mitochondrial bioenergetic function compared to respective control. (* represents statistically significant values, p < 0.05). the MTT results indicate that 100 mM glucose, 200 μM resulted in 112.93 ± 2.71%, and the sodium azide treat- hydrogen peroxide and 50 μM sodium azide result in ment resulted in 110.77 ± 2.24% ROS levels. mitochondrial specific cell stress. To further support this After 3 days, all samples were found to have signifi- conclusion, DCFDA assays were used to investigate any cantly increased (p < 0.05) ROS levels. The glucose treat- ROS generation, a common association with mitochon- ment resulted in 123.63 ± 6.24%, the H O treatment 2 2 drial impairment. resulted in 135.36 ± 7.33%, and the sodium azide treat- ment resulted in 134.77 ± 7.15% ROS levels. DCFDA assay After 7 days, all samples were found to have signifi- After 1, 3, and 7 days in culture, cells were incubated cantly increased (p < 0.05) ROS levels. The glucose treat- with DCFDA for 1 hour. After which the fluorescence ment resulted in 179.95 ± 23.78%, the H O treatment 2 2 was read, and results were normalised to the H O absent resulted in 304.56 ± 29.02%, and the sodium azide treat- 2 2 control. Results were interpreted as a percentage of ROS ment resulted in 279.13 ± 43.82% ROS levels. levels relative to the control group, as seen in Figure 4. The DCFDA assay results show that increasing the time After 24 hours, all samples were found to have signifi- of exposure to each of the treatments increases the degree cantly increased (p < 0.05) ROS levels. The glucose treat- of intracellular ROS generation, supporting the conclusion ment resulted in 109.06 ± 2.27%, the H O treatment that 100 mM glucose, 200 μM hydrogen peroxide, and 2 2 Figure 4 ROS levels in the presence of glucose, H O , and sodium azide over 24 hours, 3, and 7 days. Each sample contained significantly 2 2 elevated ROS activity after 24 hours, which continued to rise over 3 and 7 days. A dramatic increase in ROS activity is observed between days 3 and 7. (* represents statistically significant values, p < 0.05). Hall and Martinus SpringerPlus 2013, 2:431 Page 7 of 10 http://www.springerplus.com/content/2/1/431 50 μM sodium azide result in mitochondrial specific HSP60 protein expression was found to be upregulated cell stress. Increased levels of intracellular ROS activity in all treatments over 3 days, as demonstrated in Figures 5a have been shown to induce a concentration-dependent and 6. The heat shocked cells had a 2.23 fold increase in transactivation and DNA-binding activity of heat shock HSP60 expression, while the 100 mM glucose, 200 μM factor-1 (HSF-1), the principal transcription factor of H O , and 50 μM sodium azide had 1.61, 1.54 and 2.12 2 2 HSP60 (Jacquier-Sarlin and Polla, 1996). Therefore it fold increases respectively. All were significantly different can be expected that if these treatments do induce a heat to the control (p < 0.05). shock response, a longer treatment period will result in Additionally, HSP60 protein expression was found to a more pronounced induction of HSP60. Western blots be further upregulated in all treatments over a 7 day were implemented to investigate the effect of 100 mM treatment period, as shown in Figures 5b and 6. The glucose, 200 μM hydrogen peroxide, and 50 μMsodium heat shocked cells had a 2.38 fold increase in HSP60 ex- azide on HSP60 & HSP70 expression. pression according to these membranes. The 100 mM glucose, 200 μMH O , and 50 μM sodium azide had 2 2 Western blotting 2.43, 3.58 and 4.74 fold increases respectively, and each After total protein was separated on a polyacrylamide was significantly different from the control (p < 0.05). gel and transferred to a PVDF membrane, Ponceau S The western blots for HSP60 show that treatment with staining was used to show the standard HSP60 band to glucose (100 mM), H O (200 μM), and sodium azide 2 2 be at 60 kDa and that protein loading was even. After (50 μM) all result in a significant increase in expression western blot, a single band for each sample was seen of HSP60. Length of exposure appears to have a pro- and the bands appeared to be the same size as the stand- nounced effect on HSP60 induction, with the 7 day ard HSP60 protein band. The bands were then quantitated treatment having a marked increase in HSP60 expres- using the Gel Quant software, and the relative expression sion. This drastic rise appears to mirror that seen in to the control was plotted. ROS activity over the same time period. HS Control Glucose H O NaN 2 2 3 HSP60 Actin HS Control Glucose H O NaN 2 2 3 HSP60 Actin Figure 5 Western blots of HSP60 expression in HeLa cells treated with heat shock, glucose, H2O2 and sodium azide over 3 and 7 days. a- Western blots of HSP60 and actin in cells treated with heat shock, glucose (100 mM), H O (200 μM), and sodium azide (50 μM), respectively, 2 2 over 3 days. The greatest induction of HSP60 is observed in the sodium azide treated sample, with similar levels of HSP60 observed in the glucose and hydrogen peroxide treatments. HSP60 was tested for a minimum of three times on different blots to provide an accurate representation of its induction. b- Western blots of HSP60 and actin in cells treated with heat shock, glucose (100 mM), H O (200 μM), and 2 2 sodium azide (50 μM) over 7 days. The greatest induction of HSP60 is observed in the sodium azide treated sample, followed by the hydrogen peroxide treatment, with samples treated with glucose having a lesser degree of induction. HSP60 was tested for a minimum of three times on different blots to provide an accurate representation of its induction. Hall and Martinus SpringerPlus 2013, 2:431 Page 8 of 10 http://www.springerplus.com/content/2/1/431 Figure 6 Relative HSP60 expression in cells treated with heat shock, glucose (100 mM), H2O2 (200 μM), and sodium azide (50 μM) over both 3 and 7 days. All treatments and time periods give a significantly increased level of expression of HSP60 compared to control samples. (* represents statistically significant values, p < 0.05). The fold change in HSP60 expression is even across all treatments after 3 days followed by a dramatic rise in expression levels after 7 days, a similar trend to that seen in the DCFDA assay. Even though the Gel Quant results for HSP60 induc- the western blots were re-probed with antibodies against tion are an average of three different blots, the band in- HSP70. Figures 7a,b, and 8 clearly provide additional tensities seen in the images in Figure 5a and b are not evidence that a general cellular stress response is being entirely convincing. To further support the conclusion evoked, resulting in a heat shock response from at least that the cellular stressors being investigated was indeed HSP60 and HSP70. The degree of induction of HSP70 resulting in the induction of molecular stress proteins, closely mirrors that of HSP60. HS Control Glucose H O NaN 2 2 3 HSP70 Actin HS Control Glucose H O NaN 2 2 3 HSP70 Actin Figure 7 Western blots of HSP70 expression in HeLa cells treated with heat shock, glucose, H2O2 and sodium azide over 3 and 7 days. a- Western blots of HSP70 and actin in cells treated with heat shock, glucose (100 mM), H O (200 μM), and sodium azide (50 μM) over 3 days. 2 2 The greatest induction of HSP70 is observed in the sodium azide treated sample, followed by the hydrogen peroxide and glucose treated samples. HSP70 was tested for a minimum of three times on different blots to provide an accurate representation of its induction. b- Western blots of HSP70 and actin in cells treated with heat shock, glucose (100 mM), H O (200 μM), and sodium azide (50 μM) over 7 days. The greatest 2 2 induction of HSP70 is observed in the sodium azide treated sample, followed by the hydrogen peroxide treatment, with samples treated with glucose having a lesser degree of induction. HSP70 was tested for a minimum of three times on different blots to provide an accurate representation of its induction. Hall and Martinus SpringerPlus 2013, 2:431 Page 9 of 10 http://www.springerplus.com/content/2/1/431 Figure 8 Relative HSP70 expression in cells treated with heat shock, glucose (100 mM), H O (200 μM), and sodium azide (50 μM) over 2 2 both 3 and 7 days. All treatments and time periods give a significantly increased level of expression of HSP70 compared to control samples. (* represents statistically significant values, p < 0.05). The fold change in HSP70 expression is even across all treatments after 3 days followed by a dramatic rise in expression levels after 7 days, a similar trend to that seen in the DCFDA assay. HSP60 plays a critical role in the molecular cellular and Polla, 1996). Sensing of the oxidative stress requires stress response targeted at the level of the mitochondrion. two cysteine residues within the HSF-1 DNA-binding do- The primary role of HSPs is that of a molecular chap- main that are engaged in redox-sensitive disulfide bonds. erone, where they act to mediate the folding, assembly or HSF-1 derivatives in which either or both of these cysteine translocation across the intracellular membranes of other residues are mutated have been found to be defective polypeptides; and a role in protein degradation, making in stress-inducible trimerization and DNA binding, stress- up some of the essential components of the cytoplasmic inducible nuclear translocation and HSP gene trans- ubiquitin-dependent degradative pathway (Burel et al., activation, and in the protection of mouse cells from 1992). Additionally, when exposed to a various proteotoxic stress-induced apoptosis (Ahn and Thiele, 2002). In stressors, the expression of HSPs is induced in order to this way, H O is thoughttoexertstwo effectson the 2 2 minimise cellular damage, as well as to stave off apoptosis, activation and the DNA-binding activity of HSF; H O 2 2 by stabilising compromised proteins (Santoro, 2000). favours the nuclear translocation of HSF, while also alter- The inducible HSP component of a cell’s total HSP ing the HSFs DNA-binding activity, which is achieved by pool is regulated by HSFs, of which HSF-1 is the major oxidizing the two critical cysteine residues within the regulator. In the absence of cellular stress, HSF-1 is DNA-binding domain (Jacquier-Sarlin and Polla, 1996). inhibited due to its association with HSPs and is therefore Hyperglycaemia has been reported to increase oxidative maintained in an inactive state. If and when a cellular stress by increasing the rate of glycolysis while inhibiting stress does occur, the HSPs bind to any misfolded pro- oxidative phosphorylation (Crabtree, 1928). The build-up, teins, and subsequently dissociate from HSF-1. This allows and subsequent auto-oxidation, of glyceraldehyde-6-phos- the HSF-1 monomers to oligomerise and form active phate which ensues from increased glycolysis results in trimers, regaining their DNA binding activity. The trimers the generation of H O . As a result, the mitochondrion 2 2 undergo stress-induced serine phosphorylation and are experiences an increase in ROS generation. As mentioned translocated to the nucleus (Prahlad and Morimoto, earlier, activation of HSF-1, the major regulator of HSPs, 2008). Upon nuclear localisation, HSF-1 binds to the HSE is redox dependent. situated upstream of heat shock responsive genes, which results in HSP gene transcription. However, the mecha- Conclusion nisms for stress sensing and signalling to activate HSF-1 This study demonstrated that hyperglycaemic conditions have not been fully elucidated. However, there is growing and oxidative stress can lead to the induction of HSP60 evidence in the literature that this mechanism is mediated and HSP70expression, suggesting that the increased serum by ROS, and in particular H O (Ahn and Thiele, 2002). levels of these molecular stress proteins observed in 2 2 H O has been previously documented to induce a T2DM patients could also be due to uncontrolled hyper- 2 2 concentration-dependent transactivation and DNA-bind- glycaemia and oxidative stress. Interestingly there was also ing activity of HSF-1, although to a lesser extent than a significant and concomitant increase in the intracellular that of the classical heat shock treatment (Jacquier-Sarlin levels of ROS generated during the exposure of the HeLa Hall and Martinus SpringerPlus 2013, 2:431 Page 10 of 10 http://www.springerplus.com/content/2/1/431 cells to the stressors being investigated; suggesting that Ritossa FM (1996) Discovery of the heat shock response. Cell Stress Chaperones 1(2):97–98 the induction of HSP70 & HSP60 was related to ROS Santoro MG (2000) Heat shock factors and the control of the stress response. mediated processes. Biochem Pharmacol 59(1):55–63 Yuan J, Dunn P, Martinus RD (2011) Detection of HSP60 in saliva and serum from type 2 diabetic and Non-diabetic control subjects. Cell Stress Competing interests Chaperones 16(6):689–693 The authors declare that they have no competing interests. doi:10.1186/2193-1801-2-431 Authors’ contribution Cite this article as: Hall and Martinus: Hyperglycaemia and oxidative LH carried out the experiments described in the study as part of his MSc stress upregulate HSP60 & HSP70 expression in HeLa cells. SpringerPlus 2013 2:431. research program at The University of Waikato under the supervision of RDM. All authors have read and approved the final manuscript. Received: 26 August 2013 Accepted: 30 August 2013 Published: 3 September 2013 References Aguilar-Zavala H, Garay-Sevilla ME, Malacara JM, Perez-Luque EL (2008) Stress, inflammatory markers and factors associated in patients with type 2 diabetes mellitus. Stress Health 24(1):49–54 Ahn SG, Thiele DJ (2002) Redox regulation of mammalian heat shock factor 1 is essential for Hsp gene activation and protection from stress. 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Eur J Biochem 240:98–103 Mulder H, Ling C (2009) Mitochondrial dysfunction in pancreatic β-cells in type 2 diabetes. Mol Cell Endocrinol 297:34–40 Nakhjavani M, Morteza A, Khajeali L, Esteghamati A, Khalilzadeh O, Asgarani F, Submit your manuscript to a Outeiro TF (2010) Increased serum HSP70 levels are associated with the journal and benefi t from: duration of diabetes. Cell Stress Chaperones 15:959–964 Pockley AG, Georgiades A, Thulin A, de Faire U, Frostegard J (2003) Serum heat 7 Convenient online submission shock protein 70 levels predict the development of atherosclerosis in 7 Rigorous peer review subjects with established hypertension. J Hypertens 42(3):235–238 Prahlad V, Morimoto RI (2008) Integrating the stress response: lessons for 7 Immediate publication on acceptance neurodegenerative diseases from C. Elegans. Trends Cell Biol 19(2):52–61 7 Open access: articles freely available online Radak Z, Chung HY, Koltai E, Taylor AW, Goto S (2008) Exercise, oxidative stress 7 High visibility within the fi eld and hormesis. Ageing Res Rev 7:34–42 7 Retaining the copyright to your article Submit your next manuscript at 7 springeropen.com

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