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β-Elemene Inhibits Cell Proliferation by Regulating the Expression and Activity of Topoisomerases I and IIα in Human Hepatocarcinoma HepG-2 Cells.

Gong M, Liu Y, Zhang J, Gao YJ, Zhai PP, Su X, Li X, Li Y, Hou L, Cui XN - Biomed Res Int (2015)

Bottom Line: To investigate the effects of β-Elemene (β-ELE) on the proliferation, apoptosis, and topoisomerase I (TOPO I) and topoisomerase IIα (TOPO IIα) expression and activity of human hepatocarcinoma HepG-2 cells.After treatment with β-ELE, morphological alterations of HepG-2 cells were observed under an inverted microscope.Supercoiled pBR322 and kDNA were also used to determine the direct effect of β-ELE on DNA breaks. β-ELE significantly inhibited HepG-2 cell proliferation in a dose- and time-dependent manner. β-ELE also induced tumor cell arrest at S phase, induced cell apoptosis, and downregulated the protein expression of TOPO I and TOPO IIα in a dose-dependent manner. β-ELE also inhibited TOPO I- and TOPO IIα-mediated DNA relaxation but did not directly induce DNA breakage at any concentration. β-ELE could inhibit the proliferation of HepG-2 cells and interfere with the expression and activity of TOPO I and TOPO IIα.

View Article: PubMed Central - PubMed

Affiliation: Department of Oncology, The First Affiliated Hospital of Dalian Medical University, Dalian, Liaoning 116011, China.

ABSTRACT

Objective: To investigate the effects of β-Elemene (β-ELE) on the proliferation, apoptosis, and topoisomerase I (TOPO I) and topoisomerase IIα (TOPO IIα) expression and activity of human hepatocarcinoma HepG-2 cells.

Methods: After treatment with β-ELE, morphological alterations of HepG-2 cells were observed under an inverted microscope. Cell proliferation was assessed using an MTT assay, cell cycles were analyzed using flow cytometry, and apoptosis was detected by Annexin V/PI staining. The expression of TOPO I and TOPO IIα was analyzed by Western blot techniques, and their activity was measured using the TOPO I-mediated, supercoiled pBR322 DNA relaxation and TOPO IIα-mediated Kinetoplast DNA (kDNA) decatenation assays, respectively. Supercoiled pBR322 and kDNA were also used to determine the direct effect of β-ELE on DNA breaks.

Results: β-ELE significantly inhibited HepG-2 cell proliferation in a dose- and time-dependent manner. β-ELE also induced tumor cell arrest at S phase, induced cell apoptosis, and downregulated the protein expression of TOPO I and TOPO IIα in a dose-dependent manner. β-ELE also inhibited TOPO I- and TOPO IIα-mediated DNA relaxation but did not directly induce DNA breakage at any concentration.

Conclusion: β-ELE could inhibit the proliferation of HepG-2 cells and interfere with the expression and activity of TOPO I and TOPO IIα.

No MeSH data available.


Related in: MedlinePlus

TOPO IIα catalytic activity inhibited by β-ELE. (a) The effect of β-ELE (β-Elemene) on the decatenation of TOPO IIα-mediated kDNA. As shown in (a), lane 1 is kDNA and served as a control group; lane 2 is the free DNA minicircles that were produced by the decatenation of TOPO IIα, in this case, without β-ELE. Lanes 3 and 4 are the positive control groups. VP16 (a typical inhibitor of TOPO IIα) at a concentration of 5 μg/mL had no inhibitory effect on the DNA decatenation activity of TOPO IIα, whereas DNA minicircles disappeared at a concentration of 1 μg/μL, which suggests that VP16 inhibits the catalytic activity of TOPO IIα. Lane 5 shows the effect of 1 μg/μL VP16 with 40 μg/mL β-ELE on the decatenation of TOPO IIα-mediated kDNA. As shown in the figure, the activity of TOPO IIα was inhibited. Lanes 6 to 11 show the effects of different concentrations (40, 60, 80, and 100 μg/mL) of β-ELE on the decatenation of TOPO IIα-mediated kDNA. (b) β-ELE (β-Elemene) has no direct effect on kDNA. As shown in (b), DNA minicircles, which form after DNA double-strand breakage, did not appear in the figure, suggesting that β-ELE has no direct effect on DNA.
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fig7: TOPO IIα catalytic activity inhibited by β-ELE. (a) The effect of β-ELE (β-Elemene) on the decatenation of TOPO IIα-mediated kDNA. As shown in (a), lane 1 is kDNA and served as a control group; lane 2 is the free DNA minicircles that were produced by the decatenation of TOPO IIα, in this case, without β-ELE. Lanes 3 and 4 are the positive control groups. VP16 (a typical inhibitor of TOPO IIα) at a concentration of 5 μg/mL had no inhibitory effect on the DNA decatenation activity of TOPO IIα, whereas DNA minicircles disappeared at a concentration of 1 μg/μL, which suggests that VP16 inhibits the catalytic activity of TOPO IIα. Lane 5 shows the effect of 1 μg/μL VP16 with 40 μg/mL β-ELE on the decatenation of TOPO IIα-mediated kDNA. As shown in the figure, the activity of TOPO IIα was inhibited. Lanes 6 to 11 show the effects of different concentrations (40, 60, 80, and 100 μg/mL) of β-ELE on the decatenation of TOPO IIα-mediated kDNA. (b) β-ELE (β-Elemene) has no direct effect on kDNA. As shown in (b), DNA minicircles, which form after DNA double-strand breakage, did not appear in the figure, suggesting that β-ELE has no direct effect on DNA.

Mentions: KDNA is a strong network consisting of thousands of double-stranded, circular DNA. When one of the DNA minicircles is freed from the network structure, it must transfer a transiently double-stranded break to the main chain of the DNA. The decatenation assay is specific for measuring TOPO IIα activity because it is based on the conversion of catenated DNA to its decatenated form, which requires the DNA double-strand break that is uniquely performed by TOPO IIα [15]. The removal of these kDNA by the enzyme can be observed in agarose gels. In addition, with respect to TOPO IIα, the decatenation of kDNA was induced by TOPO IIα and inhibited by treatment with β-ELE, as shown in Figure 7(a). Lane 1 is the control kDNA; lane 2 is the free DNA minicircles that are produced by the decatenation of TOPO IIα, in this case, without β-ELE; and lanes 3 and 4 are the positive control groups. VP16 (a typical inhibitor of TOPO IIα) at a concentration of 5 μg/mL had no inhibitory effect on the DNA decatenation activity of TOPO IIα, whereas DNA minicircles disappeared at a concentration of 1 μg/μL, which suggested that VP16 inhibited the catalytic activity of TOPO IIα. Lane 5 shows the effect of 1 μg/μL VP16 with 40 μg/mL β-ELE on the decatenation of TOPO IIα-mediated kDNA. As shown in Figure 7(a), the activity of TOPO IIα was inhibited. Lanes 6 to 11 are the effects of different concentrations of β-ELE (40, 60, 80, and 100 μg/mL) on the decatenation of TOPO IIα-mediated kDNA.


β-Elemene Inhibits Cell Proliferation by Regulating the Expression and Activity of Topoisomerases I and IIα in Human Hepatocarcinoma HepG-2 Cells.

Gong M, Liu Y, Zhang J, Gao YJ, Zhai PP, Su X, Li X, Li Y, Hou L, Cui XN - Biomed Res Int (2015)

TOPO IIα catalytic activity inhibited by β-ELE. (a) The effect of β-ELE (β-Elemene) on the decatenation of TOPO IIα-mediated kDNA. As shown in (a), lane 1 is kDNA and served as a control group; lane 2 is the free DNA minicircles that were produced by the decatenation of TOPO IIα, in this case, without β-ELE. Lanes 3 and 4 are the positive control groups. VP16 (a typical inhibitor of TOPO IIα) at a concentration of 5 μg/mL had no inhibitory effect on the DNA decatenation activity of TOPO IIα, whereas DNA minicircles disappeared at a concentration of 1 μg/μL, which suggests that VP16 inhibits the catalytic activity of TOPO IIα. Lane 5 shows the effect of 1 μg/μL VP16 with 40 μg/mL β-ELE on the decatenation of TOPO IIα-mediated kDNA. As shown in the figure, the activity of TOPO IIα was inhibited. Lanes 6 to 11 show the effects of different concentrations (40, 60, 80, and 100 μg/mL) of β-ELE on the decatenation of TOPO IIα-mediated kDNA. (b) β-ELE (β-Elemene) has no direct effect on kDNA. As shown in (b), DNA minicircles, which form after DNA double-strand breakage, did not appear in the figure, suggesting that β-ELE has no direct effect on DNA.
© Copyright Policy - open-access
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4499621&req=5

fig7: TOPO IIα catalytic activity inhibited by β-ELE. (a) The effect of β-ELE (β-Elemene) on the decatenation of TOPO IIα-mediated kDNA. As shown in (a), lane 1 is kDNA and served as a control group; lane 2 is the free DNA minicircles that were produced by the decatenation of TOPO IIα, in this case, without β-ELE. Lanes 3 and 4 are the positive control groups. VP16 (a typical inhibitor of TOPO IIα) at a concentration of 5 μg/mL had no inhibitory effect on the DNA decatenation activity of TOPO IIα, whereas DNA minicircles disappeared at a concentration of 1 μg/μL, which suggests that VP16 inhibits the catalytic activity of TOPO IIα. Lane 5 shows the effect of 1 μg/μL VP16 with 40 μg/mL β-ELE on the decatenation of TOPO IIα-mediated kDNA. As shown in the figure, the activity of TOPO IIα was inhibited. Lanes 6 to 11 show the effects of different concentrations (40, 60, 80, and 100 μg/mL) of β-ELE on the decatenation of TOPO IIα-mediated kDNA. (b) β-ELE (β-Elemene) has no direct effect on kDNA. As shown in (b), DNA minicircles, which form after DNA double-strand breakage, did not appear in the figure, suggesting that β-ELE has no direct effect on DNA.
Mentions: KDNA is a strong network consisting of thousands of double-stranded, circular DNA. When one of the DNA minicircles is freed from the network structure, it must transfer a transiently double-stranded break to the main chain of the DNA. The decatenation assay is specific for measuring TOPO IIα activity because it is based on the conversion of catenated DNA to its decatenated form, which requires the DNA double-strand break that is uniquely performed by TOPO IIα [15]. The removal of these kDNA by the enzyme can be observed in agarose gels. In addition, with respect to TOPO IIα, the decatenation of kDNA was induced by TOPO IIα and inhibited by treatment with β-ELE, as shown in Figure 7(a). Lane 1 is the control kDNA; lane 2 is the free DNA minicircles that are produced by the decatenation of TOPO IIα, in this case, without β-ELE; and lanes 3 and 4 are the positive control groups. VP16 (a typical inhibitor of TOPO IIα) at a concentration of 5 μg/mL had no inhibitory effect on the DNA decatenation activity of TOPO IIα, whereas DNA minicircles disappeared at a concentration of 1 μg/μL, which suggested that VP16 inhibited the catalytic activity of TOPO IIα. Lane 5 shows the effect of 1 μg/μL VP16 with 40 μg/mL β-ELE on the decatenation of TOPO IIα-mediated kDNA. As shown in Figure 7(a), the activity of TOPO IIα was inhibited. Lanes 6 to 11 are the effects of different concentrations of β-ELE (40, 60, 80, and 100 μg/mL) on the decatenation of TOPO IIα-mediated kDNA.

Bottom Line: To investigate the effects of β-Elemene (β-ELE) on the proliferation, apoptosis, and topoisomerase I (TOPO I) and topoisomerase IIα (TOPO IIα) expression and activity of human hepatocarcinoma HepG-2 cells.After treatment with β-ELE, morphological alterations of HepG-2 cells were observed under an inverted microscope.Supercoiled pBR322 and kDNA were also used to determine the direct effect of β-ELE on DNA breaks. β-ELE significantly inhibited HepG-2 cell proliferation in a dose- and time-dependent manner. β-ELE also induced tumor cell arrest at S phase, induced cell apoptosis, and downregulated the protein expression of TOPO I and TOPO IIα in a dose-dependent manner. β-ELE also inhibited TOPO I- and TOPO IIα-mediated DNA relaxation but did not directly induce DNA breakage at any concentration. β-ELE could inhibit the proliferation of HepG-2 cells and interfere with the expression and activity of TOPO I and TOPO IIα.

View Article: PubMed Central - PubMed

Affiliation: Department of Oncology, The First Affiliated Hospital of Dalian Medical University, Dalian, Liaoning 116011, China.

ABSTRACT

Objective: To investigate the effects of β-Elemene (β-ELE) on the proliferation, apoptosis, and topoisomerase I (TOPO I) and topoisomerase IIα (TOPO IIα) expression and activity of human hepatocarcinoma HepG-2 cells.

Methods: After treatment with β-ELE, morphological alterations of HepG-2 cells were observed under an inverted microscope. Cell proliferation was assessed using an MTT assay, cell cycles were analyzed using flow cytometry, and apoptosis was detected by Annexin V/PI staining. The expression of TOPO I and TOPO IIα was analyzed by Western blot techniques, and their activity was measured using the TOPO I-mediated, supercoiled pBR322 DNA relaxation and TOPO IIα-mediated Kinetoplast DNA (kDNA) decatenation assays, respectively. Supercoiled pBR322 and kDNA were also used to determine the direct effect of β-ELE on DNA breaks.

Results: β-ELE significantly inhibited HepG-2 cell proliferation in a dose- and time-dependent manner. β-ELE also induced tumor cell arrest at S phase, induced cell apoptosis, and downregulated the protein expression of TOPO I and TOPO IIα in a dose-dependent manner. β-ELE also inhibited TOPO I- and TOPO IIα-mediated DNA relaxation but did not directly induce DNA breakage at any concentration.

Conclusion: β-ELE could inhibit the proliferation of HepG-2 cells and interfere with the expression and activity of TOPO I and TOPO IIα.

No MeSH data available.


Related in: MedlinePlus