Cosilencing of PKM-2 and MDR-1 Sensitizes Multidrug-Resistant Ovarian Cancer Cells to Paclitaxel in a Murine Model of Ovarian Cancer

Abstract

Tumor multidrug resistance (MDR) is a serious clinical challenge that significantly limits the effectiveness of cytotoxic chemotherapy. As such, complementary therapeutic strategies are being explored to prevent relapse. The altered metabolic state of cancer cells, which perform aerobic glycolysis, represents an interesting target that can enable discrimination between healthy cells and cancer cells. We hypothesized that cosilencing of genes responsible for aerobic glycolysis and for MDR would have synergistic antitumor effect. In this study, siRNA duplexes against pyruvate kinase M2 and multidrug resistance gene-1 were encapsulated in hyaluronic acid-based self-assembling nanoparticles. The particles were characterized for morphology, size, charge, encapsulation efficiency, and transfection efficiency. In vivo studies included biodistribution assessment, gene knockdown confirmation, therapeutic efficacy, and safety analysis. The benefit of active targeting of cancer cells was confirmed by modifying the particles' surface with a peptide targeted to epidermal growth factor receptor, which is overexpressed on the membranes of the SKOV-3 cancer cells. To augment the studies involving transplantation of a paclitaxel-resistant cell line, an in vivo paclitaxel resistance model was developed by injecting repeated doses of paclitaxel following tumor inoculation. The nanoparticles accumulated significantly in the tumors, hindering tumor volume doubling time (P < 0.05) upon combination therapy in both the wild-type (2-fold) and resistant (8-fold) xenograft models. Although previous studies indicated that silencing of MDR-1 alone sensitized MDR ovarian cancer to paclitaxel only modestly, these data suggest that concurrent silencing of PKM-2 improves the efficacy of paclitaxel against MDR ovarian cancer.

Figure 1

Characteristics of HA derivative/siRNA particles. (A) Schematic representation of HA-PEI/PEG/EGF nano-assemblies with negatively charged siRNA. (B) The self-assembling nanoparticles showed a spherical morphology in TEM. (C) Electrophoretic retardation analysis of siRNA binding by HA-PEI with the release of intact siRNA by poly(acrylic acid). (D) Cell uptake studies in SKOV-3 WT and TR model. HA-rhodamine-123 and scramble siRNA labeled with fluorescein were incubated with the cells for 5, 15 and 30 min. The cells showed uptake within 15 min of incubation with the dual targeted system showing higher fluorescence signal compared to the CD44 targeted system. The cell nuclei were stained with DAPI (blue).

Figure 2

HA-PEI/siRNA mediated MDR-1 and PKM-2 gene silencing in SKOV-3_WT (A) and SKOV-3_TR (B). Cells were treated with scramble, naked siRNA, siRNA formulated in Lipofectamine and siRNA formulated in HA-PEI/PEG nanoparticles. MDR-1 and PKM-2 expression was measured by qPCR. Data represented as a mean SD (n=3). (C) Cytotoxicity assay following single and combination therapy in SKOV-3_WT (left) and SKOV-3_TR cells (right).

Figure 3

In vivo (A-B) and ex vivo (C) biodistribution of ICG/HA-PEI/PEG and ICG/HAPEI/PEG-EGF nanoparticles in SKOV-3_WT and SKOV-3_TR tumor bearing mice over 1, 2, 4, 6 and 24 h. Quantitative biodistribution studies to assess distribution of siPKM-2 following intravenous dosing of HA-PEI-siPKM-2 as CD44-targeted and dual targeted delivery systems. Quantitative biodistribution studies to assess distribution of siPKM-2 following intravenous dosing of HA-PEI-siPKM-2 as HA-PEI/PEG and dual targeted delivery systems in SKOV-3_WT (D) and SKOV-3_TR (E) tumor models.

Figure 4

In vivo gene silencing studies of MDR-1 and PKM-2 in (A) SKOV-3_WT and (B) SKOV-3_TR tumor bearing mice n=6. (C) Effect of the combination of paclitaxel treatment and MDR1 and PKM2 silencing on growth of SKOV3_WT tumors.

Figure 5

In vivo PTX resistance model development: (A) qPCR studies to assess MDR-1 and PKM-2 expression (B) Immunofluorescence of tumor tissues from Pg-P and PKM-2 expression in PBS treated and PTX treated animals. (C) Schematic representation of the dosing schedule adopted to develop the SKOV-3TR mouse model followed by nanoparticle treatment. (D) Effect of the combination of paclitaxel treatment and MDR1 and PKM2 silencing on growth of SKOV3_TR tumors.