Fluorescence Micrographs of cells stained with rhodamine and DAPI after 72 h (A) with Doxorubicin 0.4 µg/ml , Noscapine 30 µM, and, Noscapine and Doxorubicin combination in MDA-MB-231 cells and (B) Quantitation of apoptotic MDA-MB-231 cells from TUNEL assay.

<p>DNA fragmentation indicated by positive staining (red) and nuclear condensation indicated by DAPI nuclear staining (blue). Micron bar = 100 µm. Cells were quantitated by counting 100 cells from 6 random microscopic fields. Data are expressed as mean+SD (N = 6). One-way ANOVA followed by post Tukey test was used for statistical analysis to compare control and treated groups. * <i>P</i><0.01; all treatments significantly different from control and ** <i>P</i><0.01; significantly different from Noscapine and Doxorubicin single treatments.</p

Combination Index (CI) values of the interaction between Nos with Dox against human MDA-MB-231 and MDA-MB-468 TNBC cells.

<p>The human lung cancer cell lines MDA-MB-231 and MDA-MB-468 breast cancer cells were obtained from American Type Culture Collection (Rockville, MD). Different concentrations of Nos were employed to study the effect on IC50 of Dox. Variable ratios of drug concentrations and mutually non-exclusive equations were used to determine the CI. The CI values represent mean of four experiments. CI>1.3: antagonism; CI 1.1–1.3: moderate antagonism; CI 0.9–1.1: additive effect; CI 0.8–0.9: slight synergism; CI 0.6–0.8: moderate synergism; CI 0.4–0.6: synergism; CI 0.2–0.4: strong synergism.</p

Progression profile of tumor growth kinetics of in-vivo antitumor effect of different doses of Noscapine alone (A) and in combination with Doxorubicin (B) on human MDA-MB-231 tumor xenograft model (tumor volumes, mm³ ± SEM), and measurement of body weight following Noscapine alone (C) and combination with Doxorubicin (D).

<p>Female nude mice with xenograft MDA-MB-231 tumor tumors received various treatments for 38 days starting on day 7 post tumor implantation. The mice were treated with Noscapine (150–550 mg/kg/day), Doxorubicin 1.5 mg/kg i.v. bolus, q3d×7 schedule, and Noscapine 300 mg/kg/day+Doxorubicin 1.5 mg/kg i.v. bolus, q3d×7 schedule. Control group received vehicle only. Statistical significance of the difference in tumor volume of treatment groups compared with control. <i>P</i><0.01 (*, significantly different from untreated controls; ^**, significantly different from Noscapine and Doxorubicin single treatments). Data presented are means and SE (n = 8). This experiment was repeated twice.</p

Western blotting of tumor tissue lysates to determine expressions angiogenesis-related proteins expression of (A) VEGF and (B) survivin proteins in tumors and quantitation of protein expression.

<p>Whole-cell lysates from control-untreated and treated tumors were analyzed by western blotting for protein expressions. Lane 1 = control; Lane 2 = Noscapine 150 mg/kg/day ; Lane 3 = Noscapine 300 mg/kg/day ; Lane 4 = Noscapine 450 mg/kg/day; Lane 5 = Noscapine 550 mg/kg/day; Lane 6 = Doxorubicin 1.5 mg/kg i.v. bolus, q3d×7 schedule; Lane 7 = Combination (Noscapine 300 mg/kg/day+Doxorubicin ). 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. <i>P</i><0.01 (*, significantly different from untreated controls; ^**, significantly different from Noscapine and Doxorubicin single treatments).</p

Immunohistochemical staining of MDA-MB-231 tumor tissues for (A) VEGF expression.

<p>Tumor sections were stained using the ABC staining kit as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0017733#s2" target="_blank">Materials and Methods</a>. Cells showing positive VEGF expression are stained brown. Original magnification ×40. (Micron bar = 100 µm). Immunohistochemical staining of MDA-MB-231 tumor tissues for (<b>B</b>) CD31 expression. Tumor angiogenesis was assessed by immunohistochemical staining with anti-CD31 antibody (brown) on paraffin-embedded sections. Original magnification ×40. (Micron bar = 100 µm). (<b>C</b>) Quantitation of apoptotic cells from VEGF staining. (<b>D</b>) Assessment of microvessel density. Microvessel density (MVD) was calculated by selecting three most vascularised areas of the tumour (‘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; **, <i>P</i><0.01;* <i>P</i><0.05 relative to control.</p

Western blotting of tumor tissue lysates to determine expressions apoptosis-related proteins (A) expression of NF-kβ, IKBα, P-IKBα, Bax, Bcl2, caspase 3, cleaved caspase 3, activated caspase 8 and activated caspase 9 proteins in tumor lysates by western blotting and (B) quantitation of apoptotic protein expression.

<p>Tumor tissue lysates harvested tumor tissues from control-untreated and treated groups were analyzed by western blotting for protein expressions. Lane 1 = control; Lane 2 = Noscapine 150 mg/kg/day; Lane 3 = Noscapine 300 mg/kg/day; Lane 4 = Noscapine 450 mg/kg/day; Lane 5 = Noscapine 550 mg/kg/day; Lane 6 = Doxorubicin 1.5 mg/kg i.v. bolus, q3d×7 schedule; Lane 7 = Combination (Noscapine 300 mg/kg/day+Doxorubicin1.5 mg/kg i.v. bolus, q3d×7 schedule). Similar results were observed in triplicate 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. <i>P</i><0.01 (*, significantly different from untreated controls; ^**, significantly different from Noscapine and Doxorubicin single treatments).</p

Pharmacokinetic profile of BA-SD and BBA-SD.

<p>The pharmacokinetic parameters of BA free drug and BA-SD formulations groups after oral administration of 100 mg/kg.</p

Anticancer effects of BA-SD formulations in orthotopic and metastatic lung cancer models.

<p>A) Lung tumor weights and tumor volumes after treatment with BA free drug and BA-SD formulations in A549 orthotopic and metastatic lung tumor models, B) Number of lung tumor nodules in peripheral, medial and central lobes in A549 metastatic models after treatment with BA free drug and BA-SD formulations. C) Quantification of TUNEL positive cells in orthotopic lung tumors D) Representative TUNEL assay images of orthotopic lung tumor images from control, BA free drug and BA-SD treated groups. Each data point was represented as mean±sem (n = 6–8). **p<0.01 and ***p<0.001 Vs respective untreated control groups.</p

Approaches to Improve the Oral Bioavailability and Effects of Novel Anticancer Drugs Berberine and Betulinic Acid

<div><p>Background</p><p>The poor bioavailability of Berberine (BBR) and Betulinic acid (BA) limits the development of these promising anticancer agents for clinical use. In the current study, BBR and BA in spray dried (SD) mucoadhesive microparticle formulations were prepared.</p><p>Methods</p><p>A patented dual channel spray gun technology established in our laboratory was used for both formulations. Gastrointestinal (GI) permeability studies were carried out using Caco-2 cell monolayer grown in in-vitro system. The oral bioavailability and pharmacokinetic profile of SD formulations were studied in Sprague Dawley rats. A549 orthotopic and H1650 metastatic NSCLC models were utilized for the anticancer evaluations.</p><p>Results</p><p>Pharmacokinetic studies demonstrated that BBR and BA SD formulations resulted in 3.46 and 3.90 fold respectively, significant increase in plasma C_max concentrations. AUC levels were increased by 6.98 and 7.41 fold in BBR and BA SD formulations, respectively. Compared to untreated controls groups, 49.8 & 53.4% decrease in the tumor volumes was observed in SD formulation groups of BBR and BA, respectively. Molecular studies done on excised tumor (A549) tissue suggested that BBR in SD form resulted in a significant decrease in the survivin, Bcl-2, cyclin D1, MMP-9, HIF-1α, VEGF and CD31 expressions. Cleaved caspase 3, p53 and TUNEL expressions were increased in SD formulations. The RT-PCR analysis on H1650 tumor tissue suggested that p38, Phospho-JNK, Bax, BAD, cleaved caspase 3&8 mRNA expressions were significantly increased in BA SD formulations. Chronic administration of BBR and BA SD formulations did not show any toxicity.</p><p>Conclusions</p><p>Due to significant increase in oral bioavailability and superior anticancer effects, our results suggest that spray drying is a superior alternative formulation approach for oral delivery of BBR and BA.</p></div

Immunohistochemical (IHC) analysis of orthotopic lung tumor sections.

<p>IHC analysis of tumor sections collected from untreated control, BBR free drug and BBR-SD formulation treated animals. First row of images shows the cleaved caspase-3 expression in different groups. The brown color stained cells indicate the cleaved caspase-3 specific positive cells. Second row shows the CD31 expression, the brown colored cells suggest the MVD positive cells. Third row shows the TUNEL assay, brown color stained cells indicate the apoptotic positive cells.</p