Increase of P-glycoprotein-mediated drug resistance by hsp 90 beta.

Abstract

The expression of heat shock proteins hsp27, hsp60, hsp70, hsp90 alpha and hsp90 beta in extracts of three cell lines (LoVo DxR, KBChR8-5 and S180 DxR) expressing the MDR (multidrug resistance) positive phenotype as well as in the sensitive parental lines has been investigated. We present evidence that heat shock protein hsp90 beta is associated with the P-glycoprotein (Pgp or P170) one of the most prominent components of the drug resistance machinery. In the doxorubicin-resistant cell line LoVo DxR, but not in the sensitive parental line, hsp90 beta is expressed constitutively as shown by Northern blotting. The expression of hsp90 beta in the sensitive LoVo cell line, however, can be induced by exposure of the doxorubicin-sensitive parental cell line to different stress factors (dexamethasone, doxorubicin, heat treatment or cadmium chloride). We were able to demonstrate that hsp90 beta can be co-precipitated along with Pgp and vice versa. In native agarose gels both proteins migrated together as one single band as shown by Western blot analysis. This intracellular protein-protein interaction may present a mechanism for the modulation of Pgp function possibly by a stabilization of the protein which seems to be attributed to hsp90 beta (in the human colon carcinoma cell line and in the murine cell line S180). Antisense experiments with oligonucleotides directed against hsp90 beta and Pgp, respectively, showed a synergistic effect of the selected hsp90 beta antisense oligonucleotide in combination with the previously described Pgp antisense oligonucleotide in reducing the doxorubicin resistance. The hsp90 beta antisense oligonucleotide when applied in addition to the Pgp antisense oligonucleotide increased the doxorubicin sensitivity of the resistant human colon carcinoma cell line 2-fold. On the contrary, the hsp90 beta antisense oligonucleotide alone in contrast to the Pgp antisense oligonucleotide alone did not cause a reduction of the chemoresistance. Moreover, Pgp half-life was reduced in the presence of both antisense oligonucleotides as compared with an incubation with an anti-Pgp antisense oligonucleotide alone.