Editor-in-Chief
Prof. Yi-Fei WANG,
Asian J Androl 2005; 7 (4): 375-380 |
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This web only provides the extract of this article. If you want to read the figures and tables, please reference the PDF full text on Blackwell Synergy. Thank you. - Original Article - Change of the cell cycle after flutamide treatment in prostate cancer cells and its molecular mechanism Yong Wang1, Chen Shao2, Chang-Hong Shi3, Lei Zhang4, Hong-Hong Yue5, Peng-Fei Wang2, Bo Yang2, Yun-Tao Zhang2, Fan Liu1, Wei-Jun Qin2, He Wang2, Guo-Xing Shao2 1Department of Urology, Tangdu Hospital,
2Department of Urology, 3Department of Microbiology, Abstract Aim: To explore the effect of androgen receptor (AR) on the expression of the cell cycle-related genes, such as CDKN1A and BTG1, in prostate cancer cell line LNCaP. Methods: After AR antagonist flutamide treatment and confirmation of its effect by phase contrast microscope and flow cytometry, the differential expression of the cell cycle-related genes was analyzed by a cDNA microarray. The flutamide treated cells were set as the experimental group and the LNCaP cells as the control. We labeled cDNA probes of the experimental group and control group with Cy5 and Cy3 dyes, respectively, through reverse transcription. Then we hybridized the cDNA probes with cDNA microarrays, which contained 8 126 unique human cDNA sequences and the chip was scanned to get the fluorescent values of Cy5 and Cy3 on each spot. After primary analysis, reverse transcription polymerase chain reaction (RT-PCR) tests were carried out to confirm the results of the chips. Results:After AR antagonist flutamide treatment, three hundred and twenty-six genes (3.93%) expressed differentially, 97 down-regulated and 219 up-regulated. Among them, eight up-regulated genes might be cell cycle-related, namely CDC10, NRAS, BTG1, Wee1, CLK3, DKFZP564A122, CDKN1A and BTG2. The CDKN1A and BTG1 gene mRNA expression was confirmed to be higher in the experimental group by RT-PCR, while p53 mRNA expression had no significant changes. Conclusion: Flutamide treatment might up-regulate CDKN1A and BTG1 expression in prostate cancer cells. The protein expressions of CDKN1A and BTG1 play an important role in inhibiting the proliferation of cancer cells. CDKN1A has a great impact on the cell cycle of prostate cancer cells and may play a role in the cancer cells in a p53-independent pathway. The prostate cancer cells might affect the cell cycle-related genes by activating AR and thus break the cell cycle control. (Asian J Androl 2005 Dec; 7: 375-380) Keywords: prostate cancer; LNCaP; p21; androgen receptor; CDKN1A; BTG1; cell cycle genes; flutamide Correspondence to: Dr Chen Shao, Department of Urology, Xijing
Hospital, Fourth Military Medical University, Xi'an 710032,
Shaanxi, China.
1 Introduction
Prostate cancer is a worldwide disease. The incidence of prostate cancer in Europe was 103.5 cases per 100 000 men in the year 2000 [1]. In USA, the incidence of this disease increased steadily between 1981 and 1989, with a steep increase in the early 1990s. In 1996, 317 000 new cases of prostate cancer have been detected, while 41 400 patients died of this disease in the USA [2]. In China, some well-developed areas such as Shanghai also have seen a dramatic increase in the incidence of prostate cancer due to changes of lifestyles and dietary patterns [3]. Clinical therapy of prostate cancer involves radical prostatectomy followed by adjuvant hormonotherapy or chemotherapy. Administration of antiandrogens provides only partial remission because prostate cancer cells acquire a hormone-independent phenotype and the disease relapses within a few years. Therefore, the development of androgen independency of prostate cancer is the main cause of therapeutic failure. Nevertheless, the underlying molecular mechanisms involved in this situation were not clearly understood [4]. In this study, we used the well-characterized, hormone-sensitive human prostate cancer cell line LNCaP to identify early mechanisms involved in the acquisition of hormone independence.
2 Materials and methods
2.1 Materials LNCaP cells were cultured in RPMI-1640 medium sup-plemented with 10% fetal bovine serum (FBS) (Hyclone Inc., Savannah, USA)[5]. Stock solutions of the androgen receptor (AR) antagonist flutamide (Sigma Inc., St. Louis, USA) (10-4 mol/L) were made in absolute ethanol, while working solutions were further made in phosphate-buffered saline (PBS) (pH 7.2) [6]. Flutamide was routinely used at a final concentration of 10-7 mol/L. In the control (non-treated) group, an equal volume of pure ethanol, dissolved in PBS was used to eliminate any effect of the vehicle. After 15 days of treatment, with a change of the medium every 2 days, when the experimental group cells reached 90% confluence, we isolated mRNA and analyzed it after reverse transcription. Cy3-dCTP and Cy5-dCTP were purchased from Amersham Phamacia Biotech Inc. (Piscataway, NJ, USA) and Oligotex mRNA Midi Kit from Quigen Inc. (Valencia, CA, USA). ScanArray 4 000 laser scanner was from GSI Lomonics (Ottawa, Ontario, Canada). GenePix Pro 3.0 software from Axon Instruments Inc. (Sunnyvale, CA, USA). 2.2 Methods 2.2.1 Analysis of cell cycle changes The control cells and cells that had been treated with flutamide for 15 days were analyzed by microscopy, using a microscope (Olympus, Tokyo, Japan) provided with a camera (Olympus, Tokyo, Japan). Additionally, cells were collected and placed into 6-well plates at a density of 1 × 105/mL, washed with 0.01 mol/L PBS (pH 7.2), fixed in 70% ethanol for 18 h, resuspended in PBS and stained with propidium iodide (100 ìg/mL) for 30 min. Flow cytometer was explored by using blue light Argon-Ion laser (excitation wavelength, 488 nm; laser power, 200 mW; ELITE ESP, Beckman-Coulter, Fuller-ton, CA, USA) and red fluorescence from the PI/DNA was recorded. Cell cycle analysis was performed using a DNA-Prep Reagent System, with the following settings: one cycle analysis, no apoptosis.
2.2.2 Microarray assay mRNA was extracted by Trizol and purified by Oli-gotex Midi Kit (Quigen Inc., Valencia, CA, USA)[7]. Microarray analysis was performed by using the Human Gene Expression CHIP (version H80s, Biostar, Shanghai, China) containing 8 126 human genes. A fluorescent probe was synthesized by reverse transcription of 100 ìg of the above mRNA with 50 U AMV reverse transcriptase (Takara Shuzo, Kyoto, Japan) in the presence of Cy3- or Cy5-dCTP (Amersham, Arlington Heights, USA). Then Cy3- and Cy5-labeled probes were prepared and incubated in the cDNA chip at 42 °C for 6 h, washed twice with 2 × Standard Saline Citrate(SSC)/0.2% SDS at 60 °C for 30 min and then washed again with the same buffer for 5 min. Finally, the chip was washed with 0.05 × SSC at room temperature for 10 min and signals were quantified with the ScanArray 4000 (GSI Lomonics, Ottawa, Canada) and the Quant Array Software (GSI Lomonics, Ottawa, Canada). All the Cy3 fluorescent units were normalized according to the normalized factor [6] and Cy5 fluorescent intensity was counted as 200 if it was below 200 fluorescent units. The expression changes of genes were considered as up-regulated if the Cy5/Cy3 signal ratio was higher than 2.0 and down-regulated if the ratio was lower than 0.5.
2.2.3 Semi-quantitative RT-PCR Total RNA was extracted by the Qiagen RNA Isolation Kit (GIBCO Co., New York, USA). For the first-strand cDNA synthesis, 5 mg/mL of RNA (Takara Co., Dalian, China) were used. In each reaction, a 100-mL solution containing 3 mmol random hexamers, 25 mmol/L Tris-HCl, 37 mmol/LKCl, 1.5 mmol/LMgCl2, 10 mmol/LDTT, 0.25 mmol/L dNTP, 40 units of RNasin, a RNase inhibitor, 50 U /mL Super Taq DNA polymerase, and 200 units of reverse transcriptase were used. The annealing mixture was incubated at room temperature for 15 min, and then incubated in a water bath at 41 °C for 60 min. The reverse transcriptase enzyme was inactivated by heating the solution to 95 °C for 5 min. PCR was then carried out using PCR kit (Perkin-Elmer, Foster City, CA, USA) and primers. The PCR was performed for 30 cycles consisting of denaturation at 94 °C for 1 min, annealing at 57 °C for 1 min, and extension at 72 °C for 2 min. The PCR products were analyzed on 1.5% agarose gel. The primers used for PCR were as follow: CDKN1A sense (5¡¯-GAC ACC ACT GGA GGG TGA CT-3¡¯), CDKN1A antisense (5¡¯-TAC AGG TCC ACA TGG TCT TCC-3¡¯); b-actin sense (5¡¯-GAT TGC CTC AGG ACA TTT CTG-3¡¯), b-actin antisense (5¡¯-GAT TGC TCA GGA CAT TTC TG-3¡¯) [8]. And another experiment of KLK3 (its gene product is PSA) expression was also carried out, with the primers set as follow: KLK3 sense (5¡¯-AGC GTG ATC TTG CTG GGT CG-3¡¯), KLK3 antisense (5¡¯-CGT CAT TGG AAA TAA CAT GGA GG-3¡¯). Gene primers were synthesized by Beijing Oake Company (Beijing, China).
3 Results
3.1 Morphological and cell cycle changes induced by flutamide Flutamide treatment of LNCaP cells for 15 days resulted in dramatic changes of cell morphology (Figures 1, 2). Antiandrogen-treated cells became smaller, while less mitoses and cell contacts were found. The density of cells also diminished. The results of flow cytometer indicated that (71.47 ± 0.96)% of LNCaP cells sustained at G1 phase after the flutamide treatment while that of control cells was only (66.87 ± 1.50) % (Table 1).
3.2 The changes of gene expression after flutamide treatment Detection of RNA purity was assayed by electrophoresis in agarose gels, and absorption spectrometry. As shown in Figure 3, 18S and 28S bands were clean and clear, while absorbance measurements revealed A260/A280 >2.0, indicating that the extracted mRNA was suitable for cDNA microarray assay. According to the criteria offered by the chip manufacturer (Cy5/Cy3*>2.0, up-regulated; Cy5/Cy3*<0.5, down-regulated), there were 326 genes (3.93%) which were expressed differentially after the treatment with flutamide. Ninety-seven genes were up-regulated and 219 were down-regulated (Figure 4). It was very interesting that there was an increased expression of eight genes related to cell cycle, namely CDC10, NRAS, BTG1, Wee1, CLK3, DKFZP564A122, CDKN1A and BTG2 (Table 2). Especially CDKN1A and BTG1 who have relatively high Cy3 value. Meanwhile, p53 and its related genes did not change much after flutamide treatment (Table 3). The induction of PSA mRNA may reflect the ability of AR to stimulate transcription, for the 5¡¯-regulatory region of the PSA gene contains multiple androgen responseelements [5]. PSA was obviously down-regulated (Cy5/Cy3=0.202) in our chip assay, while AR did not change (Cy5/Cy3=0.65). 3.3 Semi-quantitative RT-PCR confirmation In the control (non-treated) group, the CDKN1A expression in the LNCaP cells was merely faintly detected by RT-PCR. In contrast, in flutamide-treated LNCaP cells, the expression of this gene product was significantly up-regulated (Figure 5). In contrast, KLK3 (PSA) expression was down-regulated (as expected) after flutamide, indicating an effective blockage of AR by chronic flutamide treatment (Figure 6).
4 Discussion
AR plays a great role in regulating the proliferation of both normal and neoplastic prostate cells. The activated DNA-bound AR homodimer complex recruits several kinds of co-regulatory proteins to stimulate or inhibit target gene transcription, thus promotes or represses cell proliferation, apoptosis or angiogenesis [9]. In this study, we found that eight cell cycle-related genes, namely CDC10, NRAS, BTG1, Wee1, CLK3, DKFZP564A122, SLC31A1 and CDKN1A, were up-regulated in prostate cancer cells after flutamide treatment. In contrast, p53 and its downstream genes, such as tumor protein p53-binding protein TOPORS (Gene alias: TP53BPL), TP53BP1, PA2, TP53BP2 and p53DINP1, remain unchanged. CDKN1A is an important cell cycle regulator [10]. Its gene product p21 (waf1/cip1) can inhibit the proliferation of cancer cells via both p53-dependent and p53-independent CDK inhibition. In many tumors, CDKN1A could be activated through the p53-indpendent pathway[11, 12], in which mitogens as platelet-derived growth factor (PDGF), fibroblast growth factor (FGF), and epidermal growth factor (EGF) may function[13]. Choi et al. [12]reported that in prostate cancer cell line PC-3, the p53-independent pathway to activate CDKN1A may be related with over-expression of CDC2 and CDK2. Our results showed that there was no significant change of p53 gene and its downstreamgenes after flutamide treatment; we assumed that the high expression of CDKN1A may occur through a p53-independent pathway in this experiment. Kokontis et al.[14] found that androgen may inhibit androgen refractory prostate cancer cell line LNCaP-104R1 proliferation by a transient p21 (waf1/cip1) induction and following p27 (Kip1) induction as a result of a drop in c-myc expression. However, in our research, we found neither p27 (Kip1) nor c-myc¡¯s expression changed (data not shown), suggesting a different pathway in androgen prolific cell line. Since there have been reports that CDKN1A expression was associated with tumor progression to androgen-independent prostate carcinoma [4], we supposed that p21 may play a key role in molecular events of the initiation of AIPC because of its anti-apoptotic effect. Eder et al.[15] found in 2003 that after AR blockage by antisense oligonucleotide, the expression of IGFBP2, PIP5KIA, PTOV1 and S100P changed. However, in this study, we did not find the same kind of results. The other genes we discovered in this experiment were also of interest. BTG1, for example, may play a coordinate role in a general transduction pathway that was induced in response to DNA damage[16]. BTG1 expression was maximal in the G0/G1 phases of the cell cycle and was down-regulated when cells progress throughout G1[17]. It affected the proliferation by phosphorylating a putative p34cdc2 kinase site on BTG1, Ser-159, thus modulated CCR4 expression, and then induced the formation of hCAF-1/BTG1, which was of great consequence in the signaling events of cell division that lead to changes in cellular proliferation associated with cell-cell contact[18]. However, a shortage of correlated reports hinder further research of this gene on the prostate cancer cells¡¯ proliferation and cell contact. In conclusion, we found that eight cell cycle-related genes may be involved in the process of flutamide-induced cell growth inhibition, especially CDKN1A and BTG1. CDKN1A may function on the prostate cancer cells in a p53-independent mode. The clarification of exact mechanism of flutamide¡¯s inhibitive effect on prostate cancer cells is of great importance in clinical hormone therapy. Designing a new way with biotechnology to mimic and maximize flutamide¡¯s anti-cancer effect while avoiding its notorious hepatotoxicity may be an interesting idea to pursue in the future.
Acknowledgment
We owe our thanks to Professor Jian-Guang Zhou, the Academy of Military Medical Sciences (Beijing, China), for providing LNCaP cells. This project was granted by the National Nature Science Foundation of China (No. 30100185). References 1. Herranz Amo F, Arias Funez F, Arrizabalaga Moreno M, Calahorra Fernandez FJ, Carballido Rodriguez J, Diz Rodriguez R, et al. The prostate cancer in the community of Madrid in 2000 I.- Incidence. Actas Urol Esp 2003; 27: 323-34. 2. Sarma AV, Schottenfeld D. Prostate cancer incidence, mortality, and survival trends in the United States: 1981-2001. Semin Urol Oncol 2002; 20: 3-9. 3. Hsing AW, Devesa SS, Jin F, Gao YT. Rising incidence of prostate cancer in Shanghai, China. Cancer Epidemiol Biomarkers Prev 1998; 7: 83-4. 4. Feldman BJ, Feldman D. The development of androgen-independent prostate cancer. Nat Rev Cancer 2001; 1: 34-45. 5. Kanaya J, Takashima M, Koh E, Namiki M. Androgen-independent growth in LNCaP cell lines and steroid uridine diphosphate-glucuronosyltransferase expression. Asian J Androl 2003; 5: 9-13. 6. Kumar VL, Majumder PK, Kumar V. In vivo modulation of androgen receptor by androgens. Asian J Androl 2002; 4: 229-31. 7. Kappler-Hanno K, Kirchhoff C. Rodent epididymal cDNAs identified by sequence homology to human and canine counterparts. Asian J Androl 2003; 5: 277-86. 8. Kuwano K, Hagimoto N, Nomoto Y, Kawasaki M, Kunitake R, Fujita M, et al. P53 and p21 (Waf1/Cip1) mRNA expression associated with DNA damage and repair in acute immune complex alveolitis in mice. Lab Invest 1997; 76: 161-9. 9. McKenna NJ, Lanz RB, O'Malley BW. Nuclear receptor coregulators: cellular and molecular biology. Endocr Rev 1999; 20: 321-44. 10. Harper JW, Adami GR, Wei N, Keyomarsi K, Elledge SJ. The p21 Cdk-interacting protein Cip1 is a potent inhibitor of G1 cyclin-dependent kinases. Cell 1993; 75: 805-16. 11. Zeng YX, el-Deiry WS. Regulation of p21WAF1/CIP1 expression by p53-independent pathways. Oncogene 1996; 12: 1557-64. 12. Choi YH, Lee WH, Park KY, Zhang L. p53-independent induction of p21 (WAF1/CIP1), reduction of cyclin B1 and G2/M arrest by the isoflavone genistein in human prostate carcinoma cells. Jpn J Cancer Res 2000; 91: 164-73. 13. Michieli P, Chedid M, Lin D, Pierce JH, Mercer WE, Givol D. Induction of WAF1/CIP1 by a p53-independent pathway. Cancer Res 1994; 54: 3391-5. 14. Kokontis JM, Hay N, Liao S. Progression of LNCaP prostate tumor cells during androgen deprivation: hormone-independent growth, repression of proliferation by androgen, and role for p27Kip1 in androgen-induced cell cycle arrest. Mol Endocrinol 1998; 12: 941-53. 15. Eder IE, Haag P, Basik M, Mousses S, Bektic J, Bartsch G, et al. Gene expression changes following androgen receptor elimination in LNCaP prostate cancer cells. Mol Carcinog 2003; 37: 181-91. 16. Rouault JP, Prevot D, Berthet C, Birot AM, Billaud M, Magaud JP, et al. Interaction of BTG1 and p53-regulated BTG2 gene products with mCaf1, the murine homolog of a component of the yeast CCR4 transcriptional regulatory complex. J Biol Chem 1998; 273: 22563-9. 17. Rouault JP, Rimokh R, Tessa C, Paranhos G, Ffrench M, Duret L, et al. BTG1, a member of a new family of anti-proliferative genes. EMBO J 1992; 11: 1663-70. 18. Bogdan JA, Adams-Burton C, Pedicord DL, Sukovich DA, Benfield PA, Corjay MH, et al. Human carbon catabolite repressor protein (CCR4)-associative factor 1: cloning, expression and characterization of its interaction with the B-cell translocation protein BTG1. Biochem J 1998; 336: 471-81. |
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