Novel epigenetic target therapy for prostate cancer: a preclinical study

Abstract

Epigenetic events are critical contributors to the pathogenesis of cancer, and targeting epigenetic mechanisms represents a novel strategy in anticancer therapy. Classic demethylating agents, such as 5-Aza-2'-deoxycytidine (Decitabine), hold the potential for reprograming somatic cancer cells demonstrating high therapeutic efficacy in haematological malignancies. On the other hand, epigenetic treatment of solid tumours often gives rise to undesired cytotoxic side effects. Appropriate delivery systems able to enrich Decitabine at the site of action and improve its bioavailability would reduce the incidence of toxicity on healthy tissues. In this work we provide preclinical evidences of a safe, versatile and efficient targeted epigenetic therapy to treat hormone sensitive (LNCap) and hormone refractory (DU145) prostate cancers. A novel Decitabine formulation, based on the use of engineered erythrocyte (Erythro-Magneto-Hemagglutinin Virosomes, EMHVs) drug delivery system (DDS) carrying this drug, has been refined. Inside the EMHVs, the drug was shielded from the environment and phosphorylated in its active form. The novel magnetic EMHV DDS, endowed with fusogenic protein, improved the stability of the carried drug and exhibited a high efficiency in confining its delivery at the site of action in vivo by applying an external static magnetic field. Here we show that Decitabine loaded into EMHVs induces a significant tumour mass reduction in prostate cancer xenograft models at a concentration, which is seven hundred times lower than the therapeutic dose, suggesting an improved pharmacokinetics/pharmacodynamics of drug. These results are relevant for and discussed in light of developing personalised autologous therapies and innovative clinical approach for the treatment of solid tumours.

Figure 1. Qualitative and quantitative analysis of the distribution and effect of EMHVs treatment in DU145 prostate cancer cells.

Representative CLSM images of EMHVs internalization and released nanoparticle distribution (green fluorescence signal, B–E) into the host cell cytoplasm at several time points are depicted. Naïve cells (A) are reported as control. In (B), after 6 hours of treatment, an intact EMHV is present in the cytoplasm (arrow). At 24 hours (C) the particles appeared to have been released from the EMHVs. In the pictures taken at 48 (D) and 96 (E) hours, nanoparticles appear to have homogeneously distributed throughout the cytoplasm. Lack of toxic effect of erythrocyte drug delivery system treatment was evaluated by FACS analysis (F) where cell profile of the treated cells (EMHVs) at selected time points show no significant changes in sub-phase distributions with respect to control (CTRL).

Figure 2. HPLC-MS chromatogram of the phosphorylated forms of 5-Aza-2′-dC.

(A) chromatograms for 5-Aza-2′-dC mono-phosphate; (B) di-phosphate (RT: 16.6); (C) tri-phosphate (RT: 16.5) and (D) internal standard CTP (RT: 16.5).

Figure 3. The effect of A-EMHVs on the cell cycle arrest of LNCap, hormone sensitive and DU145 hormone refractory cells.

1.5×10⁸ A-EMHVs treatment, containing 120 ng internal 5-Aza-2'-dC, was compared with 6.8 µg free 5-Aza-2'-dC and 120 ng free 5-Aza-2'-dC treatments at 24 (top) 48 (middle) 96 (bottom) hours. Untreated cells were used as control (CTRL). In both LNCap (A) and DU145 (B) cell lines, A-EMHVs treatment induced a significant enrichment in sub G1 cell distribution (black bars) already detectable at 48 hours after treatment (A and B middle panel, respectively) that last up to 96 hours (*ANOVA p<0.05). Similar shift toward sub G1 distribution was also obtained using 6.8 µg of free 5-Aza-2'-dC (§ANOVA p<0.05) but only detected at 96 hours. No shift in cell cycle distribution was detected for free 120 ng 5-Aza-2'-dC, not differing from controls.

Figure 4. The pro-apoptotic effect of A-EMHVs on LNCap, hormone sensitive and DU145, hormone refractory cells.

1.5×10⁸ A-EMHVs treatment, containing 120 ng internal 5-Aza-2'-dC, was compared with 6.8 µg free 5-Aza-2'-dC and 120 ng free 5-Aza-2'-dC treatments at 96 hours. Untreated cells were used as control (CTRL). In both LNCap (A) and DU145 (B) cell lines, A-EMHVs treatment induced a significant enrichment in early apoptosis (*ANOVA p<0.05 and **ANOVA p<0.01, respectively). Using 6.8 µg or 120 ng of free 5-Aza-2'-dC, no effect in early apoptosis was detected in any cell lines at 96 hours. Significant increase in late apoptosis response was observed in LNCap (C) and DU145 (D) after treatment with A-EMHVs as well as with 6.8 µg of free 5-Aza-2'-dC (*ANOVA p<0.05 and ***ANOVA p<0.001, respectively), whereas no change in late apoptosis was detected after lower free 5-Aza-2'-dC treatment at 96 hours in any of the cell lines.

Figure 5. Localized targeted in vivo distribution of EMHVs after systemic tail vein administration in mice.

Representative X-ray images of mice injected with EMHVs and treated with null (A and C) or 1,000 KOersted external static magnetic field (B and D) applied for 30 min in correspondence of the abdomen, are depicted. After 1 hour from the injection, a rather sparse distribution of nanoparticles (white irregular shadow/dots) was found inside metabolic organs and tissues exposed to null magnetic field (EMHVs-NMF, A sagittal and C frontal planes, respectively). After exposure to the magnetic field (EMHVs-MF, B sagittal and D frontal planes, respectively), nanoparticles concentrated in the target tissue (abdomen) resulting in a sharp intense signal (arrow).

Figure 6. The anticancer effect of A-EMHVs treatment in vivo.

Mass tumour growth curves for all experimental group are depicted in panel A and B. The treatments are Control (CTRL, cyan); therapeutic dose (A1, red); low dose equivalent to that inside the erythrocytes (A2, purple); EMHVs and no magnetic field applied (EMHVs-NMF, blue); EMHVs undergoing magnetic field (EMHVs-MF, yellow); loaded EMHVs and no magnetic field applied (A-EMHVs-NMF, orange); loaded EMHVs undergoing magnetic field application (A-EMHVs-MF, green). In (A) LNCap xenograft, the most significant mass reduction was measured both in A-EMHVs-MF and A1, while other treatments exerted intermediate effect with respect to controls (One-Way ANOVA p<0.05 at time of the sixth injection). In (B) DU145 xenograft, mass showed a similar trend with A-EMHVs-MF and A1 treatments being the most effective (One-Way ANOVA p<0.05). In C and D, Kaplan-Meier estimator curve was used to determine the percentage of treated mice showing a reduction of tumour mass volume (≥50%) during treatment. In (C) the trend of reduction is depicted for LNCap xenograft model: 100% of animals show ≥50% tumour volume reduction after A-EMHVs-MF (day 7^th) and A1 (day18^th) treatments. In DU145 xenograft model (D), A-EMHVs-MF (green) is the most effective anti-tumour treatment (e.g. CTRL or A2 vs A-EMHVs-MF, log rank = 4.52 at day 21^st).

Figure 7. Histological and immunohistochemical features of explanted xenografts.

Representative sections of controls (CTRL), 120 ng of free 5-Aza-2'-dC (A2) and A-EMHVs-MF treated animals. Top panels (A) show H&E stain at low power (2x original magnification) and high power (40x original magnification, insets) micrographs. In B, immunohistological reactivity, positive for nuclear DNMT3b methyltrasferase (DNA methyltransferase 3b) as a marker of tumour cells de novo methylation is shown (positive nuclei: dark brown, negative nuclei: blue. Original magnification 10x and insets at 40x). In the bottom panels (C) proliferating tumour cells are visualized using Ki67 marker (positive nuclei: dark brown, negative nuclei: blue. Original magnification 10x and insets at 40x).