Multifunctional Polymeric Micelles Co-loaded with Anti-Survivin siRNA and Paclitaxel Overcome Drug Resistance in an Animal Model of Ovarian Cancer

Abstract

Ovarian cancer is a dreadful disease estimated to be the second most common gynecologic malignancy worldwide. Its current therapy, based on cytoreductive surgery followed by the combination of platinum and taxanes, is frequently complicated by the onset of multidrug resistance (MDR). The discovery that survivin, a small antiapoptotic protein, is involved in chemoresistance provided a new prospect to overcome MDR in cancer, because siRNA could be used to inhibit the expression of survivin in cancer cells. With this in mind, we have developed self-assembly polymeric micelles (PM) able to efficiently co-load an anti-survivin siRNA and a chemotherapeutic agent, such as paclitaxel (PXL; survivin siRNA/PXL PM). Previously, we have successfully demonstrated that the downregulation of survivin by using siRNA-containing PM strongly sensitizes different cancer cells to paclitaxel. Here, we have evaluated the applicability of the developed multifunctional PM in vivo. Changes in survivin expression, therapeutic efficacy, and biologic effects of the nanopreparation were investigated in an animal model of paclitaxel-resistant ovarian cancer. The results obtained in mice xenografed with SKOV3-tr revealed a significant downregulation of survivin expression in tumor tissues together with a potent anticancer activity of survivin siRNA/PXL PM, while the tumors remained unaffected with the same quantity of free paclitaxel. These promising results introduce a novel type of nontoxic and easy-to-obtain nanodevice for the combined therapy of siRNA and anticancer agents in the treatment of chemoresistant tumors.

Figure 1

(A) In vivo antitumor activity of survivin siRNA/PXL PM in SKOV3-tr xenografts. Survivin siRNA/PXL PM were administered at a final concentration of anti-survivin siRNA and PXL of 1 and 10 mg/kg, respectively, once per week for 5 consecutive weeks. Relative tumor volume (RTV) values (tumor volume in mm³ on day ‘n’ (Vn) / tumor volume at the start of the treatment (Vo) plotted versus time in days) are reported. Data were given as mean ± SD for each treatment group. *p < 0.05, **p < 0.01, and ***p < 0.005 were obtained by comparing each treatment group with Taxol group. (B) Post-mortem tumor weights. On day 30, eterotopically implanted tumors were weighed and plotted. *p < 0.05 and **p < 0.01 were considered significant and very significant, respectively, and were obtained by comparing each treatment group with survivin siRNA/PXL group. (C) Evaluation of repeated dosing toxicity in mice by measurement of changes in serum levels of transaminase (AST/ALT). Data were given as mean ± SD for each treatment group. (D) Changes in body weight by measurement of the body weight of the mice three times a week for 5 consecutive weeks. Data were given as mean ± SD for each treatment group. N.s. means no statistical significance between the different groups.

Figure 2. Apoptosis analysis on tumor sections by the TUNEL assay

Pictures were taken by the confocal microscopy (10× magnification). The nuclei were stained for Hoechst 33342 (blue) and apoptotic cells (green) for Tunel. Representative images of (a) Untreated, (b) Scrambled siRNA/PXL PM,(c) Taxol, (d) Survivin siRNA PM, (e) Survivin siRNA/PXL PM groups.

Figure 3. Survivin mRNA levels in tumor tissues by rt-PCR analysis

Data were given as mean ± SD for each treatment group. ***p < 0.005, **p < 0.01 were obtained by comparing each treatment group with survivin siRNA/PXL group.

Figure 4

(A). Immunohystochemistry analysis. Survivin protein levels were evaluated by the fluorescent microscopy (50 ×). Representative images of three independent experiments showing survivin expression (in red) and nuclei staining with Hoechst 33342 (blue). Untreated group (a); Scrambled siRNA/PXL PM group (b); Taxol group (c); Survivin siRNA PM group (d); Survivin siRNA/PXL PM group (e). Scale bar 20 µm. (B). Cell fluorescent intensity of the survivin protein levels measured with ImageJ. ***p < 0.005 was obtained by comparing the cell fluorescence of each treatment group with PBS group (C). Simultaneous down-regulation of survivin expression and PXL penetration in tumor tissues by using survivin siRNA/PXL PM. At the same time, the intra-tumor accumulation of Oregon Green labeled PXL (left) and survivin protein expression (middle) were evaluated on tumor sections by confocal microscopy (magnification 63×). Scale bar 20 µm.

Figure 5. Microtubule organization after treatment with survivin siRNA/PXL PM in vivo

SKOV3-tr tumor sections were stained for β-tubulin (green). a-e: Representative images of three independent experiments showing the organization of microtubules (magnification 63×). Untreated group (a); scrambled siRNA/PXL PM group (b); Taxol group (c); survivin siRNA PM group (d); survivin siRNA/PXL PM group (e). Scale bar 10 µm.