活化p53調控人類非小型肺癌細胞凋亡的藥物與機制

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(1)國立臺灣師範大學生命科學系博士論文. 活化 p53 調控人類非小型肺癌細胞凋亡的藥物與機制 Drugs in tumor suppressor gene p53-activated apoptosis of human non-small cell lung cancer cells. 研究生：王景平 Jing-Ping Wang. 指導教授：方剛博士 Kang Fang, Ph.D.. 中華民國104年8月.

(2) Contents Part I. Page. 中文摘要.…………………………………………………………………………………......1. Abstract ……………………………………………………………………………………...2. Introduction………………………………………………………………………………….3 Lung cancer.………………………………………………………………………..………3 Apoptosis.……………………………………………………………………….……........4 Tumor suppressor gene p53.………………………………………………………….............5 Teroxirone.……………………………………………………….…….……………...…...6 Production of ROS trigged the activation of apoptosis.…………………………..……………...7. Purpose.………………………………………………………………………………………8. Materials and methods..……………………………………………………………………..8 Chemicals.………………………………………………………………..……………..….8 Cell culture.………………………………………………………………………….….….8 Cell viability determination.…………………………………………………………..…….…9 Comet assay……………………………………………………………………..…………10 Flow cytometry and determinations for cell cycle analysis and apoptosis determination…..…….....11 Analysis of cytochrome c release…………………………………………………………….11 Western blot analysis…………………….………………………………………………....12. Results…………………………………………………………………………………….....13 Teroxirone-damaged DNA suppressed cell proliferation in A549 and H460 cells, but not in H1299 cells.…………………………………………………………………...…13.

(3) The increased annexin V and PI-positive cells by teroxirone in both H460 and A549 cells.………...13 The inducible PARP cleavage and expression mitosis regulators, p53 and p21 Waf1/Cip1 (p21), accounted for the apoptotic cell death in H460 and A549 cells.…………………………...…….14 Release of cytochrome c in H460 and A549 cells when treated with teroxirone..………………….14 Caspase-3 inhibitor blocked teroxirone-mediated apoptosis..………………………………...….15 Teroxirone-induced apoptosis is dependent on p53 status...……………………………………..15 Down-regulation of p53 proteins attenuated the onset of teroxirone-induced cell death in NSCLC cells..…………………………………………………………………..........….16 Teroxirone enhances mitochondrial membrane potential drop in NSCLC cells ..……………...…..16 To evaluated the intracellular ROS production by teroxirone in human NSCLC cells….…….……..17 NAC suppressed the effect of ROS generation on cell cycle distribution in NSCLC cells.………….17 Teroxirone induces the p53-dependent apoptosis of NSCLC cells in a ROS-dependent manner. ……18 Production of ROS mediated the release of cytochrome c in NSCLC cells………………...……..18. Figures and legends…..……………..……………………………………………………....20 Figure 1……………………………………………………………………………….......20 Figure 2……………………………..…………………………………………………….22 Figure 3………………………………………………………………………………..….24 Figure 4-1.………………………………………………………………………………...26 Figure 4-2.………………………………………………………………………………...27 Figure 5..………………………………………………………………………………….29 Figure 6..………………………………………………………………………………….31 Figure 7..………………………………………………………………………………….32 Figure 8..………………………………………………………………………………….34 Figure 9……………..…………………………………………………………………….35 Figure 10…………..………………………………………………………………..…….37 Figure 11…………..………………………………………………………………..…….38 Figure 12…………..………………………………………………………………..…….39.

(4) Discussion…………………………………………………………………………………...40. Part II 中文摘要………………………………………………………………………………….....46. Abstract …………………………………………………………………………………….47. Introduction………………………………………………………………………………...48 Topoisomerace II inhibitor.………………………………………….………………………48 Ellipticine………………………………………………………….……………………...49 PI3K/Akt signaling pathway…………………………….…………………………………..49. Purpose……………………………………………………………………………………...50. Materials and methods …………………………………………………………………….51 Chemicals……...………………………………………………………………………….51 Transfection of constructs.………………………….…………………………………….....51 Immunofluorescence analysis………………….…………………………………………....52. Results…………………………………………………………………………………….....52 The suppressed cell viabilities and the increased sub-G1 cell populations can be reverted by dominant-negative AktS473A………………………………………………...52 AktS473A abrogated ellipticine sensitivity in H1299 cells with ectopic p53….………………….…..53 Dominant-negative AktS473A inhibited ellipticine induced nucleus translocation of p53 and Akt in A549 cells.…………………………………………………………...…….…...54 Knock-down of p53 inhibited ellipticine-induced nucleus translocation of Akt……………….…..55 The enhanced autophagy during ellipticine-induced apoptosis was deactivated by AktS473A………...55.

(5) Figures and legends………………………………………………………………………...57 Figure 1…………………………………………………………………………………...57 Figure 2……………………………………..…………………………………………….59 Figure 3-1………………………………………………………………………………....61 Figure 3-2…………………………………….…………………………………………...62 Figure 4-1…………………………………….…………………………………………...64 Figure 4-2………………………………….……………………………………………...66 Figure 5…………………………………………………………..……………………….67 Figure 6…………………………………………..……………………………………….69. Discussion…………………………………………………………………………………...71. References,……………………………………………………………………..….………..75.

(6) List of Abbreviation. NSCLC cells：.......Non-small cell lung cancer cells ROS：..…………...Reactive oxygen species MMP (ΔΨm)：.…..Mitochondrial membrane potential DMEM：…………Dulbecco’s modified eagle medium DMSO：.…………Dimethyl sulfoxide MTT：..…………..3- (4, 5-Dimethylthiazol-2-yl)-2, 5-diphenyl tetrazolium bromide Akt：……………..Protein kinase B PI3K：……………Phosphoinositide 3-kinase PARP：.………..…Poly (ADP-ribose) polymerase DEVD-CHO：.…...Ac-Asp-Glu-Val-Asp-CHO NAC：.…………...N-acetyl-cysteine G418：...…………Geneticin siRNA：……..…...Small interfering RNA shRNA：…………Short hairpin RNA DAPI：…………...4', 6-diamino-2-phenylindole TRITC：………….Tetraethyl rhodamine isothiocyanate FITC：.…………...Fluoresceine isothiocyanate SO：…………........Scrambled control PBS：……………..Phosphate buffered saline PI：………………..Propidium iodide BSA：……………..Bovine serum albumin ECL：……………..Entry-level peroxidase substrate for enhanced chemiluminescence.

(7) Part I 中文摘要. 本論文發現三環化合物 (Teroxirone) 在低濃度下能抑制人類非小型肺癌細胞 (NSCLC cells) 的增生，不論在體外實驗或體內實驗都能具有顯著的效果。藥物所誘發的 p53 依賴型的細胞凋亡 (p53-dependent apoptosis) 是經由破壞癌細胞內的 DNA 結構所引發的，讓癌細胞 p53 上升，進而活化下游 caspase-3，導致最終的細胞凋亡。而 caspase-3 的抑制劑 (DEVD-CHO) 或是利用 si-RNA p53 抑制細胞內的 p53，皆能抑制藥物所引發的細胞凋亡現象。另外，此藥物在缺失 p53 的細胞 H1299 cells 中，也能引起些微的細胞毒性。在體內實驗中，此藥物對於裸鼠身上的癌細胞也具有顯著抑制生長的功效。進一步發現，此藥物會引發癌細胞內的氧化壓力、產生活性氧化物 (ROS)、讓細胞內的粒腺體外膜崩壞，進而產生 p53 依賴型的內生性細胞凋亡 ( intrinsic apoptosis pathway)。而利用 ROS 的抑制劑 (NAC) 進行處理後，也會抑制藥物所引發的細胞凋亡現象。由結果顯示，此藥物在低濃度時就能引發癌細胞的細胞凋亡並會顯著抑制癌細胞增生的結果，在未來可以是一個具有潛力治療人類非小型肺癌細胞的藥物。. 關鍵字： p53、Akt、teroxirone、ellipticine、NSCLC cells (非小型肺癌細胞)、ROS. 1.

(8) Abstract In this study, we demonstrated that the growth of human non-small cell lung cancer (NSCLC) cells H460 and A549 cells can be inhibited by low concentrations of an epoxide derivative, teroxirone, in both in vitro and in vivo models. The cytotoxicity was mediated by apoptotic cell death through DNA damage. The onset of ultimate apoptosis is dependent on the status of p53. Teroxirone caused transient elevation of p53 that activates downstream p21 and procaspase-3 cleavage. The presence of caspase-3 inhibitor reverted apoptotic phenotype. Furthermore, we showed the cytotoxicity of teroxirone in H1299 cells with stable ectopic expression of p53, but not those of mutant p53. A siRNA-mediated knockdown of p53 expression attenuated drug sensitivity. The in vivo experiments demonstrated that teroxirone suppressed growth of xenograft tumors in nude mice. Being a potential therapeutic agent by restraining cell growth through apoptotic death at low concentrations, teroxirone provides a feasible perspective in reversing tumorigenic phenotype of human lung cancer cells. The results indicated that low concentrations of teroxirone suppressed the growth of human non-small cell lung cancer cells. The induced apoptotic cell death can be reverted by caspase-3 inhibitor, DEVD-CHO. The reduced cell viability is closely related to p53-activated apoptosis. Furthermore, we also found that teroxirone-induced p53-dependent apoptosis was through regulating intrinsic pathway via ROS generation and mitochondria dysfunction, which can reverted by antioxidant NAC. Teroxirone provides a good candidate for lung cancer treatment by suppressing cellular proliferation.. Key Words： p53、Akt、teroxirone、ellipticine、NSCLC cells、ROS 2.

(9) Introduction. Lung cancer. Lung cancer is one of the most prevalent cancers, it cause of worldwide cancer related death in approximately 1.2 million patients each year, and the leading cause of cancer mortality among all types of cancers because of its poor prognosis, high therapeutic resistance and low postoperative survival rate [1,2]. In addition, adenocarcinoma, squamous cell carcinoma and large-cell carcinoma were included in human non-small cell lung cancer (NSCLC) cells, which is ranked the majority for lung cancer patients that accounts for over 85 % of clinical lung cancer cases [3,4]. It is reported that higher than 60 % of NSCLC patients are diagnosed to have advanced or metastasis tumors and is too late for surgical resection [5]. Chemoprevention is considered a promising strategy to controlling cancer mortality because it potentially reduces the incidence of malignant cancer by reversing and/or delaying carcinogenesis [6]. Previous studies indicated that the therapy of advanced NSCLC patients were treated with an anthracycline, doxorubicin given by inhalation can increase survival rate of 30-50 %, but it was also cause acute and cumulative dose-related toxicity [7]. Oncologists have been embraced carboplatin, paclitaxel, docetaxel and gemcitabine was chosen as the standard drugs for the first-line treatment of patients with metastatic NSCLC in the United States [8]. It has been showed that an increase in survival benefit for the nonsquamous NSCLC patients was treated with the bevacizumab combined cisplatin plus gemcitabine [9]. However, we are hopeful that less toxic drugs targeted at the unique molecular mechanisms that drive NSCLC to grow will lead to larger incremental improvements in the future. Thus, there is a crucial need to develop a new potent and less toxic drug, which target 3.

(10) specific chemo-intervention to retard cancer cell proliferation or to induce apoptosis or both to manage the problems of NSCLC.. Apoptosis. Apoptosis is a programmed cell death pathway that is essential in the development of multicellular organisms and functions in the maintenance of tissue homoeostasis [10]. The biochemical machinery required for apoptotic cell death is constitutively present in virtually all mammalian cells and can be activated by a variety of extra- and intracellular signals [11]. It was initially described by its morphological characteristic, including cell shrinkage, cell membrane blebbing, chromatin condensation and nuclear fragmentation [12]. A characteristic biochemical feature of the process is double-strand cleavage of the nuclear DNA at the linkage regions between nucleosomes, leading to the production of oligonucleosomal fragments [13]. In addition, the mechanism of apoptotic pathway is activation of a family of cell death proteases called the caspases, which are themselves activated by catalytic cleavage, thereby setting up a cascade of proteolytic cleavage that disrupts the function of essential regulatory proteins and commit the cell to the suicide pathway [14]. Activation of the caspase cascade is followed by nuclear condensation, and the activation of an apoptotic nuclease results in the destruction of the nuclear DNA [15]. The realization that apoptosis is a gene-directed program has had profound implications for our understanding of developmental biology and tissue homeostasis, for it implies that the numbers of cell can be regulated by factors that influence cell survival as well as those that control proliferation and differentiation [11]. In fact, defects in apoptosis pathway are now thought to contribute to a number of human diseases, ranging from neurodegenerative to malignancy [16]. Apoptosis has been 4.

(11) noted as the normal response of some cell types, notably T lymphocytes, to the induction of DNA damage, and this has been shown to be dependent on the presence of wild-type p53 gene [10]. However, p53 transactivation can contribute to the induction of apoptotic cell death in varies cell types, and a number of genes trans activated by it have been implicated in the apoptosis response, but not all p53 target genes have been linked with a role in apoptosis [14].. Tumor suppressor gene p53. Tumor suppressors regulate one or more intracellular processes to control the number and behavior of cells in a particular tissue within an organism to prevent aberrant proliferation for maintain cell or tissue homeostasis [17]. The p53 gene, first described in 1979, was the first tumor suppressor gene to be identified. It was originally believed to be an oncogene, but genetic and functional data obtained 10 years after its discovery proved it to be a tumor suppressor gene, which located at the short arm of chromosome 17 (17p13). It contains 11 exons spanning 20 kilobases and encodes a nuclear phosphorylation protein of 53 kDa. The p53 protein contains distinct functional domains: the N-terminus transactivation domain, the sequence-specific DNA-binding domain, the oligomerization domain, and the C-terminus negative regulatory domain [18]. The activities of p53, both transcription dependent and independent, are regulated via its mRNA and protein levels, cellular localization and control the expression of thousands of potential target genes. The p53 pathway is activated by UV-induced DNA damage, hypoxia, or aberrant oncogene expression to promote cell-cycle checkpoints, DNA repair, cellular senescence, and drug-mediated apoptosis that alter the activation, stabilization and nucleus accumulation of p53 [19]. P53 apparently promotes cellular apoptosis through 5.

(12) transcription dependent or -independent mechanisms that act in concert to ensure that the cell death program proceeds efficiently [20]. Moreover, the apoptotic activity of p53 and its family members were strictly controlled, and are influenced by a series of intracellular quantitative and qualitative events that affect the ability of p53 activation [19]. Under normal condition, p53 can rapidly degraded by mdm2 and thus, not present at the detectable levels within the cell [10]. It also has been shown that the wild type p53 is required for the apoptotic cell death as induced by γ-irradiation or a variety of anticancer drugs in several cancer cell types [11].. Teroxirone. Teroxirone (1, 3, 5-triazine-2, 4, 6 (1H, 3H, 5H)-tri-one-1, 3, 5-tri-(oxiranylmethyl), Fig 1A) is a triepoxide derivative with alkylating properties [21,22]. This antitumor agent was originally synthesized by Budnowski. It consists of two stereoisomer α- and β- isomer were both showed significant activity against model tumors including lymphocytic leukemia P-388, early and advanced lymphoid leukemia L1210 and the Lewis lung carcinoma by resistant to cyclophosphamide [23]. The α-stereoisomer has been found to be greater water solubility and activity against B-16 melanoma and L1210 lymphoid leukemia [24]. As an alkylator, teroxirone has reportedly underdone phase I evaluation of clinical studies [23-25]. The drug toxicity has been implicated in causing local phlebitis and myelosuppression at high dosage test [25]. However, the chemotherapeutic agent effective on antitumorigenic in several tumors models, it has also been known helping patients recovering from leukemia and lymphomas [22,26]. It has been demonstrated an anticarcinogenic effects in patients and in animal models, but the intracellular target signal molecules for this antitumorigenesis mechanism of teroxirone remains to be identified. 6.

(13) Production of ROS trigged the activation of apoptosis. Reactive oxygen species (ROS) are widely generated in biological systems, include organelles, cells, tissues and organs. In aerobic cells, the most important sources of O2‧- are the electron transport chains of mitochondria and endoplasmic reticulum [27]. In mitochondria, ROS formation is significantly increased by uncouples of oxidative phosphorylation, hyperbaric O2 treatment, pathologic conditions such as ischemia/reperfusion syndrome, ageing, etc and alterations of mitochondrial lipids occurring during deficiency of polyunsaturated fatty acids and lipoperoxidation processes [28]. In the endoplasmic reticulum (RE), the NADPH cytochrome P450 reductase can leak electrons onto O2 generating O2‧-. Intracellular production of active oxygen species such as hydroxyl radicals ( ‧OH), superoxide anions (O2‧-), singlet oxygen (1O2) and hydrogen peroxide (H2O2) is associated with the arrest of cell proliferation [28]. If oxidative stress persists, oxidative damage to critical biomolecules (including oxidant induced damage to the genome) can leading to the disruption of electron transport, loss of mitochondrial membrane potential (MMP, ΔΨm), decline in ATP levels, production of reactive oxygen species (ROS), and loss of mitochondrial structural integrity [29]. Previous reports indicated that in cancer cell lines, ROS production in response to various external stimuli significantly contributed to apoptosis triggering, while in normal human lymphocytes and fibroblasts treated by cytostatic or cytotoxic drugs, necrosis as well as apoptosis occurred and large heterogeneity of ROS production was measured [30]. Mitochondrial may play an important role in ROS-induced apoptosis. Release of cytochrome c appears to be the central event, which induced the oligomerization of adapter molecule (Apaf1), which in turn recruits and activates the proapoptotic members of the Bcl-2 family, Bax and the initiator caspase caspase-9. The resulting 7.

(14) apoptosome complex contains active caspase-9, which cleaves and activates the caspase-3 and caspase-7. One of the most important caspases, caspase-3, that cleave specific substrates within the cell to produce the changes associated with apoptosis [31].. Purpose. The present study was to evaluate the effects of antitumorigenesis mechanism on human non-small cell lung cancer (NSCLC) cells by teroxirone treatment.. Materials and methods. Chemicals. The available synthetic teroxirone is of more than 98% purity. A stock solution of 10 mM in dimethyl sulfoxide (DMSO) was stored at −20°C, and freshly dissolved in media. The DMEM medium, penicillin and streptomycin antibiotic mixture, sodium pyruvate and glutamine supplements were obtained from Sigma (St. Louis, MO). Fetal bovine serum was acquired from Invitrogen (Grands Islands, NY).. Cell culture. The human lung cell carcinoma cell lines, H460, H1299 and A549 were obtained from ATCC. The acquired cells were thawed, grown and maintained in DMEM. All cells were supplemented with L-glutamine, sodium pyruvate, and supplemented with 10% heat-inactivated FBS in the humidified atmosphere with 5% CO2 at 37°C. All 8.

(15) cell lines were periodically examined and found free of mycoplasma contamination using a MycoTect kit (Invitrogen, Grands Islands, NY). The selected stable H1299 clones transfected with cytomegalovirus promoter-driven pcDNA-p53 that encodes full-length wild type p53 (H1299/p53) or mutant p53R267L (H1299/p53R267L) were maintained in 10% serum-supplemented DMEM.. Cell viability determination. Cell cytotoxicity was measured using the MTT assay [32]. For this assay, 1.5103 cells per well were seeded on 96-well plates and cultured in 2% serum-supplemented DMEM. The suspension was incubated at 37°C for 24 h to allow cell attachment. The mononuclear cells were incubated with different concentrations of teroxirone for the specified time. DMSO was added to the cell culture used as control at final concentration of 0.1%. After the specific exposure time, the medium was removed and then MTT assays were performed. Cells from each well were incubated with 10 μl of MTT (5 mg/ml) in PBS at 37°C for 3 h. After this, the MTT was removed from wells and 100 μl DMSO was added into each well. The amount of formazan formed was determined by measuring the absorbance at 540 nm using a 96-well microplate reader (Thermo Fisher Scientific, USA). The viability assays were performed in triplicate in three separate experiments. Cell viability was expressed as percentage of the vehicle controls. The results were presented as mean ±standard deviation. Statistical differences between two groups were determined by a two-tailed unpaired Student’s t-test. P < 0.05 was considered significantly different.. 9.

(16) Comet assay. The comet assay was performed according to the method of the published work with minor modifications [33,34]. Briefly, conventional slides were covered with a layer of 70 μl 0.5% normal agarose and 0.5% low melting point agarose (GIBCO-BRL). An amount of 70 μl of low melting point agarose (0.5%, w/v) (GIBCO-BRL) was mixed with approximately 2 × 104 cells suspended in 15 μl; the mixture was then layered onto the slides, and immediately overlaid with coverslips. After agarose solidification at 25°C for 30 min, the coverslips were removed and the slides were immersed 60 min at 4°C in fresh lysing solution (2.5 M NaCl; 100 mM Na2EDTA, 10 mM Tris, pH 10 containing 1% Triton X-100). The slides were equilibrated in alkaline solution (1 mM Na2EDTA, 300 mM NaOH) for 20 min and placed in alkaline electrophoresis solution (200 mM NaOH, 1 mM EDTA). The slides were neutralized by immersing in Tris buffer (0.4 M, pH 7.5) followed by distilled water and then soaked in methanol for 5 min. The air-dry slides were then stained with 5 μg/ml propidium iodide (PI) and viewed under an epifluorescence microscope (Nikon, Japan) at 460 nm for visual scoring. Visual image analyses of DNA damage were carried out in accordance with previously reported protocols [35]. A total of at least 20 non-overlapping comet images per gel were visually assigned a score on an arbitrary scale of 0 (round and intact without discernible tail) to 4 (almost all DNA migrated towards the tail without apparent head) based on the perceived comet tail length migration and relative proportion of DNA in the comet tail. A mean DNA damage score for each slide was obtained by dividing the total damage score gained with the total number of comets analyzed by CometScoreTM software.. 10.

(17) Flow cytometry and determinations for cell cycle analysis and apoptosis assay. Cells in the early and late phases of apoptosis were quantitated using annexin V-FITC/PI Apoptosis Detection Kit (BD, Mansfield, MA). Briefly, 2 × 10 5 cells cultured in 1% serum-supplemented DMEM were incubated with different concentrations of teroxirone for 36 h before double staining. The collected cells were washed twice with PBS. After centrifugation at 1,200 rpm for 5 min, cell pellet was stained with 0.5 µl PI (50 µg/ml, BD) plus 0.5 µl annexin-V FITC (20 µg/ml, Becton-Dickinson) in annexin-V binding buffer for 30 min at room temperature in the dark. Analysis was performed with FACSCalibur system (BD). The cell distributions were analyzed by Modfit software (Becton-Dickinson, Mansfield, MA). To determine cell cycle distribution, the cells were analyzed using FACS CaliburTM (BD Biosciences). A total of 2 × 106 cells were plated in different culture conditions. For sample preparation, both medium and trypsinized cells were centrifuged and then supernatant removed. The collected cells were washed twice with PBS and then treated with 70% alcohol containing PBS for 24 h at -20°C. Right before analysis, the sample cells were treated with 10 µg/ml PI (Sigma; St. Louis, MO), 10 µg/ml RNase A (ICN Pharmaceutical; Costa Mesa, CA) containing PBS for 30 min in darkness. Data were analyzed by Modfit LT (Ver 2.0, Becton-Dickinson; Mountain View, CA).. Analysis of cytochrome c release. Cells (1 × 105) were harvested and treated with 100 μl digitonin (50 μg/ml in PBS supplemented with 100 mM KCl and 1 mM EDTA) for 5 min on ice until more than 95% were permeabilized as assessed by trypan blue exclusion. Cells were fixed 11.

(18) in 3.7% formaldehyde in PBS for 20 min at room temperature, washed thrice in PBS, and incubated in blocking buffer (3% bovine serum albumin, 0.05% saponin in PBS) for 1 h. The cells were incubated overnight at 4°C with anti-cytochrome c mouse monoclonal antibody (BD PharMingen) diluted 1:200 in blocking buffer, washed thrice, and incubated for 1 h at room temperature with TRITC-conjugated goat anti-mouse (Santa Cruz) diluted 1:200 in blocking buffer. The cells were then counterstained with Mitotracker Green (Invitrogen Life Technologies). The samples were observed using a Leica TCS SP5 Confocal Spectral Microscope.. Western blot analysis. Cells treated with teroxirone were washed with PBS and scraped in lysate buffer containing 1% triton X-100, 150 mM NaCl, 5 mM EDTA, 1% aprotonin, 5 mM PMSF and 10 µg/ml leupeptin in 20 mM sodium phosphate. Protein concentrations were determined by the BCA assay (Pierce Biotechnology, Rockford, IL) and 20 µg of total protein was performed for Western blots analysis. Protein samples were electrophoresed on SDS-PAGE gels, transferred to nitrocellulose filters, and immunoblotted with the antisera indicated. The immuno-active bands were visualized using horseradish peroxidase-conjugated secondary antiserum and enhanced chemiluminescence system ECL system (Amersham, Arlington Heights, IL). The blots are then incubated in fresh blocking solution and probed for 1 h with 1:3,000 dilutions of p21Waf1/Cip1, PARP, GAPDH, caspase-3 and human p53 antibodies, respectively. Blots are washed twice in PBS-T and then incubated with a 1:4,000 dilution of peroxidase-conjugated secondary antibody (Kirkegaard and Perry Laboratories, Inc., Gaithersburg, MD) in PBS-T for 1 h at 22℃. Blots are again washed twice for 10 min in PBS-T and then developed by ECL detection system 12.

(19) (Amersham).. Results. Teroxirone-damaged DNA suppressed cell proliferation in A549 and H460 cells, but not in H1299 cells. Human NSCLC cells exhibited different sensitivities against teroxirone. The cell growth of both A549 and H460 cells was inhibited in dose-dependent manners when treated with different concentrations of teroxirone, while the growth rates of H1299 cells were unaffected at all concentrations (Fig. 1B). The sensitive growth inhibition in A549 and H460 cells was also shown in colony formation assay (Fig. 1C and 1D). However, discrete DNA damages by teroxirone were detected in all cell lines (Fig. 1E) and the excluded lengths of DNA trail dose-dependent (Fig. 1F). The results proved that DNA lesions assisted in suppressing cell proliferation in H460 and A549 cells; while damaged DNA did not affect growth of H1299 cells.. The increased annexin V and PI-positive cells by teroxirone in both H460 and A549 cells. The induced apoptosis by teroxirone was quantified and confirmed by FACS analysis after staining cells with annexin-V/PI. Exposure to 2 M teroxirone for 36 h resulted in about 8.3% of H460 and 17.0% of A549 cells that entered early apoptotic phase and 27% and 6.0% late apoptosis (P < 0.05). As the drug concentrations were increased to 5 M, both early and late apoptotic cells in H460 and A549 were elevated, respectively (P < 0.01), but not H1299 cells (Fig. 2). Taken together, the 13.

(20) results suggested that teroxirone does trigger apoptotic cell death in NSCLC cells.. The inducible PARP cleavage and expression mitosis regulators, p53 and p21Waf1/Cip1 (p21), accounted for the apoptotic cell death in H460 and A549 cells. The levels of tumor suppressor p53, its corresponding downstream regulator p21 and the cleaved PARP were elevated by teroxirone in both H460 and A549 cell lines and the enhanced intensities associated with the increased concentrations of the drug. No detectable p53, p21, active PARP fragment and cleaved caspase-3 was observed in H1299 cells. The maximal intensities of p53 and p21 were reached by 2 M of teroxirone after 18 h of treatment (Fig. 3A and 3B). Being required to activate intrinsic apoptosis pathway, the cytotoxic mediator poly(ADP-ribose)polymerase (PARP) has been known to play a key role in base excision repair following DNA damage and in the maintenance of genome integrity [36,37]. On the other hand, the appearance of 89-kDa PARP fragment in western blot means the commitment of apoptosis and the onset of cell death. The increased proteolytic cleavage of the precursor PARP implied the ultimate commitment of apoptotic cell death after drug-induced DNA damage. Thus, elevated p53, p21 and cleaved PARP signaled the onset of apoptotic cell death in H460 and A549 cells that was absent in H1299 throughout the time course studied and various concentrations of teroxirone used.. Release of cytochrome c in H460 and A549 cells when treated with teroxirone. We next examined the effects of the drug on the release of cytochrome c from mitochondria by immunocytochemistry (Fig. 3C). In control cells, cytochrome c remained predominantly in mitochondria as evidenced by green color mitochondria. 14.

(21) By treating 2 M of teroxirone for 24 h, both H460 and A549 cells started to release of cytochrome c from mitochondria as shown by the appearance of yellow color of the coalesced green color of mitochondria and the diffused red color of cytochrome c.. Caspase-3 inhibitor blocked teroxirone-mediated apoptosis. Caspase-3 has been reported to be an effective downstream regulator of the signaling pathway during apoptosis. The synchronized cells under minimal serum supplementation were first treated with caspase-3 inhibitor. The appearance of sub-G1 cells after teroxirone treatment was offset by 24 h pretreatment with DEVD-CHO. In H460 and A549 cells, pretreatment with the peptide inhibitor prior to exposing cells to teroxirone for 36 h blocked the development of sub-G1 and G2/M population cells in exchange of G0/G1 cell accumulations (Fig. 4A). DEVD-CHO markedly attenuated the formation of cleaved caspase-3 and PARP that differed from those without pretreatment. The results indicated that p53 activation triggered formation of active caspase-3 fragment that repressed cell viabilities in both H460 (Fig. 4B) and A549 (Fig. 4C) cells. Activation of cyclin B1 with DEVD-CHO pretreatment allowed H460 and A549 cells to pass G2/M phase without apoptosis that attenuated the effectiveness of the drug.. Teroxirone-induced apoptosis is dependent on p53 status. To learn how the critical DNA binding site Arg267 of p53 affects cell sensitivity to teroxirone [38], we constructed H1299 cell lines with stably expressing p53 or mutant p53R267L. Both H1299 cell clones with stable expression of ectopic p53 (H1299/p53) and p53R267L (H1299/p53R267L) were tested with teroxirone. The 15.

(22) viabilities of H1299/p53 cells were decreased in dose-dependent manner; whereas those of H1299/p53R267L cells were unaffected by teroxirone (Fig. 5A). The decreased viability in H1299/p53 was caused by apoptosis as shown in flow cytometry analysis (Fig. 5B) and annexin-V and PI staining (Fig. 5C). In western blot analysis, the p53 levels were first elevated and dropped later in H1299/p53 clone upon teroxirone treatment, and the turnover of p53 in H1299/p53R267L cells remained undetected (Fig. 5D). On the other hand, the increased 89-kDa PARP fragment caused by teroxirone in H1299/p53 cells was time- and dose-dependent manner; the effect was not noticeable in H1299/p53R267L cells. The increased proteolytic cleavage of caspase-3 in H1299/p53 cells indicated that the intrinsic apoptotic activity by teroxirone is dependent on p53 status (Fig. 5E).. Down-regulation of p53 proteins attenuated the onset of teroxirone-induced cell death in NSCLC cells. To further verify p53 was a necessary determinant in the inducing cell death, a complemented experiment using siRNA against p53 along with scrambled control (SO). In teroxirone-treated H460 and A549 cells, transfection of p53 siRNA reduced drug sensitivity that differed from that of SO control (Fig. 6A). Western blot analysis showed that cells with or without SO induced expression p53 by teroxirone, while significant down-regulated p53 in cells transfected with p53 siRNA (Fig. 6B). The results altogether suggested that p53 status elicits drug effectiveness.. Teroxirone enhances mitochondrial membrane potential drop in NSCLC cells. The first purpose of this study was to determine whether the teroxirone-induced 16.

(23) p53-dependent apoptotic cell death in human NSCLC cells could be associated with the changes of the MMP (ΔΨm). The levels of MMP were detected by flow cytometry with JC-1 kit as a fluorescent probe. Our results showed that the decreased levels in the MMP in both H460 and A549 cells after teroxirone treatment at 2 μM for 12 h, but not in H1299 cells (Fig. 7). However, this data indicated that NAC reversed the effect on the teroxirone-mediated MMP disruption (Fig. 7).. To evaluated the intracellular ROS production by teroxirone in human NSCLC cells. We next decided to analyze whether the production of ROS were following with the disruption of MMP. The intracellular levels and production of ROS were evaluated by flow cytometry with DCFH-DA as a fluorescent and oxidation-sensitive probe. Our results showed that the generation of ROS was upregulated in both NSCLC cells H460 and A549 by teroxirone at 2 μM for 18 h (Fig. 8A). However, it was showed a significant inhibitory effect on ROS production by pretreatment with antioxidant NAC at 10 μM for 1 h (Fig. 8A). In addition, we found that pretreatment with NAC can reversed the levels of ROS and the inhibitory effect on the cell growth rate via reduced the teroxirone-induced ROS production (Fig. 8B).. NAC suppressed the effect of ROS generation on cell cycle distribution in NSCLC cells. In addition, we evaluated whether the cell growth rate inhibitory effect on ROS generation was involves with the influence of cell cycle distribution. Cell cycle distribution was determined by flow cytometry analysis after PI staining. Our results 17.

(24) showed that the accumulation of sub-G1 phase and the arrest of G2/M phase were following with concomitant decrease in the percentage of cell cycle G0/G1 and S phase both in H460 and A549 cells were treated with teroxirone for 24 h, but not in H1299 cells (Fig. 9). However, we also found that pretreatment with NAC can recover the regulation of cell cycle progression by suppressing the ROS production (Fig. 9).. Teroxirone induces the p53-dependent apoptosis of NSCLC cells in a ROS-dependent manner. We hypothesized the generation of ROS might lead to the cell cycle arrest and cell death through the teroxirone-induced apoptosis. Furthermore, we examined the mechanisms of mitochondrial-mediated apoptosis. The results showed that the expression of intrinsic apoptosis related protein p53, Bax, caspase-3 and cleavage form of PARP were upregulated by teroxirone treatment at 2 μM for 24 h (Fig. 10A, 10B). In contrast, the expression of oncogene such as Akt and Bcl-2 were downregulated after this drug treatment (Fig. 10A, 10B). However, the dose-dependent effect of teroxirone-induced apoptosis on H460 and A549 cells can be reversed by pre-treatment with NAC for 1h before teroxirone treatment (Fig. 10A, 10B).. Production of ROS mediated the release of cytochrome c in NSCLC cells. Finally, we next evaluated the effects of teroxirone on the levels of cytochrome c release from mitochondria by immunofluorescence analysis. Our results showed that the teroxirone increased the levels of cytochrome c release in a dose-dependent 18.

(25) manner both in H460 and A549 cells were treated with teroxirone for 24 h (Fig. 11). Additionally, we also found the pretreatment of NAC can inhibit the accumulation of cytochrome c released into the cytoplasm (Fig. 11).. 19.

(26) Figures and legends. Figure 1. (A) The chemical structure of the teroxirone (1,3,5-triazine-2,4,6(1H,3H,5H)-tri-one-1,3,5-tri-(oxiranylmethyl)). (B) Growth curves of H460, A549 and H1299 cells in media containing teroxirone. A total of 1.5  103 cells were cultured in 1% serum-supplemented DMEM. Twenty-four hours later, the cells were incubated with 2, 5 or 20 μM teroxirone for 48 h. The cells were 20.

(27) then stained with MTT and converted into viability as specified in Materials and Methods. Results are indicated as cell viability with teroxirone against controls, as determined from three independent experiments. Data are represented as the mean values ± standard deviation (SD). The asterisk (***) indicated a significant difference compared with the vehicle control DMSO (P < 0.005). (C) Colony formation assay. The trypsinized cells were plated in 12-well dishes of different densities (1,000 cells of H460/well, 500 cells of A549/well and 200 cells of H1299/well). The attached cells were treated with 0, 0.5 and 1µM of teroxirone, respectively, for 48 h. The media were replaced with fresh media and incubated at 37°C. Eleven days later, the cells were fixed and stained with 10% methylene blue in 70% ethanol. (D) Statistical analysis of colony formation. The numbers of colonies, defined as more than 50 cells/colony were counted and the remaining fractions were converted as the ratio of the numbers of colonies in the samples with treatment vs. those with vehicle control DMSO. Triplicate wells were set up for each condition. (E) Comet assay analysis Cells were treated for 12 h with teroxirone at the concentrations as specified. DNA tail formation was determined in H460, H1299 cells and A549 cells after teroxirone treatment according to the protocol as specified in Materials and Methods. Teroxirone caused DNA damage in all cell lines studied. (F) Relative scores of DNA damage in teroxirone-treated cells. Mean values of the average comet tail moment (percentage of DNA in tail × tail length) for the three fields were calculated. The relative scores of average comet tail were obtained by converting tail moments lengths of DNA tail formation in all cell lines of teroxirone treatment at different concentrations and compared those with vehicle control. Data represented three independent experiments of the mean values ± standard deviation (SD).. 21.

(28) Figure 2. Apoptosis assay by annexin V-FITC and PI staining. Cells cultured in 1% serum-supplemented DMEM were incubated with different concentrations of teroxirone for 36 h before double staining with annexin-V and PI and the percentage of each quadrant determined by WinDM. The numbers on top right quadrant meant percentage distributions of late apoptosis; while those on the bottom right correspond 22.

(29) to those of early apoptosis (top). The results on the right represented mean values ± SD of three individual experiments all experiments were done independently in triplicate per experimental point, and representative results shown indicating early and late apoptosis, respectively, of different cells (bottom).. 23.

(30) Figure 3. (A) Western blot analysis in cells treated with different concentrations of teroxirone. Cells were analyzed by western blot to determine gene expression at each concentration ranging 0, 1, 2 and 5 µM after 24 h of treatment. Equal amounts of 24.

(31) cell lysates protein were separated by SDS-PAGE separating gel and electro-blotted. The blots were then incubated in fresh blocking solution and probed for 1 h with 1:3,000 dilution of p21, PARP, p53 or Bcl-2 antibody, followed by incubation with a 1:4,000 dilution of horseradish peroxidase-conjugated secondary antibody and then developed by ECL detection system. (B) Western blot analysis in cells treated with teroxirone for different time points. Cells were analyzed by western blot to determine gene expression at time points (0, 12, 18 and 24 h) with 2 µM of teroxirone. (C) Mitochondrial release of cytochrome c. The induced release of cytochrome c from mitochondria in cells treated with 2 or 5 μM teroxirone or vehicle control DMSO for 24 h. Cells were fixed, permeabilized and stained with anti-cytochrome c antibody at 4°C for 18 h. After washing, cells were stained with Mitotracker Green (mitochondrial staining), DAPI (nuclear staining) and secondary antibody conjugated with TRITC for cytochrome c following the description in Materials and Methods. The pointed arrow signified the co-localization of red color cytochrome c and green color mitochondria, while blue color stood for nucleus.. 25.

(32) Figure 4-1. (A) Cell cycle analysis by flow cytometry. Both H460 and A549 cells cultured in 1% serum-supplemented media were incubated with 10 M of DEVD-CHO for 24 h before being treated with 5 µM of teroxirone for 36 h. The cells were collected and stained according to the description in Materials and Methods for phase distribution analysis.. 26.

(33) Figure 4-2. Western blot analysis of (B) H460 and (C) A549 cells. The proteins were analyzed by western blotting by incubating with PARP, p53, cyclin B1, caspase-3 or GAPDH antibodies using lysates collected from cells with (+) or without (-) pretreatment of 10 µM DEVD-CHO before exposing to 1, 2 and 5 µM of teroxirone 27.

(34) for 24 h. Protein levels were monitored based on a densitometer and expressed as a fold-change relative to protein level in untreated cells, in which changes of the cleaved PARP were labeled underneath. Data are representative of two independent experiments.. 28.

(35) Figure 5. (A) Growth curves. H1299 cells with stable expression of ectopic p53 (H1299/p53) and p53R267L (H1299/p53R267L) were treated with different concentrations (0, 1, 2 and 5 μM) of teroxirone for 48 h and the viabilities determined by MTT assay. (B) Cell cycle analysis. Both H1299/p53 and H1299/p53R267L cells were incubated with different concentrations (0, 1, 2 and 5 μM) of teroxirone for 36 h 29.

(36) for cell cycle determination after being stained with PI. (C) Apoptosis assay. Both H1299/p53 and H1299/p53R267L cells were incubated with 0 and 5 μM of teroxirone for 48 h. Cells were double-stained with annexin V-FITC and PI, and then analyzed by ﬂow cytometry (left). All experiments were done independently in triplicate per experimental point, and representative results are shown indicating early and late apoptosis, respectively (right). (D) Western blot analysis. Protein lysates of H1299/p53 and H1299/p53R267L cells treated with different concentrations (0, 1, 2 and 5 μM) of teroxirone for 24 h were analyzed by western blotting blot using different antibodies with GAPDH as loading control. (E) Western blot analysis. Both H1299/p53 and H1299/p53R267L cells were treated with 2 μM of teroxirone for the time indicated and proteins analyzed.. 30.

(37) Figure 6. Effects of p53-specific siRNA on teroxirone sensitivity in both A549 and H460 cells. (A) A total of 1.5  103 cells were either transfected with p53 siRNA or scrambled control (SO) for 18 h and then treated with 2 μM teroxirone for 48 h. The cells were then stained with MTT and converted into viability as specified in Materials and Methods. Results are indicated as cell viability against DMSO vehicle controls in SO as determined from three independent experiments. Data are represented as the mean values ± SD. *P < 0.05, compared with drug alone in SO-transfected cells. (B) Expression of p53 in SO and p53 siRNA transfected cells after drug treatment as monitored by Western blot using p53 antibody with β-actin expression as a loading control.. 31.

(38) Figure 7. Teroxirone enhances mitochondrial membrane potential drop in NSCLC cells. Cells were seeded onto 6-well plates (3 ×105 cells/well). After 24 h for complete adherence, the cells were treated with teroxirone (2 μM) for 12 h. After 12 h of treatment, the cells were stained with JC-1 kit as a fluorescent probe and detected by flow cytometry, which converted into mitochondrial membrane potential (MMP, ΔΨm). Cells were incubated with vehicle control (0.2 % DMSO).. 32.

(39) 33.

(40) Figure 8. (A) NAC can suppress teroxirone-induced ROS production. Cells were seeded onto 12-well plates (5 ×104 cells/well). After 24 h for complete adherence, the cells were pre-treated with NAC (10 μM) for 1 h and following by treatment with teroxirone (2 μM) for 18 h. After 18 h of treatment, the cells were trypsinized and evaluated by flow cytometry with DCFH-DA as a fluorescent and oxidation-sensitive probe. Cells were incubated with vehicle control (0.2 % DMSO). (B) Cell proliferation and viability of A549 and H460 cells pre-treated with NAC following by teroxirone. Cells were seeded onto 96-well plates (5 ×103 cells/well). After 24 h for complete adherence, the cells were pre-treated with NAC (10 μM) for 1 h and following by treatment with teroxirone (2 μM) for 24 h. After 24 h of treatment, the cells were trypsinized and counted by trypsin-exclusion assay and stained with MTT and converted into viability as specified in materials and methods. Cells were incubated with vehicle control (0.2 % DMSO).. 34.

(41) Figure 9. NAC suppressed the effect of ROS generation on cell cycle distribution in NSCLC cells. Cells were seeded onto 12-well plates (5 ×104 cells/well). After 24 h for complete adherence, the cells were pre-treated with NAC (10 μM) for 1 h and following by treatment with teroxirone (2 μM) for 24 h. The cells were collected and stained according to the description in Materials and Methods for phase distribution analysis.. 35.

(42) 36.

(43) Figure 10. (A) NAC recovered ROS-dependent apoptosis in NSCLC A549 cells. Cells were analyzed by western blot to determine gene expression after NAC (10 µM) pre-treated for 1 h and following teroxirone (0, 1, 2 and 5 µM) treatment for 24 h. Equal amounts of cell lysates protein were separated by SDS-PAGE separating gel and electro-blotted. The blots were then incubated in fresh blocking solution and probed for 1 h with 1:2,000 dilution of PARP, Akt, p53, Bax, Bcl-2, caspase-3 and GAPDH antibody, followed by incubation with a 1:3,000 dilution of horseradish peroxidase-conjugated secondary antibody and then developed by ECL detection system. (B) NAC recovered ROS-dependent apoptosis in NSCLC H460 cells. Cells were analyzed by western blot to determine gene expression after NAC (10 µM) pre-treated for 1 h and following teroxirone (0, 1, 2 and 5 µM) treatment for 24 h. Equal amounts of cell lysates protein were separated by SDS-PAGE separating gel and electro-blotted. The blots were then incubated in fresh blocking solution and probed for 1 h with 1:2,000 dilution of PARP, Akt, p53, Bax, Bcl-2, caspase-3 and GAPDH antibody, followed by incubation with a 1:3,000 dilution of horseradish peroxidase-conjugated secondary antibody and then developed by ECL detection system.. 37.

(44) Figure 11. Production of ROS mediated the release of cytochrome c in NSCLC cells. The induced release of cytochrome c from mitochondria in cells after NAC (10 µM) pre-treated for 1 h and following teroxirone with 2 or 5 μM teroxirone or vehicle control DMSO for 24 h. Cells were fixed, permeabilized and stained with anti-cytochrome c antibody at 4°C for 18 h. After washing, cells were stained with Mitotracker Green (mitochondrial staining), DAPI (nuclear staining) and secondary antibody conjugated with TRITC for cytochrome c following the description in Materials and Methods. The pointed arrow signified the co-localization of red color cytochrome c and green color mitochondria, while blue color stood for nucleus. (scale bar 100 μm). 38.

(45) Figure 12. Proposed model of molecular mechanism of apoptosis induced by teroxirone is mediated through ROS-driven p53 pathway in NSCLC cells. Teroxirone treatment activates p53 and suppresses Akt expression, disruption of MMP to increases ROS generation, upregulates Bax, downregulates Bcl-2 expression and activates mitochondria-mediated apoptotic pathway, which in turn causes the release of cytochrome c, the activation of caspase-3, and the cleavage of PARP and fragmentation of inter-nucleosomal DNA, resulting in NSCLC cell apoptosis.. 39.

(46) Discussion. The work here showed that NSCLC cells with wild-type p53 are sensitive to teroxirone treatment (Fig. 1B and 6A). Previous reported work using human tumor cell line rhabdomyosarcoma indicated that 20 M of teroxirone is needed to achieve 50% colony inhibition [21]. The work here showed that the decreased viable cells by low concentrations of teroxirone in H460 and A549 cells can be attributed to apoptosis. Transient induction of p53, activation of p21 and caspase-3 fragment accounted for the final cell death by the drug. Tumor suppressor p53 can be activated transiently in response to a variety of cellular stresses, including DNA damage [39]. Being regulated by p53 and regarded as an inherited property of cells, the development of cell death by treating with chemotherapeutic reagents can be characterized by DNA damage and apoptotic cell death [40]. The distinct apoptosis is an intrinsic biological feature to maintain tissue homeostasis [41,42]. Cells normally respond to exogenous stress by cell cycle arrest and/or programmed cell death [43]. DNA damage in NSCLC cells and the subsequent p53-dependent apoptosis are closely associated with tumor eradication. While the potent anticancer drug activity may involve different intracellular targets, there is ample evidence that the decreased cell viability is mediated by apoptosis following DNA damage. Cells carrying wild-type p53 are more sensitive to genotoxic injury by anticancer agents than those carrying mutant p53 [44]. Teroxirone caused DNA damage that began at 12 h in all NSCLC cell lines studied (Fig. 1E and 1F). The onset of apoptosis by teroxirone appeared in cells carrying wild-type p53 alleles only. The p53 pathway is stimulated by a very small number of DNA strand breaks or single-stranded gaps that trigger signals in eliminating DNA lesions in tumors [45]. Once activated, p53 regulates the expression of an array of downstream effector genes, 40.

(47) including those relating to DNA repair, growth arrest or apoptosis [38]. By inducing DNA repair that permits the continuation of the cell cycle, p53 may mediate an exit from the cell cycle by inducing growth arrest or apoptosis [46]. In the work as described, the induced p53 after 24 h drug treatment returned to their pretreatment levels and the characteristic checkpoint markers p21 varied accordingly (Fig. 3A and 3B). Serving as effective cyclin-dependent kinase (cdk) inhibitors by coordinating with p53, it is likely that activated p21 participated in mitotic arrest following drug induction [47]. As a p53 transcription target, p21 is implicated a potent inhibitor of the key cdk and has been regarded as the main intermediate of p53-dependent cell cycle arrest [48]. Since many therapeutic agents during management of cancer treatment ultimately target damaged DNA, the inhibitor p21 exerts vital role in preventing cell cycle exit and mitosis. The work here showed that, under stress from teroxirone, both H460 and A549 cells reversed their mitotic progress that is likely the result of p21 activation and the suppressed pathway prevented cell proliferation. To prevent further cycling on the damaged DNA template, the checkpoint blockage by p21 is regarded as a form of cellular resistance to proliferation by chemotherapeutic regimen that allows the DNA repair mechanisms to be activated and reduces the potential for aberrant mitosis [49]. Some evidence of DNA damage induced cell death, such as senescent fibroblasts that underwent extended cell arrest before undergo apoptosis [36]. In human colon cancer cells of wild-type p53, treatment with low concentrations of camptothecin induced cell arrest that lead to immediate apoptosis [50]. On the other hand, human glioblastoma cells responded to DNA-methylating agents were arrested at G2/M phase and the cell fate depends on the status of p53 [51]. While no significant change of cell viabilities in H1299 cells, the resulted apoptotic cell death in H460 and A549 cells was characterized by gradual cleavage of PARP. Having an active site and 41.

(48) DNA-binding domain, the intense 89-kDa PARP fragment plus p53 and p21 activation committed cells to apoptotic death by teroxirone that corroborates with the attenuation of prosurvival signal Bcl-2. The absence of PARP cleavage and the intact Bcl-2 levels in H1299 cells signified the role of p53 in teroxirone-mediated apoptotic cell death. The cleavage of procaspase-3 after 24 h drug treatment suggested that activated caspase-3 participated in apoptotic cascade (Fig. 4B and 4C). The apoptotic cell death was offset in cultured cells following incubation with caspase-3 inhibitor, DEVD-CHO, by suppressing the accumulated G2/M phase cells. Inhibition of caspase activation in cancer cells is a potentially efficient mechanism to promote cell survival that contributes to chemo-resistance. The accumulated G0/G1 phase cells helped in reversing final apoptosis (Fig. 4A) and the inhibited caspase-3 activities by DEVD-CHO blocked the final apoptotic cell death. The reduced procaspase-3 cleavage, suppressed p53 activation and inhibited PARP cleavage by the inhibitor indicated that teroxirone-induced apoptosis in NSCLC cells is caspase-3-dependent (Fig. 4B and 4C). Inclusion of caspase-3 inhibitors blocked the effects of a diverse range of cytotoxic drugs that reflected the essential role of the specific caspase in mediating their anticancer activity. Similar results were reported, in which paclitaxel-induced apoptotic morphological changes were reverted by the caspase-3 inhibitor, DEVD-CHO that accompanied with attenuated p53 turnover and increasing viable cells [52]. Our work here showed that caspase-3 activation is directly associated in p53-mediated apoptosis following DNA damage. In human NSCLC cells with wild-type p53, down-regulated cyclin B1 is associated with G2/M phase arrest and apoptosis of NSCLC cells [53]. We have demonstrated that, by up-regulating cyclin B1, casapse-3 inhibitor arrested cells at G0/G1 phase and thereby attenuated drug sensitivities. 42.

(49) In H460 and A549 cells, the coordination of different regulators prevented cell cycle exit and PARP cleavage marked for final cell apoptosis. Absence of molecular determinants such as p53 arrested cells without committing to apoptotic death after teroxirone treatment in H1299 cells. To underscore the importance of p53, H1299 cells with stable ectopic expression p53 became sensitive to teroxirone. The accompanied with caspase-3 activation and PARP cleavage accounted for the final apoptotic cell death. Activation of downstream regulator caspase-3 prevented cell cycle exit and the enhanced PARP cleavage signaled final apoptosis in H1299/p53 cells. It is known Arg267 contributes transcriptional activity by stabilizing DNA binding domain of p53 [38]. Thus, the attenuated sensitivities in H1299 cells expressing ectopic mutant p53R267L accounted for the importance of DNA binding domain of p53 during teroxirone-mediated apoptosis. The results also corroborated with the findings that teroxirone sensitivity was offset by knocking down p53 (Fig. 6A and 6B). In summary, we showed that teroxirone caused DNA damage in NSCLC cells after 12 h treatment. Activation of p53 and p21 beginning at 18 h and the subsequent cytochrome c release accompanied with procaspase-3 and PARP cleavage committed cells to final apoptotic cell death. We identified the toxic effect can also be attained in H1299 cells with ectopic p53, but not those with ectopic mutant p53. Our findings asserted that regulation of cell proliferation by teroxirone in human lung cancer cells depend on the status of p53. The observed antitumor effect strongly suggested that teroxirone provide a feasible and alternative treatment strategy for cancer prevention. In our previous report, we have found that teroxirone-induced apoptosis in human NSCLC cells was depends on the wild-type p53 [54]. Additionally, p53 participates directly in the intrinsic apoptosis pathway by triggering permeabilization of the outer mitochondrial membrane in a transcription-independent fashion through 43.

(50) direct activation of proapoptotic Bcl-2 proteins Bax or through binding and inactivation of anti-apoptotic Bcl-2 proteins such as Bcl-2 and Bcl-XL [55]. This permeabilization is regulated by proteins from the Bcl-2 family, mitochondrial lipids, proteins that regulate bioenergetic metabolic flux and components of the permeability transition pore [28,55]. Upon disruption of outer mitochondrial membrane, a set of proteins normally found in the space between the inner and outer mitochondrial membranes is released, including cytochrome c [29]. The release of cytochrome c from mitochondria directly triggers caspase-3 activation through formation of the cytochrome c/Apaf-1/caspase-9 containing apoptosome complex, which was followed by the activation of caspase cascade and appears to be a crucial role in mediated by direct or indirect ROS production [56]. In this study, we found that teroxirone exerted an oxidative stress on human NSCLC cells by elevating the disruption of mitochondrial membrane and following the production of intracellular ROS, which was associated with the apoptotic cell death. In this work as described, the downregulation of mitochondrial membrane potential (MMP, ΔΨm) and the generation of ROS were observed after drug treatment for 12 h and 18 h, respectively (Fig. 7, 8A). Moreover, pretreatment with N-acetyl-cysteine (NAC) significantly reduced the accumulation of ROS production and the suppressive effects of teroxirone on MMP (Fig. 7, 8A). In the cell viability test (MTT assay and cell counting method), teroxirone showed a inhibitory effect on the proliferation of H460 and A549 cells for 24 h, while pre-treatment with NAC resulted in rescue of cells with an increased survival rate and the arrest of subG1,G2/M cell cycle phase distribution (Fig. 8B, 9). Furthermore, western blot analysis revealed that teroxirone-induced intrinsic apoptosis showed a significantly increased expression in p53, Bax, caspase-3 and PARP fragment and reduced expression in Akt and Bcl-2 after drug treatment for 24 h (Fig. 10). In the immunofluorescence analysis, 44.

(51) we also found that NAC can recover the effects of teroxirone on cytoplasmic accumulation of cytochrome c release (Fig. 11). Similarly, antioxidant NAC pre-treatment showed a significantly inhibition of the stimulating effects on teroxirone-induced ROS-dependent apoptosis (Fig. 10). Teroxirone-induced ROS production can lead to substantial changes in the cytotoxicity in human NSCLC cells. Therefore, teroxirone possesses the ability to induce cellular oxidative stress, offering a promising potential therapeutic for NSCLC lung cancer. PI3k/Akt signal pathway, one of the main mediators of cell survival and proliferation signals preventing cells from undergoing apoptosis, tends to be a potential target for cancer therapy [57]. Moreover, evidence from earlier reports showed that there seems to be a hierarchical relationship between ROS and PI3K/Akt signal pathway, in which ROS tends to function as an upstream signal for Akt [58]. The antitumor effect of teroxirone is mediated through the ROS-mediated reduced the expression of Akt, which indicates the potential of this drug in the targeted therapy of ROS dependent PI3k/Akt pathway. However, there is a significant difference between the presence and absence of NAC treatment, this partial reversal effect of NAC indicated that the intracellular production of ROS plays a crucial role in the anticancer effects of teroxirone.. 45.

(52) Part II 中文摘要. 玫瑰樹鹼 (Ellipticine) 是一種 DNA 拓樸異構酶 II 抑制劑，能夠有效的抑制人類非小型肺癌細胞 (NSCLC cells) 的增生。之前研究指出，此藥物能讓 p53 及 Akt 活化且共同轉移至細胞核內，並可藉由磷酸化 AktS473 去誘導癌細胞產生細胞自噬 (autophagy)，進而引發細胞死亡。另外，此藥物也可抑制能穩定表現 p53 功能的細胞株 (利用轉殖 wild-type p53 質體進入原本缺失 p53 的細胞株 (H1299 cells) 內，並讓其穩定表現 p53 功能的細胞株) 的生長。在本研究中，我們進一步發現到，將 AktS473 的磷酸化位點突變成 alanine 以及利用 shRNA p53 去 knockdown p53 後，皆能抑制玫瑰樹鹼對於 p53 和 Akt 的活化以及轉移至細胞核內的程度，也能同時減少細胞凋亡的產生。因此，p53 能夠讓此誘導細胞凋亡，並且能協助磷酸化 AktS473 進入細胞核內，進而引發細胞自噬作用，導致細胞死亡。本研究指出，玫瑰樹鹼能活化 p53 及磷酸化 AktS473，並藉由這兩者間的協同作用去抑制癌細胞的生長。對於原本只有 DNA 拓樸異構酶 II 抑制功能的藥物，能提供另一個治療癌症抑制的新思維。. 關鍵字： p53、Akt、teroxirone、ellipticine、NSCLC cells (非小型肺癌細胞)、ROS. 46.

(53) Abstract Topoisomerase II inhibitor ellipticine effectively suppressed the growth of human non-small cell lung cancer (NSCLC) epithelial cells. Previously, we reported the drug activity was consummated through parallel nucleus migration of p53 and Akt in A549 cells. While inducing cell death, the drug activity was proved related to autophagy through phosphorylated Akt at S473. In addition, ellipticine induced cytotoxicity in p53-null H1299 cells with stable expression of ectopic p53. In this work, we further demonstrated that dominant-negative AktS473A or p53 shRNA inhibited ellipticine-mediated translocalization of p53 and Akt and attenuated apoptotic cell death in A549 cells. The presence of p53 predates ellipticine-mediated apoptotic cell death, assists in nucleus translocation of phosphorylated Akt and activation of autophagy pathway. Growth inhibition through collaborating p53 and phosphorylated Akt473 in lung epithelial cancer cells provided a new perspective of the topoisomerase inhibitor as an effective cancer therapy agent.. Key Words： p53、Akt、teroxirone、ellipticine、NSCLC cells、ROS. 47.