Enhanced proteotoxic stress: one of the contributors for hyperthermic potentiation of the proteasome inhibitor bortezomib using magnetic nanoparticles.

The induction of hyperthermia using nanoparticles, known as magnetic fluid hyperthermia (MFH) in combination with anti-cancer drugs is an attractive method because of the potential for enhanced anti-cancer effects. Recent studies have shown that cells treated with MFH are more sensitive to the proteasome inhibitor bortezomib (BZ) than cells treated by hot water hyperthermia (HWH) under the same temperature conditions. We hypothesized that enhanced proteotoxic stress, caused by a combination of microtubule damage and an increase in the amount of aggregated proteins, may be partially responsible for this observation. To test this hypothesis MCF-7 cells were exposed to hyperthermic treatment (MFH or HWH) at 43 °C or 45 °C for 30 minutes. Then, aggresome formation and microtubule disruption studies at 30 minutes or 2.5 hours of recovery time were performed to evaluate the progressive effects induced by the two treatments. Cell viability at short and long times was evaluated. Aggresome formation and microtubule disruption results suggested that one of the mechanisms by which MFH enhances BZ cytotoxicity is the formation and subsequent accumulation of aggregated proteins in the cytosol due to the interruption of their transport to the perinuclear area through microtubules. Our data show evidence that MFH induces a more toxic and unmitigated proteotoxic stress than HWH under similar temperature conditions.

Magnetic fluid hyperthermia enhances cytotoxicity of bortezomib in sensitive and resistant cancer cell lines.

The proteasome inhibitor bortezomib (BZ) has shown promising results in some types of cancer, but in others it has had minimal activity. Recent studies have reported enhanced efficacy of BZ when combined with hyperthermia. However, the use of magnetic nanoparticles to induce hyperthermia in combination with BZ has not been reported. This novel hyperthermia modality has shown better potentiation of chemotherapeutics over other types of hyperthermia. We hypothesized that inducing hyperthermia via magnetic nanoparticles (MFH) would enhance the cytotoxicity of BZ in BZ-sensitive and BZ-resistant cancer cells more effectively than hyperthermia using a hot water bath (HWH). Studies were conducted using BZ in combination with MFH in two BZ-sensitive cell lines (MDA-MB-468, Caco-2), and one BZ-resistant cell line (A2780) at two different conditions, ie, 43°C for 30 minutes and 45°C for 30 minutes. These experiments were compared with combined application of HWH and BZ. The results indicate enhanced potentiation between hyperthermic treatment and BZ. MFH combined with BZ induced cytotoxicity in sensitive and resistant cell lines to a greater extent than HWH under the same treatment conditions. The observation that MFH sensitizes BZ-resistant cell lines makes this approach a potentially effective anticancer therapy platform.

Hyperthermic potentiation of cisplatin by magnetic nanoparticle heaters is correlated with an increase in cell membrane fluidity.

Magnetic fluid hyperthermia as a cancer treatment method is an attractive alternative to other forms of hyperthermia. It is based on the heat released by magnetic nanoparticles subjected to an alternating magnetic field. Recent studies have shown that magnetic fluid hyperthermia-treated cells respond significantly better to chemotherapeutic treatment compared with cells treated with hot water hyperthermia under the same temperature conditions. We hypothesized that this synergistic effect is due to an additional stress on the cellular membrane, independent of the thermal heat dose effect that is induced by nanoparticles exposed to an alternating magnetic field. This would result in an increase in Cis-diammine-dichloroplatinum (II) (cDDP, cisplatin) uptake via passive transport. To test this hypothesis, we exposed cDDP-treated cells to extracellular copper in order to hinder the human cell copper transporter (hCTR1)-mediated active transport of cDDP. This, in turn, can increase the passive transport of the drug through the cell membrane. Our results did not show statistically significant differences in surviving fractions for cells treated concomitantly with magnetic fluid hyperthermia and cDDP, in the presence or absence of copper. Nonetheless, significant copper-dependent variations in cell survival were observed for samples treated with combined cDDP and hot water hyperthermia. These results correlated with platinum uptake studies, which showed that cells treated with magnetic fluid hyperthermia had higher platinum uptake than cells treated with hot water hyperthermia. Changes in membrane fluidity were tested through fluorescence anisotropy measurements using trimethylamine-diphenylhexatriene. Additional uptake studies were conducted with acridine orange and measured by flow cytometry. These studies indicated that magnetic fluid hyperthermia significantly increases cell membrane fluidity relative to hot water hyperthermia and untreated cells, and hence this could be a factor contributing to the increase of cDDP uptake in magnetic fluid hyperthermia-treated cells. Overall, our data provide convincing evidence that cell membrane permeability induced by magnetic fluid hyperthermia is significantly greater than that induced by hot water hyperthermia under similar temperature conditions, and is at least one of the mechanisms responsible for potentiation of cDDP by magnetic fluid hyperthermia in Caco-2 cells.

2D protrusion but not motility predicts growth factor-induced cancer cell migration in 3D collagen.

Growth factor-induced migration is a critical step in the dissemination and metastasis of solid tumors. Although differences in properties characterizing cell migration on two-dimensional (2D) substrata versus within three-dimensional (3D) matrices have been noted for particular growth factor stimuli, the 2D approach remains in more common use as an efficient surrogate, especially for high-throughput experiments. We therefore were motivated to investigate which migration properties measured in various 2D assays might be reflective of 3D migratory behavioral responses. We used human triple-negative breast cancer lines stimulated by a panel of receptor tyrosine kinase ligands relevant to mammary carcinoma progression. Whereas 2D migration properties did not correlate well with 3D behavior across multiple growth factors, we found that increased membrane protrusion elicited by growth factor stimulation did relate robustly to enhanced 3D migration properties of the MDA-MB-231 and MDA-MB-157 lines. Interestingly, we observed this to be a more reliable relationship than cognate receptor expression or activation levels across these and two additional mammary tumor lines.

Structural dynamics and single-stranded DNA binding activity of the three N-terminal domains of the large subunit of replication protein A from small angle X-ray scattering.

Replication protein A (RPA) is the primary eukaryotic single-stranded DNA (ssDNA) binding protein utilized in diverse DNA transactions in the cell. RPA is a heterotrimeric protein with seven globular domains connected by flexible linkers, which enable substantial interdomain motion that is essential to its function. Small angle X-ray scattering (SAXS) experiments with two multidomain constructs from the N-terminus of the large subunit (RPA70) were used to examine the structural dynamics of these domains and their response to the binding of ssDNA. The SAXS data combined with molecular dynamics simulations reveal substantial interdomain flexibility for both RPA70AB (the tandem high-affinity ssDNA binding domains A and B connected by a 10-residue linker) and RPA70NAB (RPA70AB extended by a 70-residue linker to the RPA70N protein interaction domain). Binding of ssDNA to RPA70NAB reduces the interdomain flexibility between the A and B domains but has no effect on RPA70N. These studies provide the first direct measurements of changes in orientation of these three RPA domains upon binding ssDNA. The results support a model in which RPA70N remains structurally independent of RPA70AB in the DNA-bound state and therefore freely available to serve as a protein recruitment module.