In vitro cytotoxicity of novel pro-apoptotic agent DM-PIT-1 in PEG-PE-based micelles alone and in combination with TRAIL

Abstract

The purpose of this study was to develope and characterize a micellar formulations of N-{[(2-hydroxy-5- nitrophenyl)amino]carbonothioyl}-3,5-dimethylbenzamide (DM-PIT-1)-a new small molecule non-lipid antagonist of phopshotidylinositol-3.4.5-triphopshate and inhibitor of the PI3-kinase pathway. Micelle-forming PEG(2000)-PE was used to solubilize DM-PIT-1. To improve the specificity of the micellar DM-PIT-1, cancer-targeting anti-nucleosomal mAb2C5 antibodies as well as Tumor necrosis factor- Related Apoptosis-Inducing Ligand (TRAIL) were attached to the surface of polymeric micelles. DM-PIT-1 was effectively incorporated (> 70%) into 14-16 nm micelles, which had a negative surface zeta potential of 4-5 mV. Micellar DM-PIT-1 demonstrated high in vitro cytotoxicity against various cancer cells. An improved potency of the dual-activity DM-PIT-1/TRAIL combination nanoparticles in inducing death of TRAIL-resistant cancer cells was shown. Efficacy of the TRAIL therapy was enhanced by combining it with the 2C5 antibody cancer-targeted micellar form of DM-PIT-1. In conclusion, DM-PIT-1 micellar preparations can be used for targeted combination therapy against TRAIL-resistant cancers.

FIG. 1

Reverse phase HPLC profile of N-{[(2-hydroxy-5-nitrophenyl)amino]carbonothioyl}-3,5-dimethylbenzamide (DM-PIT-1) in polymeric micelles made of PEG-PE. Analysis was performed on a C-18 column with photodiode-array detection. DM-PIT-1 was detected by the UV absorbance (320 nm). The retention time for DM-PIT-1 was aprox. 6.3 min.

FIG. 2

ELISA results for different micellar preparations compared to the non-modified mAb 2C5. Data represent the mean ± SD of triplicate samples.

FIG. 3

Gel-electrophoresis of TRAIL preparations. 1 – TRAIL attached to micelles via pNP-PEG-PE moities, 2 – soluble TRAIL (control). Ten microliters of TRAIL-modified PEG-PE micelles were loaded on 12% SDS-PAGE and stained using GelCode® Blue Stain Reagent. Control lane contains 50 ng of the recombinant TRAIL used for conjugation.

FIG. 4

In vitro cytotoxicity of micellar DM-PIT-1 against murine B-16 melanoma (left) and 4T1 metastatic breast cancer cells (right). Cancer cells were incubated with micellar DM-PIT-1 for 24, 48 and 72 hrs. Data represent the mean ± SD of three experiments and are expressed relative to untreated controls.

FIG. 5

Toxicity of the micellar DM-PIT-1 and its combination with TRAIL against TRAIL-resistant U87MG cells. Cells were treated with indicated amounts (in µM) of plain micelles modified with TRAIL (1); micellar DM-PIT-1 (2), or micellar DM-PIT-1 modified with TRAIL (3). The amount of lipid in all wells was kept constant at 2.5 mg/ml by adding empty micelles. Cell viability was determined after 24 hr using the MTT assay. Data represent the mean ± SD of three independent experiments and are expressed relative to the untreated control (*P<0.05, **P<0.01).

FIG. 6

(A) The cytotoxicity towards U87MG cells of mAb 2C5-modified DM-PIT-1-loaded PEG-PE micelles (1) compared to mAb 2C5-free preparation (2); (B) Same as in (A) but in the presence of the soluble TRAIL at 50 ng/ml. Cell viability was determined using the MTT assay. Viability of cells in the presence of plain micelles, immunomicelles and TRAIL was observed at comparable concentrations of drug-loaded particles. Data represent the mean ± SD of three independent experiments and are expressed relative to the untreated control (* P< 0.05, ** P< 0.01).