消除人類癌腫瘤幹細胞的藥物

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(1)國立臺灣師範大學生命科學系博士班研究生論文. 消除人類癌腫瘤幹細胞的藥物 Human cancer stem cell tumor alleviation by therapeutic agents. 研究生：金承勳 Seung-Hun Kim. 指導教授：方剛博士 Kang Fang, Ph.D.. 中華民國一○六年二月.

(2) 消除人類癌腫瘤幹細胞的藥物 Human cancer stem cell tumor alleviation by therapeutic agents. 第一部分: 利用鴉膽子水溶液萃取物清除突變上皮細胞生長因子接受體人類肺癌細胞. 第二部分: 發展 teroxirone 成為有效治療肝癌細胞的藥物. Part-I. The alleviated tumorigenicity of human lung cancer cells carrying mutated epidermal growth factor receptor by aqueous extract of Brucea javanica. Part-II. The development of teroxirone as an effective therapeutic agent against human hepatocellular carcinoma cells.

(3) ACKNOWLEDGEMENTS. I would like to express my special appreciation and thanks to my advisor Professor Dr. Kang Fang, you have been a tremendous mentor for me. I would like to thank you for encouraging my research and for allowing me to grow as a research scientist. Your advice on both research as well as on my career have been priceless. I would also like to thank my all of lab members, especially Hsieh Chang Heng, for letting my defense be an enjoyable moment, and for your brilliant comments and suggestions, thanks to you. All of you have been there to support me when I recruited patients and collected data for my Ph.D. thesis. A special thanks to my family. Words cannot express how grateful I am to my mother, father, my mother-in law and father-in-law for all of the sacrifices that you’ve made on my behalf. Your prayer for me was what sustained me thus far. At the end I would like express appreciation to my beloved wife Hsiao-Lin Wei who spent sleepless nights with me and was always my support in the moments when there was no one to answer my queries.. 2.

(4) LIST OF ABBREVIATIONS ABCG2 ALDH1A1 BJ BrdU CHM CSCs DAPI DMEM EDTA EGF EGFR EMT ESI FACS FADD FBS bFGF FITC H&E LC/MS NSCLC PARP PBS PCNA PI poly-HEMA PS RIPA RNase A RPMI SE shRNA Teroxirone TKI TRITC TUNEL. ATP-binding cassette sub-family G member 2 Aldehyde dehydrogenase Brucea javanica 5-bromo-3′-deoxyuridine Chinese herbal medicine Cancer stem cells 4',6-Diamidino-2-Phenylindole Dulbecco's Modified Eagle Medium Ethylenediaminetetraacetic acid Epidermal growth factor Epidermal growth factor receptor Epithelial–mesenchymal transition Electrospray ionization Fluorescence-activated cell sorting Fas-Associated protein with Death Domain Fetal bovine serum Basic-Fibroblast growth factor Fluorescein isothiocyanate Hematoxylin and eosin Liquid chromatography/mass spectrometry Non-small cell lung cancer Poly (ADP-ribose polymerase) Phosphate-buffered saline Proliferating cell nuclear antigen Propidium iodide Poly(2-hydroxyethyl methacrylate) Phosphatidyl serine Radioimmunoprecipitation assay Ribonuclease A Roswell Park Memorial Institute medium Standard errors Small-hairpin RNA 1,3,5-tris((oxiran-2-yl)methyl)-1,3,5-triazinane-2,4,6-trione Tyrosine kinase inhibitor Tetramethylrhodamine Terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling. 3.

(5) ACKNOWLEDGEMENTS .................................................................... 2 LIST OF ABBREVIATIONS ................................................................. 3 Abstract .................................................................................................. 10 中文摘要 ................................................................................................. 11 1. Introduction ....................................................................................... 12 1.1 The benefits of CHM .................................................................... 12 1.2 BJ as a conventional medicine..................................................... 13 1.3 The prevalent lung cancer ........................................................... 14 1.4 EGFR in lung cancer.................................................................... 14 1.5 The CSCs hypothesis and drug resistance ................................. 16 1.6 Cancer stem cell markers in common cancers .......................... 17 2. Materials and methods ...................................................................... 19 2.1 Cell culture conditions ................................................................. 19 2.2 The spheroid culture .................................................................... 19 2.3 Chemicals and reagents ............................................................... 20 2.4 Liquid chromatography/mass spectrometry analysis and instrumental conditions ...................................................................... 20 2.5 Protein extraction and western blot analyses ............................ 21 2.6 Soft-agar colony formation assay ............................................... 22 2.7 Immunofluorescence Staining ..................................................... 22 2.8 BrdU incorporation of spheroids ................................................ 22 2.9 Tumor xenograft study ................................................................ 23 2.10 TUNEL assay .............................................................................. 24 4.

(6) 2.11 PCNA antibody staining for marker of proliferating cells in vivo ........................................................................................................ 25 2.12 Statistical analysis ...................................................................... 25 3. Results ................................................................................................. 26 3.1 LC/MS analysis of composition in aqueous BJ extract ............ 26 3.2 The aqueous BJ extract induces apoptosis and reduced EGFR in H1975 cells....................................................................................... 26 3.3 Effects of BJ on apoptotic features in H1975 cells .................... 27 3.4 EGFR shRNA suppresses BJ sensitivities by inhibiting apoptosis .............................................................................................. 28 3.5 Oral administration of aqueous BJ extract inhibits xenograft tumor growth ...................................................................................... 28 3.6 Histological and fluorescence examination of the suppressed tumors .................................................................................................. 28 3.7 The aqueous BJ extract affects forming ability and proliferation in H1975 CSCs ............................................................. 29 3.8 The fluorescence cancer stem cell markers examination of the affected H1975 spheroids by aqueous BJ extract ............................ 30 3.9 The aqueous BJ extract induces apoptotic characteristics in H1975 spheroids .................................................................................. 31 3.10 The aqueous BJ extract affects xenograft tumor growth in animal models nude mice ................................................................... 31 3.11 Fluorescence examination of the affected H1975 spheroid tumor by aqueous BJ extract............................................................. 32. 5.

(7) 3.12 EGFR and cancer stem cell markers fluorescence examination of the affected H1975 spheroid tumor by aqueous BJ extract ....... 33 3.13 The relapse of distinct stem cell signatures and drug resistance marker in spheroid tumors .............................................. 33 3.14 The attenuated EMT and c-Met in tumors of mice fed with BJ .............................................................................................................. 34 3.15 Histological examination of the collected organs .................... 34 4. Discussion ........................................................................................... 35. Part-II. The development of teroxirone as an effective therapeutic agent against human hepatocellular carcinoma cells......................... 40 Abstract .................................................................................................. 40 中文摘要 ................................................................................................. 41 1. Introduction ....................................................................................... 42 1.1 Overview of liver cancer .............................................................. 42 1.2 An anticancer compounds teroxirone ........................................ 43 2. Materials and methods ...................................................................... 44 2.1 Cell culture conditions and treatments ...................................... 44 2.2 The spheroid culture of HCC cells ............................................. 44 2.3 MTT assay ..................................................................................... 45 2.4 Flow cytometry ............................................................................. 45 2.5 Determination of apoptosis by double staining with Annexin VFITC and PI ........................................................................................ 46 2.6 Protein extraction and western blot analyses ............................ 46 6.

(8) 2.7 The effect of caspase-3 by z-DEVD-FMK .................................. 47 2.8 Spheroid formation assay ............................................................ 47 2.9 Soft agar colony formation assay ................................................ 47 2.10 Immunofluorescence staining assay for spheres ..................... 48 2.11 Statistical analysis ...................................................................... 48 3. Results ................................................................................................. 49 3.1 Dose-response growth curves of teroxirone in human hepatocellular carcinoma cells .......................................................... 49 3.2 Teroxirone increases apoptotic population of Huh7 cells by dose-dependent appearance of sub-G1 cells ..................................... 49 3.3 Effects of teroxirone on apoptotic features in Huh7 cells......... 50 3.4 Inhibition of caspase-3 activity reduces apoptosis in HCC cells after treatment teroxirone ................................................................. 50 3.5 Teroxirone suppresses the growth of Huh7 spheroids ............. 51 3.6 Teroxirone reduces stemness markers of Huh7 spheroids ...... 51 3.7 Teroxirone induces apoptotic characteristics in Huh7 spheroids .............................................................................................................. 52 4. Discussion ........................................................................................... 53 5. Conclusion .......................................................................................... 54 Figures .................................................................................................... 55 Figure 1. Chromatographic fingerprint analysis of the aqueous BJ extract .................................................................................................. 55 Figure 2. The aqueous BJ extract induces apoptosis and reduces EGFR in H1975 cells .......................................................................... 59 7.

(9) Figure 3. Effects of teroxirone on apoptotic features in H1975 cells .............................................................................................................. 61 Figure 4. EGFR shRNA suppresses BJ sensitivities by inhibiting apoptosis .............................................................................................. 63 Figure 5. Oral administration of aqueous BJ extract inhibited xenograft tumor growth ..................................................................... 65 Figure 6. Histological and fluorescence examinations of the suppressed tumors .............................................................................. 68 Figure 7. BJ reduces tumor growth by apoptosis ........................... 71 Figure 8. BJ suppresses H1975 spheroids growth ........................... 73 Figure 9. BJ reduces expression of stemness markers of H1975 spheres ................................................................................................. 76 Figure 10. BJ induces apoptotic characteristics of H1975 spheroids .............................................................................................................. 79 Figure 11. Oral administration of BJ suppresses growth of tumor spheroid xenograft .............................................................................. 82 Figure 12. BJ reduces spheroid tumor growth by apoptosis ......... 85 Figure 13. Fluorescence examinations of the suppressed spheroid tumors .................................................................................................. 88 Figure 14. Fluorescence examination of the collected adherent cells and spheroid tumors ........................................................................... 92 Figure 15. Fluorescence examination of the collected tumors ....... 94 Figure 16. Histological examinations by H&E staining of the collected specimens ............................................................................. 97. 8.

(10) Figure 17. Dose-response growth of Huh7, HepG2 and Hep3B cells treated teroxirone ............................................................................... 99 Figure 18. Teroxirone increases apoptotic population of Huh7 cells ............................................................................................................ 102 Figure 19. Effects of teroxirone on apoptotic features in Huh7 cells ............................................................................................................ 105 Figure 20. Teroxirone’s effects are reverted by z-DEVD-FMK in Huh7 cells .......................................................................................... 107 Figure 21. Caspase-3 inhibitor z-DEVD-FMK inhibits teroxironeinduced apoptosis in Huh7 cells ...................................................... 110 Figure 22. Model of FADD-mediated apoptosis induced by teroxirone in Huh7............................................................................ 111 Figure 23. Teroxirone suppresses the growth of HCC spheroids 114 Figure 24. Teroxirone reduces stemness and EMT markers of Huh7 spheres ..................................................................................... 117 Figure 25. Teroxirone induces apoptotic characteristics in Huh7 spheroids ............................................................................................ 120 References............................................................................................. 122 Published paper 1 ................................................................................ 128 Published paper 2 ................................................................................ 139. 9.

(11) Part-I. The alleviated tumorigenicity of human lung cancer cells carrying mutated epidermal growth factor receptor by aqueous extract of Brucea javanica. Abstract The traditional herbal medicine has been used as convenient medicine by the majority of the Chinese population the since recorded history and is still a widely accepted medicine in oriental countries. NSCLC (non-small cell lung cancer) is the most prevalent type of lung cancers which is hard to be diagnosed early. The mutated EGFR (epidermal growth factor receptor) has become a target in the development of novel anti-cancer therapeutic approaches. In this study, the aqueous BJ (Brucea javanica) extract has found effectively attenuated the growth of human NSCLC with mutant EGFR L858R/T790M. CSCs (cancer stem cells) are involved in drug resistance, metastasis and relapse of cancers and can influence the tumor therapy development. Importantly, drug resistance makes tumors more malignant. The work investigated the underlying mechanisms of aqueous BJ extract against CSCs of NSCLC. The study showed potential implication of aqueous BJ extract as new target therapy to human lung cancer. Key words: Brucea javanica, epidermal growth factor receptor, human lung cancer, herbal medicine, stem cells. 10.

(12) 第一部分: 利用鴉膽子水溶液萃取物清除突變上皮細胞生長因子接受體人類肺癌細胞. 中文摘要從古以來，傳統草藥已經被大部分的中國人作為常用藥物，也是東方國家廣泛接受的藥物。非小細胞肺癌是最常見的肺癌類型，早期很難被診斷出。突變的表皮生長因子受體已經成為新型抗癌治療的分子標靶。本論文所使用鴉膽子水溶液萃取物有效地減弱了在非小細胞肺癌具有突變 EGFR L858R/T790M 的生長。癌幹細胞具有抗藥性，轉移和癌症復發，並且可以影響腫瘤治療的發展。重要的是，抗藥性使腫瘤更惡化。本論文也探討了鴉膽子水溶液萃取物對非小細胞肺癌的癌幹細胞的療效與反應機制。鴉膽子水溶液提取物是具可以成為非小細胞肺癌放射性治療有潛力的藥物。. 關鍵詞: 鴉膽子,表皮生長因子受體,肺癌,中草藥,幹細胞. 11.

(13) 1. Introduction. 1.1 The benefits of CHM The CHM herbs can be classified according to their properties. The use of specific herb(s) to treat diseases depends on the sign and symptom of patients or their conditions. The herbalists believe that the illness is the imbalance condition of the body. Based on their various characteristics which are in accordance with the law of nature, can neutralize the sign and the symptoms thereby keeping an overall balanced status in patient’s body [1]. CHM has been used as a front line medicine by the majority of the world’s population since before the recorded history, and is still the most widely utilized medical system in the world today. Adverse reactions to Chinese herbs are generally rare when compared with those commonly produced by pharmaceutical drugs. CHM is a medical system that is capable of treating disease in various forms [2]. The clinical aspects of CHM include differential diagnosis and treatments based on the analysis of signs and symptoms and differentiation of syndromes. The treatment is known for its sophistication in addressing clinical concerns while remedying an individual’s particular needs. The art of selecting the appropriate base herbal formula is dependent on this analysis and the modifications made according to the presenting signs and symptoms of the patient [3]. Upon completing the study of the fundamental theories of CHM, diagnosis, pathology, students and new practitioners become concerned with how to apply their knowledge in clinical settings, with the selection of appropriate formulae, and with the modification process according to the manifestations or sometimes to the conventional diagnoses [4].. 12.

(14) 1.2 BJ as a conventional medicine BJ, a shrub original from south-east Asia and northern Australia and known in CHM for anti-cancer properties. In traditional herbal medicine, the seed of this plant also has been used for other treatment of different diseases including diabetes in Malaya peninsula [5]. Recently the medicine Fructus Bruceae (Yadanzi in Chinese) in NSCLC treatment has been practiced in China by many clinical treatment cases. Yadanzi (Fructus Bruceae) is the dried ripe fruit of Brucea javanica (L.) Merr Simaroubaceae [6]. As an efficient and safe traditional Chinese herbal medicine, it has been used to treat parasite infections or malarial in China for thousands years. It also shows huge effects in cancer therapy [7]. For instance, recipe of Yadanzi and its oil emulsions have been studied and already commercialized in China to treat various types of cancer such as brain cancer, lung cancer and gastrointestinal cancer, especially NSCLC [8]. It was already determined that the oleic acids or linoleic acids, two major components of Yadanzi oil, may cause anti-cancer activities. These two major components suppressed DNA Topoisomerases-I or -II [1]. Also these components could induce cancer cell death by up-regulation of caspase-3 and -9 or inhibition of NF-kappa B and cyclooxygenase 2 [2]. Moreover, some kusulactones of Yadanzi extract have been shown with anticancer activities. For example, brusatol was found as an effective compound in anti-inflammation, anti-leukaemia and enhancement of neoplastic chemotherapy [3, 6, 7]. The anti-cancer activities were anticipated involving nuclear factor (erythroid-derived 2)-like 2-mediated defense mechanism and the activation and translocation of NF-kappa B into the nucleus. Bruceantin is another Yadanzi ingredient that has been studied for Phase II trials in treating metastatic breast carcinoma and malignant melanoma respectively. It was determined that bruceantin inhibited protein synthesis at the stage of 13.

(15) ribosomal translation by targeting ribosome peptidyltransferase [9]. It was also found that bruceantin could induce apoptotic cell death by down-regulation of c-MYC and activate of caspase activities in mitochondria. Brusatol, bruceantin and several active ingredients like bruceine B and bruceine D were investigated for their potential activities in anti-cancers and anti-inflammation [10, 11].. 1.3 The prevalent lung cancer Human lung cancer is the leading cause of cancer-related deaths worldwide and NSCLC is the most prevalent type of lung cancers which is hard to be diagnosed in patients with advanced-stage disease. Recently, about 16 different drugs have been used in NSCLC treatment including gefitinib, erlotinib. Unfortunately, the protein target-based chemotherapy sometimes failed to work because of the problem of drug resistance [12].. 1.4 EGFR in lung cancer NSCLC is responsible for ≤ 80% of all cases of lung cancer, and advanced diseases are commonly found at the time of diagnosis. Almost 10% of patients with NSCLC in the US and 35% in East Asia have tumor associated with EGFR mutations. Lung cancer is characterized with frequent aberrance in driver genes, specifically EGFR [13]. Patients with mutations in the EGFR gene are sensitive to treatment with EGFR TKIs. The first generation EGFR TKIs, gefitinib and erlotinib, reversibly suppress binding of ATP to the intracellular catalytic domain of EGFR tyrosine kinase, preventing EGFR activation and subsequent downstream signaling. However, despite an initial response to treatment with first-generation EGFR TKIs, most EGFR mutation-positive 14.

(16) patients will develop resistance after 9–12 months of treatment [14]. Also, resistance will eventually develop in all patients after various time periods. The acquired resistance to gefitinib is most usually conferred upon a patient by the EGFR T790M mutation, which has been detected in 50% of NSCLC cases in cell line models. This mutation causes steric hindrance, thus preventing binding of EGFR TKIs, by inducing the affinity of the kinase for ATP and reducing inhibitor efficacy. The second-generation irreversible EGFR TKIs (e.g., afatinib and dacomitinib) have shown preclinical therapeutic value to overcoming EGFR T790M [15]. However, they are ineffective in preventing the physiologic action or the emergence of T790M and have shown limited clinical efficiency due to their non-selective inhibition of wild-type EGFR in causing dose-limiting toxicity. Due to the limited treatment options available for individuals with advanced lung cancer, a requirement exists for the identification of novel therapeutic strategies. The emulsified formula of BJ has been approved to treat various mutated EGFR frequently occurred in lung adenocarcinoma patients, especially those of Asian ethnicity, females, and nonsmokers. Both of gefitinib and erlotinib serve as the first-line TKI therapy to treat advanced NSCLC patients harboring EGFR mutation by preventing dimerization and phosphorylation of the receptor [16]. As first choice for target therapy, gefitinib and erlotinib bind to L858R EGFR of lung cancer cells, thereby making treatment successful [16, 17]. Treatment of TKI significantly prolonged survival rates of lung cancers patients with the specifically mutated EGFR [18]. Despite their distinct response, lung cancer patients receiving target therapy eventually develop resistance due to the evolved secondary T790M mutation. The rate arises at a median of 10–13 months that occurs in more than 50% of the patients [19]. In order to overcome the high concentration toxicity and resistance because of the accumulated 15.

(17) somatic mutation of T790M EGFR, new development of safer and more useful medication following target therapy is needed.. 1.5 The CSCs hypothesis and drug resistance Tissue stem cells form a small population of cells (typically much less than 1% within tissue), have self-renewing potential and display significant plasticity. The stem cells are undifferentiated biological cells that can differentiate into specialized cells and divide through mitosis to produce more stem cells. Human stem cells represent a subpopulation of tumor cells endowed with self-renewal and multilineage differentiation capacity and with an innate resistance to cytotoxic agents. The feature is a major clinical challenge to eradicate of the residual cancer cells in cancer patients. Human stem cells are defined by their tumor-propagating ability when transplanted into immunecompromised mice and by their capacity to fully recapitulate the original heterogeneity in all cancer cell types [20]. Like all stem cells, oncogenically transformed CSCs are defined by their ability for self-renewal and multipotency. The abnormal stem cells contributed to the development and recurrence of cancer. These tumorigenic stem/progenitor cell populations are collectively named CSCs. It was explained the causes of cancer drug resistance and recurrence of cancer after curative resection. The CSC hypothesis states that, although it represent a rare population of cells within a tumor, their high tumorigenic capacity drives tumorigenesis [21]. Anticancer drugs kill cancer cells and cause a decrease in tumor mass. Frequently, however, the cancer re-arises after a few months or even years because the therapy failed to kill all of the cancer stem cells. Future anticancer 16.

(18) therapies need to be tested for their efficacy in killing CSCs rather than the population of cancer cells. New generations of anticancer drugs will be developed to selectively and specifically find and destroy various different populations of CSCs [22, 23].. 1.6 Cancer stem cell markers in common cancers CSCs are cells within tumors that possess the capacity to self-renew and to generate the heterogeneous lineages of cancer cells that comprise the tumor. First studies discovered that subpopulation of leukemic cells that express a specific surface marker CD34, but lack the CD38 marker [24]. It is the first conclusive evidence for cancer stem cells. Later studies discovered that other malignant tumors, such as cancers of the brain, breast, colon, ovary, pancreas and prostate, can also be composed of morphologically and phenotypically heterogeneous cell populations with varying self-renewal capacities, degrees of differentiation, and clonogenic and tumorigenic potentials [25]. In most cases, such cells have been identified through their expression of specific cell surface markers. For example, cancer stem cell markers frequently used to identify adult stem cells within the prostate, breast and intestine include CD44, CD133, ESA, CD69, p63, as well as some stem cell antigen, such as CD34, c-kit, fms related tyrosine kinase-3, neural cell adhesion molecule, and Thy-1 [26-28]. Also, the transcription factors Oct4, SOX2 and Nanog have been identified as main regulators that maintain the self-renewal of embryonic stem cells. They are overexpressed in many types of cancers and are associated with malignant progression and poor prognosis including NSCLC [29].. 17.

(19) The study aims to find out if BJ of CHM inhibited to growth of cancer cells and the derived CSCs in a hope that a new generation of drugs be developed against somatically mutated EGFR become of target therapy.. 18.

(20) 2. Materials and methods. 2.1 Cell culture conditions Human NSCLC cells including H1975 (wild-type p53, two mutations in EGFR (L858R/T790M, erlotinib-insensitive)), H3255 (mutant p53, one mutation in EGFR L858R, erlotinib-sensitive), A549, H1299 and H460 were acquired from American Type Culture Collection (Manassas, VA) and cultured in 75 cm2 tissue culture flasks. The cells were grown in RPMI (Roswell Park Memorial Institute medium) or DMEM with supplementation of 10% FBS (Thermo Fisher Scientific, Waltham, MA), 100 unit/mL penicillin and 100 μg/mL streptomycin and cultured at 37°C in the environment of humidified incubator with 5% CO2. The cell media was replaced every three or four days and sub-cultured. Cells reaching 80-90% confluence were used for experiments.. 2.2 The spheroid culture Spheroid cultures were initiated by seeding a total of 1 × 106 cells in 4 mL of medium per 60 mm petri dish pre-coated with poly-HEMA. This provides a coat of sufficient homogeneity to completely inhibit cell attachment. The cultures were kept at 37℃ in a humidified CO2 incubator with weekly change of 50% of the medium. Spheroid bodies were derived by placing the parental cells into DMEM/F12 (DMEM Nutrient Mixture F-12) culture medium containing 2% B-27 supplement, human bFGF (10 ng/ml) and EGF (20 ng/ml, PEPROTECH).. 19.

(21) 2.3 Chemicals and reagents Sun Ten Pharmaceutical (Taichung, Taiwan) provided the aqueous extracts of the whole plant following the published procedures. Briefly, the collected materials samples were mixed with sterile water before boiling. The supernatant following centrifugation was filtered, concentrated and adjusted to a final concentration of 1 g/mL before storage. The chemicals including PI, RNase A, trypan blue, Tris-HCl and Triton X-100, were from Sigma-Aldrich Chemical (St. Louis, MO) and penicillin– streptomycin, glutamine, trypsin-EDTA and DMEM medium (Thermo Fisher Scientific).. 2.4 Liquid chromatography/mass spectrometry analysis and instrumental conditions The LC/MS (Liquid chromatography/mass spectrometry) method was used to identify the major markers of bioactive substances. The system for analysis consisted of a LC-20AD UFLC system (Shimdzu, Kyoto, Japan) linked to a LCMS-8040 triple quadrupole mass spectrometer. The running condition was designed as follows: gradient elution by the mixture of mobile phases A (0.1% formic acid and 1 g/L solution of ammonium acetate in water) and B (0.1% formic acid and 1 g/L solution of ammonium acetate in methanol) at minutes 0– 40 with the ratio of 100%–70% in A and 0%–30% in B; at minutes 40–70 with the ratio of 70%–0% in A and 30%–100% in B; at minutes 70–70.1 with the ratio of 0%–100% in A and 100%–0% in B; and at minutes 70.1–80 with the ratio of 100% in A and 0% in B. The flow rate was fixed at 0.4 mL/min and column temperature kept at 40°C. The injection volume was adjusted at 30 μL and the analytical column used a Shim-pack XR-ODS II column (2.2 μm, 20.

(22) 2×100 mm, Shimadzu). Dual ion modes (ESI (+) and (-)) were used in MS detection and the transmission of (M+H)+ and (M-H)- was set as the optimum condition. The MS detection was arranged as full scan range (400–800 amu). The interface voltages were set at 4.5 kV for ESI (electrospray ionization) (+) and -3.5 kV for ESI (-). With nitrogen as nebulizing and drying gas, the flow was set at 3.0 and 10 L/min, respectively. Argon as collision-induced dissociation gas was kept at 230 kPa. Desolvation lines temperature was set at 150°C and heat block temperature maintained at 400°C.. 2.5 Protein extraction and western blot analyses Human NSCLC sphere were plated (1 × 106 cells/well), cultured in above-mentioned spheroid culture conditions, and then treated for an additional 12 h with aqueous BJ (5, 10 and 15 mg/ml). Spheres collected by pipetting were lysed by RIPA (Radioimmunoprecipitation assay) buffer (1 % Triton X-100, 150 mM NaCl, 5 mM EDTA, 20 mM sodium phosphate and protease inhibitor cocktail for use with mammalian cell and tissue extracts (Sigma-Aldrich)). Samples were heated for 5 min at 95°C. Lysates from cells were electrophoresed in 10% polyacrylamide gel. The proteins in the gel were transferred to nitrocellulose blotting membrane. The membrane was saturated for 1 h with 5% non-fat dry milk in Tris-buffered saline (pH 7.6). The blots were then incubated with apoptosis pathway antibodies caspase-3, PARP (Genetex) and EGFR, pEGFRY1068 (Genetex). The spheroid protein was incubated with CD133, ALDH1A1 (aldehyde dehydrogenase) and Nanog (Genetex) and then was exposed to secondary antibodies and visualized by LAS-4000 (FUJIFILM). Molecular weights are determined by comparison to known markers. 21.

(23) 2.6 Soft-agar colony formation assay Anchorage-independent growth was determined in agarose (0.33%), as described [30]. Spheroids were plated in a semisolid medium (DMEM, supplemented with 10% FBS and 0.5% agar). Dishes were incubated at 37°C in a humidified atmosphere containing 5% CO2 and colonies were stained by 0.002% crystal violet and counted after 25-28 days.. 2.7 Immunofluorescence Staining Spheroids were cultured under the spheroid conditions for 7-10 days. Spheroids were fixed in 4% paraformaldehyde and incubated with primary antibody for two days at and with secondary antibody overnight at 4°C. The primary antibodies were used include Nanog, ALDH1A1 and BrdU (Genetex).. 2.8 BrdU incorporation of spheroids The assays to measure the proliferation of spheres were BrdU Labeling and Detection Kit II (Roche, Mannheim, Germany). The enriched spheroids treated with various concentrations of BJ for 12 h were incubated in media containing 10 μM BrdU for 1 h. Fixatives of 3:7 mixture of 50 mM glycine solution (pH 2.0) and ethanol were added, followed by incubation overnight at 20°C. The slides were washed with PBS and incubated at 37°C in solution containing diluted anti-BrdU antibody (1:1,000 dilution) for 30 min. The cells were suspended in a mixture of 1% BSA containing Alexa Fluor 488-conjugated secondary antibody (1:250 dilutions) for 2 h in darkness. An inverted fluorescence microscope was used to record the fluorescent images. All colored images were converted to black and white by Photoshop software before being 22.

(24) quantitated with Multi Gauge software (version 2.1, FUJIFILM). The green fluorescence at each concentration was obtained and compared with that of water control as BrdU intensity ratio. Three independent experiments were carried out.. 2.9 Tumor xenograft study A subcutaneous xenograft animal model was used to evaluate tumorigenesis variations. The female nu/nu mice of three- or four-week old were obtained from National Applied Research Laboratories (Taipei, Taiwan). The animals were housed under aseptic and ventilated conditions free of pathogens with a 12-hour light-dark cycle. The study was approved by the Animal Committee of National Taiwan Normal University, Taipei, Taiwan. Firstly, a total of 1 × 106 exponentially growing H1975 cells were seed on poly HEMA coated dish under the stem cell culture conditions as above mentioned. After 5-7days later, a total of 8 × 104 H1975 cancer spheroid cells in 0.2 mL (1:1 mixture ratio of PBS and 100 μL Matrixgel™ Basement Membrane Matrix (BD Biosciences) were subcutaneously injected into the dorsal area of nude mice. Each group consists of two or three mice. When the xenograft tumors reached 50-100 mm3 in size following cell inoculation, the mice were given an injection of 2 and 4 g/kg of BJ or vehicle control every other day to each group of mice for six days in a row. The volumes of tumors that formed were measured by a caliper and calculated according to the equation: volume in mm3 = (A × B2)/2, where values of A and B corresponded to the largest and the smallest diameters of the implanted tumor, respectively. Mice were anesthetized by ether and tumors were subsequently removed and fixed by 5% (w/v) formaldehyde for overnight in the refrigerator. 23.

(25) Formaldehyde is always freshly prepared from paraformaldehyde just before use. For sectioning (tissue freezing methods for cryostat sectioning), firstly tumor was sliced into 5 to 8 mm thick slabs (Cryostat for frozen section, LEICA, CM0350S). And then the slabs were washed several times in PBS (pH 7.4), passed through a series of increasing concentrations (w/v) of sucrose (10%, 20%, and 30%) in phosphate-buffered saline. The slabs were frozen, equilibrated in a cryostat at -20°C and then sectioned serially at thicknesses of 10 μm. For immunostaining, tumor sections were incubated overnight in the presence of 5% FBS in PBS with 0.2% Triton X-100. The tumor sections were washed several times in PBS, then incubated primary and secondary antibodies.. 2.10 TUNEL assay The apoptotic cell death was evaluated by TUNEL staining. Spheroids were treated with different concentrations of BJ extract for 12 h were permeabilized in 10 mM citrate buffer, pH 6.0. After blocking the nonspecific labeling with PBS mixture containing 2% BSA and 0.5% NP-40, cells were incubated in TUNEL reaction solution mixed with 9 mM dUTP, 1 mM digoxigenin-labelled dUTP (Roche, Mannheim, Germany), 2.5 mM cobalt chloride, 100 mM Tris pH 7.6 and 0.3 U/l terminal deoxynucleotidyl transferase for 1 h at 37°C in a humidified atmosphere. The spheroids were washed with PBS and incubated with a 1 : 200 dilution of horseradish peroxidase-conjugated digoxigenin antibody (Roche). With removal of unbound antibody, cell images were taken by fluorescence or confocal microscope. All colored TUNEL images were converted to black and white ones by Photoshop software before being quantitated with MultiGauge software (version 2.1, FUJIFILM). Five slides of. 24.

(26) each concentration and control sections were recorded and three independent experiments carried out. To identify apoptotic cells in vivo, tumor sections (10 μm) were stained with a TUNEL assay kit (Roche) according to the manufacturer’s instructions. Under the fluorescence microscope, the apoptotic cells were exhibited green fluorescence in the nucleus after excitation with blue light. Quantitative number of TUNEL-positive cells was counted total positive cells in 100 H1975 tumor cells at each concentration. Five slides of each concentration and control sections were recorded and three independent experiments carried out.. 2.11 PCNA antibody staining for marker of proliferating cells in vivo To determine proliferating cells in vivo, tumor sections were incubated with anti-PCNA (proliferating cell nuclear antigen) antibody. Under the fluorescence microscope, the proliferating cells exhibited green fluorescence in the nucleus after excitation with blue light. Quantitative number of PCNApositive cells was counted total positive cells in 100 H1975 tumor cells at each concentration.. 2.12 Statistical analysis The student’s t-test was applied to evaluate the differences between treated and control animal groups. The statistical analysis was performed by paired Student’s t-test using SigmaPlot 12.0 software. All values were presented as mean ± SE (Standard errors) unless otherwise noted. For all the tests, the level of significance was set at *p<0.05 and **p<0.01. 25.

(27) 3. Results. 3.1 LC/MS analysis of composition in aqueous BJ extract A typical LC/MS chromatographic fingerprint profile of aqueous BJ extract was shown in Figure 1. Twelve major components were identified from the chromatogram. The well-separated peaks with retention times of 40 minutes were identified as 1) Bruceoside D (PubChem CID: 10484578), 2) Bruceine E (PubChem CID: 122785), 3) Bruceine F (17.5 minutes), 4) Bruceine D (PubChem CID: 441788), 5) Bruceine B (PubChem CID: 161496), and 6) Bruceine I (PubChem CID: 196839), respectively. The distinct peaks above 40 minutes included 7) Bruceine J (PubChem CID: 23656476), 8) Yadanzioside F (PubChem CID: 3000798), 9) Bruceantinol B (PubChem CID: 23656477), 10) Brusatol (PubChem CID: 73432), 11) Bruceine A (PubChem CID: 160006), and 12) Bruceoside E (PubChem CID: 3000803).. 3.2 The aqueous BJ extract induces apoptosis and reduced EGFR in H1975 cells The cell viabilities have determined in human NSCLC cells treated with aqueous BJ extract at 1, 2 and 5 mg/mL. H1975 cell line is an ideal model system for studying mutated L858R/ T790M EGFR-related TKI resistance in human NSCLC cells. Among the cell lines studied, H1975 cells were sensitive to BJ treatment after 12 h and showed concentration dependent growth suppression. The acquired IC50 value of 2 mg/mL of BJ in H1975 cells (Figure 2A) was not detected in erlotinib-sensitive H3255 cells that carry EGFR of mutated L858R. Wild-type EGFR cells were unaffected by aqueous BJ extract. 26.

(28) These findings suggested that the effectiveness of BJ extract is specific in NSCLC cells harboring L858R/ T790M mutated EGFR. Cell-cycle analysis by flow cytometry in human NSCLC cells showed that aqueous BJ extract induced sub-G1 cell population in H1975 cells (Figure 2B and C). Thus, the induced cytotoxicity in H1975 cells was caused by apoptotic cell death. Furthermore, analysis of Annexin V-FITC/PI doubly labeled H1975 cells, as the extract concentration was raised to 5 mg/mL, populations of early and late apoptotic phase were increased to 11 % and 15 %, respectively, after 12 h treatment (Figure 2C and D). The data implied that the induced apoptotic cell death contributed to the reduced viable H1975 cells when exposed to aqueous BJ extract.. 3.3 Effects of BJ on apoptotic features in H1975 cells Protein lysates from H1975 cells were subjected to western blot analysis. The aqueous BJ extract inhibited EGFR, phosphorylated EGFRY1068 and phosphorylated AktS473 in H1975 cells (Figure 3A). The inhibition of EGFR and pEGFRY1068 was dose-dependent (Figure 3B). Cleavage of PARP and procaspase-3 together with formation of active caspase-3 fragment in H1975 cells with increasing aqueous BJ extract concentrations implied emergent apoptotic features. The results demonstrated that the accelerated apoptotic cell death in H1975 cells arose at the expense of mutant EGFR carrying L858R/T790M.. 27.

(29) 3.4 EGFR shRNA suppresses BJ sensitivities by inhibiting apoptosis To understand how apoptotic death started, H1975 cells were transfected with shRNA (small hairpin RNA) targeting exon 4 of EGFR (RNA sequence: GAGAAU GUGGAAUACCUAAGG) along with nonspecific shRNA as control (RNA sequence: CCGGACACUCGAGCAC UUUUUG). The recovered viable cells transfected with EGFR shRNA were more than cells with nonspecific shRNA following BJ treatment (Figure 4A). Aqueous BJ extract accentuated decrease of pEGFRY1068 in H1975 cells following EGFR shRNA transfection (Figure 4B). The reduced PARP cleavage signified that knocking down EGFR incapacitated BJ sensitivity. The results implied that BJ targeted EGFR with double mutation and orchestrated cell death.. 3.5 Oral administration of aqueous BJ extract inhibits xenograft tumor growth The sizes of xenograft tumors of H1975 cells were diminished when feeding mice with BJ extracts. The continuous tumor development was repressed in animals when fed with 2 g BJ/kg compared with those with water (Figure 5A). No comparable body weight loss in nude mice orally administered with aqueous BJ extract was seen relative to those administered water treatment during experiment durations (Figure 5B). The increased BJ dosage proportionately reduced weights of the dissected tumors (Figure 5C).. 3.6 Histological and fluorescence examination of the suppressed tumors As shown in H&E staining of the histological sections, mice administered with aqueous BJ extract induced apoptotic cell death in xenograft 28.

(30) tumors. The microscopic images of tumor sections in mice fed with water showed packed viable cells, whereas those with 2 g BJ/kg exhibited increasingly apoptotic characteristics, including cytoplasm condensation, pyknotic nuclei, and interstitial space enlargement. Some apparent engulfed apoptotic bodies were visible (Figure 6A). The increasingly administered BJ depleted fluorescent EGFR and pEGFRY1068 (Figure 6B) as evidenced by antibody detection of tumor dissections. Cytochrome c released from mitochondria was visualized by confocal microscopy. In tumor section images, the increased fluorescent puncta with merged green color mitochondria marker mitotracker and the released red color cytochrome c suggested progressive apoptotic cell death of H1975 tumors in mice given increasing BJ extract (Figure 6C). BJ extract suppressed tumor proliferation as suggested by the reduced mitotic index PCNA (Figure 7A). The decreased nucleus PCNA positive staining as shown suggested that tumor growth alleviation is attributed to apoptosis (Figure 7B). On the other hand, the enriched TUNEL positive fluorescence in the resected specimen implied induced apoptotic death in H1975 xenograft tumors in mice administered with aqueous BJ extract (Figure 7C). The increased fluorescence intensities proposed enhanced apoptotic index relative to groups with water (Figure 7D).. 3.7 The aqueous BJ extract affects forming ability and proliferation in H1975 CSCs The spheroids of H1975 were established under the spheroid culture condition after 7-10 days. The sizes of H1975 spheroid cells were reduced significantly by aqueous BJ extract (5 and 15 mg/mL) after 12 h treatment 29.

(31) (Figure 8A) as indicated by spheroid formation assay. The suppression of spheroid growth was dose-dependent. Soft agar colony formation assay shows that the self-renewal ability is affected. Pre-treated aqueous BJ extract (5, 10 and 15 mg/mL) of H1975 spheroid showed that the morphology of colony was significantly reduced (Figure 8B). The H1975 spheroid colonies were stained by 0.002% crystal violet and visualized by soft agar colony formation assay. The visualized images were re-formatted and then measured colony formation ratio (%).. 3.8 The fluorescence cancer stem cell markers examination of the affected H1975 spheroids by aqueous BJ extract The levels of cancer stem cell marker during treatment aqueous BJ extract in H1975 spheroids were examined. Tumor spheroids had enriched expression of the cancer stem cell markers and CD133 and Nanog are cancer stem cell markers in H1975. The expression of cancer stem cell markers, CD133 and Nanog, were determined in H1975 tumor spheres treated with aqueous BJ extract for 12 h (Figure 9A and B). The expression of CD133 and Nanog was decreased after treatment aqueous BJ extract as indicated by immunofluorescence staining. Protein lysates from H1975 spheroids were subjected to western blot analysis (Figure 9C). Cleavage of PARP and procaspase-3 together with formation of active caspase-3 fragment in H1975 spheroids with increasing aqueous BJ extract concentrations implied emergent apoptotic features. The aqueous BJ extract inhibited phosphorylated AktS473 and cancer stem cell markers including Nanog and CD133 in H1975 spheroids on dose-dependent, but not ALDH1A1. 30.

(32) 3.9 The aqueous BJ extract induces apoptotic characteristics in H1975 spheroids To understand the cytotoxicity of aqueous BJ extract towards H1975 spheroids, cell proliferation was demonstrated by BrdU fluorescence assay. BrdU is a thymidine analog that is incorporated into cell genome as it replicates [31]. Thus, the level of BrdU in cell nucleus shows the level of cell division and proliferation. The BrdU assay was carried out 12 h after aqueous BJ extract treatment to allow enough accumulation of aqueous BJ extract in cells and to determine its impact on cellular DNA synthesis. The level of BrdU in cell nuclei was measured using a fluorescent anti-BrdU conjugate with cell nuclei counterstained by DAPI as shown in Figure 10A. The level of BrdU was significantly decreased by dose-dependent (Figure 10B). Appearance of TUNEL is often used as a marker for apoptosis. In order to test whether aqueous BJ extract induces apoptosis in H1975 spheroids after treatment, TUNEL assay was used, followed by aqueous BJ extract treatment (Figure 10C and D). H1975 spheroids at 12 h after aqueous BJ extract treatment (5 and 15 mg/mL) were positive for TUNEL staining, suggesting a massive induction of DNA fragments.. 3.10 The aqueous BJ extract affects xenograft tumor growth in animal models nude mice To perform analysis the properties of CHM in vivo, alteration of tumor growth was investigated after feeding aqueous BJ extract in H1975 spheroids xenograft model. Firstly, cultured H1975 spheroids were injected into nude mice. Once tumor was established, aqueous BJ extract was started to feed to mice orally. After stop to feed aqueous BJ extract, mice were observed 7 days more for in vivo purposes. The continuous tumor development was repressed 31.

(33) significantly by feeding mice with 4 g BJ/kg compared to those with water (Figure 11A and 11C). There was no comparable body weight loss in nude mice orally administered with aqueous BJ extract relative to those with water (Figure 11B). The H&E staining assays showed that mice fed with aqueous BJ extract induced apoptotic cell death as discernible in histological sections of xenograft tumors. The microscopic images of tumor sections in mice fed with water shown packed viable cells, whereas those with aqueous BJ extract of 4 g/kg exhibited increasing apoptotic characters including cytoplasm condensation, pyknotic nuclei and interstitial space enlargement. Some apparent engulfed apoptotic bodies appeared (Figure 11D). The diminished tumor weight with increasing aqueous BJ extract concentrations (2 and 4 g/kg) was proportional to the inhibited growth of xenograft H1975 spheroid tumors.. 3.11 Fluorescence examination of the affected H1975 spheroid tumor by aqueous BJ extract The mitotic index was assessed by analysis of PCNA expression in tumor specimens. Aqueous BJ extracts effectively inhibited tumor proliferation as proposed by the decreased PCNA staining (Figure 12A) that can be quantitated (Figure 12B). The escalated TUNEL fluorescence of the resected tumors was visible in H1975 spheroid xenograft tumor-bearing mice orally fed with aqueous BJ extracts from 2 to 4 g/kg (Figure 12C). The significantly enhanced fluorescence quantitation (2 and 4 g/kg of aqueous BJ extract) indicated increasing apoptotic index compared with groups of water treatment (Figure 12D).. 32.

(34) 3.12 EGFR and cancer stem cell markers fluorescence examination of the affected H1975 spheroid tumor by aqueous BJ extract To find out whether aqueous BJ extract affect EGFR and stem cell characteristics in vivo, the expression of EGFR and cancer stem cell markers was next examined in spheroid tumors. Firstly, Aqueous BJ extracts effectively suppressed fluorescent EGFR and pEGFRY1068 (Figure 13A and B) by antibody detection. In tumor section images, the ascending fluorescent puncta comprised of coalesced mitotracker and the released cytochrome c from mitochondria were visualized by confocal microscopy. The results suggested incessant apoptotic cell death of H1975 spheroid tumors in mice receiving increment BJ extract (Figure 13C). In addition, the cell surface stem cell indicator ALDH1A1 remained undamaged in the dissected tissues of H1975 spheroid tumors at all BJ extract concentrations (Figure 13D).. 3.13 The relapse of distinct stem cell signatures and drug resistance marker in spheroid tumors The fluorescent cancer stem cell markers including extracellular CD133 and nucleus Nanog were visibly decreased in the spheroid tumors (Figure 14A). The two stem cell markers were not palpable in the dissected tumor sections from adherent H1975 cell tumors (Figure 14B). In spheroid tumors, the increased BJ concentrations reduced coalesced fluorescence of embryonic stem cell transcription factors SOX2 and Nanog (Figure 14C) that were inconspicuous in tumors established by adherent H1975 cells (Figure 14D). The depleted dual staining of cell surface ABCG2 and nuclear Nanog by BJ extract meant elimination of drug resistance and growth in tumors of the enriched. 33.

(35) H1975 spheres (Figure 14E). The two markers were indistinct in tissue specimen as obtained from tumors of adherent H1975 cells (Figure 14F).. 3.14 The attenuated EMT and c-Met in tumors of mice fed with BJ NSCLC have developed resistance to EGFR TKIs, including gefitinib and erlotinib, are clinically linked to an EMT (epithelial–mesenchymal transition) phenotype [32]. The results suggested that the amplified EMT of spheroid tumors of mutant EGFR T790M/L858R be impaired by increasing BJ concentrations. Mutated EGFR T790M and Met enlargement have been known closely associated for the developed resistance to EGFR-TKI [33]. BJ reverted the enriched gefitinib-resistant lung cancer spheroid tumors by inhibiting c-Met signaling pathway (Figure 15A). Moreover, in the implanted spheroid tumors, the increased BJ extract concentrations gradually debilitated the enriched EMT indicators, β-catenin (Figure 15B) and Vimentin (Figure 15C) in the spheroid tumors.. 3.15 Histological examination of the collected organs The study determined if BJ influenced any effects to other organs such as liver, lung, brain or kidney in gavage fed BJ mice. As shown in H&E staining of the histological sections (Figure 16), there were not visibly differences between gavage fed water or BJ in mice organs.. 34.

(36) 4. Discussion BJ increased sub-G1 cells and Annexin V-positive populations in H1975 cells (Figure 2).The study showed that BJ suppressed cell viabilities only in H1975 cells (Figure 2). The diminished EGFR intensities and the repressed EGFRY1068 phosphorylation account for the growth rate reduction (Figure 3). More work showed that the aqueous extracts attenuated Akt pro-survival and induced procaspase-3 cleavage. Active caspase-3 is required to elicit and execute apoptosis by cleaving cellular proteins at appointed aspartate residues [34]. Western blot analysis showed that both PARP and procaspase-3 were cleaved following BJ treatment that triggered subsequent apoptosis (Figure 3). The appearance of apoptotic caspase-3 fragment corroborated the decreased viable cells. Knocking-down EGFR in H1975 cells inhibited PARP cleavage, reduced subsequent apoptotic cell death and suppressed BJ sensitivity (Figure 4). The results supported the vital role of the mutant EGFR in H1975 cells during BJ-induced apoptosis. More experiments showed that orally administered aqueous BJ extract inhibited the growth of the established xenograft H1975 tumors without affecting the healthiness of mice (Figure 5). Aqueous BJ administration diminished EGFR intensities and depressed phosphorylation levels in the collected tumor resections. The increment of cytochrome c (Figure 6C) and TUNEL staining plus the declined PCNA marker as shown in confocal images further validated that BJ eliminated H1975 tumor growth by apoptotic cell death (Figure 7). Overall, the aqueous extract of BJ effectively overcomes drug resistance by targeting EGFR with secondary mutation during therapy. The results suggested that aqueous BJ extract rich in various forms of quassinoids prompted apoptosis in H1975 cells with L858R/T790M EGFR. 35.

(37) Another report also showed that the cationic nano-emulsions of BJ oil mixture can be an effective delivery system that enhances the oral bioavailability with promising anticancer prospect [35]. A recent study has proven that the aqueous extract of the BJ plant suppressed the growth of liver cancer by reducing cell proliferation and activating apoptosis [3]. The work described here illuminates more aspects of the herbal extract as an effective therapeutic method to treating lung cancer. The study further exemplified that, as an alternative medicine, BJ complements the existing cancer therapy by targeting cells with mutant EGFR. One of the major components in the aqueous BJ extract, brusatol, is capable of overcoming drug resistance by decreasing nuclear factor erythroid 2 p45-related factor 2, thereby enhancing the cytotoxic effect of numerous chemotherapeutic agents [36]. CSCs have the capability to self-renew and differentiate into different types of cancer cells. The CSCs have a tumorigenic potential and they are resistant to radiotherapy and chemotherapy. The conventional treatment approach cannot eliminate CSCs that often lead to the tumor recurrence [37]. The study also showed that aqueous BJ extract inhibited tumor sphere formation. Inhibition of 50% of spheroid colony forming capacity was approximately 10 mg/mL (Figure 8A) that also reduced the stemness characteristics and induced apoptosis of the cultured spheres. More works showed that the aqueous BJ extract attenuated Akt signaling and induced procaspase-3 cleavage. Active caspase-3 is required to induce cleaved cellular proteins at appointed aspartate residues [34]. Western blot analysis showed that both PARP and procaspase-3 were cleaved following aqueous BJ extract treatment by triggering subsequent apoptosis. The appearance of apoptotic caspase-3 fragment corroborated the decreased viable spheroids (Figure 9C).. 36.

(38) ABCG2 is one of the human ATP-binding cassette (ABC) transporters that have been determined in multidrug resistance in cancer chemotherapy [38, 39]. ABCG2 gene was cloned from drug-selected model cell lines and human cDNA library independently in 1998 [40]. ABCG2 is an important molecule in essential multidrug resistance and in protecting cancer stem cells. Although ABCG2 is generally expressed in normal tissues, overexpression of ABCG2 has been often found in various drug-selected cancer cell lines [41] and contributes to the clinical multidrug resistance of hematopoietic malignancies and solid tumors [42]. Increased ABCG2 expression has also been linked to cancer stem cells [43, 44]. The expression of cancer stem cell markers Nanog (Figure 14), CD133 (Figure 14A), SOX2 (Figure 14C) and ABCG2 (Figure 14E) were decreased by aqueous BJ extract and indicating that BJ reduced stemness of H1975 spheroids. Xenograft tumors in nude mice were established by inoculating 8 × 104 tumor spheres as enriched from H1975 cells. Like previous findings with adherent cells (Figure 5), orally administered BJ successfully eased H1975 spheroid tumor burden, albeit at higher concentrations, without affecting the health of the animals (Figure 11). BJ extract reduced fluorescent transmembrane protein EGFR (Figure 13A) and inhibited phosphorylation of the receptor altogether (Figure 13B). Evidence of H&E staining (Figure 11D), TUNEL assay (Figure 12C) and mitochondrial release of cytochrome c (Figure 13C) proved the induced apoptosis in tumor tissues. However, the specific surface marker for therapeutic resistance involving detoxification [45], ALDH1A1, was not drastically affected in cultured spheres (Figure 9C) and in the established tumors (Figure 13D) at all experimental conditions. It is thus reasonably concluded that the tumors were indeed 37.

(39) stemmed from the enriched tumor spheres that can be debilitated by increasing concentrations of BJ extract. Erlotinib-refractive EGFR-mutant NSCLC cells are responsive to herbal mixtures because of high ALDH activity [46]. The extract effectively diminished various stem cell markers except ALDH1A1 of H1975 spheroids. The findings corroborated previous finding in liver cancer tumors pheres in which ALDH1A1 was not the targeted by BJ [3]. Lung cancer cells acquired mesenchymal characteristics and stem cell properties altogether were known insensitive to environmental intervention [47]. There is evidence suggesting the direct link between less differentiated stem cells and EMT expression in cancer cells [48]. More important, cancer cells with bestowed EMT, intensified c-Met and stem cells traits tend to resist apoptosis [49]. The current work showed that the growth of spheroid tumors with amplified drug resistance markers ABCG2 were overcome by depleting stem-like traits, dissipating EMT markers, knocking out c-Met and promoting apoptosis (Figure 14 and 15). The aqueous extract of the plant was proved effective in eliminating tumors from stem-like cancer cells bearing mutant EGFR. Previous findings disclosed that aqueous BJ suppressed the growth of the liver cancer stem-like cells by attenuating EGFR expression [50]. The current study asserted that the arising resistance in the tumor mass from the spheroids of lung adenocarcinoma cells can be overtaken by debilitating the doubly mutated EGFR and its phosphorylated form. The findings consolidated the notion that the extract composing of mixtures of quassinoids [51] abolished EGFR expression and repressed tumor growth by apoptosis. The decreased intensities of markers concerning stem cell development including EMT, c-Met and drug resistance not only signaled the opportunities of aqueous extract of the prevalent BJ for lung cancer therapy, but also broadened the scope in delineating the mechanisms of lung cancer CSCs eradication. 38.

(40) Conclusions The work has demonstrated that the aqueous extract of BJ induced apoptotic cell death in H1975 cells and suppress growth of H1975 tumor in vivo. Moreover, BJ inhibited the growth of the enriched H1975 spheroids by apoptosis. The findings promised that aqueous BJ extract is a potent anti-cancer medicine. The findings promised more innovative prospect of the conventionally available herbal medicine to counter tumor relapse that promises to prolong survival rates of lung cancer patients.. 39.

(41) Part-II. The development of teroxirone as an effective therapeutic agent against human hepatocellular carcinoma cells. Abstract Teroxirone is an experimental triepoxide antitumor agent. The purpose of this study is to determine how teroxirone regulates growth of liver cancer cell. Three hepatocellular carcinoma cell lines, Huh7 (mutant p53), HepG2 (wildtype p53) and Hep3B (p53-null) cells, were used for experiments. The study included MTT assay for assessing cell viability, flow cytometry for evaluating apoptotic cell death and Western blot analysis for determining signal pathway activation. The findings suggested that teroxirone induced apoptotic cell death in p53-mutated Huh7 by stimulating extrinsic pathway that was reverted by caspase-3 inhibitor. Teroxirone also induced apoptosis and suppresses the stemness properties of the enriched Huh7 spheroids. The study implied that the small molecular weight molecule teroxirone is a potentially valuable agent to treat human hepatocellular carcinoma.. Key words: Teroxirone, extrinsic pathway, human liver cancer, stem cells. 40.

(42) 第二部分: 發展 teroxirone 成為有效治療肝癌細胞的藥物. 中文摘要 Teroxirone (1,3,5-tris((oxiran-2-yl)methyl)-1,3,5-triazinane-2,4,6trione)是三環氧化物抗腫瘤劑，此研究目的是確定 teroxirone 如何調控與凋亡相關的細胞死亡。利用 MTT 檢測三種肝細胞癌細胞系：Huh7（突變體 p53），HepG2（野生型 p53）和 Hep3B（p53-缺失型） assay(細胞存活檢測) 細胞存活率，利用流式細胞儀及西方點墨法觀察凋亡所導致的細胞死亡。本研究發現在 Huh7 細胞所引起凋亡的細胞死亡是透過外在途徑。Teroxirone 可以誘導從 Huh7 腫瘤球體所純化出來的腫瘤幹細胞的凋亡並抑制 Huh7 腫瘤球體幹細胞特性。. 關鍵詞: 三環氧化物, 外源性途徑, 肝癌, 幹細胞. 41.

(43) 1. Introduction. 1.1 Overview of liver cancer Liver cancer is the fifth most prevalent cancer in the world and the third most frequent cause of cancer-related death. Both the incidence and associatedmortality of liver cancer are rising. HCC is the major form of liver cancer, accounting for 90% of all liver cancers, and resulting in at least 500,000 deaths per year [30]. The overall 5-year relative survival rate for patients with liver cancer is about 15% in the US (www.cancer.org). Liver cancer is usually resistant to most chemotherapy drugs [52]. HCC is one of the most frequent malignancies and common cause of death from cancer in the world. Despite great advances in the diagnosis and treatment of HCC, its prognosis is difficult. Postoperative recurrence or metastasis is quite common and the main factor related to poor prognosis in patients with HCC. Further insight into the molecular mechanisms underlying HCC recurrence and metastasis might help identify novel therapeutic targets and consequently improve the prognosis [53]. It is refractory to most chemotherapeutic treatment strategies, and there is a lack of an effective molecularly targeted therapy. Sorafenib, for example, extends survival poorly by approximately 2 months, according to two large multicenter randomized clinical studies. Furthermore, there have been no oncogenic addiction loops discovered for HCC so far, in contrast to other cancers such as lung and breast (BRCA1/2 mutations) cancers, which likely reflects the complexity of gene interactions and diverse pathways involved in HCC [54]. While the approval of Sorafenib was a significant step forward, the benefit is at best modest and confers a rather transient clinical benefit. Moreover, 42.

(44) patients who are resistant, refractory or intolerant to Sorafenib, no effective therapeutic options currently are existed. Therefore, additional treatment options are warranted [55].. 1.2 An anticancer compounds teroxirone Teroxirone was synthesized and used as an experimental antitumor agent in human clinical trials in the 1980s. Initially, teroxirone was used against a series of murine transplantable tumors, and marked cytotoxicity was observed including substantial retardation in tumor growth and significant prolongation of animal survival [56]. A broad spectrum of preclinical activity of teroxirone has been observed. In a phase I clinical trial, adult patients with a variety of solid tumors were entered, and anticancer effect was observed in one patient with adenocarcinoma [57]. The anticancer mechanism of teroxirone has been suggested via binding to nuclear proteins which are involved in DNA repair mechanism and replication, which is different from the direct binding of platinum-based anticancer drugs to DNA [58]. Teroxirone is also structurally different from platinum- based anticancer drugs. Since oxaliplatin, a third generation platinum (II) based anticancer drug, is the standard treatment in conjunction for locally advanced and metastatic cancer of the colon or rectum, three platinum-based anticancer drugs were used as controls in the present study to determine if teroxirone exhibit better anticancer effect than these drugs used for patients [58, 59]. In treating human lung cancer cells, teroxirone has been known to induce a programmed cell death [56]. However, the molecular mechanisms by which it induces apoptosis in HCC liver cancer cells have not been explored.. 43.

(45) 2. Materials and methods. 2.1 Cell culture conditions and treatments The HCC lines, Huh7 (mutant p53) cells were from Japanese Collection of Research Bioresources. And HepG2 (HB-8065, wild-type p53) and Hep3B (HB-8064, p53-null) are obtained from ATCC (American Type Culture Collection, Massachusetts, MA, USA). The cell lines were cultured in DMEM supplemented with L-glutamine, sodium pyruvate and 10% FBS at 37℃ in a humidified atmosphere with 5% CO2. HCC cells were treated with teroxirone at 2, 5, 10 and 20 μM. All measurements were conducted in duplicate in three independent experiments.. 2.2 The spheroid culture of HCC cells Human liver carcinoma cells were supplemented to acquire enough target spheroid cells. To maintenance growing undifferentiated spheroids, cells were cultured at 1×106 cells/mL in serum-free DMEM-F12 (Thermo Fisher Scientific). The media in ultra-low attachment dishes (Corning Incorporated, Corning, NY, USA) contained B-27 supplement (Thermo Fisher Scientific), 20 ng/mL of EGF (PeproTech, Rocky Hill, NJ, USA), and 10 ng/mL of bFGF (PeproTech). Floating sphere cultures were expanded by mechanical dissociation, followed by replanting of single cells in complete fresh medium every 3 days. The cultures were kept at 37℃ in a humidified CO2 incubator. For clonal expansion, single cells were added to each well of 96-well ultra-low attachment tissue culture plates (Corning Incorporated) and clonal expansion monitored at the indicated time points. The collected spheroid cells were subject 44.

(46) to experiments in non-adherent culture conditions. Spheroid diameters were measured using Image-Pro plus 6.3 (Media Cybernetics, Bethesda, MD, USA).. 2.3 MTT assay The MTT assay is conducted as described [60]. Briefly, following the treatment of cells with teroxirone, medium was removed and replaced with fresh medium and MTT solution and cells were incubated for 3 h at 37°C. As the cells were rinsed with PBS and formazan was solubilized with DMSO. Absorbance was measured at 570 nm using a multiwell scanning spectrophotometer. The inhibitory concentration at 50% growth (IC50) was determined.. 2.4 Flow cytometry The flow cytometry was using FACS (fluorescence-activated cell sorting ) Calibur™ flow cytometer (BD Biosciences, San Jose, CA, USA). The cells were plated (2 × 106/well), cultured in above-mentioned culture conditions, and then treated for an additional 48 h with teroxirone (2, 5 and 10 μM). For flow cytometry preparation, supernatant and cells were collected by pipetting and were lysed by Trypsin-EDTA. Samples were centrifuged, washed twice with PBS and then treated 70% alcohol with PBS at -20°C for 24 h. After that, samples were treated with 10 g/ml PI, 10 g/ml RNase A with PBS for 30 min in darkness. Data is analyzed by FlowJo software (Tree Star, Ashland, OR, USA).. 45.

(47) 2.5 Determination of apoptosis by double staining with Annexin V-FITC and PI Externalization of PS was measured by Annexin V-FITC staining according to the manufacturer's protocol. Briefly, 1 × 106 cells were treated with indicated doses of teroxirone for 48 h, washed twice in PBS, and re-suspended in 1× binding buffer (0.01 M HEPES, pH 7.4; 0.14 M NaCl; 2.5 mM CaCl2) and incubated for 30 minutes in the dark at room temperature with 5 μL Annexin VFITC and 10 μL of 50 μg/mL PI. The flow cytometer FACS Calibur™ was employed to determine the early and late phases of apoptotic cell distribution, and the data were analyzed by FlowJo software.. 2.6 Protein extraction and western blot analyses HCC cells were plated (2 × 106 /well), cultured in above-mentioned culture conditions, and then treated for an additional 24 and 48 h with teroxirone (2, 5 and 10 μM). Cells collected by pipetting were lysed by RIPA buffer (1 % Triton X100, 150 mM NaCl, 5mM EDTA, 20mM sodium phosphate and protease inhibitor cocktail for use with mammalian cell and tissue extracts (sigma)). Samples were heated for 5 min at 95°C and electrophoresed in 10% polyacrylamide gel. The proteins in the gel were transferred to nitrocellulose blotting membrane. The membrane was saturated for 1 h with 5% nonfat dry milk in Tris-buffered saline (pH 7.6). The blots were then incubated with extrinsic related apoptosis pathway antibodies FADD, caspase-3, -6 and -8, PARP and intrinsic related apoptosis pathway antibodies p53, BAX and caspase-9 (Gentex). The spheroid proteins were incubated with CD133, Nanog 46.

(48) and Vimentin (Gentex) and then exposed to TRITC-conjugated secondary antibodies. All proteins were visualized by LAS-4000.. 2.7 The effect of caspase-3 by z-DEVD-FMK The assay was based on evaluating the effect of cleaved caspase-3 after treatment with caspase-3 inhibitor z-DEVD-FMK. Huh7 cells were prepared with z-DEVD-FMK (20 μM) for 24 h, then cultured Huh7 cells were treated with teroxirone as same conditions (2, 5 and 10 μM) for 48h. The effect of caspase-3 was demonstrated by MTT assay, flow cytometry, Annexin V-FITC and PI and western blot analysis.. 2.8 Spheroid formation assay To further investigate the spheroid formation capability, suspended adherent cells (700 cells/well) were seeded in 24-well ultra-low attachment plates (Corning Incorporated) in spheroid culture medium and incubated at 37°C for 7-10 days and then treated with teroxirone at 10, 20 and 30 μM. Spheroids images were taken at 24, 48 and 72 h by light microscopy.. 2.9 Soft agar colony formation assay The anchorage-independent growth was determined in agarose (0.33%), as described [30]. Huh7 cells (200 cells/well) were seeded in 12-well ultra-low attachment tissue culture plates, and then cultured in growth-factor supplemented medium for 7 to 14 days. Numbers of cultured Huh7 spheroids were reached to 50 spheroids, the spheres were treated with various 47.

(49) concentrations of teroxirone (10, 20 and 30 μM) for 24 and 48 h. Then, the spheroid mixtures were cultured in a semisolid medium (agarose (0.5%) on top and agar (0.3%) on bottom layers). After 25-28 days, the plates were stained with 0.002% crystal violet and incubated at 37°C for 18 h. Following the removal of the dye solution, colonies with more than 50 cells were counted as positive. Three individual experiments were conducted for each condition.. 2.10 Immunofluorescence staining assay for spheres Spheroids were cultured for 7-10 days and fixed in 4% paraformaldehyde and then incubated with primary antibody for two days at and with secondary antibody overnight at 4°C. The primary antibodies used include CD133, Nanog and Vimenin (Gentex) and then exposed to TRITC-conjugated secondary antibodies. With removal of unbound antibody, spheroids images were taken by fluorescence or confocal microscope.. 2.11 Statistical analysis The student’s t-test was applied to evaluate the differences between treated and control animal groups. The statistical analysis was performed by paired Student’s t-test using SigmaPlot 12.0 software. All values were presented as mean ± SE unless otherwise noted. For all the tests, the level of significance was set at *p<0.05 and **p<0.01.. 48.

(50) 3. Results. 3.1 Dose-response growth curves of teroxirone in human hepatocellular carcinoma cells The cell viability of three HCC cells was determined by MTT assay. Huh7, HepG2 and Hep3B cells were treated with teroxirone (2, 5 and 20 μM) for 48 h. As shown in Figure 17, the value of IC50 was indicated that inhibition of cell growth rate in Huh7 cells by teroxirone, but not in Hep3B and HepG2. Thus, concentrations of 2, 5 and 10 μM of teroxirone were used in the subsequent experiments.. 3.2 Teroxirone increases apoptotic population of Huh7 cells by dosedependent appearance of sub-G1 cells Flow cytometry analysis of PI-stained cells revealed that the induced sub-G1 cell population in Huh7 cells was dose-dependent (Figure 18A and B). Furthermore, analysis of Annexin V-FITC/PI doubly labeled Huh7 cells showed that, as the concentration 10 μM following 48 h treatment, population of early and late apoptotic phase were increased to 7% and 23%, respectively (Figure 18C and D). The data implied that the induced apoptotic cell death contributed to the reduced viable Huh7 cells by teroxirone.. 49.