Agar either with or without cucurbitacin B, clonal growth of the BRCA1 knocked-down cells was inhibited significantly in the presence of cucurbitacin B compared with the untreated control cells. The clonal growth, as determined by the number of colonies formed in soft agar, was reduced by cucurbitacin B (Fig. 3A, 3B) and decrease in the size of colonies was also observed in the cucurbitacin B treated culture (not shown). Cucurbitacin B significantly inhibited cellular migration and invasion in the shRNA-BRCA1 transfected cells but had no effect upon parental cells at concentration of 12 mg/ml (Fig. 3C?F). These results indicate that the biological action of cucurbitacin B in Microcystin-LR site cancer cells could be associated with the BRCA1 SPI 1005 web function.Cucurbitacin B in BRCA1 Defective Breast CancerFigure 8. Cucurbitacin B treatment in exogenously induced BRCA1 expressing cells. (A), Western blot analysis for BRCA1 from BRCA1defective MDA-MB-436 cells which either transfected with vector containing BRCA1 full length (pCEP4-BRCA1) or the splice variant (skip exon 9?0; pCEP4-BRCA1-Delta(9,10)). pCEP4 was used as empty vector control. (B), Cells were grown for 5 days and cell viability was tested by using MTS assay. (C), MDA-MB-436 parental cells, empty vector control cells and cells with transfected BRCA1 were treated with 12 mg/ml cucurbitacin B for 48 h and cell viability was analyzed. BRCA1 expressing cells showed significant higher resistance to cucurbitacin B when compared to the BRCA1 defective parental cells, (* p,0.01). doi:10.1371/journal.pone.0055732.gCucurbitacin B induced expression of p27Kip1 and p21/Waf1 but suppressed the expression of survivin in BRCA1 dependent mannerKnocking down BRCA1 in breast cancer cells resulted in an increase in the expression of survivin which associated with malignant progression and drug resistance [26]. In the absence of cucurbitacin B treatment, knocking down of BRCA1 expression could result in an increased anti-apoptotic molecule survivin expression with a concurrent reducdion of paclitaxel sensitivity (Fig. 4A, 4B and 4C). Treatment of the BRCA1 knocked-down cells with 15 mg/ml cucurbitacin B could induce cell cycleinhibitors p27Kip1 and p21/Waf1 expression but down modulate survivin expression (Fig. 4A, 4B). Reduced expression of survivin in these cucurbitacin B treated cells could be an important sign of increased apoptotic process, as a significant increased sensitivity to 18325633 cucurbitacin B was observed (Fig. 4C).BRCA1 mutant cells are more sensitive to cucurbitacin B than the non-mutant counterpartThe two BRCA1-defective breast cancer cells (HCC1937 and MDA-MB-436) shown to express low BRCA1 compared to the wild type cells (Fig. 5A). Similar to the BRCA1 knocked-down cells mentioned earlier, cucurbitacin B could suppress the growth of theCucurbitacin B in BRCA1 Defective Breast CancerFigure 9. Cucurbitacin B treatment in exoenously induced wt-BRCA1 and mutant BRCA1 expressing cells. (A), Western blot analysis for BRCA1 from BRCA1 defective MDA-MB-436 cells transfected with either wt-BRCA1 vector (pCEP4-BRCA1) or the mutant BRCA1 (3300delA) vector (pCEP4-BRCA1-3300delA). (B), Proliferative rate of wild type and mutant BRCA1 expressing cells. The cells were grown and MTS assay was assessed at indicated times. (C), MDA-MB-436 parental cells, empty vector control cells and cells with wild type or mutant BRCA1 expression were treated with 5, 10, 15, 20 and 25 mg/ml cucurbitacin B for 48 h. Control cells were treated.Agar either with or without cucurbitacin B, clonal growth of the BRCA1 knocked-down cells was inhibited significantly in the presence of cucurbitacin B compared with the untreated control cells. The clonal growth, as determined by the number of colonies formed in soft agar, was reduced by cucurbitacin B (Fig. 3A, 3B) and decrease in the size of colonies was also observed in the cucurbitacin B treated culture (not shown). Cucurbitacin B significantly inhibited cellular migration and invasion in the shRNA-BRCA1 transfected cells but had no effect upon parental cells at concentration of 12 mg/ml (Fig. 3C?F). These results indicate that the biological action of cucurbitacin B in cancer cells could be associated with the BRCA1 function.Cucurbitacin B in BRCA1 Defective Breast CancerFigure 8. Cucurbitacin B treatment in exogenously induced BRCA1 expressing cells. (A), Western blot analysis for BRCA1 from BRCA1defective MDA-MB-436 cells which either transfected with vector containing BRCA1 full length (pCEP4-BRCA1) or the splice variant (skip exon 9?0; pCEP4-BRCA1-Delta(9,10)). pCEP4 was used as empty vector control. (B), Cells were grown for 5 days and cell viability was tested by using MTS assay. (C), MDA-MB-436 parental cells, empty vector control cells and cells with transfected BRCA1 were treated with 12 mg/ml cucurbitacin B for 48 h and cell viability was analyzed. BRCA1 expressing cells showed significant higher resistance to cucurbitacin B when compared to the BRCA1 defective parental cells, (* p,0.01). doi:10.1371/journal.pone.0055732.gCucurbitacin B induced expression of p27Kip1 and p21/Waf1 but suppressed the expression of survivin in BRCA1 dependent mannerKnocking down BRCA1 in breast cancer cells resulted in an increase in the expression of survivin which associated with malignant progression and drug resistance [26]. In the absence of cucurbitacin B treatment, knocking down of BRCA1 expression could result in an increased anti-apoptotic molecule survivin expression with a concurrent reducdion of paclitaxel sensitivity (Fig. 4A, 4B and 4C). Treatment of the BRCA1 knocked-down cells with 15 mg/ml cucurbitacin B could induce cell cycleinhibitors p27Kip1 and p21/Waf1 expression but down modulate survivin expression (Fig. 4A, 4B). Reduced expression of survivin in these cucurbitacin B treated cells could be an important sign of increased apoptotic process, as a significant increased sensitivity to 18325633 cucurbitacin B was observed (Fig. 4C).BRCA1 mutant cells are more sensitive to cucurbitacin B than the non-mutant counterpartThe two BRCA1-defective breast cancer cells (HCC1937 and MDA-MB-436) shown to express low BRCA1 compared to the wild type cells (Fig. 5A). Similar to the BRCA1 knocked-down cells mentioned earlier, cucurbitacin B could suppress the growth of theCucurbitacin B in BRCA1 Defective Breast CancerFigure 9. Cucurbitacin B treatment in exoenously induced wt-BRCA1 and mutant BRCA1 expressing cells. (A), Western blot analysis for BRCA1 from BRCA1 defective MDA-MB-436 cells transfected with either wt-BRCA1 vector (pCEP4-BRCA1) or the mutant BRCA1 (3300delA) vector (pCEP4-BRCA1-3300delA). (B), Proliferative rate of wild type and mutant BRCA1 expressing cells. The cells were grown and MTS assay was assessed at indicated times. (C), MDA-MB-436 parental cells, empty vector control cells and cells with wild type or mutant BRCA1 expression were treated with 5, 10, 15, 20 and 25 mg/ml cucurbitacin B for 48 h. Control cells were treated.