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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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Cell cycle analysis of HepG2 cancer cells treated with oleanolic acid. The distribution of cells undergoing apoptosis and in various phases of the cell cycle was determined in HepG2 cells following treatment with oleanolic acid at various concentrations. (A) G0/G1–5%, S-30%, G2M-65%; (B) G0/G1–15%, S-25% G2M-60%; (C) G0/G1–38%, S-20%, G2M-42%; (D) G0/G1–55%, S-15%, G2M-30%; representing treatment with 0, 5, 25 and 50 µM, respectively. Values are presented as the mean ± standard error of mean of three determinations. PI, propidium iodide.
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f2-mmr-12-04-5012: Cell cycle analysis of HepG2 cancer cells treated with oleanolic acid. The distribution of cells undergoing apoptosis and in various phases of the cell cycle was determined in HepG2 cells following treatment with oleanolic acid at various concentrations. (A) G0/G1–5%, S-30%, G2M-65%; (B) G0/G1–15%, S-25% G2M-60%; (C) G0/G1–38%, S-20%, G2M-42%; (D) G0/G1–55%, S-15%, G2M-30%; representing treatment with 0, 5, 25 and 50 µM, respectively. Values are presented as the mean ± standard error of mean of three determinations. PI, propidium iodide.

Mentions: In order to demonstrate whether oleanolic acid induces cell cycle disturbances in HepG2 cells, flow cytometric analysis using propidium iodide as a staining agent was performed, following oleanolic acid treatment at various concentrations (5, 25 and 50 µM) for 24 h. As shown in Fig. 2, following treatment with oleanolic acid at 0, 5, 25 and 50 µM concentrations for 24 h, significant G0/G1 cell cycle growth arrest was observed, while the number of cells in the S and G2-M phases was markedly reduced. At a low concentration of oleanolic acid, apoptosis was not observed. However, at 25 and 50 µM concentrations, the fraction of cells undergoing apoptosis increased significantly up to 31.7 and 54.2% compared with untreated cells at 4.5%.


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)

Cell cycle analysis of HepG2 cancer cells treated with oleanolic acid. The distribution of cells undergoing apoptosis and in various phases of the cell cycle was determined in HepG2 cells following treatment with oleanolic acid at various concentrations. (A) G0/G1–5%, S-30%, G2M-65%; (B) G0/G1–15%, S-25% G2M-60%; (C) G0/G1–38%, S-20%, G2M-42%; (D) G0/G1–55%, S-15%, G2M-30%; representing treatment with 0, 5, 25 and 50 µM, respectively. Values are presented as the mean ± standard error of mean of three determinations. PI, propidium iodide.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f2-mmr-12-04-5012: Cell cycle analysis of HepG2 cancer cells treated with oleanolic acid. The distribution of cells undergoing apoptosis and in various phases of the cell cycle was determined in HepG2 cells following treatment with oleanolic acid at various concentrations. (A) G0/G1–5%, S-30%, G2M-65%; (B) G0/G1–15%, S-25% G2M-60%; (C) G0/G1–38%, S-20%, G2M-42%; (D) G0/G1–55%, S-15%, G2M-30%; representing treatment with 0, 5, 25 and 50 µM, respectively. Values are presented as the mean ± standard error of mean of three determinations. PI, propidium iodide.
Mentions: In order to demonstrate whether oleanolic acid induces cell cycle disturbances in HepG2 cells, flow cytometric analysis using propidium iodide as a staining agent was performed, following oleanolic acid treatment at various concentrations (5, 25 and 50 µM) for 24 h. As shown in Fig. 2, following treatment with oleanolic acid at 0, 5, 25 and 50 µM concentrations for 24 h, significant G0/G1 cell cycle growth arrest was observed, while the number of cells in the S and G2-M phases was markedly reduced. At a low concentration of oleanolic acid, apoptosis was not observed. However, at 25 and 50 µM concentrations, the fraction of cells undergoing apoptosis increased significantly up to 31.7 and 54.2% compared with untreated cells at 4.5%.

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