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

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 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

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

Regulation of MAR binding tumor regulatory proteins by n6/n3 ratios in breast cancer and non-cancerous cells.

<p>Expression of SMAR1 and Cux/CDP proteins was analyzed in MDA-MB-231 (A, B); MCF7 (C,D) and MCF10A (E,F). The effect of low (A, C, E) and high (B, D, F) n6/n3 FA ratios has been shown in MDAMB231, MCF7 and MCF10A cell lines, respectively. The bands were quantified by densitometry using ImageJ 1.44p (National Institutes of Health, USA, <a href="http://imagej.nih.gov/ij" target="_blank">http://imagej.nih.gov/ij</a>) and have been presented as mean±SEM of three different experiments. Ώp<0.05, @p<0.01 and *p<0.001 compared to UC; αp<0.05, ¥p<0.01 and ¤p<0.001 compared to 1:1; ₭p<0.01and Θp<0.001 compared to 1:2.5; #p<0.05 and Øp<0.001 compared to 1:4; $p<0.05 compared to 1:5;ρp<0.05, ∞p<0.01 and Δp<0.001 compared to 2.5:1; ‡p<0.01 and Σp<0.001 compared to 4:1, μp<0.01 and πp<0.001 compared to 5:1.</p

Differential Ratios of Omega Fatty Acids (AA/EPA+DHA) Modulate Growth, Lipid Peroxidation and Expression of Tumor Regulatory MARBPs in Breast Cancer Cell Lines MCF7 and MDA-MB-231

<div><p>Omega 3 (n3) and Omega 6 (n6) polyunsaturated fatty acids (PUFAs) have been reported to exhibit opposing roles in cancer progression. Our objective was to determine whether different ratios of n6/n3 (AA/EPA+DHA) FAs could modulate the cell viability, lipid peroxidation, total cellular fatty acid composition and expression of tumor regulatory Matrix Attachment Region binding proteins (MARBPs) in breast cancer cell lines and in non-cancerous, MCF10A cells. Low ratios of n6/n3 (1:2.5, 1:4, 1:5, 1:10) FA decreased the viability and growth of MDA-MB-231 and MCF7 significantly compared to the non-cancerous cells (MCF10A). Contrarily, higher n6/n3 FA (2.5:1, 4:1, 5:1, 10:1) decreased the survival of both the cancerous and non-cancerous cell types. Lower ratios of n6/n3 selectively induced LPO in the breast cancer cells whereas the higher ratios induced in both cancerous and non-cancerous cell types. Interestingly, compared to higher n6/n3 FA ratios, lower ratios increased the expression of tumor suppressor MARBP, SMAR1 and decreased the expression of tumor activator Cux/CDP in both breast cancer and non-cancerous, MCF10A cells. Low n6/n3 FAs significantly increased SMAR1 expression which resulted into activation of p21^WAF1/CIP1 in MDA-MB-231 and MCF7, the increase being ratio dependent in MDA-MB-231. These results suggest that increased intake of n3 fatty acids in our diet could help both in the prevention as well as management of breast cancer.</p></div

Different ratios of n6 and n3 regulate the lipid peroxidation in breast cancer and non-cancerous cells.

<p>(A) MDA-MB-231, (B) MCF7 and (C) MCF10A cells were treated with low and high n6/n3 ratios for 24h. Next day, lipid peroxidation was analyzed by using cis-parinaric acid and the values have been plotted in terms of percentage fluorescent intensity. Decrease of cis-parinaric acid fluorescence is proportional to increase in lipid peroxidation. Data has been presented as mean±SEM of three independent experiments, each conducted in triplicates. ƍp<0.01 and *p<0.001 compared to UC; ¥p<0.01 and ¤p<0.001 compared to 1:1; ₭p<0.01, Φp<0.01 and Θp<0.001 compared to 1:2.5; #p<0.05, Λp<0.01 and Øp<0.001 compared to 1:4; $p<0.05, ϒp<0.01 and ϙp<0.001 compared to 1:5; βp<0.05, ßp<0.001 as compared to 1:10; ∞p<0.01 and Δp<0.001 compared to 2.5:1; ‡p<0.01 as compared to 4:1; Ŧp<0.05 compared to 5:1; μp<0.01 compared to 5:1; €p<0.01 compared to 10:1.</p

Effect of different concentrations of EPA, DHA and AA on viability of non-cancerous transformed cells.

<p>(A) MCF10A, (B) HEK293 and (C) HaCaT cells were exposed to different concentrations (0–320μM) of either EPA, DHA, AA and MTT assay was performed. Data has been presented as mean±SEM of three independent experiments, performed in 96 well plates. Statistical significance was assayed by one-way ANOVA, followed by a Dunnett's test. ***p<0.01.</p

Differential ratios of n6 and n3 fatty acids regulate the viability of breast cancer and non-cancerous cells.

<p>Breast cancer cell lines, (A) MDA-MB-231 and (B) MCF7 as well as immortalized non-tumorigenic human breast epithelial, MCF10A cells (C) were treated with low and high n6/n3 ratios and analyzed for cell viability by MTT assay. Data has been presented as mean±SEM of five independent experiments, each conducted in triplicates. ƍp<0.01 and *p<0.001 compared to UC; ¥p<0.01 and ¤p<0.001 compared to 1:1; Θp<0.001 compared to 1:2.5; #p<0.05, Λp<0.01 and Øp<0.001 compared to 1:4;ϒp<0.01 and ϙp<0.001 compared to 1:5; βp<0.05, ×p<0.01 and ßp<0.001 compared to 1:10; ∞p<0.01 and Δp<0.001 compared to 2.5:1.</p