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Anticancer and apoptotic activities of oleanolic acid are mediated through cell cycle arrest and disruption of mitochondrial membrane potential in HepG2 human hepatocellular carcinoma cells.

Zhu YY, Huang HY, Wu YL - Mol Med Rep (2015)

Bottom Line: Following acridine orange and ethidium bromide staining, treatment with various doses (0, 5, 25 and 50 µM) of oleanolic acid induced typical morphological changes associated with apoptosis, including cell shrinkage, membrane blebbing, nuclear condensation and apoptotic body formation.Oleanolic acid treatment also resulted in fragmentation of nuclear DNA in a dose‑dependent manner, producing the typical features of DNA laddering on an agarose gel.The results also demonstrated that oleanolic acid treatment resulted in a potent loss of mitochondrial membrane potential, which also occurred in a dose‑dependent manner.

View Article: PubMed Central - PubMed

Affiliation: Liver Center, The First Affiliated Hospital of Fujian Medical University, Fuzhou, Fujian 350005, P.R. China.

ABSTRACT
Hepatocellular carcinoma (HCC) is an aggressive form of cancer, with high rates of morbidity and mortality, a poor prognosis and limited therapeutic options. The objective of the present study was to demonstrate the anticancer activity of oleanolic acid in HepG2 human HCC cells. Cell viability was evaluated using an MTT assay, following administration of various doses of oleanolic acid. The effect of oleanolic acid on cell cycle phase distribution and mitochondrial membrane potential was evaluated using flow cytometry with propidium iodide and rhodamine‑123 DNA‑binding cationic fluorescent dyes. Fluorescence microscopy was employed to detect morphological changes in HepG2 cells following oleanolic acid treatment. The results revealed that oleanolic acid induced a dose‑dependent, as well as time‑dependent inhibition in the growth of HepG2 cancer cells. Following acridine orange and ethidium bromide staining, treatment with various doses (0, 5, 25 and 50 µM) of oleanolic acid induced typical morphological changes associated with apoptosis, including cell shrinkage, membrane blebbing, nuclear condensation and apoptotic body formation. Cell cycle analysis revealed that oleanolic acid induced cell cycle arrest in HepG2 cells at the sub‑G1 (apoptotic) phase of the cell cycle, in a dose‑dependent manner. Staining with Annexin V‑fluorescein isothiocyanate and propidium iodide revealed that apoptosis occurred early in these cells. Oleanolic acid treatment also resulted in fragmentation of nuclear DNA in a dose‑dependent manner, producing the typical features of DNA laddering on an agarose gel. The results also demonstrated that oleanolic acid treatment resulted in a potent loss of mitochondrial membrane potential, which also occurred in a dose‑dependent manner. Therefore, oleanolic acid may be used as a therapeutic agent in the treatment of human HCC.

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Apoptotic analysis of HepG2 cells treated with oleanolic acid at various concentrations. (A–D) represent treatment with 0, 5, 25 and 50 µM oleanolic acid, respectively. The distribution of cells undergoing early and late apoptosis, together with cells not undergoing apoptosis (intact cell) and the total extent of apoptosis, was determined in HepG2 cells, following treatment with oleanolic acid, using Annexin V/fluorescein isothiocyanate and propidium iodide flow cytometric analysis. *P<0.05, compared with control.
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f4-mmr-12-04-5012: Apoptotic analysis of HepG2 cells treated with oleanolic acid at various concentrations. (A–D) represent treatment with 0, 5, 25 and 50 µM oleanolic acid, respectively. The distribution of cells undergoing early and late apoptosis, together with cells not undergoing apoptosis (intact cell) and the total extent of apoptosis, was determined in HepG2 cells, following treatment with oleanolic acid, using Annexin V/fluorescein isothiocyanate and propidium iodide flow cytometric analysis. *P<0.05, compared with control.

Mentions: An essential feature of apoptosis is the flipping of phosphatidyl serine (PS) from the inner surface to outer surface of the plasma membrane of the cells. PS is a phospholipid component usually positioned on the cytoplasmic surface of the cell membrane in viable normal cells. When an apoptotic event is induced in a cell, PS is no longer restricted to the cytosolic region of the membrane, but is exposed on the cell surface. As such, PS translocation is considered to be a biochemical marker of apoptosis. Annexin V staining is able to detect PS and may therefore be used for apoptosis analysis. When cells are stained with Annexin V in tandem with propidium iodide, this reagent enters the cell only once the plasma cell membrane has deteriorated. In the current study, flow cytometric analysis revealed that a higher number of Annexin V-positive cells were present in the oleanolic acid-treated HepG2 cells than in the control group (Figs. 3 and 4). The percentage of viable cells was low in the samples treated with lower concentration of oleanolic acid. However, at higher doses (25 and 50 µM), the total number of apoptotic cells significantly increased. The present study confirmed that oleanolic acid induces apoptosis in HepG2 cells.


Anticancer and apoptotic activities of oleanolic acid are mediated through cell cycle arrest and disruption of mitochondrial membrane potential in HepG2 human hepatocellular carcinoma cells.

Zhu YY, Huang HY, Wu YL - Mol Med Rep (2015)

Apoptotic analysis of HepG2 cells treated with oleanolic acid at various concentrations. (A–D) represent treatment with 0, 5, 25 and 50 µM oleanolic acid, respectively. The distribution of cells undergoing early and late apoptosis, together with cells not undergoing apoptosis (intact cell) and the total extent of apoptosis, was determined in HepG2 cells, following treatment with oleanolic acid, using Annexin V/fluorescein isothiocyanate and propidium iodide flow cytometric analysis. *P<0.05, compared with control.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4581805&req=5

f4-mmr-12-04-5012: Apoptotic analysis of HepG2 cells treated with oleanolic acid at various concentrations. (A–D) represent treatment with 0, 5, 25 and 50 µM oleanolic acid, respectively. The distribution of cells undergoing early and late apoptosis, together with cells not undergoing apoptosis (intact cell) and the total extent of apoptosis, was determined in HepG2 cells, following treatment with oleanolic acid, using Annexin V/fluorescein isothiocyanate and propidium iodide flow cytometric analysis. *P<0.05, compared with control.
Mentions: An essential feature of apoptosis is the flipping of phosphatidyl serine (PS) from the inner surface to outer surface of the plasma membrane of the cells. PS is a phospholipid component usually positioned on the cytoplasmic surface of the cell membrane in viable normal cells. When an apoptotic event is induced in a cell, PS is no longer restricted to the cytosolic region of the membrane, but is exposed on the cell surface. As such, PS translocation is considered to be a biochemical marker of apoptosis. Annexin V staining is able to detect PS and may therefore be used for apoptosis analysis. When cells are stained with Annexin V in tandem with propidium iodide, this reagent enters the cell only once the plasma cell membrane has deteriorated. In the current study, flow cytometric analysis revealed that a higher number of Annexin V-positive cells were present in the oleanolic acid-treated HepG2 cells than in the control group (Figs. 3 and 4). The percentage of viable cells was low in the samples treated with lower concentration of oleanolic acid. However, at higher doses (25 and 50 µM), the total number of apoptotic cells significantly increased. The present study confirmed that oleanolic acid induces apoptosis in HepG2 cells.

Bottom Line: Following acridine orange and ethidium bromide staining, treatment with various doses (0, 5, 25 and 50 µM) of oleanolic acid induced typical morphological changes associated with apoptosis, including cell shrinkage, membrane blebbing, nuclear condensation and apoptotic body formation.Oleanolic acid treatment also resulted in fragmentation of nuclear DNA in a dose‑dependent manner, producing the typical features of DNA laddering on an agarose gel.The results also demonstrated that oleanolic acid treatment resulted in a potent loss of mitochondrial membrane potential, which also occurred in a dose‑dependent manner.

View Article: PubMed Central - PubMed

Affiliation: Liver Center, The First Affiliated Hospital of Fujian Medical University, Fuzhou, Fujian 350005, P.R. China.

ABSTRACT
Hepatocellular carcinoma (HCC) is an aggressive form of cancer, with high rates of morbidity and mortality, a poor prognosis and limited therapeutic options. The objective of the present study was to demonstrate the anticancer activity of oleanolic acid in HepG2 human HCC cells. Cell viability was evaluated using an MTT assay, following administration of various doses of oleanolic acid. The effect of oleanolic acid on cell cycle phase distribution and mitochondrial membrane potential was evaluated using flow cytometry with propidium iodide and rhodamine‑123 DNA‑binding cationic fluorescent dyes. Fluorescence microscopy was employed to detect morphological changes in HepG2 cells following oleanolic acid treatment. The results revealed that oleanolic acid induced a dose‑dependent, as well as time‑dependent inhibition in the growth of HepG2 cancer cells. Following acridine orange and ethidium bromide staining, treatment with various doses (0, 5, 25 and 50 µM) of oleanolic acid induced typical morphological changes associated with apoptosis, including cell shrinkage, membrane blebbing, nuclear condensation and apoptotic body formation. Cell cycle analysis revealed that oleanolic acid induced cell cycle arrest in HepG2 cells at the sub‑G1 (apoptotic) phase of the cell cycle, in a dose‑dependent manner. Staining with Annexin V‑fluorescein isothiocyanate and propidium iodide revealed that apoptosis occurred early in these cells. Oleanolic acid treatment also resulted in fragmentation of nuclear DNA in a dose‑dependent manner, producing the typical features of DNA laddering on an agarose gel. The results also demonstrated that oleanolic acid treatment resulted in a potent loss of mitochondrial membrane potential, which also occurred in a dose‑dependent manner. Therefore, oleanolic acid may be used as a therapeutic agent in the treatment of human HCC.

Show MeSH
Related in: MedlinePlus