Fig 1.
Texas-Red-immunofluorescence patterns of inducible Hsp70 in fixed/permeabilized cells of different cell lines.
The cells were immunostained as untreated (control) samples or after 20 h incubation with 100 nM 17AAG without quercetin or in the presence of 40 μM quercetin, or 20 h after hyperthermia (43°C, 60 min); bar = 10 μm. It is clearly seen that the marked Hsp70 induction (the brightly stained cytoplasm) takes place in all cell samples exposed to hyperthermia as well as in HeLa cells and MCF-7 cells incubated with 17AAG, whereas 17AAG-treated HBL-100 cells and 293 cells are not stained. Very similar results were obtained with 50–200 nM geldanamycin or 30–100 nM radicicol, or 20–100 nM NVP-AUY922 instead of 17AAG and with 3–10 nM triptolide or 100–200 μM KNK437, or 5–20 μM NZ28 instead of quercetin (not shown). Similar variability in the expression of inducible Hsp70 was found in other cell cultures (Table 1).
Fig 2.
Western blots showing the diversity in dose-dependent effects of 17AAG on the Hsp70 induction in cancer MCF-7 cells and non-cancer 293 cells.
The cells were lysed at different time points (indicated in hours along the upper sides of blots) of incubation with graded concentrations 17AAG and then analyzed with antibodies to inducible Hsp70 and β-Actin (load control). The values of Hsp70/Actin band ratio are presented along the lower sides of blots and reflect the relative amount of Hsp70 in cell samples. As it is seen, Hsp70 is induced in MCF-7 cells by much lower concentrations of 17AAG as compared to 293 cells. The similar difference was observed with other Hsp90 inhibitors in other cell cultures (see Table 1).
Table 1.
Comparative data on the expression of inducible Hsp70 in cancer and non-cancer cell cultures treated with hyperthermia or inhibitors of the Hsp90 activity.
Fig 3.
The diversity in effects of 17AAG on the post-radiation clonogenicity of various cell cultures.
The cells were incubated with various concentrations of the Hsp90 inhibitor for 24 h before γ-irradiation at clinically relevant doses. Here it is demonstrated the drug-conferred radiosensitization of HeLa, MCF-7, KTC-1, PC-3, Myc-CaP, A549 and HT 1080 cancer cells and also of actively proliferating (pre-confluent) endothelial cells, whereas no radiosensitizing effect takes place on HBL-100, FRO and B16 cancer cell lines, non-cancerous 293 and BJ cell lines and quiescent (tight monolayer) endothelial cells. The presented bars express mean ± SEM of 4–5 independent experiments. *—significant difference from the neighboring unmarked bars, p<0.05; **—significant difference from the unmarked bars and bars marked with *, p<0.05. ***—significant difference from all the neighboring bars in each group of samples, p<0.01.
Table 2.
The enhanced radiosensitization of cancer cells and proliferating endothelial cells (pr EC) by combinations of inhibitors of the Hsp90 activity (17AAG, NVP-AUY922) and inhibitors of the Hsp70 induction (quercertin, triptolide).
Fig 4.
The enhanced radiosensitization of certain cell cultures with combination of 17AAG with inhibitors of the Hsp70 induction.
The curves of post-radiation survival of colony-forming cells show the considerable decrease in the survival fractions when the up-regulation of inducible Hsp70 in 17AAG-treated cells was blocked by quercetin (Querc) or triptolide. The similarly enhanced radiosensitization was also found in other cell cultures (A549, HT 1080 and Myc-CaP) with 50–200 nM geldanamycin or 30–100 nM radicicol, or 50–200 nM NVP-AUY922 instead of 17AAG and with 100–200 μM KNK437, or 5–20 μM NZ28 instead of quercetin and triptolide (see also Table 2). *—significant difference from control, p<0.05. **—significant difference from control and values marked with *, p<0.05.
Fig 5.
The data of Western blotting and MTT assay demonstrating the enhanced radiosensitization of 17AAG-treated HeLa cells by preventing the Hsp70 induction with triptolide or quercetin.
The presented blots (A) demonstrate that both inhibitors of the Hsp induction completely abrogated up-regulation of inducible Hsp70 in response to the 17AAG treatment. (The values of Hsp70/Actin band ratio are presented along the lower sides of blots and reflect the relative amount of Hsp70 in cell samples. Of note, such co-treatments with the Hsp70 induction inhibitors did not decrease the basal level of constitutively expressed Hsp70 in target cells). The presented curves (B) show that in contrast to the action of 17AAG alone, the two-inhibitor combinations prevented the post-radiation recovery of proliferative activity in the drug-treated cells. Very similar results were obtained with 50–200 nM geldanamycin or 30–100 nM radicicol, or 50–200 nM NVP-AUY922 instead of 17AAG and with 100–200 μM KNK437, or 5–20 μM NZ28 instead of quercetin and triptolide (not shown). MTT assay also revealed the enhanced radiosensitization of MCF-7, KTC-1, PC-3, Myc-CaP and HT 1080 cancer cells pretreated with combination of the Hsp90 activity inhibitors and inhibitors of the Hsp70 induction (not shown). The presented data express mean ± SEM of 5 independent experiments. *—significant difference from the respective unmarked values, p<0.05; **—significant difference from the respective unmarked or marked with * values, p<0.05.
Fig 6.
Various cell cultures can exhibit the different susceptibility of their Hsp90 chaperone machine and their HSF1-mediated Hsp70 induction to inhibitors of the Hsp90 activity.
Here it is seen that much lower concentrations of 17AAG are required to repress the Hsp90 chaperone function-dependent refolding of luciferase (A) and stimulate the HSF1 phosphorylation (B) and the Hsp70 induction (C) in MCF-7 breast cancer cells as compared with non-cancerous 293 cells. Numbers under the blots represent the expression of phosphorylated HSF1 (pHSF1) or inducible Hsp70 relative to β-actin. Both cell cultures were treated with 17AAG for 20 h before analyses.
Fig 7.
The diversity in dynamics of appearance and disappearance of the γH2AX foci in nuclei of non-cancerous 293 cells and MCF-7 breast cancer cells pretreated with 17AAG before radiation exposure.
The presented bars show average amounts (mean ± SEM) of the γH2AX foci per cell nucleus at different time points following irradiation (4 Gy). A–MCF-7 cells: at all time points following irradiation, the effects of 17AAG (marked by *) significantly differ from respective values in control, p<0.05. B– 293 cells: no significant differences between the 17AAG-treated cells and control.
Fig 8.
Targeting the Hsp70 induction in Hsp90 inhibitor-treated cancer cells enhances their apoptotic death following radiation exposure.
MCF-7 breast cancer cells were either untreated (control) or exposed to γ-photons (6 Gy) without any drug pretreatment or after 24 h incubation with 50 nM 17AAG alone or in combination with 40 μM quercetin (Q). After 48 h, the cells were stained with FITC-annexin V/propidium iodide (PI) and analyzed by flow cytometry. The presented distribution of stained cell subpopulations demonstrates the considerable enhancement of post-radiation apoptosis (FITC-annexin V-positive, PI-negative cells) and secondary necrosis (PI-positive cells) in samples where the 17AAG-induced up-regulation of Hsp70 was fully blocked by quercetin (see Table 3). Analogous effects were also observed on HeLa, PC-3 and Myc-CaP cancer cells and actively proliferating vascular endothelial cells (not shown).
Table 3.
Effects of the Hsp90 activity inhibition with 17AAG and suppression of the Hsp70 induction with quercetin (Querc) on post-radiation apoptosis and necrosis in MCF-7 breast cancer cells.