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

Karimian H, Fadaeinasab M, Zorofchian Moghadamtousi S, Hajrezaei M, Razavi M, Safi SZ, Ameen Abdulla M, Mohd Ali H, Ibrahim Noordin M - PLoS ONE (2015)

Bottom Line: Ferulago angulata leaf hexane extract (FALHE) was found to be a potent inducer of MCF7 cell apoptosis.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 F. angulata chemical constituents as breast cancer chemopreventive agents.

View Article: PubMed Central - PubMed

Affiliation: Department of Pharmacy, Faculty of Medicine, University of Malaya, Kuala Lumpur, Malaysia.

ABSTRACT
Ferulago angulata 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 in vivo 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 in vivo 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 in vitro 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 F. angulata chemical constituents as breast cancer chemopreventive agents.

No MeSH data available.


Related in: MedlinePlus

Time-dependent apoptosis rates of IC50 concentration of polycerasoidin-treated MCF7 cells after (B) 12, (C) 24 and (D) 48 h.(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 *P<0.05.
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pone.0127434.g009: Time-dependent apoptosis rates of IC50 concentration of polycerasoidin-treated MCF7 cells after (B) 12, (C) 24 and (D) 48 h.(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 *P<0.05.

Mentions: To determine whether the cytotoxic effect of polycerasoidin is via apoptosis induction, we performed an Annexin-V-FITC/PI double staining assay using a flow cytometer system. Apoptotic programmed cell death is accompanied by several biochemical characterizations, including changes in the plasma membrane asymmetry as an early feature of apoptosis [26]. Thus, Annexin-V-FITC, a recombinant probe for phosphatidylserine externalization, was employed to determine the effect of polycerasoidin on the induction of early apoptosis (Annexin-V-FITC+, PI-). The DNA-binding ability of PI was employed to detect the incidence of secondary apoptosis (Annexin-V-FITC+, PI+) and necrosis (Annexin-V-FITC+, PI+) in the MCF7 cells treated with polycerasoidin. As shown in Fig 9, the number of viable MCF7 cells (Annexin-V-FITC-, PI-) was time-dependently decreased after exposure to an IC50 dose of polycerasoidin. This was accompanied by significant accumulations of early and late apoptotic populations after 12 h. After 24 h of treatment, the percentage of early apoptotic cells was decreased. However, the number of necrotic cells, which represented the percentage of dead cells, showed significant elevation after 48 h as a result of a long exposure of MCF7 cells to polycerasoidin. These findings showed that the cytotoxic effect of polycerasoidin toward MCF7 cells is mediated via the induction of apoptosis.


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

Karimian H, Fadaeinasab M, Zorofchian Moghadamtousi S, Hajrezaei M, Razavi M, Safi SZ, Ameen Abdulla M, Mohd Ali H, Ibrahim Noordin M - PLoS ONE (2015)

Time-dependent apoptosis rates of IC50 concentration of polycerasoidin-treated MCF7 cells after (B) 12, (C) 24 and (D) 48 h.(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 *P<0.05.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4440818&req=5

pone.0127434.g009: Time-dependent apoptosis rates of IC50 concentration of polycerasoidin-treated MCF7 cells after (B) 12, (C) 24 and (D) 48 h.(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 *P<0.05.
Mentions: To determine whether the cytotoxic effect of polycerasoidin is via apoptosis induction, we performed an Annexin-V-FITC/PI double staining assay using a flow cytometer system. Apoptotic programmed cell death is accompanied by several biochemical characterizations, including changes in the plasma membrane asymmetry as an early feature of apoptosis [26]. Thus, Annexin-V-FITC, a recombinant probe for phosphatidylserine externalization, was employed to determine the effect of polycerasoidin on the induction of early apoptosis (Annexin-V-FITC+, PI-). The DNA-binding ability of PI was employed to detect the incidence of secondary apoptosis (Annexin-V-FITC+, PI+) and necrosis (Annexin-V-FITC+, PI+) in the MCF7 cells treated with polycerasoidin. As shown in Fig 9, the number of viable MCF7 cells (Annexin-V-FITC-, PI-) was time-dependently decreased after exposure to an IC50 dose of polycerasoidin. This was accompanied by significant accumulations of early and late apoptotic populations after 12 h. After 24 h of treatment, the percentage of early apoptotic cells was decreased. However, the number of necrotic cells, which represented the percentage of dead cells, showed significant elevation after 48 h as a result of a long exposure of MCF7 cells to polycerasoidin. These findings showed that the cytotoxic effect of polycerasoidin toward MCF7 cells is mediated via the induction of apoptosis.

Bottom Line: Ferulago angulata leaf hexane extract (FALHE) was found to be a potent inducer of MCF7 cell apoptosis.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 F. angulata chemical constituents as breast cancer chemopreventive agents.

View Article: PubMed Central - PubMed

Affiliation: Department of Pharmacy, Faculty of Medicine, University of Malaya, Kuala Lumpur, Malaysia.

ABSTRACT
Ferulago angulata 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 in vivo 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 in vivo 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 in vitro 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 F. angulata chemical constituents as breast cancer chemopreventive agents.

No MeSH data available.


Related in: MedlinePlus