Schedule-Dependent Synergy between the Heat Shock Protein 90 Inhibitor 17-(Dimethylaminoethylamino)-17-Demethoxygeldanamycin and Doxorubicin Restores Apoptosis to p53-Mutant Lymphoma Cell Lines

Abstract

Purpose: Loss of p53 function impairs apoptosis induced by DNA-damaging agents used for cancer therapy. Here, we examined the effect of the heat shock protein 90 (HSP90) inhibitor 17-(dimethylaminoethylamino)-17-demethoxygeldanamycin (DMAG) on doxorubicin-induced apoptosis in lymphoma. We aimed to establish the optimal schedule for administration of both drugs in combination and the molecular basis for their interaction. Experimental Design: Isogenic lymphoblastoid and nonisogenic lymphoma cell lines differing in p53 status were exposed to each drug or combination. Drug effects were examined using Annexin V, active caspase-3, cell cycle, and cytotoxicity assays. Synergy was evaluated by median effect/combination index. Protein expression and kinase inhibition provided insight into the molecular mechanisms of drug interaction. Results: Presence of mutant p53 conferred increased survival to single agents. Nevertheless, DMAG showed synergistic toxicity with doxorubicin independently of p53 status. Synergy required exposure to doxorubicin before DMAG. DMAG-mediated down-regulation of CHK1, a known HSP90 client, forced doxorubicin-treated cells into premature mitosis followed by apoptosis. A CHK1 inhibitor, SB-218078, reproduced the effect of DMAG. Administration of DMAG before doxorubicin resulted in G 1 -S arrest and protection from apoptosis, leading to additive or antagonistic interactions that were exacerbated by p53 mutation. Conclusions: Administration of DMAG to doxorubicin-primed cells induced premature mitosis and had a synergistic effect on apoptosis regardless of p53 status. These observations provide a rationale for prospective clinical trials and stress the need to consider schedule of exposure as a critical determinant of the overall response when DMAG is combined with chemotherapeutic agents for the treatment of patients with relapsed/refractory disease.

Novel HSP90 inhibitors, NVP-AUY922 and NVP-BEP800, radiosensitise tumour cells through cell-cycle impairment, increased DNA damage and repair protraction

“…For that reason, the drugs were used at the same concentration of 200 nM in subsequent experiments. Besides this, the selected drug concentration is consistent with the previously reported 100 nM for 17-DMAG (Robles et al, 2006;Koll et al, 2008).…”

“…The growth inhibition assay was carried out essentially as described elsewhere (Robles et al, 2006). Serial dilutions of Hsp90 inhibitors (0 -5 mM) in CGM were added to cell cultures in duplicates.…”

“…Serial dilutions of Hsp90 inhibitors (0 -5 mM) in CGM were added to cell cultures in duplicates. The cytotoxicity of each drug was determined 24 h later (Robles et al, 2006) using the Cell Titer 96 Aqueous One Solution Cell Proliferation Assay (Promega, Madison, WI, USA) according to the manufacturer's instructions. Control samples contained the respective concentrations of DMSO.…”

Geldanamycin promotes premature mitotic entry and micronucleation in irradiated p53/p21 deficient colon carcinoma cells

“…Previous reports demonstrated that HSP90 inhibitors can sensitize cells to the cytotoxic effects of DNAdamaging agents, including IR, primarily through downregulation of cell survival and cytoprotective factors (Arlander et al, 2003;Bisht et al, 2003;Rahmani et al, 2003;Bull et al, 2004;Dote et al, 2006;Robles et al, 2006). However, GA has also been shown to abrogate G 2 arrest in doxorubicin-treated lymphoma and irradiated carcinoma cells (Bull et al, 2004;Dote et al, 2006;Robles et al, 2006;Sugimoto et al, 2007). Mechanistic studies on the enhancement of IR cytotoxicity by HSP90 inhibition, particularly those focusing on G 2 checkpoint abrogation, are limited.…”

Heat Shock Proteins in Chronic Lymphocytic Leukaemia

“…However, this could be due to activation of wild-type p53 and consequent p53-dependent apoptosis. Further research has shown a synergistic effect between 17-DMAG and Doxorubicin, demonstrating a sensitisation of p53 mutated cells to Doxorubicin-induced cell death (Robles et al 2006). …”