The Role of Heat Shock Proteins and Autophagy in Mechanisms Underlying Effects of Sulforaphane on Doxorubicin-Induced Toxicity in HEK293 Cells

Abstract

Doxorubicin (DOX) is a cytostatic agent belonging to anthracycline group. Important role in mechanism associated with negative effects of DOX plays an oxidative stress. Heat shock proteins (HSPs) are part of mechanisms initiated in response to stressful stimuli and play an important role in cellular responses to oxidative stress through interaction with components of redox signaling. The present work was aimed to study the role of HSPs and autophagy in mechanisms underlying effects of sulforaphane (SFN), a potential activator of Nrf-2, on doxorubicin-induced toxicity in human kidney HEK293 cells. We investigated effects of SFN and DOX on proteins associated with regulation of heat shock response, redox signaling, and autophagy. Results show that SFN significantly reduced cytotoxic effects of DOX. The positive effects of SFN on DOX-induced changes were associated with up-regulation of Nrf-2 and HSP60 protein levels. In the case of another heat shock protein HSP40, SFN increased its levels when was administered alone but not in conditions when cells were exposed to the effects of DOX. Sulforaphane also reversed negative effects of DOX on activities of superoxide dismutases (SODs) and up-regulation of autophagy markers (LC3A/B-II, Atg5, and Atg12). In conclusion, the changes observed in HSP60 are of particular importance in terms of protecting cells from the effects of DOX. Finding that under conditions where SFN reduced cytotoxic effects of DOX were significantly increased protein levels of both Nrf-2 and HSP60 point to the role of HSP60 in mechanisms of redox signaling underlying effects of SFN on DOX-induced toxicity in HEK293 cells. Moreover, data confirmed an important role of autophagy in effects of SFN on DOX-induced toxicity.

Fig. 1

Effect of sulforaphane on changes in cytotoxicity of DOX. Quantitative analysis of changes in the IC₅₀ value for DOX after precultivation with sulforaphane. The IC₅₀ value indicates the concentration of the DOX at which 50 % of the cells survive. Statistical significance was analyzed by one-way ANOVA analysis. Each bar represents mean ± SEM. * p<0.05 compared to DOX. DOX-doxorubicin, DOX+SFN2.5 – doxorubicin with sulforaphane in a concentration of 2.5 μM, DOX+SFN10 – doxorubicin with sulforaphane in a concentration of 10 μM. Data were obtained from three independent experiments performed in triplicate.

Fig. 2

Effect of doxorubicin and/or sulforaphane on protein levels of Nrf-2 and Keap-1 in kidney HEK293 cells. (A) Quantitative analysis of changes in protein levels of Nrf-2 and (B) Keap-1. Values are expressed as the intensity of the reaction compared to the control (in %). (C) Western blot records showing the protein levels of Nrf-2, Keap-1, and housekeeping protein GAPDH. Statistical significance was analyzed by one-way ANOVA. Each bar represents mean ± SEM, n=4–6, * p<0.05 compared to control, ^& p<0.05 compared to SFN, ^# p<0.05 compared to DOX. C – Control (0.1 % DMSO), SFN – 10 μM sulforaphane, DOX – 2.5 μM doxorubicin, SFN+DOX – sulforaphane + doxorubicin.

Fig. 3

Effect of doxorubicin and/or sulforaphane on SOD activities in kidney HEK293 cells. SOD activities were determined using colorimetric assay kit (Abcam). The data represents the percentage of inhibition of superoxide production by SOD activity. Statistical significance was analyzed by one-way ANOVA. Each bar represents mean ± SEM, n=4–6, * p<0.05 compared to vehicle control, ^# p<0.05 compared to DOX. C – Control (0.1 % DMSO), SFN – 10 μM sulforaphane, 2.5 μM DOX – doxorubicin, SFN+DOX – sulforaphane + doxorubicin.

Fig. 4

Effect of doxorubicin and/or sulforaphane on protein levels of HSP40 and HSP60 in HEK293 cells. (A) Quantitative analysis of changes in protein levels of HSP40 and (B) HSP60. Values are expressed as the intensity of the reaction compared to the control (in %). (C) Western blot record showing the protein levels of HSP40 and HSP60. Statistical significance was analyzed by one-way ANOVA. Each bar represents mean ± SEM, n=4–6, * p<0.05 compared to vehicle control, ^& p<0.05 compared to SFN, ^# p<0.05 compared to DOX. C – Control (0.1 % DMSO), SFN – 10 μM sulforaphane, DOX – 2.5 μM doxorubicin, SFN+DOX – sulforaphane + doxorubicin.

Fig. 5

Effect of doxorubicin and/or sulforaphane on protein levels of HSP90 and HSP70 in kidney HEK293 cells. (A) Quantitative analysis of changes in protein levels of HSP90 and (B) HSP70. Values are expressed as the intensity of the reaction compared to the control (in %). (C) Western blot record showing the protein levels of HSP90 and HSP70. Statistical significance was analyzed by one-way ANOVA. Each bar represents mean ± SEM, n=4–6, C – Control (0.1 % DMSO), SFN – 10 μM sulforaphane, DOX – 2.5 μM doxorubicin, SFN+DOX – sulforaphane + doxorubicin.

Fig. 6

Effect of doxorubicin and/or sulforaphane on protein levels of Beclin-1 and LC3A/B-II in HEK293 cells. (A) Quantitative analysis of changes in protein levels of Beclin-1 and (B) LC3A/B-II. Values are expressed as the intensity of the reaction compared to the control (in %). (C) Western blot records showing the protein levels of Beclin-1 and LC3A/B-II. Statistical significance was analyzed by one-way ANOVA. Each bar represents mean ± SEM, n=4–6, * p<0.05 compared to vehicle control, ^& p<0.05 compared to SFN, ^# p<0.05 compared to DOX. C – Control (0.1 % DMSO), SFN – 10 μM sulforaphane, DOX – 2.5 μM doxorubicin, SFN+DOX – sulforaphane + doxorubicin.

Fig. 7

Effect of doxorubicin and/or sulforaphane on protein levels of Atg5 and Atg12 in HEK293 cells. (A) Quantitative analysis of changes in protein levels of Atg5 and (B) Atg12. Values are expressed as the intensity of the reaction compared to the control (in %). (C) Western blot records showing the protein levels of Atg5 and Atg12. Statistical significance was analyzed by one-way ANOVA. Each bar represents mean ± SEM, n=4–6, * p<0.05 compared to vehicle control, ^& p<0.05 compared to SFN, ^# p<0.05 compared to DOX. C – Control (0.1 % DMSO), SFN – 10 μM sulforaphane, DOX – 2.5 μM doxorubicin, SFN+DOX – sulforaphane + doxorubicin.