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

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

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

Expression of VEGF, SP1, SP3, pAKT, cyclin D1, p53, p21, and survivin proteins in tumor lysates by western blotting (A) and (B) quantitation of apoptotic protein expression.

<p>Lane 1, untreated control tumors; lane 2, oral Nos 300 mg/kg; lane 3, Gem 30 mg/kg i.v. bolus, q3d ×7 schedule ; lane 4; NGC. β-actin protein acts as a loading control. Similar results were observed in triplicate experiments. Protein expression levels (relative to β-actin) were determined. Mean ± SE for three replicate determinations.</p

Isobolograms (A) and (B) Combination Index (CI) values of the interaction between Gem with Nos against human lung Cancer cells.

<p>Different concentrations of Nos were employed to study the effect on IC50 of Gem. 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

Immunohistochemical staining of H460 tumor tissues for VEGF, CD31, TUNEL assay and cleaved caspase 3 expression (A) and (B) quantitation of quantization of VEGF, assessment of microvessel density, quantitation of TUNEL positive cells and cleaved caspase 3 positive cells.

<p>Data are expressed as mean + SD (N = 6).</p

Tube formation assay with HUVEC cells after 6 h (A); quantification of branching points (B); micrographs of cells stained with TUNEL after 72 h (C) and quantitation of apoptotic H460 (D) and A549 cells from TUNEL assay (E).

<p>For tube formation assay, HUVEC cells were incubated with Nos (30 µM), Gem (0.4 µg/ml) and NGC on polymerized Matrigel at 37°C. After 6 h, tube formation by endothelial cells was photographed and the capillary tube branch point formation were quantified (n = 3). For TUNEL assay, H460 cells were treated with Gem 0.4 µg/ml, Nos 30 µM, and, NGC and A549 cells were treated with Gem 0.3 µg/ml, Nos 50 µM, and, NGC. Control cells were untreated. Micron bar  = 10 µm. Cells were quantitated by counting 100 cells from 6 random microscopic fields. Data are expressed as mean + SD (n = 6).</p