Contribution of HSP90 Cleavage to the Cytotoxic Effect of Suberoylanilide Hydroxamic Acid In Vivo and the Involvement of TXNIP in HSP90 Cleavage

Abstract

Heat shock protein (HSP) 90 is expressed in most living organisms, and several client proteins of HSP90 are necessary for cancer cell survival and growth. Previously, we found that HSP90 was cleaved by histone deacetylase (HDAC) inhibitors and proteasome inhibitors, and the cleavage of HSP90 contributes to their cytotoxicity in K562 leukemia cells. In this study, we first established mouse xenograft models with K562 cells expressing the wild-type or cleavage-resistant mutant HSP90β and found that the suppression of tumor growth by the HDAC inhibitor suberoylanilide hydroxamic acid (SAHA) was interrupted by the mutation inhibiting the HSP90 cleavage in vivo. Next, we investigated the possible function of thioredoxin interacting protein (TXNIP) in the HSP90 cleavage induced by SAHA. TXNIP is a negative regulator for thioredoxin, an antioxidant protein. SAHA transcriptionally induced the expression of TXNIP in K562 cells. HSP90 cleavage was induced by SAHA also in the thymocytes of normal mice and suppressed by an anti-oxidant and pan-caspase inhibitor. When the thymocytes from the TXNIP knockout mice and their wild-type littermate control mice were treated with SAHA, the HSP90 cleavage was detected in the thymocytes of the littermate controls but suppressed in those of the TXNIP knockout mice suggesting the requirement of TXNIP for HSP90 cleavage. We additionally found that HSP90 cleavage was induced by actinomycin D, β-mercaptoethanol, and p38 MAPK inhibitor PD169316 suggesting its prevalence. Taken together, we suggest that HSP90 cleavage occurs also in vivo and contributes to the anti-cancer activity of various drugs in a TXNIP-dependent manner.

Keywords: Anti-cancer; Caspase; Heat shock protein 90 cleavage; Histone deacetylase inhibitor; Reactive oxigen species; Thioredoxin interacting protein.

Fig. 1

Modulation of the anti-cancer activity of SAHA by HSP90 cleavage in a mouse xenograft model. (A) Experimental schedule. A xenograft mouse model was established by implantation of K562-HSP90β WT or K562-HSP90β D294A cells in the immune deficient NOD/ShiLtJ-Rag2^em1AMCIl2rg^em1AMC (NRGA) mice. From ten days after the injection, DMSO or SAHA was intraperitoneally injected into the mice every day, and various characteristics such as the tumor volume and mice weight were monitored (n=8/group). (B) Macroscopic appearance of the tumor tissues derived from the K562-HSP90β WT and K562-HSP90β D294A cells. (C) Tumor volumes were calculated as (length x width²)/2 (n=8/group). (D) Tumor weight (n=8/group). (E) Body weight. Values are the mean ± SEM (n=8/group). ***p<0.001 vs. DMSO control. SAHA, suberoylanilide hydroxamic acid.

Fig. 2

SAHA-induced expression of TXNIP mRNA and protein in K562 cells. (A, B) K562 cells were treated with DMSO or the indicated dose of SAHA. (A) The expression of mRNA was determined by RT-qPCR. Values are the mean ± SD. *p<0.05, **p<0.01 vs. 0 h (n=3). (B) The protein expression was determined by western blot analysis. (C) K562 cells were treated with the indicated dose of cycloheximide for 1 h, followed by treatment with DMSO or 5 μM of SAHA for 24 h. The cell lysates were subjected to western blot analysis using the indicated antibodies. GAPDH was used as a loading control. CHX, cycloheximide. SAHA, suberoylanilide hydroxamic acid.

Fig. 3

SAHA-induced HSP90 cleavage in mouse thymocytes. (A) The thymocytes and splenocytes were isolated from BALB/c mice and treated with DMSO or indicated dose of SAHA for 24 h. (B, C) The thymocytes were isolated from C57BL/6 mice and treated with 10 mM of NAC (B) or 2.5 μM of Z-VAD-FMK (C) for 1 h, followed by treatment with DMSO or 10 μM of SAHA for 24 h. The cell lysates were subjected to western blot analysis using the indicated antibodies. GAPDH or actin were used as a loading control. NAC, N-acetylcysteine. Z-VAD-FMK, pan-caspase inhibitor. Con, control. SAHA, suberoylanilide hydroxamic acid.

Fig. 4

Decrease of the SAHA-induced HSP90 cleavage in TXNIP knockout mice. (A, B) Thymocytes were isolated from TXNIP knockout and wild-type littermate control mice. (A) The expression of TXNIP mRNA in the thymocytes was determined by RT-PCR analysis. (B) The thymocytes were treated with DMSO or 10 μM of SAHA for 24 h. The cell lysates were subjected to western blot analysis using the indicated antibodies. Actin was used as a loading control. SAHA, suberoylanilide hydroxamic acid.

Fig. 5

HSP90 cleavage was induced by various compounds via caspase 10 activation. (A) K562 cells were treated with the indicated dose of actinomycin D (ActD), PD169316 or β-mercaptoethanol (2-ME) for 24 h. DMSO or distilled water (DW) were used as a control. The cell lysates were subjected to western blot analysis using the indicated antibodies. GAPDH was used as a loading control. (B, C) K562 cells were treated with DMSO or 50 ng/mL of ActD, 50 μM of PD169316 or 20 mM of 2-ME for 24 h. The cell lysates were incubated with each colorimetric caspase substrate (B (n=6)) or caspase 10 substrate (C (n=8)), and the activation of caspase was monitored by measuring the color development at a wavelength of 405 nm. Values are the mean ± SD. ***p<0.001 vs. DMSO control.