Ficus regulates the growth of cervical cancer cells.

<p>SiHa (<b>A</b>) and HeLa (<b>B</b>) were treated with FR<b>_aq</b>(0–80 µg/ml) for 24–72 h and the number of viable cells were counted using the trypan blue dye exclusion method. Data represent mean ± SD of three independent experiments. (<b>C</b>) The cervical cancer cell lines (SiHa and HeLa) were treated with FR<b>_aq</b>(0–80 µg/ml) for one week. The colonies were stained with crystal violet and photographed. The experiments were repeated three times. (<b>D</b>) Both SiHa and HeLa (5×10³) along with FR<b>_aq</b> (0–80 µg/ml) were grown in soft agar for two weeks. Colonies were counted from at least 10 different areas and the average of each has been plotted. The data represents mean ± SD of five independent experiments.</p

Ficus induces apoptosis in HeLa through mitochondrial dependent pathway.

<p>(<b>A</b>) Representative FACS pictograms of cells treated with FR<b>_aq</b>(0–80 µg/ml) are shown. Percent of annexin V-positive (early-apoptotic cells, lower right quadrant) and Annexin V/PI-double-positive cells (late-apoptotic cells, upper right quadrant) are indicated. (<b>B</b>) Flow cytometric analysis of the rapid calcium release in HeLa cells after treatment with FR<b>_aq</b>(0–80 µg/ml) has been shown. Ionomycin was used as a positive control. The data represents mean ± SD of three independent experiments. (<b>C</b>) FACS analysis following JC-1 staining of HeLa showed alteration of the mitochondrial membrane potential after FR<b>_aq</b>(0–80 µg/ml) treatment compared to untreated control cells. The data represents mean ± SD of three independent experiments. (<b>D</b>) Western blot shows the expression of cytochrome c from cytosolic fraction. Tubulin was used as a loading control. (<b>E</b>) Total protein was isolated and analysed for expression of p53 and caspase 3 by immunoblotting. Tubulin was used as a loading control. (<b>F and G</b>) Densitometric analysis of the western blot showing fold change in protein levels. The bands were quantified by using ImageJ 1.44p (Wayne Rasband, National Institutes of Health, USA, <a href="http://imagej.nih.gov/ij" target="_blank">http://imagej.nih.gov/ij</a>).</p

Ficus arrests the cell cycle in SiHa at G₁/S phase and modulates the expression of cell cycle regulatory proteins.

<p>SiHa cells were treated with different concentrations of FR_aq (0–80 µg/ml) for 24 h. (<b>A</b>) Enhanced accumulation of the cells in G₁ phase with a concomitant decrease in S-phase population was observed after treatment with Ficus (as indicated by histograms). Western blot shows the expression levels of p53 and pRb (<b>B</b>) as well as p21 and ppRb (<b>C</b>). Tubulin was used as a loading control. (<b>D, E</b>) Densitometric analysis of the western blot showing fold change in protein levels upon FR_aqtreatment. The bands were quantified by densitometry scanning using ImageJ 1.44p (Wayne Rasband, National Institutes of Health, USA, <a href="http://imagej.nih.gov/ij" target="_blank">http://imagej.nih.gov/ij</a>). The data represents mean ± SD of three independent experiments.</p

The Aqueous Extract of <i>Ficus religiosa</i> Induces Cell Cycle Arrest in Human Cervical Cancer Cell Lines SiHa (HPV-16 Positive) and Apoptosis in HeLa (HPV-18 Positive)

<div><p>Natural products are being extensively explored for their potential to prevent as well as treat cancer due to their ability to target multiple molecular pathways. <i>Ficus religiosa</i> has been shown to exert diverse biological activities including apoptosis in breast cancer cell lines. In the present study, we report the anti-neoplastic potential of aqueous extract of <i>F. religiosa</i> (FR_aq) bark in human cervical cancer cell lines, SiHa and HeLa. FR_aq altered the growth kinetics of SiHa (HPV-16 positive) and HeLa (HPV-18 positive) cells in a dose-dependent manner. It blocked the cell cycle progression at G₁/S phase in SiHa that was characterized by an increase in the expression of p53, p21 and pRb proteins with a simultaneous decrease in the expression of phospho Rb (ppRb) protein. On the other hand, in HeLa, FR_aq induced apoptosis through an increase in intracellular Ca²⁺ leading to loss of mitochondrial membrane potential, release of cytochrome-c and increase in the expression of caspase-3. Moreover, FR_aq reduced the migration as well as invasion capability of both the cervical cancer cell lines accompanied with downregulation of MMP-2 and Her-2 expression. Interestingly, FR_aq reduced the expression of viral oncoproteins E6 and E7 in both the cervical cancer cell lines. All these data suggest that <i>F. religiosa</i> could be explored for its chemopreventive potential in cervical cancer.</p></div

Ficus regulates invasion and migration of cervical cancer cells.

<p>Analysis of cell migration in SiHa (<b>A</b>) and HeLa (<b>B</b>) treated with FR_aq (0–80 µg/ml) was measured by wound-healing assay. The upper panel of the image shows the wound made at 0 h. The lower panel shows the migration of cells corresponding to the distance travelled at 16 h. (<b>C</b>) Graphical representation of wound closure in SiHa and HeLa cells at 16 h after FR_aq treatment has been shown. Values were represented as the percent wound closure and expressed as mean ± SD for three independent experiments. (<b>D</b>) Cell invasion assay showing the percentage of cells invaded per field in the presence or absence of FR<b>_aq</b>. The invaded cells were counted in ten random fields and the values have been expressed as mean ± SD for three independent experiments. (<b>E</b>) Gelatin zymography showing downregulation of MMP-2 expression in FR<b>_aq</b> (0–80 µg/ml) treated SiHa and HeLa. (<b>F</b>) Western blot analysis showing decrease in Her-2 expression in SiHa and HeLa treated with FR<b>_aq</b> (0–80 µg/ml). Tubulin was used as a loading control. (<b>G</b>) Densitometric analysis of the western blot showing fold change in HER-2 protein levels in SiHa and HeLa. The bands were quantified by densitometry using ImageJ 1.44p (Wayne Rasband, National Institutes of Health, USA, <a href="http://imagej.nih.gov/ij" target="_blank">http://imagej.nih.gov/ij</a>).</p