Article pubs.acs.org/jnp
Cephalochromin Induces G0/G1 Cell Cycle Arrest and Apoptosis in A549 Human Non-Small-Cell Lung Cancer Cells by Inflicting Mitochondrial Disruption Che-Jen Hsiao,† George Hsiao,‡ Wei-Lin Chen,‡ Shih-Wei Wang,§ Chun-Ping Chiang,‡ Li-Ya Liu,‡ Jih-Hwa Guh,⊥ Tzong-Huei Lee,*,∥ and Chi-Li Chung*,†,▽ †
School of Respiratory Therapy, College of Medicine, Taipei Medical University, Taipei 110, Taiwan Graduate Institute of Medical Sciences and Department of Pharmacology, College of Medicine, Taipei Medical University, Taipei 110, Taiwan § Department of Medicine, Mackay Medical College, New Taipei City 252, Taiwan ⊥ School of Pharmacy, National Taiwan University, Taipei 110, Taiwan ∥ Graduate Institute of Pharmacognosy, Taipei Medical University, Taipei 110, Taiwan ▽ Division of Pulmonary Medicine, Department of Internal Medicine, Taipei Medical University Hospital, Taipei 110, Taiwan ‡
S Supporting Information *
ABSTRACT: The fungus-derived compound cephalochromin, isolated from the fermented broth of Cosmospora vilior YMJ89051501, shows growth-inhibitory and apoptotic activity against human lung cancer A549 cells in a concentration-dependent manner with an IC50 value of 2.8 μM at 48 h. Cephalochromin induced cell cycle arrest at the G0/G1 phase through down-regulation of cyclin D1, cyclin E, Cdk 2, and Cdk 4 expressions. Cephalochromin markedly increased the hypodiploid sub-G1 phase (apoptosis) of the cell cycle at 48 h as measured by flow cytometric analysis. Reactive oxygen species generation and loss of the mitochondrial membrane potential (MMP) were also markedly induced by cephalochromin. Moreover, the immunoblotting assays showed that cephalochromin reduced survivin and Bcl-xL expression and induced the activation of caspase-8, -9, and -3 and the cleavage of poly(ADP-ribose) polymerase, indicating the involvement of a caspase signaling cascade. The caspase inhibitor Z-VAD-fmk significantly suppressed cephalochromin-induced apoptosis. Cephalochromin also triggered LC3 II, autophagic marker, expression. Taken together, this is the first report that cephalochromin induced an antiproliferative effect on human lung cancer cells through mitochondrial disruption and down-regulation of survivin, leading to cell cycle arrest at the G0/G1 phase, loss of MMP, and subsequently apoptotic cell death.
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reported that many anticancer compounds induce G1, S, or G2/M phase arrest, depending on their selectivity.11−13 Apoptosis is an important process for the maintenance of tissue homeostasis.14 The caspases are a family of cystine proteases that provide significant roles in apoptotic pathways.15 Caspase activation is regulated through either extrinsic death receptor-mediated or intrinsic mitochondrial signaling pathways.16 The activated initiator caspases trigger downstream effector caspase activation, thereby executing apoptosis.17 For counteracting regulation, survivin, a member of the inhibitors of apoptosis family, suppresses apoptosis by preventing activation of effector caspases in both intrinsic and extrinsic apoptotic pathways.14 Survivin is usually undetectable in normal adult tissues but is abundantly expressed in fetal tissue and many human tumors including lung cancer.18
ung cancer is the leading cause of cancer deaths in the world.1 Non-small-cell lung cancer (NSCLC) constitutes around 80% of lung malignancies, and the 5-year survival of this highly aggressive disease is only 15%.2,3 Current chemotherapy protocols for NSCLC are known to induce chemoresistance and have a range of adverse effects.4 Therefore, the discovery and development of effective and less toxic antitumor treatments for NSCLC is strongly needed. Especially relevant to this issue are natural fungus-derived products, which have provided abundant candidates with antitumor activity.5 The dysregulation of the tumor cell cycle is governed by altered activation of various cyclin/Cdk complexes.6,7 In general, the G1 phase is subtly regulated by cyclin D1 and its partners Cdk4 and Cdk6. Cyclin E and Cdk2 play a critical role in the G1 to S phase transition. Specifically, cyclin D overexpression is frequently observed in various cancers and is associated with tumorigenesis and metastasis.8 Additionally, aberrant expression of cyclin E is also involved in cancer development and is often found in lung cancer.9,10 It has been © 2014 American Chemical Society and American Society of Pharmacognosy
Received: July 15, 2013 Published: March 3, 2014 758
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Figure 1. Inhibitory effects of cephalochromin on cell growth. (A) Chemical structure of cephalochromin. (B) A549 cells were incubated in the absence (control) or presence of various concentrations of cephalochromin for 48 h. Then, the cells were fixed and stained with sulforhodamine B (SRB). Data are expressed as the mean ± SEM of three independent determinations. (C) Cells were incubated in the presence or absence of the indicated concentrations of cephalochromin for 24 and 48 h. Cellular viability was determined using the MTT assay. Data are expressed as the mean ± SEM of three determinations (each in triplicate). (D) Various types of human cancer cells were treated with a range of cephalochromin for 48 h. The cell number was determined using the SRB assay. The IC50 of each cell line was expressed as the mean ± SEM of three independent determinations.
Table 1. Effects of Cephalochromin on Cell Cycle Phase Distribution in A549 Cellsa treatment control (24 h) cephalochromin (24 h) control (36 h) cephalochromin (36 h) control (48 h) cephalochromin (48 h)
sub-G1 2.0 3.8 1.7 9.8 1.5 15.5
± ± ± ± ± ±
0.3 0.2 0.4 1.4d 0.1 0.7d
G0/G1 51.0 59.6 53.1 53.7 57.3 52.1
± ± ± ± ± ±
1.1 0.5c 1.2 1.7 0.9 1.0
S 12.0 3.9 9.6 4.2 9.5 4.4
± ± ± ± ± ±
G2/M 1.4 1.1c 0.9 1.5b 0.7 1.0
35.2 32.7 35.8 32.3 31.9 27.9
± ± ± ± ± ±
2.3 1.4b 1.8 1.5 1.4 0.4
A549 cells were incubated in the absence (control) or presence of cephalochromin (10 μM) for 24 to 48 h. Cells were then fixed and stained with propidium iodide to analyze DNA content using FACScan flow cytometry. Data are expressed as mean ± SEM of three independent determinations. b p < 0.05 compared with control. cp < 0.01 compared with control. dp < 0.001 compared with control. a
Mitochondria are necessary mediators that play a vital role in the execution of both extrinsic and intrinsic apoptotic pathways.19 Excess mitochondrial reactive oxygen species (ROS) generation causes cellular damage, induces apoptosis, and plays a critical role in caspase-3 activation.20,21 In ROSinduced autophagy, Bcl-xL and Bax may be included in this process, suggesting that ROS may have synergistic effects in apoptosis and autophagy.22 Evidence shows that the proteins of the Bcl-2 family play a pivotal role in regulation of mitochondrial membrane permeability and the mitochondrial pathway.23 Several previous studies also indicate that anticancer agents such as camptothecin, paclitaxel, and doxorubicin induce apoptosis of human cancer cells through generation of ROS, modulation of the Bcl-2 family of proteins, and inhibition of the
mitochondrial membrane potential and the mitochondrial pathway.24−26 A variety of products from fungi have been found to exhibit antitumor activities.27−30 Cephalochromin (Figure 1A) is a bis(naphtho-γ- pyrone) derivative, previously isolated from several fungi including Cephalosporium sp., Pseudoanguillospora sp., and Verticillium sp.31−33 It has been found that cephalochromin is the selective bacterial enoyl-acyl carrier reductase (FabI) inhibitor in both Staphylococcus aureus and Escherichia coli and shows antibacterial activity against several Gram-positive bacteria.34 It has been reported that cephalochromin inhibits nitric oxide production by activated macrophages32 and has antitumor and cytotoxic activities against human KB cells.35 The aim of the present study was to 759
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use cephalochromin isolated by us from Cosmospora vilior to investigate its growth inhibitory effect on human lung cancer A549 cells and to determine the molecular mechanisms of action.
proteins were evaluated using Western blot analysis. A549 cells were treated with cephalochromin (10 μM) for the indicated times (6, 12, 24, and 36 h). Cephalochromin markedly downregulated the protein levels of cyclin D1, cyclin E, Cdk 2, and Cdk 4 (Figure 3A). Furthermore, the A549 cells treated with
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RESULTS AND DISCUSSION Effect of Cephalochromin on Cellular Growth and Cellular Viability. Cephalochromin inhibited cell proliferation of lung cancer A549 cells in a concentration-dependent manner with an IC50 of 2.80 ± 0.26 μM as measured by sulforhodamine B assay (Figure 1B). Furthermore, the effect of cephalochromin on cellular viability was examined using the mitochondrial MTT reduction assays. After 48 h of treatment, cephalochromin inhibited cellular viability on A549 cells in a concentrationdependent manner with an IC50 value of 5.22 ± 0.22 μM (Figure 1C). Cephalochromin was also active against Hep3B, Caco-2, HT1080, Huh7, SW1353, and A549 cells with IC50 values of 14.31 ± 0.14, 12.40 ± 0.05, 4.98 ± 1.49, 4.64 ± 0.29, 3.54 ± 0.36, and 2.72 ± 0.42 μM, respectively (Figure 1D). Effects of Cephalochromin on the Cell Cycle. To examine whether cephalochromin-induced growth inhibition was associated with regulation of the cell cycle, the cell cycle distribution in the presence of cephalochromin was analyzed by FACScan flow cytometry. As shown in Table 1, treatment with 10 μM cephalochromin for 24 h resulted in a significant accumulation of A549 cells in the G0/G1 phase. Furthermore, the accumulation of cells with G0/G1 DNA content was followed by an increase in hypodiploid cells of the sub-G1 phase at later time points (36 and 48 h, Table 1). Apoptotic cell counting showed cephalochromin-induced apoptosis of A549 cells in a concentration (3, 10, and 30 μM)- and time (24, 36, and 48 h)-dependent manner (Figure 2). These results suggest
Figure 3. Effect of cephalochromin on cyclin D1, cyclin E, Cdk 2, and Cdk 4 protein expression in A549 cells. (A) The cells were treated with 10 μM cephalochromin for 6, 12, 24, and 36 h. (B) The cells were treated with various concentrations of cephalochromin for 24 h and evaluated for the expression of cyclin D1, cyclin E, Cdk2, and Cdk4 proteins. The cellular proteins were separated using SDS-polyacrylamide gels and transferred onto PVDF membranes. The membranes were then probed with the indicated antibodies. Proteins were visualized using an ECL detection system. Data are representative from three independent experiments.
various concentrations (1, 3, and 10 μM) of cephalochromin for 24 h decreased the expression of cyclin D1, cyclin E, Cdk2, and Cdk4 in a concentration-dependent manner (Figure 3B). Consistently, these results also suggested that cephalochromin induced cell cycle arrest at the G0/G1 phase. Effects of Cephalochromin on Apoptosis-Related Proteins. In order to investigate the mechanism by which cephalochromin induces apoptosis, changes in the level of apoptosis-related proteins were examined. A549 cells were treated with 10 μM cephalochromin for 6, 12, 24, and 36 h. At each time point, total protein was isolated, and caspase-3, caspase-8, caspase-9, poly(ADP-ribose) polymerase (PARP), survivin, Bad, and Bcl-xL immunoreactivity levels were measured by Western blot analysis. As shown in Figure 4A, cephalochromin induced time-dependent (12, 24, and 36 h)
Figure 2. Effect of cephalochromin on apoptosis. A549 cells were treated without or with cephalochromin for the indicated concentrations and times. Then, the cells were fixed and stained with propidium iodide to analyze DNA content by FACScan flow cytometer. Data are expressed as the mean ± SEM of three determinations. *p < 0.05, **p < 0.01, and ***p < 0.001 compared with the vehicle-treated control group.
that cephalochromin inhibited cell proliferation through arrest of cell cycle progression in the G0/G1 phase and subsequent induction of apoptosis in A549 cells. Effects of Cephalochromin on the Regulation of Cell Cycle Regulators. To further determine the effect of cephalochromin on the regulation of cell cycle, several cyclin 760
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cephalochromin (10 and 20 μM). As shown in Figure 5, ZVAD-fmk effectively and significantly inhibited cephalochromin (10 and 20 μM)-mediated apoptosis.
Figure 5. Effect of Z-VAD-fmk on cephalochromin-induced apoptosis. A549 cells were preincubated with or without 50 μM Z-VAD-fmk for 2 h, then were treated with indicated concentrations of cephalochromin. After 48 h incubation, cells were analyzed for their hypodiploid DNA content by flow cytometry to evaluate apoptosis. Data are expressed as mean ± SEM of three independent determinations. *p < 0.05, **p < 0.01, and ***p < 0.001 compared with cephalochromin-treated cells.
Effects of Cephalochromin on Cellular Mitochondrial Membrane Potential and ROS Generation. To estimate the role of mitochondria in cephalochromin-induced cell death, we examined whether cephalochromin caused a loss of mitochondrial membrane potential (MMP). The flow cytometric analysis showed that compared with controls, cephalochromin (10 and 30 μM) caused significant loss of mitochondrial membrane potential after treatment for 12 and 24 h (Figure 6A). Moreover, the addition of protonophore carbonyl cyanide m-chlorophenylhydrazone (50 μM) to depolarize the mitochondria resulted in a decrease in the mitochondrial membrane potential in A549 cells (Figure 6A). On the other hand, a loss of mitochondrial membrane potential may be correlated with the production of reactive oxygen species.36 Therefore, we evaluated the level of ROS in A549 cells treated with cephalochromin. As shown in Figure 6B, exposure of A549 cells to cephalochromin (1, 3, 10, and 30 μM) for 12 h led to a significant and concentration-dependent increase in fluorescent intensity by 1.24 ± 0.03-, 1.89 ± 0.05-, 2.87 ± 0.12-, and 4.65 ± 0.08-fold compared with the control group, respectively. Furthermore, the fluorescent intensity of A549 cells with 5 mM H2O2 markedly increased by 7.20 ± 0.27-fold compared to the vehicle-treated control (data not shown). To further evaluate ROS formation, the involvement of superoxide in the fluorescence intensity was also measured after the treatment with cephalochromin (10 μM) or Tiron (1 mM), a superoxide scavenger, for the indicated times (1, 3, and 5 h). The level of ROS in cephalochromin-treated A549 cells was significantly increased in a time-dependent manner, and pretreatment with Tiron notably inhibited ROS generation induced by 5 h treatment of cephalochromin (Figure 6C). These findings indicated that cephalochromin may induce mitochondrial disruption and cellular dysfunction through generation of ROS, such as superoxide. Effects of Cephalochromin on Autophagy. Previous studies indicated that some natural anticancer compounds could induce autophagy through ROS generation in various
Figure 4. Effect of cephalochromin on caspase-3, caspase-8, caspase-9, PARP, survivin, Bcl-xL, and Bad protein expression in A549 cells. (A) The cells were treated with or without 10 μM cephalochromin for different times. Cells were harvested and lysed for the detection of the indicated protein expression by Western blot. (B) The expression levels of survivin were quantified using the computerized image analysis system BIO-PROFIL Bio-1D light. The data are representative of three independent experiments. *p < 0.05 compared with the vehicle-treated control group.
proteolytic cleavage of inactive procaspase-8, -9, and -3 into active caspase-8, -9, and -3, respectively. Similarly, treatment of A549 cells with cephalochromin for 24 and 36 h also significantly induced cleaved PARP protein expression. In addition, the expression of survivin was examined in A549 cells and cephalochromin-treated cells. As shown in Figure 4A and B, cephalochromin resulted in a significant and timedependent inhibition of survivin expression. Moreover, the results also revealed that the cephalochromin significantly down-regulated Bcl-xL expression, but cephalochromin did not affect the expression of pro-apoptotic Bad proteins in A549 cells (Figure 4A). To evaluate whether cephalochromin-mediated apoptosis is caspase-dependent, A549 cells were pretreated with Z-VADfmk (50 μM), a potent caspase inhibitor, prior to treatment of 761
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Figure 7. Effect of cephalochromin on LC3 protein expression in A549 cells. (A) The cells were treated without or with 10 μM cephalochromin for the indicated times. After treatment, the cells were harvested and lysed for the detection of LC3 protein expression by Western blot. (B) The expression levels of LC3 II were quantified using the computerized image analysis system BIO-PROFIL Bio-1D light. The data are representative of three independent experiments. ***p < 0.001 compared with the vehicle-treated control group.
dependent manner (6, 12, 24, and 36 h), suggesting that cephalochromin could also induce autophagy. Discussion. In this work, we found that cephalochromin, an active component from the fungus C. vilior, displayed antiproliferative activity in several types of cancer cell lines and was more potent against human lung adenocarcinoma A549 cells. Cephalochromin showed a concentration-dependent inhibitory effect on the proliferation of A549 cells. In the present study, we investigated the mechanisms of action of cephalochromin on A549 non-small-lung cancer cells in relation to cell growth inhibition and death. Many conventional anticancer agents have been reported to arrest the cell cycle at the G0/G1, S, or G2/M phase and then induce apoptotic cell death.39,40 Other studies indicate that natural compounds such as paclitaxel, silibinin, or xanthoxyletin could inhibit cell proliferation by blocking cell cycle progression at the G2/M, G1, or S phase, respectively.11,13,41 Cephalochromin induced G0/G1 cell cycle arrest after 24 h and a subsequent increase in the sub-G1 phase of secondary apoptotic cell death as measured by flow cytometric analysis at 36 and 48 h. These results suggest that the G0/G1 phase arrest resulted in the inhibition of cell proliferation and consequent apoptosis. Evidence revealed that cell cycle progression is subtly controlled by the interactions of various regulators, cyclins, and their catalytic partner, Cdks.42 In addition, cyclin D1 interacts with Cdk4 and Cdk6 and drives a cell cycle progression across the G1 phase. Cyclin E and Cdk2 are involved in the G1 to S phase progression.43 Particularly, elevated levels of the cyclin D1 or cyclin E protein are observed
Figure 6. Effect of cephalochromin on the mitochondrial membrane potential and reactive oxygen species generation in A549 cells. (A) The cancer cells were treated with the indicated concentrations of cephalochromin for 12 and 24 h, stained with rhodamine 123, and incubated at 37 °C for 30 min. The mean rhodamine 123 fluorescence intensity was detected using FACScan flow cytometric analysis. (B) A549 cells were treated with various concentrations of cephalochromin for 12 h. Intracellular ROS levels were then measured using flow cytometry with DCF-DA as fluorescent probes. (C) Cells were pretreated with 1 mM Tiron for 1 h before treatment with or without 10 μM cephalochromin for the indicated time, and then the intracellular ROS levels were detected as described in (B). Data are expressed as mean ± SEM of three independent determinations. *p < 0.05, **p < 0.01, and ***p < 0.001 compared with the vehicle-treated control group. #p < 0.05 compared with cephalochromin-treated cells.
cancer cells.37,38 To further confirm that autophagy is also induced by cephalochromin, we detected the autophagic marker LC3 II by Western blot analysis. As shown in Figure 7A and B, treatment of cephalochromin (10 μM) significantly increased the expression of LC3 II in A549 cells in a time762
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in lung tumors.10 In our study, the G0/G1 phase arrest was associated with a decrease of cyclin D1, cyclin E, and Cdk4 proteins in cephalochromin-treated A549 cells. The crucial effector survivin is a multifunctional protein that regulates mitotic progression, enhances proliferation, inhibits apoptosis, and promotes angiogenesis.44 Survivin is overexpressed in many cancers, including lung, breast, colon, and pancreatic cancer.45 The overexpression of survivin in tumors might play critical roles in metastasis and tumorigenesis.46 Recent studies also suggest that survivin plays a role in tumor angiogenesis.47 Furthermore, high expression of survivin in the primary tumor is frequently associated with a poor prognosis for patients.44 Previous studies also indicate that survivin mRNA or protein is a useful marker of diagnosis and prognosis for NSCLC patients.48,49 Furthermore, survivin inhibits apoptosis primarily through targeting the terminal effector caspases in the apoptotic pathway.14 Our findings illustrate that the induction of apoptosis of lung cancer A549 cells by cephalochromin is associated with down-regulation of survivin expression. It was also shown that cephalochromin induced a dramatic down-regulation of survivin that could explain the inhibition of caspase activation. Apoptosis is executed by the interaction of various initiator and effector caspases.50 Caspase-3 is a key effector caspase and is activated through either the death-receptor or mitochondriamediated pathway.17 In order to understand the cause of the antitumor effects of cephalochromin in A549 cells, we examined apoptotic signaling effector proteins. Our data showed that cephalochromin can induce the activation of caspase-3, caspase-8, caspase-9, Bcl-xL, and PARP cleavage of A549 cells. Moreover, the potent caspase inhibitor Z-VAD-fmk could obviously prevent the hypodiploid DNA content phase induced by cephalochromin. These results suggest that caspase3 is involved in cephalochromin-induced apoptosis in A549 cells. Furthermore, apoptosis may progress via both intrinsic and extrinsic caspase pathways by cephalochromin. It is well known that mitochondria play a critical role in the regulation of apoptosis.36 Moreover, the members of the Bcl-2 family of proteins are the key regulators of the mitochondrial apoptotic pathway. Several reports indicated that overexpression of Bcl-xL and Mcl-1 has been detected in many human cancers.51 In the present study, we examined the effect of cephalochromin on the expression of the Bcl-2 family of proteins, such as Bcl-xL and Bad expression. Cephalochromin exerted down-regulation of Bcl-xL, one of the inhibitors of the apoptosis protein family in A549 cells. Furthermore, exposure of A549 cells to various concentrations of cephalochromin for 12 and 24 h led to a significant loss of the mitochondrial membrane potential in a concentration-dependent manner. These results suggest that cephalochromin may induce apoptosis by the disruption of mitochondrial-related mechanisms. Reactive oxygen species play important roles in mediating apoptosis of cancer cells.52 Anticancer drugs such as paclitaxel or etoposide are believed to stimulate ROS as part of their antitumor activities and cytotoxic effects.21 Our data indicated that cephalochromin significantly increased the ROS levels in lung A549 cells. It is known that Tiron, a superoxide scavenger, could significant attenuate apoptosis in cancer cells through an NF-κB-mediated event.53 Similarly, the cephalochromininduced effect was obviously reversed by Tiron, indicating the involvement of superoxide in ROS generation by cephalochromin. We also examined whether a ROS scavenger prevent
cephalochromin-induced A549 cell death. Flow cytometric analysis indicated that Tiron (1 mM) did not decrease the subG1 cell population in cephalochromin (10 μM)-treated A549 cells (data not shown). Similarly, the report also showed that Tiron reduced the intracellular H2O2 level in DNP-treated As4.1 cells, but did not affect the level of apoptosis.54 It was proposed that apoptosis in DNP-treated As4.1 cells is correlated with the rapid change of intracellular GSH levels rather than ROS levels. Also, 7,8-dihydroxy-4-methylcoumarin induces apoptosis of human lung adenocarcinoma (A549) cells by a ROS-independent mitochondrial pathway.55 From these points we proposed that the rapid depletion of intracellular GSH or direct disruption of mitochondria might play a role in cephalochromin-induced apoptosis. However, the related mechanisms involved need to be further investigated. Recently, it was shown that ROS generation could also trigger cellular autophagy in lung and other tumor cells.56,57 The antitumor action of harmol (a β-carboline alkaloid) was evaluated by activation of autophagy with increased LC3 II expression as a well-established marker of autophagy.58 Because autophagy can result in both survival and cell death, we found an autophagic inhibitor, chloroquine (25 μM), did not reverse the decrease of cell viability with cephalochromin (10 μM) (data not shown). Moreover, flow cytometric analysis showed that chloroquine did not inhibit the increased level of sub-G1 population with cephalochromin (data not shown). Likewise, the report also showed that 3-MA, an autophagic inhibitor, enhances 5-FU-induced cell death in A549 cells.59 These results indicated that autophagy inhibition enhanced the antitumor effects of cephalochromin in A549 cells, and it implied autophagy could play a protective role in cephalochromintreated cancer cells. Additionally, Bcl-xL and Bcl-2 might be involved in the regulation of autophagy.60 The down-regulation of Bcl-xL observed in our study is also consistent with the finding that sodium selenite induced autophagy in human lung cancer A549 cells.56 In conclusion, our study demonstrates that cephalochromin possesses a novel antiproliferative and cytotoxic activity through cell cycle arrests at the G0/G1 phase and induces apoptosis in human lung cancer A549 cells. These related mechanisms included loss of MMP, down-regulation of survivin and Bcl-xL, activation of caspase-8, -9, and -3, and cleavage of PARP. However, autophagic cell death might not be involved in the antitumor effect of cephalochromin. These findings indicate that cephalochromin should be tested in animal models as a potential therapeutic agent against human lung cancer.
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EXPERIMENTAL SECTION
General Experimental Procedures. RPMI-1640 medium, fetal bovine serum (FBS), penicillin, streptomycin, and all other tissue culture regents were obtained from GIBCO/BRL Life Technologies (Grand Island, NY, USA). Sulforhodamine B (SRB), dithiothreitol (DTT), sodium dodecyl sulfate (SDS), propidium iodide, leupeptin, rhodamine 123, trichloroacetic acid (TCA), 2′,7′-dichlorofluorescin diacetate (DCF-DA), ethylenediaminetetraacetate (EDTA), citric acid, Triton X-100, RNase, aprotinin, sodium orthovanadate, dimethyl sulfoxide (DMSO), Z-VAD-fmk, Tiron, and all of the other chemical reagents were obtained from Sigma−Aldrich (St. Louis, MO, USA). Antibodies to cyclin D1, cyclin E, PARP, caspase-3, caspase-8, and caspase-9 were obtained from Cell Signaling Technologies (Boston, MA, USA). Antibody to α-tubulin was obtained from LabVision/ NeoMarkers (Fremont, CA, USA). Antibodies to Cdk4, Bad, Bcl-xL, and survivin were obtained from GeneTex Inc. (Irvine, CA, USA). Antibody to LC3 was obtained from Sigma−Aldrich. Antibodies to
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150 mM NaCl, 1 mM EGTA, 0.5 mM PMSF, 10 μg/mL aprotinin, 10 μg/mL leupeptin, and 0.1% Triton X-100). Total proteins were quantified, mixed with sample buffer, and boiled at 95 °C for 5 min. An equal amount of protein (40 μg) was separated by electrophoresis in 8−12% SDS−polyacrylamide gels and then transferred onto polyvinylidene difluoride (PVDF) membranes. The membranes were blocked with 5% nonfat milk in PBS−0.1% Tween 20. Afterward, the membranes were washed with PBS−0.1% Tween 20 for 1 h and incubated with the primary antibodies for 1 h at room temperature. After three washings with PBS−0.1% Tween 20, the anti-mouse or anti-rabbit IgG (dilute 1:3000) were incubated with the membranes for 1 h at room temperature. The membranes were washed with PBS− 0.1% Tween 20 for 1 h, and immunoreactive proteins were detected with an enhanced chemiluminescence detection kit (Amersham Biosciences, Buckinghamshire, UK). Statistical Analysis. The experimental results are expressed as the means ± SEM and are accompanied by the number of observations. One-way analysis of variance (ANOVA) was performed using SigmaStat v3.5 software. When group comparisons showed a significant difference, the Student−Newman−Keuls test was used. A p value of
