Kaempferol induces DNA damage and inhibits DNA repair associated protein expressions in human promyelocytic leukemia HL-60 cells
Lung-Yuan Wu1,#,*, Hsu-Feng Lu2,3,#, Yu-Cheng Chou4,5, Yung-Luen Shih6,7,8, Jaw-Chyun Chen9, Shu-Chun Hsu10, and Jing-Gung Chung10,11,*
1I-Shou University, the School of Chinese Medicine for Post Baccalaureate, Kaohsiung, Taiwan, R.O.C.
2Departments of Clinical Pathology, Cheng Hsin General Hospital, Taipei, Taiwan, R.O.C.
3Department of Restaurant, Hotel and Institutional Management, Fu-Jen Catholic University, Taipei, Taiwan, R.O.C.
4Division of Neurosurgical Oncology, Neurological Institute, Taichung Veterans General Hospital, Taichung, Taiwan, R.O.C.
5Institute of Medical Sciences, Tzu Chi University, Hualien, Taiwan, R.O.C. 6School of Medicine, Fu-Jen Catholic University, Taipei, Taiwan, R.O.C.
7Department of Pathology and Laboratory Medicine, Shin Kong Wu Ho-Su Memorial Hospital, Taipei, Taiwan, R.O.C.
8School of Medical Laboratory Science and Biotechnology, Taipei Medical University, Taipei, Taiwan, R.O.C.
9Department of Medicinal Botany and Health Applications, Da-Yeh University, Changhua, Taiwan, R.O.C.
10Department of Biological Science and Technology, China Medical University, Taichung, Taiwan, R.O.C.
11Department of Biotechnology, Asia University, Taichung, Taiwan, R.O.C.
Running Title: Kaempferol induces DNA damage and inhibits DNA repair in HL-60 cells.
*Correspondence to:
Jing-Gung Chung, Ph.D., Department of Biological Science and Technology, China Medical University. No 91, Hsueh-Shih Road, Taichung 404, Taiwan. Tel: +886 4 2205 3366 ext. 2161, Fax: +886 4 2205 3764, e-mail: [email protected] Lung-Yuan Wu, Ph.D. & MD, Adjunct Assistant Professor, The School of Chinese Medicine for Post-Baccalaureate, I-Shou University No.8, Yida Rd., Jiaosu Village, Yanchao District, Kaohsiung City 82445,Taiwan, R.O.C. (Yanchao Campus). Tel: +886-2-23512321; FAX:+886-2-23515220
Abstract. Numerous evidence have shown that plant flavonoids (naturally occurring substances) have been reported to have chemopreventive activities and protect against experimental carcinogenesis. Kaempferol, one of the flavonoids, is widely distributed in fruits and vegetables, and may have cancer chemopreventive properties. However, the precise underlying mechanism regarding induced DNA damage and suppressed DNA repair system are poorly understood. In this study, we investigated whether kaempferol induced DNA damage and affected DNA repair associated protein expression in human leukemia HL-60 cells in vitro. Percentages of viable cells were measured by flow cytometry assay. DNA damage was examined by Comet assay and DAPI staining. DNA fragmentation (ladder) was examined by DNA gel electrophoresis. The changes of protein levels associated with DNA repair were examined by Western blotting. Results showed that kaempferol decreased the viable cells in a dose-dependently. Comet assay indicated that kaempferol induced DNA damage (Comet tail) dose-dependent manner and DAPI staining also showed increased doses of kaempferol which led to increased DNA condensation. These effects are of dose-dependent manners. Western blotting indicated that kaempferol decreased protein expression associated with DNA repair system such as phosphate-ataxia telangiectasia mutated (p-ATM), phosphate-ataxia-telangiectasia and Rad3-related (p-ATR), 14-3-3 proteins sigma (14-3-3σ), DNA-dependent serine/threonine protein kinase (DNA-PK), O6-methylguanine-DNA methyltransferase (MGMT), p53 and MDC1 protein expressions but increased the protein expression of p53 and p-H2A.X. The protein translocation was examined by confocal laser microscopy and we found that kaempferol increased the levels of p-H2A.X and p-p53 in HL-60 cells. Taken together in, the present study, we found that kaempferol induced DNA damage and suppressed DNA repair and inhibited DNA repair associated protein expression in
HL-60 cells that may be the factors for kaempferol-induced cell death in vitro.
Keywords: Kaempferol; DNA damage; Comet assay; DNA repair; human leukemia HL-60 cells.
Introduction
Majority of anticancer drugs generate reactive oxygen species (ROS) to form oxidative stress for inducing cancer cell DNA damage and apoptosis and genomic instability . DNA damage leading to cancer cell death has been recognized to be a critical mechanism in cancer chemotherapy. It was reported that oxidizing agents can induce structural alterations and DNA mutations that lead to cancer and age-related disorders . Cells have developed systems to detect DNA damage, especially the DNA double-strand breaks (DSBs) and they also can repair these lesions. It was reported that tumor cells can go through DNA repair pathways to survive from chemotherapeutic caused DNA damage. Thus, it provides prognostic and/or predictive value .
Kaempferol is one of the flavonoids that have been studied for their chemopreventive functions and the vegetables and fruits that contain significant amounts of kaempferol are edible berries, grapefruit, and ginkgo biloba . Numerous studies have shown that the chemopreventive functions of kaempferol are involved in the induced cell death through the induction of apoptosis in many cancer cells such as breast cancer , lung Cancer , ovarian cancer , prostate cancer , oral cancer , glioblastoma and osteosarcoma cells . Moreover, it was reported that the consumption of kaempferol significantly reduced the risk of ovarian cancer in American female nurses .
It has been reported that kaempferol inhibits growth of human leukemia mast cells . Kaempferol induced significant concentration-dependent nuclear DNA degradation concurrent with lipid peroxidation . However, there is no available information to show that kaempferol induced DNA damage and affected DNA repair associated protein expression in human leukemia cells. Thus, in these studies, we investigated
the effect of kaempferol on DNA damage and repair associate protein expression in human leukemia HL-60 cells. The results indicated that kaempferol induced DNA damage and affected DNA repair associated protein expression in HL-60 cells.
Materials and methods Chemicals and reagents
Kaempferol, dimethyl sulfoxide (DMSO) (with final concentration never exceeding 0.1%), propidium iodide (PI) and Trypsin-EDTA were purchased from Sigma Chemical Co. (St. Louis, MO, USA). RPMI-1640 medium, fetal bovine serum (FBS), L-glutamine and penicillin-streptomycin were purchased from GIBCO®/Invitrogen Life Technologies (Carlsbad, California, USA). Primary antibody (p-ATM, anti-p53, anti-p-anti-p53,,anti-DNA-PK and anti-MGMT were obtained from Sigma Chemical Co. (St. Louis, MO, USA), anti-p-ATR was obtained from Cell Signaling (Danvers, MA, USA), anti-14-3-3σ was obtained from Merck and anti-DNA-PK was obtained from Calbiochem (San Diego, CA, USA), anti-p-H2A.X was obtained from GeneTex (Irvine, CA, USA). Kaempferol was dissolved in DMSO at a concentration of 100 mg/ml and were stored frozen under light-protected conditions at -20°C.
Cell culture
The HL-60 human promyelocytic leukemia cells were obtained from the Food Industry Research and Development Institute (Hsinchu, Taiwan) and were cultured in RPMI-1640 media supplemented with 10% FBS, 2 mM L-glutamine, 100 Units/ml penicillin and 100 μg/ml streptomycin in a 75 cm2 tissue culture flasks and grown at 37°C and 5% CO2 .
The HL-60 cells (2 x 105 cells/well) were placed in 12-well plates and then were incubated with 0, 25, 50, 75, 100 and 125 μM of kaempferol for 48 h. Cells from each treatment were harvested, centrifuged, washed and stained with PI (5 μg/ml) and then were analyzed by flow cytometry (Becton-Dickinson, San Jose, CA, USA) as described previously .
Comet assay
HL-60 cells (2 x 105 cells/well) were placed in 12-well plates and then were treated with 75 μM of kaempferol, vehicle (1 μl DMSO) and 0.1% of H2O2 (positive control) for 0, 6, 12, 24 and 48 h in RPMI-1640 medium. After incubation, all cells were harvested for examining the cell DNA damage by using Comet assay as described previously and then were examined and photographed using a fluorescence microscope at 200 x. The extent of DNA damage in individual cells from each treatment was evaluated by the percentage of tail DNA. Three independent experiments were conducted.
4',6-diamidino-2-phenylindole dihydrochloride (DAPI) staining
HL-60 cells (2 x 105 cells/well) were placed in 12-well plates and then were treated with 75 μM of kaempferol for 0, 6, 12, 24 and 48 h. Then, cells from each treatment were washed 2 times with PBS and fixed with 3.7% formaldehyde in PBS for 10 min at room temperature. After washing twice with PBS and staining with DAPI solution as described previously were then examined and photographed using a fluorescence microscope at 200 x.
DNA gel electrophoresis
previously described . HL-60 cells were incubated with or with kaempferol at final concentration 75 μM for 0, 6, 12, 24 and 48 h. Then, cells were harvested and lysed in 400 μl of ice-cold lysis buffer (containing 50 mM Tris–HCl, pH 7.5, 10 mM EDTA and 0.3% Triton X-100) for 30 min and then centrifuged and RNase A (100 μg/ml) was added to the supernatant and incubated at 50°C for 30 min. The 200 μg/ml proteinase K was added to supernatant and then incubated at 50°C for 1 h. DNA was extracted with phenol/chloroform and precipitated at -20°C with ethanol/sodium acetate and then were electrophoresed on a 1.5% agarose gel containing 0.1 μg/ml ethidium bromide (EtBr), and they were examined by photographed.
Western blotting
HL-60 cells (2 x 106 cells/well) were placed in 10 cm dish and treated with 75 μM of kaempferol for 0, 6, 12, 24 and 48 h. Cells were harvested and the total proteins from each treatment were determined by Bio-Rad assay Kit. Protein expressions from each treatment were measured by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblotting as described previously . After electrophoresis, samples were transferred to membrane followed by staining by primary antibodies such as anti-DNA-PK, MDC1, MGMT, p53, p53, ATM, p-ATR, 13-3-3σ and p-H2A.X, and then were washed and stained by horseradish peroxidase-conjugated anti-mouse secondary antibody. The membrane were then visualized with a chemiluminescent detection system as described by the manufacturer .
Confocal laser system microscopy
HL-60 cells (5 × 104 cells/well) were placed on 4-well chamber slides and then were incubated with 0 and 75 μM of kaempferol for 48 h. After incubation, cells were
fixed in 4% formaldehyde in PBS for 15 min and then were incubated with 0.3% Triton-X 100 in PBS for 1 h to permeable the cells. Then, cells were blocked non-specific binding sites by using 2% BSA as described previously . They by contained green fluorescence anti-p-p53 and anti-p-H2A.X were individually stained overnight. Then, all samples were washed twice with PBS followed by staining with FITC-conjugated goat anti-mouse IgG (secondary antibody) followed by mitotracker (red fluorescence) staining the nuclein. Then samples were examined and photomicrograph under a Leica TCS SP2 Confocal Spectral Microscope as described previously .
Statistical analysis
All samples were analyzed in three replications and results (data) are shown as mean ± S.D. The comparisons between kaempferol-treated and untreated (control) groups were performed by Student’s t-test. Statistical significant was determined *p < 0.05 and ***p < 0.001, which were considered significant.
Results
Kaempferol decrease the percentage of viable HL-60 cells
To investigate the effects of kaempferol on HL-60 cells, cells were treated with various concentration of kaempferol for 48 h and the percentage of viable cells were measured by flow cytometric assay. Results are shown in Figure 1, and data showed that kaempferol decreased the percentage of viable cells in HL-60 cells and these effects are dose-dependent.
Kaempferol-induced DNA damage in HL-60 cells
cell via the induction of DNA damage in HL-60 cells, DNA damage was examined by Comet assay and the results are shown in Figure 2A and B. Results from Figures 2A and B indicated that kaempferol induced DNA damage in HL-60 cells and these effects are dose-dependent. The higher concentrations of kaempferol, the longer comet tail (DNA migration smear) in HL-60 cells (Fig. 2A). Furthermore, when comet tail from each treatment were elevated, increased doses of kaempferol leading to more DNA damage were observed (Fig. 2B).
Kaempferol-induced DNA damage and condensation in HL-60 cells
Due to kaempferol decreasing the viable cells and inducing DNA in HL-60 cells being observed, we further assessed the DNA condensation in HL-60 cells after exposed to kaempferol by using fluorescent microscopy using the DNA-binding fluorescent dye DAPI and the results are shown in Figure 3. Results indicated that kaempferol induced DNA condensation in NCI-H460 cells and these effects are dose-dependent manners. Control cells displayed nuclei with homogeneous chromatin distribution and light staining by DAPI. However, kaempferol treated cells shown higher DAPI staining and lower cell number when compared to kaempferol untreated cells.
Kaempferol induced DNA fragmentation in HL-60 cells
After treatment of HL-60 cells with 75 μM kaempferol for 0, 6, 12, 24 and 48 h, the characteristic pattern of apoptotic internucleosomal DNA cleavage (DNA fragmentation) was visualized on agarose gel and the results are shown in Figure 4. DNA ladder (DNA fragmentation) was observed in HL-60 cells treated with various time doses of kaempferol for 75 μM (Fig. 4). However, it seems that higher time doses of kaempferol leading to higher DNA fragmentation indicated that kaempferol induced DNA damage in HL-60 cells.
Kaempferol affect DNA damage and repair associated proteins expressions in HL-60 cells
Results from the Figures 1, 2 and 3 indicated that kaempferol induced cell death are involved in induced DNA damage and condensation in HL-60 cells. Thus, to further investigate protein expressions associated with DNA damage and repair and whether or not they are affected by kaempferol, HL-60 cells were treated with 0 and 75 μM of kaempferol for various timer periods. All samples were examined by Western blotting and the results were shown in Figure 5, which showed that kaempferol inhibited the protein expressions of DNA-PK, MDC1, MGMT and p53 (Fig. 5A), p-ATM, p-ATR and 14-3-3σ (Fig. 5B) but increased p-p53 (Fig. 5A) and p-H2A.X (Fig. 5B) in 6-48 h treatment.
Kaempferol affect p-p53 and p-H2AX expression and translocation in HL-60 cells Reults from Western blotting (Fig. 5A and B) indicated that kaempferol increased the protein expression of p-p53 and p-H2A.X in HL-60 cells, and we further investigated whether or not they involved the translocation of those proteins. Thus, confocal laser microscope was used and the results shown in Figure 6A and B. The results demonstrated that kaempferol increased the expression of the p53 (Fig. 6A) and p-H2A.X (Fig. 6B) in HL-60 cells when compared to control groups.
Discussion
Although numerous studies have shown that kaempferol induced cell death through the induction of apoptosis in different types of human cancer cells, furthermore, it was reported that kaempferol inhibits growth of human leukemia mast cells and it induced significant concentration-dependent nuclear DNA degradation concurrent with lipid
peroxidation . However, the effects of kaempferol on DNA damage and repair in human leukemia cells are still unclear. Thus, in the present studies, we investigate the effects of kaempferol on DNA damage and repair in human leukemia cells in vitro. At first, we found that kaempferol induced cell death dose-dependently (Fig. 1) just as in other reports. Then, we used Comet assay and DAPI staining method to examine the occurrence of DNA damage and condensation and the result indicated that kaempferol induced DNA damage (Fig. 2) and condensation (Fig. 3), respectively, and these effects are dose-dependent.
HL-60 cells were treated with various doses of kaempferol for 48 h and then were examined for DNA damage by Comet assay based on the comet tail production and results from Figure 2 indicated a significant increase in the tail moment of the comets of HL-60 cells, with the longer comet tail meaning higher DNA damage (Fig. 2). Comet assay is a single cell electrophoresis which has been recognized to be a significant and sensitive technique for measuring DNA damage and trend break formation . It is well document that DAPI staining is a suitable measurement of DNA condensation and the results from figure 3 indicated increased doses of kaempferol led to increase the DNA condensation.
DNA repair systems are critical for cells maintaining to survival after agent induced DNA damage in cells and these systems can go through eliminating DNA lesions or added new DNA based . The ataxia-telangiectasia mutated (ATM) protein is recognized to be the major regulator of the cellular response to DNA double-strand breaks . Furthermore, when double-stranded DNA breaks occur in cells, it then can activate two master checkpoint kinases (ATM and ATR) for cells to maintain genomic integrity. Herein, we found that kaempferol inhibited the protein expressions such as p-ATM, p-ATR, 14-3-3σ, DNA-PK, MGMT, p53 and MDC1 that are
associated with DNA damage and repair in examined HL-60 cells (Fig. 5). Several reports have shown that ATM can be activated by double-stranded DNA breaks occur in DNA and ATM also can be phosphorylated by p53, H2A.X, breast cancer susceptibility protein 1 (BRCA1) and p53 binding protein 1 (53BP1). It then will lead to cell cycle arrest, DNA repair or induction of cell apoptosis . Figure 5 showed that kaempferol promoted the levels of p-p53 in HL-60 cells that is reasonable because it is well documented that when agent induced cell DNA damage, it will induce p-p53 expression . Thus, the expression of BRCA1, and p53 proteins significantly decreased after treatment with kaempferol in HL-60 cells. H2A.X is a critical factor for the efficient accumulation of DNA repair factors at the break site of DNA and H2A.X deficient mice will have higher radiosensitivity . Tumor suppressor BRCA-1 is also involved in DNA repair and maintenance of genomic stability .
Results from Figure 5 also showed that kaempferol significantly suppressed the protein expression of DNA-dependent protein kinase (DNA-PK) in HL-60 cells and DNA-PK has been reported to involve DNA damage repair . Furthermore, DNA damage can elicit the pan-nuclear activation of ATM and DNA–PK . Based on those observations, we suggest that kaempferol induced cell death my via the inhibition of DNA-PK and p-ATM in HL-60 cells. It was reported that O6-methylguanine DNA methyltransferase (MGMT) reduces cytotoxicity of therapeutic agents and promoted MGMT activity will decrease the responses to temozolomide drug and overexpressed human MGMT in tumorigenic and mice will lead to being against methylating agent-induced tumor formation .
Taken together, kaempferol induced DNA damage and inhibited DNA repair protein expression in HL-60 cells such as p-ATM, p-ATR, BRCA-1, 14-3-3σ, DNA-PK and MGMT and then led to DNA damage and these effects are summarized in
Figure 7.
Acknowledgement
This study is supported in part by a research grant from China Medical University [CMU101-ASIA-09]. Experiments and data analysis were performed in part through the use of the Medical Research Core Facilities Center, Office of Research & Development at China medical University, Taichung, Taiwan, R.O.C.
References
Aiyer, H.S., Warri A.M., Woode D.R., Hilakivi-Clarke L. and Clarke R. Influence of Berry Polyphenols on Receptor Signaling and Cell-Death Pathways: Implications for Breast Cancer Prevention. J Agric Food Chem. 60: 5693-5708, 2012.
Alexandrakis, M., Letourneau R., Kempuraj D., Kandere-Grzybowska K., Huang M., Christodoulou S., Boucher W., Seretakis D. and Theoharides T.C. Flavones Inhibit Proliferation and Increase Mediator Content in Human Leukemic Mast Cells (Hmc-1). Eur J Haematol. 71: 448-454, 2003.
Anderson, D., Yu T.W., Phillips B.J. and Schmezer P. The Effect of Various Antioxidants and Other Modifying Agents on Oxygen-Radical-Generated DNA Damage in Human Lymphocytes in the Comet Assay. Mutat Res. 307: 261-271, 1994.
Celeste, A., Fernandez-Capetillo O., Kruhlak M.J., Pilch D.R., Staudt D.W., Lee A., Bonner R.F., Bonner W.M. and Nussenzweig A. Histone H2ax Phosphorylation Is Dispensable for the Initial Recognition of DNA Breaks.
Nat Cell Biol. 5: 675-679, 2003.
Celeste, A., Petersen S., Romanienko P.J., Fernandez-Capetillo O., Chen H.T., Sedelnikova O.A., Reina-San-Martin B., Coppola V., Meffre E., Difilippantonio M.J., Redon C., Pilch D.R., Olaru A., Eckhaus M., Camerini-Otero R.D., Tessarollo L., Livak F., Manova K., Bonner W.M., Nussenzweig M.C. and Nussenzweig A. Genomic Instability in Mice Lacking Histone H2ax. Science. 296: 922-927, 2002.
Chang, Y.-M., Velmurugan B.K., Kuo W.-W., Chen Y.-S., Ho T.-J., Tsai C.-T., Ye C.-X., Tsai C.-H., Tsai F.-J. and Huang C.-Y. Inhibitory Effect of Alpinate Oxyphyllae Fructus Extracts on Ang Ii-Induced Cardiac Pathological
Remodeling-Related Pathways in H9c2 Cardiomyoblast Cells. BioMedicine. 3: 148-152, 2013.
Choi, J.H., Sancar A. and Lindsey-Boltz L.A. The Human Atr-Mediated DNA Damage Checkpoint in a Reconstituted System. Methods. 48: 3-7, 2009. Dejmek, J., Iglehart J.D. and Lazaro J.B. DNA-Dependent Protein Kinase
(DNA-Pk)-Dependent Cisplatin-Induced Loss of Nucleolar Facilitator of Chromatin Transcription (Fact) and Regulation of Cisplatin Sensitivity by DNA-Pk and Fact. Mol Cancer Res. 7: 581-591, 2009.
Dumenco, L.L., Allay E., Norton K. and Gerson S.L. The Prevention of Thymic Lymphomas in Transgenic Mice by Human O6-Alkylguanine-DNA Alkyltransferase. Science. 259: 219-222, 1993.
Friedman, H.S., McLendon R.E., Kerby T., Dugan M., Bigner S.H., Henry A.J., Ashley D.M., Krischer J., Lovell S., Rasheed K., Marchev F., Seman A.J., Cokgor I., Rich J., Stewart E., Colvin O.M., Provenzale J.M., Bigner D.D., Haglund M.M., Friedman A.H. and Modrich P.L. DNA Mismatch Repair and O6-Alkylguanine-DNA Alkyltransferase Analysis and Response to Temodal in Newly Diagnosed Malignant Glioma. J Clin Oncol. 16: 3851-3857, 1998. Gates, M.A., Tworoger S.S., Hecht J.L., De Vivo I., Rosner B. and Hankinson S.E. A
Prospective Study of Dietary Flavonoid Intake and Incidence of Epithelial Ovarian Cancer. Int J Cancer. 121: 2225-2232, 2007.
Hakkinen, S.H., Karenlampi S.O., Heinonen I.M., Mykkanen H.M. and Torronen A.R. Content of the Flavonols Quercetin, Myricetin, and Kaempferol in 25 Edible Berries. J Agric Food Chem. 47: 2274-2279, 1999.
Hegi, M.E., Diserens A.C., Gorlia T., Hamou M.F., de Tribolet N., Weller M., Kros J.M., Hainfellner J.A., Mason W., Mariani L., Bromberg J.E., Hau P., Mirimanoff R.O., Cairncross J.G., Janzer R.C. and Stupp R. Mgmt Gene Silencing and Benefit from Temozolomide in Glioblastoma. N Engl J Med. 352: 997-1003, 2005.
Hsia, T.C., Lin J.H., Hsu S.C., Tang N.Y., Lu H.F., Wu S.H., Lin J.G. and Chung J.G. Cantharidin Induces DNA Damage and Inhibits DNA Repair-Associated Protein Levels in Nci-H460 Human Lung Cancer Cells. Environ Toxicol. 2014.
Huang, W.W., Chiu Y.J., Fan M.J., Lu H.F., Yeh H.F., Li K.H., Chen P.Y., Chung J.G. and Yang J.S. Kaempferol Induced Apoptosis Via Endoplasmic Reticulum Stress and Mitochondria-Dependent Pathway in Human Osteosarcoma U-2 Os Cells. Mol Nutr Food Res. 54: 1585-1595, 2010.
Jesien-Lewandowicz, E., Jesionek-Kupnicka D., Zawlik I., Szybka M., Kulczycka-Wojdala D., Rieske P., Sieruta M., Jaskolski D., Och W., Skowronski W.,
Sikorska B., Potemski P., Papierz W., Liberski P.P. and Kordek R. High Incidence of Mgmt Promoter Methylation in Primary Glioblastomas without Correlation with Tp53 Gene Mutations. Cancer Genet Cytogenet. 188: 77-82, 2009.
Kang, J.W., Kim J.H., Song K., Kim S.H., Yoon J.H. and Kim K.S. Kaempferol and Quercetin, Components of Ginkgo Biloba Extract (Egb 761), Induce Caspase-3-Dependent Apoptosis in Oral Cavity Cancer Cells. Phytother Res. 24 Suppl 1: S77-82, 2010.
Kaur, P., Shukla S. and Gupta S. Plant Flavonoid Apigenin Inactivates Akt to Trigger Apoptosis in Human Prostate Cancer: An in Vitro and in Vivo Study.
Carcinogenesis. 29: 2210-2217, 2008.
Kelly, J.A., Lucia M.S. and Lambert J.R. P53 Controls Prostate-Derived Factor/Macrophage Inhibitory Cytokine/Nsaid-Activated Gene Expression in Response to Cell Density, DNA Damage and Hypoxia through Diverse Mechanisms. Cancer Lett. 277: 38-47, 2009.
Khansari, N., Shakiba Y. and Mahmoudi M. Chronic Inflammation and Oxidative Stress as a Major Cause of Age-Related Diseases and Cancer. Recent Pat
Inflamm Allergy Drug Discov. 3: 73-80, 2009.
Lai, W.W., Hsu S.C., Chueh F.S., Chen Y.Y., Yang J.S., Lin J.P., Lien J.C., Tsai C.H. and Chung J.G. Quercetin Inhibits Migration and Invasion of Sas Human Oral Cancer Cells through Inhibition of Nf-Kappab and Matrix Metalloproteinase-2/-9 Signaling Pathways. Anticancer Res. 33: 1941-1950, 2013.
Lau, A.T., Wang Y. and Chiu J.F. Reactive Oxygen Species: Current Knowledge and Applications in Cancer Research and Therapeutic. J Cell Biochem. 104: 657-667, 2008.
Lee, H.Z., Liu W.Z., Hsieh W.T., Tang F.Y., Chung J.G. and Leung H.W. Oxidative Stress Involvement in Physalis Angulata-Induced Apoptosis in Human Oral Cancer Cells. Food Chem Toxicol. 47: 561-570, 2009.
Lin, M.-C., Tsai S.-Y., Wang F.-Y., Liu F.-H., Syu J.-N. and Tang F.-Y. Leptin Induces Cell Invasion and the Upregulation of Matrilysin in Human Colon Cancer Cells. BioMedicine. 3: 174-180, 2013.
Liu, K.C., Ho H.C., Huang A.C., Ji B.C., Lin H.Y., Chueh F.S., Yang J.S., Lu C.C., Chiang J.H., Meng M. and Chung J.G. Gallic Acid Provokes DNA Damage and Suppresses DNA Repair Gene Expression in Human Prostate Cancer Pc-3 Cells. Environ Toxicol. 28: 579-587, 2013.
Lou, Z., Chini C.C., Minter-Dykhouse K. and Chen J. Mediator of DNA Damage Checkpoint Protein 1 Regulates Brca1 Localization and Phosphorylation in DNA Damage Checkpoint Control. J Biol Chem. 278: 13599-13602, 2003.
Lu, C.C., Yang J.S., Chiang J.H., Hour M.J., Lin K.L., Lin J.J., Huang W.W., Tsuzuki M., Lee T.H. and Chung J.G. Novel Quinazolinone Mj-29 Triggers Endoplasmic Reticulum Stress and Intrinsic Apoptosis in Murine Leukemia Wehi-3 Cells and Inhibits Leukemic Mice. PLoS One. 7: e36831, 2012.
Luo, H., Rankin G.O., Li Z., Depriest L. and Chen Y.C. Kaempferol Induces Apoptosis in Ovarian Cancer Cells through Activating P53 in the Intrinsic Pathway. Food Chem. 128: 513-519, 2011.
Meyer, B., Voss K.O., Tobias F., Jakob B., Durante M. and Taucher-Scholz G. Clustered DNA Damage Induces Pan-Nuclear H2ax Phosphorylation Mediated by Atm and DNA-Pk. Nucleic Acids Res. 41: 6109-6118, 2013. Mi, J., Dziegielewski J., Bolesta E., Brautigan D.L. and Larner J.M. Activation of
DNA-Pk by Ionizing Radiation Is Mediated by Protein Phosphatase 6. PLoS
One. 4: e4395, 2009.
Miean, K.H. and Mohamed S. Flavonoid (Myricetin, Quercetin, Kaempferol, Luteolin, and Apigenin) Content of Edible Tropical Plants. J Agric Food
Chem. 49: 3106-3112, 2001.
Nguyen, T.T., Tran E., Ong C.K., Lee S.K., Do P.T., Huynh T.T., Nguyen T.H., Lee J.J., Tan Y., Ong C.S. and Huynh H. Kaempferol-Induced Growth Inhibition and Apoptosis in A549 Lung Cancer Cells Is Mediated by Activation of Mek-Mapk. J Cell Physiol. 197: 110-121, 2003.
Olive, P.L., Banath J.P. and Durand R.E. Detection of Etoposide Resistance by Measuring DNA Damage in Individual Chinese Hamster Cells. J Natl Cancer
Inst. 82: 779-783, 1990.
Pan, M.H. and Ho C.T. Chemopreventive Effects of Natural Dietary Compounds on Cancer Development. Chem Soc Rev. 37: 2558-2574, 2008.
Pool-Zobel, B.L., Lotzmann N., Knoll M., Kuchenmeister F., Lambertz R., Leucht U., Schroder H.G. and Schmezer P. Detection of Genotoxic Effects in Human Gastric and Nasal Mucosa Cells Isolated from Biopsy Samples. Environ Mol
Mutagen. 24: 23-45, 1994.
Sahu, S.C. and Gray G.C. Kaempferol-Induced Nuclear DNA Damage and Lipid Peroxidation. Cancer Lett. 85: 159-164, 1994.
Santarpia, L., Iwamoto T., Di Leo A., Hayashi N., Bottai G., Stampfer M., Andre F., Turner N.C., Symmans W.F., Hortobagyi G.N., Pusztai L. and Bianchini G. DNA Repair Gene Patterns as Prognostic and Predictive Factors in Molecular Breast Cancer Subtypes. Oncologist. 18: 1063-1073, 2013.
Sathyamoorthy, N., Wang T.T. and Phang J.M. Stimulation of Ps2 Expression by Diet-Derived Compounds. Cancer Res. 54: 957-961, 1994.
Induces Apoptosis in Glioblastoma Cells through Oxidative Stress. Mol
Cancer Ther. 6: 2544-2553, 2007.
Shiloh, Y. Atm and Related Protein Kinases: Safeguarding Genome Integrity. Nat
Rev Cancer. 3: 155-168, 2003.
Shiotani, B. and Zou L. Single-Stranded DNA Orchestrates an Atm-to-Atr Switch at DNA Breaks. Mol Cell. 33: 547-558, 2009.
Stiff, T., O'Driscoll M., Rief N., Iwabuchi K., Lobrich M. and Jeggo P.A. Atm and DNA-Pk Function Redundantly to Phosphorylate H2ax after Exposure to Ionizing Radiation. Cancer Res. 64: 2390-2396, 2004.
Tice, R.R., Andrews P.W. and Singh N.P. The Single Cell Gel Assay: A Sensitive Technique for Evaluating Intercellular Differences in DNA Damage and Repair. Basic Life Sci. 53: 291-301, 1990.
Venkitaraman, A.R. Cancer Susceptibility and the Functions of Brca1 and Brca2.
Cell. 108: 171-182, 2002.
Wang, B., Matsuoka S., Carpenter P.B. and Elledge S.J. 53bp1, a Mediator of the DNA Damage Checkpoint. Science. 298: 1435-1438, 2002.
Wu, C.L., Huang A.C., Yang J.S., Liao C.L., Lu H.F., Chou S.T., Ma C.Y., Hsia T.C., Ko Y.C. and Chung J.G. Benzyl Isothiocyanate (Bitc) and Phenethyl Isothiocyanate (Peitc)-Mediated Generation of Reactive Oxygen Species Causes Cell Cycle Arrest and Induces Apoptosis Via Activation of Caspase-3, Mitochondria Dysfunction and Nitric Oxide (No) in Human Osteogenic Sarcoma U-2 Os Cells. J Orthop Res. 29: 1199-1209, 2011.
Wu, S.H., Wu T.Y., Hsiao Y.T., Lin J.H., Hsu S.C., Hsia T.C., Yang S.T., Hsu W.H. and Chung J.G. Bufalin Induces Cell Death in Human Lung Cancer Cells through Disruption of DNA Damage Response Pathways. Am J Chin Med. 42: 729-742, 2014.
Yu, C.S., Huang A.C., Yang J.S., Yu C.C., Lin C.C., Chung H.K., Huang Y.P., Chueh F.S. and Chung J.G. Safrole Induces G0/G1 Phase Arrest Via Inhibition of Cyclin E and Provokes Apoptosis through Endoplasmic Reticulum Stress and Mitochondrion-Dependent Pathways in Human Leukemia Hl-60 Cells.
Anticancer Res. 32: 1671-1679, 2012.
Zaidi, N.H., Pretlow T.P., O'Riordan M.A., Dumenco L.L., Allay E. and Gerson S.L. Transgenic Expression of Human Mgmt Protects against Azoxymethane-Induced Aberrant Crypt Foci and G to a Mutations in the K-Ras Oncogene of Mouse Colon. Carcinogenesis. 16: 451-456, 1995.
Figure legends
Figure 1. Kaempferol decreased the viablility of human leukemia HL-60 cells. Cells (2x105cells/well) were placed in 24-well plates and then were incubated with kaempferol 0, 25, 50, 75, 100 and 125 μM and vehicle (1 μl DMSO) for 24 h. All samples were stained with PI (5 μg/ml) and analyzed by flow cytometry as previously described. *P<0.05 was considered significant.
Figure 2. Kaempferol -induced DNA damage in HL-60 cells was examined by Comet assay. Cells (2x105cells/well) were placed in 24-well plates and then were incubated with kaempferol (75 μM) and vehicle (1 μl DMSO) for 0, 6, 12, 24 and 48 h and DNA damage was determined by Comet assay as described in Materials and Methods. A: representative picture of Comet assay; B: Comet length (folds of control). The 0.1%H2O2 was acted as positive control. Arrow showing the comet tail (DNA damage). *P<0.05 and ***P<0.001 were considered significant.
Figure 3. Kaempferol-induced DNA condension in HL-60 cells was examined by 4,6-diamidino-2-phenylindole dihydrochloride (DAPI) staining. Cells (1×105 cells/well) were placed in 12-well plates for 24 h and were treated with 75 μM of kaempferol for 0, 6, 12, 24 and 48 h. Then cells in each treatment were DAPI stained as described in Materials and Methods. Cells were examined and photographed using a fluorescence microscope at 200x. *P<0.05 and ***P<0.001 were considered significant.
Figure 4. Kaempferol induced DNA fragmentation in HL-60 cells. Cells (5×105 cells/well) were placed in 12-well plates for 24 h and were treated with 75μM of kaempferol for 0, 6, 12, 24 and 48 h DNA was extract and DNA gel electrophoresis was performed and the characteristic pattern of apoptotic internucleosomal DNA cleavage (DNA fragmentation) was visualized.
Figure 5. Kaempferol affected the protein expression associated with DNA damage and repair in HL-60 cells. Cells (5x105 cells/well) were placed in 12-well plates and then were incubated with 75 μM of kaempferol for 0, 6, 12, 24 and 48 h. The total proteins were collected and determined by Bio-Rad assay Kit. The amounts of proteins from each treatment were measured by SDS-PAGE and immunoblotting as in Materials and methods. A: DNA-PK, MDC1, MGMT, p53 and p53; B: ATM, p-ATR, 13-3-3a and p-H2A.x
cells were examined by Confocal laser system microscopy. Cells (5× 104 cells/well) were kept on 4-well chamber slides and were incubated with 75 μM of kaempferol for 24 h and then were fixed in 4% formaldehyde in PBS for 15 min and incubated with 0.3% Triton-X 100 in PBS for 1 h and then immunostaining were performed as described in Materials and Methods. A: p-PH2AX; B: p-p53 examined and photo-micrographed under a Leica TCS SP2 Confocal Spectral Microscope.
Figure 7. The possible flow chart for kaempferol-induced DNA damage and inhibit DNA repair associated proteins expression in human leukemia HL-60 cells.
Figure 5 Figure 4
(B) (A)
