miRNA-205 Nanoformulation Sensitizes Prostate Cancer Cells to Chemotherapy

Abstract

The therapeutic application of microRNA(s) in the field of cancer has generated significant attention in research. Previous studies have shown that miR-205 negatively regulates prostate cancer cell proliferation, metastasis, and drug resistance. However, the delivery of miR-205 is an unmet clinical need. Thus, the development of a viable nanoparticle platform to deliver miR-205 is highly sought. A novel magnetic nanoparticle (MNP)-based nanoplatform composed of an iron oxide core with poly(ethyleneimine)-poly(ethylene glycol) layer(s) was developed. An optimized nanoplatform composition was confirmed by examining the binding profiles of MNPs with miR-205 using agarose gel and fluorescence methods. The novel formulation was applied to prostate cancer cells for evaluating cellular uptake, miR-205 delivery, and anticancer, antimetastasis, and chemosensitization potentials against docetaxel treatment. The improved uptake and efficacy of formulations were studied with confocal imaging, flow cytometry, proliferation, clonogenicity, Western blot, q-RT-PCR, and chemosensitization assays. Our findings demonstrated that the miR-205 nanoplatform induces significant apoptosis and enhancing chemotherapeutic effects in prostate cancer cells. Overall, these study results provide a strong proof-of-concept for a novel nonviral-based nanoparticle protocol for effective microRNA delivery to prostate cancer cells.

Keywords: chemosensitivity and EMT; docetaxel; miR-205; prostate cancer.

Figure 1

Generation of miR-205-NPs formulations (A) Preparative approach and hypothetical structure of miR-205 nanoformulations. PEI-PEG: gray line belongs to PEI and dotted greens belong to PEG. miR-205 binds to PEI. (B–C) Nanocomplexation assessment with miR-205. Fluorescence-based quenching study of fluorescein amidite (FAM)-labeled miRNA mimic MNP and MNP-MPEI-PEG (MPEI-PEG) nanoparticles. Note: MNP alone is also able to bind smaller amounts of miR-205, due to physical deposition on the nanoparticles but not due to binding. (D) Determination of nanocomplexation of miR-205 with MNP and MPEI-PEG through agarose gel electrophoresis. Data represent that 5 µg of nanocarrier is sufficient to hold 1 µg of miR-205 for delivery applications. (E) Evaluation of miR-205 binding with MNP and MPEI-PEG nanoparticles by circular dichroism (CD) spectral analysis.

Figure 2

miR-205 delivery characteristics of miR-205-NPs. (A,B) Hemolytic activity of MNP-PEI-PEG nanocarrier. Brightfield microscopy at 20× magnification was employed for capturing hemolytic characteristic of human red blood cells. Scale bar: 50 µm. Note: NPs are not toxic but lipofectamine is at the tested concentrations. Data indicate that nanocarrier is hemocompatible, unlike lipofectamine. (C) Dissociation studies of miR-205 in presence of poly(anion) (heparin). (D) miRNA-205 stability in the presence of 0–50% Fetal Bovine Serum (FBS) concentration. Equal amount of each sample was incubated with 10 μL of FBS at 37 °C for 24 h prior to gel electrophoresis. Note: “No NPs” lane did not show any band due to the absence of miRNA. (E) FAM-miRNA release profile from the FAM-miR through fluorescence spectral analysis at variable pH solutions (7.4, 6.5 and 3.5). The significance level was * p < 0.05. Each experiment has been repeated three times.

Figure 3

Cellular fate of miR-205-NPs formulation. (A) Cellular uptake of coumarin 6-labeled MPEI-PEG nanoformulation. Representative cellular internalized nanoparticles images were captured at 40× magnification using confocal microscopy. Green color arise from coumarin 6 in MPEI-PEG. Scale bar: 50 µm. (B) Quantitative measurement of FAM-miRNA mimic in cells treated with FAM-labeled miR-MPEI-PEG formulation. (C) Restoration of miR-205 through miR-205-NPs in PrCa cells. q-PCR gene expression studies reveals that treatments with miR-205-NPs reconstitutes the gene expression of miR-205. (D) miR-205-NPs influence cell growth in PrCa cells. Proliferation assays after miR-205 and miR-205-NPs transfection treatments in C4-2 and PC-3 cell lines using 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt (MTS) assays. The significance level was * p < 0.05 with respect to NC/NPs. Each individual experiment has been repeated three times.

Figure 4

miR-205-NPs treatment chemosensitizes PrCa cells towards docetaxel therapy. (A) NC (No Dtxl), NPs, NC (non-targeting control), miR-205 naïve, miR-205-Lipo, and miR-205-NPs-treated PrCa cells (5 × 10³/well in 96-well plate) were treated with 0–25 nM Dtxl or respective control for 48 h and proliferation of cells was assessed by MTS assay. (B) NC, NPs, miR-205-Lipo, and miR-205-NPs-treated PrCa cells with 2.5 nM Dtxl. On day 14, cells were PBS-rinsed and stained with hematoxylin. Photographs of clonogenic pattern (in Multimage™ light cabinet) represent inhibition of clonogenic formation with miR-205-NPs formulation. Note: Figure 4B quantification was provided in Figure S3.

Figure 5

miR-205-NPs induce enhanced apoptotic potential of docetaxel in PrCa cells. (A) Representative microscopic images of various treatment groups after 24 h. Images were captured at 20× magnification. Scale bar: 100 µm. (B) Western blot analysis confirms the significant induction of apoptotic signaling proteins after the delivery of miR-205-NPs and Dtxl treatment. Cl-PARP and Cl-casp. 3 indicates cleaved PARP and cleaved caspase 3, respectively.

Figure 6

miR-205-NPs inhibits EMT signaling and sensitizes Dtxl treatment in PrCa cells. (A) miR-205-NPs treatment suppresses the migratory ability of C4-2 and PC-3 cells in presence/absence of the drug through Boyden chamber study. Images were captured at 20× magnification. Transwell assay with matrigel was performed to detect invasion activity of PrCa cells transfected with miR-205. Docetaxel treatments at 5 nM concentration. Scale bar: 200 µm. (B) Protein profiling studies of Control, miR-205 transfected and miR-205-NPs treated PrCa cells for 24 h for EMT signaling. Note: miR-205-L or miR-205-Lipo represents same and indicates to miR-205 transfected with lipofectamine.

Figure 7

miR-205-NPs effectively inhibit Pgp activity and facilitates Dtxl chemosensitization. (A,B) Rh123 Dye exclusion studies were performed in cells through morphological and flow cytometric methods. miR-205-NPs formulation facilitates Rh123, demonstrating its chemosensitizing potential. (C) miR-205-NPs inhibited MDR1/ABCB1 expression in both C4-2 and PC-3 cells and densitometry studies were calculated. (D) miR-205-NPs promote microtubule stabilization in PrCa cells. Except for non-targeting control (NC), 5 nM Dtxl was used in all treatment groups. Treatment period was 8 h. Images were captured at 40× magnification using confocal microscopy. Scale bar: 50 µm. Corrected total cell fluorescence (CTCF) values were calculated using ImageJ and showed as insets. The level of significance was * p < 0.05. Each individual experiment has been repeated three times.

Figure 8

Schematic representation of possible chemosensitization mechanism of action for miR-205-NPs formulation on prostate cancer cells.