Immunohistochemical results for Bcl-2, Bax, p53 and caspase 3.

<p>Tumor control (A), FALHE low-dose treatment (B), FALHE high-dose treatment (C), and tamoxifen treatment (D). Dark brown particles are representing the expression of Bax, Bcl-2, Caspase 3 and p53 proteins. Microscopic observation of the FALHE-treated group compared with the control tumor showing high expression levels of Bax, p53 and caspase 3 and a low expression level of Bcl-2 protein. This result demonstrates the activation of the intrinsic pathway of apoptosis. Magnification, 40×.</p

Real-time qPCR analysis of apoptosis and cell cycle-related proteins normalized against the β-actin gene.

<p>Effect of polycerasoidin on mRNA expression of treated MCF7 cells. MCF7 cell were treated with IC₅₀ concentration of polycerasoidin for 12, 24 and 48 h. Quantitative analysis showed a time-dependent downregulation of Bcl-2 and upregulation of Bax, caspase 9, caspase 7, p21 and p27 in polycerasoidin-treated MCF7 cells. The data are shown as the means ± SEM. Values are statistically significant at *<i>P</i><0.05.</p

Immunohistochemical analysis of tumor proliferation markers.

<p>Tumor control (Ki67, A), low-dose FALHE treatment (Ki67, B), high-dose FALHE treatment (Ki67, C), tamoxifen-treated (Ki67, D) and tumor control (PCNA, E and F). Brown particles irregular shapes indicating PCNA and Ki67 expression. The quantification of PCNA and Ki67 represent the reduction of tumor cells after treatment with low- and high-dose FALHE (G). The data are shown as the means ± SEM. Values are statistically significant at *<i>P</i><0.05. Magnification, 100×. Magnification, 20× (E).</p

¹H NMR (500 MHz) and ¹³C NMR (125 MHz) spectral data of polycerasoidin in CDCl₃ (δ in ppm, <i>J</i> in Hz).

<p>¹H NMR (500 MHz) and ¹³C NMR (125 MHz) spectral data of polycerasoidin in CDCl₃ (δ in ppm, <i>J</i> in Hz).</p

Time-dependent apoptosis rates of IC₅₀ concentration of polycerasoidin-treated MCF7 cells after (B) 12, (C) 24 and (D) 48 h.

<p>(A) 0.1% DMSO treatment for 48 h was used as a vehicle control. (E) Representative bar chart of quadrant statistical analysis showing a significant elevation in the number of cells undergoing early and late apoptosis after 12 h. The data are shown as the means ± SEM. Values are statistically significant at *<i>P</i><0.05.</p

H & E staining of normal and breast cancer tissues.

<p>Histopathological observation of normal and tumor sections represents the mammary tissues from various experimental rat groups. Arrow shows a breast normal duct. (A), LA7-induced breast tumor (B), FLAHE low-dose treatment (C), FLAHE high-dose treatment (D) and tamoxifen-treated group (E). Histological examination of normal breast and tumor breast cancer before and after FALHE treatment. The normal breast shows normal duct tissues, but the LA7-induced breast tumor shows a disruption in morphology and an invasion of ductal cells throughout the breast tissues. Magnification, 40×.</p

Cell cycle distribution in polycerasoidin-treated MCF7 cells.

<p>(A) 0.1% DMSO treatment for 48 h was used as a vehicle control. The percentage of IC₅₀ concentration of polycerasoidin-treated cells at the G₀/G₁, S and G₂/M phases was examined after (B) 12, (C) 24 and (D) 48 h. (E) Analysis of the cell cycle distribution showed increase in G₁ population which revealed time-dependent cell cycle arrest at the G₀/G₁ phase. The data are shown as the means ± SEM. Values are statistically significant at *<i>P</i><0.05.</p

Chemopreventive Activity of <i>Ferulago angulate</i> against Breast Tumor in Rats and the Apoptotic Effect of Polycerasoidin in MCF7 Cells: A Bioassay-Guided Approach

<div><p> <i>Ferulago angulata </i>leaf hexane extract (FALHE) was found to be a potent inducer of MCF7 cell apoptosis. The aims of the present study were to investigate the <i>in vivo</i> chemopreventive effect of FALHE in rats, to identify the contributing anticancer compound in FALHE and to determine its potential mechanism of action against MCF7 cells. Thirty rats harboring LA7-induced breast tumors were divided into five groups: tumor control, low-dose FALHE, high-dose FALHE, treatment control (tamoxifen) and normal control. Breast tissues were then subjected to histopathological and immunohistochemical analyses. A bioassay-guided investigation on FALHE was performed to identify the cytotoxic compound and its mechanism of action through flow cytometry, real-time qPCR and western blotting analyses. An <i>in vivo</i> study showed that FALHE suppressed the expression of the tumor markers PCNA and Ki67. The tumor size was reduced from 2031 ± 281 mm³ to 432 ± 201 mm³ after FALHE treatment. FALHE administration induced apoptosis in breast tumor cells, and this was confirmed by high expression levels of Bax, p53 and caspase 3. Cell cycle arrest was suggested by the expression of p21 and p27. The <i>in vitro</i> experimental results resulted in the isolation of polycerasoidin as a bioactive ingredient of FALHE with an IC₅₀ value of 3.16 ± 0.31 μg/ml against MCF7 cells. Polycerasoidin induced mitochondrial-dependent apoptosis in breast cancer cells via caspase activation and changes in the mRNA and protein expression of Bax and Bcl-2. In addition, flow cytometric analysis demonstrated that the treated MCF7 cells were arrested at the G₁ phase, and this was associated with the up-regulation of p21 and p27 at both the mRNA and protein levels. The results of the present study reinforce further investigations scrutinizing the promising potential of the <i>F</i>. <i>angulata</i> chemical constituents as breast cancer chemopreventive agents.</p></div

Immunohistochemical results for p21 and p27.

<p>Various groups of mammary tumor were screen as: Tumor control (A), FALHE low-dose treatment (B), FALHE high-dose treatment (C), and tamoxifen treatment (D). Dark brown particles are indicating the expression of p21 and p27. High expression of p21 and p27 was observed after FALHE treatment compared with the tumor control, revealing cell cycle arrest.</p

Western blotting analysis of apoptosis and cell cycle-related proteins normalized against the positive control β-actin.

<p>Polycerasoidin effects on protein expression of MCF7 treated cells. MCF7 cells were treated with IC₅₀ concentration of polycerasoidin for 12, 24 and 48 h. Total protein were extracted from the cells and western blot was performed. The results showed a downregulation of Bcl-2 and upregulation of Bax, caspase 9, caspase 7, p21 and p27 in polycerasoidin-treated MCF7 cells after 12, 24 and 48 h.</p