Efficacy of aerosolized celecoxib encapsulated nanostructured lipid carrier in non-small cell lung cancer in combination with docetaxel

Abstract

Purpose: Evaluation of in-vivo anticancer activity of aerosolized Celecoxib encapsulated Nanolipidcarriers (Cxb-NLC) as a single therapeutic agent and combined with intravenously administered Docetaxel (Doc) against non-small cell lung cancer.

Methods: Cxb-NLC were prepared by high-pressure homogenization and were characterized for its physicochemical characteristics. Metastatic A549 tumor model in Nu/Nu mice was used to evaluate response of aerosolized Cxb-NLC & Doc. Isolated lung tumor samples were analyzed for: a) DNA fragmentation and cleaved caspase-3 by immunohistochemistry, b) apoptotic and angiogenic protein markers by western blot, c) global proteomic alterations by an isobaric labeling quantitative proteomic method and d) toxicity studies of NLC.

Results: The particle size of Cxb-NLC was 217 ± 20 nm, while entrapment efficiency was more than 90%. Cxb-NLC and Doc alone and in combination showed 25 ± 4%, 37 ± 5%, and 67 ± 4% reduction in tumor size respectively compared to control. Proteomic analysis with combination treatment further revealed significantly decreased expression of multiple pro-survival and pro-metastasis proteins as well as tumor invasion markers and the expression of S100 family proteins, such as S100A6 and S100P were decreased by 2.5 and 1.6 fold.

Conclusions: Combination therapy with Cxb-NLC and Doc showed significant reduction in tumor growth which was further confirmed by proteomic analysis.

Fig. 1. Isobolograms (A) and (B) Combination Index (CI) values of the interaction between Cxb with Doc against human lung Cancer cells

Different concentrations of Cxb were employed to study the effect on IC50 of Doc. Variable ratios of drug concentrations and mutually non-exclusive equations were used to determine the CI. The CI values represent mean of four experiments. CI >1.3: antagonism; CI 1.1–1.3: moderate antagonism; CI 0.9–1.1: additive effect; CI 0.8–0.9: slight synergism; CI 0.6–0.8: moderate synergism; CI 0.4–0.6: synergism; CI 0.2–0.4: strong synergism.

Fig. 2. Effects of Cxb-NLC and Doc on metastatic A549 lung tumor weight (A); metastatic A549 lung tumor volume (B); mice body weight (C)

A549 cells (2 × 10⁶) were injected into the nude mice by tail vein. Tumors were established for 7 days before therapy. Tumors from animals treated with Cxb-NLC aerosol (3 times a week), 10 mg/kg Doc (days 14, 18, 22), or combination were harvested after 28 days. Lung weights and tumor volumes were determined for measurement of therapeutic activity of the treatments. One-way ANOVA followed by post Tukey test was used for statistical analysis. P < 0.05 (*, significantly different from untreated controls; **, significantly different from single treatments). Data presented are means ± SD (n =8).

Fig. 3. Western blotting of tumor tissue lysates to determine (A) expression of Bax, Bcl2, caspase-3, caspase-9, VEGF, Survivin and Vimentin proteins in tumor lysates by western blotting and (B) quantitation of apoptotic protein expression

Tumor tissue lysates harvested tumor tissues from control-untreated and treated groups were analyzed by western blotting for protein expressions. Lane 1 = control; Lane 2 = Cxb-NLC; Lane 3 = Doc i.v.; Lane 4 = Cxb-NLC + Doc i.v. Protein expression levels (relative to β-actin) were determined. Mean ± SE for three replicate determinations. One-way ANOVA followed by post Tukey test was used for statistical analysis. P<0.01 (*, significantly different from untreated controls; ^**, significantly different from single treatments).

Fig. 4. Immunohistochemical staining of lung tumor tissues for induction of apoptosis using TUNEL assay (A); for expression of cleaved caspase-3 (B); quantitation of apoptotic cells from TUNEL staining (C); and quantitation of caspase-3 positive cells apoptotic cells (D)

Percentages of TUNEL-positive and cleaved caspase 3-positive cells were quantitated by counting 100 cells from 6 random microscopic fields. Data are expressed as mean+SD (N = 6). One-way ANOVA followed by post Tukey test was used for statistical analysis to compare control and treated groups. P<0.01 (*, significantly different from untreated controls; ^**, significantly different from single treatments). Original magnification ×40 (Micron bar = 100 μm).