Aerosol azacytidine inhibits orthotopic lung cancers in mice through Its DNA demethylation and gene reactivation effects - PubMed (original) (raw)

Aerosol azacytidine inhibits orthotopic lung cancers in mice through Its DNA demethylation and gene reactivation effects

Xuan Qiu et al. PLoS One. 2014.

Abstract

We devised an aerosol based demethylation therapy to achieve therapeutic efficacy in premalignant or in situ lesions of lung cancer, without systemic toxicity. Optimum regimens of aerosolized azacytidine (Aza) were designed and used in orthotopic human non-small cell lung cancer xenograft models. The therapeutic efficacy and toxicity of aerosol Aza were compared with intravenously administered Aza. We observed that 80% of the droplets of the aerosol Aza measured ∼0.1-5 microns, which resulted in deposition in the lower bronchial airways. An animal model that phenocopies field carcinogeneisis in humans was developed by intratracheal inoculation of the human lung cancer cells in mice, thus resulting in their distribution throughout the entire airway space. Aerosolized Aza significantly prolonged the survival of mice bearing endo-bronchial lung tumors. The aerosol treatment did not cause any detectable lung toxicity or systemic toxicity. A pre-pharmacokinetic study in mice demonstrated that lung deposition of aerosolized Aza was significantly higher than the intravenous route. Lung tumors were resected after aerosol treatment and the methylation levels of 24 promoters of tumor-suppresser genes related to lung cancer were analyzed. Aerosol Aza significantly reduced the methylation level in 9 of these promoters and reexpressed several genes tested. In conclusion, aerosol Aza at non-cytotoxic doses appears to be effective and results in DNA demethylation and tumor suppressor gene re-expression. The therapeutic index of aerosol Aza is >100-fold higher than that of intravenous Aza. These results provide a preclinical rationale for a phase I clinical trial of aerosol Aza to be initiated at our Institution.

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Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1

Figure 1. Orthotopic NSCLC model.

The model was created by intratracheally injecting NSCLC H460 cells into nude mice. Top: H & E stained lung sections from IT inoculated mice on day 35. The tumors (T) arise within the airways and grow vicinally into the lung parenchyma. Bottom: H & E stained lung sections from the IV inoculated mice on day 35. Tumors arise within small vessels in the lung parenchyma. The objective magnification was 40X. The scale bars were 50 µm.

Figure 2

Figure 2. Aerodynamic size of Aerosol Aza.

T determined with extrusion-precipitation method using a 7-Stage Cascade Impactor linked to PARI’s personal compressor and LC star nebulizer system. Aza solution (4 ml) was aerosolized at an airflow rate of 5 L/min. The condensed aerosol samples were collected at 3 different intervals, from 1 to 1.5 min, from 3 to 3.5 min, and from 5 to 5.5 min. Aerodynamic size and fraction of aerosol with a particular size range were measured and calculated as per manufacturer’s protocol. The data was mean ± standard deviation of the aerodynamic size based on weight (solid bars) and cumulative weight (empty bars) from 3 independent experiments.

Figure 3

Figure 3. Toxicity of aerosol Aza.

Lung sections of mice treated with aerosol Aza at 2.5 mg/m2×7 (A), IT Aza at 75 mg/m2×7 (B), and IT Aza at 270 mg/m2×5 (C), respectively show no toxicity at 2.5 and 75 2.5 mg/m2 but pneumonitis at 270 mg/m2. The objective magnification was 40X. The scale bars were 50 µm. White blood cell count ratio (after treatment vs. before treatment, n = 6) (D) and percentage of viable cells of the sorted airway epithelial cells (n = 6) (E), respectively.

Figure 4

Figure 4. Aerosol administration of Aza significantly prolongs the survival of mice with orthotopic human NSCLC xenografts (A∼C).

Mice intratracheally inoculated with the cell lines H226 (A), H358 (B), or H460 (C) were treated with aerosol Aza (red thick line) at 2.5 mg/m2 daily for 7 days or aerosol vehicle (blue thin line) with the same volume. Treatment with IV Aza (dash line) at the optimal dose of 75 mg/m2 daily for 5 days was used as control. The statistical comparison of the survival curves was performed with Log-rank (Mantel-Cox) test. The p values for H266, H358, and H460 models were 0.0135, 0.0150, and 0.0719, respectively. Pre-pharmacokinetics of the aerosol Aza (D). ICR mice were treated with aerosol Aza at 2.5 mg/m2. The data at each time point was the average ± standard deviation of the percent of given/initial dose from lungs of 3 mice. The equation for each curve was simulated curve with the best fitting (the highest R2 value) under the Microsoft Excel program.

Figure 5

Figure 5. Methylation changes in the lung tumors.

Tumor-bearing mice from 3 orthotopic lung cancer models (H226-xeno, H358-xeno, and H460-xeno) were treated with aerosol Aza (A) or IV Aza (B) at 2.5 mg/m2 daily×7. The lungs were resected 14 days after the final treatment and tumor nodules larger than 1.5 mm were isolated for methylation detection by qPCR array technology at Qiagen (SA Biosciences). The data is %methylation of the total CG cytosine of the particular promoter region.

Figure 6

Figure 6. Protein expression of the TSGs in the lung tumors (A).

Western blotting assay was used to determine the protein expression of the TSGs with significant promoter demethylation after aerosol Aza treatment identified by the methylation q-PCR array. The tumor samples were the same as used in Figure 5; Histogram of the Western blots (B). The western blot photo films were scanned and the density ratio of targeted protein vs. actin loading control of each sample was presented as the protein expression level.

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