Heat shock protein 90 inhibitors suppress aryl hydrocarbon receptor-mediated activation of CYP1A1 and CYP1B1 transcription and DNA adduct formation

Abstract

The aryl hydrocarbon receptor (AhR), a client protein of heat shock protein 90 (HSP90), plays a significant role in polycyclic aromatic hydrocarbon (PAH)-induced carcinogenesis. Tobacco smoke, a source of PAHs, activates the AhR, leading to enhanced transcription of CYP1A1 and CYP1B1, which encode proteins that convert PAHs to genotoxic metabolites. The main objectives of this study were to determine whether HSP90 inhibitors suppress PAH-mediated induction of CYP1A1 and CYP1B1 or block benzo(a)pyrene [B(a)P]-induced formation of DNA adducts. Treatment of cell lines derived from oral leukoplakia (MSK-Leuk1) or esophageal squamous cell carcinoma (KYSE450) with a saline extract of tobacco smoke, B(a)P, or dioxin induced CYP1A1 and CYP1B1 transcription, resulting in enhanced levels of message and protein. Inhibitors of HSP90 [17-allylamino-17-demethoxygeldanamycin (17-AAG); celastrol] suppressed these inductive effects of PAHs. Treatment with 17-AAG and celastrol also caused a rapid and marked decrease in amounts of AhR protein without modulating levels of HSP90. The formation of B(a)P-induced DNA adducts in MSK-Leuk1 cells was inhibited by 17-AAG, celastrol, and alpha-naphthoflavone, a known AhR antagonist. The reduction in B(a)P-induced DNA adducts was due, at least in part, to reduced metabolic activation of B(a)P. Collectively, these results suggest that 17-AAG and celastrol, inhibitors of HSP90, suppress the activation of AhR-dependent gene expression, leading, in turn, to reduced formation of B(a)P-induced DNA adducts. Inhibitors of HSP90 may have a role in chemoprevention in addition to cancer therapy.

Figure 1

Structures of 17-AAG, celastrol, and gedunin.

Figure 2. HSP90 inhibitors suppress TS-mediated induction of CYP1A1 and CYP1B1 protein

MSK-Leuk1 and KYSE450 cells were treated with the indicated concentrations of 17-AAG, celastrol or gedunin for 2 h. Subsequently, cells received vehicle or TS for 5 h, and were then harvested for Western blot analysis. Cellular lysate protein (100 μg/lane) was loaded onto a 10% SDS–polyacrylamide gel, electrophoresed and subsequently transferred onto nitrocellulose. Immunoblots were probed with antibodies specific for CYP1A1 (panel A), CYP1B1 (panel B) and β-actin.

Figure 3. HSP90 inhibitors suppress TS-mediated induction of CYP1A1 and CYP1B1 mRNA

MSK-Leuk1 and KYSE450 cells were treated with the indicated concentrations of 17-AAG or celastrol for 2 h. Subsequently, cells received vehicle or TS for 3 h. RT-PCR was used to determine the amounts of CYP1A1 (panel A), CYP1B1 (panel B) and β-actin mRNAs.

Figure 4. HSP90 inhibitors suppress B[a]P- and TCDD-mediated induction of CYP1A1 and CYP1B1 protein

MSK-Leuk1 cells were treated with the indicated concentrations of 17-AAG or celastrol for 2 h. Subsequently, cells received vehicle, 1 μmol/L B[a]P or 5 nmol/L TCDD for 5 h, and were then harvested for Western blot analysis. Cellular lysate protein (100 μg/lane) was loaded onto a 10% SDS–polyacrylamide gel, electrophoresed and subsequently transferred onto nitrocellulose. Immunoblots were probed with antibodies specific for CYP1A1 (panel A), CYP1B1 (panel B) and β-actin.

Figure 5. HSP90 inhibitors suppress TS-, B[a]P-, and TCDD-mediated induction of AhR-dependent transcription

In panels A and B, MSK-Leuk1 cells were transfected with 1.8 μg of pGudLuc6.1 (XRE-luciferase) and 0.2 μg of pSVβgal. After transfection, cells were treated with vehicle or the indicated concentration of 17-AAG or celastrol for 2 h, followed by treatment with vehicle (control), TS (panel A), B[a]P (panel B) or TCDD (panel B). Reporter activities were measured in cellular extract 12 h after treatment. Luciferase activity represents data that have been normalized to β-galactosidase activity. Values of luciferase activity are means ± SD; n=6/group. *p<0.05. C–F, MSK-Leuk1 cells were pretreated with vehicle, 17-AAG (panels C and D) or celastrol (panels E and F) for 2h, followed by treatment with TS for 30 min. Chromatin fragments were immunoprecipitated with antibodies against AhR and then the CYP1A1 and CYP1B1 promoters were amplified by PCR. DNA sequencing was carried out, and the PCR products were confirmed to be the correct promoters. The CYP1A1 and CYP1B1 promoters were not detected when normal IgG was used or when antibody was omitted from immunoprecipitation step (data not shown).

Figure 6. HSP90 inhibitors suppress amounts of AhR protein and inhibit the formation of B[a]P induced DNA adducts

A, MSK-Leuk1 cells were treated with 0.1 μmol/L 17-AAG or 2.5 μmol/L celastrol for the indicated periods of time, and were then harvested for Western blot analysis. Cellular lysate protein (100 μg/lane) was loaded onto a 10% SDS–polyacrylamide gel, electrophoresed and subsequently transferred onto nitrocellulose. Immunoblots were probed with antibodies specific for AhR, HSP90, p23, XAP-2, and β-actin. In panels B and C, MSK-Leuk1 cells were treated with the indicated concentration of 17-AAG, celastrol or vehicle (0.1% DMSO) for 2 h. Radio-labeled B[a]P was added and 12 h later, the cells and media were harvested for analysis of DNA adducts (panel B) and B[a]P-tetrol formation (panel C), respectively. In B and C, values are means +/− SD, n=3 *P<0.05, **P<0.001.