Figure 1.
AE and quercetin treatment reduces cell proliferation and alters morphology of OC cells.
A. Dose dependent effect of AE on proliferation of OVCAR3 cells. B. Dose dependent effect of AE on cell death in normal placental cells (HS-799 pl). C–D. Time and Dose dependent effects of AE on proliferation OVCAR3 (C) and SW626 (D) cells. E. Dose dependent effect of quercetin on proliferation of OVCAR3 cells. OVCAR3 and SW626 cells were cultured and grown for 2 days in DMEM in presence of 10% serum as described under Materials and Methods. After this period, the cultures were fed with medium containing 10% serum and different doses of AE for 24 hours except time dependent study. Data are the mean + S.E.M. from 6 independent observations. *, p<0.05, significantly different from vehicle-treated control group.
Figure 2.
AE treatment alters morphology of OC cells.
A–D. Dose dependent effect of AE on morphology and cell density of OVCAR3: A = Control, B=100 µg/ml AE, C=200 µg/ml AE, D=300 µg/ml AE. E–H. Dose dependent effect of AE on morphology and cell density of SW626 cells. E = Control, F=100 µg/ml AE, G=200 µg/ml AE, H=300 µg/ml AE. I–J, M–N. AE increased cytoplasmic vacuoles in OVCAR3. I and M = Control, J and N = 300 µg/ml AE. K–L and O–P. AE increased cytoplasmic vacuoles in SW626. K and O = Control, L and P=300 µg/ml AE. OVCAR3 and SW626 cells were cultured and grown for 2 days in DMEM in presence of 10% serum as described under Materials and Methods. After this period, the cultures were fed with medium 10% serum and different doses of AE for 24 hours. Some of the vacuoles are indicated by arrows (N and P). Bar=50 µm.
Figure 3.
AE treatment does not cause apoptotic cell death.
A–B. Representative photographs of DNA fragmentation in OVCAR3 cells (A) and SW626 cells (B). OVCAR3 and SW626 cells were treated with 300 µg/ml AE as described in Materials and Methods. After treatment, DNA was extracted and electrophoresed on 1% agarose gel. The size in base pairs of the molecular weight markers (lane) is indicated alongside the gel. The number on the right side indicates the length in nucleotides for several of the bands. C24 = Control 24 hour, C96=Control 96 hour, T24=AE 24 hour treatment, T96=AE 96 hour treatment, +C=Positive control, S=DNA size standard. C–L. Expressions of apoptotic proteins after AE treatment (300 µg/ml) in OVCAR3 and SW626 cells. Representative photographs of Western blot of Bax (C, D), Bcl2 (E, F), Caspase 3 (G, H), cleaved Caspase 3 (I, J), Caspase 7 (K, L) in OVCAR3 (C, E, G, I, K) and SW626 (D, F, H, J, L) cells after AE treatment are shown on the top. β-actin was detected as a control for each blot. The histogram corresponding to each photograph represents the ratio of densitometric analysis of each band to the respective β-actin band. The culture was treated with 0 or 300 µg/ml AE for 24 hour. After treatment, protein from OVCAR3 and SW626 cells was extracted and 30 µg proteins were electrophoresed on SDS-PAGE. The protein was then transferred to nitrocellulose membrane and immunoblotted against apoptosis related antibodies - Bax, Bcl2, Caspase 3, cleaved Caspase 3 or Caspase 7. The values are means + S.E.M. of 4 independent experiments. *, p<0.05, as compared with control group.
Figure 4.
AE treatment increases beclin1 and LC3B-II expression in OVCAR3 and SW626 cells.
Immunostaining of beclin1 and LC3B-II in OVCAR3 and SW626 control cells and after being treated with 300 µg/ml AE (A, B, E and F). The histograms show the percentage of beclin1 (C and D) and LC3B-II (G and H) immunopositive cells as a percentage of total cells. OVCAR3 and SW626 cells were cultured and grown for 2 days in DMEM in the presence of 10% serum as described under Materials and Methods. After this period, the cultures were fed with medium containing 10% serum and AE for 24 hours. Cells were photographed at 400 x magnification. Expression of beclin1 in total protein of OVCAR3 (I) and SW626 (J) cells and expression of LC3B-II in total protein of OVCAR3 (M) and SW626 (N) cells after being treated with AE (300 µg/ml) for 24 hrs. Representative photograph of western blot for beclin1 and LC3B-II are shown on the top. β-actin was detected as control for each blot. Mean + S.E.M. values of densitometric ratio of beclin1 (K and L) and LC3B-II (O and P) with β-actin are shown on the bottom of each respective gel. Q–T: Quercetin treatment (5 µg/ml for 48 hour) induces expression of beclin1 (Q, R) and LC3B-II (S, T) in OVCAR3 cells. After treatment, protein from OVCAR3 and SW626 cells was extracted and 50 µg proteins were electrophoresed on SDS-PAGE. The protein was then transferred to a nitrocellulose membrane and immunoblotted against beclin1 and LC3B-II antibodies. After immunodetection, beclin1 and LC3B-II positive bands were measured densitometrically and normalized with β-actin values. The values are means + S.E.M. of 6 independent experiments. *, p<0.05, as compared with control group. Bar=50 µm.
Figure 5.
AE treatment reduces expression of angiogenesis related genes in OVCAR3 cells.
A. The representative photographs of micro array from control and AE-treated culture. The culture was treated with 0 or 300 µg/ml AE for 24 h. After treatment, RNA was isolated using trizol extraction method. The superarray membranes were hybridized with biotin labeled cDNA, incubated with alkaline phosphatase-conjugated streptavidin, the gene expression was detected with the chemiluminescent substrate CDP-Star. B. Representative scatterplot of AE-treated vs control cell cultures. Many genes (green *) are under expressed in AE-treated group. C. Left panel: Heat map (clustergram) of control and AE-treated cultures. Nine genes which are reduced by more than 70% are indicated by arrowheads on the right side of the heat map. Middle panel: An enlarged heat map showing reduced expression of Hif-1α in the AE-treated group. Right panel: The magnitude of gene expression. D. Graphic representation of the relative gene expression using global background and GAPDH as reference gene and converted to fold-change values (AE versus control).
Figure 6.
AE treatment reduces the expression of Hif-1α in OVCAR3 cells in vitro, and with cisplatin synergistically reduces cell proliferation and induces autophagy in vitro.
A. Photomicrograph showing reduced expression of Hif-1α immunostatining in OVCAR3 cells after AE treatment. The histogram shows the percentage of Hif-1α immunopositive cells compared to total cells. OVCAR3 cells were cultured and grown and treated with 0 or 300 µg/ml of AE for 24 hours. Cells were immunostained with Hif-1α antibody and photographed at 400X magnification. Bar=50 µm. The values are means + S.E.M. of 4 independent experiments. *, p<0.05, as compared with control group. B. A representative photograph of Western blot for Hif-1α is shown on the top. β-actin was detected as a control for each blot. Mean + S.E.M. values of densitometric ratio of Hif-1α and β-actin are shown on the bottom of the gel. After immunodetection, the volume of Hif-1α positive bands was measured densitometrically and normalized with β-actin values. The values are means + S.E.M. of 4 independent experiments. *, p<0.05, as compared with control group. C. The histogram shows the synergistic effect of AE in presence of different doses of cisplatin on cell proliferation in OVCAR3 cells. OVCAR3 cells were cultured and grown for 2 days in DMEM in presence of 10% serum as described under Materials and Methods. After this period, the cultures were fed with medium containing 10% serum and 300 µg/ml AE with or without different doses of cisplatin for 24 hours. Data are the mean + S.E.M. from 6 independent observations. a, p<0.05, significantly different from vehicle-treated control group, b, p<0.05, significantly different from cisplatin group. D and E. The synergistic effect of AE with cisplatin (5 µg/ml) on autophagy. D. Representative photograph of a Western blot showing increased expression of beclin1 in AE, cisplatin and AE with cisplatin treated groups. E. Representative photograph of a Western blot showing increased expression of LC3B-II in AE, cisplatin and AE with cisplatin treated groups. OVCAR3 cells were treated with 0, AE (300 µg/ml), cisplatin (5 µg/ml) and AE (300 µg/ml) with cisplatin (5 µg/ml) for 24 hours. β-actin was used as a control for each blot. Mean + S.E.M. values of densitometric ratio of respective protein (beclin1 and LC3B-II) and β-actin are shown on the bottom of each gel. a, p<0.05, significantly different from vehicle-treated control group; b, p<0.05, significantly different from AE group; c, p<0.05, significantly different from cisplatin group.
Figure 7.
AE treatment inhibits growth of xenografted tumors in vivo.
A. Top: Nude mice bearing tumors, left – control, right – treated. Bottom left. Size of tumors. Bottom right. Wet weight of tumor at 24 days of injection. B. Decreased immunohistochemical expression of Ki67 positive cells in mouse tumor xenograft after AE treatment. Histogram showing the decreased percentage of Ki67 immunopositive cells in AE-treated tumors. The tissue sections were photographed at 400X magnification. Bar=50 µm. The values are means + S.E.M. of 5 different mice. *, p<0.05, as compared with control group.
Figure 8.
AE treatment induces autophagy, and inhibits angiogenesis and Hif-1α expression in xenografted tumors.
A. Photograph showing increased immunoexpression of beclin1 in xenografted tumors after being treated with AE. The histogram shows the percentage of beclin1 immunopositive cells compared to total cells in control and AE treated groups. B. Representative photograph of a Western blot showing increased expression of beclin1 in AE treated group. C. Representative photograph of a Western blot showing increased expression of LC3B-II in AE treated group. β-actin was used as a control for each blot. Mean + S.E.M. values of densitometric ratio of respective protein (beclin1 and LC3B-II) and β-actin are shown on the bottom of each gel. D. AE treatment decreased immunohistochemical expression of CD31 positive cells in mouse xenograft tumors. Histogram showing decreased microvessel density in xenografts of AE-treated mice. E. Photomicrograph showing reduced expression of HIF-1α immunostaining in xenograft tumors after AE treatment. The histogram shows the percentage of Hif-1α immunopositive cells compared to total cells. F. Representative photograph of a Western blot for Hif-1α is shown on the top. β-actin was used as a control for each blot. Mean + S.E.M. values of densitometric ratio of Hif-1α and β-actin are shown on the bottom of the gel. After immunodetection, the density of Hif-1α positive bands was measured and normalized with β-actin values. Tissue sections were photographed at 400X magnification. The values are means + S.E.M. of 5 different mice. *, p<0.05, as compared with control group. Bar=50 µm.