The Hsp70 chaperone binds and inhibits proteins implicated in apoptotic signaling including Caspase-3. Induction of apoptosis is an important mechanism of anti-cancer drugs, therefore Hsp70 can act as a protective system in tumor cells against therapeutic agents. In this study we present an assessment of candidate compounds that are able to dissociate the complex of Hsp70 with Caspase-3, and thus sensitize cells to drug-induced apoptosis. Using the PASS program for prediction of biological activity we selected a derivative of benzodioxol (BT44) that is known to affect molecular chaperones and caspases. Drug affinity responsive target stability and microscale thermophoresis assays indicated that BT44 bound to Hsp70 and reduced the chaperone activity. When etoposide was administered, heat shock accompanied with an accumulation of Hsp70 led to an inhibition of etoposide-induced apoptosis. The number of apoptotic cells increased following BT44 administration, and forced Caspase-3 processing. Competitive protein–protein interaction and immunoprecipitation assays showed that BT44 caused dissociation of the Hsp70–Caspase-3 complex, thus augmenting the anti-tumor activity of etoposide and highlighting the potential role of molecular separators in cancer therapy.

Cancer cells are known to contain high levels of the heat shock protein 70 kDa (Hsp70), which mediates increased cell proliferation, escape from programmed cell death, enhanced invasion, and metastasis. A part of Hsp70 molecules may release from cancer cells and affect the behavior of adjacent stromal cells. To explore the effects of Hsp70 on the status of monocytes/macrophages in the tumor locale, we incubated human carcinoma cells of three distinct lines with normal and reduced content of Hsp70 with THP1 monocytes. Using two methods, we showed that the cells with knock-down of Hsp70 released a lower amount of protein in the extracellular medium. Three cycles of the co-cultivation of cancer and monocytic cells led to the secretion of several cytokines typical of the tumor microenvironment (TME) and to pro-cancer activation of the monocytes/macrophages as established by elevation of F4/80 and arginase-1 markers. Unexpectedly, the efficacy of epithelial–mesenchymal transition and resistance of carcinoma cells to anticancer drugs after incubation with monocytic cells were more pronounced in cells with lower Hsp70, e.g., releasing less Hsp70 into the extracellular milieu. These data suggest that Hsp70 released from tumor cells into the TME is able, together with the development of an anti-cancer immune response, to limit the conversion of a considerable part of monocytic cells to the pro-tumor phenotype.

Molecular chaperones, especially 70 kDa heat shock protein, in addition to their intracellular localization in cancer cells, can be exposed on the surface of the plasma membrane. We report that the membrane-associated chaperone mHsp70 of malignant brain tumors is required for high migratory and invasive activity of cancer cells. Live-cell inverted confocal microscopy of tumor samples from adult (n = 23) and pediatric (n = 9) neurooncologic patients showed pronounced protein expression on the membrane, especially in the perifocal zone. Mass spectrometry analysis of lipid rafts isolated from tumor cells confirmed the presence of the protein in the chaperone cluster (including representatives of other families, such as Hsp70, Hsc70, Hsp105, and Hsp90), which in turn, during interactome analysis, was associated with proteins involved in cell migration (e.g., Rac1, RhoC, and myosin-9). The use of small-molecule inhibitors of HSP70 (PES and JG98) led to a substantial decrease in the invasive potential of cells isolated from a tumor sample of patients, which indicates the role of the chaperone in invasion. Moreover, the use of HSP70 inhibitors in animal models of orthotopic brain tumors significantly delayed tumor progression, which was accompanied by an increase in overall survival. Data demonstrate that chaperone inhibitors, particularly JG98, disrupt the function of mHsp70, thereby providing an opportunity to better understand the diverse functions of this protein and offer aid in the development of novel cancer therapies. Significance: Membrane-bound mHsp70 is required for brain tumor cell migration and invasion and therefore could be employed as a target for anticancer therapies.

Quantum dots (QDs) have attracted great attention as tools for theranostics that combine the possibility of simultaneous biological target visualization and medicine delivery. Here, we address whether core/shell InP/ZnS QDs (InP-QDs) may be an alternative to toxic Cd-based QDs. We analyze InP-QD photophysical characteristics in cell culture medium, salt solutions, and directly in the cells. It was demonstrated that InP-QDs were internalized into endolysosomes in HeLa and A549 cells with dynamics similar to Cd-based QDs of the same design, but the two cell lines accumulated them with different efficiencies. InP-QDs were reliably detected in the endosomes despite their low quantum yields. Cell culture medium efficiently decreased the InP-QD photoluminescence lifetime by 50%, acidic pH (4.0) had a moderate effect (20–25% reduction), and quenching by salt solutions typical of intra-endosomal medium composition resulted in a decrease of about 10–15%. The single-vesicle fluorescence-lifetime imaging microscopy analysis of QDs inside and outside the cells shows that the scatter between endosomes in the same cell can be significant, which indicates the complex impact of the abovementioned factors on the state of InP-QDs. The PI test and MTT test demonstrate that InP-QDs are toxic for both cell lines at concentrations higher than 20 nM. Possible reasons for InP-QD toxicity are discussed.