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Transcription Inhibition of Heat Shock Proteins: A Strategy for Combination of 17-Allylamino-17-Demethoxygeldanamycin and Actinomycin D

Departments of ¹ Experimental Therapeutics and ² Leukemia, The University of Texas M. D. Anderson Cancer Center; ³ Graduate School of Biomedical Sciences, The University of Texas Health Science Center, Houston, Texas; and ⁴ Department of Pathobiology, College of Veterinary Medicine, Nursing, and Allied Health, Tuskegee University, Tuskegee, Alabama

Requests for reprints: Varsha Gandhi, Department of Experimental Therapeutics, The University of Texas M. D. Anderson Cancer Center, Unit 71, 1515 Holcombe Boulevard, Houston, TX 77030. Phone: 713-792-2989; Fax: 713-794-4316; E-mail: vgandhi{at}mdanderson.org.

Key Words: multiple myeloma • 17-AAG • actinomycin D • heat shock proteins • transcription

The heat shock protein (HSP) 90 inhibitor 17-allylamino-17-demethoxygeldanamycin (17-AAG) is currently in clinical trials because of its unique mechanism of action and antitumor activity. However, 17-AAG triggers the transcription and elevation of antiapoptotic HSP90, HSP70, and HSP27, which lead to chemoresistance in tumor cells. We hypothesized that inhibiting HSP90, HSP70, and HSP27 transcription may enhance 17-AAG–induced cell death in multiple myeloma cell lines. Actinomycin D (Act D), a clinically used agent and transcription inhibitor, was combined with 17-AAG. The concentrations for 17-AAG and Act D were selected based on the target actions and plasma levels during therapy. Inducible and constitutive HSP27, HSP70, and HSP90 mRNA and protein levels were measured by real-time reverse transcription-PCR and immunoblot assays. Compared with no treatment, Act D alone decreased HSP mRNA levels in MM.1S and RPMI-8226 cell lines. Combining Act D with 17-AAG did not attenuate 17-AAG–mediated increases in transcript levels of inducible HSP70; however, constitutive HSP mRNA levels were decreased. In contrast to its effect on mRNA levels, Act D was able to abrogate 17-AAG–mediated increases in all HSP protein levels. The cytotoxicity of combined Act D and 17-AAG was assessed. Treatment with Act D alone caused <40% cell death, whereas the combination of 17-AAG and Act D resulted in an increase of cell death in both multiple myeloma cell lines. In conclusion, these results indicate that 17-AAG–mediated induction of HSP70 and HSP27 expression can be attenuated by Act D and therefore can potentially improve the clinical treatment of multiple myeloma. [Cancer Res 2009;69(9):3947–54]