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Cytotoxic Activities, Cellular Uptake, Gene Regulation, and Optical Imaging of Novel Platinum(II) Complexes Jian Gao,*,†,‡ Ya-Guang Liu,‡ and Ralph A. Zingaro*,† Department of Chemistry, Texas A&M UniVersity, College Station, Texas 77843-3255, and School of Medicine, UniVersity of Texas Health Science Center, San Antonio, Texas 78229-3900 ReceiVed May 29, 2009
A new class of platinum(II) coordination complexes and their dye tagged conjugates has been synthesized from N-substituted diaminocyclohexane ligands. The in vitro anticancer activities of the platinum compounds have been validated against the breast cancer cell-line MCF-7 and the normal cell-line MCF10A via sulforhodamine B and colony formation assay. The platinum compounds and the corresponding metal-free ligands exhibited higher drug efficiencies than cisplatin and oxaliplatin against MCF-7 cells. Cellular uptake and DNA-bound Pt were demonstrated by atomic absorption spectroscopy. The platinum complexes displayed increased cellular accumulation and DNA binding as compared with cisplatin. Realtime reverse transcription polymerase chain reaction assay was employed to investigate drug effects on mRNA expression in MCF-7 cells. The results indicated that the study compounds are effective in regulating cyclin D1, Bcl-2, and p53 genes; yet, oxaliplatin is less effective in manipulating those genes. The luminescent probe that was integrated into the platinum complexes made it possible to monitor cellular drug distribution using optical imaging. Targeting of tumor cell nuclei by the study compounds was confirmed by confocal microscopy. Taken together, these new platinum(II)-based antitumor agents are different from marketed platinum drugs in several critical aspects and could have potential in cancer therapy. Introduction In 1965, cisplatin was rediscovered by Barnett Rosenberg during his study of mitosis in Escherichia coli bacteria (1). Noting that cisplatin inhibited cell division, Rosenberg was interested in its potential as an anticancer drug. Following several years of testing, the FDA approved cisplatin for human use as an anticancer agent in 1978. Although it has been successfully used in the treatment of testicular, ovarian, lung, and head neck carcinomas, cisplatin is less than an ideal drug due to its toxic side effects. It does not discriminate between normal and cancerous tissues, which results in pronounced side effects including potentially fatal renal failure, bone marrow suppression, and compromised immunity, severe nausea, and toxicity (2). Numerous platinum-based compounds have been developed and tested in animals and humans during the past three decades in an effort to minimize toxic side effects and a propensity for acquired drug resistance. Carboplatin is an example of such a modified platinum agent, which went into clinical use in the early 1980s (3). It possesses decreased side effects and greater water solubility. However, the high dose needed for antitumor activity diminished the advantages obtained by the use of the cyclobutane-1,1-dicarboxylate as a leaving group. Randomized trials of cisplatin versus carboplatin in ovarian cancer demonstrated similar responses and a recurrence of tumor growth. To overcome this cross-resistance, the so-called third generation of platinum complexes was synthesized and the most promising drug molecule is oxaliplatin (4), which bears a 1,2-diaminocy* To whom correspondence should be addressed. E-mail: gaojiantamu@ yahoo.com (J.G.) or
[email protected] (R.A.Z.). † Texas A&M University. ‡ University of Texas Health Science Center.
clohexane (DACH) ligand and oxalate as a leaving group. While oxaliplatin has only mild hematologic and gastrointestinal side effects, its dose-limiting toxicity is a cumulative sensory neurotoxicity that resembles that of cisplatin (5). In searching for better platinum compounds, a wide variety of carrying ligands and leaving groups have been screened. Vicinal diamines, and particularly DACH, appear to be useful carrying ligands (6-8). Our previous study (9) indicated that bis-salicyl DACH and DPEN compounds are active anticancer agents, and these compounds have demonstrated significant specificity toward cancer cells. Our next study focused on the questions of whether the corresponding platinum(II) complexes possess an optimal anticancer activity, and if so, how they differ from the existing platinum(II) anticancer agents, such as oxaliplatin, at the chemical and cell biological levels. In this paper, we report the syntheses of new N,N′substituted-DACH platinum(II) complexes. Meaningful in vitro anticancer activities were observed in MCF-7 cells. The normal breast cell-line MCF-10A was also tested to determine the specificity toward cancer cells. Many molecular biological methods have been used in finding the targeted biomolecules in cytotoxic platinum complexes (10, 11). Cisplatin’s mechanism of action has been accepted as covalent binding to DNA and ultimately resulting in cell death. New evidence suggests that proteins and other biomolecules can also be the target of Pt ions (12, 13). Using atomic absorption spectroscopy, we found that at least a few percent of total cellular accumulated platinum ions are bound to DNA. The synthetic compounds were validated in the regulation of cyclin D1, p21, p27, p53, Bcl-2, Bcl-xl, Bad, and Bax genes by real time reverse transcription polymerase chain reaction (RT-PCR). Research aimed at increasing the body of knowledge about compounds’ role in gene expression
10.1021/tx900180v CCC: $40.75 2009 American Chemical Society Published on Web 08/20/2009
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should be critical with respect to increasing their utility as anticancer agents. Many biochemical and biophysical methods have been used to elucidate the governing dynamics in platinum compounds. It is well understood that the existing platinum(II) drugs are nonfluorescent in aqueous solution. To date, a few fluorescent platinum compounds have been explored (14-16). The ability to dynamically visualize the intracellular penetration behavior of platinum(II) drug candidates required the development of nondestructive methods for coupling platinum(II) drugs with luminescent dyes. In this study, novel conjugation techniques were developed to allow the reaction of fluorescent dye-tagged platinum analogues. The value of the method was subsequently confirmed by confocal cell imaging showing the synthetic drugs to be effective in penetrating cell membranes and targeting cell nuclei.
Experimental Procedures Material and Measurements. All chemical reagents and solvents were of analytical grade and obtained from the Sigma-Aldrich Chemical Co. Methanol and acetonitrile were dried over molecular sieves (4 Å) prior to use. Analyses for C, H, and N were carried out on a Perkin-Elmer analyzer, model 240. Positive ion electrospray ionization-mass spectrometry (ESI-MS) spectra were recorded using a LCQ electrospray mass spectrometer. The spectra were recorded over the mass range m/z 200-1000. 1H NMR spectra were measured using a Unix-VMR-500 MHz spectrometer. Preparation of 2,2′-(1R,2R)-Cyclohexane-1,2-diylbis(azanediyl)bis(methylene) Diphenol-dichloroplatinum(II) (4). Compound 1 was synthesized by following a published procedure (9). Compound 1, 65.2 mg (0.2 mmol) in methanol (5 mL), was added dropwise to an aqueous solution of equimolar K2PtCl4. The reaction solution was stirred for 2 h, and a yellow-colored microcrystalline product was obtained in 46% yield. Elemental analysis calcd (%) for C20H26N2O2PtCl2: C, 40.55; H, 4.42; N, 4.73. Found: C, 40.52; H, 4.36; N, 4.83%. ESI-MS calculated for [C20H26N2O2PtCl2 + H]+, 592.1; found, 592.2. 1H NMR (CDCl3): δ ) 1.28-1.45 (m, 8H, -CH2- of cyclohexane), 2.82 (s, 2H, -CH- of cyclohexane), 3.73 (m, 4H, -CH2-Ar), 6.55-7.12 (m, 8H, H-Ar). Preparation of 2,2′-(1S,2S)-Cyclohexane-1,2-diylbis(azanediyl)bis(methylene) Diphenol-dichloroplatinum(II) (5). Compound 2, 65.2 mg (0.2 mmol) in methanol (5 mL), was added dropwise to an aqueous solution of equimolar K2PtCl4. The reaction solution was stirred for 2 h, and a yellow-colored microcrystalline product was obtained in 48% yield. ESI-MS calculated for [C20H26N2O2PtCl2 + H]+, 592.1; found, 592.1. Elemental analysis calcd (%) for C20H26N2O2PtCl2: C, 40.55; H, 4.42; N, 4.73. Found: C, 40.46; H, 4.32; N, 4.65%. 1H NMR (CDCl3): δ ) 1.28-1.48 (m, 8H, -CH2of cyclohexane), 2.95 (s, 2H, -CH- of cyclohexane), 3.74 (m, 4H, -CH2-Ar), 6.60-7.15 (m, 8H, H-Ar). Preparation of 2,2′-(1R,2S)-Cyclohexane-1,2-diylbis(azanediyl)bis(methylene) Diphenol-dichloroplatinum(II) (6). Compound 3, 65.2 mg (0.2 mmol) in methanol (5 mL), was added dropwise to an aqueous solution of equimolar K2PtCl4. The reaction solution was stirred for 2 h, and a yellow-colored microcrystalline product was obtained in 36% yield. ESI-MS calculated for [C20H26N2O2PtCl2 + H]+, 592.1; found, 592.2. Elemental analysis calcd (%) for C20H26N2O2PtCl2: C, 40.55; H, 4.42; N, 4.73. Found: C, 40.47; H, 4.41; N, 4.80%. 1H NMR (CDCl3): δ ) 1.18-1.45 (m, 8H, -CH2of cyclohexane), 3.02 (s, 2H, -CH- of cyclohexane), 3.84 (m, 4H, -CH2-Ar), 6.73-7.26 (m, 8H, H-Ar). Preparationof4-Hydroxy-3-{[(1R,2R)-2-(2-hydroxybenzylamino)cyclohexyl-amino]methyl}benzoic Acid (7). A solution of 3-formyl4-hydroxy-benzoic acid (0.10 mmol) and 2-hydroxy-benzaldehyde (0.10 mol) was added dropwise to a solution containing R,R-DACH (0.1 mmol) in 10 mL of EtOH, and this was followed by the addition of 0.40 mmol of Et3N to the system. After 2 h of stirring, a yellow-colored Schiff-base product was obtained. To this system
Gao et al. was added NaBH4 (2.0 mmol, 80 mg) in solid form to reduce the Schiff base compound. The solution was magnetically stirred for an additional 2 h at room temperature. The solvent was removed using a rotary evaporator. The reduced product was purified by column chromatography to remove honosubstituted impurity. Yield, 70%. Anal. calcd for C21H26N2O4: C, 68.09; H, 7.07; N, 7.56. Found: C, 68.12; H, 7.11; N, 7.72%. ESI-MS calculated for [C21H26N2O4 + H]+, 371.19; found, 371.18. 1H NMR (CDCl3): δ ) 1.29-1.37 (m, 8H, CH2 of cyclohexane), 2.82 (s, 2H, CH of cyclohexane), 3.84 (m, 4H, CH2-Ar), 6.61-6.90 (m, 4H, H-Ar-OH), 7.51-7.92 (m, 3H, H-Ar-COOH), 11.6 (s, 1H, HOOC-Ar). Preparationof4-Hydroxy-3-{[(1S,2S)-2-(2-hydroxybenzylamino)cyclohexyl-amino]methyl}benzoic Acid (8). A solution of 3-formyl4-hydroxy-benzoic acid (0.10 mmol) and 2-hydroxy-benzaldehyde (0.10 mol) was added dropwise to a solution containing S,S-DACH (0.1 mmol) in 10 mL of EtOH, and this was followed by the addition of 0.40 mmol of Et3N to the system. After 2 h of stirring, a yellow-colored Schiff base product was obtained. To this system was added NaBH4 (2.0 mmol, 80 mg) in solid form to reduce the Schiff base compound. The solution was magnetically stirred for an additional 2 h at room temperature. The solvent was removed using a rotary evaporator. The reduced product was purified by column chromatography and was identified as the desired product. Yield, 65%. Anal. calcd for C21H26N2O4: C, 68.09; H, 7.07; N, 7.56. Found: C, 68.07; H, 7.03; N, 7.64%. ESI-MS calculated for [C21H26N2O4 + H]+, 371.19; found, 371.18. 1H NMR (CDCl3): δ ) 1.29-1.35 (m, 8H, CH2 of cyclohexane), 2.84 (s, 2H, CH of cyclohexane), 3.80 (m, 4H, CH2-Ar), 6.64-7.01 (m, 4H, H-Ar), 7.70-7.82 (m, 3H, H-Ar-COOH), 12.6 (s, 1H, HOOC-Ar). Preparationof4-Hydroxy-3-{[(1R,2S)-2-(2-hydroxybenzylamino)cyclohexyl-amino]methyl}benzoic Acid (9). A solution of 3-formyl4-hydroxy-benzoic acid (0.10 mmol) and 2-hydroxy-benzaldehyde (0.10 mol) was added dropwise to a solution containing R,S-DACH (0.1 mmol) in 10 mL of EtOH, and this was followed by the addition of 0.40 mmol of Et3N to the system. After 2 h of stirring, a yellow-colored Schiff base product was obtained. To this system was added NaBH4 (2.0 mmol, 80 mg) in solid form to reduce the Schiff base compound. The solution was magnetically stirred for an additional 2 h at room temperature. The solvent was removed using a rotary evaporator. The reduced product was purified by column chromatography and was identified as the desired product. Yield, 62%. Anal. calcd for C21H26N2O4: C, 68.09; H, 7.07; N, 7.56. Found: C, 68.15; H, 7.01; N, 7.60%. ESI-MS calculated for [C21H26N2O4 + H]+, 371.19; found, 371.18. 1H NMR (CDCl3): δ ) 1.28-1.37 (m, 8H, CH2 of cyclohexane), 2.80 (s, 2H, CH of cyclohexane), 3.88 (m, 4H, CH2-Ar), 6.24-6.95 (m, 4H, H-Ar), 7.75-7.87 (m, 1H, H-Ar-COOH), 10.9 (s, 1H, HOOC-Ar). Synthesis of Conjugate 10. Compound 7, 74.0 mg (0.2 mmol), was dissolved in 5 mL of methanol. To this was added 1,1′carbonyldiimidazole (CDI; 1 equiv), and the reaction mixture was stirred at room temperature for 2 h. To this system was added a tetrahydrofuran (THF) solution containing 0.2 mmol of 5-carboxyfluorescein with a hexane-diamine modified terminal. The reaction proceeded at room temperature for 4 h, and the dye-tagged ligand was purified by gel chromatography. Yield, 56%. ESI-MS calculated for [C48H49N3O9 + H]+, 812.35; found, 812.35. 1H NMR (D2O): δ ) 1.22-1.62 (m, 18H, -CH2-), 2.82 (s, 2H, -CH- of cyclohexane), 2.98 (s, 2H, -CH2-CO), 3.84 (m, 4H, CH2-Ar), 6.61-7.76 (m, 14H, H-Ar), 8.02 [s, 1H, H-Ar(COOH)(COR)], 8.29 [m, 1H, H-Ar(COR)], 10.6 (s, 1H, HOOC-Ar). A solution of the above conjugate (0.1 mmol) in 5 mL of methanol was added dropwise to an aqueous solution containing an equivalent of K2PtCl4. The reaction at room temperature for 2 h afforded a yellowcolored precipitate. The final product was obtained by recrystallization from a methanol/ethanol solution. Yield, 36%. ESI-MS calculated for [C48H49N3O9PtCl2 + H]+, 1077.25; found, 1077.26. Anal. calcd for C48H49N3O9PtCl2: C, 53.48; H, 4.58; N, 3.90. Found: C, 53.44; H, 4.45; N, 3.88%. 1H NMR (D2O): δ ) 1.20-1.68 (m, 18H, -CH2-), 2.82 (s, 2H, -CH- of cyclohexane), 3.12 (s, 2H, -CH2-CO), 3.80
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Scheme 1
Table 1. IC50 (µM)a Values ((SD) of Compounds 1-6, the Mixture of 4 and 5, Cisplatin, and Oxaliplatin IC50 (µM) compounds
MCF-7
MCF-10A
TRc ) IC50 (MCF-10A)/IC50 (MCF-7)
1 2 3 4 5 6 4 + 5b cisplatin oxaliplatin
0.4 ( 0.1 0.6 ( 0.1 1.8 ( 0.1 0.2 ( 0.1 0.5 ( 0.1 0.7 ( 0.1 0.3 ( 0.1 11.2 ( 0.2 16.4 ( 0.2
19.8 ( 0.1 17.6 ( 0.1 12.7 ( 0.1 12.6 ( 0.1 10.0 ( 0.1 9.9 ( 0.1 10.5 ( 0.2 28.3 ( 0.3 45.8 ( 0.3
50 29 7 63 20 14 35 3 3
a Mean of two experiments ( range. b An isomeric mixture of compounds 4 and 5 in a 1:1 molar ratio. c TR represents toxicity ratio.
(m, 4H, CH2-Ar), 6.52-7.81 (m, 14H, H-Ar), 8.13 [s, 1H, H-Ar(COOH)(COR)], 8.24 [m, 1H, H-Ar(COR)], 10.5 (s, 1H, HOOC-Ar). Synthesis of Conjugate 11. Compound 8, 74.0 mg (0.2 mmol), was dissolved in 5 mL of methanol. To this was added CDI (1 equiv), and the reaction mixture was stirred at room temperature for 2 h. To this system was added a THF solution containing 0.2 mmol of 5-carboxyfluorescein with a hexane-diamine modified terminal. The reaction proceeded at room temperature for 4 h, and the dye-tagged ligand was purified by gel chromatography. Yield, 62%. ESI-MS calculated for [C48H49N3O9 + H]+, 812.35; found, 812.35. 1H NMR (D2O): δ ) 1.20-1.71 (m, 18H, -CH2-), 2.76 (s, 2H, -CH- of cyclohexane), 3.00 (s, 2H, -CH2-CO), 3.82 (m, 4H, CH2-Ar), 6.54-7.80 (m, 14H, H-Ar), 8.16 [s, 1H, H-Ar(COOH)(COR)], 8.25 [m, 1H, H-Ar(COR)], 9.8 (s, 1H, HOOC-Ar). A solution of the above-synthesized conjugate (0.1 mmol) in 5 mL of methanol was added dropwise to an aqueous solution containing an equivalent of K2PtCl4. The reaction at room temperature for 2 h afforded the formation of a yellow-colored precipitate. Pure product was obtained by recrystallization from a methanol/ethanol solution. Yield, 44%. ESI-MS calculated for [C48H49N3O9PtCl2 + H]+, 1077.25; found, 1077.25. Anal. calcd for C48H49N3O9PtCl2: C, 53.48; H, 4.58; N, 3.90. Found: C, 53.52; H, 4.41; N, 3.92%. 1H NMR (D2O): δ ) 1.18-1.54 (m, 18H, -CH2-), 2.78 (s, 2H, -CH- of cyclohexane), 2.96 (s, 2H, -CH2-CO), 3.81 (m, 4H, CH2-Ar), 6.58-7.70 (m, 14H, H-Ar), 8.05 [s, 1H, H-Ar(COOH)(COR)], 8.31 [m, 1H, H-Ar(COR)], 10.6 (s, 1H, HOOC-Ar). Synthesis of Conjugate 12. Compound 9, 74.0 mg (0.2 mmol), was dissolved in 5 mL of methanol. To this was added CDI (1 equiv), and the reaction mixture was stirred at room temperature for 2 h. To this system was added a THF solution containing
Figure 1. MCF-7 (A) and MCF-10A (B) cells at a density of ∼250 cells per well were seeded in 12-well plates. On the second day, cells were treated with the indicated concentration of compounds 4-6. The same treatments were repeated every 3 days. After 10 days, the plate was stained for the formation of cell colonies with SRB dye.
0.2 mmol of 5-carboxyfluorescein with a hexane-diamine modified terminal. The reaction proceeded at room temperature for 4 h, and the conjugate was purified by gel chromatography, Yield, 71%. ESI-MS calculated for [C48H49N3O9 + H]+, 812.35; found, 812.36. 1H NMR (D2O): δ ) 1.21-1.57 (m, 18H, -CH2-), 2.63 (s, 2H, -CH- of cyclohexane), 3.07 (s, 2H, -CH2-CO), 3.83 (m, 4H, CH2-Ar), 6.53-7.80 (m, 14H, H-Ar), 8.10 [s, 1H, H-Ar(COOH)(COR)], 8.27 [m, 1H, H-Ar(COR)], 10.2 (s, 1H, HOOC-Ar). A solution of the above-synthesized conjugate (0.1 mmol) in 5 mL of methanol was added dropwise to an aqueous solution containing an equivalent of K2PtCl4. The reaction at room temperature for 2 h afforded a yellow-colored precipitate. The final product was obtained by recrystallization from a methanol/ethanol solution. Yield, 35%. ESI-MS calculated for [C48H49N3O9PtCl2 + H]+, 1077.25; found, 1077.25. Anal. calcd for C48H49N3O9PtCl2: C, 53.48; H, 4.58; N, 3.90. Found: C, 53.53; H, 4.63; N, 3.95%. 1H NMR (D2O): δ ) 1.17-1.62 (m, 18H, -CH2-), 2.79 (s, 2H, -CH- of cyclohexane), 2.98 (s, 2H, -CH2-CO), 3.84 (m, 4H, CH2-Ar),6.61-7.70(m,14H,H-Ar),8.06[s,1H,H-Ar(COOH)(COR)], 8.27 [m, 1H, H-Ar(COR)], 10.3 (s, 1H, HOOC-Ar). Cell Culture and Growth Inhibition Assay. Human cancer cell lines MCF-7 and MCF-10A were obtained from American Type Culture Collection (Manassas, VA). Cells were maintained in RPMI medium supplemented with 10% fetal bovine serum, penicillin (100 U/mL), and streptomycin (100 µg/mL) at 37 °C in an atmosphere humidified with 5% CO2 and 95% air. Cells were seeded in 96-well cell culture plates and treated on the second day with the drug candidates. Experimental compounds 1-6 were dissolved in ethanol at 2.0 mM and diluted immediately with PBS to the desired final maximum test concentrations (10 and 5.0 µM, respectively). Cisplatin and oxaliplatin were predissolved in DMSO (10 mM) and diluted with PBS to the required concentration, such that the total DMSO
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Figure 2. (A) Cellular accumilation of compounds 4-6, oxaliplatin, and cisplatin in MCF-7 cells after drug exposure for 2 h. (B) DNA-bound Pt for compounds 4-6, oxaliplatin, and cisplatin in MCF-7 cells after drug exposure for 24 h.
Figure 3. Relative expression of cell cycle and apoptosis-related genes. MCF-7 cells were treated with 5.0 µM compounds 4-6 for 24 h. The mRNA expression was determined by real-time RT-PCR and normalized with GAPDH expression in each samples. *P < 0.05, statistically significant differences from the control group (error bars were given as STDEV of triplicate measurements).
concentration did not exceed 0.8%. At this concentration, DMSO was found to be nontoxic to the cells tested. At the end of a 2 day treatment, the cell number was estimated by the SRB assay as described in the literature (15). The percentage of growth inhibition was calculated by using the equation: % growth inhibition ) (1 - At/Ac) × 100%, where At and Ac represent the absorbance in treated and control cultures, respectively.
In Vitro Uptake of Pt and Measurement of DNA-Bound Pt. The Pt content was measured using a graphite furnace atomic absorption spectrometer (AA-700 Perkin-Elmer). The standard for calibration curves was established using compound 4, oxaliplatin, and cisplatin. Five × 106 cells were seeded, and after 24 h, cells were treated with Pt complexes at 1, 2, 3, 4, and 5 µM and the corresponding IC50 value for each compound. After
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Figure 4. Relative expression of cell cycle and apoptosis-related genes. MCF-7 cells were treated with 5.0 µM compounds 1-3 for 24 h. The mRNA expression was determined by real-time RT-PCR and normalized with GAPDH expression in each samples. *P < 0.05, statistically significant differences from the control group (error bars were given as STDEV of triplicate measurements).
2 h of exposure to the Pt compounds, the drug-containing medium was removed, and the cells were washed with ice-cold PBS. The cells were than trypsinized and centrifuged into a pellet. The isolated cells were digested in concentrated HNO3 and H2O2. Data represented in Figure 2 were obtained from triplicate runs from a single sample at a given concentration. For DNA-Pt binding measurements, 5 × 106 cells were seeded, and after 24 h, cells were treated with the Pt compounds at 1, 2, 3, 4, and 5 µM and the corresponding IC50 value for 24 h. After the treatment, cells were washed with ice-cold PBS. The cells were trypsinized and centrifuged into a pellet. Cells were then subjected to DNA extraction using a Wizard SV DNA purification kit (Promega). The quantity of DNA was determined by UV-vis instrument from the absorption at 260 nm. Triplicate runs were performed to determine the content of Pt in DNA. Real-Time RT-PCR Analysis. The total RNA from MCF-7 cells was isolated, following homogenization of the tissue, with the Tri Reagent (Sigma, St. Louis, MO). After DNA-free DNase treatment (Ambion, Austin, TX), 200 ng of the total RNA was used in RT by the GeneAmp RNA PCR kit (Perkin-Elmer, Foster City, CA), Real-time PCR was then performed on cDNA from RT reactions, using OmniMix HS bead (Cepheid, Sunnyvale, CA) according to the manufacturer’s protocol. SYBR green dye (1×; Fisher Scientific, Atlanta, GA) was added to the reaction mixture to detect amplicon synthesis in the SmartCycler real-time PCR thermal cycler (Cepheid). Specific primers of genes were designed on different exons, and used were the following primers:
Cyclin D1: forward, 5′-ATGGAACACCAGCTCCTGTGCT3′; reverse, 5′-GCGGCCAGGTTCCACTTGAGCT-3′ P27: forward, 5′-CTGTGGAGCAGACGCCCAAGAAG-3′; reverse, 5′-CCTGCCCTCCCTTCCCCAAAGTT-3′ Bcl-2: forward, 5′-CAGCTGCACCTGACGCCCTTCACC3′; reverse, 5′-CTGAGCAGAGTCTTCAGAGACAGC-3′ Bcl-xL: forward, 5′-GCACTGTGCGTGGAAAGCGTAGAC3′; reverse, 5′-CTGAAGAGTGAGCCCAGCAGAACC-3′ Bad: forward, 5′-CCTTTCGGGGCCGCTCGCGCTCG-3′; reverse, 5′-CACTGGGAGGGGGCGGAGCTTCC-3′ Bax: forward, 5′-GACAGGGGCCCTTTTGCTTCA-3′; reverse, 5′-AACCCGGCCCCAGTTGAAGTT-3′ P53: forward, 5′-GCGCACAGAGGAAGAGAATC-3′; reverse, 5′-CTCTCGGAACATCTCGAAGC-3′ P21: forward, 5′-CTGGCACCTCACCTGCTCTGCT-3′; reverse, 5′-GCTTCCAGGACTGCAGGCTTCC-3′ The specificity of the PCR-amplified product was verified by sequencing the product (data not shown). The thermal cycling conditions included an initial denaturation step at 95 °C for 2 min and 45 cycles at 95 °C for 10 s, 65 °C for 15 s, and 72 °C for 30 s. For quantification, the cycle threshold number (Ct) that exhibits the maximum curve growth rates was determined using the Cepheid SmartCycler software. The relative gene expression of each sample
was normalized to that of glyceraldehyde-3-phosphate dehydroge(GAPDH)-C(gene) t nase (GAPDH) and calculated by the formula 2Ct . Statistical Analysis. Results are presented as mean ( standard deviations (STDEV) for at least three repeated individual experiments for each group. Statistical analyses were performed using SPSS software (version 10.0.5). Fisher’s exact test was employed for independent samples. A P value of
