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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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Gel electrophoresis of DNA samples isolated from HepG2 cancer cells following treatment with oleanolic acid. Cells were incubated with various concentrations of oleanolic acid for 72 h. M, 100 bp DNA ladder marker; lane A, HepG2 cells treated with 5 µM of oleanolic acid; lane B, HepG2 cells treated with 25 µM of oleanolic acid; and lane C, HepG2 cells treated with 50 µM of oleanolic acid. Each experiment was conducted in triplicate. DNA fragments were separated using 1.5% agarose gel electrophoresis and visualized under UV light following staining with ethidium bromide.
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f7-mmr-12-04-5012: Gel electrophoresis of DNA samples isolated from HepG2 cancer cells following treatment with oleanolic acid. Cells were incubated with various concentrations of oleanolic acid for 72 h. M, 100 bp DNA ladder marker; lane A, HepG2 cells treated with 5 µM of oleanolic acid; lane B, HepG2 cells treated with 25 µM of oleanolic acid; and lane C, HepG2 cells treated with 50 µM of oleanolic acid. Each experiment was conducted in triplicate. DNA fragments were separated using 1.5% agarose gel electrophoresis and visualized under UV light following staining with ethidium bromide.

Mentions: DNA fragmentation is a process, which damages DNA, leading to cell death that occurs via the activation of certain intrinsic agents, such as caspase 3 and 9. This cleavage produces ladders of DNA fragments that are the size of integer multiples of a nucleosome length (180–200 bp). The DNA fragmentation is initiated by caspase-3 activation of inactive caspase-activated deoxyribonuclease (CAD), through removal of its inhibitors, such as inhibitor of CAD (ICAD) (9). As a biochemical hallmark of intrinsic apoptotic cell death, DNA fragmentation was used to determine whether the anticancer effect of oleanolic acid on cells occurs via the activation of caspases, mainly caspase-3. As shown in Fig. 7, marked DNA fragmentation was observed in HepG2 cancer cells following treatment with 5, 25 and 50 µM treatments of oleanolic acid for 72 h. However, the control cells did not exhibit evident DNA laddering (data not shown). The treatment of HepG2 cells with oleanolic acid resulted in the induction of intrinsic apoptotic effects as low as 5 µM. Higher concentrations of oleanolic acid (lane 1–3; Fig. 7) for 72 h resulted in the presence of the typical features of DNA laddering on an agarose gel.


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)

Gel electrophoresis of DNA samples isolated from HepG2 cancer cells following treatment with oleanolic acid. Cells were incubated with various concentrations of oleanolic acid for 72 h. M, 100 bp DNA ladder marker; lane A, HepG2 cells treated with 5 µM of oleanolic acid; lane B, HepG2 cells treated with 25 µM of oleanolic acid; and lane C, HepG2 cells treated with 50 µM of oleanolic acid. Each experiment was conducted in triplicate. DNA fragments were separated using 1.5% agarose gel electrophoresis and visualized under UV light following staining with ethidium bromide.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f7-mmr-12-04-5012: Gel electrophoresis of DNA samples isolated from HepG2 cancer cells following treatment with oleanolic acid. Cells were incubated with various concentrations of oleanolic acid for 72 h. M, 100 bp DNA ladder marker; lane A, HepG2 cells treated with 5 µM of oleanolic acid; lane B, HepG2 cells treated with 25 µM of oleanolic acid; and lane C, HepG2 cells treated with 50 µM of oleanolic acid. Each experiment was conducted in triplicate. DNA fragments were separated using 1.5% agarose gel electrophoresis and visualized under UV light following staining with ethidium bromide.
Mentions: DNA fragmentation is a process, which damages DNA, leading to cell death that occurs via the activation of certain intrinsic agents, such as caspase 3 and 9. This cleavage produces ladders of DNA fragments that are the size of integer multiples of a nucleosome length (180–200 bp). The DNA fragmentation is initiated by caspase-3 activation of inactive caspase-activated deoxyribonuclease (CAD), through removal of its inhibitors, such as inhibitor of CAD (ICAD) (9). As a biochemical hallmark of intrinsic apoptotic cell death, DNA fragmentation was used to determine whether the anticancer effect of oleanolic acid on cells occurs via the activation of caspases, mainly caspase-3. As shown in Fig. 7, marked DNA fragmentation was observed in HepG2 cancer cells following treatment with 5, 25 and 50 µM treatments of oleanolic acid for 72 h. However, the control cells did not exhibit evident DNA laddering (data not shown). The treatment of HepG2 cells with oleanolic acid resulted in the induction of intrinsic apoptotic effects as low as 5 µM. Higher concentrations of oleanolic acid (lane 1–3; Fig. 7) for 72 h resulted in the presence of the typical features of DNA laddering on an agarose gel.

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