HSP90 inhibitor, celastrol, arrests human monocytic leukemia cell U937 at G0/G1 in thiol-containing agents reversible way

Abstract

Background: Because some of heat shock protein 90's (HSP90) clients are key cell cycle regulators, HSP90 inhibition can affect the cell cycle. Recently, celastrol is identified both as a novel inhibitor of HSP90 and as a potential anti-tumor agent. However, this agent's effects on the cell cycle are rarely investigated. In this study, we observed the effects of celastrol on the human monocytic leukemia cell line U937 cell cycle.

Results: Celastrol affected the proliferation of U937 in a dose-dependent way, arresting the cell cycle at G0/G1 with 400 nM doses and triggering cell death with doses above 1000 nM. Cell cycle arrest was accompanied by inhibition of HSP90 ATPase activity and elevation in HSP70 levels (a biochemical hallmark of HSP90 inhibition), a reduction in Cyclin D1, Cdk4 and Cdk6 levels, and a disruption of the HSP90/Cdc37/Cdk4 complex. The observed effects of celastrol on the U937 cell cycle were thiol-related, firstly because the effects could be countered by pre-loading thiol-containing agents and secondly because celastrol and thiol-containing agents could react with each other to form new compounds.

Conclusions: Our results disclose a novel action of celastrol-- causing cell cycle arrest at G0/G1 phase based upon thiol-related HSP90 inhibition. Our work suggests celastrol's potential in tumor and monocyte-related disease management.

Figure 1

Effects of celastrol on U937 culture proliferation and viability in vitro. Cells were seeded at 2 × 10⁵/ml in 96-well culture plate and treated with indicated doses of celastrol for 1 d. A: Dose-dependent effects of celastrol on the number of total, living and dead cells. After treatment, the numbers of living and dead cells in each sample was determined by single-tube platform flow cytometry using self-made cell-Beads as an internal standard (detailed in Methods). Each data point represents the mean value of three test repetitions. B: Dot plot for flow cytometric analysis of living and apoptotic cells. The samples were labeled with Annexin V- FITC and PI double staining, and then detected by FCM. Living cells tested negative for both Annexin V and PI. Populations testing Annexin V positive/PI negative were in early-stage apoptosis, and double positives were in late-stage apoptosis. The percentages of each population are labeled in the corresponding plot region. C. Bar graph showing percentage of living, early, and late-stage apoptotic cells in samples with varying treatments. The values shown are the mean of three independent experiments.

Figure 2

Celastrol arrests cell cycle at G0/G1 phase in U937. Following the treatment, cells were incubated with RNase A and PI, cell cycle was assayed by FCM. A: Panel of flow cytometric histograms showing DNA content in samples treated with various doses of celastrol. X-axis represents DNA content and Y-axis shows cell number. B: Percentages of sample cells in each cell cycle phase. The dose-effects of celastrol on the percentages of U937 cells at each phase of cell cycle are shown. Each value is found as the mean of three independent experiments.

Figure 3

Celastrol decreases the level of Cyclin D1 and some Cdks. Following treatment, cells were incubated with indicated antibodies and the expressions of proteins were detected by FCM detailed in Methods. A: Celastrol induces reduction of Cyclin D1 in a dose-dependent manner. The left panel shows the histogram for FCM detection of Cyclin D1 expression with X-axis as fluorescence intensity and Y-axis representing cell number. The right panel shows the detected intensities of this protein. Each value represents the mean of three independent experiments. B: Effects of celastrol on Cdk4, Cdk6, and Cdk2 expressions. After exposure to 600 nM celastrol for 1 d, the proteins were detected by FCM. Y-axis represents the relative levels of each protein in different treatments, with the protein level in DMSO-control sample being set at 1.0. Each value is the mean of three independent experiments.

Figure 4

Effects of celastrol on HSP90's ATPase activity, HSP70 expression and the HSP90-Cdc37-Cdks complex. A: Celastrol inhibits the activity of ATPase. Co-immunoprecipition of HSP90 was performed on untreated cells, and the beads-bound immunoprecipites were separated into three equal portions before incubation with celastrol, 17-AAG, or DMSO. ATPase activity was determined as the decrease of the absorbance at 340 nm, detailed in Methods. B: Celastrol induces the increase of HSP70. Cells treated with 600 nM celastrol for 1 d and HSP70 levels were deteceted by flow cytometry, as detailed in Methods. X-axis shows channel number and Y-axis shows cell number. C: Celastrol disrupts the HSP90/Cdc37/Cdks complexes. Cells treated with 600 nM celastrol for 1 d, then were used for immunoprecipitation by anti-HSP90 (H9010), detailed as in Methods. The left column (input) displays the detection based on total proteins of cells, while the right column (IP) shows detection of immunoprecipitation using the anti-HSP90 (H9010) antibody.

Figure 5

Reversing effects of thiol-containing agents on the actions of celastrol in U937. Cells were seeded at 2 × 10⁵/ml in a 24-well plate. After being pre-treated with 2 mM NAC, 0.1 mM Vit C, 2 mM GSH or 2 mM GSSG for 1 h, cells were exposed to 600 nM celastrol for 1 d. At the end of the indicated time points, cells were analysed for cell cycle location and protein detection by FCM. A: Flow cytometric histogram of HSP70 expressions in U937 with various treatments. B: Cell cycle distributions of drug treated U937. The dark gray, light gray and white areas in the circle track represent the ratio of cells at G0/G1, S, and G2/M phases, respectively. C: Expressions of selected proteins in U937 with different pre-treatments. The vertical axis represents the relative levels of each protein, which were determined by dividing individual protein intensity by its levels in the DMSO-treated sample. The DMSO-control sample was set at 1.0. The plotted data represents the mean results of three repetitions. The values under the figure are mean ± SD for each sample. * P < 0.05, **P < 0.01 when compared with DMSO-control.

Figure 6

Reactions between celastrol and thiol-containing agents and between celastrol and non-thiol small molecules. A: Absorption spectra of celastrol mixed with different agents. Celastrol was mixed with different small molecules in ratio of 1:2, the absorption spectra then detected. B-D: Mass spectrum analysis of the following agents or reactions: (B) celastrol; (C) the mixture of celastrol and DTT at a 1:2 molar ratio in DMSO; (D) celastrol and DTT reaction result with formic acid added (to save space, the detection patterns presented have been truncated. The non-truncated view can be found in this manuscript's additional file 2).