Abstract A214: A novobiocin analogue HSP90 inhibitor induces apoptosis and has potent antitumor effects in head and neck squamous cell cancer in vivo
Authors: Samadi Abbas K. , Mukerji Ridhwi , Blagg Brian S.J. , Cohen Mark S. 
Published: June 2012

Introduction: Small molecule n-terminal inhibitors of heat shock protein 90 (HSP90i) have been evaluated as anticancer agents in various stages of human clinical trials. This study evaluates a novel C-terminal HSP90 inhibitor derived as an analogue of novobiocin for its anti-tumor activity against head and neck squamous cell carcinoma (HNSCC) in vitro and in vivo.

Methods: Head and neck squamous cell carcinoma cells MDA1986 and JMAR were used to examine the efficacy of KU174 as an anti-cancer agent. Cell viability assay MTS and cell proliferation analysis were used to examine the concentration-dependent effect of KU174 on HNSCC cells. Cells were stained with propidium iodide and evaluated on flow cytometry (FC) to study the effects on cell cycle progression. Analysis of KU174 - induced apoptosis of HNSCC was evaluated first by annexin V/PI staining on FC after treatment with drug and confirmed by Western blot analysis for caspase activation and PARP cleavage. KU174 - induced modulation of kinases Akt and ERK1/2 was studied using Western blot analysis for levels of protein expression following treatment. Finally in vivo efficacy of KU174 was evaluated in an orthotopic mouse model of HNSCC following 3 weeks of daily i.p. injection with 5 mg/kg/d of drug.

Results: KU174 inhibited cell viability in both MDA1986 and JMAR cells (IC50 = 7.5 and 4.3 M respectively; novobiocin IC50&gt;700 M for each; p&lt;0.01). In addition, treatment of JMAR and MDA1986 cells by KU174 induced a 30% shift from G0/G1 arrest to G2/M cell cycle arrest. Cells treated with KU174 strongly stained with annexin V/PI indicating induction of apoptosis in these cells, quantitatively measured on FC at 80% with &lt;3% necrosis. Apoptosis was confirmed by Western blot analysis demonstrating induced activation of caspase 3 and cleavage of PARP substrate in MDA1986 cells treated with KU174. HSP90 levels are reduced with drug treatment and both cell lines demonstrated downregulation of Akt activation without significant activation of ERK1/2. In vivo efficacy analysis demonstrates an 80% response rate with a 60% sustained complete response with treatment. Ex vivo immunohistochemistry staining indicate caspase 3 cleavage in animal tumor cells validating the role of apoptosis induction by this inhibitor.

Conclusion: These results show that the c-terminal HSP90 inhibitor KU174 is significantly more potent in antiproliferative activity in HNSCC cells than its parent compound novobiocin. Mechanistic studies suggest that the HNSCC cells treated with KU174 are likely dying by a combination of apoptosis, cell cycle arrest and modulation of proteins such as prosurvival kinases chaperoned by HSP90. Additionally this compound has potent antitumor efficacy in an orthotopic mouse model of HNSCC in vivo lending support for future preclinical proof-of-concept studies to translate its application as a potential human therapy in HNSCC.

Citation Information: Mol Cancer Ther 2009;8(12 Suppl):A214.