Cell type-specific activation of AKT and ERK signaling pathways by small negatively-charged magnetic nanoparticles

Abstract

The interaction of nanoparticles (NPs) with living organisms has become a focus of public and scientific debate due to their potential wide applications in biomedicine, but also because of unwanted side effects. Here, we show that superparamagnetic iron oxide NPs (SPIONs) with different surface coatings can differentially affect signal transduction pathways. Using isogenic pairs of breast and colon derived cell lines we found that the stimulation of ERK and AKT signaling pathways by SPIONs is selectively dependent on the cell type and SPION type. In general, cells with Ras mutations respond better than their non-mutant counterparts. Small negatively charged SPIONs (snSPIONs) activated ERK to a similar extent as epidermal growth factor (EGF), and used the same upstream signaling components including activation of the EGF receptor. Importantly, snSPIONs stimulated the proliferation of Ras transformed breast epithelial cells as efficiently as EGF suggesting that NPs can mimic physiological growth factors.

Figure 1. Scheme for the ultra-uniform synthesis of monodisperse SPIONs and their coating process.

Iron–oleate precursor was prepared from the reaction of iron chlorides and sodium oleate. The monodisperse SPIONs in various sizes (i.e. 9 and 15 nm) were prepared by thermal decomposition of the metal–oleate precursors in high boiling solvent. The coating processes were performed by ligand exchange method in DMSO. TEM images show the formation of uniform bare- and dextran-coated-SPIONs with two different sizes. (The scale bars are 10 nm and 20 nm, respectively).

Figure 2. Regulation of ERK and AKT signaling by SPIONs in HCT116 and Hke3 cells.

HCT116 and Hke3 cells were incubated with SPIONs or EGF after serum-withdrawal for 18 hours. Activation of ERK (A) and activation of AKT (B) was assessed at the indicated time-points by immunoblotting. Results represent the mean of 3 independent experiments with SD.

Figure 3. Regulation of ERK and AKT signaling by SPIONs in MCF10A/CA1 cells.

MCF10A and CA1 cells were incubated with SPIONs or EGF after serum-withdrawal for 18 hours. Activation of ERK (A) and activation of AKT (B) was assessed at the indicated time-points by immunoblotting. Results represent the mean of 3 independent experiments with SD.

Figure 4. Signaling-specific inhibitors inhibit activation by snSPIONs.

(A) Schematic representation of the small-molecule inhibitors used and (B) the time course. MCF10A and CA1 cells were incubated with SPIONs or EGF after serum-withdrawal for 18 hours and pre-treatment with inhibitors. Activation of ERK (A) and activation of AKT (B) was assessed at the indicated time-points by immunoblotting. Results represent the mean of 3 independent experiments with SD.

Figure 5. snSPIONs activate the EGF receptor.

(A) Serum starved MCF10A and CA1 cells were incubated with snSPIONs or EGF.Activation of the EGFR was assessed with phosphospecific antibodies. Activation of EGFR by SPIONs can be inhibited by EGFR-specific inhibitors. Serum starved MCF10A and CA1 cells were treated with EGFR inhibitors before stimulation. Activation of ERK (B) and activation of AKT (C) was assessed at the indicated time-points by immunoblotting. (D) Cell numbers of MCF10A and CA1 cells were determined at the indicated timepoints following incubation with snSPIONs, lnSPIONs, spSPIONs or EGF. Results represent the mean of 3 independent experiments with SD. * - p<0.05. ** - p<0.01.