RNAi-mediated silencing of the Bmi-1 gene causes growth inhibition and enhances doxorubicin-induced apoptosis in MCF-7 cells

The oncogene Bmi-1 is a member of the Polycomb group gene family. Its expression is found to be greatly increased in a number of malignant tumors including breast cancer. This could suggest Bmi-1 as a potent therapeutic target. In this study, RNAi was introduced to down-regulate the expression of Bmi-1 in a highly malignant breast adenocarcinoma cell line, MCF-7. A thorough study of the biological behavior and chemosensitivity changes of the MCF-7 cells was carried out in context to the therapeutic potential of Bmi-1. The results obtained indicated that siRNA targeting of Bmi-1 could lead to an efficient and specific inhibition of endogenous Bmi-1 activity. The mRNA and protein expression of Bmi-1 were determined by RT-PCR and Western blot, respectively. Furthermore, silencing of Bmi-1 resulted in a drastic inhibition of the growth of MCF-7 cells as well as G(1) /S phase transition. The number of target cells was found to increase in phase G (0) /G (1) and decrease in the S phase, but no increase in the basal level of apoptosis was noticed. On the other hand, a reduction in the expression of cyclin D1 and an increase in the expression of p21 were also noticed. Silencing of Bmi-1 made the MCF-7 cells more sensitive to the chemotherapeutic agent doxorubicin and induced a significantly higher percentage of apoptotic cells. Here, we report on a study regarding the RNAi-mediated silencing of the Bmi-1 gene in breast cancer.     

沉默Bmi-1基因表达稳定细胞株的建立及其生物学特性的研究

B细胞特异性莫洛尼鼠白血病病毒插入位点1(B-cell-specific moloney murine leukemia virus insertionsite 1,Bmi-1)基因是多梳基因家族成员,参与细胞增殖调控.研究发现Bmi-1基因可能参与肿瘤的形成,可能成为肿瘤潜在的治疗靶点.用RNA干扰(RNA interference,RNAi)沉默Bmi-1基因表达观察其对乳腺癌细胞株MCF-7侵袭和转移等生物学特性的影响,以探讨Bmi-1在乳腺癌发生发展中的作用.PT67细胞包装质粒后产生的逆转录病毒感染MCF-7细胞,嘌呤霉素筛选建立稳定细胞株,稳定抑制Bmi-1的细胞株命名为MCF-7/Bmi-1si.通过RT-PCR和Western blot分别从mRNA和蛋白水平检测Bmi-1的表达量;平板克隆形成实验检测细胞克隆形成能力;Transwell侵袭小室模型检测细胞体外侵袭和转移能力.MCF-7/Bmi-1si组与MCF-7和MCF-7/GFPsi组相比,Bmi-1 mRNA和蛋白表达量明显减少,克隆形成数及形成率也明显减少(P<0.05).侵袭和转移实验表明:与MCF-7和MCF-7/GFPsi组相比,MCF-7/Bmi-1si组细胞在Transwell侵袭小室中24 h穿膜细胞数明显减少(P<0.05).结果表明沉默Bmi-1基因表达稳定细胞株构建成功,Bmi-1基因表达的沉默能显著降低MCF-7细胞的体外增殖及侵袭转移能力.     

miR-424-5p通过下调BCL-2的表达抑制人肺癌A549细胞增殖

microRNAs(miRNAs)是一类在真核生物中广泛存在的长度约为20～22个核苷酸的单链非编码小RNA,通过与其靶基因mRNA的3′非翻译区（3′UTR）结合发挥转录后抑制作用,参与调节细胞生长增殖、细胞代谢、细胞凋亡以及肿瘤的发生发展等过程。为研究microRNA-424-5p（miR-424-5p）在肺癌细胞中的作用及机理,利用lipo2000转染试剂将miR-424-5p mimics转染入人的非小细胞型肺癌细胞(NSCLC)A549中,流式细胞术检测A549细胞的周期变化及凋亡情况,发现细胞生长阻滞于G1/G0期且凋亡率显著上升。利用克隆形成实验和CCK-8法分别检测,发现miR-424-5p导致A549细胞增殖能力及活力降低。用在线数据库预测出抗凋亡基因BCL-2可能是miR-424-5p的靶基因,随后扩增BCL-2 mRNA 的3′UTR,采用双荧光素酶报告实验及Western印迹检测证明BCL-2确为miR-424-5p的靶基因。构建BCL-2的真核表达载体pCMV-HA-BCL-2,与空载分别转染A549细胞后发现过表达BCL-2可抵消miR-424-5p引起的细胞周期阻滞及细胞凋亡。以上结果提示,miR-424-5p可以通过下调BCL-2的表达来抑制肺癌细胞增殖。     

CBX4 promotes the proliferation and metastasis via regulating BMI‐1 in lung cancer

Proliferation and metastasis are significantly malignant characteristics of human lung cancer, but the underlying molecular mechanisms are poorly understood. Chromobox 4 (CBX4), a member of the Polycomb group (PcG) family of epigenetic regulatory factors, enhances cellular proliferation and promotes cancer cell migration. However, the effect of CBX4 in the progression of lung cancer is not fully understood. We found that CBX4 is highly expressed in lung tumours compared with adjacent normal tissues. Overexpression of CBX4 significantly promotes cell proliferation and migration in human lung cancer cell lines. The knockdown of CBX4 obviously suppresses the cell growth and migration of human lung cancer cells in vitro. Also, the proliferation and metastasis in vivo are blocked by CBX4 knockdown. Furthermore, CBX4 knockdown effectively arrests cell cycle at the G0/G1 phase through suppressing the expression of CDK2 and Cyclin E and decreases the formation of filopodia through suppressing MMP2, MMP9 and CXCR4. Additionally, CBX4 promotes proliferation and metastasis via regulating the expression of BMI‐1 which is a significant regulator of proliferation and migration in lung cancer cells. Taken together, these data suggest that CBX4 is not only a novel prognostic marker but also may be a potential therapeutic target in lung cancer.     

弱碱性消癌液抑制乳腺癌细胞生长、转移并介导其消亡

为了研究新的肿瘤治疗方法,设计了1种弱碱性消癌液（weak alkaline cancer-eliminating liquid,WACEL）,测试WACEL是否抑制癌细胞的生长转移及消亡.在体外,检测WACEL对3种细胞株,即子宫颈鳞癌细胞SiHa、非小细胞肺鳞癌细胞H1299、人乳腺癌细胞MDA-MB-231的生长增殖、细胞周期、细胞凋亡、侵袭转移的影响. 在体内,建立人乳腺癌裸鼠颈背皮下移植瘤模型,观察WACEL对裸鼠皮下肿瘤生长转移的作用. 研究结果发现,WACEL明显抑制3种细胞系的生长增殖,G2/M期细胞增多,出现G2/M期阻滞,阻止细胞周期的进程. 细胞形态呈圆状,细胞核浓缩,caspase-3活性检测增加,线粒体的膜电位降低,细胞凋亡;活细胞数目减少,细胞膜破裂,发生消亡现象,癌细胞迁移明显减少（P﹤0.001）.目标基因SCCA1、cyclinB1、MMP-2以及MMP-9 的mRNA表达水平下调,caspase-3的mRNA表达水平上调;SCCA1、cyclinB1和MMP-2蛋白表达下调,caspase-3蛋白表达上调.体内动物实验发现,处理组的肿瘤生长较对照组明显缓慢,肺组织HE染色未见明显癌细胞转移,而对照组可见癌细胞转移. WACEL能抑制乳腺癌细胞的生长、转移并介导其消亡.本研究系统分析WACEL与癌细胞本身及其pH微环境之间的相互作用机制,发现其改变癌细胞所处的微环境的重要性.     

B-Myb稳定过表达H1299细胞株的构建与分析

B—Myb是Myb家族的成员之一,在细胞周期和癌变过程中具有重要作用。但其在肺癌中的作用及其分子机制仍不清楚。为了研究B—Myb在肺癌中的作用,构建了B-Myb稳定过表达的H1299肺癌细胞株。流式细胞术和MTT检测的结果表明,B—Myb稳定过表达导致G1期细胞减少,S期细胞增加进而促进细胞增殖：克隆形成实验及Transwell的结果表明,B—Myb稳定过表达显著增强H1299细胞的克隆形成、侵袭及迁移能力。定量RT-PCR检测结果表明,B—Myb稳定过表达显著提高了细胞周期基因CCNA1的表达水平;对CD97和MTSS1等细胞运动相关下游基因的表达则无明显影响。该研究成功构建了B-Myb稳定过表达细胞株,发现了B-Myb过表达可促进肺癌细胞的增殖、侵袭迁移及克隆形成能力,为进一步研究奠定了基础。     

Lentivirus-mediated silencing of SCIN inhibits proliferation of human lung carcinoma cells

SCIN (scinderin) is a calcium-dependent actin severing and capping protein. Homologue in zebrafish has been found to be related with cell death. In the present study, we found that SCIN is highly expressed in human lung cancer specimens. However, the role of SCIN in lung cancer has not yet been determined. To investigate the function of SCIN in lung carcinoma cells, we took advantage of lentivirus-mediated RNA interference (RNAi) to knockdown SCIN expression in two lung carcinoma cell lines A549 and H1299. Silencing of SCIN significantly inhibited the proliferation and colony formation ability of both cell lines in vitro. Moreover, flow cytometry analysis showed that knockdown of SCIN led to G0/G1 phase cell cycle arrest as well as an excess accumulation of cells in the sub-G1 phase. Furthermore, depletion of SCIN resulted in a significant increase in Cyclin B1, p21 and PARP expression, and a little decrease in Cyclin D1 expression. These results suggest that SCIN plays an important role in lung carcinoma cell proliferation, and lentivirus-mediated silencing of SCIN might be a potential therapeutic approach for the treatment of lung cancer.     

沉默PRR14基因抑制结肠癌HCT116细胞增殖

已有报道显示,富脯氨酸蛋白 14（proline-rich protein 14,PRR14）促进肿瘤的发生发展,但具体作用机制仍不清楚。本文以结肠癌细胞为模型,探索其对细胞增殖和细胞周期进程的影响。qPCR和Western 印迹检测发现,PRR14在4个结肠癌细胞系中呈现高水平表达。合成特异靶向PRR14基因的siRNA,转染结肠癌HCT116细胞。检测发现,PRR14基因表达下调约70%。CCK8结果显示,沉默PRR14后各时间点细胞增殖能力均显著降低,克隆形成实验细胞克隆数减少约40%;流式细胞仪结果显示,沉默PRR14后,G1期细胞比例升高约10%,S期细胞比例降低约14%;BrdU标记免疫荧光检测结果显示,BrdU阳性细胞比例减少约50%,表明细胞DNA合成速率显著降低。机制分析表明：促G1/S期转换基因周期蛋白依赖性激酶2（cyclin dependent kinase 2, CDK2）mRNA水平降低约85%,对应的蛋白质水平也明显降低,G1/S期转换抑制因子周期蛋白依赖性激酶抑制因子1A（cyclin dependent kinase inhibitor 1A,CDKN1A/P21）和周期蛋白依赖性激酶抑制因子1B（cyclin dependent kinase inhibitor 1B,CDKN1B/P27）mRNA水平分别升高约1.8倍和5倍,对应的蛋白质水平也明显升高。沉默PRR14表达,G1/S期相关基因表达紊乱,导致细胞G1期阻滞并抑制细胞增殖。结肠癌细胞中PRR14高表达可促进癌细胞恶性增殖。     

沉默PRR14基因抑制结肠癌HCT116细胞增殖

已有报道显示,富脯氨酸蛋白 14（proline-rich protein 14,PRR14）促进肿瘤的发生发展,但具体作用机制仍不清楚。本文以结肠癌细胞为模型,探索其对细胞增殖和细胞周期进程的影响。qPCR和Western 印迹检测发现,PRR14在4个结肠癌细胞系中呈现高水平表达。合成特异靶向PRR14基因的siRNA,转染结肠癌HCT116细胞。检测发现,PRR14基因表达下调约70%。CCK8结果显示,沉默PRR14后各时间点细胞增殖能力均显著降低,克隆形成实验细胞克隆数减少约40%;流式细胞仪结果显示,沉默PRR14后,G1期细胞比例升高约10%,S期细胞比例降低约14%;BrdU标记免疫荧光检测结果显示,BrdU阳性细胞比例减少约50%,表明细胞DNA合成速率显著降低。机制分析表明：促G1/S期转换基因周期蛋白依赖性激酶2（cyclin dependent kinase 2, CDK2）mRNA水平降低约85%,对应的蛋白质水平也明显降低,G1/S期转换抑制因子周期蛋白依赖性激酶抑制因子1A（cyclin dependent kinase inhibitor 1A,CDKN1A/P21）和周期蛋白依赖性激酶抑制因子1B（cyclin dependent kinase inhibitor 1B,CDKN1B/P27）mRNA水平分别升高约1.8倍和5倍,对应的蛋白质水平也明显升高。沉默PRR14表达,G1/S期相关基因表达紊乱,导致细胞G1期阻滞并抑制细胞增殖。结肠癌细胞中PRR14高表达可促进癌细胞恶性增殖。     

Erythropoietin enhancement of rat pancreatic tumor cell proliferation requires the activation of ERK and JNK signals

Erythropoietin (EPO) regulates the proliferation and differentiation of erythroid cells by binding to its specific transmembrane receptor EPOR. Recent studies, however, have shown that the EPOR is additionally present in various cancer cells and EPO induces the proliferation of these cells, suggesting a different function for EPO other than erythropoiesis. Therefore, the purpose of the present study was to examine EPOR expression and the role of EPO in the proliferation and signaling cascades involved in this process, using the rat pancreatic tumor cell line AR42J. Our results showed that AR42J cells expressed EPOR, and EPO significantly enhanced their proliferation. Cell cycle analysis of EPO-treated cells indicated an increased percentage of cells in the S phase, whereas cell numbers in G0/G1 phase were significantly reduced. Phosphorylation of extracellular regulatory kinase 1/2 (ERK1/2) and c-Jun NH₂ terminal kinase 1/2 (JNK1/2) was rapidly stimulated and sustained after EPO addition. Treatment of cells with mitogen-activated protein/ERK kinase (MEK) inhibitor PD98059 or JNK inhibitor SP600125 significantly inhibited EPO-enhanced proliferation and also increased the fraction of cells in G0/G1 phase. Furthermore, the inhibition of JNK using small interference RNA (siRNA) suppressed EPO-enhanced proliferation of AR42J cells. Taken together, our results indicate that AR42J cells express EPOR and that the activation of both ERK1/2 and JNK1/2 by EPO is essential in regulating proliferation and the cell cycle. Thus both appear to play a key role in EPO-enhanced proliferation and suggest that the presence of both is required for EPO-mediated proliferation of AR42J cells. erythropoietin receptor; cell signaling; mitogen-activated protein kinase induction     

siRNA对乳腺癌细胞Cyclin E表达和生长抑制作用

研究siRNA对乳腺癌MCF-7细胞株cyclin E表达的抑制及对细胞生长的影响。化学合成针对cyclin E基因的小干扰RNA（siRNA）,转染MCF-7细胞株;分别应用荧光定量PCR和免疫印迹测定cyclin E mRNA和蛋白质的表达,CCK-8测定细胞的增殖活性,流式细胞仪检测细胞周期,软琼脂培养检测细胞克隆形成能力。10、50、100nmol／L siRNA-cyclin E分别使MCF-7细胞cyclin E基因表达降低了24．7％、62．5％和71．0％,蛋白质表达降低了40．8％、66．5％和71．3％。转染siRNA-cyclin E后,G1期细胞增多,S期减少,增殖受到抑制,软琼脂克隆形成率降低。结果提示,在MCF-7细胞株中,导入针对cyclin E的siRNA,可有效抑制cyclin E的表达,进而使细胞增殖减缓,逆转其恶性表型。     

Effect of ARHI on lung cancer cell proliferation, apoptosis and invasion in vitro

The purposes of this study were to elucidate the effects of ARHI (aplysia ras homolog I) on several biological features of lung cancer cells, including growth, proliferation and invasion, to collect experimental evidence for the future biological treatment of human lung cancer. The eukaryotic expression vector, pcDNA3.1–ARHI, was constructed and transfected into the human lung cancer cell line SK-MES-1. The biological properties of the resulting ARHI-expressing lung cancer cell line were evaluated using methyl thiazolyl tetrazolium assay, flow cytometry, and a Transwell invasion assay. Additionally, the influence of ARHI on the gene expression levels of cyclin D1, p27^KIP1, death-associated protein kinase 1 (DAPK1), and matrix metalloproteinases1/2 (MMP-1/2) was determined. Compared to the non-transfected SK-MES-1 cells and the cells transfected with the empty pcDNA3.1 plasmid, the ARHI-transfected cells displayed significantly reduced growth rates and decreased viability (P < 0.05). The ARHI-transfected cells also displayed a significantly higher percentage of cells in G1 phase (P < 0.05) and a lower percentage of cells in S phase (P < 0.05); a higher percentage of apoptosis (P < 0.05); and finally, a notable reduction in the basement membrane-penetration rate in the Transwell invasion assay (P < 0.05). Furthermore, it was determined that ARHI is capable of inhibiting the expression of cyclin D1, MMP-1, and MMP-2; however, ARHI promotes the expression of both p27^KIP1 and DAPK1 in SK-MES-1 cells. In conclusion, overexpression of ARHI gene might be associated with the inhibition of lung cancer cell growth, proliferation and invasion, and the promotion of apoptosis.     

顺铂对HepG2细胞增殖、细胞周期及肝癌干细胞标志物的影响

目的:研究顺铂对HepG2细胞增、细胞周期及肝癌干细胞标志物(CD133、ICAM-1和ABCG2)的影响。方法:选取HepG2作为研究对象,分别采用MMT比色法、PI染色法及免疫荧光法检测不同浓度顺铂对其增殖、细胞周期、CD133、ICAM-1和ABCG2表达的影响。结果:每个浓度顺铂作用后均可以显著抑制HepG2细胞增殖力(F=12.23,P=0.004);顺铂对HepG2细胞增殖力的抑制作用和浓度可能与时间成正比。0 mg/L组静息期(G0/G1期)细胞比例为(50.25±0.79)%、2 mg/L组G0/G1期细胞比例为(89.24±0.41)%、4 mg/L组G0/G1期细胞比例为(29.54±3.02)%,2 mg/L组和4 mg/L组分别比0 mg/L组显著上升和下降,差异明显有统计学意义(t=6.53、-3.65,均P0.05)。0 mg/L组DNA合成期(S期)细胞比例为(47.13±0.74)%、2 mg/L组S期细胞比例为(5.65±0.42)%、4 mg/L组S期细胞比例为(67.46±3.24)%,2 mg/L组和4 mg/L组分别比0 mg/L组显著下降和上升,差异明显有统计学意义(t=-7.35、3.79,均P0.05)。结果提示2 mg/L组和4 mg/L组顺铂可让HepG2在G0/G1期与S期显著阻滞,差异有统计学意义(P0.01);顺铂处理后,剩余的HepG2细胞的CD133、ICAM-1和ABCG2呈现高表达水平。结论:HepG2细胞系中会有很少部分肝癌干细胞避开了顺铂的杀灭作用,是造成临床上肝癌反复发作的重要因素之一,临床上应予以重视。     

p53 Regulates Cellular Responses to Environmental Carcinogen Benzo[a]pyrene-7,8-diol-9,10-epoxide in Human Lung Cancer Cells

The p53 tumor suppressor is a mutational target of environmental carcinogen anti-benzo[a]pyrene-7,8-diol-9,10-epoxide (BPDE). We now demonstrate that p53 plays an important role in regulation of cellular responses to BPDE. Exposure of p53-null H1299 human lung cancer cells to BPDE resulted in S and G2 phase cell cycle arrest, but not mitotic block, which correlated with induction of cyclin B1 protein expression, down-modulation of cell division cycle 25C (Cdc25C) and Cdc25B protein levels, and hyperphosphorylation of Cdc25C (S216), cyclin-dependent kinase 1 (Cdk1; Y15), checkpoint kinase 1 (Chk1; S317 and S345) and Chk2 (T68). The BPDE-induced S phase block, but not the G2/M phase arrest, was significantly attenuated by knockdown of Chk1 protein level. The BPDE-mediated accumulation of sub-diploid fraction (apoptotic cells) was significantly decreased in H1299 cells transiently transfected with both Chk1 and Chk2 specific siRNAs. The H460 human lung cancer cell line (wild-type p53) was relatively more sensitive to BPDE-mediated growth inhibition and enrichment of sub-diploid apoptotic fraction compared with H1299 cells. The BPDE exposure failed to activate either S or G2 phase checkpoint in H460 cells. Instead, the BPDE-treated H460 cells exhibited a nearly 8-fold increase in sub-diploid apoptotic cells that was accompanied by phosphorylation of p53 at multiple sites. Knockdown of p53 protein level in H460 cells attenuated BPDE-induced apoptosis but enforced activation of S and G2 phase checkpoints. In conclusion, the present study points towards an important role of p53 in regulation of cellular responses to BPDE in human lung cancer cells.     

IscU2对非小细胞肺癌增殖、迁移、侵袭能力的影响及其作用机制的研究

该文通过shRNA干扰技术敲低IscU2干扰细胞IscU2的表达,研究了干扰IscU2对非小细胞肺癌(NSCLC)细胞NCI-H520增殖、迁移及侵袭能力的影响。构建了稳定低表达IscU2的非小细胞肺癌细胞系NCI-H520;采用CCK-8和平板克隆实验检测细胞的增殖能力;流式细胞仪检测细胞周期、凋亡、ROS、线粒体膜电位变化情况;Transwell实验检测细胞迁移及侵袭能力;Western blot检测相关蛋白的表达。结果表明,干扰IscU2后,非小细胞肺癌细胞的增殖及克隆形成能力降低;细胞周期停滞在G1/G0期,同时伴随有p-AKT和Cyclin D1蛋白含量的下降;细胞晚期凋亡率明显增加,凋亡蛋白Cleaved-caspase3和Cleaved-PARP表达上调;细胞迁移和侵袭能力降低,上皮标志物E-Cadherin表达上调,间质标志物N-Cadherin和Snail表达下调;细胞ROS积累和线粒体膜电位下降。该研究结果表明,干扰IscU2显著抑制非小细胞肺癌的增殖、迁移、侵袭能力和上皮–间质转化,这为非小细胞肺癌的诊断和治疗提供了新的潜在靶点和视角。     

Silencing of Bmi-1 gene by RNA interference enhances sensitivity to doxorubicin in breast cancer cells

The oncogene Bmi-1 is highly up-regulated in breast carcinoma and is found to be efficient in preventing apoptosis of the cancer cells. Doxorubicin is an important chemotherapeutic agent against breast carcinoma. However, the effective therapeutic response to doxorubicin is often associated with severe toxicity. The present study is targetted at developing a strategy to increase doxorubicin sensitivity to lower doses without compromising its efficacy. A stable cell line with a persistent silencing of Bmi-1 was established. MTT assay was performed to evaluate 50% inhibitory concentration (IC50) values of doxorubicin. Apoptosis was detected by FCM and the expression of related genes [phosphor-Akt (pAkt), totle-Akt (tAkt), Bcl-2 and Bax] was studied by Western blot. In vivo, the sensitivity of the tumor tissues against doxorubicin was evaluated by transplanted MCF-7 nude mice model and the apoptosis of tissue cells was detected by TUNEL assay. The expression of pAkt and Bcl-2 was down-regulated, whereas Bax was up-regulated in Bmi-1 silencing cells. The results obtained indicated that silencing of Bmi-1 can render MCF-7 cells more sensitive to doxorubicin which induced a significantly higher percentage of apoptosis cells in vitro and in vivo. All together these results clearly demonstrate that Bmi-1 siliencing combined treatment of doxorubicin might be a new strategy for biological treatment on breast cancer.     

Ectopic expression of 10-formyltetrahydrofolate dehydrogenase in A549 cells induces G1 cell cycle arrest and apoptosis

We have recently shown that transient expression of 10-formyltetrahydrofolate dehydrogenase (FDH) strongly inhibits proliferation of several cancer cell lines and ultimately results in cell death. In the present studies using Tet-On system, we have generated a stable A549 lung carcinoma cell line capable of inducible FDH expression. Using this system, we were able to express FDH at different levels depending on concentration of the inducer, doxycycline, and we have observed that inhibition of proliferation depends on FDH intracellular levels. We have further shown that induction of FDH expression results in initiation of apoptosis beginning 24 h post-induction. Apoptotic cells revealed cleavage of poly-(ADP-ribose) polymerase and general caspase inhibitor zVAD-fmk protected cells against FDH-induced apoptosis. FDH-expressing cells showed accumulation of cells in G(0)-G(1) phase and a sharp decrease of cells in S phase. Accumulation of intracellular FDH was followed by accumulation of the tumor suppressor protein p53 and its downstream target p21. These results indicate that FDH antiproliferative effects on A549 cells include both G(1) cell cycle arrest and caspase-dependent apoptosis.