Cationic lipid guided short-hairpin RNA interference of annexin A2 attenuates tumor growth and metastasis in a mouse lung cancer stem cell model.

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 (199nm) with a slight positive charge (21.82mV); CLG-shAnx2 was of similar size (217nm) with decreased charge (12.11mV), and entrapment and loading efficiencies of 97% and 6.13% respectively. Fluorescence microscopy showed high uptake of CLG-shAnxA2 in H1650 SP cells after 2h 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 4h 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 ß-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 ß-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.

Epithelial transport of noscapine across cell monolayer and influence of absorption enhancers on in vitro permeation and bioavailability: implications for intestinal absorption.

The purpose of this study was to investigate the permeation of Noscapine (Nos) across the Caco-2 and Madin-Darby canine kidney (MDCK) cell monolayers and to evaluate the influence of absorption enhancers on in vitro and in vivo absorption of Nos. The bidirectional transport of Nos was studied in Caco-2 and MDCK cell monolayers at pH 5.0-7.8. The effect of 0.5% w/v chitosan (CH) or Captisol (CP) on Nos permeability was investigated at pH 5.0 and 5.8. The effect of 1-5% w/v of CP on oral bioavailability of Nos (150 mg/kg) was evaluated in Sprague-Dawley rats. The effective permeability coefficients (Peff) of Nos across Caco-2 and MDCK cell monolayers was found to be in the order of pH 5.0 > 5.8 > 6.8 > 7.8. The efflux ratios of Peff < 2 demonstrated that active efflux does not limit the absorption of Nos. The use of CH or CP have shown significant (***, p < 0.001) enhancement in Peff of Nos across cell monolayer compared with the control group. The CP (1-5% w/v) based Nos formulations resulted in significant (***, p < 0.001) increase in the bioavailability of Nos compared with Nos solution. The use of CP represents viable approach for enhancing the oral bioavailability of Nos and reducing the required dose.

1,1-Bis (3'-indolyl)-1-(p-substitutedphenyl)methane compounds inhibit lung cancer cell and tumor growth in a metastasis model.

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 invivo. Respirable aqueous formulations of C-DIM-5 (mass median aerodynamic diameter of 1.92±0.22µm and geometric standard deviation of 2.31±0.12) and C-DIM-8 (mass median aerodynamic diameter of 1.84±0.31µm 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, ß-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.

Inhalation delivery of Telmisartan enhances intratumoral distribution of nanoparticles in lung cancer models.

The purpose of the present study was to evaluate the effect of Telmisartan (Tel) and Losartan (Los) on nanoparticle intratumoral distribution and anticancer effects in lung cancer. A549 lung tumor cells were orthotopically and metastatically administered to Nu/nu mice. Fluorescent polystyrene nanoparticles (FPNPs, size ~200 nm) beads were used to study their intratumoral distribution after Tel and Los treatments. Animals were administered with FPNPs and after 2h, FPNPs intratumoral distribution was studied by fluorescent microscopy. Tel (~1.12 mg/kg) and Los (~4.5mg/kg) were administered by inhalation delivery at alternative days for 4 weeks to tumor bearing animals. Collagen-1, transforming growth factor beta 1 (TGF-ß1), cleaved caspase-3, Vimentin and E-Cadherin expressions were studied by western blotting. To correlate the AT1 receptor blockage to anticancer effects, VEGF levels and microvessel densities (MVD) were quantified. Los and Tel treated group resulted in the 5.33 and 14.33 fold increase respectively in the FPNPs intratumoral distribution as compared to the controls. Tel treatment attenuated 2.23 and 1.70 fold Collagen 1 expression compared to untreated control and Los groups, respectively. Further, in Tel and Los treated groups, the TGF-ß1 active levels were significantly (p<0.05) decreased. Tel (at four times less dose) was 1.89 and 1.92 fold superior in anticancer activity to Los respectively in A549 orthotopic and metastatic tumor models (p<0.05) when given by inhalation route. Tel, by virtue of its dual pharmacophoric nature could be an ideal candidate for combination therapy to improve the nanoparticle intratumoral distribution and anticancer effects.

Evaluation of the behavioral and pharmacokinetic profile of SYA013, a homopiperazine analog of haloperidol in rats.

SYA013, a homopiperazine analog of haloperidol, was further evaluated for antipsychotic potential using additional animal models. Previously, SYA013 was tested in mice with an antipsychotic screening model in which it inhibited apomorphine induced climbing behavior, indicating antagonism of the dopaminergic system and the potential for use in the treatment of schizophrenia. In this study, SYA013 was shown to inhibit both d-amphetamine-induced locomotor activity in rats and conditioned avoidance response (CAR) in rats in a dose dependent manner and in the case of CAR, without producing any escape failure responses (EFRs), two tests predictive of antipsychotic action. The selective 5HT(1A) antagonist WAY100,635 was used to determine if binding of SYA013 to the 5HT(1A) receptor contributed to suppression of CAR. The results indicated that 0.63mg/kg WAY100,635 did not have a significant effect on the inhibition of CAR by SYA013. Pharmacokinetic parameters in brain and plasma were determined for SYA013. A log brain/plasma concentration ratio at a t(max) of 1.48 suggests that SYA013 readily crosses the blood brain barrier (BBB). The hypothesis that binding of SYA013 to the 5HT(1A) receptor contributed to the lack of significant catalepsy was investigated using the 5HT(1A) antagonist WAY100,635. The results of acute and semi-chronic tests suggest that binding to the 5HT(1A) receptor alone did not significantly account for the lack of catalepsy. Lack of catalepsy was preserved after the semi-chronic challenge with SYA013. These tests further indicate that SYA013 has a pharmacological profile with the potential for use in the treatment of neuropsychiatric diseases. In addition, the 5HT(1A) receptor does not appear to play a significant role in the pharmacological profile of SYA013.

Enhanced anticancer activity of gemcitabine in combination with noscapine via antiangiogenic and apoptotic pathway against non-small cell lung cancer.

BACKGROUND: The aim of this investigation was to evaluate the anticancer activity of Noscapine (Nos) and Gemcitabine (Gem) combination (NGC) against non-small cell lung cancer (NSCLC) and to elucidate the underlying mechanism of action. METHODS: Isobolographic method was used to calculate combination index values from cytotoxicity data. In vitro antiangiogenic and apoptotic activity of Nos, Gem and NGC was evaluated. For in vivo studies, female athymic Nu/nu mice were xenografted with H460 tumors and the efficacy of Nos, Gem, or NGC was determined. Protein expressions by immunohistochemical staining were evaluated in harvested tumor tissues. RESULTS: The CI values (<0.59) were suggestive of synergistic behavior between Nos and Gem. NGC treatment showed significantly inhibited tube formation and increased percentage of apoptotic cells. NGC, Gem and Nos treatment reduced tumor volume by 82.9±4.5 percent, 39.4±5.8 percent and 34.2±5.7 percent respectively. Specifically, NGC treatment decreased expression cell survival proteins; VEGF, CD31 staining and microvessel density and enhanced DNA fragmentation and cleaved caspase 3 levels compared to single agent treated and control groups. CONCLUSION: Nos potentiated the anticancer activity of Gem in an additive to synergistic manner against lung cancer via antiangiogenic and apoptotic pathways. These findings suggest potential benefit for use of NGC chemotherapy for treatment of lung cancer.

Antitumor activity of Noscapine in combination with Doxorubicin in triple negative breast cancer.

BACKGROUND: The aim of this study was to investigate the anticancer activity and mechanism of action of Noscapine alone and in combination with Doxorubicin against triple negative breast cancer (TNBC). METHODS: TNBC cells were pretreated with Noscapine or Doxorubicin or combination and combination index values were calculated using isobolographic method. Apoptosis was assessed by TUNEL staining. Female athymic Nu/nu mice were xenografted with MDA-MB-231 cells and the efficacy of Noscapine, Doxorubicin and combination was determined. Protein expression, immunohistochemical staining were evaluated in harvested tumor tissues. RESULTS: Noscapine inhibited growth of MDA-MB-231 and MDA-MB-468 cells with the IC(50) values of 36.16±3.76 and 42.7±4.3 µM respectively. The CI values (<0.59) were suggestive of strong synergistic interaction between Noscapine and Doxorubicin and combination treatment showed significant increase in apoptotic cells. Noscapine showed dose dependent reduction in the tumor volumes at a dose of 150-550 mg/kg/day compared to controls. Noscapine (300 mg/kg), Doxorubicin (1.5 mg/kg) and combination treatment reduced tumor volume by 39.4±5.8, 34.2±5.7 and 82.9±4.5 percent respectively and showed decreased expression of NF-KB pathway proteins, VEGF, cell survival, and increased expression of apoptotic and growth inhibitory proteins compared to single-agent treatment and control groups. CONCLUSIONS: Noscapine potentiated the anticancer activity of Doxorubicin in a synergistic manner against TNBC tumors via inactivation of NF-KB and anti-angiogenic pathways while stimulating apoptosis. These findings suggest potential benefit for use of oral Noscapine and Doxorubicin combination therapy for treatment of more aggressive TNBC.

Anticancer activity of Noscapine, an opioid alkaloid in combination with Cisplatin in human non-small cell lung cancer.

The purpose of this study was to examine the efficacy of Noscapine (Nos) and Cisplatin (Cis) combination treatment in vitro in A549 and H460 lung cancer cells, in vivo in murine xenograft model and to investigate the underlying mechanism. The combination index values (< 0.6) suggested synergistic effects of Nos+Cis and resulted in the highest increase in percentage of apoptotic NSCLC cells and increased expression of p53, p21, caspase 3, cleaved caspase 3, cleaved PARP, Bax, and decreased expression of Bcl2 and surviving proteins compared with treatment with either agent. Nos+Cis treatment reduced tumor volume by 78.1 ± 7.5% compared with 38.2 ± 6.8% by Cis or 35.4 ± 6.9% by Nos alone in murine xenograft lung cancer model. Nos+Cis treatment decreased expression of pAkt, Akt, cyclin D1, survivin, PARP, Bcl2, and increased expression of p53, p21, Bax, cleaved PARP, caspase 3, cleaved caspase 3, cleaved caspase 8, caspase 8, cleaved caspase 9 and caspase 9 compared to single-agent treated and control groups. Our results suggest that Nos enhanced the anticancer activity of Cis in an additive to synergistic manner by activating multiple signaling pathways including apoptosis. These findings suggest potential benefit for use of Nos and Cis combination in treatment of lung cancer.

Inhalation delivery of a novel diindolylmethane derivative for the treatment of lung cancer.

The purpose of this study was to determine the anticancer efficacy of 1,1-bis (3'-indolyl)-1-(p-biphenyl) methane (DIM-C-pPhC6H5) by inhalation delivery alone and in combination with i.v. docetaxel in a murine model for lung cancer. An aqueous DIM-C-pPhC6H5 formulation was characterized for its aerodynamic properties. Tumor-bearing athymic nude mice were exposed to nebulized DIM-C-pPhC6H5, docetaxel, or combination (DIM-C-pPhC6H5 plus docetaxel) using a nose-only exposure technique. The aerodynamic properties included mass median aerodynamic diameter of 1.8 ± 0.3 µm and geometric SD of 2.31 ± 0.02. Lung weight reduction in mice treated with the drug combination was 64% compared with 40% and 47% in mice treated with DIM-C-pPhC6H5 aerosol and docetaxel alone, respectively. Combination treatment decreased expression of Akt, cyclin D1, survivin, Mcl-1, NF-κB, IκBα, phospho-IκBα, and vascular endothelial growth factor (VEGF) and increased expression of c-Jun NH2-terminal kinase 2 and Bad compared with tumors collected from single-agent treatment and control groups. DNA fragmentation was also enhanced in mice treated with the drug combination compared with docetaxel or DIM-C-pPhC6H5 alone. Combination treatment decreased expressions of VEGF and CD31 compared with single-agent treated and control groups. These results suggest that DIM-C-pPhC6H5 aerosol enhanced the anticancer activity of docetaxel in a lung cancer model by activating multiple signaling pathways. The study provides evidence that DIM-C-pPhC6H5 can be used alone or in combination with other drugs for the treatment of lung cancer using the inhalation delivery approach.

Formulation, characterization and pulmonary deposition of nebulized celecoxib encapsulated nanostructured lipid carriers.

The aim of the current study was to encapsulate celecoxib (Cxb) in the nanostructured lipid carrier (Cxb-NLC) nanoparticles and evaluate the lung disposition of nanoparticles following nebulization in Balb/c mice. Cxb-NLC nanoparticles were prepared with Cxb, Compritol, Miglyol and sodium taurocholate using high-pressure homogenization. Cxb-NLC nanoparticles were characterized for physical and aerosol properties. In-vitro cytotoxicity studies were performed with A549 cells. The lung deposition and pharmacokinetic parameters of Cxb-NLC and Cxb solution (Cxb-Soln) formulations were determined using the Inexpose system and Pari LC star jet nebulizer. The particle size and entrapment efficiency of the Cxb-NLC formulation were 217+/-20nm and >90%, respectively. The Cxb-NLC released the drug in controlled fashion, and in-vitro aerosolization of Cxb-NLC formulation showed an FPF of 75.6+/-4.6%, MMAD of 1.6+/-0.13microm and a GSD of 1.2+/-0.21. Cxb-NLC showed dose and time dependent cytotoxicity against A549 cells. Nebulization of Cxb-NLC demonstrated 4 fold higher AUC(t)/D in lung tissues compared to the Cxb-Soln. The systemic clearance of Cxb-NLC was slower (0.93l/h) compared to the Cxb-Soln (20.03l/h). Cxb encapsulated NLC were found to be stable and aerodynamic properties were within the respirable limits. Aerosolization of Cxb-NLC improved the Cxb pulmonary bioavailability compared to solution formulation which will potentially lead to better patient compliance with minimal dosing intervals.

Simultaneous RP-HPLC-DAD quantification of bromocriptine, haloperidol and its diazepane structural analog in rat plasma with droperidol as internal standard for application to drug-interaction pharmacokinetics.

A simple and rapid RP-HPLC-DAD method was developed and validated for simultaneous determination of the dopamine antagonists haloperidol, its diazepane analog, and the dopamine agonist bromocriptine in rat plasma, to perform pharmacokinetic drug-interaction studies. Samples were prepared for analysis by acetonitrile (22.0 microg/mL) plasma protein precipitation with droperidol as an internal standard, followed by a double-step liquid-liquid extraction with hexane : chloroform (70:30) prior to C-18 separation. Isocratic elution was achieved using a 0.1% (v/v) trifluoroacetic acid in deionized water, methanol and acetonitrile (45/27.5/27.5, v/v/v). Triple-wavelength diode-array detection at the lambda(max) of 245 nm for haloperidol, 254 nm for the diazepane analog and droperidol, and 240 nm for bromocriptine was carried out. The LLOQ of DAL, HAL, and BCT were 45.0, 56.1, and 150 ng/mL, respectively. In rats, the estimated pharmacokinetic parameters (i.e., t(1/2), CL, and V(ss)) of HAL when administered with DAL and BCT were t(1/2) = 16.4 min, V(ss) = 0.541 L/kg for HAL, t(1/2) = 28.0 min, V(ss) = 2.00 L/kg for DAL, and t(1/2) = 24.0 min, V(ss) = 0.106 L/kg for BCT. The PK parameters for HAL differed significantly from those previously reported, which may be an indication of a drug-drug interaction.

Enhancement of docetaxel anticancer activity by a novel diindolylmethane compound in human non-small cell lung cancer.

PURPOSE: This study was conducted to examine the cytotoxic effects of a peroxisome proliferator-activated receptor gamma (PPARgamma) agonist, 1,1-bis (3'-indolyl)-1-(p-biphenyl) methane (DIM-C-pPhC(6)H(5)), alone and in combination with docetaxel in vitro in A549 lung cancer cells and in vivo in nude mice bearing A549 orthotopic lung tumors. EXPERIMENTAL DESIGN: Isobolographic method was used to calculate combination index values from cell viability data. Apoptosis was evaluated in A549 cells by terminal deoxynucleotidyl transferase-mediated nick end labeling assay and measurement of cleaved poly(ADP-ribose) polymerase level. Expression of proteins was studied by Western blotting. A549 cells were implanted to induce orthotopic lung tumors in nude mice and the efficacy of docetaxel, DIM-C-pPhC(6)H(5), or combination was determined. Apoptosis and cleaved caspase-3 expression in the harvested tissues were studied by terminal deoxynucleotidyl transferase-mediated nick end labeling and immunohistochemistry, respectively. RESULTS: The combination index values (0.36-0.9) suggested synergistic to additive effects of docetaxel + DIM-C-pPhC(6)H(5) and resulted in the highest increase in percentage of apoptotic cells and expression of cleaved poly(ADP-ribose) polymerase, Bax, and N-cadherin compared with treatment with either agent. The combination also enhanced procaspase-3 and -9 cleavage. In vivo, docetaxel + DIM-C-pPhC(6)H(5) reduced lung weights by 57% compared with 39% by docetaxel or 22% by DIM-C-pPhC(6)H(5) alone, induced apoptosis in 43% of the tumor cells compared with 29% and 22% in tumors treated with docetaxel and DIM-C-pPhC(6)H(5), respectively, and increased procaspase-3 cleavage compared with either agent alone. CONCLUSIONS: These findings suggest potential benefit for use of docetaxel and DIM-C-pPhC(6)H(5) combination in lung cancer treatment.

Identification of a butyrophenone analog as a potential atypical antipsychotic agent: 4-[4-(4-chlorophenyl)-1,4-diazepan-1-yl]-1-(4-fluorophenyl)butan-1-one.

The synthesis and exploration of novel butyrophenones have led to the identification of a diazepane analogue of haloperidol, 4-[4-(4-chlorophenyl)-1,4-diazepan-1-yl]-1-(4-fluorophenyl)butan-1-one (compound 13) with an interesting multireceptor binding profile. Compound 13 was evaluated for its binding affinities at DA subtype receptors, 5HT subtype receptors, H-1, M-1 receptors and at NET, DAT, and SERT transporters. At each of these receptors, compound 13 was equipotent or better than several of the standards currently in use. In in vivo mouse and rat models to evaluate its efficacy and propensity to elicit catalepsy and hence EPS in humans, compound 13 showed similar efficacy as clozapine and did not produce catalepsy at five times its ED(50) value.

Antitumor activity of noscapine in human non-small cell lung cancer xenograft model.

PURPOSE: An antitussive plant alkaloid, Noscapine HCl (Nos) displays anticancer activity and has a safe pharmacological profile in humans. The current study was aimed to investigate the in vitro and in vivo anti tumor activity of Nos to determine possible mechanisms of anti tumor activity for treatment of non-small cell lung cancer (NSCLC). METHODS: In vitro cytotoxicity of Nos was studied in H460 cells treated with different doses of Nos (10-160 microM) for 72 h and cell viability was determined using crystal violet assay. Apoptosis in H460 cells was evaluated by TUNEL assay after treatment of cells for 72 h with 30 and 40 microM doses of Nos. For in vivo studies, female athymic Nu/nu mice were xenografted with H460 tumors and on day 4 onwards Nos was administered orally at dose of 300, 450 and 550 mg/kg/day for 24 days. As a control, xenografted tumors were separately treated with Docetaxel (10 mg/kg i.v. bolus on day 5, 11, 17, 23). The tumor volumes were measured every five days. Expression of PARP, Bcl(2, )Bax, and caspase-3 families of proteins was measured by Western Blotting (WB), while TUNEL and Immunohistochemical methods were utilized to determine DNA fragmentation and cleaved caspase-3 levels respectively. RESULTS: Nos inhibited growth of H460 cells with the IC50 values of 34.7 +/- 2.5 microM. Nos at 30 and 40 microM doses caused apoptosis as evidenced by nuclear condensation in treated H460 cells. Nos caused 49, 65 and 86% reduction in the xenografted tumor volumes at a dose of 300 (P < 0.05), 450 (P < 0.01), 550 mg/kg/day (P < 0.01), respectively, when compared to controls. Nos-dependent suppression of xenografted tumor growth involved up regulation of PARP, Bax, caspase-3 and repression of Bcl(2) expression. An increase in Bax/Bcl(2) ratio suggests involvement of a mitochondrial mediated apoptotic processes. Our studies revealed a non significant (P > 0.05) increase in Bax/Bcl(2) ratio with Nos at a dose of 300 mg/kg/day, while a significant (P < 0.001) increase in Bax/Bcl(2) ratio was observed with Nos doses of 450 and 550 mg/kg/day. Further, Nos caused elevated apoptosis in tumor xenografts as evidenced by enhanced expression of caspase-3 and positive TUNEL staining in regressed tumor tissues, thus suggesting induction of apoptosis by mitochondrial pathway. CONCLUSION: Our studies suggest that potent antitumor activity of Nos against NSCLC cells. Oral administration of Nos showed significant reduction in tumor volume in human non-small cell lung tumor xenograft in nude mice in a dose dependant manner. Thus, Nos is a promising novel chemotherapeutic agent for the treatment of human lung cancer.

15-Deoxy-Delta12,14-prostaglandin J2 enhances docetaxel anti-tumor activity against A549 and H460 non-small-cell lung cancer cell lines and xenograft tumors.

15-Deoxy-Delta-prostaglandin J2 is a naturally occurring endogenous ligand for peroxisome proliferator-activated receptor-gamma. The current study was aimed to determine the mechanism of anti-proliferative effect of 15-deoxy-Delta-prostaglandin J2+docetaxel against A549 and H460 non-small-cell lung cancer cell lines and xenograft tumors. In-vitro cytotoxicity of 15-deoxy-Delta-prostaglandin J2 alone and in combination with docetaxel was studied against A549 and H460 cell lines. For in-vivo studies, female athymic nu/nu mice were xenografted with A549 and H460 tumors and treated with 15-deoxy-Delta-prostaglandin J2 (1 mg/kg/day; intraperitoneal), docetaxel (10 mg/kg; intravenous on days 14, 18 and 22) and 15-deoxy-Delta-prostaglandin J2+docetaxel. Apoptosis was measured in A549 cells and tumor tissues, following various treatments. Peroxisome proliferator-activated receptor-gamma, caspases, Bcl2 and p53 family proteins or their mRNA expressions were measured by Western blotting, reverse transcription-polymerase chain reaction and real-time polymerase chain reaction in A549 tumors. A possible role of a peroxisome proliferator-activated receptor-gamma-independent mechanism was studied in A549 cells treated with peroxisome proliferator-activated receptor-gamma antagonist, GW9662. Isobolographic analysis demonstrated synergistic interaction (combination index <1.0) between 15-deoxy-Delta-prostaglandin J2 and docetaxel against A549 and H460 cells in vitro. 15-Deoxy-Delta-prostaglandin J2+docetaxel significantly reduced the tumor volume compared with control (P<0.05), 15-deoxy-Delta-prostaglandin J2 (P<0.05) and docetaxel (P<0.05, P<0.01) in both A549 and H460 tumors. 15-Deoxy-Delta-prostaglandin J2+docetaxel showed a significant increase in apoptosis associated with inhibition of the Bcl2 and cyclin D1 expression and overexpression of caspase and p53 pathway genes. Further, enhanced expression of caspase 3 and inhibition of cyclin D1 by 15-deoxy-Delta-prostaglandin J2+docetaxel was not reversed by GW9662, thus suggesting a possible peroxisome proliferator-activated receptor-gamma-independent mechanism. In conclusion, 15-deoxy-Delta-prostaglandin J2 enhanced the anti-tumor action of docetaxel by peroxisome proliferator-activated receptor-gamma-dependent and -independent mechanisms mediated by induction of apoptosis.

Inhalation delivery and anti-tumor activity of celecoxib in human orthotopic non-small cell lung cancer xenograft model.

PURPOSE: To determine the in vivo anti-tumor effect of aerosolized Celecoxib (Cxb) in combination with i.v Docetaxel (Doc) and compare the anti-tumor effect with oral Cxb combined with i.v Doc in human orthotopic non-small cell lung cancer (NSCLC) xenograft model. MATERIALS AND METHODS: Female Nu/Nu mice were implanted with orthotopic tumors by injecting A549 cells into the lung parenchyma. Seven day after tumor implantation the mice were treated with aerosolized Cxb (30 min exposure/day, 5 mg/ml solution) + i.v Doc (10 mg/kg) and the effect was compared with oral Cxb (150 mg/kg/day) + i.v Doc (10 mg/kg), for 28 days. Small-animal nose only inhalation chamber (CH Technologies, Westwood, NJ) was utilized for aerosol exposure. Therapeutic activity of Cxb (aerosol/oral) + Doc was estimated by differences in lung weight, tumor area and animal body weight. Lung tumor samples isolated from mice were analyzed for (a) PGE2 levels by enzyme immunoassay (EIA) (b) expression of Fas and Factor VIII by immunohistochemistry (c) IL-8 expression using EIA kits and (d) mRNA expression for caspase-3 by Real-Time PCR. RESULTS: Mice treated with Cxb (aerosol/oral) + Doc showed significant reduction (P < 0.001) in lung weight and tumor area as compared to Cxb or Doc treatments. Cxb (aerosol/oral) + Doc showed increased apoptosis mediated via increased Fas and caspase-3 (P < 0.001) expression as compared to untreated control. Further, the combination treatment showed antiangiogenic effect as demonstrated by reduced expression of Factor VIII, IL-8 (P < 0.001) and PGE2 (P < 0.001) in lung tumors as compared to untreated control. Aerosolized Cxb at a significantly lower therapeutic dose (4.56 mg/kg/day) demonstrated comparable anti-tumor efficacy to orally administered Cxb (150 mg/kg/day). CONCLUSION: Cxb was formulated and effectively delivered via aerosolization to treat orthotopic lung tumors in combination with i.v Doc. Cxb when administered by aerosol produced same therapeutic effect as oral Cxb, but at lower therapeutic dose and thus shows promise for the treatment of lung cancer.

Enhancement of antitumor activity of docetaxel by celecoxib in lung tumors.

Our study investigates the effect of a highly selective cyclooxygenase-2 (COX-2) inhibitor, celecoxib, on the cytotoxicity of docetaxel in nude mice bearing A549 tumor xenografts and elucidates the molecular mechanisms of the antitumor effect of this combination. Female nu/nu mice, xenografted with s.c. A549 tumors were treated with either celecoxib (150 mg/kg/day), docetaxel (10 mg/kg) or a combination of both. The tumor tissues were quantified for the induction of apoptosis, intratumor levels/expressions of prostaglandin E2 (PGE2), 15 deoxy prostaglandin J2 (15-d PGJ2), microsomal prostaglandin E synthase (mPGES) and cytoplasmic phospholipase A2 (cPLA2). The combination of celecoxib with docetaxel significantly inhibited the tumor growth (p < 0.03) as compared to celecoxib or docetaxel alone, decreased the levels of PGE2 by 10-fold and increased the 15-d PGJ2 levels by 4-fold as compared to control. The combination also enhanced the peroxisome proliferator-activated receptor (PPAR)-gamma expression, decreased the expression of cPLA2, mPGES and vascular endothelial growth factor (VEGF), but had no effect on the expression of COX-1 or COX-2 in tumor tissues. TUNEL staining of the tumor tissues showed a marked increase in the apoptosis in the combination group as compared to the celecoxib- or docetaxel-treated groups and this was associated with an increase in the intratumor p53 expression. In conclusion, the combination of celecoxib with docetaxel produces a greater antitumor effect in s.c. A549 tumors as compared to celecoxib or docetaxel alone and this effect is associated with concomitant alterations in the intratumor levels of PGE2 and 15-d PGJ2.

Effect of monensin liposomes on the cytotoxicity of anti-My9-bR immunotoxin.

The purpose of the study was to evaluate the utility of monensin liposomes in the enhancement of in-vitro cytotoxicity, apoptosis and in-vivo antitumour activity of anti-My9-bR immunotoxin. Monensin liposomes were prepared and studied for the enhancement of in-vitro cytotoxicity and apoptotic response of anti-My9-bR immunotoxin against both sensitive and resistant human promyelocytic leukemia HL-60 cells by MTS/PES method and acridine orange staining, respectively. Further, the in-vivo cytotoxicity enhancement of anti-My9-bR immunotoxin by monensin liposomes was studied in a survival model of severe combined immunodeficient (SCID) mice bearing intraperitoneal HL-60 tumours. The in-vitro cytotoxicity of anti-My9-bR immunotoxin was enhanced 580 fold and 4.7 fold against sensitive and resistant HL-60 cells, respectively, by monensin liposomes (5 x 10(-8) M). The combination of anti-My9-bR immunotoxin (50ng mL(-1)) with monensin liposomes (5 x 10(-8) M) produced apoptosis in 40% of cells, whereas the apoptotic response was minimal (< 10%) in anti-My9-bR immunotoxin- or monensin liposome (alone)-treated HL-60 (resistant) cells. In SCID mice bearing HL-60 tumours, anti-My9-bR immunotoxin (75 microg kg(-1) administered intravenously every other day for a total of five courses) showed a median survival time of 20 days, which was no different than that of vehicle control- or monensin liposome-treated mice. However, anti-My9-bR immunotoxin (75 microg kg(-1)) in combination with monensin liposomes (4 microg kg(-1) monensin), administered every other day for a total of five courses, was found to prolong the survival of 20% of mice for more than 46 days. Our results indicate that, despite anti-My9-bR immunotoxin being ineffective in the HL-60 tumour model, its combination with monensin liposomes could improve the antitumour response.