Curcumin Affects HSP60 Folding Activity and Levels in Neuroblastoma Cells

Abstract

The fundamental challenge in fighting cancer is the development of protective agents able to interfere with the classical pathways of malignant transformation, such as extracellular matrix remodeling, epithelial-mesenchymal transition and, alteration of protein homeostasis. In the tumors of the brain, proteotoxic stress represents one of the main triggering agents for cell transformation. Curcumin is a natural compound with anti-inflammatory and anti-cancer properties with promising potential for the development of therapeutic drugs for the treatment of cancer as well as neurodegenerative diseases. Among the mediators of cancer development, HSP60 is a key factor for the maintenance of protein homeostasis and cell survival. High HSP60 levels were correlated, in particular, with cancer development and progression, and for this reason, we investigated the ability of curcumin to affect HSP60 expression, localization, and post-translational modifications using a neuroblastoma cell line. We have also looked at the ability of curcumin to interfere with the HSP60/HSP10 folding machinery. The cells were treated with 6, 12.5, and 25 µM of curcumin for 24 h, and the flow cytometry analysis showed that the compound induced apoptosis in a dose-dependent manner with a higher percentage of apoptotic cells at 25 µM. This dose of curcumin-induced a decrease in HSP60 protein levels and an upregulation of HSP60 mRNA expression. Moreover, 25 µM of curcumin reduced HSP60 ubiquitination and nitration, and the chaperonin levels were higher in the culture media compared with the untreated cells. Furthermore, curcumin at the same dose was able to favor HSP60 folding activity. The reduction of HSP60 levels, together with the increase in its folding activity and the secretion in the media led to the supposition that curcumin might interfere with cancer progression with a protective mechanism involving the chaperonin.

Keywords: HSP60; brain tumors; extracellular HSP60; heat shock proteins; molecular chaperones; post-translational modifications; protein folding.

Figure 1

Effects of curcumin on cell proliferation (A) MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) test analysis. LAN-5 cell viability treated with different curcumin doses (1.5–200 µM) for 24 h using MTT assay. Vertical axis percentage of cell viability; horizontal axis, the concentration of curcumin in micrometers. A dose-dependent decrease in LAN-5 cell viability was observed (growth 50% index = 37.5 μM). * p < 0.05 vs. untreated cells (UT). (B) Representative flow cytometry graphs are shown for control cells (CTRL, cells treated with dimethyl sulfoxide, DMSO) and for UT and treated with 6, 12.5, and 25 µM of curcumin (P.I.: propidium iodide). The histograms are representative of three independent experiments and show the effect of different doses of curcumin on LAN-5 apoptosis (* p < 0.0001 vs. UT; ** p = 0.04 vs. UT).

Figure 2

Effect of curcumin treatment on HSP60 expression level (A) Representative Western blots and graph of densitometry of the corresponding bands for HSP60 protein expression level in UT, 6 µM, 12.5 µM, and 25 µM of curcumin. β-actin was used as an internal control (* Different than UT, 6 µM, and 12.5 µM, p < 0.01). Statistical analysis was performed using ANOVA analysis of variance followed by a Bonferroni post-hoc test. Experiments were performed in quadruplicate. (B) Immunofluorescence images confirming the data (Bar: 30 µm). The chaperonin seems to be confined to mitochondria. The insets were obtained using the NIH Image J 1.40 analysis program (National Institutes of Health, Bethesda, MD, USA). (C) Representative graph showing real-time PCR analysis of HSPD1, (heat shock protein family D, HSP60 member 1) gene expression. The data were normalized to reference genes according to the Livak Method (2^−ΔΔCt). (^* Different than UT p < 0.05).

Figure 3

Effects of curcumin treatment on HSP60 post-translational modification and localization (A) 25 µM of curcumin does not promote HSP60 ubiquitination and nitrosylation after 24 h of treatment. (B) HSP60 levels in cells supernatant, detected by enzyme-linked immunoadsorbent assay (ELISA). The difference between HSP60 levels in cells supernatant after 24 h of treatment (25 µM of curcumin) versus untreated cells was significant (* p < 0.001).

Figure 4

Folding test: At indicated times 30–60 min, aliquots were taken from each of the reactions and added to assay wells containing 50 µL of Luciferin reagent. Luminescence measurements were taken using a GloMax^® 96 Microplate Luminometer (Promega Corporation, Madison, WI, USA) (Substrate: Glow-Fold™ Substrate Protein). (A) The graph shows the average of three independent experiments. The folding test demonstrated that curcumin (25 µM) significantly promoted the refolding activity of HSP60/HSP10 complex after 60 min of reaction (* p < 0.01 vs. Substrate + HSP60/HSP10 + ATP, Substrate + HSP60 + ATP + 25 µM curcumin, Substrate + HSP10 + ATP + 25 µM curcumin, and Substrate only; no heat shock treatment). Furthermore, the test showed that curcumin had no effects on the activity of the complexes, which were not present, respectively, HSP10 and HSP60. After 30 min of reaction, we observed no significant changes in folding activity in the presence of curcumin (Substrate + HSP60/HSP10 + ATP + 25 µM curcumin) and in the absence of HSP10 (Substrate + HSP60 + ATP + 25 µM curcumin) or HSP60 (Substrate + HSP10 + ATP + 25 µM curcumin), when compared with the reaction without curcumin (Substrate + HSP60/HSP10 + ATP, at 30 min). (B) The graph shows the percentage of the refolding in all conditions used, compared with the luciferase activity in the presence of the substrate not heated (Substrate only; no heat shock treatment), considered 100% of activity. (* p < 0.01 vs. Substrate + HSP60/HSP10 + ATP, Substrate + HSP60 + ATP + 25 µM curcumin, Substrate + HSP10 + ATP + 25 µM curcumin, and Substrate only; no heat shock treatment).