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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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Morphological observation following acridine orange and ethidium bromide staining (magnification, ×400). HepG2 cells were treated (A) without and (B) with oleanolic acid at 5 µM, (C) 25 µM and (D) 50 µM for 48 h. Arrows indicate nuclei size and membrane integrity.
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f6-mmr-12-04-5012: Morphological observation following acridine orange and ethidium bromide staining (magnification, ×400). HepG2 cells were treated (A) without and (B) with oleanolic acid at 5 µM, (C) 25 µM and (D) 50 µM for 48 h. Arrows indicate nuclei size and membrane integrity.

Mentions: Furthermore, AO and EB double staining was conducted in the HepG2 cells in order to observe cell apoptosis, with the assistance of a fluorescence microscope. Following staining with a mixture of AO and EB, viable cells (0 µM; Fig. 6A) were observed to have large green nuclei, indicating that their cell membranes had remained intact. However, when treated with 5 or 25 µM of oleanolic acid, the number of cells with large green nuclei reduced significantly (Fig. 6). Furthermore, at a concentration of 50 µM, almost all cells exhibited signs of nuclear condensation and apoptotic body formation.


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)

Morphological observation following acridine orange and ethidium bromide staining (magnification, ×400). HepG2 cells were treated (A) without and (B) with oleanolic acid at 5 µM, (C) 25 µM and (D) 50 µM for 48 h. Arrows indicate nuclei size and membrane integrity.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f6-mmr-12-04-5012: Morphological observation following acridine orange and ethidium bromide staining (magnification, ×400). HepG2 cells were treated (A) without and (B) with oleanolic acid at 5 µM, (C) 25 µM and (D) 50 µM for 48 h. Arrows indicate nuclei size and membrane integrity.
Mentions: Furthermore, AO and EB double staining was conducted in the HepG2 cells in order to observe cell apoptosis, with the assistance of a fluorescence microscope. Following staining with a mixture of AO and EB, viable cells (0 µM; Fig. 6A) were observed to have large green nuclei, indicating that their cell membranes had remained intact. However, when treated with 5 or 25 µM of oleanolic acid, the number of cells with large green nuclei reduced significantly (Fig. 6). Furthermore, at a concentration of 50 µM, almost all cells exhibited signs of nuclear condensation and apoptotic body formation.

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