Ritonavir causes G1 arrest in non-small cell lung cancer (NSCLC), in part, by binding hsp90 and down-regulating Cdk4 and other Hsp90 client proteins

Abstract

2275

The HIV protease inhibitor, ritonavir, has been implicated as an inhibitor of cancer cell growth, but the mechanisms of its action are not established. Ritonavir inhibits the growth of the NCI-H522 human lung adenocarcinoma line, exhibiting an IC₅₀ of 42±2 (mean±SEM) microM. Ritonavir causes a G0/G1 arrest, with an increase in the G0/G1 population from 38 to 73% and corresponding reduction of the G2/M population. Apoptosis is induced 3-fold by ritonavir compared to vehicle control at 24 h, from 3.8 to 11%, as measured by propidium iodide/Annexin-FITC staining, and increases with exposure time. In contrast, the proteasome inhibitor lactacystin fails to induce cell cycle arrest or apoptosis, suggesting that the effects of ritonavir are independent of proteasome inhibition. Surface EGFR is down-regulated 50% by ritonavir, as measured by flow cytometric analysis, as is total cellular EGFR. Other Hsp90 clients, including the glucocorticoid receptor, ErbB2, c-Raf1, ERK, Akt, mutant p53, and Cdk4 are similarly down-regulated by ritonavir. Phospho-ERK and phospho-Akt are also down-regulated by ritonavir, while Hsp90 is itself reduced by 50%. In contrast, p27 levels are not affected. These results suggest that ritonavir-induced G1 arrest of the H522 line is mediated, in part, by down-regulation of Hsp90 and resulting reduction of Cdk4. Similar down-regulation of Hsp90 and Cdk4 by ritonavir is observed for the A549 and H460 NSCLC lines. Surface plasmon resonance biosensor analysis revealed that ritonavir binds to human Hsp90, exhibiting a K_D of 7.8 microM, in contrast to a K_D of 0.65 microM for 17-allyamino-17-demethoxygeldanamycin (17-AAG). Because the K_D of ritonavir for Hsp90 is well below the median C_max of clinical serum ritonavir levels, 27 microM, clinical ritonavir dosing may potentially inhibit Hsp90 function. Ritonavir dramatically sensitizes the H522, A549 and H460 lines to the S-phase active chemotherapeutic agent, gemcitabine, exhibiting Chou-Talalay combination indices of 0.16±0.09, 0.57±0.33 and 0.19±0.05 (mean±SEM), respectively. Together, these results suggest that Hsp90 and Cdk4 are novel ritonavir targets in NSCLC and that ritonavir inhibition of Cdk4 may sensitize NSCLC lines to DNA replication-associated stress.