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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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(A) Effect of oleanolic acid on the mitochondrial ΔΨm of HepG2 cells. Cells treated with oleanolic acid for 48 h were incubated with rhodamine-123 and measured using flow cytometry. The percentages of cells in the right section of the histogram indicate the number of ΔΨm-collapsed cells. (B) Percentage loss of ΔΨm in the oleanolic acid-treated cells. ΔΨm, membrane potential.
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f8-mmr-12-04-5012: (A) Effect of oleanolic acid on the mitochondrial ΔΨm of HepG2 cells. Cells treated with oleanolic acid for 48 h were incubated with rhodamine-123 and measured using flow cytometry. The percentages of cells in the right section of the histogram indicate the number of ΔΨm-collapsed cells. (B) Percentage loss of ΔΨm in the oleanolic acid-treated cells. ΔΨm, membrane potential.

Mentions: An important and indicative stage in the intrinsic apoptotic pathway is the depolarization of the mitochondrial membrane and the subsequent increase in permeability of the outer membrane, following pore formation. This is accompanied by the release of proapoptotic molecules and cytochrome c. The fluorescent dye, rhodamine-123, is a specific probe for the detection of alterations in ΔΨm in living cells. The present results demonstrated that oleanolic acid induced a significant reduction in the number of cells with an intact membrane potential and increased the number of cells with low ΔΨm after 48 h. Loss of ΔΨm is an essential event in the mitochondrial pathway of apoptosis. The ΔΨm level was observed to be reduced following treatment with increasing concentrations of oleanolic acid in HepG2 cells over 48 h (Fig. 8A and B). Disruption of ΔΨm was relatively low in untreated 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)

(A) Effect of oleanolic acid on the mitochondrial ΔΨm of HepG2 cells. Cells treated with oleanolic acid for 48 h were incubated with rhodamine-123 and measured using flow cytometry. The percentages of cells in the right section of the histogram indicate the number of ΔΨm-collapsed cells. (B) Percentage loss of ΔΨm in the oleanolic acid-treated cells. ΔΨm, membrane potential.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f8-mmr-12-04-5012: (A) Effect of oleanolic acid on the mitochondrial ΔΨm of HepG2 cells. Cells treated with oleanolic acid for 48 h were incubated with rhodamine-123 and measured using flow cytometry. The percentages of cells in the right section of the histogram indicate the number of ΔΨm-collapsed cells. (B) Percentage loss of ΔΨm in the oleanolic acid-treated cells. ΔΨm, membrane potential.
Mentions: An important and indicative stage in the intrinsic apoptotic pathway is the depolarization of the mitochondrial membrane and the subsequent increase in permeability of the outer membrane, following pore formation. This is accompanied by the release of proapoptotic molecules and cytochrome c. The fluorescent dye, rhodamine-123, is a specific probe for the detection of alterations in ΔΨm in living cells. The present results demonstrated that oleanolic acid induced a significant reduction in the number of cells with an intact membrane potential and increased the number of cells with low ΔΨm after 48 h. Loss of ΔΨm is an essential event in the mitochondrial pathway of apoptosis. The ΔΨm level was observed to be reduced following treatment with increasing concentrations of oleanolic acid in HepG2 cells over 48 h (Fig. 8A and B). Disruption of ΔΨm was relatively low in untreated 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