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OncoTargets and Therapy Volume 8, 2015 - Issue
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Original Research

Echinacoside induces apoptotic cancer cell death by inhibiting the nucleotide pool sanitizing enzyme MTH1

Liwei Dong1 Key Laboratory for Molecular Enzymology and Engineering of the Ministry of Education, National Engineering Laboratory for AIDS Vaccine, School of Life Sciences, Jilin University, Changchun, Jilin Province, People’s Republic of China
,
Hongge Wang1 Key Laboratory for Molecular Enzymology and Engineering of the Ministry of Education, National Engineering Laboratory for AIDS Vaccine, School of Life Sciences, Jilin University, Changchun, Jilin Province, People’s Republic of China
,
Jiajing Niu1 Key Laboratory for Molecular Enzymology and Engineering of the Ministry of Education, National Engineering Laboratory for AIDS Vaccine, School of Life Sciences, Jilin University, Changchun, Jilin Province, People’s Republic of China
,
Mingwei Zou2 Department of Psychology, College of Liberal Arts and Social Sciences, University of Houston, Houston, TX, USA
,
Nuoting Wu1 Key Laboratory for Molecular Enzymology and Engineering of the Ministry of Education, National Engineering Laboratory for AIDS Vaccine, School of Life Sciences, Jilin University, Changchun, Jilin Province, People’s Republic of China
,
Debin Yu1 Key Laboratory for Molecular Enzymology and Engineering of the Ministry of Education, National Engineering Laboratory for AIDS Vaccine, School of Life Sciences, Jilin University, Changchun, Jilin Province, People’s Republic of China
,
Ye Wang1 Key Laboratory for Molecular Enzymology and Engineering of the Ministry of Education, National Engineering Laboratory for AIDS Vaccine, School of Life Sciences, Jilin University, Changchun, Jilin Province, People’s Republic of China
&
Zhihua Zou1 Key Laboratory for Molecular Enzymology and Engineering of the Ministry of Education, National Engineering Laboratory for AIDS Vaccine, School of Life Sciences, Jilin University, Changchun, Jilin Province, People’s Republic of ChinaCorrespondence[email protected]
 show all
Pages 3649-3664 | Published online: 08 Dec 2015

Figures & data

Figure 1 In vitro screening of natural compounds.

Notes: (A) A dose–response curve of MTH1 inhibition by a positive control: (S)-crizotinib inhibited MTH1 with an IC50 of 0.5 μM; (B) the inhibition of MTH1 by individual herbal compounds: 15 μM Echinacoside inhibited the activity of MTH1 by >80%; (C) the chemical structure of Echinacoside: a polyphenol glycoside consisting of a phenylpropanoid (red box) and a phenylethanoid (blue box) glycosidically linked to a trisaccharide moiety (a central rhamnose and two side glucose); and (D) a dose–response curve of MTH1 inhibition by a natural compound: Echinacoside inhibited MTH1 with an IC50 of 7.01±2.13 μM.
Abbreviations: IC50, inhibitory concentration; dGTP, deoxyguanosine triphosphate.
Figure 1 In vitro screening of natural compounds.

Figure 2 Examination of cellular 8-oxoG, ROS, and DNA damages.

Notes: (A) Detection of 8-oxoG by staining with Cy3-conjugated avidin, (B) quantification of data from three independent experiments: treatment of MG-63 cells with 60 μM Echinacoside for 24 h significantly increased the level of cellular 8-oxoG (Cy3-avidin reactive substance) (***P<0.001 vs vehicle control), (C) measurement of cellular ROS by flow cytometry, (D) quantification of data from three independent experiments: treatment of MG-63 cells with 60 μM Echinacoside for 5 h, 12 h, or 24 h did not change cellular ROS level, (E) immunofluorescent staining of nuclear 53BP1, and (F) quantification of data from three coverslips: significant increase in the number of cells with five or more bright 53BP1 foci was seen as early as 5 h after initiation of treatment with 60 μM Echinacoside (*P<0.05, ***P<0.001 vs vehicle control).
Abbreviations: 8-oxoG, 8-oxoguanine; ROS, reactive oxygen series; DAPI, 4′,6-diamidino-2-phenylindole; DCFH-DA, dichloro-dihydro-fluorescein diacetate; h, hours.
Figure 2 Examination of cellular 8-oxoG, ROS, and DNA damages.

Figure 3 Analysis of cell growth and proliferation.

Notes: (A and B) Colony formation assay: MG-63 cells treated with 60 μM or 80 μM Echinacoside for 7 days formed significantly fewer and smaller colonies (***P<0.001 vs vehicle control); (C and D) MTT assay: Echinacoside dose dependently inhibited the growth of MG-63 cells with an IC50 of 45.11 μM (C), and significant inhibition of proliferation was seen 12 h after initiation of treatment with 60 μM Echinacoside (D) (**P<0.01, ***P<0.001 vs vehicle control).
Abbreviations: MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; IC50, inhibitory concentration; h, hours; OD, optical density.
Figure 3 Analysis of cell growth and proliferation.

Figure 4 Analysis of cell cycle.

Notes: (A and B) Western blot analysis of p21 and quantification of data from three independent experiments: significantly upregulated p21 was seen in MG-63 cells treated with 60 μM Echinacoside for 5 h and longer (***P<0.001 vs vehicle control), (C–E) cell cycle analysis: MG-63 cells were treated with 0 μM (C) and 60 μM (D) Echinacoside and quantitative data from three independent experiments are shown (E); Echinacoside significantly reduced the percentage of cells in both S and G2/M phases, while the percentage of cells in G1 phase increased (**P<0.01, ***P<0.001 vs vehicle control).
Abbreviations: h, hours; GAPDH, glyceraldehyde 3-phosphate dehydrogenase.
Figure 4 Analysis of cell cycle.

Figure 5 Analysis of apoptosis.

Notes: (A) Western blot detection of cleaved PARP and active caspase-3 proteins, (B) quantification of data from three independent experiments: treatment with 60 μM or 80 μM Echinacoside for 24 h increased the levels of cleaved PARP and active caspase-3 proteins in MG-63 cells (***P<0.001 vs vehicle control), and (C) images of DAPI-stained nuclei: MG-63 cells were treated with 0 μM, 60 μM, and 80 μM Echinacoside for 24 h, some apoptotic cells were marked by white arrows.
Abbreviations: PARP, poly (ADP-ribose) polymerase; h, hours; DAPI, 4′,6-diamidino-2-phenylindole; GAPDH, glyceraldehyde 3-phosphate dehydrogenase.
Figure 5 Analysis of apoptosis.

Figure 6 Induction of apoptosis by Echinacoside.

Notes: (A) Analysis of Annexin V-FITC- and propidium iodide-stained cells by flow cytometry, (B) time-course analysis of apoptosis induction: significant apoptosis was seen in MG-63 cells treated with 60 μM or higher concentrations of Echinacoside for 12 h (***P<0.001 vs control), and (C) measurement of mitochondrial membrane potential: MG-63 cells were treated with 60 μM Echinacoside for 5 h, 12 h, and 24 h; increase in the intensity of green at the 12 h point showed significant disruption of mitochondrial membrane potential.
Abbreviations: h, hours; PI, propidium iodide; FITC, fluorescein isothiocyanate.
Figure 6 Induction of apoptosis by Echinacoside.

Figure S1 Examination of cellular 8-oxoG and apoptosis.

Notes: (A) Detection of 8-oxoG by a monoclonal anti-8-oxoG antibody: 8-oxoG signal was revealed by an Alexa 488-conjugated secondary antibody, and treatment of MG-63 cells with 60 μM and 80 μM Echinacoside for 24 h increased the level of 8-oxoG; (B) analysis of cancer cell DNA by electrophoresis on 2% agarose gel: treatment of MG-63 cells with 80 μM Echinacoside for 24 h produced a typical apoptosis ladder pattern; and (C) immunofluorescent staining of active caspase-3: strongly activated caspase-3 signals were detected in MG-63 cells treated with 60 μM Echinacoside for 24 h.

Abbreviations: 8-oxoG, 8-oxoguanine; h, hours; DAPI, 4′,6-diamidino-2-phenylindole.

Figure S1 Examination of cellular 8-oxoG and apoptosis.Notes: (A) Detection of 8-oxoG by a monoclonal anti-8-oxoG antibody: 8-oxoG signal was revealed by an Alexa 488-conjugated secondary antibody, and treatment of MG-63 cells with 60 μM and 80 μM Echinacoside for 24 h increased the level of 8-oxoG; (B) analysis of cancer cell DNA by electrophoresis on 2% agarose gel: treatment of MG-63 cells with 80 μM Echinacoside for 24 h produced a typical apoptosis ladder pattern; and (C) immunofluorescent staining of active caspase-3: strongly activated caspase-3 signals were detected in MG-63 cells treated with 60 μM Echinacoside for 24 h.Abbreviations: 8-oxoG, 8-oxoguanine; h, hours; DAPI, 4′,6-diamidino-2-phenylindole.
Figure S1 Examination of cellular 8-oxoG and apoptosis.Notes: (A) Detection of 8-oxoG by a monoclonal anti-8-oxoG antibody: 8-oxoG signal was revealed by an Alexa 488-conjugated secondary antibody, and treatment of MG-63 cells with 60 μM and 80 μM Echinacoside for 24 h increased the level of 8-oxoG; (B) analysis of cancer cell DNA by electrophoresis on 2% agarose gel: treatment of MG-63 cells with 80 μM Echinacoside for 24 h produced a typical apoptosis ladder pattern; and (C) immunofluorescent staining of active caspase-3: strongly activated caspase-3 signals were detected in MG-63 cells treated with 60 μM Echinacoside for 24 h.Abbreviations: 8-oxoG, 8-oxoguanine; h, hours; DAPI, 4′,6-diamidino-2-phenylindole.

Figure S2 Flow cytometry analysis of apoptosis in noncancer cells.

Notes: (A) Normal human liver L-O2 cells, (B) human embryonic kidney HEK 293 cells, and (C) mouse NIH/3T3 fibroblast cells. Treatment with 60 μM and 80 μM Echinacoside for 24 h did not change the percentage of apoptotic cells in these noncancer cell lines.

Abbreviations: h, hours; PI, propidium iodide; FITC, fluorescein isothiocyanate.

Figure S2 Flow cytometry analysis of apoptosis in noncancer cells.Notes: (A) Normal human liver L-O2 cells, (B) human embryonic kidney HEK 293 cells, and (C) mouse NIH/3T3 fibroblast cells. Treatment with 60 μM and 80 μM Echinacoside for 24 h did not change the percentage of apoptotic cells in these noncancer cell lines.Abbreviations: h, hours; PI, propidium iodide; FITC, fluorescein isothiocyanate.

Figure S3 Time-course analysis of apoptosis.

Notes: MG-63 cells were treated with 60 μM Echinacoside for 2 h, 5 h, and 12 h, and significant apoptosis was seen after treatment for 12 h.

Abbreviations: h, hours; PI, propidium iodide.

Figure S3 Time-course analysis of apoptosis.Notes: MG-63 cells were treated with 60 μM Echinacoside for 2 h, 5 h, and 12 h, and significant apoptosis was seen after treatment for 12 h.Abbreviations: h, hours; PI, propidium iodide.

Table S1 List of herbal compounds used in the in vitro screening

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