Cancer ranks as the second leading cause of death in developed countries. Due to advances in early detection and therapeutic options, the cancer mortality rate has been decreasing in recent years, but some malignant diseases remain incurable or develop resistance. Cancer cells overproduce heat shock proteins (Hsps), which makes them a promising target for cancer treatment. As part of a Master's thesis, we investigated the effect of Hsp90 inhibitors on breast cancer cells and Ewing's sarcoma cells. We tested two different inhibitors of the C-terminal domain of Hsp90 (1, 2) and a selective inhibitor of the Hsp90β N-terminal domain (compound 3), on a breast cancer cell model MCF-7. We also tested three different Hsp90 inhibitors (1, 2, and 4) on Ewing's sarcoma cells – SK-N-MC. They all bind to the C-terminal domain of Hsp90, with one of them (compound 4) also inhibiting the N-terminal domain of topoisomerase IIⱺ. Cells were exposed to compounds at different concentrations, which were determined based on the results of previous proliferation screening assays. Compounds were tested on MCF-7 cells at the following concentrations: compound 1 at 0.7 µM; 1.5 µM and 7.0 µM, compound 2 at 7.0 µM, and compound 3 at 7.0 µM and 37.5 µM. On SK-N-MC cells, we tested compounds at the following concentrations: compound 1 at 0.7 µM; 1.5 µM; 7.0 µM; 37.5 µM; and 70 µM, compound 2 at 7.0 µM; 37.5 µM; and 70 µM, and compound 4 at 0.7 µM; 1.5 µM; 7.0 µM; and 37.5 µM. We compared the efficacy of each compound on specific cells. Apoptosis was measured in selected cells by flow cytometry using a combination of fluorescently labelled annexin V and SytoxBlue dye. Cell cycle arrest was measured using propidium iodide. The results were obtained at three time points: 24 hours, 48 hours, and 72 hours. Results showed that all investigated Hsp90 inhibitors were effective in induction of apoptosis in both MCF-7 and SK-N-MC cells. The compounds 1, 2 and 3 triggered cell cycle arrest in the G2/M phase after 24 hours of treatment. In the cell line SK-N-MC, we also observed cell cycle arrest in the G2/M phase after 24 hours of treatment with compound 2. A compound acting on both the C-terminal domain of Hsp90 and the N-terminal domain of topoisomerase IIⱺ (compound 4) induced apoptosis on SK-N-MC cells to a greater extent than selective inhibitors of the C-terminal domain. Compound 1 proved to be the most effective against breast cancer cells. Based on our results, future studies could address the in vitro evaluation of selected Hsp90 inhibitors in combination with other inhibitors of different signaling pathways in cancer to investigate synergistic effects. This approach would allow lower concentrations of Hsp90 inhibitors to be used, potentially reducing the associated side effects.
|
