Induction of Hsp70 in tumor cells treated with inhibitors of the Hsp90 activity: A predictive marker and promising target for radiosensitization.

We studied a role of the inducible heat shock protein 70 (Hsp70) in cellular response to radiosensitizing treatments with inhibitors of the heat shock protein 90 (Hsp90) chaperone activity. Cell lines derived from solid tumors of different origin were treated with the Hsp90 inhibitors (17AAG, geldanamycin, radicicol, NVP-AUY922) or/and γ-photon radiation. For comparison, human cells of the non-cancerous origin were subjected to the same treatments. We found that the Hsp90 inhibitors yielded considerable radiosensitization only when they cause early and pronounced Hsp70 induction; moreover, a magnitude of radiosensitization was positively correlated with the level of Hsp70 induction. The quantification of Hsp70 levels in Hsp90 inhibitor-treated normal and cancer cells enabled to predict which of them will be susceptible to any Hsp90-inhibiting radiosensitizer as well as what concentrations of the inhibitors ensure the preferential cytotoxicity in the irradiated tumors without aggravating radiation damage to adjacent normal tissues. Importantly, the Hsp70 induction in the Hsp90 inhibitor-treated cancer cells appears to be their protective response that alleviates the tumor-sensitizing effects of the Hsp90 inactivation. Combination of the Hsp70-inducing inhibitors of Hsp90 with known inhibitors of the Hsp induction such as quercetin, triptolide, KNK437, NZ28 prevented up-regulation of Hsp70 in the cancer cells thereby increasing their post-radiation apoptotic/necrotic death and decreasing their post-radiation viability/clonogenicity. Similarly, co-treatment with the two inhibitors conferred the enhanced radiosensitization of proliferating rather than quiescent human vascular endothelial cells which may be used for suppressing the tumor-stimulated angiogenesis. Thus, the easily immunodetectable Hsp70 induction can be a useful marker for predicting effects of Hsp90-inhibiting radiosensitizers on tumors and normal tissues exposed to ionizing radiation. Moreover, targeting the Hsp70 induction in Hsp90 inhibitor-treated cancer cells and tumor vasculature cells may beneficially enhance the radiosensitizing effect

Characterization of nitrogen and carbon stable isotopes in epiphytic foraminiferal morphotypes

Epiphytic foraminifera are important components of the seagrass-meadow biota. These foraminifera previously were categorized, based upon their ecological and feeding strategies, into four morphotypes that were subsequently modified to include a new morphotype for the symbiont-bearing foraminifera. We propose further modifications to increase the ecological resolution. Thus, the A* morphotype splits into leaf-encrusting forms (AF*) and rhizome encrusting taxa (AR*). Similarly, the symbiont-bearing morphotype has been separated into Large Miliolids (LM) that host a variety of algal symbionts, and Large Rotalids (LR) that exclusively host diatoms. B and C morphotypes remain as they were originally defined, whilst D* morphotype does not include symbiont-bearing taxa and represents opportunistic forms. To determine the trophic strategy of the epiphytic morphotypes, the cytoplasmic nitrogen and carbon stable-isotope signals from two localities of Mallorca (Sa Foradada and Sant Elm) and one from Madagascar were analysed. The most abundant morphotype reported in Mallorca localities was B (38% ± 4.3 in Sa Foradada and 45% ± 4.2 in Sant Elm), followed by AF* (34% ± 4.6 in Sa Foradada and 41% ± 1.0 in Sant Elm). In Madagascar, the most abundant morphotype is D* (45% ± 10), and symbiont-bearing morphotypes (LM and LR) were considerably more abundant than at the Mediterranean locations. Among all samples, the δ15N values ranged between 0.5 and 3‰; δ13C values varied between −18 and −0.9‰. An MDS statistical analysis showed that variability in the δ15N and δ13C isotopes is associated with differences among the morphotypes and likely reflects their feeding strategies. A SIMPER analysis of the isotopic composition revealed minimal differences within the sessile (AF* and AR*) and within the symbiont-bearing (LM and LR) morphotypes, indicating similar trophic strategies within each pair, largely based upon cyanobacteria as a food source. These foraminifera perform “farming” of (cyano)bacteria, fungi and diatoms, which constitute the essential components of their diet. The LM-LR morphotypes also receive organic carbon from their algal symbionts. The δ15N and δ13C values of the motile B and D* morphotypes are highly variable, indicative of diverse food sources, including cyanobacteria, fungi, microalgae and particulate organic matter (phytodetritus). The δ15N in the C morphotype are more enriched and δ13C more depleted (3‰ and −10‰, respectively) than in the sessile morphotypes. Consistent with observations of other epiphytic, sessile organisms, cyanobacteria seem to be a very important food sourceVersión del editor1,617

Targeting the Hsp70 induction in Hsp90 inhibitor-treated cancer cells enhances their apoptotic death following radiation exposure.

<p>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 (<b>Q</b>). 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 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0173640#pone.0173640.t003" target="_blank">Table 3</a>). Analogous effects were also observed on HeLa, PC-3 and Myc-CaP cancer cells and actively proliferating vascular endothelial cells (not shown).</p

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.

<p>Here it is seen that much lower concentrations of 17AAG are required to repress the Hsp90 chaperone function-dependent refolding of luciferase (<b>A</b>) and stimulate the HSF1 phosphorylation (<b>B</b>) and the Hsp70 induction (<b>C</b>) 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.</p

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.

<p>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 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0173640#pone.0173640.t001" target="_blank">Table 1</a>).</p

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.

<p>The presented blots (<b>A</b>) 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>B</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.</p