利用非小細胞肺癌細胞以及動物模式探討中草藥之治療潛力

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(1)國立臺灣師範大學生命科學系碩士論文. 利用非小細胞肺癌細胞以及動物模式探討中草藥之治療潛力 Investigation of potential Chinese herbs in non-small cell lung carcinoma in vitro and in vivo. 研究生：韋會妤 Hui-Yu Wei 指導教授：謝秀梅博士 Hsiu-Mei Hsieh, Ph. D.. 中華民國一○二年七月.

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(3) Index. Abbreviation…………………………………………............ 1 Abstract……………………………………………………. 2. Abstract (Chinese)………………………………………...... 3. Introduction……………………………………..................... 4. Material and method………………………………………... 13. Result…………………………………………...................... 20. Discussion………………………………………................... 30. Reference……………………………………….................... 36. Table…………………………………………….................... 43. Figure…………………………………………...................... 47. Appendix……………………………………………………. 74.

(4) ABBREVIATION LIST. Abbreviation. Full name. ALB ALT AST BUN CDKi CDKs CHM CREA CSC EGFR EMT GOT GPT H&E stain HLJDT IC50 NLAC NSCLC SCID TCM TP. Albumin Alanine transaminase Aspartate transaminase Blood urea nitrogen Cyclin-dependent kinase inhibitors Cyclin dependent kinases Chinese herbal medicine Creatinine Cancer stem cell Epidermal growth factor receptor Epithelia-mesenchymal transition Glutamic oxaloacetic transaminase Glutamic-pyruvic transaminase Hematoxylin and Eosin Y stain Huang-Lian-Jie-Du-Tang The half maximum inhibitory concentration National Laboratory Animal Center Non-small cell lung carcinoma Severely compromised immunodeficient Traditional Chinese medicine Total protein. 1.

(5) ABSTRACT Non-small cell lung carcinoma (NSCLC) is lung cancer with the highest incidence and mortality rate in the world. Some Chinese herb medicines (CHM) have been reported to have potential anti-cancer activity through reducing tumor recurrence, angiogenesis, metastasis, and drug-resistance. Our previous studies have identified Formulae 3, 5, and14, showing potential anti-cancer activity in NSCLC cell lines. These formulae were found to have anti-proliferative activity through G1 and G2 arrest toward the A549 cells. We further investigated the effect of these formulae toward another two NSCLC cancer cell lines. Using MTT assay, we have determined the IC50 of Formula 14 in NSCLC H460 and H520 to be 4.5% and 3.8%, respectively. Furthermore, Formula 14 reduced the ability of colony formation, and induced G1 and G2 arrest. In addition, we characterized their therapeutic potential in vivo using mouse xenograft tumor model. We confirmed that all the 3 formula treatment had no notable toxicity for the mice though the pathological examination of mouse kidney and liver. We found that tumor size was decreased by Formulae 3 and 14. Further investigation showed that these formulae may induce G2 arrest and apoptosis to reduce the tumor size. In conclusion, we found that Formulae 3 and 14 showed potential anti-cancer activity from both the in vitro and in vivo analyses.. Key word: NSCLC, CHM, xenograft model, cell cycle, apoptosis. 2.

(6) 摘要非小細胞肺癌在肺癌中具有高發生率與致死率。文獻報導有許多中草藥含抑制腫瘤生長、血管新生和轉移的能力，同時伴隨較少的副作用。我們先前從十五種臨床上常用的中藥複方中，篩檢出三種對非小細胞肺癌細胞具有抑制能力的複方，分別為三號、五號及十四號藥方。已知此三種複方會造成 A549 細胞週期停滯進而抑制細胞的生長。我們更進一步使用非小細胞肺癌細胞株 H460 以及 H520 為細胞模式，評估這三種複方是否對非小細胞肺癌細胞具有一致的抑制效果，並以 A549 細胞株於雄性裸鼠進行腫瘤異種移植動物模式，以評估藥方的抗癌效果。在細胞模式中，我們利用細胞存活率(MTT assay) 檢測十四號複方對非小細胞肺癌細胞株 H460 以及 H520 的半致死率，分別為 4.5% 跟 3.8%。此外，我們也發現十四號複方具有抑制細胞群落形成，以及造成細胞週期停滯在 G1 與 G2 的現象。在異種移植動物模式中，我們投予三種複方後發現三號以及十四號複方可減少腫瘤的大小，以西方墨點轉漬法進一步探討複方藥物處理的結果，我們發現這兩種複方可能藉由將腫瘤內細胞停滯在 G2 細胞週期以及誘導細胞凋亡的發生。本實驗的結果提供細胞以及動物實驗模式的結果顯示三號以及十四號複方可能具有抗腫瘤的潛力。關鍵字: 非小細胞肺癌、中藥、異種移植、細胞週期、細胞凋亡 3.

(7) INTRODUCTION Lung Cancer Lung cancer is a group of uncontrolled cell growing in lung tissues, including bronchus, lung epithelium and peripheral lung tissues. Lung cancer, with high incidence and mortality, has long been the most deadly cancer disease. Based on the statistics in WHO, lung cancer is the 3rd leading cause of death all over the world, especially in the high-income countries, and it causes 1.3 million deaths every year(Murray and Lopez, 1997). According to the data of the Department of Health, Executive Yuan, R.O.C, lung cancer causes 8541 Taiwanese to dead, which is almost 20.1% of death, and also the 1st deadly cancer disease (DOH, 2011).. Classification of lung cancer Most of the lung cancers are derived from the alternation of epithelial cells of lung tissues. Lung cancer could be at least divided into two groups by their different pathologies, size and appearance of the tumor cells, small-cell and non-small cell lung carcinoma (NSCLC), which account for about 16.8% and 80.4% of all kinds of lung carcinoma respectively (Travis et al., 1995).NSCLCs can also be classified into adenocarcinoma, squamous cell lung carcinoma, and large cell lung carcinoma in detail by where they originate from (Travis, 2002). In 2004 WHO also reported several new types of non-small cell carcinoma, including sarcomatoid carcinoma, salivary gland tumor, carcinoid tumor and adenosquamous carcinoma. In contrast, small-cell lung carcinoma is 4.

(8) more sensitive to chemotherapy and radiotherapy, and strongly associated with smoking (Herbst et al., 2008).. Etiology of lung cancer There are many factors that may cause lung cancer disease, including smoke, secondhand smoke (Moritsugu, 2007), radon gas (Catelinois et al., 2006), air pollution (Chiu et al., 2006; Coyle et al., 2006; Kabir et al., 2007), asbestos (O'Reilly et al., 2007), and genetic factors (Hackshaw et al., 1997). Cigarette , containing more than 60 known carcinogens (Hecht, 2003), has been considered to be a major cause of lung cancer (Biesalski et al., 1998). Furthermore, nicotine and its derivative can activate immune activity or promote tumorigenesis (Sopori, 2002; Lin et al., 2010). However, studies show that people with passive smoking will suffering higher incidence of lung cancer than nonsmokers. Radon gas was considered as the 2nd leading cause of lung cancer (Catelinois et al., 2006), which was generated by decaying of radioactive radium. The radon gas causes mutation by ionizing radiation. It could ionize DNA causing DNA damage and mutation which will further result in tumorgenesis. Asbestos is another material which can cause lung cancer and has been reported to have a synergistic effect on lung cancer formation with cigarette smoke (O'Reilly et al., 2007). Virus ,like human papillomavirus (Cheng et al., 2001), John Cunningham virus (Zheng et al., 2007), and cytomegalovirus (Giuliani et al., 2007), can also result uncontrolled cell growth from interfering cell cycle.. 5.

(9) Symptoms and treatment of lung cancer Lung cancer result in coughing, loss of weight, and shortness of breath (Hamilton et al., 2005). Nevertheless, it is not easy to have a specific signs or symptoms in the beginning of onset. Patients were diagnosed to have lung cancer accidentally, due to their regular physical examination, and it is usually found in an advance stage, which means the cancer may have already spread out to other sites in their body in many cases.. Lung cancer can be classified by its primary tumor size (T), regional lymph node metastasis (N) and distant metastasis (M), which is also called the TNM system. Lung cancer is divided into five stages at least in clinical diagnosis, 0, I, II, III, and IV, which describe the physical condition of patients. Different approaches or combinatory treatments will be performed by different stages or types of lung cancer. For instance, solid tumors on early stage, with no metastasis, are easily to localize to have surgery, however, some reports show that it would readily relapse recurrence. Radiotherapy and chemotherapy are used on advanced stage of lung cancer. Cisplatin as an example, it forms a platinum complex inside of a cell which binds to DNA and cross-links DNA. When DNA is cross-linked in this manner, it causes the cells to undergo apoptosis. The causes of apoptosis through cross-linking is by damaging the DNA so that the repair mechanisms for DNA are activated, and once the repair mechanisms are activated and the cells are found not salvageable, the death of those cells is triggered instead. In addition, this drug gives 6.

(10) paitient some side effects including diarrhea, vomiting, renal toxicity and neurotoxicity (Albers et al., 2011).Target therapy arises because of some scientific progress, which may specifically target to some specific molecular, has been used on clinical treatments. Gefitinib (also called Iressa® ), for example, can inhibit epidermal growth factor receptor (EGFR) by targeting the tyrosine kinase domain (Wakeling et al., 2002), which has been used on many cases of lung cancers that overexpressing EGFR (Herbst et al., 2008).Target therapy is more effective and less side effects than current treatment concerned recently. However, drug-resistance is also one problem need to be sloved for this type of treatment.. There are lots of therapeutic methods toward lung cancer diseases, however, none of them provide radical treatment until now. Therefore, it is still a very important problem on developing new therapies toward lung cancer.. Chinese herbal medicines Traditional Chinese medicine (TCM), including lots of traditional medical methods, like acupuncture, Tuina, etc., has prevailed throughout East Asia for centuries. Based on these traditional medical methods, Chinese herbal medicine (CHM) is one of the most important therapeutic modality of internal medicine. Chinese herbology made from variety of ingredients including plants, animals, minerals and marine materials. Products of different herbs are divided to cold, cool, warm and hot nature 7.

(11) groups, which represent the degree of yin and yang, in Chinese it is representing cold and hot. A formula is made by CHMs, and may contain more than 3 herbs. Generally there will be only one or two major ingredients to treat the disease and the other herbs adjust the yin and yang to balance the patient’s physical conditioning.. Lung cancer and TCM theory In East Asia CHMs have been used to treat lung cancers for several centuries. There are around 130 CHMs have been reported to have anti-lung cancer ability (Liu and Peng, 1994) and about 40 CHMs (Koops et al., 1998) and 30 formulae have been frequently used for lung cancer patients (Guo and Song, 2002). CHMs use with chemotherapies or radiotherapies to reinforce patients’ immune activity and ameliorating prognosis. The use of CHMs as therapeutic approaches is very common in East Asia, though the specific mechanisms of how CHMs affects the disease still remain largely unknown.. In the previous studies, herbs use to treat lung cancer in vivo and in vitro model were conducted to evaluate how the herbs affect lung cancers. Plumbagin, phenylbutenoid, and parthenolide had been demonstrated to inhibit proliferation of NSCLC cells A549 (Hsu et al., 2006; Lee et al., 2007; Parada-Turska et al., 2007). Cantharidin, curcumin, and gambogic acid can induce apoptosis of A549 cells (Zhang et al., 2005; Chen et al., 2010; Li et al., 2010). Especially gambogic acid is in potential as an excellent cytotoxicity against a variety of malignanttumors, as well as 8.

(12) cancers of the breast, lung and liver (Gu et al., 2008; Qi et al., 2008; Qiang et al., 2008). Ginsenoside inhibits the potential of metastasis toward in lung cancer (Park et al., 2008). However, most studies focus on the effects of one single herb toward cancer cells, not multiple herb formula which patients actually taken according to the prescription.. We obtained CHM formulae which are frequently used for lung cancer patients from Yu-Sheng Chinese Medical Clinic. Our previous study show that three formulae (Formulae 3, 5 and 14) substantially decreased the survival of A549 cells but did not affect MRC5 normal lung tissue cells (Wang et al., 2012). In this study, we used both in vivo and in vitro models to investigate the mechanism of these formulae toward NSCLC.. Cell cycle machinery In cancer development cell cycle plays an important role which induced uncontrolled cell growth. Lots evidences indicate that there is high correlation between cancer development and abnormal expression of cell cycle regulatory genes. Eukaryotic cell cycle is divided into G1, S, G2 and M phase during cell cycle processing which the cell splits itself into two distinct cells.. Two main protein families direct affect the cell cycle progression in cell cycle machinery are cyclins and cyclin dependent kinases (CDKs). Cyclin proteins possess a homology region called cyclin box which binds 9.

(13) CDKs to become an active complex (Murray, 2004). Different combinations of cyclins and CDKs of cell cycle drive cells to go on to next phase. For instance, cyclin D/CDK4 or cyclin D/CDK6 promote G1 phase to S phase. This transition is made by phosphorylating retinoblastoma protein (Rb), which detach Rb from transcription factor E2F then activate the transcriptional activity of E2F (Blagosklonny and Pardee, 2002). In addition, cyclin B/CDK1 is an essential check point protein, which checks cell can entry into M phase and is involved in mitosis. Cylin B/CDK1 complex phosphorylates and activates cohesion to help condense chromosomes (Kimura et al., 1998). Moreover, phosphorylate nuclear laminin to break down the nuclear envelope allowing formation of mitotic spindle (Heald and McKeon, 1990). In addition to these cell cycle regulators, there are also cyclin-dependent kinase inhibitors (CDKi) which negatively regulate the CDK activities. P15 (CDKN2B) can bind to CDK4 or CDK6 to prevent the activation of cyclin D (Khleif et al., 1996). We therefore performed western blot to identify whether the cell cycle machinery was affected under the herb formulae treatment.. Epithelia-mesenchymal transition (EMT) Epithelial and mesenchymal describe different status of cell morphology. Transition of epithelial to mesenchymal were usually connected with disease developmental process (Thiery, 2003). Generally, cells in mesenchymal state were more likely involved in tissue repair and wound healing. In the other hand, epithelial cells are usually regarded as 10.

(14) barrier cells with adherence and tight junctions. Recently, the trans-differentiation programs are concerned in cancer studies by their specific character, for example, cancer cells which are mesenchymal look loosely, and have potential on invasion of extracellular matrix. In contrast, epithelial cancer cells performed tight connection with each others, with relatively small leak in the migration (Acloque et al., 2009).. It was also suggested that EMT could induce the capacity of renewal and maintenance of cancer cells by several studies (Huber et al., 2005; Hermann et al., 2007; Mani et al., 2008; Peacock and Watkins, 2008). In this study, we also investigate whether HCs induce the mesenchymal to epithelialtrans-differentiation during the cancer stem cell inhibition.. Xenograft tumor model Numerous murine models have been developed to study human cancer.. Transplantation models offer another strategy for cancer researchers. These methods include several systems and techniques to disseminate tumor tissues in different hosts for controlled studies in vivo.Some of these methods have been used for decades and are well-established models. These models are used to investigate the factors involved in malignant transformation, invasion and metastasis, as well as to examine response to therapy. One of the most widely used models is the human tumor xenograft. In this model, human tumor cells are transplanted, either under the skin or into the organ type in which the tumor originated, into immunocompromised mice that do not reject human cells. For example, 11.

(15) the xenograft will be readily accepted by athymic nude mice, severely compromised immunodeficient (SCID) mice, or other immunocompromised mice(Morton and Houghton, 2007).Depending upon the number of cells injected, or the size of the tumor transplanted, the tumor will develop over 1–8 weeks (or in some instances 1–4 months, or longer), and the response to appropriate therapeutic regimes can be studied in vivo. There are several key advantages of using human tumor xenografts to examine therapeutic responses to drugs: (1) one can use the actual human tumor tissue, featuring the complexity of genetic and epigenetic abnormalities; (2) human tumor xenografts can be used to aid in the development of individualized molecular therapeutic approaches; (3) results can be obtained in a matter of a few weeks from a human tumor biopsy regarding response to therapy; (4) xenografts using NOD/SCID mice that have been ‘humanized’ by injection of peripheral blood or bone marrow cells, allow for an almost complete reconstitution of the immune response to the tumor. Xenografts using human cell lines to test drug responses do not often correlate with clinical activity in patients(Kerbel, 2003).. 12.

(16) MATERIAL AND METHOD Cell culture NSCLC cell lines A549, NCI-H460 and NCI-H520 were obtained from Bioresource Collection and Research Center (BCRC, Taiwan). A549 is a human lung adenocarcinoma cell line, with the adhesion of dish and variant cell morphology. They were maintained in Ham's F12K (GIBCO, Carlsbad, CA, USA) containing 10% Fetal bovine serum (FBS, GIBCO) and 10000 units Penicillin and 10000 µg Streptomycin (GIBCO). NCI-H460 (H460) is a human large cell lung cancer cell line, established from the pleural effusion tumor tissues (large cell carcinoma) (Banks-Schlegel et al., 1985). NCI-H520 (H520) is a squamous cell carcinoma cell line, which was also established in 1985 (Banks-Schlegel et al., 1985). Both of H460 and H520 were maintained in 85% RPMI1640 (GIBCO) supplemented with 5% FBS and 10% horse serum (HS), containing 2 mM L-glutamine, 10 units Penicillin and 10 µg Streptomycin, 1 mM sodium pyruvate (GIBCO), 4.5 g/L D-glucose, 1.5 g/L sodium bicarbonate, 1 M HEPES (Sigma, St. Louis, MO, USA). All cultures were maintained in 37℃ 5% CO2 incubator (Thermo forma 370, Waltham, USA.), and culture medium was changed every 48-72 hr.. Chinese Herbal decoctionspreparation Herbal decoctions were prepared by TCM clinics. Formulae were boiled in 1.2 L water for 1 hr to get the final decoction (around 450 ml) and then aliquoted and stored at 4℃. Before experiments, decoctions were filtered with 0.22 μm (in vitro) or 3 mm (in vivo) membrane to 13.

(17) remove insoluble particles.. Cell viability assay (MTT assay) Different amount of herbal decoctions were used to investigate the dose response of different cell lines. We adjust the dosing volume to 10% v/v with ddH2O to prevent the dilution effect of medium, while 10% v/v ddH2O was used in the control group. A549, H460, and H520 cells were seeded at 1×104, 6×103, 4×104 cells respectively per well in a 24 well-plate to ensure cells grow logarithmically during total treatment period. Twenty-four hours after seeding, different decoctions were directly added into the 24 well-plate and the plate was incubated in 37℃ incubator before MTT assays.. After treatment for 72 hr, 50 μl of thiazolyl blue tetrazolium bromide (MTT, m5655, Sigma) were added into each well of the 24-well-plate and further incubated the plate in 37℃ for 2 hr. The plates were then move out from incubator and the medium was discarded. Five hundred μl of DMSO was then added into each well to dissolve the purple black formazan, the products of MTT under the reaction of dehydrogenase in mitochondria of live cells. Vigorous pipetteing was performed to fully dissolve the formazan, and then 200 μl of formazan solution was transferred into a 96-well ELISA plate. OD570 absorption was detected with Microplate Autoreader EL311 (uQuant, Bio-Tek Instruments Inc, Winooski, USA.).. 14.

(18) Cell cycle analysis Cells were cultivated in 6-cm plate for 24 hr and then treated with different formulae. Cells were harvested by trypsinization at different time course (24, 48 and 72 hr, respectively) and fixed with cold 70% EtOH at -20℃ overnight after PBS wash once. Excess EtOH was removed by PBS wash in the next day. PI staining buffer (PBS : RNase (10μg/ml, R6513, Sigma) : PI (1μg/ml, P4170, Sigma) = 97 : 1 : 2) 1 mL was added into 1×106 cells to guarantee the DNA staining was fully saturated. Incubate cells with the PI staining buffer for 30 mins at 37℃ in dark with frequently shaking. Cells were thieved with 35 μm nylon mesh into round bottom tubes to avoid cell clumps and analyze with FACSCalibur (BD Biosciences, San Jose, USA) as soon as possible. Single cell gate was set to exclude aggregated cells. Fifteen thousand cells per sample were collected to show cell cycle distribution. Percentages of different cell cycle phases were calculated with Mofit software (Verity Software House). Western blot Cells and tumor tissues were scraped and lyzed with RIPA buffer [(10 mM Tris (pH7.5), 150 mM sodium chloride, 5 mM ethylenedinitrilotetracetic acid (pH8.0), 0.1% SDS, 1% sodium deoxycholate, 10% NP40], 1% protease inhibitors (Thermo Scientific, Rockford, IL) and 1% phosphatase inhibitor (Sigma) following by centrifuge at 13300 g for 30 min at 4℃ to spin down the cell debris. Protein-containing supernatants were collected and stored at -80℃ until use. Total protein concentration was quantified with BCA protein assay 15.

(19) kits (23250, Thermo Scientific) according to manufacturer’s instruction. Ten μg protein of each sample was electrophoresed with 10% SDS-PAGE and then electrotransfered onto nitrocellulose membrane at 400 mA for 1 hr. Blot membrane was blocked with 5% nonfat dry milk in TBST (Tris Buffered Saline with 0.1% Tween-20) for 1 hr at room temperature. At least three times wash with TBST were performed to wash out excess milk. Then the PVDF membrane was incubated with proper diluted primary antibody overnight at 4℃ with frequent shaking. Corresponding HRP-conjugated secondary antibody was applied to the membrane incubating for 1 hr after three times wash with TBST. The membranes were washed in TBST three times again to remove excess antibody. ELC substrate (WBKLS0050, Billerica, Millipore) was added onto blot membrane to show the hybridization pattern and the images were acquired with Fuji LAS-4000 mini imaging system (GE Healthcare). Blot images were quantified with Gelpro 31 software (Media cybernetics). Xenotransplantation and herbal treatment in vivo Male athymic nude (CAnN.Cg-Foxn1nu/CrlBltw, BALB/cnu/nu ) mice were obtained from BioLASCO Taiwan Co., Ltd. All animals were maintained in individually ventilated cages (IVC, LASCO, Taiwan) on a 12 hr light/dark cycle. Food and water were autoclaved. A549 cells (7×106) in 75 μL PBS was mixed with 25 μL matrigel (356234, BD) and injected subcutaneously into the right flank of mice. Tumor-bearing mice was divided randomly into 4 groups, with each group having 10 mice. After the solid tumor volume has reached about 100 mm3, each mouse was treated with Formula 3, 5, 14 or water by gavage daily for 4 weeks. 16.

(20) After tumor xenograft transplantation, mouse tumors were monitored and tumor size was measured every 7 days using a Vermier’s caliper (Mytutoyo Co., Japan) across its two perpendicular diameters. The solid tumor volume was calculated by the following formula: tumor volume (mm3) = 0.5 × (W2×L) (where W is the smaller perpendicular diameter and L is the larger perpendicular diameter).. Blood biochemistry analysis Blood biochemistry was used to determine physiological and biochemical condition of animals. Here we used it for drug toxicity evaluation. We first conducted blood collection from orbital sinus of mice. This method consistently yielded less blood volume (0.2–0.5 mL). Therefore we performed the blood collection through cardiac puncture. Briefly, we first inserted a 21 gauge needle into the mouse ventricle and slowly drew out the mouse blood. This method consistently yielded a higher blood volume (0.5-0.7 mL). Blood collected was stayed at room temperature for 30 min to 1 hr. The whole blood was then centrifuged at 13300 g at 4℃for 5 min to obtain the serum-containing supernatant. Serum was kept at -80℃until shipping to National Library Animal Center for biochemical examination.. Tissue sampling Immediately after the tumor was dissected out from the mouse, kidney and liver tissues was immediately fixed in 4% buffered para-formaldehyde (PFA, P6148, Sigma) for 16 hr. Further tissue 17.

(21) processing and embedding was performed by conventional tissue processors from National library animal center with the following programming: 2 hr 4% PFA, dehydrating ethanol series for 30 or 60 min, isopropanol for 2 hr and xylene for 3 hr. The tissue was kept in paraffin wax for at least 4 hr until manual paraffin embedding. The paraffin-embedded blocks were stored at room temperature until further sectioning.. Hematoxylin and Eosin staining Three μm sections were cut from each paraffin-embedded block. Hematoxylin and Eosin Y stain (H&E stain) method is used in histology for displaying morphological features. The staining consisted of sequential incubations of slides at 60℃for 1 min to melt paraffin, and then dewaxing in 100% Xylene for 3 min twice. 100% EtOH for 3 min twice, 95% EtOH for 10 sec, 70% EtOH for 10 sec, 50% EtOH for 10 sec respectively. Before stained by hematoxylin for 4 min 20 sec, slides were washed for 1 min to remove the EtOH. After stained with Eosin Y, slides were rehydrated in increasing EtOH concentration, 50% EtOH for 1.5 min, 70% EtOH for 1.5 min, 95% EtOH for 1.5 min, 100% EtOH for 2.5 min twice respectively. At least, slides were immersed in 100% Xylene to completely remove the hydrate. The tissue histology were examined by NLAC.. Statistic analysis In vitro and in vivo data were presented as mean ± SEM. One way 18.

(22) anova was performed with SPSS software to evaluate the significance. The p-value < 0.05 was considered to be significant.. 19.

(23) RESULT Effects of CHM formulae on the NSCLC viability In our previous study, we found that Formulae 3, 5, and 14 decreased the survival rate of NSCLC A549 cells but not significantly affected the viability of non-tumor MRC5 cells (Wang et al., 2012). These three formulae were chosen to investigate their survival inhibitory effect on other NSCLC cell lines. To investigate whether these potential Chinese herb directly induce cytotoxicity of another two NSCLC cell lines H460 and H520. We applied different concentration of these formulae to the cells and performed the MTT assay to examined the cell viability after a 72 hr treatment. As shown in Figure 1, Formula 3 significantly decreased the viability of H460 cells. The half maximum inhibitory concentration (IC50) of Formula 3 calculated is 5% (v/v) in H460 cells (Fig. 1A). In addition, Formula 14 significantly decrease the cell viability of both H460 and H520, and the IC50 are 4.5% (v/v), and 3.8 % (v/v) for H460 and H520, respectively (Figs.1B and 1C).. Effects of CHM Formula 14 on the inhibition of H460 cell colony formation We also conducted colony forming assay to determine whether Formula 14 could affect the tumorigenesis ability of H460 cells, and the results showed that the number of colonies after cultured for 14 days was significantly reduced in cells with Formula 14 compared to that of the control group (Figs. 1D and 1E). In addition, no colony could be formed in H520 cells with the same experimental condition (data not shown). 20.

(24) Formula 14 reduced H520 proliferation without inducing massive cell death events Cell death resulted from the Formula 14 treatment could only explain a marginal portion of the decline of survival rate. We thus conducted cell cycle analysis to identify the possible reason to cause the reduction of cell survival rate. In accordance with the time course response of MTT assays, the proliferative population (S phase) was significantly decreased and G2/M phase population was significantly increased under the treatment of Formula 14 for 24 hr (Figs 2A-B). These results indicate that the formula treatment reduced the proliferation rate of H520 cells.. In addition, S phase cell population was significantly decreased and G0/G1 phase population was significantly increased under the treatment of Formula 14 for 48 hr (Figs 2A-B). Moreover, G0/G1 phase population was significantly increased and S and G2/M phase populations were significantly decreased under the treatment of Formula 14 for 72 hr (Figs 2A-B). These data further confirmed the retardation of H520 cell cycle progression owing to the treatment of Formula 14.. Expression level of cell cycle regulators were altered after Formula 14 treatment To further confirm the results of cell cycle analysis, we examined the expression levels of several cell-cycle regulators. We found that G1/S transition promoting protein, including D-type cyclin dependent kinase 21.

(25) CDK4, and the substrate of cyclin D/CDK4/6 complex, phosphorylated Rb, were all decreased significantly after Formula 14 treatment (Figs. 2C, 2D and 2E). These results revealed that these cells are under G1 arrest, which are consistent to the cell cycle profiling identified from the flow cytometry analysis. Cyclin B1was also checked to ensure the situation of G2/M transition. We found that cyclin B1 was substantially decreased after Formula 14 treatment (Fig. 2f). All these results confirm the data obtained from cell cycle analysis.. Establishment of NSCLC xenograph tumor model Our previous studies have identified three of the fifteen Chinese herb formulae, Formulae 3, 5, and 14, showing potential anti-cancer activity in NSCLC cell lines. These formulae were found to have anti-proliferative activity through G1 and G2 arrest toward the A549 cells. Based on the data from the cell model, we characterized their therapeutic potential in vivo using xenograft model. When the tumor size reached to 100 mm3, each mouse was treated 400 μL Formulae 3, 5, and 14, or water (Fig. 3A). This dose applied to the mice was the same as the clinical prescription, 800 mL per day for patients weighting 60 kg. We found the tumor size was decreased after Formula 3 treatment for 7 days. After Formula 3 and Formula 5 treatment for 14 and 21 days, tumor size was reduced than water treatment group (Figs. 3B and 3C). Moreover, there was no difference in body weight among these groups (Fig. 3D). In addition, blood biochemistry analysis was conducted to investigate the cytotoxicity effect of formulae. Creatinine (CREA) was an important indicator of 22.

(26) renal health because it was an easily-measured by-product of muscle metabolism (Taylor et al., 1989). CREA was mainly filtered out of the blood by the kidneys based on glomerular filtration and proximal tubular secretion. Blood urea nitrogen (BUN) was a waste from the urea cycle of the digestion of protein. If the filtering of the kidney was deficient, CREA and BUN levels will be increased in the blood. Therefore, CREA and BUN levels in blood could be used as markers of kidney function. From our result, we didn’t find any difference in CREA, BUN, total protein (TP), and Albumin (ALB) among different groups. This indicated that this concentration of formulae applied to the mice had no notable side effect on kidney (Figs. 4A - 4D).. Evaluation of acute toxicity of high concentration treatment on mice Our studies have shown that 400 μL Formulae 3, 5, and 14 by gavage daily for 4 weeks had no significant effect on tumor size regression. Therefore, we intended to conduct a treatment with higher concentration of formulae. We first investigated the toxic effect of high dose formulae in liver and kidney. Eight weeks old mice were treated with 900μL of formulae by gavage twice a day for 14 days. There was no difference identified in body weight among these groups (Fig. 5B).. Mouse tissues were examined by the help of pathologist in NLAC. Two types of inflammation cells (mononuclear and neutrophilic) were identified in livers of control group (Fig. 6A). Inflamatory cells were found in both Formula 3 (Fig. 6B) and Formula 5 (Fig. 6C)-treated mouse 23.

(27) livers. In addition, focal necrosis was also present in liver after Formula 14 treatment (Fig. 6D). Minimal to slight infiltrates of inflammation cells occasionally with necrosis were sometimes found in mice. The lesions were observed in both control and experimental groups and no other significant lesions were identified in each group.. Moreover, in the kidney pathology examination, normal renal tissues were shown in control (Fig. 7A), Formulae 3 (Fig. 7B), and 14 (Fig. 7C) mice. Tubular dilation with hyaline cast was found in the kidney after Formula 5 treatment (Fig. 7D). Hyaline casts are typically composed of proteins, which associate with increased proteins in the glomerular filtrate. To sum up the liver and kidney pathology report, high concentration of Formulae 3, 5, and 14 used in this study showed no significant toxicity in animals. The results from microscopic observation were presented on Table 2. A histopathological evaluation was performed in submitted section. Severity of lesions was graded according to the methods described by Shackelford (Shackelford et al., 2002). Degrees of lesions were graded histopathologically from one to five depending on severity: 1= minimal (< 1%); 2= slight (1-25%); 3= moderate (26-50%); 4= moderately severe (51-75%); 5= severe/high (76-100%).. Effect of high concentration treatment on NSCLC xenograph tumor model After confirmed that high concentration of formula treatment had no noteable toxicity for the mice, we thus conducted the treatment with 24.

(28) higher concentration of formulae on the mice with xenographed tumor. To reduce the volume of formula for gavage feeding, we used freeze-drying method to concentrate the original formulae into a 4-fold concentration. We then gave mice 250 μL the concentrated Formula by gavage daily for 4 weeks.. Using the same protocol, we treated the mice with Formulae 3, 5, and 14, or water (vehicle control group) when the tumor size reached to 100 mm3 (Fig. 8A). There was no difference in body weight among these groups (Fig. 8B). We found the tumor size was decreased after treatment with Formulae 3 and 5 for 14 days. After treatment Formula 3 and 5 for 21 and 28 days, tumor size was reduced than water treatment group (Fig. 8C). In addition, blood biochemistry analysis was conducted to investigate the cytotoxicity of Formulae. Aspartate transaminase (AST), also called aspartate aminotransferase or serum glutamic oxaloacetic transaminase (GOT), was a pyridoxal phosphate (PLP)-dependent transaminase enzyme. AST catalyzed the reversible transfer of an α-amino group between aspartate and glutamate and, as such, was an important enzyme in amino acid metabolism. AST was found in the liver, heart, skeletal muscle, kidneys, brain, and red blood cells, and it was commonly measured clinically as a marker for liver health. Alanine transaminase (ALT) was a transaminase enzyme. It was also called serum glutamic-pyruvic transaminase (GPT) or alanine aminotransferase. ALT was found in plasma and in various bodily tissues, but was most 25.

(29) commonly associated with the liver. From our result, we didn’t find any difference in ALT, AST, CREA, BUN, total protein (TP), and Albumin (ALB) among different groups. This indicated that this concentration of formulae applied to the mice had no notable side effect on kidney (Figs. 9A – 9F).. Mouse liver and kidney pathologies were also examined by the help of pathologist in NLAC. The normal liver tissue is shown in Fig. 10A. Perivascular inflammation was also identified in control mice (Fig. 10B). Slight necrosis with mononuclear and neutrophil infiltration was shown after Formula 3 treatment (Fig. 10C). Perivascular inflammation was also found in Formula 5-treated mice (Fig. 10D). Formula 14-treated mouse liver showed focal necrosis (Fig. 10E). Randomly distributed aggregates of mononuclear cells (primarily lymphocutes) without any accompanying hepatocyte degeneration or necrosis are common in animal. However, the lesions were observed in both control and treatment groups and no other significant lesions were identified in any group.. Moreover, for the kidney pathology examination, normal renal tissue were found in control (Fig. 11A), and mice treated with Formulae 3 (Fig. 11B), 5 (Fig. 11C), and 14 (Fig. 11D).To sum up the liver and kidney pathology examination, high concentration of Formulae 3, 5, and 14 showed no significant toxicity on the mouse health. The results from microscopic observation were presented on Table 3. A histopathological evaluation was performed in submitted section. Severity of lesions was 26.

(30) graded according to the methods described by Shackelford (Shackelford et al., 2002). Degrees of lesions were graded histopathologically from one to five depending on severity: 1= minimal (< 1%); 2= slight (1-25%); 3= moderate (26-50%); 4= moderately severe (51-75%); 5= severe/high (76-100%).. Expression level of cell cycle regulators were altered under high concentration formula treatment on NSCLC xenograph tumor model We investigated the expression of cell cycle regulatory proteins to uncover more clues in explaining the formulae effects in vivo. With the result of cell cycle analysis, the expressions of G1/S transition promoting proteins were increased under Formula 3, 5, 14 treatment, including cyclin D1 (Fig. 12A), D-type cyclin dependent kinase CDK4 and CDK6 (Fig. 12B and 12C), the substrate of cyclin D/CDK4/CDK6 complex, thus leading to G1phase transition to S phase. We also checked G2/M transition regulating proteins, cyclin B1 (Fig. 12D) and cdc2 (cdk1). All three formulae increased the expression of cyclin B1 and the phosphorylation of cyclin B dependent kinase cdc2 at Tyr15 (Fig. 12E), but not the phosphorylation of cdc2 at Thr161 (Fig. 12F). Phosphorylation of cdc2 at Tyr-15 negatively regulates the activity of the cdc2/cyclin B complex (Parker and Piwnica-Worms, 1992; McGowan and Russell, 1993), whereas Thr-161 phosphorylation is essential for kinase activity (King et al., 1994; O'Leary et al., 1998). The result of cell cycle analysis showing the down regulation of cdc2/cyclin B complex might explain the G2 phase arrest effect of Formula 3. 27.

(31) Formulae effect on the expression level of tumor suppressor proteins and oncoproteins A tumor suppressor gene, or anti-oncogene, is a gene that protects a cell from one step on the path to cancer. Recently, a family of low molecular weight cyclin-dependent kinase inhibitors had been shown to play essential roles in arresting cell cycle progression. These CDK inhibitors, which include p21, p27, and p16, physically associate with their target cyclin-CDK complexes to inhibit their activities (Harper et al., 1993; Xiong et al., 1993; Dulic et al., 1994). We examined the expressions of tumor suppressor proteins, p15 (Fig. 13A), p21 (Fig. 13B), and p53 (Fig. 13C). All three Formulae do not alter the expression of p15 but increased the expression of p21 and p53. On the other hand, we also examined the expression of proto-oncogene, c-Myc and c-Jun, after formula treatment (Figs. 13D-E). The results show that c-Myc was notably increased and c-Jun was not altered after the treatment.. High concentration formula treatment induced apoptosis rather than autophagy on NSCLC xenograph tumor model We investigated the effect of formulae on the expression of pro-apoptotic proteins. From the result of western analysis, we found the expressions of apoptotic proteins were up-regulated under Formula 3, 5, 14 treatment, which included Bax (Fig. 14a), and cleaved-caspase 3 (Fig. 14b), especially significantly under the treatments of Formulae 3 and 5. In the other hand, anti-apoptotic protein, Bcl-2 was decreased after all the 28.

(32) three Formulae treatment (Fig. 14c). These data reveal that the Formula treatment induced tumor cell apoptosis. Moreover, we also analysis the autophagy associated proteins, mammalian target of rapamycin (mTOR), which inhibited autophagy pathway (Schmelzle and Hall, 2000), and Beclin 1, which is required for the initiation of the formation of the autophagosome (Kihara et al., 2001). The expression of both mTOR and Beclin 1 were not notably altered after treatment (Figs. 14d and 14e). These results suggest that tumor regression might be resulted from formulae treatment induced apoptosis rather than autophagy.. Formula treatment effect on the xenograph cancer stemness We then evaluated whether treatments of these formulae have any effect on lung cancer stem cell (CSC) related marker, ALDH1(Jiang et al., 2009), β-catenin (Ireland et al., 2004), Sox2(Xu et al., 2013), vimentin and ABCG2 (Sung et al., 2008) , as shown in Figures 15, Formulae treatment did not significantly alter the expression levels of all these 5 markers. It indicated that these three Formulae can not influence xenograph cancer stemness under the treatment concentration.. 29.

(33) DISCUSSION Our previous study had identified that Formulae 3, 5, and 14 could substantially reduce the proliferation of NSCLC cell lung A549 (Wang et al., 2012). In this study, we further identified that formulae 3, 5, and 14 could also inhibit the proliferation of NSCLC cell lines H460 and H520. As shown in Fig. 16, substantial decline of the expression levels of G1/S regulatory proteins was identified by western blot analysis and which is in accordance with the G1 arrest results obtained by the cell cycle analysis after treatment of Formula 14. From the analysis of G2 regulatory protein expression, we found cells treated with Formula 14 were also arrested in G2 phase. Formula 14 was also found to inhibit colony formation of H520. The results discussed showed that Formula 14 could inhibit tumorigenesis.. After the formulae effect was identified in vitro, we developed a mouse NSCLC xenograph tumor model to further characterize these formulae. In the in vivo experiments, oral administration of Formulae3, 5, and 14, 400 μL daily for 28 days, did not cause significant delay in tumor growth.. Side effect profiles such as changes in weight and toxicity levels of the formulae were tested on mice, and minimal side effects were observed. Therapeutic potential of higher doses of the formulae were evaluated by using the xenograph tumor model. Before increasing the formula concentration, an acute toxicity test was conducted with 4.5 folds daily amount of formulae for 2 weeks on the nude mice. No notable toxicity on 30.

(34) these mice was observed. We then did a second test using an increased formula dose of 2.5 folds. Both Formulae 3 and 14 reduced tumor growth significantly, and the tumor growth curve was similar in Formulae 3 and 14. On the other hand, Formula 5 treatment showed no effect on tumor growth when compared to the control group. These data are consistent to the results from western blot analyses of some specific markers related to the tumor growth. One of the possible explanation is that Formulae 3 and 14 could reduce the tumor size in xenograph mouse model and that the two formulae were not toxic in the high dose and they had the ability to inhibit cell cycle progression, mainly an effect on G2/M arrest.. Formulae 3 and 14 further demonstrated to be able to increase the expression level of tumor suppressor proteins, which cause repressive effect on the regulation of cell cycle or promote apoptosis (Tammemagi et al., 1999; Sherr, 2004).First of all, tumor suppressor p53 expression was up-regulated after the treatment of Formulae 3 and 14.The tumor suppressor gene p53 played a major role in regulating the mammalian cellular stress response, in part through the transcriptional activation of genes involved in cell cycle control and apoptosis (Leonard et al., 1995). In cell cycle control, p53-regulated genes participate in the regulation of G2 arrest. Inducible systems have demonstrated that p53 overexpression can result in G2 arrest, and that prolonged overexpression of p53 could down-regulate cyclinB (Agarwal et al., 1998; Badie et al., 2000). In addition, after formula treatment, the upregulation of p21 expression showed a similar trend as p53. Cyclin-dependent kinase inhibitor p21 is 31.

(35) required or a sustained G2 arrest after activation of the DNA damage checkpoint(Li et al., 1994).Previous studies had addressed the mechanism by which p21 can contribute to this arrest in G2. They showed that p21 blocks the activation phosphorylation of cdc2 on Thr161 (Yun et al., 1999; Smits et al., 2000). Our results also showed that cdc2Tyr 25 was phosphorylated which may inactivate cyclin B, but did not cause G1 phase arrest.. Furthermore, we also investigated whether the oncoprotein expressions were altered after formula treatment. Studies suggested that the absence of c-Jun results in elevated expression of the tumor suppressor gene p53 and its target gene, the CDK inhibitor p21, whereas overexpression of c-Jun represses p53 and p21 expression and accelerates cell proliferation (Schreiber et al., 1999). In a study of NSCLC, c-Jun was found to be overexpressed in 31% of the cases in primary and metastatic lung tumors (Szabo et al., 1996).. The role of c-Myc was not clear-cut as this proto-oncogene has been demonstrated to be a potent inducer of both apoptosis and cellular proliferation, suggesting that these processes are mechanistically related (Evan et al., 1995). Inappropriate expression of c-Myc and bcl-2 genes also modulated the function of p53 and triggered cell proliferation (Ryan et al., 1994). Up-regulation of Bax protein expression and induction of apoptosis was modulated by c-Myc overexpression (Mitchell et al., 2000). Our data showed that Formulae 3 and 14 increased c-Myc levels 32.

(36) significantly and decreased c-Jun levels.. On the other hand, several studies indicated that sequence specific transactivation was a required function for p53-mediated apoptosis in some experimental systems (Sabbatini et al., 1995; Attardi et al., 1996). Further investigation would be required to determine the mechanism by which these genes contribute to the control of apoptosis, but some p53-regulated genes played known roles in the apoptotic pathway. For instance, Bax is a p53-inducedmember of the Bcl-2 family of apoptosis promoting and preventing factors (Miyashita et al., 1994; Swisher et al., 1999; Pearson et al., 2000; Wu et al., 2012). Leading to the idea that the relative level of these two proteins in a stressed cell determines life or death (Oltvai et al., 1993). Scutellaria baicalensis crude ethanol extract treated A549 cells, speciﬁc immunoblotting showed an increased expression of p53 and Bax (Gao et al., 2011). We found that the expression of Bax was increased under treatment of Formulae 3, 5, and 14, and Bcl-2 was decreased under treatment of Formulae 3 and 14. The more solid evidence was that the cleaved-caspase 3 was increased after treatment. In addition, we also checked autophagy related proteins and did not find any differences among the treatment and control groups. These data suggest these formulae might inhibit tumor size by inducing apoptosis rather than autophagy.. We also examined the MET process-related proteins by comparing the expression of two classic markers, epithelial and mesenchymal cell 33.

(37) type respectively, used to show the status of MET process. However, our results indicate no difference among the 4 groups. Further studies are needed to uncover the proliferation inhibitory mechanism.. Formula 3 Formula 3 is Huang-Lian-Jie-Du-Tang (HLJDT) with two warm nature herbs Atroclytes Rhizoma and Glycyrrhizae Uralensis added to compromise the extremely cold nature of the original formula. With the cold nature, HLJDT is often used to treat inflammation related disease including gastritis, dermatitis, and pneumonia. In Hsu’s study, selectivity of HLJDT was also shown in their liver cancer model which induced apoptosis and arrest of liver cancer cells and without significantly affecting normal liver cells(Hsu et al., 2008).. Lots investigations on constituent herbs of HLJDT have revealed several pure compounds possessing various biological activities. The different effects between HLJDT and formula 3 may result from the addition of the two herbs, Atroclytes Rhizoma and Glycyrrhizae Uralensis, into the formula which are thought to have boosting and nurturing effects. Indeed, Glycyrrhizae Uralensis had been reported to have the ability to suppress doxorubicin induced apoptosis (Choi et al., 2008).. Formula 5 Formula 5 is a modified formula of Qing-Wei-San and it’s mostly composed of heat clearing, blood cooling, and toxin resolving herbs thus 34.

(38) possessing a cool nature. Therefore this formula is also used to treat heat toxin or inflammation related symptoms. Crude extracts of Rhizoma Cimicifugae could induce cell cycle arrest and apoptosis of mammary cancer cells (Hostanska et al., 2004; Gaube et al., 2007). Rehmanniae Glutinosae crude extracts were shown to have pro-apoptosis ability in hepatoma cells (Chao et al., 2006).. Formula 14 Formula 14 is Gui-Zhi-Tang combined with Scutellariae Baicalensisto form another formula Yang-Dan-Tang. Different from formulae 3 and 5, this formula owns a warm nature, but nonetheless also suppresses tumor size in vivo. There are also many reports defined effective compounds of herbs in formula 14. Scutellariae Baicalensis and Glycyrrhizae Uralensis had been discussed to have anti-proliferation and pro-apoptosis ability in the section of formula 3.. Advantage of Chinese herbal medicine Formulae Using CHMs are popular in East Asia for centuries to treat diseases recently, and was considered not to have the side effect relative to western medicines. First of all, CHMs are usually used as formulae which usually are composed of three or more herbs. Complex components of the CHM formulae make it adjustable dependent on the status of each patient.. 35.

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(46) Table 1. Antibodies used in this study. Antibody MW ABCG 2. Actin. ALDH1. Bax. Beclin 1. Bcl-2. Caspase 3. Cdc2. CDK4. CDK6. c-Jun. c-Myc. Cyclin B1. Cyclin D1. Titer. Source. Manufacturer. 1st 1:250. Mouse. Abcam. 2nd 1:5000. Sheep. GE healthcare. 1st 1:5000. Mouse. Millipore. 2nd 1:5000. Sheep. GE healthcare. 1st 1:1000. Rabbit. Abcam. 2nd 1:5000. Donkey. GE healthcare. 1st 1:1000. Mouse. BD Biosciences. 2nd 1:5000. Sheep. GE healthcare. 1st 1:1000. Rabbit. Cell Signaling Technology. 2nd 1:2000. Goat. Cell Signaling Technology. 1st 1:1000. Rabbit. 2nd 1:2000. Goat. 1st 1:200. Mouse. Santa Cruz Biotechnology. 2nd 1:5000. Sheep. GE healthcare. 1st 1:1000. Rabbit. Cell Signaling Technology. 2nd 1:2000. Goat. Cell Signaling Technology. 1st 1:1000. Mouse. Cell Signaling Technology. 2nd 1:2000. Horse. Cell Signaling Technology. 1st 1:1000. Mouse. Cell Signaling Technology. 2nd 1:2000. Horse. Cell Signaling Technology. 1st 1:1000. Rabbit. Abcam. 2nd 1:5000. Donkey. GE healthcare. 1st 1:200. Mouse. Santa Cruz Biotechnology. 2nd 1:5000. Sheep. GE healthcare. 1st 1:1000. Mouse. Upstate. 2nd 1:5000. Sheep. GE healthcare. 1st 1:1000. Mouse. Cell Signaling Technology. 2nd 1:2000. Horse. Cell Signaling Technology. 72.3 kDa. 43 kDa. 55 kDa. 21 kDa. 60 kDa. 26 kDa. GeneTex. 18, 35 kDa. 34 kDa. 30 kDa. 36 kDa. 40 kDa. 41 kDa. 58 kDa. 36 kDa. 43.

(47) Antibody MW p15. p21. p53. Titer. Source. Manufacturer. 1st 1:1000. Rabbit. Cell Signaling Technology. 2nd 1:2000. Goat. Cell Signaling Technology. 1st 1:1000. Mouse. Cell Signaling Technology. 2nd 1:2000. Horse. Cell Signaling Technology. 1st 1:1000. Mouse. Cell Signaling Technology. 2nd 1:2000. Horse. Cell Signaling Technology. 1st 1:1000. Rabbit. Cell Signaling Technology. 2nd 1:2000. Goat. Cell Signaling Technology. 1st 1:1000. Rabbit. Cell Signaling Technology. 2nd 1:2000. Goat. Cell Signaling Technology. 1st 1:1000. Mouse. Millipore. 2nd 1:5000. Sheep. GE healthcare. 1st 1:500. Mouse. Santa Cruz Biotechnology. 2nd 1:5000. Sheep. GE healthcare. 1st 1:1000. Rabbit. Abcam. 2nd 1:5000. Donkey. GE healthcare. 15 kDa. 21 kDa. 53 kDa. p-cdc2 34 kDa Thr161 p-cdc2 Tyr15. Sox2. Vimentin. β-catenin. 34 kDa. 34 kDa. 57 kDa. 94 kDa. 44.

(48) Table 2. No significant lesions were identified in mouse liver and kidney after acute concentration formula treatment. 45.

(49) Table 3. No significant lesions were identified in mouse liver and kidney after high concentration formula treatment. 46.

(50) Figure 1.. A 2.5. OD570. 2.0. 1.5. 1.0. 0.5. 0.0 0%. 4%. 8%. 14%. F3. C 2.5. 2.5. 2.0. 2.0. 1.5. 1.5. OD570. OD570. B. 1.0. 0.5. 1.0. 0.5. 0.0. 0.0 0%. 4%. 8%. 14%. 0%. F14. 4%. 8%. F14. D. 47. 14%.

(51) Figure 1. Effects of CHM formulae on the decrease of cell proliferation To determine the IC50 of these formulae toward different NSCLC cell lines, we applied different concentration of these formulae to the NSCLC cells for the MTT assay. (A) Formula 3 significantly decreased the viability of H460 cells and the IC50 calculated by MTT assay is 5% (v/v). (B) Formula 14 significantly decreased the viability of H460 cells and the IC50 calculated is 4.5% (v/v). (C) Formula 14 significantly decreased the viability of H520 cells and the IC50 calculated is 3.8 % (v/v). (D and E) The colony forming assay was conducted to show Formula 14 could affect the tumorigenesis ability of H460 cells. Statistical significance was evaluated by Student’s t test. *, p < 0.05; **, p <0.01; ***, p <0.001, respectively.. 48.

(52) Figure 2.. A. B. C. D. 1.2. 2.5. 1.5. pRb (relative to actin). CDK4 (relative to actin). 1.0 2.0. *. 1.0. 0.5. 0.8. 0.6. **. 0.4. 0.2. 0.0. 0.0. control. F14. control. E. Cyclin B1 (relative to actin). 1.2. 1.0. *. 0.8. 0.6. 0.4. 0.2. 0.0. control. F14. 49. F14.

(53) Figure 2. Formula 14 reduced H520 proliferation without inducing massive cell death event (A and B) Flow cytometric analysis of H520 cells under the Formula 14 treatment. Cell population in S phase was significantly decreased and cells in G2/M phase population were significantly increased under the treatment of Formula 14 for 24 hr. After 48hr of treatment, S phase population was significantly decreased and G0/G1 phase population was significantly increased. After 48 hr of treatment, cells in G0/G1 phase were significantly decreased and cells in S phase and G2/M phase were significantly increased. (C-D) Western blot analysis of the G1/S transition promoting protein of H520 cells after treatment of Formula 14 for 72 hr. Both the cyclin D dependent kinase CDK4, and the substrate of cyclin D/CDK4/6 complex, phosphorylated Rb, were decreased significantly after Formulae 14 treatment. (E) Western blot analysis of the G2/M transition protein, cyclin B1, after H520 cells treated with Formula 14 for 72hr. Cyclin B1was substantially decreased after treatment. Statistical significance was evaluated by Student’s t test. *, p < 0.05; **, p <0.01; ***, p <0.001, respectively.. 50.

(54) Figure 3.. A. Experimental timeline Mouse age (week) 3. 4. 7. 11. Sacrifice body weight, tumor size, hepatotoxicity. Treatment When the tumor volume has reached about 100 mm3, each mouse was treated with 400 μL formula by gavage daily for 4 weeks.. Xenegragt Inplantation A549 cells (5×106) in RPMI 1640 medium were injected subcutaneously into one flank of mouse.. BALB/cnu/nu mice Acclimatize. B. C. weight. tumor size. 20 weight (g). 800. size (mm3). Ctrl F3 F5 F14. 25. Ctrl F3 F5 F14. 1000. 600 400 200. 15 10 5. 0 0. 7. 14. 21. 0. 28. 0. Days after treatment. 7. 14. 21. Days after treatment. 51. 28.

(55) Figure 3. Evaluation of the formula effect on NSCLC xenograph tumor model We characterized the therapeutic potential of formulae in vivo using xenograft model. (A) The experimental timeline of the xenograft and treatment. (B) The tumor size was examined every week during the treatment. (C) The mouse body weight was monitored during the treatment (n = 6 for each group).. 52.

(56) Figure 4.. A. B. C. D. Figure 4. Analysis of the kidney cytotoxicity effect of formulae treatment The concentration of (A) CREA, (B) BUN, (C) TP, and (D) ALB in mouse serum were determined by biochemical methods. No significant difference was found among different groups (n = 6 for each group).. 53.

(57) Figure 5.. A. B Weight Ctrl F3 F5 F14. 25. weight (g). 20 15 10 5 0 0. 7. 14. Days after treatment. Figure 5. Evaluation of the acute toxicity of of high concentration formulae on mice (A) The experimental timeline of the treatment. (B) The mouse body weight was monitored after treatment. No significant difference was identified among the 4 groups (n = 1 for control alone and n = 3 for others). 54.

(58) Figure 6.. A. B. C. D. 55.