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Free Radical Scavenging Activity: Antiproliferative and Proteomics Analyses of the Differential Expression of Apoptotic Proteins in MCF-7 Cells Treated with Acetone Leaf Extract of Diospyros lycioides (Ebenaceae).

Pilane MC, Bagla VP, Mokgotho MP, Mbazima V, Matsebatlela TM, Ncube I, Mampuru L - Evid Based Complement Alternat Med (2015)

Bottom Line: The acetone leaf extract of Diospyros lycioides was evaluated qualitatively and quantitatively for its antioxidant potential using DPPH assay and nitric oxide radical scavenging effect, while the viability of MCF-7 cells was evaluated using the MTT.Cytotoxicity was demonstrated to be due to apoptosis, with 70% of the extract-treated cells being annexin-V-positive/PI negative at 48 hours.The extract was also shown to upregulate the expression of p53 gene with concomitant downregulation of the Bcl-2 antiapoptotic gene while differentially expressed proteins were identified as enolase, pyruvate kinase, and glyceraldehyde-3-phosphate.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry, Microbiology and Biotechnology, Faculty of Science and Agriculture, University of Limpopo, Turfloop Campus, Private Bag X1106, Sovenga, Limpopo 0727, South Africa.

ABSTRACT
Breast cancer is the most common cancer in South Africa. The acetone leaf extract of Diospyros lycioides was evaluated qualitatively and quantitatively for its antioxidant potential using DPPH assay and nitric oxide radical scavenging effect, while the viability of MCF-7 cells was evaluated using the MTT. MCF-7 treated cells were stained with Hoechst 335258 dye and annexin-V-FITC to be evaluated for apoptotic effect of the extract, while mRNA expression levels of apoptotic genes were assessed by quantitative real-time PCR and deferential protein expression levels using 2D gel electrophoresis and mass spectrometry. Results revealed presence of antioxidant constituents in the extract. Extract was shown to be cytotoxic in a concentration- and time-dependent manner. Cytotoxicity was demonstrated to be due to apoptosis, with 70% of the extract-treated cells being annexin-V-positive/PI negative at 48 hours. The extract was also shown to upregulate the expression of p53 gene with concomitant downregulation of the Bcl-2 antiapoptotic gene while differentially expressed proteins were identified as enolase, pyruvate kinase, and glyceraldehyde-3-phosphate. The extract in this study was shown to induce apoptosis at an early stage which makes it an ideal source that can be explored for compounds that may be used in the treatment and management of cancer.

No MeSH data available.


Related in: MedlinePlus

Percentage of apoptotic MCF-7 cells after treatment with 65 μg/mL of the extract for 24 and 48 h. The 0 μg/mL represents untreated cells, while curcumin (7.36 μg/mL) represents the positive control.
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fig8: Percentage of apoptotic MCF-7 cells after treatment with 65 μg/mL of the extract for 24 and 48 h. The 0 μg/mL represents untreated cells, while curcumin (7.36 μg/mL) represents the positive control.

Mentions: To quantitatively ascertain the percentage of cells within the population that were actively undergoing apoptosis, untreated and treated MCF-7 cells were stained using annexin V-FITC/propidium iodide (PI) and evaluated by flow cytometry. As shown in Figures 6(a) and 7(a), only a small percentage of untreated cells were annexin V-FITC positive after 24 and 48 h of incubation. In contrast, the percentage of annexin V-FITC positive cells increased after treatment with 65 μg/mL of the extract after 24 h (Figure 6(b)) and 48 h (Figure 7(b)) of incubation. The results demonstrated that the extract induced apoptosis of MCF-7 cells in a time-dependent manner. A graphical representation of the percentage of annexin V-FITC positive cells after treatment with 65 μg/mL of extract for 24 and 48h is presented in Figure 8.


Free Radical Scavenging Activity: Antiproliferative and Proteomics Analyses of the Differential Expression of Apoptotic Proteins in MCF-7 Cells Treated with Acetone Leaf Extract of Diospyros lycioides (Ebenaceae).

Pilane MC, Bagla VP, Mokgotho MP, Mbazima V, Matsebatlela TM, Ncube I, Mampuru L - Evid Based Complement Alternat Med (2015)

Percentage of apoptotic MCF-7 cells after treatment with 65 μg/mL of the extract for 24 and 48 h. The 0 μg/mL represents untreated cells, while curcumin (7.36 μg/mL) represents the positive control.
© Copyright Policy - open-access
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC4589632&req=5

fig8: Percentage of apoptotic MCF-7 cells after treatment with 65 μg/mL of the extract for 24 and 48 h. The 0 μg/mL represents untreated cells, while curcumin (7.36 μg/mL) represents the positive control.
Mentions: To quantitatively ascertain the percentage of cells within the population that were actively undergoing apoptosis, untreated and treated MCF-7 cells were stained using annexin V-FITC/propidium iodide (PI) and evaluated by flow cytometry. As shown in Figures 6(a) and 7(a), only a small percentage of untreated cells were annexin V-FITC positive after 24 and 48 h of incubation. In contrast, the percentage of annexin V-FITC positive cells increased after treatment with 65 μg/mL of the extract after 24 h (Figure 6(b)) and 48 h (Figure 7(b)) of incubation. The results demonstrated that the extract induced apoptosis of MCF-7 cells in a time-dependent manner. A graphical representation of the percentage of annexin V-FITC positive cells after treatment with 65 μg/mL of extract for 24 and 48h is presented in Figure 8.

Bottom Line: The acetone leaf extract of Diospyros lycioides was evaluated qualitatively and quantitatively for its antioxidant potential using DPPH assay and nitric oxide radical scavenging effect, while the viability of MCF-7 cells was evaluated using the MTT.Cytotoxicity was demonstrated to be due to apoptosis, with 70% of the extract-treated cells being annexin-V-positive/PI negative at 48 hours.The extract was also shown to upregulate the expression of p53 gene with concomitant downregulation of the Bcl-2 antiapoptotic gene while differentially expressed proteins were identified as enolase, pyruvate kinase, and glyceraldehyde-3-phosphate.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry, Microbiology and Biotechnology, Faculty of Science and Agriculture, University of Limpopo, Turfloop Campus, Private Bag X1106, Sovenga, Limpopo 0727, South Africa.

ABSTRACT
Breast cancer is the most common cancer in South Africa. The acetone leaf extract of Diospyros lycioides was evaluated qualitatively and quantitatively for its antioxidant potential using DPPH assay and nitric oxide radical scavenging effect, while the viability of MCF-7 cells was evaluated using the MTT. MCF-7 treated cells were stained with Hoechst 335258 dye and annexin-V-FITC to be evaluated for apoptotic effect of the extract, while mRNA expression levels of apoptotic genes were assessed by quantitative real-time PCR and deferential protein expression levels using 2D gel electrophoresis and mass spectrometry. Results revealed presence of antioxidant constituents in the extract. Extract was shown to be cytotoxic in a concentration- and time-dependent manner. Cytotoxicity was demonstrated to be due to apoptosis, with 70% of the extract-treated cells being annexin-V-positive/PI negative at 48 hours. The extract was also shown to upregulate the expression of p53 gene with concomitant downregulation of the Bcl-2 antiapoptotic gene while differentially expressed proteins were identified as enolase, pyruvate kinase, and glyceraldehyde-3-phosphate. The extract in this study was shown to induce apoptosis at an early stage which makes it an ideal source that can be explored for compounds that may be used in the treatment and management of cancer.

No MeSH data available.


Related in: MedlinePlus