Journal: Oncology Reports

Article Title: Reactive oxygen species induced by icaritin promote DNA strand breaks and apoptosis in human cervical cancer cells

doi: 10.3892/or.2018.6864

Figure Lengend Snippet: Cytotoxicity of icaritin against cervical cancer cells. (A) Chemical structure of icaritin. (B) HeLa and SiHa cervical cancer cells treated with 0, 3, 6, 9, 12, 15, 18, 21, 24, 30, 50 or 80 µM of icaritin for 48 h and cell viability determined by MTT assay. The results were analyzed using the GraphPad Prism software by nonlinear regression (curve fit). (C) Sensitivity of different types of cells: HeLa and SiHa cancer cells, and CCD-1095Sk and 293 non-cancerous cells, were treated with 12.5 µM or 25 µM of icaritin for 48 h and cell viability was determined by the MTT assay (n=3, mean ± standard deviation; **P<0.01; ***P<0.001). (D) Time- and dose-dependency; representative results of HeLa cells. IC 50 , half maximal inhibitory concentration.

Article Snippet: The human HeLa and SiHa cervical cancer cell lines, and the non-cancerous 293 and CCD-1095Sk cell lines were purchased from the American Type Culture Collection (ATCC; Manassas, VA, USA).

Techniques: MTT Assay, Software, Standard Deviation, Concentration Assay

Journal: Oncology Reports

Article Title: Reactive oxygen species induced by icaritin promote DNA strand breaks and apoptosis in human cervical cancer cells

doi: 10.3892/or.2018.6864

Figure Lengend Snippet: Colony formation and wound healing assay. (A) Colony formation assay. HeLa or SiHa cells were treated with icaritin for 8 days and stained with crystal violet (representative images of HeLa cells; scale bar, 200 µm). (B) Quantification of colony formation assay. Crystal violet-stained cells were dissolved in 70% alcohol, and absorbance at 595 nm (O.D.) was measured using a microplate reader and presented as mean ± standard error from three independent experiments (***P < 0.001). (C) Wound healing assay. Representative images of HeLa cells. (D) Quantification of wound healing assay. Closure rate was defined as follows: (original wound size-new wound size)/original wound size ×100 (n=3, mean ± standard error; ***P<0.001). O.D., optical density.

Article Snippet: The human HeLa and SiHa cervical cancer cell lines, and the non-cancerous 293 and CCD-1095Sk cell lines were purchased from the American Type Culture Collection (ATCC; Manassas, VA, USA).

Techniques: Wound Healing Assay, Colony Assay, Staining

Journal: Oncology Reports

Article Title: Reactive oxygen species induced by icaritin promote DNA strand breaks and apoptosis in human cervical cancer cells

doi: 10.3892/or.2018.6864

Figure Lengend Snippet: Measurement of total cellular ROS. (A) Flow cytometry analysis: HeLa or SiHa cells with or without 1 h pre-incubation with 5 mM NAC were treated by icaritin for 12 h and a representative result of HeLa cells was shown. (B) Data from three independent experiments in HeLa cells were processed by the GraphPad Prism software (***P<0.001). (C) Time-dependent ROS accumulation. HeLa or SiHa cells were treated by icaritin for 3 h or longer (***P<0.001). (D) Inhibition of icaritin cytotoxicity by NAC. HeLa or SiHa cells with or without pre-incubation (1 h) with 5 mM NAC were treated by icaritin for 48 h and cell survival was analyzed by the MTT assay (*P<0.05; **P<0.01; ***P<0.001). ROS, reactive oxygen species; NAC, N-acetyl cysteine; DCF, dichlorofluorescin; NS, not significant.

Article Snippet: The human HeLa and SiHa cervical cancer cell lines, and the non-cancerous 293 and CCD-1095Sk cell lines were purchased from the American Type Culture Collection (ATCC; Manassas, VA, USA).

Techniques: Flow Cytometry, Incubation, Software, Inhibition, MTT Assay

Journal: Oncology Reports

Article Title: Reactive oxygen species induced by icaritin promote DNA strand breaks and apoptosis in human cervical cancer cells

doi: 10.3892/or.2018.6864

Figure Lengend Snippet: Comet assay. (A) HeLa and SiHa cells were treated by 1× IC 50 or 2× IC 50 icaritin and/or vehicle control for 24 h and a comet assay was used to identify DNA strand breaks (scale bar, 200 µm). (B) Quantification of tail moment. Tail moment was defined as percentage of tail DNA × tail length and was quantified using the CASP software (n=40; ***P < 0.001). (C) Inhibition of DNA strand breaks by NAC. HeLa and SiHa cells with or without pre-incubation (1 h) with 5 mM NAC were treated by icaritin for 24 h (***P<0.001). IC 50 , half maximal inhibitory concentration; OGG, 8-oxoguanine glycosylase; NAC, N-acetyl cysteine; NS, not significant.

Article Snippet: The human HeLa and SiHa cervical cancer cell lines, and the non-cancerous 293 and CCD-1095Sk cell lines were purchased from the American Type Culture Collection (ATCC; Manassas, VA, USA).

Techniques: Single Cell Gel Electrophoresis, Control, Software, Inhibition, Incubation, Concentration Assay

Journal: Oncology Reports

Article Title: Reactive oxygen species induced by icaritin promote DNA strand breaks and apoptosis in human cervical cancer cells

doi: 10.3892/or.2018.6864

Figure Lengend Snippet: Immunostaining of γH2AX and 53BP1. (A) HeLa cells were treated with 12.5 µM icaritin and/or vehicle control for 12 h (scale bar, 10 µm). Time course of γH2AX and 53BP1 induction in (B) HeLa and (C) SiHa cells; the number of cells with five or more bright 53BP1 or γH2AX foci in three cover slips was counted manually (*P<0.05; **P<0.01; ***P<0.001). 53BP1, TP53-binding protein 1; γH2AX, γH2AX histone; NS, not significant.

Article Snippet: The human HeLa and SiHa cervical cancer cell lines, and the non-cancerous 293 and CCD-1095Sk cell lines were purchased from the American Type Culture Collection (ATCC; Manassas, VA, USA).

Techniques: Immunostaining, Control, Binding Assay

Journal: Oncology Reports

Article Title: Reactive oxygen species induced by icaritin promote DNA strand breaks and apoptosis in human cervical cancer cells

doi: 10.3892/or.2018.6864

Figure Lengend Snippet: Cell cycle analysis. (A) Cell cycle analysis was performed by flow cytometry. HeLa or SiHa cells with or without pre-incubation (1 h) with 5 mM NAC were treated by 25 µM or 34 µM of icaritin for 24 h. (B) Quantification of flow cytometry data from three independent experiments (*P<0.05 vs. 0 µM group; ***P<0.001 vs. 0 µM group). NAC, N-acetyl cysteine.

Article Snippet: The human HeLa and SiHa cervical cancer cell lines, and the non-cancerous 293 and CCD-1095Sk cell lines were purchased from the American Type Culture Collection (ATCC; Manassas, VA, USA).

Techniques: Cell Cycle Assay, Flow Cytometry, Incubation

Journal: Oncology Reports

Article Title: Reactive oxygen species induced by icaritin promote DNA strand breaks and apoptosis in human cervical cancer cells

doi: 10.3892/or.2018.6864

Figure Lengend Snippet: Analysis of apoptosis. (A) Representative images of nuclei stained with DAPI. HeLa cells were treated with 1× IC 50 or 2× IC 50 icaritin for 24 h (arrows point to nuclei of apoptotic cells). (B) Analysis of Annexin V-fluorescein isothiocyanate and propidium iodide-stained cells by flow cytometry. HeLa or SiHa cells were treated by 1× IC 50 or 2× IC 50 icaritin for 24 h or 48 h. (C) Quantification of flow cytometry data from three independent experiments. HeLa or SiHa cells with or without pre-incubation (1 h) with 5 mM NAC were treated with 1× IC 50 or 2× IC 50 icaritin for 24 h or 48 h (**P<0.01; ***P<0.001). (D) Measurement of mitochondrial membrane potential by JC-1. HeLa or SiHa cells were treated by 1× IC 50 or 2× IC 50 for 24 h. IC 50 , half maximal inhibitory concentration.

Article Snippet: The human HeLa and SiHa cervical cancer cell lines, and the non-cancerous 293 and CCD-1095Sk cell lines were purchased from the American Type Culture Collection (ATCC; Manassas, VA, USA).

Techniques: Staining, Flow Cytometry, Incubation, Membrane, Concentration Assay

Journal: Oncology Reports

Article Title: Reactive oxygen species induced by icaritin promote DNA strand breaks and apoptosis in human cervical cancer cells

doi: 10.3892/or.2018.6864

Figure Lengend Snippet: Western blot analysis. HeLa and SiHa cells with or without pre-incubation (1 h) with 5 µM NAC were treated by icaritin for 24 h. Multiple SDS-PAGE gels were prepared for each sample, and each gel was used for the detection of one protein. Thus, one loading control, β-actin, was used for all proteins of each sample. (A) Detection of MMP-9. (B) Detection of cell cycle-associated proteins. (C) Detection of apoptosis-associated proteins. (D) Quantification of apoptosis-associated proteins in the 0 µM, 2× IC 50 and 2× IC 50 + NAC groups. Relative protein levels were quantified from western blots using ImageJ and presented as ratios of each protein band relative to the loading control. (*P<0.05, **P<0.01, ***P<0.001 vs. 0 µM). NAC, N-acetyl cysteine, MMP-9, matrix metalloproteinase-9; CDK1, cyclin-dependent kinase 1; CDC25C, cell division cycle 25C; XIAP, X-linked inhibitor of apoptosis protein; Bax, apoptosis regulator Bax; Bcl-2, B-cell lymphoma-2.

Article Snippet: The human HeLa and SiHa cervical cancer cell lines, and the non-cancerous 293 and CCD-1095Sk cell lines were purchased from the American Type Culture Collection (ATCC; Manassas, VA, USA).

Techniques: Western Blot, Incubation, SDS Page, Control

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: Targeting HMGB3/hTERT axis for radioresistance in cervical cancer

doi: 10.1186/s13046-020-01737-1

Figure Lengend Snippet: Cervical cancer RR Siha and RR Hela cells possesses radioresistant abilities. a The ability of colony formation in the radioresistant and parallel cervical cancer cells after radiation. b Biological parameters of radioresistant and parallel cervical cancer cells after radiation. c Immunoblotting of γH2AX at different time point in SiHa, HeLa and RR SiHa, RR HeLa cells after treated with 4 Gy radiation. d Comet assay was performed to detect DNA damage after exposed to 4 Gy radiation in HeLa, RR HeLa, SiHa and RR SiHa cells at the different time point. e 24 h after 4Gy radiation, apoptosis in the cells was detected by FACS analysis. f HeLa, SiHa, RR HeLa and RR SiHa were exposed to 4 Gy radiation. 24 h later, the cytochrome-c release was detected by immunofluorescence assay. g The expression level of caspase-3,caspase-9 and cleaved-PARP were detected by western blot

Article Snippet: The human cervical cancer cell lines HeLa, SiHa, C33A, DoTc2, HeLa S3, and CaSki were obtained from American Type Culture Collection (ATCC, VA).

Techniques: Western Blot, Single Cell Gel Electrophoresis, Immunofluorescence, Expressing

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: Targeting HMGB3/hTERT axis for radioresistance in cervical cancer

doi: 10.1186/s13046-020-01737-1

Figure Lengend Snippet: HMGB3was identified as a new transcriptional factor of hTERT in cervical cancer radioresistant cells. a Differential gene expression between radioresistant and parental cervical cancer cells from RNA-Seq. b A heatmap showing 42 significantly up-regulated genes in the RR SiHa and RR HeLa. c A demonstration map of hTERT promoter probe with a biotin from − 1645 to + 15. d HMGB3 was identified as a new transcriptional factor of hTERT by streptavidin-agarose and LC/MS. e Biotin-streptavidin pulldown was perform to further verify the binding of HMGB3 on the hTERT promoter. f Cervical cancer cells were transfected with HMGB3-overexpressing plasmids or HMGB3 specific siRNAs. The expression of HMGB3 and hTERT were detected by western blot. g SiHa was transfected with HMGB3 specific siRNAs, and then the binding ability of HMGB3 on hTERT promoter (− 1645 to + 15) was detected by pulldown assay. h SiHa and HeLa S3 cells were transfected with HMGB3 specific siRNAs, and the activity of different fragments of hTERT promoter was detected by Luciferase Reporter assay. i CHIP assay was used to verify the binding of HMGB3 and H3K4me3 on region − 902 to − 321 of hTERT promoter

Article Snippet: The human cervical cancer cell lines HeLa, SiHa, C33A, DoTc2, HeLa S3, and CaSki were obtained from American Type Culture Collection (ATCC, VA).

Techniques: Gene Expression, RNA Sequencing, Liquid Chromatography with Mass Spectroscopy, Binding Assay, Transfection, Expressing, Western Blot, Activity Assay, Luciferase, Reporter Assay

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: Targeting HMGB3/hTERT axis for radioresistance in cervical cancer

doi: 10.1186/s13046-020-01737-1

Figure Lengend Snippet: Knockdown of HMGB3 increased the susceptibility to radiotherapy in cervical cancer cells and the xenograft mouse model Cervical cancer cells were transfected with HMGB3-overexpressing plasmids ( a ) or HMGB3 specific siRNAs ( b ) and then exposed to 4Gy radiation. At 24 h, colony formation assay was performed.SiHa and HeLa S3 were transfected with HMGB3 specific siRNAs and then exposed to 4Gy radiation. At 24 h, comet assay was performed to detect DNA damage ( c ), and FACS analysis was performed to detect cell apoptosis( d ). e SiHa was infected by lentivirus-HMGB3 shRNA. Cells were implanted into left armpit of the nude mice. Mice were sacrificed on day 15 and tumors were removed and photographed. f Recorded the tumor volume every two days during the course of the experiment.Tumor volume ( g ) and and weight ( h ) was measured and recorded. i Immunohistochemical staining for HMGB3, ki67, hTERT and γH2AX. j The expression of HMGB3, hTERT and γH2AX proteins in mice tumor tissues were detected by Western blot

Article Snippet: The human cervical cancer cell lines HeLa, SiHa, C33A, DoTc2, HeLa S3, and CaSki were obtained from American Type Culture Collection (ATCC, VA).

Techniques: Knockdown, Transfection, Colony Assay, Single Cell Gel Electrophoresis, Infection, shRNA, Immunohistochemical staining, Staining, Expressing, Western Blot

Journal: Oncology Reports

Article Title: Reactive oxygen species induced by icaritin promote DNA strand breaks and apoptosis in human cervical cancer cells

doi: 10.3892/or.2018.6864

Figure Lengend Snippet: Cytotoxicity of icaritin against cervical cancer cells. (A) Chemical structure of icaritin. (B) HeLa and SiHa cervical cancer cells treated with 0, 3, 6, 9, 12, 15, 18, 21, 24, 30, 50 or 80 µM of icaritin for 48 h and cell viability determined by MTT assay. The results were analyzed using the GraphPad Prism software by nonlinear regression (curve fit). (C) Sensitivity of different types of cells: HeLa and SiHa cancer cells, and CCD-1095Sk and 293 non-cancerous cells, were treated with 12.5 µM or 25 µM of icaritin for 48 h and cell viability was determined by the MTT assay (n=3, mean ± standard deviation; **P<0.01; ***P<0.001). (D) Time- and dose-dependency; representative results of HeLa cells. IC 50 , half maximal inhibitory concentration.

Article Snippet: The human HeLa and SiHa cervical cancer cell lines, and the non-cancerous 293 and CCD-1095Sk cell lines were purchased from the American Type Culture Collection (ATCC; Manassas, VA, USA).

Techniques: MTT Assay, Software, Standard Deviation, Concentration Assay

Journal: Oncology Reports

Article Title: Reactive oxygen species induced by icaritin promote DNA strand breaks and apoptosis in human cervical cancer cells

doi: 10.3892/or.2018.6864

Figure Lengend Snippet: Colony formation and wound healing assay. (A) Colony formation assay. HeLa or SiHa cells were treated with icaritin for 8 days and stained with crystal violet (representative images of HeLa cells; scale bar, 200 µm). (B) Quantification of colony formation assay. Crystal violet-stained cells were dissolved in 70% alcohol, and absorbance at 595 nm (O.D.) was measured using a microplate reader and presented as mean ± standard error from three independent experiments (***P < 0.001). (C) Wound healing assay. Representative images of HeLa cells. (D) Quantification of wound healing assay. Closure rate was defined as follows: (original wound size-new wound size)/original wound size ×100 (n=3, mean ± standard error; ***P<0.001). O.D., optical density.

Article Snippet: The human HeLa and SiHa cervical cancer cell lines, and the non-cancerous 293 and CCD-1095Sk cell lines were purchased from the American Type Culture Collection (ATCC; Manassas, VA, USA).

Techniques: Wound Healing Assay, Colony Assay, Staining

Journal: Oncology Reports

Article Title: Reactive oxygen species induced by icaritin promote DNA strand breaks and apoptosis in human cervical cancer cells

doi: 10.3892/or.2018.6864

Figure Lengend Snippet: Measurement of total cellular ROS. (A) Flow cytometry analysis: HeLa or SiHa cells with or without 1 h pre-incubation with 5 mM NAC were treated by icaritin for 12 h and a representative result of HeLa cells was shown. (B) Data from three independent experiments in HeLa cells were processed by the GraphPad Prism software (***P<0.001). (C) Time-dependent ROS accumulation. HeLa or SiHa cells were treated by icaritin for 3 h or longer (***P<0.001). (D) Inhibition of icaritin cytotoxicity by NAC. HeLa or SiHa cells with or without pre-incubation (1 h) with 5 mM NAC were treated by icaritin for 48 h and cell survival was analyzed by the MTT assay (*P<0.05; **P<0.01; ***P<0.001). ROS, reactive oxygen species; NAC, N-acetyl cysteine; DCF, dichlorofluorescin; NS, not significant.

Article Snippet: The human HeLa and SiHa cervical cancer cell lines, and the non-cancerous 293 and CCD-1095Sk cell lines were purchased from the American Type Culture Collection (ATCC; Manassas, VA, USA).

Techniques: Flow Cytometry, Incubation, Software, Inhibition, MTT Assay

Journal: Oncology Reports

Article Title: Reactive oxygen species induced by icaritin promote DNA strand breaks and apoptosis in human cervical cancer cells

doi: 10.3892/or.2018.6864

Figure Lengend Snippet: Comet assay. (A) HeLa and SiHa cells were treated by 1× IC 50 or 2× IC 50 icaritin and/or vehicle control for 24 h and a comet assay was used to identify DNA strand breaks (scale bar, 200 µm). (B) Quantification of tail moment. Tail moment was defined as percentage of tail DNA × tail length and was quantified using the CASP software (n=40; ***P < 0.001). (C) Inhibition of DNA strand breaks by NAC. HeLa and SiHa cells with or without pre-incubation (1 h) with 5 mM NAC were treated by icaritin for 24 h (***P<0.001). IC 50 , half maximal inhibitory concentration; OGG, 8-oxoguanine glycosylase; NAC, N-acetyl cysteine; NS, not significant.

Article Snippet: The human HeLa and SiHa cervical cancer cell lines, and the non-cancerous 293 and CCD-1095Sk cell lines were purchased from the American Type Culture Collection (ATCC; Manassas, VA, USA).

Techniques: Single Cell Gel Electrophoresis, Control, Software, Inhibition, Incubation, Concentration Assay

Journal: Oncology Reports

Article Title: Reactive oxygen species induced by icaritin promote DNA strand breaks and apoptosis in human cervical cancer cells

doi: 10.3892/or.2018.6864

Figure Lengend Snippet: Immunostaining of γH2AX and 53BP1. (A) HeLa cells were treated with 12.5 µM icaritin and/or vehicle control for 12 h (scale bar, 10 µm). Time course of γH2AX and 53BP1 induction in (B) HeLa and (C) SiHa cells; the number of cells with five or more bright 53BP1 or γH2AX foci in three cover slips was counted manually (*P<0.05; **P<0.01; ***P<0.001). 53BP1, TP53-binding protein 1; γH2AX, γH2AX histone; NS, not significant.

Article Snippet: The human HeLa and SiHa cervical cancer cell lines, and the non-cancerous 293 and CCD-1095Sk cell lines were purchased from the American Type Culture Collection (ATCC; Manassas, VA, USA).

Techniques: Immunostaining, Control, Binding Assay

Journal: Oncology Reports

Article Title: Reactive oxygen species induced by icaritin promote DNA strand breaks and apoptosis in human cervical cancer cells

doi: 10.3892/or.2018.6864

Figure Lengend Snippet: Cell cycle analysis. (A) Cell cycle analysis was performed by flow cytometry. HeLa or SiHa cells with or without pre-incubation (1 h) with 5 mM NAC were treated by 25 µM or 34 µM of icaritin for 24 h. (B) Quantification of flow cytometry data from three independent experiments (*P<0.05 vs. 0 µM group; ***P<0.001 vs. 0 µM group). NAC, N-acetyl cysteine.

Article Snippet: The human HeLa and SiHa cervical cancer cell lines, and the non-cancerous 293 and CCD-1095Sk cell lines were purchased from the American Type Culture Collection (ATCC; Manassas, VA, USA).

Techniques: Cell Cycle Assay, Flow Cytometry, Incubation

Journal: Oncology Reports

Article Title: Reactive oxygen species induced by icaritin promote DNA strand breaks and apoptosis in human cervical cancer cells

doi: 10.3892/or.2018.6864

Figure Lengend Snippet: Analysis of apoptosis. (A) Representative images of nuclei stained with DAPI. HeLa cells were treated with 1× IC 50 or 2× IC 50 icaritin for 24 h (arrows point to nuclei of apoptotic cells). (B) Analysis of Annexin V-fluorescein isothiocyanate and propidium iodide-stained cells by flow cytometry. HeLa or SiHa cells were treated by 1× IC 50 or 2× IC 50 icaritin for 24 h or 48 h. (C) Quantification of flow cytometry data from three independent experiments. HeLa or SiHa cells with or without pre-incubation (1 h) with 5 mM NAC were treated with 1× IC 50 or 2× IC 50 icaritin for 24 h or 48 h (**P<0.01; ***P<0.001). (D) Measurement of mitochondrial membrane potential by JC-1. HeLa or SiHa cells were treated by 1× IC 50 or 2× IC 50 for 24 h. IC 50 , half maximal inhibitory concentration.

Article Snippet: The human HeLa and SiHa cervical cancer cell lines, and the non-cancerous 293 and CCD-1095Sk cell lines were purchased from the American Type Culture Collection (ATCC; Manassas, VA, USA).

Techniques: Staining, Flow Cytometry, Incubation, Membrane, Concentration Assay

Journal: Oncology Reports

Article Title: Reactive oxygen species induced by icaritin promote DNA strand breaks and apoptosis in human cervical cancer cells

doi: 10.3892/or.2018.6864

Figure Lengend Snippet: Western blot analysis. HeLa and SiHa cells with or without pre-incubation (1 h) with 5 µM NAC were treated by icaritin for 24 h. Multiple SDS-PAGE gels were prepared for each sample, and each gel was used for the detection of one protein. Thus, one loading control, β-actin, was used for all proteins of each sample. (A) Detection of MMP-9. (B) Detection of cell cycle-associated proteins. (C) Detection of apoptosis-associated proteins. (D) Quantification of apoptosis-associated proteins in the 0 µM, 2× IC 50 and 2× IC 50 + NAC groups. Relative protein levels were quantified from western blots using ImageJ and presented as ratios of each protein band relative to the loading control. (*P<0.05, **P<0.01, ***P<0.001 vs. 0 µM). NAC, N-acetyl cysteine, MMP-9, matrix metalloproteinase-9; CDK1, cyclin-dependent kinase 1; CDC25C, cell division cycle 25C; XIAP, X-linked inhibitor of apoptosis protein; Bax, apoptosis regulator Bax; Bcl-2, B-cell lymphoma-2.

Article Snippet: The human HeLa and SiHa cervical cancer cell lines, and the non-cancerous 293 and CCD-1095Sk cell lines were purchased from the American Type Culture Collection (ATCC; Manassas, VA, USA).

Techniques: Western Blot, Incubation, SDS Page, Control