The orphan nuclear receptor TR3 (NR41A, Nur77) is overexpressed in most lung cancer patients and is a negative prognostic factor for patient survival. The function of TR3 was investigated in non-small cell lung cancer A549 and H460 cells, and knockdown of TR3 by RNA interference (siTR3) inhibited cancer cell growth and induced apoptosis. The prosurvival activity of TR3 was due, in part, to formation of a p300/TR3/Sp1 complex bound to GC-rich promoter regions of survivin and other Sp-regulated genes (mechanism 1). However, in p53 wild-type A549 and H460 cells, siTR3 inhibited the mTORC1 pathway and this was due to activation of p53 and induction of the p53-responsive gene sestrin 2 which subsequently activated the mTORC1 inhibitor AMPKα (mechanism 2). This demonstrates that the pro-oncogenic activity of TR3 in lung cancer cells was due to inhibition of p53 and activation of mTORC1. 1,1-Bis(3′-indolyl)-1-(p-hydroxyphenyl)methane (DIM-C-pPhOH) is a recently discovered inhibitor of TR3 which mimics the effects of siTR3. DIM-C-pPhOH inhibited growth and induced apoptosis in lung cancer cells and lung tumors in murine orthotopic and metastatic models, and this was accompanied by decreased expression of survivin and inhibition of mTORC1 signaling, demonstrating that inactivators of TR3 represent a novel class of mTORC1 inhibitors.

BackgroundThree-dimensional (3D) in-vitro cultures are recognized for recapitulating the physiological microenvironment and exhibiting high concordance with in-vivo conditions. Taking the advantages of 3D culture, we have developed the in-vitro tumor model for anticancer drug screening.MethodsCancer cells grown in 6 and 96 well AlgiMatrix™ scaffolds resulted in the formation of multicellular spheroids in the size range of 100–300 µm. Spheroids were grown in two weeks in cultures without compromising the growth characteristics. Different marketed anticancer drugs were screened by incubating them for 24 h at 7, 9 and 11 days in 3D cultures and cytotoxicity was measured by AlamarBlue® assay. Effectiveness of anticancer drug treatments were measured based on spheroid number and size distribution. Evaluation of apoptotic and anti-apoptotic markers was done by immunohistochemistry and RT-PCR. The 3D results were compared with the conventional 2D monolayer cultures. Cellular uptake studies for drug (Doxorubicin) and nanoparticle (NLC) were done using spheroids.ResultsIC50 values for anticancer drugs were significantly higher in AlgiMatrix™ systems compared to 2D culture models. The cleaved caspase-3 expression was significantly decreased (2.09 and 2.47 folds respectively for 5-Fluorouracil and Camptothecin) in H460 spheroid cultures compared to 2D culture system. The cytotoxicity, spheroid size distribution, immunohistochemistry, RT-PCR and nanoparticle penetration data suggested that in vitro tumor models show higher resistance to anticancer drugs and supporting the fact that 3D culture is a better model for the cytotoxic evaluation of anticancer drugs in vitro.ConclusionThe results from our studies are useful to develop a high throughput in vitro tumor model to study the effect of various anticancer agents and various molecular pathways affected by the anticancer drugs and formulations.

Breast cancer is the leading cause of malignancies among women globally. The triple negative breast cancer (TNBC) subtype is the most difficult to treat and accounts for 15% of all cases. Targeted therapies have been developed for TNBC but come short of clinical translation due to acquired tumor resistance. An effective therapy against TNBC must combine properties of target specificity, efficient tumor killing, and translational relevance. The objective of this study was to formulate a nontoxic, cationic, lipid-conjugated estrogenic derivative (ESC8), with demonstrated anticancer activity, for oral delivery in mice bearing triple negative breast cancer (TNBC) as xenograft tumors. The in vitro cell viability, Caco-2 permeability, and cell cycle dynamics of ESC8-treated TNBC cells were investigated. ESC8 was formulated as liposomes, solid lipid nanoparticles (SLNs), and nanostructured lipid carriers (NLCs) and characterized for size, zeta potential, entrapment efficiency, size stability, and tumor biodistribution. Pharmacokinetic modeling of plasma concentration-time course data was carried out following intravenous and oral administration in Sprague-Dawley rats. In vivo efficacy investigation of ESC8-SLNC was carried out in Nu/Nu mice bearing MDA-MB-231 TNBC as xenograft tumors, and the molecular dynamics modulating tumor growth inhibition was analyzed by Western blot. In vitro ESC8 inhibited TNBC and non-TNBC cell viability with IC50 ranging from 1.81 to 3.33 μM. ESC8 was superior to tamoxifen and Cisplatin in inhibiting MDA-MB-231 cell viability; and at 2.0 μM ESC8 enhanced Cisplatin cytotoxicity 16-fold. Intravenous ESC8 (2.0 mg/kg) was eliminated at a rate of 0.048 ± 0.01 h(-1) with a half-life of 14.63 ± 2.95 h in rats. ESC8 was orally bioavailable (47.03%) as solid lipid nanoparticles (ESC8-SLN). ESC8-SLN (10 mg/kg/day, ×14 days, p.o.) inhibited breast tumor growth by 74% (P < 0.0001 vs control) in mice bearing MDA-MB-231 cells as xenografts; and when given in combination with Cisplatin (2.0 mg/kg/biweekly, ×2 weeks, IV), tumor growth was inhibited by 87% (P = 0.0002, vs ESC8-SLN; 10 mg/kg/day, ×14 days, p.o). ESC8-SLN tumor growth inhibition was associated with increased expression of p21 and Caspase-9; as well as by inhibition of EGFR, Slug, p-Akt1, Vimentin, NFkβ, and IKKγ. These results show the promise of ESC8 as an oral adjuvant or neoadjuvant against triple negative breast cancer.

The role of side populations (SP) or cancer stem-like cells (CSC) in promoting the resistance phenotype presents a viable anticancer target. Human-derived H1650 SP cells over-express annexin A2 (AnxA2) and SOX2, and are resistant to conventional cytotoxic chemotherapeutics. AnxA2 and SOX2 bind to proto-oncogenes, c-Myc and c-Src, and AnxA2 forms a functional heterotetramer with S100A10 to promote tumor motility. However, the combined role of AnxA2, S100A10 and SOX2 in promoting the resistant phenotype of SP cells has not been investigated. In the current studies, we examined for the first time a possible role of AnxA2 in regulating SA100A10 and SOX2 in promoting a resistant phenotype of lung tumors derived from H1650 SP cells. The resistance of H1650 SP cells to chemotherapy compared to H1650 MP cells was investigated by cell viability studies. A short hairpin RNA targeting AnxA2 (shAnxA2) was formulated in a liposomal (cationic ligand-guided, CLG) carrier and characterized for size, charge and entrapment and loading efficiencies; CLG carrier uptake by H1650 SP cells was demonstrated by fluorescence microscopy, and knockdown of AnxA2 confirmed by qRT-PCR and Western blot. Targeting of xenograft and orthotopic lung tumors was demonstrated with fluorescent (DiR) CLG carriers in mice. The therapeutic efficacy of CLG-AnxA2, compared to that of placebo, was investigated after 2 weeks of treatment in terms of tumor weights and tumor burden in vivo. Compared to mixed population cells, H1650 SP cells showed exponential resistance to docetaxel (15-fold), cisplatin (13-fold), 5-fluorouracil (31-fold), camptothecin (7-fold), and gemcitabine (16-fold). CLG carriers were nanoparticulate (199 nm) with a slight positive charge (21.82 mV); CLG-shAnx2 was of similar size (217 nm) with decreased charge (12.11 mV), and entrapment and loading efficiencies of 97% and 6.13% respectively. Fluorescence microscopy showed high uptake of CLG-shAnxA2 in H1650 SP cells after 2 h resulting in a 6-fold reduction in AnxA2 mRNA expression and 92% decreased protein expression. Fluorescence imaging confirmed targeting of tumors and lungs by DiR-CLG carriers with sustained localization up to 4 h in mice. CLG-shAnxA2 treatment of mice significantly reduced the weights of lung tumors derived from H1650 SP cells and tumor burden was reduced to only 19% of controls. The loss in tumor weights in response to CLG-shAnxA2 was associated with a significant loss in the relative levels of AnxA2, SOX2, total β-catenin and S100A10, both at the RNA and protein levels. These results suggest the intriguing possibility that AnxA2 may directly or indirectly regulate relative levels of β-catenin, S100A10 and SOX2, and that the combination of these factors may contribute to the resistant phenotype of H1650 SP cells. Thus down-regulating AnxA2 using RNAi methods may provide a useful method for targeting cancer stem cells and help advance therapeutic efficacy against lung cancers.

1,1-Bis(3-indolyl)-1-(p-substitutedphenyl)methane (C-DIM) compounds exhibit remarkable antitumor activity with low toxicity in various cancer cells including lung tumors. Two C-DIM analogs, DIM-C-pPhOCH3 (C-DIM-5) and DIM-C-pPhOH (C-DIM-8) while acting differentially on the orphan nuclear receptor, TR3/Nur77 inhibited cell cycle progression from G0/G1 to S-phase and induced apoptosis in A549 cells. Combinations of docetaxel (doc) with C-DIM-5 or C-DIM-8 showed synergistic anticancer activity in vitro and these results were consistent with their enhanced antitumor activities in vivo. Respirable aqueous formulations of C-DIM-5 (mass median aerodynamic diameter of 1.92 ± 0.22 um and geometric standard deviation of 2.31 ± 0.12) and C-DIM-8 (mass median aerodynamic diameter of 1.84 ± 0.31 um and geometric standard deviation of 2.11 ± 0.15) were successfully delivered by inhalation to athymic nude mice bearing A549 cells as metastatic tumors. This resulted in significant (p<0.05) lung tumor regression and an overall reduction in tumor burden. Analysis of lung tumors from mice treated with inhalational formulations of C-DIM-5 and C-DIM-8 showed decreased mRNA and protein expression of mediators of tumor initiation, metastasis, and angiogenesis including MMP2, MMP9, c-Myc, β-catenin, c-Met, c-Myc, and EGFR. Microvessel density assessment of lung tissue sections showed significant reduction (p<0.05) in angiogenesis and metastasis as evidenced by decreased distribution of immunohistochemical staining of VEGF, and CD31. Our studies demonstrate both C-DIM-5 and C-DIM-8 have similar anticancer profiles in treating metastatic lung cancer and possibly work as TR3 inactivators.