Inhalation Delivery of a Novel Diindolylmethane Derivative for the Treatment of Lung Cancer

Abstract

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 (Doc) 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, Doc, or combination (DIM-C-pPhC₆H₅ plus Doc) using a nose-only exposure technique. The aerodynamic properties included mass median aerodynamic diameter of 1.8 ± 0.3 μm and geometric standard deviation of 2.31 ± 0.02. Lung weight reduction in mice treated with the drug combination was 64 % compared to 40 % and 47 % in mice treated with DIM-C-pPhC₆H₅ aerosol and Doc alone respectively. Combination treatment decreased expression of akt, cyclinD1, survivin, Mcl-1, NF-kB, IKBα, P-IkBα, VEGF, and increased expression of JNK2 and Bad compared to tumors collected from single-agent treatment and control groups. DNA fragmentation was also enhanced in mice treated with the drug combination mice compared to Doc or DIM-C-pPhC₆H₅ alone. Combination treatment decreased expressions of VEGF & CD31 compared to single agent treated and control groups. These results suggest that DIM-C-pPhC₆H₅ aerosol enhanced the anticancer activity of Doc 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.

Introduction

Lung cancer is one of the leading causes of cancer deaths (9% of all cancer deaths) in United States and non-small cell lung cancer (NSCLC) accounts for 85 % of all lung cancers (1). The lung is a common site of primary malignancy and for metastasis from other primary locations such as colon, breast, prostate, and other tumors. Response and remission in non-small cell lung cancer (NSCLC) patients remain relatively low despite advances in lung cancer treatment (1). Systemic or oral drug delivery is rarely successful because only a limited amount of the chemotherapeutic drug targets lung tumor sites even when administered at a high dose. Most chemotherapeutic drugs also act on normal cells inhibiting their growth, which results in toxic adverse effects. A poor clinical outcome in lung cancer treatment has been partly attributed to the inability to achieve therapeutic concentrations of drugs at the tumor site (2). Regional drug delivery has therefore generated interest among scientists as a strategy to achieve better efficacy for treatment of lung cancer.

Localized delivery of aerosolized drugs to the lungs offers several advantages over systemic delivery including delivery of high concentration of the compounds at the target tissue, avoiding first-pass effect, improving efficacy, requiring lower drug dosing, and minimizing systemic side effects (3). Drug absorption is enhanced by the vast surface area of the lungs and relatively low proteolytic enzymatic activity in the lungs (4).

The potential of inhalation drug delivery for lung cancer treatment has been demonstrated by several researchers in preclinical and clinical studies (5-10). For example, treatment of nude mice with liposomal 9-nitrocamptothecin aerosol for 8 or 10 weeks starting from week-9 after implantation of osteosarcoma tumors resulted in a highly significant decrease in the number of animals with disease, the total number of tumor foci in the lungs, and the size of the individual tumor nodules (7). One of the initial clinical studies evaluated the inhalation delivery of 5-fluorouracil by nebulization and reported beneficial effects in NSCLC patients (11). In recent years, phase I clinical studies using aerosolized 9-nitrocamptothecin (12), doxorubicin (13), and cisplatin (14) have been conducted in patients with primary or metastasized lung cancer who failed previous conventional treatments. Overall, aerosolized chemotherapy was found to be feasible and safe with obvious lower adverse side effects relative to systemic delivery.

Peroxisome proliferator-activated receptor γ (PPARγ) has been evaluated as a therapeutic target for the treatment of various cancer types including lung cancer (15-17). The use of PPARγ agonists in combination with taxane exhibited synergistic antitumor effects in breast cancer (18) and additive antitumor effects in thyroid carcinoma (19) cancer models. DIM-C-pPhC₆H₅ (Fig 1) is a PPARγ agonist that inhibits growth of colon (20, 21), bladder (22), prostate (23), and breast (24, 25) cancer cells and tumors.

Fig. 1. Chemical structures of DIM-C-pPhC₆H₅ (1,1-Bis (3′-indolyl)-1-(p-biphenyl) methane).

These findings suggest that PPARγ agonists may be used as chemopreventive agents and/or as an adjunct in cancer chemotherapy. Docetaxel (Doc) has been approved for the treatment of NSCLC patients and exhibits its cytotoxic effects due to decreased proliferation and induction of apoptosis through stabilization of microtubules. Several researchers have studied the combination of Doc and other agents for the treatment of lung cancer (26-30) and reported enhanced anticancer effects. Thus, like C substituted Diindolylmethane (DIM), taxanes have cytotoxic properties, but act through different biological mechanisms (28, 30, 31). Our previous studies showed that DIM-C-pPhC₆H₅ in combination with Doc exhibits synergistic activity in vitro against NSCLC (32). However, the inhalation delivery of DIM-C-pPhC₆H₅ alone and in combination with standard chemotherapeutic agents for treatment of lung cancer has not been determined.

Therefore, the purpose of this study was to examine the feasibility of aerosolizing DIM-C-pPhC₆H₅ for lung cancer treatment and to evaluate the anticancer effect of DIM-C-pPhC₆H₅ aerosol alone and in combination with i.v. Doc in an orthotopic murine lung tumor model. Our hypothesis is that inhalation delivery of DIM-C-pPhC₆H₅ will provide an enhanced antitumor effect along with intravenous administration of a traditional cytotoxic agent, such as Doc for treatment of NSCLC. This combination therapy may significantly decrease the therapeutic dose required for the cytotoxic agent (Doc) and thereby minimize adverse toxic side effects. The experimental design adapted to test this hypothesis includes evaluation of the antitumor effects of DIM-C-pPhC₆H₅ aerosol when administered alone or in combination with Doc in a murine lung tumor model. Our results show that DIM-C-pPhC₆H₅ alone or in combination with the Doc was highly potent as anticancer agents and the aerosol delivery method significantly increased the effectiveness of DIM-C-pPhC₆H₅.

Materials and Methods

Materials

DIM-C-pPhC₆H₅ was synthesized as previously described (24). Docetaxel was a gift from Aventis (Collegeville, PA, USA). The human NSCLC cell lines A549 was obtained from American Type Culture Collection (Rockville, MD, USA) and all experiments with cell cultures were performed within six months. The cell lines were characterized by American Type Culture Collection using techniques such as short tandem repeat profiling, cell morphology evaluation, karyotyping, cytochrome C oxidase I assay and also evaluated for contamination. A549 cells were grown in F12K medium (Sigma, St. Louis, MO, USA) supplemented with 10% FBS. All tissue culture media contained antibiotic antimycotic solution of penicillin (5000 U/ml), streptomycin (0.1 mg/ml), and neomycin (0.2mg/ml). The cells were maintained at 37°C in the presence of 5% CO₂. All other chemicals were either reagent or tissue culture grade.

Formulation of nebulizer solution

Aqueous formulations of DIM-C-pPhC₆H₅ suitable for nebulization were prepared by partly dissolving DIM-C-pPhC₆H₅ in 0.5 ml ethanol and 500 mg of α-tocopherol polyethylene glycol succinate (TPGS) and diluted up to 10 ml with distilled water to achieve an 0.05% w/v solution. This was used for in vitro characterization and an 0.2% w/v solution was used for animal studies.

Characterization of aerodynamic properties using cascade impactor

Particle size distribution was measured using an 8-stage Anderson cascade impactor, Mark II connected to the PARI LC STAR jet nebulizer mouthpiece. The impactor plates were coated with 10% Pluronic L10 in ethanol solution to prevent particle bounce. The aqueous formulation was nebulized using PARI LC STAR jet nebulizer at dry compressed air flow rate of L/min for 5 min into the cascade impactor which was operated at a flow rate of 28.3 L/min according to USP guidance. To determine the aerodynamic properties of DIM-C-pPhC₆H₅ formulation, the inhaled aerosol on the nebulizer, throat, jet stage, plates on impactor stages 0-7, and filter was collected by washing with 5 ml of mobile phase [acetonitrile:water (90:10)]. The analysis was performed on a Waters HPLC system using a Symmetry C18 column (5 μm, 4.6 × 250mm) with a Nova-Pack C8 guard column at a wavelength of 240 nm and flow rate of 1 ml/min. The HPLC system consisted of a Waters autosampler (model 717 plus), Waters binary pump (model 1525), and Waters UV photodiode array detector (model 996). All samples will be analyzed in triplicate. The mass median aerodynamic diameter (MMAD) and geometric standard deviation (GSD) was obtained from impactor data using established software in our lab. Impactor experiments were repeated at least two times.

In vivo orthotopic lung tumor model

The laboratory murine model has been used extensively in lung cancer research. Female, 6-week old, athymic Nu/Nu mice were purchased from Harlan Inc. (Indianapolis, IN). The mice were housed and maintained in specific pathogen-free conditions in a facility approved by the American Association for Accreditation of Laboratory Animal Care. Food and water were provided ad libium to the animals in standard cages. All experiments were performed in accordance with the guidelines of the Institutional Animal Care and Use Committee, Florida A & M University.

Mice were anesthetized and a 5 mm skin incision was made to the left chest, about 5 mm below the scapula. One-ml Hamilton syringes with 28-guage hypodermic needles were used to inject the cell inoculum through the sixth intercostal space into the left lung. The needle was quickly advanced to a depth of 3 mm and quickly removed after the injection of the A549 cells (1 × 10⁶ per mouse) suspended in 100 μl PBS into the lung parenchyma. Only cell suspensions of > 90% viability determined by trypan blue exclusion were used. Wounds from the incisions were closed with surgical skin clips (32, 33). Animals were observed for 45-60 min until fully recovered.

Administration and dosage of DIM-C-pPhC₆H₅ aerosol

DIM-C-pPhC₆H₅ formulation at a concentration of 2 mg/ml was used to generate aerosol by nebulization with a PARI LC STAR jet nebulizer using dry compressed air at a flow rate of 4 L/min. Female Nu/Nu mice (20 ± 2 g) were restrained in animal holders and placed in an inhalation chamber (SCIREQ, Montreal, Canada) such that only the nose of each mouse was exposed to the aerosol cloud. The nebulizer was connected to the top part of the inhalation chamber from which the generated aerosol flowed down the central tower to the 12 mice peripherally arranged. The duration of aerosol exposure per treatment was 30 min.

The deposition fraction was measured by estimating the amount of DIM-C-pPhC₆H₅ deposited in lungs following nebulization by HPLC analysis. Briefly, DIM-C-pPhC₆H₅ formulation (2 mg/ml) was nebulized to female Nu/Nu mice (n=12) placed in a inhalation chamber (SCIREQ, Montreal, Canada) with a PARI LC STAR jet nebulizer using dry compressed air at a flow rate of 4 L/min for 30 min. After completion of the 30 min exposure, animals were sacrificed with overdose of halothane. Lung tissues were collected and evaluated for DIM-C-pPhC₆H₅ content by an earlier validated HPLC method using solvent extraction. Lungs were weighed and homogenized with 500 μl of phosphate buffered saline (pH 7.4) and spiked with 50 μl internal standard solution (100 μg/ml nimesulide) and vortexed. To the resultant samples 1.5 ml of ter-butyl ether was added, vortexed and then centrifuged (15 min at 3500 rpm) to separate aqueous and organic phases. The organic layer was separated, evaporated to dryness and the residue was then reconstituted with 200 μl of mobile phase and 100 μl was injected onto HPLC for quantification. HPLC system comprised of an autosampler (model 717 plus), binary pump (model 1525), Waters UV photodiode array detector (model 996) and Symmetry C18 column (5 μm, 4.6 × 250mm) at a flow rate of 1.0 ml/min and the eluent was monitored at 242 nm. The gradient method was utilized with mobile phase consisting of acetonitrile, water (10:90% v/v) at 0 min, (90:10% v/v) at 8 min and remaining steady up to 17 min and changing back to (10:90% v/v) at 20 min. Quantification of DIM-C-pPhC₆H₅ was accomplished by a calibration standard curve between 0.05 and 8 μg/ml.

The deposition fraction was calculated using following equation:

$$\text{Deposition fraction}(\text{DF})=\text{A}/\text{C}\times \text{F}\times \text{T}$$ (1)

where, A is the total amount of drug deposited in mice at end of 30 min nebulization (A= 2113.593μg); C is the concentration of drug in aerosol volume (for DIM-C-pPhC₆H₅ aerosol, C = 167 μg/L); F is the flow rate used for aerosolization (F = 4.5 L/min); and T is the duration of treatment (T = 30 min).

The estimated total deposited amount of inhaled drugs (D) for the ambient air was calculated by the following formula (7-8):

$$\text{D}=\text{C}\times \text{V}\times \text{DF}\times \text{T}$$ (2)

Where, C is the concentration of drug in aerosol volume (for DIM-C-pPhC₆H₅ aerosol, C = 167 μg/L); V is the volume of air inspired by the animal during 1 min (for mice, V = 1.0 L-min/kg); DF is the measured deposition fraction using analytical method (DF = 0.093); and T is the duration of treatment (T = 30 min).

In this study, the deposition fraction and deposited dose of DIM-C-pPhC₆H₅ for duration of 30 min nebulization treatment was ∼ 0.093 and ∼ 0.47 mg/kg respectively.

Treatment of animals

Seven days after tumor implantation, the mice were randomly divided into the following groups (n=12) to receive DIM-C-pPhC₆H₅ and Doc formulations. The control group received aerosolized vehicle (vitamin E TPGS solution); the second group received Doc 10 mg/kg i.v. on days 14, 18, and 22, and 29; the third group was exposed to DIM-C-pPhC₆H₅ aerosol three times a week; the fourth group received a combination of Doc i.v. and DIM-C-pPhC₆H₅ aerosol. To check for evidence of toxicity, the animals were weighed twice weekly. On day 35, all animals were sacrificed by exposure to a lethal dose of halothane in a desiccator. After dissection and removal of the lungs, the lungs and tumor mass were washed in sterile PBS and weighed. The lung weights and tumor volume will be used for assessment of therapeutic activity of the treatments. We also evaluated efficacy of therapy in different areas by determining average number of tumor nodules in central, mid and peripheral region of lungs harvested from control and treated groups. Tumor nodules of 2-10 mm³ in volume were counted using harvested lungs for control and treated groups. For immunohistochemistry (IHC), Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL), and Hematoxylin and Eosin (H & E) staining procedures, some of the tumors were fixed in formalin while others were rapidly frozen in liquid Nitrogen and stored in -80°C.

TUNEL assay of orthotopic lung tumor tissues

Formalin-fixed tumor tissues harvested 35 days after tumor implantation were embedded in paraffin and sectioned. DeadEnd™ Colorimetric Apoptosis Detection System (Promega, Madison, WI) was used to detect apoptosis in the tumor sections placed on slides according to the manufacturer's protocol. Briefly, the equilibration buffer was added to slides and incubated for 10 min followed by incubation for 10 min in 20 μg/ml proteinase K solution. The sections were washed in PBS and incubated with TdT enzyme at 37°C for 1 hr in a humidified chamber for incorporation of biotinylated nucleotides at the 3′- OH ends of DNA. The slides were incubated in horseradish peroxidase-labeled streptavidin to bind the biotinylated nucleotides followed by detection with stable chromagen DAB. The images on the slides were visualized with an Olympus BX40 light microscope equipped with a computer-controlled digital camera (QImaging, Burnaby, BC, Canada) and imaging software (Q capture). Three slides per group were stained and apoptotic cells were identified by dark brown cytoplasmic staining.

Western blot analysis

Protein was extracted from tumor nodules collected from control-untreated and treated tumors using RIPA buffer (50mM Tris-HCL, pH 8.0, with 150 mM sodium chloride, 1.0% Igepal CA-630 (NP-40), 0.5% sodium deoxychlorate, and 0.1% sodium dodecyl sulfate) with protease inhibitor (500 mM phenylmethylsulfonyl fluoride). The lysate from normal lung tissues was also prepared in a similar manner as described above. Protein content was measured using BCA Protein Assay Reagent Kit (PIERCE, Rockford, IL). Equal amounts of supernatant protein (50 μg) from the control and different treatments were denatured by boiling for 5 min in SDS sample buffer, separated by 10% SDS-PAGE, transferred to nitrocellulose membranes for immunoblotting. Membranes were blocked with 5 % skim milk in Tris-buffered saline with Tween 20 [10mM Tris-HCL (pH 7.6), 150 mM Nacl, and 0.5% Tween 20] and probed with antibodies against Akt (1:500), Cyclin D1(1:500), JNK2 (1:500), Bad (1:500), Mcl-1 (1:500), survivin (1:500), NF-kB (1:500), IKBα (1:200), P-IkBα (1:200), VEGF (1:500), and β-actin (1:1000) (Santa Cruz Biotechnology, Santa Cruz, CA). Horseradish peroxidase-conjugated secondary antibodies (Santa Cruz Biotechnology, Santa Cruz, CA) were used. Proteins were visualized using enhanced chemiluminescent solution (Pierce, Rockford, IL) and exposed to Kodak X-OMAT AR autoradiography film (Eastman Kodak, Rochester, NY).

Immunohistochemistry for VEGF expression

Tissue sections (4–5 μm thick) mounted on poly-L-lysine–coated slide were deparaffinized by xylene and dehydrated through graded concentrations of alcohol, then incubated with 3% hydrogen peroxidase for 20 min to block endogenous peroxidase activity. Antigen retrieval for VEGF and cleaved caspase-3 staining was carried out for 10 min in 0.01 M sodium citrate buffer (pH 6) heated at 95°C in a steam bath followed by cooling for 30 min. Endogenous peroxidase was blocked by 3% hydrogen peroxide in PBS for 10 min. The slides were washed with PBS and incubated for 1 hr at room temperature with a protein blocking solution. Excess blocking solution was drained, and the samples were incubated overnight at 4°C with either 1:50 dilution of VEGF antibody incubated with biotinylated secondary antibody followed by streptavidin. The color was developed by exposing the peroxidase to a substrate-chromagen, which forms a brown reaction product. The sections were then counterstained with hematoxylin. VEGF and cleaved caspase-3 expression was identified by the brown cytoplasmic staining.

Immunohistochemistry for CD31 expression and Assessment of Microvessel Density

Tissue sections (4–5 μm thick) mounted on poly-L-lysine–coated slide were deparaffinized and blocked for peroxidase activity as described under methodology for IHC for VEGF expression. After washing with PBS, the sections were pretreated in citrate buffer in a microwave oven for 20 min at 92–98°C. After two washes with PBS, specimens were incubated in 10% normal goat serum (Atlanta Biologicals, GA, USA) for 20 min to reduce the nonspecific antibody binding. Subsequently, the sections were then incubated with a 1:500 diluted mouse CD31 monoclonal antibody (Cell Signaling Tech, MA), which is recognized as an endothelial cell surface marker, at room temperature for 1 hr, followed by a 30 min treatment with HRP rabbit/mouse (Santa Cruz Biotechnology, Santa Cruz, CA, USA). After three washes with PBS, the section was developed with diaminobenzidene-hydrogen peroxidase substrate, and lightly counterstained with hematoxylin. To calculate microvessel density (MVD), three most vascularised areas of the tumour (‘hot spots’) were selected and mean values obtained by counting vessels. A single microvessel was defined as a discrete cluster of cells positive for CD31 staining, with no requirement for the presence of a lumen. Microvessel counts were performed at ×400 (×40 objective lens and ×10 ocular lens; 0.74 mm² per field).

Statistics

One-way ANOVA followed by Tukey's Multiple Comparison Test was performed to determine the significance of differences among groups using GraphPad PRISM version 3.0 software (SanDiego, CA). Differences were considered significant in all experiments at P < 0.05 (*, significantly different from untreated controls; **, significantly different from DIM-C-pPhC₆H₅ and Doc single treatments unless otherwise stated.

Results

Aerosol characteristics

An aqueous 0.2% (2 mg/ml) solution of DIM-C-pPhC₆H₅ with suitable characteristics for nebulization was developed. The aerodynamic properties included a MMAD of 1.78 ± 0.34, GSD of 2.31 ±0.02 (Fig 2).

Fig. 2. Aerodynamic properties of DIM-C-pPhC₆H₅ aerosol using Andersen cascade impactor.

The aerodynamic particle size distribution of DIM-C-pPhC₆H₅ aerosol was measured using an 8-stage Anderson cascade impactor, Mark II following nebulization for 5 min at a flow rate of 28.3 L/min using the PARI LC STAR jet nebulizer. The total drug deposited on various stages, actuator and throat was determined using HPLC. Data was expressed as the percentage of the total drug deposited on all stages of the impactor including actuator and throat and represent mean±SD (n=3); calculated aerodynamic characteristics from impaction data, MMAD - 1.78 ± 0.34 μm, GSD - 2.31 ± 0.02.

Doc + DIM-C-pPhC₆H₅ aerosol inhibits growth of A549 orthotopic lung tumors

The anticancer activity of DIM-C-pPhC₆H₅ aerosol alone and in combination with Doc was investigated in female athymic nude mice bearing A549 orthotopic lung tumors. After 30 min of DIM-C-pPhC₆H₅ nebulization, the deposition fraction and deposited dose of DIM-C-pPhC₆H₅ was found to be ∼ 0.093 and ∼ 0.47 mg/kg respectively. Initial pilot studies showed that nude mice implanted with 10⁶ A549 cells develop fairly uniform tumors with 1 week. After 7 days of tumor inoculation the average lung weight and tumor volume were 245 ± 15.89 mg and 215 ± 21.48 mm³, respectively. Treatment was started 7 days after tumor implantation and continued for a total of 28 days. The results (Fig.3A) show that lung tumor weights were significantly (*, P<0.001) decreased after treatment with Doc, DIM-C-pPhC₆H₅ aerosol, and Doc + DIM-C-pPhC₆H₅ aerosol compared to control. Combination treatment was the most effective inhibitor of lung tumor growth compared to Doc or DIM-C-pPhC₆H₅ aerosol treatments alone. Lung tumor weight reduction in mice treated with combination of Doc + DIM-C-pPhC₆H₅ was 64 % compared to 40 and 47 % in mice treated with DIM-C-pPhC₆H₅ aerosol and Doc alone respectively. Lung tumor volume reduction (Fig. 3B) in mice treated with the combination of treatment was 90 % compared to 44 and 63 % in mice treated with DIM-C-pPhC₆H₅ aerosol and Doc alone respectively. A non-significant (P >0.05) change in average number of tumor nodules was observed among central, mid and peripheral regions of harvested lungs from each treated groups (Fig. 3C). DIM-C-pPhC₆H₅ aerosol and Doc treatment showed a significant (*, P<0.001) decrease in average number of tumor nodules in central, mid and peripheral regions compared to single agent treatment and control groups. We did not observe any weight loss or other signs of toxicity in mice treated with DIM-C-pPhC₆H₅ aerosol (data not shown). The average weight loss observed in the Doc alone treatment group was comparable to that of the combination group consistent with the expected toxicity of Doc.

Fig. 3. Effects of DIM-C-pPhC₆H₅ and Doc on human orthotopic lung tumor weight (A); human orthotopic lung tumor volume (B) and tumor nodules in central, mid and pheripheral regions of lungs (C).

A549 cells (1 × 10⁶) were injected into the lungs of nude mice. Tumors were established for 7 days before therapy. Tumors from animals treated with 2 mg/ml C-DIM aerosol (3 times a week), 10 mg/kg Doc (days 14, 18, 22, 29), or combination were harvested after 35 days. Lung weights and tumor volumes were determined for measurement of therapeutic activity of the treatments. Tumor nodules of 2-10 mm³ in volume were counted using harvested lungs for control and treated groups and the average number of tumor nodules were determined. One-way ANOVA followed by post Tukey test was used for statistical analysis. P < 0.05 (*, significantly different from untreated controls; **, significantly different from DIM-C-pPhC₆H₅ and Doc single treatments). Data presented are means ± SD (n =12).

Effects of treatments on apoptotic and angiogenic proteins in A549 orthotopic lung tumors

We compared expression of several apoptotic proteins and VEGF in normal lung tissue lysates, tumor lysates from control and treated mice by Western blot analysis using β-actin as loading control (Figs 4 & 5). DIM-C-pPhC₆H₅ aerosol and Doc treatment non-significantly (P >0.05) decreased Akt expression to 0.65 and 0.6-fold in regressed tumor samples respectively. Interestingly, expression of akt was significantly (*, P<0.001) decreased in the combination treatment group (Fig 4A & 4B). In regressed tumors, the combination (*,P<0.001), Doc (*,P<0.001), and DIM-C-pPhC₆H₅ (*,P<0.001) significantly decreased cyclin D1 expression to a non detectable level, 0.58, and 0.10-fold, respectively of controls (Fig 4A & 4B). Doc + DIM-C-pPhC₆H₅ increased JNK2 protein expression significantly (**, P<0.05) to 4.6-fold compared to 2.8-fold with DIM-C-pPhC₆H₅ (*, P<0.01) and 3.08-fold Doc (*, P<0.01) treatment, respectively of controls in regressed tumors (Fig 4A & 4B). The combination, Doc, and DIM-C-pPhC₆H₅ increased Bad expression significantly (*, P<0.001) and this protein was non-detectable in tumors from control mice (Fig 4A & 4B). DIM-C-pPhC₆H₅ and Doc treatment significantly (*, P<0.05) decreased Mcl-1 expression to 0.52 and 0.09-fold in regressed tumor samples respectively and the Mcl-1 protein expression was non-detectable in the combination treatment group (Fig 4A & 4B). The expression of survivin protein were significantly decreased by 0.13 fold (*, P<0.01), 0.43 fold (*, P<0.05) and 0.44 fold (*, P<0.05) with combination, Doc and DIM-C-pPhC₆H₅ aerosol treatment compared to control group respectively (Fig 4A & 4B). Results in Fig. 4A & 4B showed that expression of Akt, cyclin D1, Mcl-1 and survivin proteins were significantly (P<0.001) increased in tumor tissues from control group compared to normal lung tissue. The tumor tissues from the control group showed a significant (P<0.01) decrease in expression of JNK and BAD compared to normal lung tissue (Fig 4A & 4B). Results illustrated in Fig. 5 show that the combination significantly decreased expression of NF-kB (Fig 5A) to 0.06- fold (*,P<0.001), IkBα (Fig 5B) to 0.32- fold (*, p<0.01), P-IkBα (Fig 5C) to 0.37- fold (*,P<0.001), and VEGF (Fig 5D) to 0.04- fold (*,P<0.001), compared to control, whereas the effects of Doc or DIM-C-pPhC₆H₅ alone on decreasing these parameters was response –dependent. Tumor tissues from control mice exhibited a significant (P<0.001) decrease in expression of NF-kB, IkBα, P-IkBα and VEGF compared to normal lung tissue (Fig 5).

Fig. 4. Expression of apoptotic proteins (Akt, cyclin D1, JNK2, Bad, Mcl-1, and surviving) in tumor nodule and normal lung lysates by western blotting (A) and B. quantitation of apoptotic protein expression.

Lane 1, normal lung tissue; lane 2, DIM-C-pPhC₆H₅ aerosol; lane 3, Doc; lane 4, Doc + DIM-C-pPhC₆H₅; lane 5, untreated control tumors; β-actin protein acts as a loading control. Similar results were observed in replicate experiments. 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 < 0.05 (*, significantly different from untreated controls; **, significantly different from DIM-C-pPhC₆H₅ and Doc single treatments).

Fig. 5. Expression of apoptotic and angiogenic proteins in tumors. Tumor nodule lysates from control-untreated and treated tumors were analyzed by western blotting for NF-kB (A), IkB α (B) P-IkB α (C), and VEGF (D) protein expression.

Lane 1, normal lung tissue; lane 2, DIM-C-pPhC₆H₅ aerosol; lane 3, Doc; lane 4, Doc + DIM-C-pPhC₆H₅; lane 5, untreated control tumors. β-actin protein acts as a loading control. Quantitation of proteins expression relative to β-actin determined, and results are expressed as means ± SE (n=3). One-way ANOVA followed by post Tukey test was used for statistical analysis. P < 0.05 (*, significantly different from untreated controls; **, significantly different from DIM-C-pPhC₆H₅ and Doc single treatments).

Hematoxylin and eosin staining and induction of DNA fragmentation in regressed A549 lung tumors

The A549 lung tumor histology was evaluated by H & E staining of lung tumor tissue. DIM-C-pPhC₆H₅ aerosol, Doc and combination treated tumor revealed only occasional, isolated microvessels, while the section of representative images with well-formed capillaries surrounding nests of tumor cells in control (Fig 6A). Histological examination of the lungs and tracheobronchial epithelium showed no signs of inflammation or edema among all groups which suggests safer profile of DIM-C-pPhC₆H₅ aerosol and combination therapy. To investigate the role of apoptosis on tumor growth inhibition, the tumor sections harvested at the end of the study were stained with TUNEL for detection of DNA fragmentation (Fig. 6B). Single-agent therapy with either Doc or DIM-C-pPhC₆H₅ aerosol induced DNA fragmentation (brown staining) that was further increased by combination therapy.

Fig. 6. Analysis of lung tumor tissues.

(A) Immunohistochemical H & E staining of orthotopic A549 lung tumor tissues immunohistochemical staining with hematoxylin and eosin on paraffin-embedded sections. DIM-C-pPhC₆H₅ aerosol, Doc and combination treated tumor revealed only occasional, isolated microvessels, while the section of representative images with well-formed capillaries surrounding nests of tumor cells in control. Original magnification × 10. (Micron bar = 200 μm). (B) TUNEL staining of orthotopic A549 lung tumor tissues. Lungs were dissected from mice on day 35, fixed in 10% formalin, paraffin-embedded, and sectioned. Sections were stained according to the protocol specified in DeadEndTM Colorimetric Apoptosis Detection System (Promega, Madison, WI). Control cells were untreated. Original magnification × 40 (Micron bar = 20 μm). The brown staining indicates apoptotic cells.

Inhibition of angiogenesis by Doc + DIM-C-pPhC₆H₅ aerosol in A549 orthotopic lung tumors

To examine if the Doc + DIM-C-pPhC₆H₅ aerosol combination inhibited lung tumor growth through inhibition of angiogenesis, we determined VEGF expression in orthotopic A549 tumor tissue sections by IHC. The highest expression of VEGF was seen in tumor tissues harvested from untreated mice. Decreased expression of VEGF (Fig. 7A) was observed in tumors treated with the combination compared to tumors treated with Doc or DIM-C-pPhC₆H₅ alone. This response correlated with down regulation of VEGF protein observed in tumor lysates from mice treated with the same compounds (Fig. 5D).

Fig. 7. Modulation of angiogenesis by DIM-C-pPhC₆H₅ aerosol, Doc and their combination.

(A) Immunohistochemical staining of orthotopic A549 lung tumor tissues for VEGF expression. Lungs were dissected from mice on day 35, fixed in 10% formalin, paraffin-embedded, and sectioned. Sections were stained using the ABC staining kit as described in Materials and Methods. Cells showing positive VEGF expression are stained brown. Original magnification × 40. (Micron bar = 200 μm). (B) Immunohistochemical staining of orthotopic A549 lung tumor tissues for CD31 expression. Tumor angiogenesis was assessed by immunohistochemical staining with anti-CD31 antibody (brown) on paraffin-embedded sections. DIM-C-pPhC₆H₅ aerosol, Doc and combination treated tumor revealed only occasional, isolated microvessels, while the section of representative images with well-formed capillaries surrounding nests of tumor cells in control. Original magnification × 40. (Micron bar = 200 μm). (C) Assessment of microvessel density in control, DIM-C-pPhC₆H₅ aerosol, Doc and DIM-C-pPhC₆H₅+ Doc treated mice. Microvessel density (MVD) was calculated by selecting three most vascularised areas of the tumor (‘hot spots’) and mean values obtained by counting vessels. A single microvessel was defined as a discrete cluster of cells positive for CD31 staining, with no requirement for the presence of a lumen. Microvessel counts were performed at ×400 (×40 objective lens and ×10 ocular lens; 0.74 mm² per field). The MVD was significantly different between the control group and treated groups in sequential analysis; P < 0.05 (*, significantly different from untreated controls; **, significantly different from DIM-C-pPhC₆H₅ and Doc single treatments).

MVD in tumor tissue

Using the immunohistochemical technique, CD31 (+) endothelial cells were identified, as illustrated in Fig. 7B. The staining of microvessels in Doc + DIM-C-pPhC₆H₅ aerosol and DIM-C-pPhC₆H₅ aerosol groups was decreased compared to Doc treatment and control. The average number of microvessels per field in groups treated with Doc + DIM-C-pPhC₆H₅ aerosol, DIM-C-pPhC₆H₅ aerosol, Doc were found to be 58 ± 10.5 (**,P<0.001), was 95 ± 6.6 (*,P<0.05), 169.3 ± 23.7 respectively compared to 179.0 ± 28.4 in the control group (Fig. 7C).

Discussion

Despite increased interest in regional chemotherapy, relatively few studies have reported the feasibility of delivering drugs by inhalation for lung cancer treatment. Combinations of celecoxib (inhalation or oral) with i.v Doc in vivo in an orthotopic NSCLC xenograft model resulted in a significant reduction (P<0.001) in lung weight and tumor volume in mice compared to celecoxib (oral or inhalation) or Doc treatment alone (33). Koshkina et al. evaluated the effects of 9-nitrocamptothecin aerosol in B16 melanoma and osteosarcoma lung metastasis models (7). Reduced lung weights (P=0.005) and number of tumor foci (P<0.001) were observed in the B16 melanoma model. In the osteosarcoma model, the number of lung tumor foci (P<0.005) and the size of individual tumor nodules (P<0.02) were decreased. Administration of Paclitaxel liposome aerosol resulted in lung weights similar to the normal lung weights (179 ± 16 and 153 ± 19 mg, respectively; P>0.05) in a renal carcinoma lung metastasis model (8).

Previous in vitro studies with A549 and H460 lung cancer cells in our laboratory demonstrated that the combination of DIM-C-pPhC₆H₅+ Doc synergistically or additively induced apoptosis and several proapoptotic proteins (32). Moreover, in vivo studies using Doc (i.v. bolus 10 mg/kg) and DIM-C-pPhC₆H₅ (40 mg/kg) three times weekly by oral gavage showed that both compounds alone and in the combination induced apoptosis and decreased lung weights (compared to vehicle control). In this study we primarily focused in compound-induced in vivo changes in proapoptotic and kinase activities in an orthotopic murine lung tumor model using A549 cells as previously described (32). Aerosolization of poorly water-soluble drugs via nebulization and the consequent aerosol exposure to mice pose difficulties in proof-of-concept studies such as the current study. Dahl et al. used ethanolic solutions of 13-cis retinoic acid for chemoprevention in A/J mice and thus resulted in a significant (P <0.005) decrease in tumor multiplicity ranging from 56 to 80% (34). In the present study, attempts were made to prepare DIM-C-pPhC₆H₅ as an aqueous formulation using TPGS an ethoxylated derivative of Vitamin E which is suitable for inhalation delivery with a nebulizer. Characterization of the DIM-C-pPhC₆H₅ aerosol using Anderson Cascade Impactor showed MMAD of 1.78 ± 0.34 μm and GSD of 2.31 ± 0.02 respectively (Fig 2). The DIM-C-pPhC₆H₅ aerosol formulation used in this study was found to be chemically stable for one month at room temperature (data not shown).

A number of orthotopic mouse models have been developed for drug screening and this model provides tumor cells with an optimal environment for growth and progression and may reflect the clinical situation (32). Hence, we have used this orthotopic murine lung tumor model with A549 cells to study the efficacy of aerosolized DIM-C-pPhC₆H₅ alone and in combination with Doc. In this study, a nose-only inhalation chamber was used for aerosol exposure since the procedure and system is simple, does not require anaesthetization and a large number of animals can be simultaneously exposed to the aerosol (33). The aerosolized DIM-C-pPhC₆H₅ formulation at ambient air following 30 min nebulization showed deposition fraction of ∼ 0.093 which was 3.2 fold lower than deposition fraction (0.3) reported with nebulization of anticancer drugs using 5% CO₂ (7-8). DIM-C-pPhC₆H₅ was administered (alone and in combination with Doc) as an aerosol (deposited dose of ∼ 0.47 mg/kg) three times per week and this dosing regimen allowed us to directly compare the effectiveness of DIM-C-pPhC₆H₅ administered by oral gavage (32) or by an aerosol. Results summarized in Fig 3 show that the Doc & DIM-C-pPhC₆H₅ alone decreased mean lung tumor weights and volumes and the combined treatment was more effective than the single agent treatment. The efficacy of this therapy was determined from the average number of tumor nodules in central, mid and peripheral region of lungs harvested from control and treated groups. DIM-C-pPhC₆H₅ aerosol alone and in combination with Doc showed non-significant (P > 0.05) change in average number of tumor nodules among the three (central, mid and peripheral) regions (Fig. 3C) demonstrating efficient pulmonary delivery of the DIM-C-pPhC₆H₅ aerosol even into the deep lungs. Moreover, DIM-C-pPhC₆H₅ aerosol + Doc treatment also exhibited a significant (P <0.01) decrease in the average number of tumor nodules in all regions of the harvested lungs compared to single agent treated and control groups. The overall pattern of antitumorogenic activity summarized in Fig 3 using ∼ 0.47 mg/kg of aerosol treatment with DIM-C-pPhC₆H₅ was similar to that of previously observed using 40 mg/kg DIM-C-pPhC₆H₅ administered by oral gavage (32). Thus, treatment with DIM-C-pPhC₆H₅ as an aerosol was significantly effective (P<0.001) than administering the compound by oral gavage (32) and this dramatically illustrates the efficacy of the inhalation route of drug delivery.

Previous studies with DIM-C-pPhC₆H₅ and related C-substituted DIMs demonstrated that these compounds induce multiple propaoptotic responses that activate the intrinsic and extrinsic pathways (20-22, 25, 35-37). In addition other proteins such as cyclin D1 and the estrogen receptor are downregulated by c-substituted DIM through activation of the proteasome pathway (21, 24, 25) and C-substituted DIMs also enhance or modulate phosphorylation of several kinases including JNK. Result of our in vivo studies demonstrate that Doc and DIM-C-pPhC₆H₅ aerosol alone and the combination treatment induce proapototic (Bad) or decrease survival (survivin, Akt and Mcl-1) proteins and this was also accompanied by downregulation of cyclin D1 (Fig. 4A & 4B). DIM-C-pPhC₆H₅ also induced JNK2 phosphorylation in tumors (Fig. 4A & 4B) and this response has previously has been observed for C-DIMs in pancreatic and colon cancer cells where JNK phosphorylation plays a role in activation of the extrinsic proapoptotic pathway through induction of death receptor 5 (35-36). In addition we also observed that Doc, DIM-C-pPhC₆H₅ and their combination decreased two additional survival pathways in the lung tumors. Akt phosphorylation was decreased (Fig. 4A & 4B) and decreased expression of NF-kB (Fig. 5A), IkBα (Fig. 5B) and p- IkBα (Fig. 5C) proteins were also observed.

We also investigated the effects of Doc, DIM-C-pPhC₆H₅, and their combination on apoptosis in A549 lung tumors tissues determining DNA fragmentation using the TUNEL assay. DNA fragmentation was highly induced by the drug combination compared to Doc or DIM-C-pPhC₆H₅ alone thus confirming that apoptosis is an important pathway associated with the anticancer activity of these compounds (Fig 6). We also observed that treatment with Doc or oral DIM-C-pPhC₆H₅ combination increased the number of apoptotic cells compared to Doc or DIM-C-pPhC₆H₅ alone and this was consistent with results of a previous studies using oral administration of DIM-C-pPhC₆H₅ (32).

In this study, we also evaluated the effects of Doc, DIM-C-pPhC₆H₅ and combination treatment on VEGF expression which is important in formation of new blood vessels from existing vascular network and is required for providing nutrients and oxygen to rapidly proliferating cells before a tumor is established (38). VEGF and angiogenesis are critical for establishing growth, and metastasis of solid tumors. We observed that the combination treatment reduces expression of VEGF (Fig 5D) in regressed tumors and thereby inhibits angiogenesis. Other researchers have observed in vitro and in vivo antiangiogenic activities of DIM (39) and Doc (40) in MCF-7 breast and HT1080 fibrosarcoma cancers, respectively. VEGF expression in tumor tissues obtained from control and treated mice was also determined by IHC. Decreased expression of VEGF was observed in lung tumors from mice treated with Doc + DIM-C-pPhC₆H₅ aerosol compared to those treated with Doc, DIM-C-pPhC₆H₅ aerosol, or control (Fig. 7A) suggesting that induced tumor regression after treatment with Doc + DIM-C-pPhC₆H₅ aerosol may also be due, in part, to decreased expression of VEGF (Fig. 5D). MVD is a commonly used index of tumor angiogenic activity, and the density of neovessels can be counted in histological sections of the tumor and quantified (41). The tumor CD31 expression (Fig. 7B) and the average microvessels per field (Fig. 7C) in Doc + DIM-C-pPhC₆H₅ aerosol treated group were weak and significantly (p<0.001) decreased compared to the control group respectively and this correlates with VEGF expression (western blotting and IHC) in the treated and control groups. Thus, DIM-C-pPhC₆H₅ alone and in combination with Doc exhibited antiangiogenic activity, and the mechanism of VEGF downregulation is currently being investigated in our laboratories.

In summary, results of this study demonstrate that DIM-C-pPhC₆H₅ aerosol alone and in combination with Doc is highly effective for inhibiting lung tumor growth in a murine orthotopic model for lung cancer. The antitumor activities of these agents are associated with activation of growth inhibitory, proapoptotic and antiangiogenic pathways in lung tumors. Moreover, using the inhalation route of exposure we have shown that DIM-C-pPhC₆H₅ at a deposited dose of ∼ 0.47 mg/kg (inhalation) was equipotent to an oral dose of 40 mg/kg (32). Thus, the use of DIM-C-pPhC₆H₅ alone and in combination with chemotherapeutic agents could be a novel approach for treatment and possibly prevention of lung cancer in high risk patients. Currently we are carrying out additional preclinical studies to establish the benefits of administering DIM-C-pPhC₆H₅ and related compounds by the inhalation route.

Abbreviation list

DIM-C-pPhC₆H₅

1,1-bis (3′ indolyl)-1-(p-biphenyl) methane

NSCLC

non-small cell lung cancer

PPARγ

Peroxisome proliferator-activated receptor γ

Doc

docetaxel

DIM

Diindolylmethane

TPGS

α-tocopherol polyethylene glycol succinate

MMAD

mass median aerodynamic diameter

GSD

geometric standard deviation

IHC

immunohistochemistry

TUNEL

Terminal deoxynucleotidyl transferase dUTP nick end labeling

MVD

microvessel density