Cadmium Induces Cell Cycle Arrest and Change in Expression of Cell Cycle Related Proteins in Breast Cancer Cell Lines

Lee Young Joo,Kang Tae Seok,Kim Tae Sung,Moon Hyun Ju,Kang Il Hyun,Oh Ji Young,Kwon Hoonjeong,Han Soon Young Korean Society of ToxicologyKorea Environmental Mu 2005 Toxicological Research Vol.21 No.1

Cadmium is an environmental pollutant exposed from contaminated foods or cigarette smoking and known to cause oxidative damage in organs. We investigated the cadmium-induced apoptosis and cell arrest in human breast cancer cells, MCF-7 cells and MDA-MB-231 cells. Obvious apoptotic cell death was shown in CdCl₂ 100 μM treatment for 12 hr, which were determined by DAPI staining and flow cytometric analysis. In cell cycle analysis, MCF-7 cells and MDA-MB-231 cells were arrested in S phase and G2/M phase respectively. These could be explained by the induction of cell cycle inhibitory protein, p21/sup Waf1/Cip1/ and p27/sup Kip1/, expression and reduction of cyclin/Cdk complexes in both cell lines. The decreased expression of cyclin A and Cdk2 in MCF-7 cells and cyclin B1 and Cdc2 in MDA-MB-231 cells were consistent with the flow cytometric observation. p-ERK expression was increased dose-dependent manner in both cell lines. It suggests that ERK MAPK pathway are involved in cadmium-induced cell cycle arrest and apoptosis. Moreover, cotreatment of zinc (100 μM, 12 hr) recovered the cadmium-induced cell arrest in both cells, which shows cadmium-induced oxidative stress mediates apoptosis and cell cycle arrest in human breast cancer cells.

MCF-7 세포주에서 Staurosporine 투여에 의한 세포주기 분기 변화와 p53단백 발현

남정(Jung Nam),여경아(Kyung A Yea),이해남(Hae Nam Lee),조현희(Hyun Hee Jo),류기성(Ki Sung Ryu),유영옥(Young Oak Lew),나종구(Jong Gu Rha),한구택(Ku Taek Han) 대한산부인과학회 2001 Obstetrics & Gynecology Science Vol.44 No.3

N/A Objective : We investigated the effects on the cell cycle and p53 expression by the treatment of various concentrations of staurosporine to elucidate the molecular mechanism of staurosporine induced cell cycle arrest in MCF-7 cell line. Methods : Various concentrations of staurosporine were treated in MCF-7 cells cultured with RPMI 1640 media. The harvested cells were fixed and permeabilized with 1% paraformaldehyde and absolute methanol. Then the cells were stained indirectly with anti-p53 primary antibody and FITC conjugated goat anti-mouse(GAM)-IgG secondary antibody. Sequentially DNA were stained with 0.1% RNase and PI solution. These stained cells were analyzed by the standard FACScan flow cytometer. The obtained results were analyzed further with WinList 3.0, and ModiFit LT software program. Results : MCF-7 cells were arrested mostly in G1 phase of cell cycle at 5-10 nM of staurosporine, however, the cells were arrested in G2 phase at 20-100 nM of staurosporine. The expressions of p53 protein were higher in the MCF-7 cells treated with both concentrations of 10 nM and 100 nM of staurosporine compaired with the control cells. This suggests that the p53 may be involved in the mechanism of G1 and G2M arrest of cell cycle in MCF-7 cell. Conclusion : The points of arrest in cell cycle differred depending on the concentrations of staurosporine and these cell cycle arrests at G0G1 and G2M pahse were related with p53 protein expression. It suggested that these results could be extended to study for staurosporine to be usefull as a potential anti-tumor agent.

NADPH oxidase inhibitor diphenyleneiodonium induces p53 expression and cell cycle arrest in several cancer cell lines

조홍재,김강미,송주동,박영철,Jo, Hong-Jae,Kim, Kang-Mi,Song, Ju-Dong,Park, Young-Chul Korean Society of Life Science 2007 생명과학회지 Vol.17 No.6

Diphenyleneiodonium (DPI)는 NADPH oxidase 같은 flavoenzymes의 저해제로써 널리 사용되고 있다. 본 연구에서는 인간 대장암 세포주 HCT-116 (wild-type p53)와 HT-29 (p53 mutant) 및 인간 유방암 세포주인 MCF-7(wild-type p53)의 세포성장 과정에서의 DPI의 효과를 살펴보았다. DPI는 농도 및 시간 의존적으로 암세포주의성장을 막았으며 G2/M phase에서 cell cycle arrest를 일으켰다. Cell cycle arrest의 가장 높은 값은 DPI 처리후 12 시간에서 관찰할 수 있었다. 한편 DPI는 아폽토시스 그리고 cell cycle arres 에 관여하는 유전자 발현에 관여하는 p53의 표현을 크게 증가시켰으며, 이는 DPI처리 후 6시간 후 부터 관찰할 수 있었다. 그러나 NADPH oxidase의 조합을 억제하는 catechol 계인 apocynin은 p53의 발현을 유도하지 못하였다. 이것은 DPI에 의해 유도되는 p53의 발현증가는 NADPH oxidase활성의 저해와 관련되어 있지 않다는 것을 의미한다. 결론적으로 DPI는 HCT-116, HCT-15 및 MCF-7 암세포주에서 ROS에 비 의존적으로 wild-type p53 발현의 증가를 유도하며, 이 증가된 p53은 DPI에 의해 유도되는 성장 억제 및 C2/M phase에서의 cell cycle arrset과정의 조절기전에 관여한다는 것을 시사한다. The Diphenyleneiodonium (DPI) is widely used as an inhibitor of flavoenzymes, particularly NADPH oxidase. In this study, we investigated the effect of DPI on the cell growth progression of human colon cancer cells HCT-116 (wild-type p53), HT-29 (p53 mutant) and human breast cancer cells MCF-7 (wild-type p53). DPI treatment in cancer cells evoked a dose- and time-dependent growth inhibition, and also induced the cell cycle arrest in C2/M phase. The peak of cell population arrested in C2/M phase was observed at12 hr after treatment of DPI. In addition, DPI significantly induced the expression of p53, which induces proapoptotic genes in response to DNA damage or irreparable cell cycle arrest, at 6 hr in DPI-stimulated cells. However, a catechol apocynin, which inhibits the assembly of NADPH oxidase, did not induce p53 expression. This suggest that p53 expression induced by DPI is not associated with the inhibition of NADPH oxidase. In conclusion, we suggest that DPI induces the expression of wild-type p53 by ROS-in-dependent mechanism in several cancer cells, and upregulated p53 may be involved in regulatory mechanisms for growth inhibition and cell cycle arrest at C2/M phase in DPI-stimulated cells.

Bleomycin Inhibits Proliferation via Schlafen-Mediated Cell Cycle Arrest in Mouse Alveolar Epithelial Cells

( Soojin Jang ),( Se Min Ryu ),( Jooyeon Lee ),( Hanbyeol Lee ),( Seok-ho Hong ),( Kwon-soo Ha ),( Won Sun Park ),( Eun-taek Han ),( Se-ran Yang ) 대한결핵 및 호흡기학회 2019 Tuberculosis and Respiratory Diseases Vol.82 No.2

Background: Idiopathic pulmonary fibrosis involves irreversible alveolar destruction. Although alveolar epithelial type II cells are key functional participants within the lung parenchyma, how epithelial cells are affected upon bleomycin (BLM) exposure remains unknown. In this study, we determined whether BLM could induce cell cycle arrest via regulation of Schlafen (SLFN) family genes, a group of cell cycle regulators known to mediate growth-inhibitory responses and apoptosis in alveolar epithelial type II cells. Methods: Mouse AE II cell line MLE-12 were exposed to 1-10 μg/mL BLM and 0.01-100 μM baicalein (Bai), a G1/G2 cell cycle inhibitor, for 24 hours. Cell viability and levels of pro-inflammatory cytokines were analyzed by MTT and enzymelinked immunosorbent assay, respectively. Apoptosis-related gene expression was evaluated by quantitative real-time reverse transcription-polymerase chain reaction (qRT-PCR). Cellular morphology was determined after DAPI and Hoechst 33258 staining. To verify cell cycle arrest, propidium iodide (PI) staining was performed for MLE-12 after exposure to BLM. Results: BLM decreased the proliferation of MLE-12 cells. However, it significantly increased expression levels of interleukin 6, tumor necrosis factor α, and transforming growth factor β1. Based on Hoechst 33258 staining, BLM induced condensation of nuclear and fragmentation. Based on DAPI and PI staining, BLM significantly increased the size of nuclei and induced G2/M phase cell cycle arrest. Results of qRT-PCR analysis revealed that BLM increased mRNA levels of BAX but decreased those of Bcl2. In addition, BLM/Bai increased mRNA levels of p53, p21, SLFN1, 2, 4 of Schlafen family. Conclusion: BLM exposure affects pulmonary epithelial type II cells, resulting in decreased proliferation possibly through apoptotic and cell cycle arrest associated signaling.

Dehydroabietic acid inhibits the gastric cancer cell growth via induced apoptosis and cell cycle arrest

김원진,Kang Hyeon-Gu,김석준 대한독성 유전단백체 학회 2021 Molecular & cellular toxicology Vol.17 No.2

Background Gastric cancer is one of the serious malignant tumors with high incidence worldwide. The two commonly used treatment strategies for gastric cancer include chemotherapy and radiation therapy. Conventional anticancer drugs have limited effectiveness. Therefore, it is important to explore new anticancer drugs. Dehydroabietic acid (DAA) is known to have anti-bacterial, anti-inflammatory, and anticancer effects. However, there are fewer reports on the effect of DAA on human gastric cancer cell lines. Objective We investigated the gastric cell growth inhibitory effects of DAA on human gastric cancer cell lines. Gastric cancer cell proliferation, apoptosis and cell cycle arrest assay were detected by WST assay, crystal violet staining assay, Flow cytometry, RT-PCR and western blot analysis. Results These results showed that as the concentration of DAA increased, the proliferation inhibitory effect appeared in gastric cancer cells. In addition, DAA induced sub-G1 phase accumulation and G1 cell cycle arrest, and subsequently induced apoptosis in AGS and YCC-2 cells. In addition, the expression of the pro-apoptotic Bax was upregulated, and that of the anti-apoptotic Bcl-2 was downregulated in DAA-treated AGS and YCC-2 cells. Moreover, DAA induced the cleavage of caspase-3 and PARP. Conclusion These results suggest that DAA induces cell death through mitochondria-mediated apoptosis and G1 cell cycle arrest. Therefore, our results indicate the anticancer and therapeutic potential of DAA for treatment of human gastric cancer. Background Gastric cancer is one of the serious malignant tumors with high incidence worldwide. The two commonly used treatment strategies for gastric cancer include chemotherapy and radiation therapy. Conventional anticancer drugs have limited effectiveness. Therefore, it is important to explore new anticancer drugs. Dehydroabietic acid (DAA) is known to have anti-bacterial, anti-inflammatory, and anticancer effects. However, there are fewer reports on the effect of DAA on human gastric cancer cell lines. Objective We investigated the gastric cell growth inhibitory effects of DAA on human gastric cancer cell lines. Gastric cancer cell proliferation, apoptosis and cell cycle arrest assay were detected by WST assay, crystal violet staining assay, Flow cytometry, RT-PCR and western blot analysis. Results These results showed that as the concentration of DAA increased, the proliferation inhibitory effect appeared in gastric cancer cells. In addition, DAA induced sub-G1 phase accumulation and G1 cell cycle arrest, and subsequently induced apoptosis in AGS and YCC-2 cells. In addition, the expression of the pro-apoptotic Bax was upregulated, and that of the anti-apoptotic Bcl-2 was downregulated in DAA-treated AGS and YCC-2 cells. Moreover, DAA induced the cleavage of caspase-3 and PARP. Conclusion These results suggest that DAA induces cell death through mitochondria-mediated apoptosis and G1 cell cycle arrest. Therefore, our results indicate the anticancer and therapeutic potential of DAA for treatment of human gastric cancer.

Hep3B 간암세포에서 개똥쑥 추출물에 의한 Cell Cycle Arrest 효과

김은지(Eun Ji Kim),김근태(Guen Tae Kim),김보민(Bo Min Kim),임은경(Eun Gyeong Lim),김상용(Sang Yong Kim),하성호(Sung Ho Ha),김영민(Young Min Kim),유제근(Je-Geun Yoo) 한국생물공학회 2015 KSBB Journal Vol.30 No.4

Cells proliferate via repeating process that growth and division. This process is G1, S, G2 and M four phases consists. Monitoring the progression of the cell cycle is a specific step that to be a continuous process is repeated to adjust the start of the next step. At this time, this process is called a Checkpoint. Currently, there are three known checkpoints that G1-S phase, G2-M phase, and the M phase. In this study, we confirmed that cell cycle arrest effects by ethanol extracts of Artemisia annua Linne (AAE) in Hep3B liver cancer cells. AAE was regulated proteins which involved in cell cycle such as pAkt, pMDM2, p53, p21, pCDK2 (T14/Y15). AAE induced cell cycle arrest in G1 checkpoint through phosphorylation of CDK2. Akt and p53 upstream is inhibited by AAE and p53 activated by non-activated pMDM2, p53 inhibitor. Thereby, activated p53 is transcript to p21 and activated p21 protein is combined with Cyclin E-pCDK2 complex. Therefore, we confirmed that AAE-induced cell cycle arrest was occurred by p21-Cyclin E-pCDK2 complex by inhibition of pAkt signal. Because of this cell cycle can’t pass to S phase from G1 phase.

Induction apoptosis and G2/M phase cell cycle arrest by non-thermal plasma in human osteosarcoma MG-63 cell line

( Byul Bo Ra Choi ),( Gyoo Cheon Kim ) 조선대학교 구강생물학연구소 2017 Oral Biology Research (Oral Biol Res) Vol.41 No.4

Osteosarcoma is a malignant bone tumor that develops in the osteoblast cells that form the outer covering of bone. It is the most frequent malignant bone tumor found at 10-14 years and at >65 years of age. Atmospheric pressure plasma has advantages of high density and rich chemical agents without elevation of the substrate temperature. These non-equilibrium characteristics show promising applications in the biomedical field, opening a new research area called “Plasma Medicine”, which includes sterilization, coagulation, wound healing, and cancer treatment. However, the mechanism of apoptosis caused by plasma treatment in osteosarcoma is not well understood. In this study, the researchers show that microwave plasma causes selective cell cycle arrest and apoptosis in osteosarcoma cells in vitro. An inhabitation of cell growth of microwave plasma showed that this plasma has selective cytotoxic effects on MG-63 cells in comparison to hFOB 1.19 cells. Also, the resesearchers observed that microwave plasma treatmenst significantly showed disruption and aggregation of F-actin. The results demonstrated that treatment of MG-63 cells with microwave plasma induced cell cycle arrest in the G2/M phase, using flow cytometry and western blot assay. To detect PARP and DFF-45 cleavage or a decrease as a result of caspase-3 activation, MG-63 cells were treated either with various times of microwave plasma. The research studies demonstrated that microwave plasma induced G2/M cell phase arrest and triggered apoptosis.

Involvement of Cdc25c in Cell Cycle Alteration of a Radioresistant Lung Cancer Cell Line Established with Fractionated Ionizing Radiation

Li, Jie,Yang, Chun-Xu,Mei, Zi-Jie,Chen, Jing,Zhang, Shi-Min,Sun, Shao-Xing,Zhou, Fu-Xiang,Zhou, Yun-Feng,Xie, Cong-Hua Asian Pacific Journal of Cancer Prevention 2013 Asian Pacific journal of cancer prevention Vol.14 No.10

Cancer patients often suffer from local tumor recurrence after radiation therapy. Cell cycling, an intricate sequence of events which guarantees high genomic fidelity, has been suggested to affect DNA damage responses and eventual radioresistant characteristics of cancer cells. Here, we established a radioresistant lung cancer cell line, A549R, by exposing the parental A549 cells to repeated ${\gamma}$-ray irradiation with a total dose of 60 Gy. The radiosensitivity of A549 and A549R was confirmed using colony formation assays. We then focused on examination of the cell cycle distribution between A549 and A549R and found that the proportion of cells in the radioresistant S phase increased, whereas that in the radiosensitive G1 phase decreased. When A549 and A549R cells were exposed to 4 Gy irradiation the total differences in cell cycle redistribution suggested that G2-M cell cycle arrest plays a predominant role in mediating radioresistance. In order to further explore the possible mechanisms behind the cell cycle related radioresistance, we examined the expression of Cdc25 proteins which orchestrate cell cycle transitions. The results showed that expression of Cdc25c increased accompanied by the decrease of Cdc25a and we proposed that the quantity of Cdc25c, rather than activated Cdc25c or Cdc25a, determines the radioresistance of cells.

수 종의 상피기원 종양 세포주에서 방사선 조사와 표피성장인자 투여에 따른 세포 주기의 변화와 apoptosis 유발에 관한 연구

한원정,허민석,이삼선,최순철,박태원 대한구강악안면방사선학회 2000 Imaging Science in Dentistry Vol.30 No.1

Purpose : This study was aimed to evaluate the cell cycle arrest and apoptosis induction after irradiation and epidermal growth factor(EGF) treatment in three human epithelial tumor cell lines (A43l, Siha, KB). Materials and Methods : Single irradiation of 2, 5 and 10 Gy was done on three cell lines with 5.38 Gy/min dose rate using Cs-137 irradiator at room temperature. Also, EGF of 10 ng/ml was added immediately after 10 Gy irradiation. Cell growth was evaluated by counting the living cell number using a hemocytometer at 1 day, 2 days, 3 days, 4 days and 5 days after irradiation. Cell cycle arrest and apoptosis induction were assayed with the flow cytometry at 8 hours, 12 hours, 1 day, 2 days, 3 days, 4 days and 5 days after irradiation. Results : Growth of irradiated three cell lines were inhibited in proportion to radiation dose, EGF treatment after irradiation showed various results according to cell lines. On all cell lines, G2 arrest was detected after 8 hours and maximized after 12 hours or 1 day. Amount of G2 arrest was positively dose dependent. However, EGF showed no significant change on G2 arrest. G2 arrest was recovered with time at 2 Gy and 5 Gy irradiation. However, at 10 Gy irradiation, G2 arrest was continued. Apoptosis was detected at 10 Gy irradiation. On EGF treated group after irradiation, A431 and Siha cell lines showed slightly increased apoptosis but there was no statistically significant difference. KB cell line showed no marked change of apoptosis induction. Conclusion : Irradiation effects on cell cycle arrest and apoptosis induction in three human epithelial tumor cell lines, however epidermal growth factor doesn't effect on. (Korean J Oral Maxillofac Radiol 2000; 30: 71-79)

Silencing of the COPS3 Gene by siRNA Reduces Proliferation of Lung Cancer Cells Most Likely via induction of Cell Cycle Arrest and Apoptosis

Wang, Xue-Mei,Cui, Jiu-Wei,Li, Wei,Cai, Lu,Song, Wei,Wang, Guan-Jun Asian Pacific Journal of Cancer Prevention 2012 Asian Pacific journal of cancer prevention Vol.13 No.3

The COPS3 gene has stimulating effect on cell proliferation and progression of osteosarcomas and related cells. However, the features of COPS3 and its potential application as a therapeutic target in other cancers has not yet been studied. In this study, therefore, the effect of COPS3 silencing via COPS3 siRNA on lung cancer cell proliferation was examined. Expression levels of COPS3 gene in COPS3 siRNA infected cells and control siRNA infected cells were compared with real time PCR and Western blot analysis. Cell proliferation levels were comprehensively analyzed by MTT, BrdU incorporationy, and colony formation assays. For mechanistic assessment the effects of COPS3 silencing on cell cycle and apoptosis were analyzed using flow cytometry. Results showed that successful silencing of the COPS3 gene at both translational and transcriptional levels significantly reduced the proliferation and colony formation by lung cancer cells (p<0.01). Flow cytometry showed cell cycle arrest in the G0/G1 phase after COPS3 silencing, and more importantly, apoptosis was induced as a result of COPS3 knockdown, which negatively affected cell survival. Therefore, these results provide another piece of important evidence that the COPS3 gene expressed in lung cancer cells may play a critical role in stimulating proliferation. Down-regulation of COPS3 could significantly inhibit lung cancer cell growth, which was most likely mediated via induction of cell cycle arrest in G0/G1 phase and apoptosis.