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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 I catalytic activity is inhibited by β-ELE. (a) The effect of β-ELE (β-Elemene) on the relaxation of TOPO I-mediated, negative supercoiled pBR322 DNA. As shown in (a), lane 1 is negative supercoiled pBR322 DNA (supercoiled DNA, S); lane 2 is relaxed DNA that is the product of supercoiled DNA reacted with the enzyme (relaxed DNA, R); and lanes 3 and 4 are positive control groups. HCPT (a typical inhibitor of TOPO I) had no inhibitory effect on the DNA relaxation activity of TOPO I at the concentration of 5 μg/mL, whereas it completely inhibited the relaxation of pBR322 DNA mediated by TOPO I at a concentration of 1 μg/μL. Lane 5 shows the effect of 1 μg/μL HCPT combined with 40 μg/mL β-ELE on the relaxation of TOPO I-mediated negative supercoiled pBR322 DNA. As shown in (a), the combination inhibited the relaxation activity of TOPO I. Lanes 6 to 11 show the effects of different concentrations of β-ELE (40, 60, 80, and 100 μg/mL) on the relaxation of negative supercoiled pBR322 DNA mediated by TOPO I. β-ELE has no inhibitory effect on the relaxation activity of TOPO I at concentrations of 10 and 20 μg/mL; however, with increasing drug concentration, β-ELE showed an increasing inhibitory effect on the DNA relaxation activity of TOPO I at concentrations of 40, 60, 80, and 100 μg/mL. The OD of MAX was 58 ± 3, 80 ± 6, 92 ± 10, and 134 ± 12. The statistical analysis showed significant differences between the 100 μg/mL and 40 μg/mL treatment groups and the 60 μg/mL and 80 μg/mL treatment groups (P < 0.05). (b) β-ELE (β-Elemene) has no direct effect on pBR322 DNA. As shown in (b), the average OD of the control group was 22860 ± 2412, and those of the β-ELE treatment groups were 24572 ± 518, 22318 ± 651, 22781 ± 837, 20781 ± 1180, and 24284 ± 749 for 20, 40, 60, 80, and 100 μg/mL of β-ELE, respectively. There was no significant difference between the groups (P > 0.05).
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fig6: TOPO I catalytic activity is inhibited by β-ELE. (a) The effect of β-ELE (β-Elemene) on the relaxation of TOPO I-mediated, negative supercoiled pBR322 DNA. As shown in (a), lane 1 is negative supercoiled pBR322 DNA (supercoiled DNA, S); lane 2 is relaxed DNA that is the product of supercoiled DNA reacted with the enzyme (relaxed DNA, R); and lanes 3 and 4 are positive control groups. HCPT (a typical inhibitor of TOPO I) had no inhibitory effect on the DNA relaxation activity of TOPO I at the concentration of 5 μg/mL, whereas it completely inhibited the relaxation of pBR322 DNA mediated by TOPO I at a concentration of 1 μg/μL. Lane 5 shows the effect of 1 μg/μL HCPT combined with 40 μg/mL β-ELE on the relaxation of TOPO I-mediated negative supercoiled pBR322 DNA. As shown in (a), the combination inhibited the relaxation activity of TOPO I. Lanes 6 to 11 show the effects of different concentrations of β-ELE (40, 60, 80, and 100 μg/mL) on the relaxation of negative supercoiled pBR322 DNA mediated by TOPO I. β-ELE has no inhibitory effect on the relaxation activity of TOPO I at concentrations of 10 and 20 μg/mL; however, with increasing drug concentration, β-ELE showed an increasing inhibitory effect on the DNA relaxation activity of TOPO I at concentrations of 40, 60, 80, and 100 μg/mL. The OD of MAX was 58 ± 3, 80 ± 6, 92 ± 10, and 134 ± 12. The statistical analysis showed significant differences between the 100 μg/mL and 40 μg/mL treatment groups and the 60 μg/mL and 80 μg/mL treatment groups (P < 0.05). (b) β-ELE (β-Elemene) has no direct effect on pBR322 DNA. As shown in (b), the average OD of the control group was 22860 ± 2412, and those of the β-ELE treatment groups were 24572 ± 518, 22318 ± 651, 22781 ± 837, 20781 ± 1180, and 24284 ± 749 for 20, 40, 60, 80, and 100 μg/mL of β-ELE, respectively. There was no significant difference between the groups (P > 0.05).

Mentions: DNA TOPO I regulates the number of topological links between two DNA strands (i.e., the change in the number of superhelical turns) by catalyzing transient single- or double-strand breaks, crossing the strands through one another, and then resealing the breaks, so that DNA converts to a relaxed state. Therefore, TOPO I can relax supercoiled DNA. The appearance of relaxed DNA bands and the disappearance of supercoiled forms are regarded as evidence of the catalytic activity of TOPO I. The effect of β-ELE on the catalytic activity of TOPO I was examined using the TOPO I-mediated supercoiled pBR322 DNA relaxation assay. As shown in Figure 6(a), lane 1 is negative supercoiled pBR322 DNA (supercoiled DNA, S); lane 2 is relaxed DNA that is the product of supercoiled DNA that has reacted with the enzyme (relaxed DNA, R); and lanes 3 and 4 are positive control groups. HCPT (a typical inhibitor of TOPO I) at 5 μg/mL had no inhibitory effect on the DNA relaxation activity of TOPO I, whereas, at 1 μg/μL, it completely inhibited the relaxation of pBR322 DNA mediated by TOPO I. Lane 5 shows the effect of 1 μg/μL HCPT combined with 40 μg/mL β-ELE on the relaxation of TOPO I-mediated, negative supercoiled pBR322 DNA. As shown in Figure 6(a), the combination inhibited the relaxation activity of TOPO I. Lanes 6 to 11 show the effects of different concentrations of β-ELE (40, 60, 80, and 100 μg/mL) on the relaxation of negative supercoiled pBR322 DNA mediated by TOPO I. β-ELE had no inhibitory effect on the relaxation activity of TOPO I at concentrations of 10 and 20 μg/mL; however, β-ELE showed an increasingly inhibitory effect on the DNA relaxation activity of TOPO I at increasing concentrations of 40, 60, 80, and 100 μg/mL; the OD of MAX was 58 ± 3, 80 ± 6, 92 ± 10, and 134 ± 12, respectively. The statistical analysis showed significant differences between the 100 μg/mL and 40 μg/mL treatment groups and the 60 μg/mL and 80 μg/mL treatment groups (P < 0.05). These results demonstrate that β-ELE has an inhibitory effect on the catalytic activity of TOPO I, and the inhibition occurs in a dose-dependent manner.


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 I catalytic activity is inhibited by β-ELE. (a) The effect of β-ELE (β-Elemene) on the relaxation of TOPO I-mediated, negative supercoiled pBR322 DNA. As shown in (a), lane 1 is negative supercoiled pBR322 DNA (supercoiled DNA, S); lane 2 is relaxed DNA that is the product of supercoiled DNA reacted with the enzyme (relaxed DNA, R); and lanes 3 and 4 are positive control groups. HCPT (a typical inhibitor of TOPO I) had no inhibitory effect on the DNA relaxation activity of TOPO I at the concentration of 5 μg/mL, whereas it completely inhibited the relaxation of pBR322 DNA mediated by TOPO I at a concentration of 1 μg/μL. Lane 5 shows the effect of 1 μg/μL HCPT combined with 40 μg/mL β-ELE on the relaxation of TOPO I-mediated negative supercoiled pBR322 DNA. As shown in (a), the combination inhibited the relaxation activity of TOPO I. Lanes 6 to 11 show the effects of different concentrations of β-ELE (40, 60, 80, and 100 μg/mL) on the relaxation of negative supercoiled pBR322 DNA mediated by TOPO I. β-ELE has no inhibitory effect on the relaxation activity of TOPO I at concentrations of 10 and 20 μg/mL; however, with increasing drug concentration, β-ELE showed an increasing inhibitory effect on the DNA relaxation activity of TOPO I at concentrations of 40, 60, 80, and 100 μg/mL. The OD of MAX was 58 ± 3, 80 ± 6, 92 ± 10, and 134 ± 12. The statistical analysis showed significant differences between the 100 μg/mL and 40 μg/mL treatment groups and the 60 μg/mL and 80 μg/mL treatment groups (P < 0.05). (b) β-ELE (β-Elemene) has no direct effect on pBR322 DNA. As shown in (b), the average OD of the control group was 22860 ± 2412, and those of the β-ELE treatment groups were 24572 ± 518, 22318 ± 651, 22781 ± 837, 20781 ± 1180, and 24284 ± 749 for 20, 40, 60, 80, and 100 μg/mL of β-ELE, respectively. There was no significant difference between the groups (P > 0.05).
© Copyright Policy - open-access
Related In: Results  -  Collection

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fig6: TOPO I catalytic activity is inhibited by β-ELE. (a) The effect of β-ELE (β-Elemene) on the relaxation of TOPO I-mediated, negative supercoiled pBR322 DNA. As shown in (a), lane 1 is negative supercoiled pBR322 DNA (supercoiled DNA, S); lane 2 is relaxed DNA that is the product of supercoiled DNA reacted with the enzyme (relaxed DNA, R); and lanes 3 and 4 are positive control groups. HCPT (a typical inhibitor of TOPO I) had no inhibitory effect on the DNA relaxation activity of TOPO I at the concentration of 5 μg/mL, whereas it completely inhibited the relaxation of pBR322 DNA mediated by TOPO I at a concentration of 1 μg/μL. Lane 5 shows the effect of 1 μg/μL HCPT combined with 40 μg/mL β-ELE on the relaxation of TOPO I-mediated negative supercoiled pBR322 DNA. As shown in (a), the combination inhibited the relaxation activity of TOPO I. Lanes 6 to 11 show the effects of different concentrations of β-ELE (40, 60, 80, and 100 μg/mL) on the relaxation of negative supercoiled pBR322 DNA mediated by TOPO I. β-ELE has no inhibitory effect on the relaxation activity of TOPO I at concentrations of 10 and 20 μg/mL; however, with increasing drug concentration, β-ELE showed an increasing inhibitory effect on the DNA relaxation activity of TOPO I at concentrations of 40, 60, 80, and 100 μg/mL. The OD of MAX was 58 ± 3, 80 ± 6, 92 ± 10, and 134 ± 12. The statistical analysis showed significant differences between the 100 μg/mL and 40 μg/mL treatment groups and the 60 μg/mL and 80 μg/mL treatment groups (P < 0.05). (b) β-ELE (β-Elemene) has no direct effect on pBR322 DNA. As shown in (b), the average OD of the control group was 22860 ± 2412, and those of the β-ELE treatment groups were 24572 ± 518, 22318 ± 651, 22781 ± 837, 20781 ± 1180, and 24284 ± 749 for 20, 40, 60, 80, and 100 μg/mL of β-ELE, respectively. There was no significant difference between the groups (P > 0.05).
Mentions: DNA TOPO I regulates the number of topological links between two DNA strands (i.e., the change in the number of superhelical turns) by catalyzing transient single- or double-strand breaks, crossing the strands through one another, and then resealing the breaks, so that DNA converts to a relaxed state. Therefore, TOPO I can relax supercoiled DNA. The appearance of relaxed DNA bands and the disappearance of supercoiled forms are regarded as evidence of the catalytic activity of TOPO I. The effect of β-ELE on the catalytic activity of TOPO I was examined using the TOPO I-mediated supercoiled pBR322 DNA relaxation assay. As shown in Figure 6(a), lane 1 is negative supercoiled pBR322 DNA (supercoiled DNA, S); lane 2 is relaxed DNA that is the product of supercoiled DNA that has reacted with the enzyme (relaxed DNA, R); and lanes 3 and 4 are positive control groups. HCPT (a typical inhibitor of TOPO I) at 5 μg/mL had no inhibitory effect on the DNA relaxation activity of TOPO I, whereas, at 1 μg/μL, it completely inhibited the relaxation of pBR322 DNA mediated by TOPO I. Lane 5 shows the effect of 1 μg/μL HCPT combined with 40 μg/mL β-ELE on the relaxation of TOPO I-mediated, negative supercoiled pBR322 DNA. As shown in Figure 6(a), the combination inhibited the relaxation activity of TOPO I. Lanes 6 to 11 show the effects of different concentrations of β-ELE (40, 60, 80, and 100 μg/mL) on the relaxation of negative supercoiled pBR322 DNA mediated by TOPO I. β-ELE had no inhibitory effect on the relaxation activity of TOPO I at concentrations of 10 and 20 μg/mL; however, β-ELE showed an increasingly inhibitory effect on the DNA relaxation activity of TOPO I at increasing concentrations of 40, 60, 80, and 100 μg/mL; the OD of MAX was 58 ± 3, 80 ± 6, 92 ± 10, and 134 ± 12, respectively. The statistical analysis showed significant differences between the 100 μg/mL and 40 μg/mL treatment groups and the 60 μg/mL and 80 μg/mL treatment groups (P < 0.05). These results demonstrate that β-ELE has an inhibitory effect on the catalytic activity of TOPO I, and the inhibition occurs in a dose-dependent manner.

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