第二章 研究方法(METHODS)
第二節 研究材料 (M ATERIALS )
三、 藥品試劑 (Reagents)
（A） 40% 29:1 Acrylamide/Bis (BIO BASIC.INC, Canada )
（B） APS (USB, USA )
（C） Bradford ( BIO-RAD, USA )
（D） Bromophenol Blue (AMRESCO®, USA)
（E） Caffeine (SIGMA, USA）
（F） DMEM （GIBCO,USA）
（G） DMSO（SIGMA, USA）
（H） DTT (BIO-RAD, USA )
（I） Ethanol (ECHO Chemical, Taiwan)
（J） FBS（GIBCO,USA）
（K） Glycerol (GERBU, Germany )
（L） Glycine (GERBU, Germany )
（M） Methanol (ECHO Chemical, Taiwan)
（N） MTT (BIO BASIC INC., Canada)
（O） Nonfat dry milk (Anchor, New Zealanders)
（P） Potassium phosphate Monobasic,Crystal (KH2PO4) (J.T. Baker, USA)
（Q） Potassium chloride (KCl) (Scharlau, Spain)
（R） Sodium chloride ( BIO BASIC INC., USA )
（S） Sodium phosphate, dibasic anhydrous (Na2HPO4) (J.T. Baker, MALAYSIA)
（T） Sodium dihydrogen phosphate, monohydrate (NaH2PO4‧H2O) (MERCK, Germany)
（U） Sodium bicarbonate (NaHCO3) (BIO BASIC INC.,
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USA)
（V） Sodium azide (NaN3) (USB, USA)
（W） TEMED (AMRESCO, USA )
（X） Tis-HCL(GERBU, USA )
（Y） Tris-C (GERBU, USA )
（Z） Triton X-100（SIGMA, USA）
（AA） Trypan blue（GIBCO,USA）
（BB） Trypsin（GIBCO,USA）
（CC） Tween-20 (GERBU, USA )
2. 抗體 (Antibodies)
（A） 一級抗體 (Primary Antibodies)
i. Anti- phsopho-ATM (Ser 1981) (ab36810; Abcam, USA)
ii. Anti- phospho-ATR (Ser 428) (sc-109912; SENTA CRUZ, USA)
iii. Anti-phospho-Chk1 (Ser 345) (#9114; Cell Signaling, USA)
iv. Anti-phospho-Chk2 (Thr 68) (#2661; Cell Signaling, USA)
v. Anti-Chk1 (#2345; Cell Signaling, USA) vi. Anti-Chk2 (#2662; Cell Signaling, USA) vii. Anti-Cyclin B1(#4138 ;Cell Signaling, USA)
viii. Anti-phospho-cdc2 (Thr 161) (#9114 ;Cell Signaling, USA)
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ix. Anti-Cdc2 (#9112;Cell Signaling, USA)
x. Anti-Caspase 3 (AB1899; MILLIPORE, USA) xi. Anti-PARP (ab4830; Abcam, USA)
xii. Anti-β-actin (sc-47778; SENTA CRUZ, USA)
（B） 二級抗體 (Secondary Antibodies)
i. Goat-anti-mouse IgG HRP (Sc-2005; SENTA CRUZ, USA)
ii. Goat-anti-rabbit IgG HRP (NEF812001EA; PerkinElmer, USA)
3. 試劑組 (Kits)
（A） Western Lightning® Plus-ECL Enhanced
Chemiluminescence Substrate (NEL 104001EA; PerkinElmer, USA)
（B） Bio-Rad Protein Assay Kit (500-0002; Bio-Rad, USA)
（C） Pro-PREP™ Protein Extration Solution (17081.1; iNtRON BIOTECHNOLOGY, Korea)
四、 設備與器材 (Equipments)
（A） BD FACSCanto (Argon-Ion Laser 488 nm, He-Ne Lasser 633 nm) (Becton Dickinson, USA)
（B） Centrifuge (Hettich, Germany)
（C） Centrifuge 5804R (eppendorf, Germany)
（D） CO2 Incubator (NU4500, Nuaire, USA)
（E） Dry Bath (Model 110001, Boekel)
（F） Elctronic Precision Scales/ Balances (ACCULAB sartorius
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group, Germany)
（G） Freezer (Frigidaire®, USA)
（H） General-Purpose Analysis Software Multi Gauge -Ver3.X- (FUJIFILM, Japan)
（I） Hemocytometer (Boeco, Germany)
（J） Heraeus Megafuge 16R Centrifuge (Thermo Fisher Scientific Inc., USA)
（K） Hotplate Stirrer (Laboratory & Medical Supplies, Japan)
（L） Laminar Flow Hood (TSAO HSIN ENTERPRISE CO.,LTD., Taiwan)
（M） LAS-4000 luminescence/fluorescence imaging system (FUJIFILM, Japan)
（N） Microscope (Nikon, TE-2000-U, Japan)
（O） Mini Format Vertical Electrophoresis (Mini-PROTEAN®
Tetra Cell, Bio-RAD, USA)
（P） ModFit LT Analysis Software (Verity Software House, USA)
（Q） MS Orbital Shaker (Major Science, Taiwan)
（R） Orbital Shaker (GENPURE, Taiwan)
（S） pH Meter (C831, Consort, UK)
（T） PowerPac™ Basic Power Supply (Bio-RAD, USA)
（U） Spectrafuge 24D Microcentrifuge (Labnet International, Inc., USA)
（V） Tank Transfer Systems (Mini Trans-Blot® Cell, Bio-RAD, USA)
（W） Ultrapure water System (Millipore, USA)
（X） Vortex Genie-2 (Scientific Industries, Inc., USA)
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（Y） Water Bath (YIHDERN, Taiwan)
（Z） ELISA Reader (BioTeck, British)
（AA） ELISA Reader (ANTHOS-2030, Salzbrug, Austria)
（BB） Cell Culture dishes (Greiner Bio-One, USA)
（CC） Centrifuge tube (AXYGEN, USA)
五、 試劑配製 (Reagent Preparation)
Table 4 1.5M Tris-HCl, pH 8.8
Table 5 0.5M Tris-HCl, pH6.8
Final concentration Amount
Tris-base 181.71 g
HCl adjust to pH 8.8
1.5 M
d.d H2O added to total volume 1000 ml
, p
Final concentration Amount
Tris-base 60.57 mg
HCl adjust to pH 6.8
0.5 M
d.d H2O added to total volume 1000 ml
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Table 6 PI Staining Solution
Table 7 10％ SDS
HCl adjust to pH 7.4
27
Table 10 0.05％ MTT Reagent
Table 11 SDS-PAGE Preparation
Table 12 Running Buffer
Final concentration Amount
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Table 13 6× Protein Dye
Table 14 Transfer Buffer
Table 15 1×TBST Buffer
y
HCl adjust to pH 6.8
d.d H2O added to total volume 15 ml
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第三節 實驗方法 (Experimental Methods)
一、 細胞培養 (Cell Culture)
1. 人類肺癌細胞株 A549 之細胞培養 (Culture of Human Lung Cancer Cell Line, A549)
A549 human cell lung carcinoma cell line was form the American Type Culture Collection (ATCC). Cells were grown in Dulbecco’s modified Eagles’s medium (DMEM), supplemented with 10% Fetal bovine serum (FBS) and 100 units/ml penicillin G, and 100 μg/ml streptomycin sulfates.
The culture medium was replaced every two days and cells were passaged every week. Cells that became at least 80% confluent were starved for 24 h in DMEM followed by a treatment with indicated concentration of NSC746364 in DMEM containing 10% FBS for indicated time.
2. 細胞計數 (Cell Counting)
The most widely used type of cell counting chamber is called a hemocytometer. To prepare the device the surface of counting chamber is carefully cleaned with 70% ethanol and wiped out the surface with lens paper.
The cover glass is also cleaned and is placed over the counting surface prior to introducing the cell suspension. First, 100 μl of cell suspension was mixed with 10 μl of Trypan Blue dye (GIBICO) in 1.7 ml eppendorf, then placed 10 μl of the mixture in both wells of the hemocytometer. viable cells were count in both sides of the hemocytometer using the hand counter while looking through the microscope.
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二、 細胞存活檢測 (MTT Cell Viability Assay)
MTT assay was performed to measurethe cytotoxicity of NSC746364 on A549 lung cancer cells. Cells were seeded in 24-well plates with 2×104 cells/well in DMEM supplemented with 10% FBS. After 24 hr, cells were washed with phosphate-buffered saline (PBS) and then exposed to either DMSO alone or different concentrations（5, 10 and 20 μM） of NSC746364.
After 24 hr and 48 hr, the number of viable cells was determined. Briefly, MTT (0.5 mg/ml in DMEM containing 10% FBS) was added to each well (400 μl per well), and the plate was incubated at 37℃ for 4 hr. Cells were then spun at 300g for 5min, and the medium was carefully aspirated. A 400 μl aliquot of DMSO was added, and the absorbance at 595 nm was measured for each well on ELISA reader (Anthos, 2001, Anthos Labtech. Austria).
三、 流式細胞儀─細胞週期分析 (Flow Cytometry─Cell Cycle
Analysis)
The DNA content of the treated cells was assessed using flow cytometry following propidium iodide (PI) staining. Cells were seeded in 6-cm petri-dishes with 2×105 cells/dish in DMEM supplemented with 10% FBS.
After 24 hr, cells were washed with phosphate-buffered saline (PBS) and starved for 24 hr in DMEM followed by exposing to either DMSO alone or serial dilutions（5, 10 and 20 μM） of NSC746364 for 24 hr or 48 hr. After 24 hr or 48 hr, cells were harvested with trypsin-EDTA, washed twice with 10 ml ice-cold PBS, fixed in 70% ethanol, and kept at -20℃ prior to FACS analysis. For DNA content analysis, cells were centrifuged and resuspended
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in 0.4 ml of DNA staining solution [0.4 mg/dl PI, 1% Triton X-100, 0.1 mg/ml RNase A (DNase-free) in PBS]. The cell suspension was stored at 4℃
and protected from light for a minimum of 30 min and analyzed within 2 hr.
Cells were analyzed using a FACScan flow cytometer (Becton Dickinson, San Jose, CA, USA). The percentages of hypodiploid (apoptotic, sub-G1) events and percentages of cells in G0/G1, S-, and G2-M phases were determined using the DNA analysis software ModFitLT, version 2.0 (Verity Software, Topsham, ME, USA).
四、 西方墨點法 (Western Blot)
A549 cells cultured in petri dishes were incubated with 5, 10 and 20 μM of NSC746364 in DMEM containing 10% FBS for 24hr. For the time course experiments, cells were treated with 20 μM of NSC746364 in DMEM containing 10% FBS for 15, 30, and 60 min. Cells were then lysed in protein extraction buffer (iNtRON Biotechnology Inc), followed by incubation at 95°C for 5 min. Samples were separated using SDS-PAGE, transferred to PVDF membranes, blocked with 5% nonfat dry milk in TBST (Tris-base saline containing 0.05% Tween-20) for 1 h, and then probed with the desired antibodies [anti-Cyclin B1 (Cell Signaling #4138), anti-Cdc2 (Cell Signaling
#9112), anti-phsoho-Cdc2-Thr-161(Cell Signaling #9114), anti-p-ATM-Ser-1981 (ab36810), anti-ATM, anti-p-ATR-Ser-428 (sc-109912), anti-ATR, anti-p-Chk1-Ser345 (Cell Signaling #2341), anti-Chk1 (Cell Signaling #2345), anti-p-Chk2-Ser345 (Cell Signaling #2661), anti-Chk2 (Cell Signaling #2662) and anti-β-actin (sc-47778) ] overnight at 4°C. The blots were then incubated with horseradish peroxidase-linked
32
secondary antibody for 1 h followed by development with the ECL reagent and chemiluminescence signals were detected by LAS-4000 luminescence/fluorescence imaging system (FUJIFILM). The intensities were quantified by densitometric analysis software Multi Guage version 2.0 (FUJIFILM).
五、 DAPI 染色 (DAPI Staining)
A549 cells were seeded in 6-wells plate and incubated with 0, 5, 10, and 20 μ M of NSC746364 in DMEM containing 10% FBS for 24 h. After 24 h incubation time, cells were washed three times with PBS, fixed with 3%
Formaldehyde for 15–20 min, following 0.1% Triton X-100 for 15 min, and stained with DAPI (1μg/mL) for 1 min. Wash cells two times with PBS. Cells were observed by using fluorescence microscope, and then all pictures were taken at 40X.
六、 統計分析 (Statistics Analysis)
Data are expressed as mean ± S.E.M Statistical analysis was conducted using ANOVA test. A p-value ≦ 0.05 was considered significant.
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第三章 實驗結果 (Results)
一、 NSC746364 suppresses cell proliferation of A549 human lung cancer cell line
The effect of NSC746364 on cell proliferation was assessed using the MTT proliferation assay. To test the effect of NSC746364 on the proliferation of A549 cells, the cells were treated with different concentrations of NSC746364 (0 μM, 5 μM, 10 μM, 20 μM). After 24 h (A) and 48 h (B) incubation, the cell viability was determined by using the MTT assay. As shown in Figure 11 A and B, treatment with NSC746364 significantly inhibited the viability and proliferation of cells, and these effects occurred in a dose-dependent manner.
Figure 11 Cell survival inhibition of A549 human lung cancer cell lines by NSC746364 assessed using the MTT proliferation assay. NSC746364 significantly suppressed proliferation of A549 cells in concentration dependent manner. Cells were treated with NSC746364 at concentrations 5, 10, and 20 μM for 24 h (A) and 48 h (B). Untreated groups (0 μM) contained DMSO less than 0.01％. All results were obtained from at least 3 times independent experiments. Statistical significance was determined using
34
ANOVA test, (* p<0.05).
二、 NSC746364 modulates cell cycle progression
The consequences of telomere dysfunction caused by telomere-disrupting agents, such as G-quadruplex inhibitors, resulting in activation of DNA damage response signaling and leading to activation of cell cycle checkpoints [161, 205-208]. The cell cycle distribution of A549 cells was examined by flow Cytometry on cells treated with various concentrations (0 μM, 5 μM, 10 μM, 20 μM) of NSC746364 for 24 h Figure 12 (A to E) and 48 h (F to J). In the present study, flow cytometric analysis showed that NSC746364 modulated cell cycle progression through inducing cells to accumulate at G2/M-phase with concurrent decrease of cells at G0/G1 and S phase.
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Figure 12 Flow cytometric analyses elucidated the effect of NSC746364 on the cell cycle phase distribution in A549 cells. Averages from three independent experiments of 0.01% DMSO-treated cells (0 μM, as a control) and NSC736364-treated cells at doses 5, 10 and 20 μM for 24 h (A to E) and 48 h (F to J) were calculated. Statistical significance was analyzed using ANOVA test, (* p<0.05). NSC746364 induced G2/M cell cycle arrest in a dose-dependent manner.
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三、 NSC746364 modulates cell cycle progression through activation of DNA damage sensing pathways
To further elucidate the mechanism by which NSC746364 induced G2/M arrest through investigating G2/M cell cycle regulatory proteins. Two dominant regulatory proteins, Cyclin B1 and the mitotic marker protein kinase (Cdk1/Cdc2) were determined by western blot analysis. We found that NSC746364 down-regulated Cyclin B1 levels in a dose-dependent manner. However, the protein levels of Cdc2 were not affected by NSC746364. We also found that NSC746364 up-regulate both Chk1 and Chk2 phosphorylation at Ser-345 and Thr-68, respectively. However, NSC746364 markedly increases Chk1 phosphorylation at Ser-345 but not Chk2 phosphorylation at Thr-68. Furthermore, the significantly increased Chk1 phosphorylation at Ser-345 could be detected very early after 15 min of NSC746364 treatment.
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Figure 13 Effects of NSC746364 on protein expression level of Cyclin B1, p-Chk1, and p-Chk2 in A549 cells. (A) Cells were treated with 5, 10 and 20 μM for 24 h. Cell lysates were analyzed by Western blotting with anti-Cyclin B1, anti-Cdc2, and anti-β-actin antibodies. The bar graphs are results of densitometry analyses of the ratio of Cyclin B1 to β-actin. (B) A549 cells were incubated with NSC746364 for 0, 15, 30, and 60 minutes. The protein levels of p-Chk1, p-Chk2, Chk1, Chk2, and β-actin were determined by Western blot. The bar graphs are results of densitometry analyses of the ratio of p-Chk1 to β-actin and p-Chk2 to β-actin. Each value represents the mean±
S.D. *P<0.05, as compared to the control (0 μM and 0 min), n=3.
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四、 NSC746364 induces A549 cells apoptosis
As shown in our MTT results, tumor growth was strongly suppressed by NSC746364 treatment, suggesting the cytotoxic effects of NSC746364 on lung cancer cells. To test this hypothesis, we examined the potential apoptotic signaling pathways in A549 cancer cell lines. Our results show that NSC746364 induced tumor cell apoptosis by activating Caspase-3 (Figure 14A). The increasing intensity of DAPI staining demonstrates the dose dependent apoptotic effects of NSC746364 on A549 cells (Figure 14B).
Furthermore, Annexin V-FITC/propidium iodide (PI) staining was another alternative way to evaulate apoptosis. As shown in Figure 14C, NSC746364 significantly increased PI-positive and Annexin-V-positive cells in a dosel–
dependent-manner.
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Figure 14 NSC746364 induces cell apoptosis. (A) A549 cells were incubated with NSC746364 at concentration 5, 10 and 20 μM for 24 h. Cell lysates were analyzed by Western blotting with anti-Caspase-3 and anti-β-actin antibodies.
The bar graphs are results of densitometry analyses of the ratio of either pro-Caspase-3 to β-actin or active Caspase-3 to β-actin. (B) The cells were cultured in 10% FBS with or without NSC746364 for 24 h. After 24 h, the cells were fixed and incubated with DAPI for 1 min, and observed using a fluorescent microscope. White arrows indicate the apoptotic nucleus. All graphs were taken at 40X. (C) Apoptosis of A549 cells treated with different concentrations of NSC746364 (0, 5, 10 and 20 μM) for 24h was assessed using Annexxin V/PI staining and flow cytometry. Cells in the lower right quadrant (Q4) indicate early apoptotic cells. Cells in the upper right quadrant
40
(Q2) indicate late apoptotic cells.
五、 ATM/ATR-Chk1/Chk2 DNA damage sensing signaling pathways are responsible for NSC746364’s action in A549 human lung cancer cell lines We have previously demonstrated that cells treated with NSC746364 showed activated Chk1 and Chk2 protein phosphorylation levels. To further confirm the mechanism that underlies the antitumor effects of NSC746364 via ATM/ATR DNA damage sensing pathways, we examined the effect of caffeine, an inhibitor of ATM and ATR kinases [209], on tumor cells. Cells were either incubated with caffeine (10 mM) for 15 min or treated for 15 min before NSC746364 (20μM) treatment. As the data indicated (Figure 15), cells treated with NSC746364 significantly accumulated in G2/M phase. In contrast, pre-treated cells with caffeine markedly attenuated the effect of NSC746364 on cell cycle regulation. Furthermore, caffeine also significantly increased the cell viability as compared to NSC746364 alone group.
Figure 15 ATM and ATR blockage attenuated the effect of NSC746364 on cell cycle regulation and cell viability. Cells were pre-treated with caffeine (10 mM) for 15 min, following incubated with NSC746364 (20μM) for 24 h. Cell cycle phase distribution (A) and cell viability (B) were determined by flow
41
cytometry and MTT assay, respectively. Each value represents the mean±S.D.
*P<0.05, as compared to the control (0 μM). #P<0.05, as compared to the NSC746364 treated group, n=3.
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第四章 討論 (Discussion)
In the present study, we have shown that NSC746364 can inhibit the growth of A549 human lung cancer cells. One of the major mechanisms by which NSC746364 mediates its effects against lung cancers seems to be through activation of DNA damage signaling pathways, which in turn stalls cell cycle at G2/M phase and subsequently leading to cellular apoptosis.
NSC746364 is one of the novel 2,7-diamidoanthraquinone derivates. It has been shown to inhibit cell growth and telomerase activity of multiple cancer cell lines [171]. Various studies provided evidences that telomerase inhibition would induce growth arrest or apoptosis of cancers [20]. Our results demonstrate that NSC746364 can effectively inhibit cell growth (Figure 11), arrest cell cycle at G2/M phase (Figure 12) and induce cell apoptosis (Figure 14). Although caspase-3 activation and nucleus condense indicate cells are undergoing apoptotic process, we didn’t detect such alteration in our experiments. The sub-G1 peaks on DNA histograms usually represent the apoptotic cells with degraded DNA [210, 211]. However, we did not detect any sub-G1 peaks when we measured the DNA contents of cells (Figure 12).
All these results indicate that the anticancer effects of NSC746364 might mainly through inhibiting cell growth, with minor portion of cells undergoing apoptosis.
The telomere-specific protein complex, named as shelterin, is essential for genome stability and cell survival. These telomere binding proteins have been identified as TRF2, POT1, RAP1, TIN2, and TPP1. All these proteins form the shelterin complex, to prevent telomeres from triggering a DNA damage response [212]. In addition, telomerase is also one of the mechanisms
43
to maintain telomere integrity. Disrupting telomere DNA maintenance by telomerase or telomere targeting agents can be sensed as DAN damage, which in turn rapidly activated signal transducers which responded for DNA damage.
As shown in Figure 13 B, NSC746364 activates ATM/ATR signaling pathways by phosphorylating ATM at Ser-1981 and ATR at Ser-428. However, it seems that NSC746364 dominantly increases ATR/Chk1 phosphorylation but not ATM/Chk2 phosphorylation. One of the possible mechanisms to regulate ATR activation is through repression of the shelterin protein, POT1.
Recent studies showed that distinct shelterin components independently repress ATM and ATR signaling: Activation of ATM at telomeres is repressed by TRF2, while POT1 is required to prevent ATR activation [213-216]. Thus, NSC746364 might downregulate POT1, leading to ATR activation. In Figure 15, caffeine, an ATM and ATR kinase inhibitor [209], effectively abrogate the effects of NSC746364 on cell cycle regulation and cell viability. These results indicate that inhibition of cell growth by NSC746364 is through activation of ATM and ATR. However, more specific inhibitors like siRNA should be used in further experiments to evaluate the ATM/ATR signaling. In addition, the role of POT1 in regulating ATR activation in our experimental models should also be tested.
Taken together, our study demonstrates that tumor growth could be suppressed by NSC746364. Its pharmacological mechanism may be through targeting ATM/ATR/Chk1/Chk2 signaling pathways. Our results unmask the molecular mechanisms of NSC746364 in inhibiting A549 human lung cancer cells growth in vitro to shed light into the conjunctive roles of NSC746364 with some other pharmacological anticancer agents. Further studies using
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animal models or clinical evaluations need to be conducted to confirm the proposed theory in this aspect.
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