Inhalation delivery of topotecan is superior to intravenous exposure for suppressing lung cancer in a preclinical model

<p>Intravenous (IV) topotecan is approved for the treatment of various malignancies including lung cancer but its clinical use is greatly undermined by severe hematopoietic toxicity. We hypothesized that inhalation delivery of topotecan would increase local exposure and efficacy against lung cancer while reducing systemic exposure and toxicity. These hypotheses were tested in a preclinical setting using a novel inhalable formulation of topotecan against the standard IV dose. Respirable dry-powder of topotecan was manufactured through spray-drying technology and the pharmacokinetics of 0.14 and 0.79 mg/kg inhalation doses were compared with 0.7 mg/kg IV dose. The efficacy of four weekly treatments with 1 mg/kg inhaled vs. 2 mg/kg IV topotecan were compared to untreated control using an established orthotopic lung cancer model for a fast (H1975) and moderately growing (A549) human lung tumors in the nude rat. Inhalation delivery increased topotecan exposure of lung tissue by approximately 30-fold, lung and plasma half-life by 5- and 4-folds, respectively, and reduced the maximum plasma concentration by 2-fold than the comparable IV dose. Inhaled topotecan improved the survival of rats with the fast-growing lung tumors from 7 to 80% and reduced the tumor burden of the moderately-growing lung tumors over 5- and 10-folds, respectively, than the 2-times higher IV topotecan and untreated control (<i>p</i> < .00001). These results indicate that inhalation delivery increases topotecan exposure of lung tissue and improves its efficacy against lung cancer while also lowering the effective dose and maximum systemic concentration that is responsible for its dose-limiting toxicity.</p

Suppression of EGFR, Akt and ERK activation inhibits BPDE-induced cell transformation.

<p>Graphical and quantitative representation of colony formation in soft agar of BEAS-2B cells exposed to BPDE (0.1 µM) and/or the indicated inhibitors (EGFRin, LY, and U0126) every two days for 1 week. Bars show the averages of colony numbers of 6 randomly selected fields. Data shown are mean ± S.D; ** P<0.01.</p

Suppression of MUC1 inhibits BPDE-induced transformation in BEAS-2B cells.

<p><i>A</i>, BEAS-2B cells were transfected with MUC1 siRNA or negative control siRNA. The cells were then treated with BPDE (0.1 µM) for 1 wk and seeded in soft agar. Colony formation was photographed under a light microscope. <i>B</i>, Quantitative representation of the transformation experiment. Bars show the averages of colony numbers of 6 randomly selected fields. Data shown are mean ± S.D; ** P<0.01. Insert, Confirmation of MUC1 knockdown by Western blot.</p

Transient BPDE exposure activates Akt and ERK through EGFR in human bronchial epithelial cells.

<p><i>A</i>, Acute BPDE exposure induces activation of EGFR, Akt, and ERK in BEAS-2B cells. BEAS-2B cells were treated with the indicated concentrations of BPDE for 2 hr. Activation of EGFR, Akt and ERK were detected by Western blot with antibodies recognizing each phosphorylated protein (phosphorylation sites are indicated). β-Actin was detected as an input control. <i>B</i>, Suppression of EGFR attenuates the activation of Akt and ERK in BEAS-2B cells. The expression and activity of EGFR in BEAS-2B cells were suppressed with either EGFR inhibitor III or EGFR siRNA. The cells were then exposed to BPDE (0.4 µM) for 2 hr. The indicated proteins were detected by Western blot. β-Actin was detected as a loading control.</p

A model of MUC1-mediated EGFR activation and HBEC transformation.

<p>CS carcinogens such as BPDE trigger MUC1 expression in bronchial epithelial cells, facilitating EGFR-mediated cell survival signaling via Akt and ERK activation. Akt and ERK protect cells against DNA damage-mediated apoptosis to promote cell transformation, facilitating lung carcinogenesis.</p

Chronic BPDE exposure activates Akt and ERK through EGFR in human bronchial epithelial cells.

<p><i>A</i>, Induction of MUC1 expression and EGFR-, Akt- and ERK-activation in HBEC-2 cells by BPDE. Specifically, HBEC-2 cells were treated with the vehicle DMSO or BPDE (0.1 µM) for the indicated weeks. Western blot was the same as in <i>A</i>. β-Actin was detected as a loading control. <i>B</i>, Increased MUC1 expression and EGFR-, Akt- and ERK-activation in transformed HBEC-2 cells by BPDE. HBEC-2 cells were treated with BPDE (0.1 µM) for 12 wk and then seeded in soft agar. Colonies were grown up for 3 wk in transformed cells (TRANS). Wild-type (WT, exposed to sham) HBEC-2 cells were as a negative control. Expression of MUC1 and activation of Akt and ERK were detected by Western blot in both WT and the transfected cells. β-Actin was detected as a loading control.</p

Blocking EGFR, Akt and ERK activation potentiates BPDE-induced cytotoxicity.

<p>BEAS-2B cells were pretreated with the indicated inhibitors (LY (10 µM) for Akt, EGFRin (6 µM) for EGFR, U0126 (5 µM) for ERK, and SP (10 µM) for JNK) for 30 min followed by exposure to BPDE (0.2 µM) for 48 hr. Cell viability was detected by LDH release and MTT assays. Data shown are mean ± S.D; ** P<0.01, * P<0.05.</p

MUC1 stabilizes EGFR, contributes to BPDE-induced EGFR, Akt and ERK activation in BEAS-2B cells and protects cells from BPDE-induced cytotoxicity.

<p><i>A</i>, MUC1 is required for BPDE-induced EGFR, Akt and ERK activation. Cells stably transfected with MUC1 shRNA or negative control shRNA were treated with BPDE for the indicated time periods. Activation of each protein was detected with antibodies against the phosphorylated form of the proteins. The phosphorylation sites of each protein are indicated. Total EGFR, Akt and ERK were also detected. β-Actin was detected as an input control. <i>B</i>, Reduced EGFR expression in MUC1 Knockdown cells. <i>Upper left</i>, equal amounts of total RNA from the indicated cells were detected for EGFR mRNA expression. β-Actin was detected as an input control. <i>Upper right</i>, Cells stably transfected with MUC1 shRNA or negative control shRNA were treated with cycloheximide (CHX, 10 µM) for the indicated time periods. EGFR protein was detected by Western blot. β-Actin was detected as an input control. <i>Lower right</i>, quantification of the results of <i>Upper right</i>. The intensity of the individual bands was quantified by densitometry (NIH Image 1.62) and normalized to the corresponding input control (β-actin) bands. EGFR expression changes were calculated with the control taken as 100%. <i>C</i>, BPDE-induced cytotoxicity is increased in MUC1 knockdown cells. BEAS-2B WT and MUC1 knockdown cells were treated with the indicated concentrations of BPDE for 48 hr. Cell viability was detected by LDH release and MTT assays. Data shown are mean ± S.D; **P<0.01.</p

Relative expression of TOX subfamily genes in normal lung tissue.

<p>(<b>A</b>) Expression of each gene was quantified using TaqMan assays and the level of <i>TOX4</i>, which is unmethylated in all samples and expressed the highest in normal lung tissue, was used as a reference to calculate the relative level of the remaining genes. * p = 0.03, ** p<0.001, *** p<0.0001 compared to <i>TOX4</i>. (<b>B</b>) COBRA conducted as described for <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0034850#pone-0034850-g001" target="_blank">Figure 1A</a>. (<b>C</b>) <i>TOX</i> expression was measured relative to its expression in <i>MCF-7</i> (Top left) or vehicle treated <i>MDA-MB-231 (M-231)</i>, <i>T47D</i>, or <i>MCF-7</i>. (<b>D</b>) Transfection of <i>M-231</i> with siTOX reduced its expression by 75% compared to siControl (left) but this did not alter the migration potential of the cells.</p

<i>TOX2</i> expression in normal and cancer cells.

<p>(<b>A</b>) Genomic structure of <i>TOX2</i>. Top box: Predicted transcript variants of <i>TOX2</i> (var.1-4) currently used as reference sequence for <i>Homo sapiens</i> chromosome 20, GRCh37.p2, (GenBank accession number NC_000020.10). Bottom box: Transcripts sequenced from human cells (var.5 and 6). Small arrows indicate the location and direction of primer binding sites; T#F or T#R (forward or reverse primers for TaqMan assays) and G#F or G#R (forward or reverse primers for gel-based assays). (<b>B</b>) Expression of <i>TOX2</i> transcript variants 5 and 6 and the house keeping gene beta-actin in distant normal lung tissue (DNLT), HBEC, and various lung and breast cancer cell lines. In Vehicle-treated (S, for sham) lung cancer (H1838, H2009) and breast cancer (T47D) cell lines with methylated promoter CpG island, both transcripts were silenced and expression of both was primarily restored with 5-Aza-2′-deoxycytidne (D) but not trichostatin A (T) treatment. (<b>C and D</b>) TaqMan assays that use distinct primer sets from those used for gel-based assays confirmed results shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0034850#pone-0034850-g002" target="_blank">Figure 2B</a>. (<b>C</b>) Expression of TOX2 var.5 or both (var.5 & 6) in lung tumors (n = 20) relative to DNLT (n = 10) obtained from NSCLC patients. (<b>D</b>) Expression of <i>TOX2</i> var.5 or both (var.5+6) in TSA or DAC treated lung and breast cancer cell lines relative to Vehicle-treated (Sham) cell lines.</p