RESEARCH POSTER PRESENTATION DESIGN © 2012 www.PosterPresentations.com The herbs Scutellaria baicalensis (SB) and Fritillaria cirrhosa (FC) are widely used in Chinese medicine to treat several aliments and as an adjuvant to chemotherapy of lung cancer. No information is available regarding the two herbs' influence on ovarian and endometrial cancer. To fill this data gap we compared cell growth responses to SB and FC in ovarian and endometrial cancer cell lines. Dose- dependent cell growth inhibition was observed following higher doses in all cell lines while lower doses stimulated growth in only endometrial cell lines. Higher doses of SB and FC significantly decreased cell growth on soft agar and decreased the invasive potential of cancer cells. Treatment of cells with both herbs resulted in activation of caspase-3, G 0 /G 1 phase cell cycle arrest, downregulation of cyclins D1 and D3 and induction of p27. Both herbs decreased NFκB DNA binding, reduced expression of phosphorylated IκBα, abrogated NFκB activation, and downregulated NFκB-regulated metastasis-promoting proteins in cancer cells. Furthermore, knockdown of NFκB attenuated SB- and FC-induced cell growth inhibition. These results suggest that inhibition of NFκB activation may be an important mechanism for growth suppression by SB and FC. Data indicate that these herbs may represent a new source of agents for NFκB inhibition in cancer therapy. ABSTRACT (A) Cells treated with SB or FC (200 mg/mL) or vehicle for 96 h were cultured on soft agar, and 4wk later, colonies were counted. The value shown is the percentage compared with vehicle control cells (100%). (B) Cells treated with SB or FC (200mg/mL) for 96h were plated on Matrigel invasion chambers. After 22h, cells that migrated through the Matrigel were counted. Data are the means of three experiments with triplicates. Error bars are the mean±SEM. Statistically significant decreases in colony formation and migration are indicated with asterisks. Of note was the dramatic inhibition (40–60%) of invasiveness caused by SB treatment in cancer cells compared to about 15–25% inhibition observed with FC treatment SB and FS Decreased the Ability of Cancer Cells to Grow in Soft Agar and Migrate Through Matrigel (A) Ovarian and endometrial cancer cells were treated with SB or FC for 96h, after which cell lysates were prepared and analyzed by Western blotting for caspase-3. β-Actin was used as a loading control. The expression of caspase-3 was significantly higher than vehicle-treated cultures. SB and FC Inhibited Cell Proliferation Through Apoptosis and Cell Cycle Arrest at G 1 Phase SB and FC Inhibited NF-κB Regulated Proteins Cells treated with SB or FC for 96 h were analyzed by Western blotting for the expression of CXCR4 and MMP-9. β-Actin was used as a loading control. The immunoblots shown are representative of three independent experiments with similar results. The values above the bands represent relative density of the bands normalized to β-Actin. Knockdown of NFκB-p50 Attenuates SB and FC Growth Inhibitory Effects on Cancer Cells Effect of NFκB silencing on protein expression and growth of ovarian and endometrial cells. Cells were transfected with NFkB- p50 siRNA (5 mM) or control siRNA (5 mM) using Lipofectamine 2000. After 24 h, cells were treated with SB or FC for 5 d. (A) Expression of NFκB-p50 was analyzed by Western Blot. (B) Cell proliferation was evaluated by MTS assay. Data shown are means±SEM of values from three independent experiments. *P values<0.05 (statistically significant) between the control and the two treatment groups. a P values<0.05 (statistically significant) between the NFκB knockdown and the NFκB knockdown SB- and FC-treated groups. 1 Department of Obstetrics and Gynecology, Uniformed Services University of the Health Sciences, Bethesda, Maryland 2 Department of Acupuncture and Oriental Medicine, Northwestern Health Sciences University, Minneapolis, Minnesota 3 Department of Basic Sciences, Northwestern Health Sciences University, Minneapolis, Minnesota 4 Department of Cell and Molecular Biology, Uniformed Services University of the Health Sciences, Bethesda, Maryland The Chinese Herbs Scutellaria baicalensis and Fritillaria cirrhosa Target NFkB to Inhibit Proliferation of Ovarian and Endometrial Cancer Cells * 0 Control SB FC Cell Proliferation (%) 40 80 0 40 80 120 Control SB FC Ovarian Cancer Endometrial Cancer 120 HEC-1B Ishikawa OVCA 420 OVCA 429 24 h 48 h 72 h * * * * * * * * * * * * * * * 96 h * * * * * * * * * * * * * * * 0 Control SB FC Cell Proliferation (%) 40 80 0 40 80 120 Control SB FC Ovarian Cancer Endometrial Cancer 120 HEC-1B Ishikawa OVCA 420 OVCA 429 24 h 48 h 72 h * * * * * * * * * * * * * * * 96 h * * * * * * * * * * * * * * 0 40 80 120 OVCA 420 OVCA 429 HEC-1B Ishikawa Ovarian Cell Lines Endometrial Cell Lines SB FC Colony Formation (%) Control 0 40 80 120 * * * * * * * * A 0 40 80 120 OVCA 420 OVCA 429 HEC-1B Ishikawa Ovarian Cell Lines Endometrial Cell Lines SB FC Colony Formation (%) Control 0 40 80 120 * * * * * * * * A 0 40 80 120 OVCA 420 OVCA 429 HEC-1B Ishikawa Ovarian Cell Lines Endometrial Cell Lines SB FC Cell Invasion (%) Control 0 40 80 120 * * * * B * * * * 0 40 80 120 OVCA 420 OVCA 429 HEC-1B Ishikawa Ovarian Cell Lines Endometrial Cell Lines SB FC Cell Invasion (%) Control 0 40 80 120 * * * * B * * * * Ovarian Endometrial OVCA 420 OVCA 429 HOSE 642 HEC-1B Ishikawa EM-E6/E7-TERT β-Actin (42 kDa) Caspase-3 (17 kDa) β-Actin (42 kDa) β-Actin (42 kDa) 0.14 0.35 0.27 0.11 0.53 0.42 0.17 0.44 0.35 0.19 0.69 0.51 0.09 0.36 0.33 0.10 0.27 0.32 Caspase-3 (17 kDa) Caspase-3 (17 kDa) OVCA 420 OVCA 429 HEC-1B Ishikawa Ovarian Endometrial HOSE 642 EM-E6/E7-TERT Cyclin D1 (36 kDa) β-Actin (42 kDa) Cyclin D3 (31 kDa) P27 (27 kDa) 0.84 0.38 0.40 0.76 0.24 0.30 0.67 0.17 0.21 0.94 0.14 0.17 0.16 0.42 0.39 0.20 0.44 0.52 0.97 0.10 0.33 0.44 0.09 0.13 0.55 0.11 0.18 0.85 0.21 0.34 0.13 0.45 0.37 0.09 0.52 0.61 0.15 0.76 0.70 0.18 0.86 0.65 0.49 0.07 0.10 0.87 0.11 0.17 0.42 0.14 0.16 0.38 0.07 0.11 Cyclin D1 (36 kDa) β-Actin (42 kDa) Cyclin D3 (31 kDa) P27 (27 kDa) Cyclin D1 (36 kDa) β-Actin (42 kDa) Cyclin D3 (31 kDa) P27 (27 kDa) B (B) Cell cycle regulatory protein expression in SB and FC-treated cells. β-Actin was used as a loading control. The values above the bands represent relative density of the bands normalized to β-actin. In both SB- and FC-treated cells, immunoblotting showed decreased (P<0.05) expression of cyclin D1, D3 and induction of p27. These observations agreed with results of flow cytometry analysis (not shown), which indicated enrichment of cells at the G 0 /G 1 phase and concomitant decrease in the number of cells in S-phase. A Control 0 1 2 4 5 NFkB Activation (OD 405 nm) * # SB FC Control SB FC Control SB FC Control SB FC Control SB FC Control SB FC Control LPS 3 * * * * * * * # # # * A Ovarian (OVCA 420, OVCA 429) and endometrial (Ishikawa, and HEC-1B) cancer cells were treated with SB, FC, or LPS for 96h and processed for the preparation of nuclear protein extracts. The presence of activated NFκB-p50 in the nuclear extracts was examined with the TransAM NFκB-p50 Chemi Transcription Factor Assay kit. Normal ovarian surface epithelial cells (HOSE 642) and normal endometrial cells (EM-E6/E7-TERT) were used as controls. SB and FC Inhibit Activation of NF-κB as Measured in a DNA-Binding Assay SB and FS Caused Time-Dependent Suppression of Ovarian and Endometrial Cell Growth Cells were treated with SB or FC (200 mg/mL). Viability of cells was assessed 24, 48, 72, and 96 h after the treatment. SB markedly inhibited growth in a time-dependent manner. In a separate experiment (not shown), higher doses SB and FC caused marked inhibition of all cancer cell lines, but lower doses of both herbs caused marked increase of growth of endometrial but not ovarian cancer cell lines. OVCA 420 OVCA 429 HEC-1B Ishikawa NFkB p50 (50 kDa) β-Actin (42 kDa) p-IkBα (40 kDa) IkBα (41 kDa) Ovarian Endometrial B 0.10 0.54 0.62 0.21 0.64 0.46 0.86 0.13 0.21 0.53 0.12 0.31 0.51 0.08 0.26 0.73 0.20 0.37 0.12 0.36 0.31 0.10 0.47 0.39 0.71 0.12 0.23 0.64 0.16 0.25 0.39 0.19 0.23 0.89 0.15 0.32 NFkB p50 (50 kDa) β-Actin (42 kDa) p-IkBα (40 kDa) IkBα (41 kDa) SB and FC Decreases NFκB-p50, Upregulates IκBα, and Concomitantly Decreases p-IκBα Levels Ovarian and endometrial cancer cells were treated with SB or FC for 96 h. Extracts were prepared for assessing the levels of NFκB, IκBα, and phosphorylated IκBα by Western blotting. β-Actin was used as a loading control. The values above the bands represent relative density of the bands normalized to β-Actin. Ovarian Cancer MMP-9 (92 kDa) CXCR4 (47 kDa) β-Actin (42 kDa) MMP-9 (92 kDa) CXCR4 (47 kDa) β-Actin (46 kDa) Endometrial Cancer OVCA 420 OVCA 429 HEC-1B Ishikawa 0.43 0.11 0.16 0.66 0.22 0.24 0.67 0.09 0.23 0.54 0.10 0.17 0.77 0.11 0.27 0.63 0.9 0.13 0.44 0.13 0.19 0.71 0.15 0.18 OVCA 420 OVCA 429 HEC-1B Ishikawa β-Actin (42 kDa) β-Actin (42kDa) β-Actin (42 kDa) β-Actin (42 kDa) NFkB p50 (50 kDa) NFkB p50 (50 kDa) NFkB p50 (50 kDa) NFkB p50 (50 kDa) A 0.65 0.67 0.05 0.58 0.55 0.09 0.72 0.74 0.03 0.68 0.71 0.11 OVCA 420 0 20 40 60 80 100 120 0 20 40 60 80 100 120 Cell Proliferation (% of control) OVCA 429 HEC-1B Ishikawa * * * * * * * * * * * * aa a a a a a a B Mol. Carcinog. doi: 10.1002/mc.22107 © 2013 Wiley Periodicals, Inc. Leyla Kavandi, 1 Laura R. Lee, 1 Amber A. Bokhari, 1 John E. Pirog, 2 Yongping Jiang, 2 Kashif A. Ahmad, 3 and Viqar Syed 1,4