Bangladesh Journal of Pharmacology Research Article Lovastatin induces G1Lovastatin induces G1Lovastatin induces G1---phase syn-phase syn-phase syn- chronization of HECchronization of HECchronization of HEC---111---A cells and A cells and A cells and JEC cellsJEC cellsJEC cells BJP Introduction Tumor has been regarded as a group of diseases related to impairment of cell cycle (Sherr, 1996; Evan and Vousden, 2001; Go et al., 2012) and the pathogenesis of tumor attributes to the uncontrolled cell cycle. Under this condition, the cells are autonomous and uncon- trolled and can unlimitedly proliferate. The cell cycle of tumor cells has been a hot topic in studies on molecular biology of tumors. In the studies on cell cycle, cell phase synchronization is usually required to collect cells in the same phase. According to the specific phase in synchronization, synchronization is classified as G0/G1 phase synchronization, G1 phase synchronization, G2 phase synchronization, M phase synchronization and S phase synchronization. In addition, there are some methods that have been developed for synchronization. For example, for G0/G1 phase synchronization, serum starvation or amino acid starvation; for G1 phase syn- chronization, mimosine and lovastatin are used; for S phase synchronization, treatment with thymidine for arrest is usually performed. To date, few studies have conducted for the synchroni- zation of endometrial cancer cells. The present study employed endometrial cancer cell lines (HEC-1-A cells and JEC cells) to investigate the efficacy of lovastatin in G1-phase synchronization of both cell lines and to explore the optimal duration of lovastatin treatment and optimal lovastatin concentration. Subsequently, the cell cycle progression was investigated after G1-phase desynchronization. Our findings may provide evidence for the role of cell cycle regulation in the occurrence and development of endometrial cancer. Materials and Methods Materials Human endometrial cancer cell lines (HEC-1-A cells and JEC cells) were preserved in our lab. DMEM low glucose, fetal bovine serum (FBS) (GIBCO), trypsin This work is licensed under a Creative Commons Attribution 3.0 License. You are free to copy, distribute and perform the work. You must attribute the work in the manner specified by the author or licensor. Abstract Our study investigated lovastatin-induced G1 phase synchronization of HEC- 1-A cells and JEC cells and the cell cycle progression after desynchronization of these cells. HEC-1-A cells and JEC cells were treated with by lovastatin and the proportion of cells in G1 phase was detected. Both cell types were incubated with lovastatin and the synchronization was determined. The cell cycle progression of both cell types was investigated. The proportion of Both cell types in G1 phase was 87.9 ± 0.7% and 85.0 ± 0.8%, respectively. At 20 and 16 hours after G1-phase desynchronization, the proportion of cells in S phase reached a maximal level and that in G1 phase was at the minimal level for HEC-1-A cells and JEC cells, respectively. Thus, at 20 and 16 hours after desynchronization, the proportion of cells in S phase was the highest and that in G1 phase the lowest for HEC-1-A cells and JEC cells, respectively. Article Info Received: 5 December 2012 Accepted: 6 December 2012 Available Online: 24 December 2012 DOI: 10.3329/bjp.v8i1.12847 Cite this article: Shen H, Li X, Yang Y, Wang X, Zhang X. Lovastatin induces G1-phase syn- chronization of HEC-1-A cells and JEC cells. Bangladesh J Pharmacol. 2013; 8: 1-7. A Journal of the Bangladesh Pharmacological Society (BDPS) Bangladesh J Pharmacol 2013; 8: 1-7 Journal homepage: www.banglajol.info; www.bdjpharmacol.com Abstracted/indexed in Academic Search Complete, Agroforestry Abstracts, Asia Journals Online, Bangladesh Journals Online, Biological Abstracts, BIOSIS Previews, CAB Abstracts, Current Abstracts, Directory of Open Access Journals, EMBASE/Excerpta Medica, Google Scholar, HINARI (WHO), International Pharmaceutical Abstracts, Open J-gate, Science Citation Index Expanded, SCOPUS and Social Sciences Citation Index ISSN: 1991-0088 Lovastatin induces G1-phase synchronization of HEC-1-A cells and JEC cells Huimin Shen, Xiaomao Li, Yuebo Yang, Xiaoyun Wang and Xu Zhang Department of Obstetrics and Gynecology, Third Affiliated Hospital of Sun Yat-sen University, Guangzhou 510 630, China. (Genom Biotech, Co., Ltd), lovastatin, mevalonic acid (MVA), propidium iodide (PI), Ribonuclease A (RNase A) (Sigma, USA), CCK-8 (Dojindo, Japan) and FACS caliber flow cytometer (Becton Dickinson) were used in the present study. Cell culture Human endometrial cancer HEC-1-A cells and JEC cells were adherent cells and seeded in DMEM low-glucose containing 10% FBS followed by incubation at 37°C in an atmosphere with 5% CO2. Detection of doubling time by CCK-8 assay The HEC-1-A cells and JEC cells were digested, centri- fuged, suspended and counted. Then, these cells were re-suspended in DMEM low-glucose containing 10% FBS and then seeded into a 96-well plate (3,000 cells/ well). These cells were divided into 6 groups and 5 wells were included in each group. Incubation was performed at 37°C in an atmosphere with 5% CO2. CCK-8 was added at 24 hours (0 hour) and 40 hours (16 hours) after incubation and thereafter every 2~4 hours followed by incubation at 37°C in an atmosphere with 5% CO2 for 2 hours. Optical density (OD) was mea- sured on a microplate at the detection wavelength of 490 nm and reference wavelength of 630 nm. The time to doubling of OD was used as the doubling time of cell proliferation. Induction of G1-phase synchronization by lovastatin treatment The induction of G1-phase synchronization was perfor- med according to previously described with modifica- tion (Javanmoghadam-Kamrani and Keyomarsi, 2008). In brief, the HEC-1-A cells and JEC cells were digested, centrifuged, re-suspended and counted. After re- suspending in DMEM low-glucose containing 10% FBS, cells were seeded into a 6-well plate (5 × 105 cells/well). Cells were divided into 4 group and 3 wells included in each group. Incubation was done at 37°C in an atmos- phere with 5% CO2 for 24 hours. Then, cells in different groups were treated with lovastatin at 10, 20, 30, and 40 μmol/L, respectively for a doubling time. Then, these cells were collected and subjected to flow cytometry. The concentration at which the proportion of cells in G1 phase was the highest was used for further experiments and regarded as the respective optimal concentration. Then, HEC-1-A cells and JEC cells were seeded into 6- well plate, independently, and three wells included in each group. These cells were treated with lovastatin at the optimal concentration for 0.5~2-fold of doubling time. Cells were harvested every 4 hours and subjected to flow cytometry. The time when the proportion of cells in G1 phase was the highest was regarded as the optimal duration for lovastatin treatment. G1-phase desynchronization HEC-1-A cells and JEC cells were seeded into 6-well plate and 3 wells included in each group. These cells were treated with lovastatin at optimal concentration for optimal duration and the supernatant containing lovastatin was removed. These cells were washed in PBS thrice and then treated with MVA at a concentra- tion of 100-fold of the optimal lovastatin concentration followed by incubation at 37°C in an atmosphere with 5%CO2 for more than one doubling time. Cells were harvested every 4 hours followed by flow cytometry. Detection of cell cycle by flow cytometry Cells were digested with 0.25% trypsin and harvested into centrifuge tube followed by centrifugation at 1,000 rpm/min for 4 min. The supernatant was removed and cells were washed in PBS by centrifugation at 1,000 rpm/min for 4 min. The supernatant was removed and cells were re-suspended in 1 mL of 70% alcohol over- night. Following centrifugation at 1,000 rpm/min for 5 min, the supernatant was removed and cells were washed in PBS once. After centrifugation at 1,000 rpm/ min for 5 min, the supernatant was removed and cells were re-suspended in 50 μL of 0.5% RNase A and 450 μL of PI solution followed by incubation in dark at 2~8° C for 30 min. Then, flow cytometry was performed. Statistical analysis Data were expressed as mean (± SD) and statistical analysis was performed with SPSS version 13.0 statistics package. Comparisons of means were done with t-test between two groups and with one way analysis of variance among different groups. A value of p<0.05 was considered statistically significant. Results CCK-8 assay revealed, at 0, 16, 20, 22, 24 and 28 hours, the OD was 0.3447, 0.5568, 0.6188, 0.6303, 0.6782 and 0.7262, respectively, for HEC-1-A cells and 0.3778, 0.5829, 0.6670, 0.6970, 0.7423 and 0.7691, respectively, for JEC cells. At 24 hours, the OD of HEC-1-A cells and JEC cells was increased by about 100% and thus 24 hours was defined as the doubling time. HEC-1-A cells were divided into 4 groups and treated with lovastatin at 10, 20, 30 and 40 μmol/L, respec- tively. After treatment for 24 hours, the proportion of cells in G1 phase was (76.7 ± 0.5)%, (79.7 ± 0.9)%, (80.4 ± 1.6)%, and (84.6 ± 1.3)%, respectively (Figure 1). Statis- tical analysis showed the proportion of cells in G1 phase after treatment with 40 μmol/L lovastatin for 24 hours was dramatically increased when compared with other treatments (p<0.05). This suggests the optimal concentration of G1-synchronization of HEC-1-A cells was 40 μmol/L. JEC cells were divided into 4 groups and treated with lovastatin at 10, 20, 30 and 40 μmol/L, respectively. 2 Bangladesh J Pharmacol 2013; 8: 1-7 After treatment for 24 hours, the proportion of cells in G1 phase was (81.1 ± 0.7)%, (85.0 ± 0.8)%, (82.6 ± 0.7)%, and (82.9 ± 0.6)%, respectively (Figure 1). Statistical ana- lysis showed the proportion of cells in G1 phase after treatment with 20 μmol/L lovastatin for 24 hours was dramatically increased when compared with other treat -ments (p<0.05). This suggests the optimal concentra- tion of G1-synchronization of JEC cells was 20 μmol/L. HEC-1-A cells were divided into 10 groups and then treated with 40 μmol/L lovastatin for 12, 16, 20, 24, 28, 32, 36, 40, 44 and 48 hours, and the proportion of cells in G1 phase was (77.8 ± 1.8)%, (81.1 ± 0.5)%, (83.6 ± 0.9)%, (84.6 ± 1.3)%, (87.9 ± 0.7)%, (81.4 ± 1.1)%, (72.6 ± 0.8)%, (69.3 ± 0.9)%, (61.3 ± 0.6)% and (56.9 ± 0.5)%, respec- tively (Figure 2). Statistical analysis showed treatment with 40 μmol/L lovastatin for 28 hours could maximize the proportion of HEC-1-A cells in G1 phase (p<0.05). This suggests the optimal concentration of lovastatin and duration of lovastatin treatment was 40 μmol/L and 28 hours for G1-phase synchronization of HEC-1-A cells. JEC cells were divided into 10 groups and then treated with 20 μmol/L lovastatin for 12, 16, 20, 24, 28, 32, 36, 40, 44 and 48 hours, and the proportion of cells in G1 phase was (70.4 ± 1.2)%, (75.5 ± 1.1)%, (82.5 ± 1.1)%, (85.0 ± 0.8)%, (83.0 ± 0.4)%, (80.7 ± 0.5)%, (67.6 ± 0.6)%, (55.6 ± 0.5)%, (47.8 ± 1.8)% and (50.4 ± 2.1)%, respec- tively (Figure 2). Statistical analysis showed treatment with 20 μmol/L lovastatin for 24 hours could maximize the proportion of JEC cells in G1 phase (p<0.05). This suggests the optimal concentration of lovastatin and duration of lovastatin treatment was 20 μmol/L and 24 hours for G1-phase synchronization of JEC cells. HEC-1-A cells were divided into 8 groups and treated with 40 μmol/L lovastatin for 28 hours for synchro- Bangladesh J Pharmacol 2013; 8: 1-7 3 Figure 1: Optimal lovastatin concentration for G1-synchronization. (A) Flow cytometry of HEC-1-A cells and JEC cells following treatment with lovastatin at different concentrations for 24 hours. (B) The ratio of HEC-1-A cells in G1 phase induced by lovastatin for 24 hours at different concentrations. ap<0.05 vs 40 μmol/L. (C) The ratio of JEC cells in G1 phase induced by lovastatin for 24 hours at different concentrations. ap<0.05 vs 20 μmol/L % o f G 1 c el ls Concentration of lovastatin (µ mol/L) 10 µM Lov 20 µM Lov 30 µM Lov 40 µM Lov HEC-1-A JEC A 88.0 84.0 80.0 76.0 72.0 10 20 30 40 B % o f G 1 c el ls Concentration of lovastatin (µ mol/L) 86 84 82 80 78 10 20 30 40 C a a a a a a nization and then with 4 mmol/L MVA for desynchronization. Results showed the proportion of cells in G1 phase reached a minimal level (58.4 ± 0.5)% but that in S phase was at a maximal level (33.6 ± 0.6)% at 20 hours after desynchronization, which were markedly different from those in the remaining groups (4, 8, 12, 16, 24, 28 and 32 hours) (p<0.05) (Table I and Figure 3). 4 Bangladesh J Pharmacol 2013; 8: 1-7 Figure 2: Optimal duration of Lovastatin treatment for G1-synchronization. (A) Flow cytometry of HEC-1-A cells following treat- ment with 40 μmol/L lovastatin and JEC cells following treatment with 20 μmol/L lovastatin for different durations. (B) The ratio of HEC-1-A cells in G1 phase induced by 40 μmol/L lovastatin for different durations. ap<0.05 vs 28 hours (C) The ratio of JEC cells in G1 phase induced by 20 μmol/L lovastatin for different durations. ap<0.05 vs 24 hours % o f G 1 c el ls Duration of 40 µ mol/L lovastatin (hours) 90 80 70 60 50 12 16 20 24 28 32 36 40 44 48 B a a 12 hours 16 hours 20 hours 24 hours 28 hours A HEC-1-A HEC-1-A JEC JEC 32 hours 36 hours 40 hours 44 hours 48 hours 12 hours 16 hours 20 hours 24 hours 28 hours 32 hours 36 hours 40 hours 44 hours 48 hours a a a a a a a % o f G 1 c el ls Duration of 20 µ mol/L lovastatin (hours) 12 16 20 24 28 32 36 40 44 48 C a a a a a a a a a 90 80 70 60 50 40 Bangladesh J Pharmacol 2013; 8: 1-7 5 Figure 3: Cell cycle progression after G1-phase desynchronization. (A) Flow cytometric analysis of HEC-1-A cells and JEC cells desynchronized for different durations after induced by optimal lovastatin concentration and duration. (B) Cell cycle progress of HEC-1-A cells desynchronized for different durations after induced by 40 μmol/L lovastatin for 28 hours. *p<0.05 vs 20 hours (C) Cell cycle progress of JEC cells desynchronized for different durations after induced by 20 μmol/L lovastatin for 24 hours. *p<0.05 vs 16 hours JEC cells were divided into 8 groups and treated with 20 μmol/L lovastatin for 24 hours for synchronization and then with 2 mmol/L MVA for desynchronization. Results showed the proportion of cells in G1 phase reached a minimal level (35.9 ± 0.4)% but that in S phase was at a maximal level (45.3 ± 0.5)% at 16 hours after desynchronization, which were markedly different from those in the remaining groups (4, 8, 12, 16, 24, 28 and 32 hours) (p<0.05) (Table II and Figure 3). Discussion Lovastatin can inhibit the HMG-CoA reductase resul- ting in deficiency of mevalonate which is a precursor of cholesterol and also essential for the prenylation of substrates of p21 and ras (Wejde et al., 1993). Lovastatin can arrest suspended and adherent cells of a lot of types in the G1 phase and has following advantages (Keyo- marsi et al., 1991; Javanmoghadam-Kamrani and Keyo- marsi, 2008): 1) lovastatin-induced synchronization is not toxic to cells and reversed by mevalonate; 2) in the lovastatin-induced synchronization, the nutritional substances and growth factors are sufficient, which may not cause disorder of cell metabolism; 3) lovastatin- induced synchronization can be used to achieve a large number of cells in G1 phase; 4) the efficiency of lovas- tatin-induced synchronization is unrelated to the type of cells, the cell density and the culture medium; 5) synchronization can be applied repeatedly. Wu et al. applied 5 μmol/L lovastatin to treat CHO cells for 32 hours and the proportion of cells in G1 phase was 90% (Wu and Gilbert, 2000). In addition, lovastatin at 10 and 20 μmol/L was applied to treat M6 cells and MCF-1 cells for 24 and 33 hours, respectively, and 78% and 77~96% of cells were identified to be in the G1 phase (Gupta et al., 2003; Shibata et al., 2003). Treatment of Pr14 cells and Pc-3M cells with 10 μmol/L lovastatin for 24 hours and 24~48 hours can achieve 82% and 68~90% of cells in G1 phase, respectively (Park et al., 2001; Shibata et al., 2003). Treatment of T-24 cells with 30 μmol/L lovastatin may achieve 80% of cells in G1 phase and about 70~90% of Calu-1 cells were identified to be in the G1 phase following treatment with 30 μmol/L lovastatin (Jakobisiak et al., 1991; Vogt et al., 1997). In addition, the G1-phase synchronization of HL-60 cells, T47D cells, HELA cells and U2-OS cells was also favo- rable following lovastatin treatment (Kishi et al., 2001; Reimers et al., 2001; Jurchott et al., 2003; Audic and Hartley, 2004). In the present study, G1-phase synchronization of HEC- 1-A cells and JEC cells was performed by lovastatin treatment. Firstly, the optimal concentration of lovas- tatin for G1-phase synchronization was determined. Cells were treated with lovastatin at 10, 20, 30 and 40 μmol/L, independently for one doubling time (24 hours). Results showed the optimal concentration of lovastatin was 40 μmol/L for HEC-1-A cells and 20 μmol/L for JEC cells under which the proportion of cells in G1 phase was (84.6 ± 1.3)% and (85.0 ± 0.8)%, respectively. Lovastatin at this concentration was applied for further experiment. Then, the optimal time for lovastatin treatment in the G1-phase synchroni- zation was further studied. HEC-1-A cells and JEC cells were treated with lovastatin at 40 and 20 μmol/L, respectively, for 0.5~2-fold of doubling time and G1- phase synchronization was measured every 4 hours. Results showed the optimal duration for lovastatin treatment in the G1-phase synchronization was 28 hours for HEC-1-A cells and 24 hours for JEC cells under which the proportion of cells in G1 phase was (87.9 ± 0.7)% and (85.0 ± 0.8)%, respectively. Both synchronizations were favorable. Thus, we speculate that the optimal lovastatin concentration and duration of lovastatin treatment for G1-phase synchronization were 40 μmol/L and 28 hours for HEC-1-A cells and 20 μmol/L and 24 hours for JEC cells. Following desynchronization of these cells, the cell cycle pro- gression was further investigated. Results showed the proportion of HEC-1-A cells in S phase was markedly increased (33.6 ± 0.6)% and that in G1 phase was the 6 Bangladesh J Pharmacol 2013; 8: 1-7 Table I Cell cycle progress after desynchronization of HEC -1-A cells Group (hours) G1 (%) S (%) G2/M (%) 4 78.9 ± 1.8 14.2 ± 1.2 7.0 ± 0.7 8 74.5 ± 1.4 18.2 ± 0.9 7.3 ± 0.5 12 68.6 ± 2.2 20.2 ± 1.7 11.1 ± 0.6 16 63.5 ± 2.9 24.3 ± 3.5 12.6 ± 0.7 20 58.4 ± 0.5 33.6 ± 0.6 8.0 ± 0.1 24 64.0 ± 1.6 25.7 ± 1.3 10.4 ± 0.3 28 69.7 ± 0.5 19.1 ± 0.9 11.2 ± 0.5 32 72.2 ± 1.1 20.9 ± 1.5 6.9 ± 0.5 Data are (mean ± SD) Table II Cell cycle progress after desynchronization of JEC cells (mean ± SD) Group (hours) G1 (%) S (%) G2/M (%) 4 76.1 ± 0.6 14.5 ± 0.8 9.5 ± 0.2 8 68.0 ± 0.3 17.6 ± 0.4 14.5 ± 0.2 12 51.5 ± 1.3 32.2 ± 1.1 16.3 ± 0.4 16 35.9 ± 0.4 45.3 ± 0.5 18.8 ± 0.9 20 37.6 ± 1.0 39.7 ± 1.0 22.7 ± 0.9 24 55.9 ± 0.9 22.9 ± 0.7 21.2 ± 1.4 28 72.8 ± 1.7 16.4 ± 1.1 10.8 ± 1.4 32 76.3 ± 1.0 17.7 ± 2.1 5.9 ± 1.2 Data are (mean ± SD) highest (58.4 ± 0.5)% at 20 hours after desynchronization. In addition, the proportion of JEC cells in S phase was markedly increased (45.3 ± 0.5)% and that in G1 phase was the highest (35.9 ± 0.4)% at 16 hours after desynchronization. Both desynchronizations were favorable. These, these conditions for synchroni- zation with lovastatin and subsequent desynchro- nization can be used in related studies. Acknowledgements This work was supported by the National Natural Science Foundation of China (No. 30772332), Natural Science Foun- dation of Guangdong Province (No. 7001568) and Science and Technology Project of Guangdong Province (No. 2011B031800056). References Audic Y, Hartley RS. Post-transcriptional regulation in cancer. Biol Cell. 2004; 96: 479-98. Evan GI, Vousden KH. Proliferation, cell cycle and apoptosis in cancer. Nature 2001; 411: 342-48. Gupta V, Cherkassky A, Chatis P, Joseph R, Johnson AL, Broadbent J, Erickson T, DiMeo J. Directly labeled mrna produces highly precise and unbiased differential gene expression data. Nucleic Acids Res. 2003; 31: e13. Go R, Takizawa K, Hirose S, Katsuragi Y, Aoyagi Y, Mishima Y, Kominami R. Impairment in differentiation and cell cycle of thymocytes by loss of a Bcl11b tumor suppressor allele that contributes to leukemogenesis. Leuk Res. 2012; 36: 1035- 40. Jakobisiak M, Bruno S, Skierski JS, Darzynkiewicz Z. Cell cycle -specific effects of lovastatin. Proc Natl Acad Sci USA. 1991; 88: 3628-32. Javanmoghadam-Kamrani S, Keyomarsi K. Synchronization of the cell cycle using lovastatin. Cell Cycle. 2008; 7: 2434-40. Jurchott K, Bergmann S, Stein U, Walther W, Janz M, Manni I, Piaggio G, Fietze E, Dietel M, Royer HD. Yb-1 as a cell cycle- regulated transcription factor facilitating cyclin a and cyclin b1 gene expression. J Biol Chem. 2003; 278: 27988-96. Keyomarsi K, Sandoval L, Band V, Pardee AB. Synchro- nization of tumor and normal cells from g1 to multiple cell cycles by lovastatin. Cancer Res. 1991; 51: 3602-09. Kishi S, Wulf G, Nakamura M, Lu KP. Telomeric protein pin2/ trf1 induces mitotic entry and apoptosis in cells with short telomeres and is down-regulated in human breast tumors. Oncogene. 2001; 20: 1497-508. Park C, Lee I, Kang WK. Lovastatin-induced e2f-1 modulation and its effect on prostate cancer cell death. Carcinogenesis. 2001; 22: 1727-31. Reimers K, Antoine M, Zapatka M, Blecken V, Dickson C, Kiefer P. Nobp, a nuclear fibroblast growth factor 3 binding protein, is cell cycle regulated and promotes cell growth. Mol Cell Biol. 2001; 21: 4996-5007. Sherr CJ. Cancer cell cycle. Science 1996; 274: 1672-77. Shibata MA, Kavanaugh C, Shibata E, Abe H, Nguyen P, Otsuki Y, Trepel JB, Green JE. Comparative effects of lovastatin on mammary and prostate oncogenesis in transgenic mouse models. Carcinogenesis 2003; 24: 453-59. Vogt A, Sun J, Qian Y, Hamilton AD, Sebti SM. The geranylgeranyltransferase-i inhibitor ggti-298 arrests human tumor cells in g0/g1 and induces p21(waf1/cip1/sdi1) in a p53-independent manner. J Biol Chem. 1997; 272: 27224-29. Wejde J, Carlberg M, Hjertman M, Larsson O. Isoprenoid regulation of cell growth: Identification of mevalonate- labelled compounds inducing DNA synthesis in human breast cancer cells depleted of serum and mevalonate. J Cell Physiol. 1993; 155: 539-48. Wu JR, Gilbert DM. Lovastatin arrests cho cells between the origin decision point and the restriction point. FEBS Lett. 2000; 484: 108-12. Bangladesh J Pharmacol 2013; 8: 1-7 7 Author Info Xiaomao Li (Principal contact) e-mail: lixiaomao1206@163.com DatePrinted: This article was downloaded by you on: Jan 13, 2018