Efficacy of Hsp90 inhibition for the induction of apoptosis and inhibition of growth in cervical carcinoma cells in vitro and in vivo

3220

Heat shock protein 90 (Hsp90) is a conserved chaperone involved in crucial signaling events in normal and malignant cells. Previous research suggests that tumor cells are particularly dependent on Hsp90 for survival as well as malignant progression. Hsp90 inhibitors which are derivates of the natural compound geldanamycin (e.g. the orally bioavailable 17-(dimethylaminoethylamino)-17-demethoxygeldanamycin; 17-DMAG) are currently being tested in clinical trials and synthetic inhibitors are in development. In this study we investigated the response of a panel of cervical carcinoma cell lines in vitro and in vivo to determine potential factors that might influence the sensitivity towards Hsp90 inhibition.

Live-cell imaging, proliferation assays and flow cytometry were used to examine the in vitro effects of Hsp90 inhibition on cell cycle and viability in HeLa, SiHa, CaSki and ME180 cells. Drug-induced changes on Hsp90 client factors were examined with focus on G2/M cell cycle regulators, and a comparison with immortalized (Ect1/E6E7) and normal keratinocytes was performed. In vivo studies were performed with subcutaneous xenografts of ME180 and CaSki in immunodeficient mice treated with 6-10 mg/kg 17-DMAG by oral gavage 2x/day on a chronic schedule. Drug concentrations in xenografts were measured by high performance liquid chromatography.

Mitotic catastrophe was identified as one mechanism of cell death and accompanied by an increase in pHistone H3 [Ser10] in 3 carcinoma cell lines. ME180 and CaSki were more sensitive than SiHa and HeLa (~30% versus ~15% non-viable cells after 48 hours, 0.1-5 microM). IC50 values ranged between 17-37 nanoM geldanamycin (tetrazolium-based assay), and keratinocytes were at least as sensitive as carcinoma cells. All cell lines responded with an increase of the G2/M fraction. Despite in vitro effectiveness and tissue concentrations close to 1 microM, only a limited tumor growth reduction was observed with 17-DMAG given at the maximum tolerated dose level. Lower levels of Hsp90 and a lower Akt activity were detected in all xenografts (control and treatment) compared to cell cultures as well as indicators of tissue hypoxia. Proliferation assays performed under hypoxic conditions revealed a low-dose pro-proliferative effect of geldanamycin and 17-DMAG.

We show here that Hsp90 inhibition effectively induces apoptosis and growth arrest in cervical carcinoma cells in vitro. In contrast, a limited efficacy of 17-DMAG was observed in vivo. Induction of a heat shock response has been implicated in resistance towards Hsp90 inhibition. Additional factors might be (i) an altered abundance and/or activity of primary (Hsp90) and secondary (e.g. Akt) target(s), (ii) a narrow therapeutic range and (iii) dose-dependent pleiotropic effects of Hsp90 inhibition.

17-DMAG (NSC 707545, Kosan Biosciences) was supplied by CTEP/NCI (Rockville, MD).