長庚大學基礎醫學研究所生化暨細胞分子生物學組 博士論文

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1 長庚大學基礎醫學研究所生化暨細胞分子生物學組 博士論文 透過小鼠動物模式篩選及研究肝癌轉移相關基因 Identification and Characterization of the Metastasis Associated Genes via an in vivo Mouse Model 研究生 : 廖正容 學號 :D 指導教授 : 林光輝教授 中華民國九十六年二月

2 Abstract 中文摘要 : 肝癌乃世界五大癌症之ㄧ, 在台灣, 更是男性癌症死亡原因第一位 由於肝癌的高度轉移及高度復發之特性, 導致肝癌在治療成效及預後皆無法得到令人滿意的結果, 且因肝癌轉移的機制非常複雜, 到目前為止仍有許多機制有待釐清 本研究計畫, 即利用免疫不全小鼠 (SCID mice) 模式來尋找肝癌轉移的樞紐基因 我們利用雙層通透培養皿, 馴化岀轉移能力較強的肝癌細胞株 (SK-M1), 並將此細胞株利用眼窩注射的方式導入小鼠體內, 最後收集轉移到肺臟並形成癌斑的區域, 以初代培養的方法, 得到具有不同轉移能力的數種細胞株 (SK-M1-2, SK-R1-3) 接著即利用這些細胞株進行核酸微矩陣的分析, 比較不同轉移能力細胞株基因表現的型態, 進而找到一系列可能參與肝癌轉移到肺臟的候選基因 其中 Mcg1 為轉移能力較強細胞中顯著增加的基因, 但其在癌細胞侵犯, 轉移的角色目前為止並不清楚 我們藉由同步定量 PCR (Q-PCR) 系統 西方墨點法, 確認在 SK-M 及 SK-R 細胞中,Mcg1 有較高的表現 接著我們也利用穩定且過量表現目標基因的細胞株 (Mcg#1), 發現 Mcg1 的確可以促進細胞移行及侵犯的能力, 不過並不影響細胞增生 由於基質金屬蛋白酵素 (Matrix metalloproteinase, 簡稱 MMPs) 已知可以促進癌細胞的移行及侵犯能力, 我們進一步分析各細胞株所分泌之 MMP2 及 MMP9 的活性, 發現轉移能力較強的細胞株中,MMP9 活性有增加的趨勢 此外, 我們也觀察到在轉移能力較強, 及過量表現 Mcg1 的細胞中, E-cadherin β-catenin 有增加的趨勢, 相反的,Fibronectin Vimentin 的表現下降, 且不受到 Mcg1 影響 最後我們亦分析了 Mcg1 基因在臨床肝癌病人組織中表現的情況, 發現 Mcg1 在癌區組織中, 相較鄰近良性組織有較高的表現, 確立了這些基因在癌症進程過程中, 的確扮演重要的角色 綜合以上的結果, 我們利用免疫不全小鼠體內篩選模式, 發現 Mcg1 基因參與在癌細胞移行及侵犯過程種, 其中可能是透過增加 MMP9 的活性, 及影響黏附因子的表現所造成 在臨床研究 1

3 中, 我們亦觀察到 Mcg1 基因的確在癌區組織有較高的表現, 如次, 更確立此基因與癌症進程及轉移有密切關係 因此, 透過我們的發現, 可以更清楚釐清肝癌轉移的機制, 並有助於找到早期診斷的生物標籤, 以及對新治療方法研發提供依據 2

4 Abstract: Hepatocellular carcinoma (HCC) is the 5th most common cancer in the world and the 1st leading cause of cancer-related death in Taiwan. Since HCC is highly metastatic and recurrent, the treatment on metastatic HCC is un-satisfactory. Because of the complex process of metastasis, several mechanisms have remained to be distinguished. In this study, highly invasive SK-Hep-1 cells (SK-M) were isolated by the Transwell method. Subsequently, SK-R cells were established by injection the invasiveness SK-M cells into the eye socket of the SCID mice and isolate the metastatic nodules from the pulmonary alveolus one month later. This procedure was repeated three times and highly metastatic cell lineages were established. Furthermore, cdna microarray was carried out to identify the potential genes involved in HCC metastasis. Several potential genes, such as Metastasis Candidate gene 1 (Mcg1), with high expression level in SK-M and SK-R cells, were selected. However the mechanisms of Mcg1 in tumor progression, invasion, and metastasis are still unknown. By using quantitative-pcr and Western blot, the Mcg1 expression levels were elevated in SK-M and SK-R cells. The cell motility and invasive ability were increased in Mcg1 over-expressed stable cell line, but no significant difference of cell proliferation activity was observed. Matrix metalloproteinase (MMPs) are implicated in tumor cell invasion. An increasing of MMP9 activity in potential metastatic cells was observed. Besides, E-cadherin and β-catenin were upregulated in SK-M, SK-R, and Mcg1 over-expressed cell lines. However fibronectin and vimentin were downregulated and were not affected by Mcg1 over-expression. Finally, the expression level of Mcg1 gene in human HCC tissues was determined. Mcg1 was highly expressed in tumor tissues compared to the adjacent on-tumor regions. This result indicated that Mcg1 plays important roles in the processes of tumor progression and metastasis. Taken together, Mcg1 involves in tumor cell mobility and invasive 3

5 ability. This promotion may be due to the increased MMP9 activity and different adhesion molecular expression. Moreover, over-expression of Mcg1 in HCC tissues indicate that Mcg1 participating in HCC progression. This study may clarify the underlie mechanisms of metastasis. The potential biomarkers of metastasis were identified, and provided the evidences for prevention and treatment of HCC. 4

6 Introduction Hepatoma Liver cancer remains the fifth most common malignancy in male and eighth in female worldwide according to the epidemiology of hepatoma from year There are about new cases per year and continuously increasing in recent decades (1). Worldwide prevalence of hepatoma is highest in Southeast Asia and sub-saharan Africa, less in the United States. However it becomes the most rapidly increasing type of cancer in America, which promotes a new consideration of hepatoma (2). In Taiwan, hepatoma is the leading cause of death in man and second in women since 1988 (Health and national health insurance annul statistics, Department of health, Execulive -Yuan, Taiwan, R.O.C.). Therefore hepatoma is a challenge to human healthy and should be solved immediately. Primary hepatoma includes hepatocellular carcinoma (HCC>80%) and cholangio -carcinoma (CC<20%). The incidence of HCC is 15 to 20 fold higher then CC. HCC is a rapid progression, highly recurrence (70 ~ 100% recurrences after surgical 5 years), extremely malignant, and potent invasiveness tumor. This leads it lethality. HCC is often diagnosed at an advanced stage and is not amenable to curative therapies(3). When the symptoms present, the tumor often growing over 5~10 cm and the survival rate is 4~40% lower, ultimately no patient in further advanced stages survive for more than four years (4). Although there are many tumor markers have been found, such as alpha-fetaprotein (AFP), the numerous one, none of them have been proved to specific enough for early diagnosis and recurrence (5). Therefore finding the HCC tumor markers for early diagnosis is important. HCC can be classified by stages to base on the tumor node metastasis system (TNM), which provides an evaluation for treatment of HCC. T means the number and 5

7 size of primary tumor (s). N means the regional lymph nodes metastasis. And M means the distant metastasis. The stage are grouping as describe in table 1. Table 1 Stage Grouping Stage I T1 N0 M0 Stage II T2 N0 M0 Stage IIIA T3 N0 M0 Stage IIIB T1 N1 M0 T2 N1 M0 T3 N1 M0 Stage IVA T4 Any N M0 Stage IVB Any T Any N M1 HCC is a gender difference disease. HCC in male are typically 2 to 4 times higher then that in female (1). Many reports indicated that both androgen receptor (AR) and estrogen receptor (ER) were closely related to the prognosis of HCC patients (6). In addition, advancing age, increasing alcohol intake, acute (hepatitis B and C infection) and chronic liver disease (cirrhosis) are also the risk factors of HCC (1, 7). Some of these factors are geographic specific. Current estimates of the attribute fractions for the main risks factors by 3 geographic areas are shown in table2 (1). The molecular mechanism of HCC carcinogenesis seems to be more complex than that for polyp-based colon cancer. It s a multiple steps process including activation of proliferation, overexpression oncogens, impairing tumor suppressor genes, inhibition of cell death, and promoting angiogenesis (8, 9). In the recent several years, there have also been significant advances in our understanding of fundamental concepts of carcinogenesis. To go into details, the proliferation markers, such as PCNA and ki-67, have been found increasing in patients in advanced HCC stage (10). Overexpressions 6

8 of cell cycle promoting factors (such as cyclin A, cyclin D, cyclin E, and cdc2) lead to uncontrolled cell growth and resulting in the development and progression of HCC (11, 12). Many growth factors and their receptors, such as HGF/SF, c-met, and EGFR, also play the proliferation activation roles to promote tumor cell growth. On the other hand, p27and p21, the proteins bind and inhibit cyclin/cdk complexes, are negative regulators of cell cycle progression. Loss of p27 cooperates with mutations in several oncogenes (Ras, Myc, c-fos) and tumor suppressor genes can facilitate tumor growth (13, 14). p53 protein plays a central role in cell cycle arrest and cell death responses to DNA damage. Mutations in the p53 gene are the most frequently reported somatic gene alteration in human cancers, including HCC (15, 16). Finally, dysfunction of apoptosis is also an important portion of HCC progression. It has been reported that Fas expression level is significantly decreased in patients in advanced stage. To sum up, as understanding of tumor biology deepens, more effective diagnosis markers and treatment methods may be found. Table 2 Metastasis Metastasis is the process by which a tumor cell leaves the primary tumor, travels to a distant site via the circulatory system, and establishes a secondary tumor. It is the leading cause of death in cancer patients (17). The main steps in the formation of a metastasis are invasion, intravasation, extravasation, and growing of metastasis (Fig. 7

9 1). When primary tumors grow over 1 to 2 mm diameter, extensive angiogenesis must occur (18). The rich vascularization increases the chance for tumor cells to the bloodstream and colonizes secondary sites. The invasive process is translocation of tumor cells across extracellular matrix (ECM) barrier. It requires local proteolysis of the ECM and cell migration. Detachment and embolization of single or aggregates occurs next, the intravasation process. However most circulating cells being rapidly destroyed. Less than 0.01% of circulating cells survival (19). After the tumor cells have survived the circulation, they become trapped in the capillary beds of distinct organs by adhering either to capillary endothelial cells or to subendothelial basement membrane that might be exposed. Then extravasation occurs next, the mechanism might similar to invasion. Tumor proliferation at the secondary site is required to establish metastasis. Many growth factors function as regulator of tumor proliferation. For example, bfgf and IL-8 are autocrine growth factors which promote tumor growth at metastasis site. IGF-I and EGF serve as paracrine growth factors to regulate tumor cells proliferation. Finally the metastasis foci form (17). In recent years there are many genes have been discovered involved in each step of metastasis. FGF, TGF-alpha, and angiogenin are known to promote angiogenesis process. Decreased E-cadherin, main adhesion molecule of epithelia, has been found to be associated with cancer invasion. MMPs, a family of zinc-dependent endopeptidases are capable of degrading essentially all ECM components, were upregulated during tumor cell invasion. Recent studies have highlighted the importance of MMPs in tumor spread. The motility of tumor cells is also an important component of invasion. IGF-II, vitronectin, laminin, thrombospondin, IGF-I, and IL-8 are known to stimulate the mobility of tumor cells to promote the intravasation process. Then those cells survive in blood stream by decreasing the apoptosis ability. Fas/FasL signaling dysfunction, Bcl-2 upregulation, and p53 mutation are involved in 8

10 the anti-apoptosis process. When these cells arrive in the target organ, the intravasation process occurs. Finally begf, IL-8, IGF-I, EGF, TGF-beta, IL-6, and IGF-II, the metastasis growth stimulators, stimulate the growth of metastasis. It s all the process of metastasis. However more and more fundamental molecular evidences have been discovered; more considerations of metastasis prevention or treatment are provided. Fig. 1 The stapes of metastasis Intravasation Invasion Extravasation Growth of Nat Rev, Vol.3, 1~6, 2003 metastasis Non-metastasis tumors are with a satisfied prognosis and treatment effects. But when tumor metastasis to secondary organ the treatments become inefficient and with a poor prognosis. The death rate caused by metastasis tumors are about 90% higher. We have more understand about the mechanisms of tumorgenesis; however the mechanisms of metastasis are not clear. Although there are many fundamental observation of tumor metastasis have been discovered recent years, the prognosis and curative effects do not improve. Metastasis stills a challenge for HCC patients. It is important to pay more attention on defining the detailed molecular mechanisms of metastasis. 9

11 Animal models used for metastasis study The biological complexity that characterizes metastasis requires complexity experimental systems for its study. Animal model is the only strategy for metastasis research. There are three common in vivo models for metastasis studying including 1 transplantable cancer model, 2 genetically engineered mice model, and 3 spontaneously occurred cancer model. But no single metastasis model is sufficient to answer all questions. As such, the selection of the optimal model for each biological or translational question is necessary (20, 21). We choose the transplantable cancer cell mouse model. The genomic approach allows us to identify families of genes involved in a process, not just single gene, and can indicate which molecular and cellular events might be important in complex metastasis processes. The cells we generated with progressive metastatic ability are in the same genetic background. Thus we can specifically obtain the metastasis related genes by comparing the gene expression patterns of parental cell lines with highly metastatic cell lines. 10

12 Results The cell lines with increasing metastatic ability The in vitro transwell invasion assay and in vivo SCID mice model were used for selecting the cells with progression metastasis ability. The cells traversed the upper chamber of transwell term SK-M, and derived from the pulmonary metastasis term SK-R (Fig. 2). The increased metastatic ability of SK derived cell lines The metastatic ability of SK-M and SK-R cells were assayed by transwell invasion assay. As shown in fig. 3, SK-R was the most portent metastatic cell line with a large number of traversed cells (3 to 4 folds than SK cells), SK-M listed in second (fig. 3B and C). The candidate genes selected from cdna microarray SK, SK-R2, and SK-R3 cell lines were used to carry out the genomic approach (Fig. 4A). The total RNA was extracted from SK and SK-R lines, and RT-PCR was performed to obtain the cdna. cdna microarray was used to screen the difference expression of human genome library (Array1: SK-R2/SK; Array2: SK-R3/SK). 35 up-regulated genes (SKR/SK > 1.7) and 36 down-regulated genes (SKR2 / SK < 0.6) (Table 3A) were found in the array1 analyzed data. 68 up-regulated (SK-R3/SK>1.7) and 40 down-regulated (SK-R3/SK<0.6) genes were identified in array2 (Table 3B). Among them, the genes known to be involved in tumor metastasis processes, such as fibronectin-1 (22), vascular cell adhesion molecule-1 (23), and aminopeptidase-n (24) were included. According to the data of these two independent microarray experiments, Metastasis Candidate Gene 1 (Mcg1), was choose for further study. The 11

13 roles of Mcg1 gene in HCC metastasis are needed to be discovered. Mcg1 over-expressed in SK-M and SK-R cell The mrna and protein expression levels of candidate genes were analyzed in SK, SK-M and SK-R cells by Q-PCR and western blot. The mrna and protein levels of Mcg1 were 3 to 4.5 folds in SK-M and SK-R cells (Fig. 5A-C) as the result of mocroarray data (Fig. 4C). According to the observations, we speculated that Mcg1 gene maybe participate in the processes of tumor cell metastasis. Mcg1 promotes tumor cell migration Since Mcg1 was over-expressed in potent metastasis cell lines, the Mcg1 stable over-expressed cell line (Mcg#1~4, Fig. 6A and B) was established to investigate the role of Mcg1 in metastasis. The motility of tumor cells is one of the important components of invasion. The motility of SK, SK-M, SK-R, and SK-P cell lines were assayed by wound healing assay. As the result of 24 hrs incubation after scraped, SK-R3 and Mcg#1~4 cells showed more motile ability comparing with SK parental cells (Fig. 7A, B). Therefore, the enhanced cell mobility of SK-R3 cells should due to the over-exptessed Mcg1. The motile property of SK-R3 and SK-P cell line might promote the invasion step during tumor metastasis. PSG-1 plays an important role in tumor cell invasion Transwell invasion assay was used to study whether Mcg1 promotes tumor cell invasion. As the result of Fig.8B, Mcg#1~4 stable cell line was more invasiveness than SK parental cell. The traversed cell of Mcg#1~4 was about 3 folds compare to SK cell. According to this result, Mcg1 plays a critical role in the process of metastasis. 12

14 The proliferation activity is not altered in cells with different metastasis ability Growing out of control is found in almost all tumors. Whether cell growth rate correlates with invasive ability? MTT assay was used to analyze the proliferation activity of these SK derived cell lines. There was not any significant difference of cell growth rate have been found (Fig. 9 A and B). MMP9 is increased in SK derived cell lines MMPs are the main group of proteolytic enzymes that are involved in the tumor invasion, metastasis, and angiogenesis in cancer. Among the previously reported human MMPs, MMP-2 and MMP-9 are abundantly expressed in various malignant tumors(25). They are considered key enzymes for tumor invasion and metastasis. We also detected the enzyme activity of MMP2 and MMP9 by zymography. The MMP9 activity was raised in SK-R and greatly increased in SK-M cell culture medium (Fig. 10A, B). But MMP2 enzyme activity could not been detected here (Fig. 10B). Therefore, Mcg1 possible augments tumor invasion by enhancing the MMP9 enzyme activity. The exact mechanism is still needed to be discovered. Mcg1 influences the marker proteins of EMT Recent studies demonstrated that cell undergo epithelial-mesenchymal transition (EMT) during tumor progression. The characteristics of EMT are loss of epithelia markers (E-cadherin, β-catenin) and gain of mesenchymal markers (Fibronectin, Vimentin, and N-cadherin)(26). The expression levels of E-cadherin, β-catenin, fibronectin, and vimentin were test in these SK derived cell lines. The unexpected results were observed. E-cadherin and β-catenin were overexpressed in potent metastatic cell lines (Fig. 11A, B); furthermore fibronectin and vimentin were 13

15 downregulated in cells with higher metastasis ability (Fig. 11C, D). Besides, E-cadherin and β-catenin were also upregulated in Mcg1 stable over-expressed cell line (Mcg#1~4) but fibronectin and vimentin were not affected by Mcg1 over-expression (Fig. 11A-D). These puzzled results are needed to be solved. Mcg1 over-expresses in human HCC tissues The expression patterns of candidate genes were also checked clinically. The liver tumor specimens of patients with primary tumor (L2, L3) or recurrence tumor combined with lung metastasis (L1) were collected, and the mrna and protein of these tissues were isolated. As shown in fig. 12, Mcg1 was over-expressed in tumor tissues rather than the adjacent benign tissues. Nevertheless, this clinical data are still preliminary result. A large-scald screening is necessary to clarify the expression patterns of these two genes in HCC. Then the correlation between gene expressions with clinical data should be carried out. 14

16 Conclusion and discussion In this study we used the in vivo SCID mice model to select the metastasis associated genes. Clark and his collaborators identified RhoC as an essential gene involved in melanoma invasion by using the same in vivo mice model (21). We also discovered that Mcg1 gene was highly expressed in potent metastastic SK-R cell lines to promote tumor cell migration and invasion. Since MMP2 and MMP9 activity correlate with tumor cell motile and invasive ability. The MMP2 and 9 enzyme activity were also detected in SK-derived cell lines. MMP9 activity was raised highly in SK-M and slightly increased in SK-R3 cells. However, the MMP2 activity could not been detected in our system. This result indicated that MMP9 is involved in Mcg1 induced tumor invasion. Besides, it has been reported that EMT is a phenomenon of tumor cells gain of invasive property. The important molecules, β-catenin, E-cadherin, fibronectin, and vimentin, involved in EMT were also detected in these cells. An unexpected result, epithelia marker (β-catenin and E-cadherin) upregulated and mesenchymal markers (fibronectin and vimentin) downregulated in SK-M and SK-R cells, was observed. Nevertheless, it has been reported that E-cadherin and β-catenin were re-expressed in some metastasis tumor tissues, such as breast cancer(27), ovarian cancer(28), and gastric cancer(29). This adhesion molecules re-expression may facilitate tumor cell implantation, establishment of metastasis lesions, and protect cell from apoptosis. However, the exact mechanisms responsible for these genes re-expression in our model are needed to be solved. Finally, the Mcg1 expression was also checked clinically. The enhanced Mcg1 expression level was observed in human liver tumor tissues. This result implicates that Mcg1 gene must participate in HCC progression. 15

17 By using this approach may clarify the mechanisms of metastasis. The potential biomarkers of metastasis will be identified, and provided the evidences for prevention and treatment of HCC. 16

18 Materials and methods Cell culture The human hepatoma cell line SK-Hep-1 (ATCC Number: HTB-52) or J7 were obtained from American Type Culture Collection, or Kennth SS Cheng, respectively and was routinely grown in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% (v/v) fetal bovine serum. Cells were cultured at 37 degrees centigrade of in a humidified atmosphere of 95% air and 5% CO2 of. Cells were trypsination twice every week. Transwell assay The effect of the interesting genes on the invasive activity of several HCC cell lines will be examined with a rapid in vitro assay for quantifying the potential of tumor cells for metastatic invasion (Transwell assay). The upper chamber is coated with 50 l matrix-gel (2.5mg/ml; BD Biosciences Discovery Labware, Bedford, MA). Cell density was adjusted to 1x10 5 /100 µl, and 100 µl of this will be added to each chamber in triplicate. The medium in the upper chamber will be serum-free DMEM and that in the lower chamber will be supplemented with 20% fetal bovine serum. After incubation for 24 h at 37 degrees Centigrade, the viable number of cells that had traversed the filter to the lower chamber will be counted, and then expressed as a percentage of the total viable number of cells to provide an index of invasive activity. Animal model establishing We gained the moderate metastasis cells by transwell assay and isolate the cells traversed the filter. Those moderate metastatic HCC cell lines were injected of eye socked of host mice and pulmonary metastases were isolated. These metastases were 17

19 grown in tissue culture and injected into additional host mice. The procedure to selecting for highly metastatic tumor cells was repeated three times. cdna microarray The cdna microarray analysis was analyzed using GenePix Pro microarray scanning and analysis software (Axon Instruments). All of the array data will be deposited in the microarray database at bioinformatics core facility, Chang Gung University. Multiarray analytical tools available on the database will be used to apply the selection criteria. Agglomerative clustering by Euclidean distance measurement will be accomplished using S-PLUS 5.1 soft ware (MathSoft, Inc., Camb ridge, Mass), and the clustered data will be visualized using Eisen Tressview software. The interesting genes were classified by FatiGO software ( Quantitative-PCR If the samples are limited, we will use Q-RT-PCR. The RNA will be extracted and the quality of RNA will be determined by gel electrophoresis. Single-stranded cdna will be synthesized with oligo-(dt) in a 10 µl reaction from 4 µg total RNA using SuperScript (Life Technologies, Inc., Rockville, MD). The sample used for the standard curve in the Q-RT-PCR will be prepared by serial dilution of an 18s RNA sample to contain 16, 8, 4, 2, and 1 ng. The primer used, based on the cdna sequence from the gene bank and analyzed by the ABI Prism Primer Express (PE Applied Biosystems). 1 µl of cdna for 1 cycle of 94 degrees Centigrade of for 10 min of PCR will be performed with, followed by 40 cycles of 94 degrees Centigrade of for 30 s, 72 degrees Centigrade of for 60 s using gene-specific primers and Cyber green kit of and (Applied Biosystems). The fluorescence emitted by the Cybergreen will be detected by the ABI prism 7000 (PE Applied Biosystems). The relative amount of 18

20 plasma protein mrna, standardized against the amount of 18s RNA will be expressed as Ct =-[Ct (plasma protein)-ct (18 SRNA)]. The ratio of the number of plasma protein mrna copies to the number of 18 SRNA copies will be calculated as 2^Ct xk, where k is a constant. Western blot To determine the protein level of each gene, we will carry out the immunoblot. If the gene we interested is known gene and the antibody is available, cell lysates will be fractionated by SDS-polyacrylamide gel electrophoresis (PAGE) on a 10% gel, and the separated proteins will be transferred to a nitrocellulose membrane (PH 7.9 membrane, Schleicher & Schuell). The membrane will be gently shaken for 2 hrs at room temperature in 5% (w/v) nonfat dried milk in Tris-buffered saline (TBS), washed three times with TBS, and then incubated for 1 h with rabbit polyclonal antibodies (1:2000 dilution in TBS). After further washing, the membrane will be incubated for 1 h with horseradish peroxidase-conjugated, affinity-purified antibodies to either rabbit (1:3000 dilution in TBS) or mouse (1:3000 dilution in TBS) immunoglobulin (Santa Cruz Biotechnology). Immune complexes will be then visualized by chemiluminescence with an ECL detection kit (Amersham). The intensities of immunoreactive bands will be quantitated by analysis with Image Gauge software (Fuji Film). MTT assay Cell proliferation was detected by MTT method which determines cell proliferation through measuring the absorbent value at OD570 based on the fine positive linear correlation between OD570 value and cell numbers. The HCC cells in 96-well plates above were washed twice with PBS. Into each of the wells in the plates was added 19

21 200 µl of FBS-free DMEM medium and 20 µl of 5 mg/ml MTT solution (Amresco) and then was continuously incubated at 37 for 4 h. The medium in the wells was discarded and 100 µl of 4N isopropenol was then added into each of the wells. The OD570 value of each of the wells was detected by spectrophotometer. Wound healing assay For the scratch-wound assay, 3 scratches were made on each 60-mm plate of confluent HCC cell lines, using a yellow pipette tip. The cells were fixed for Giemsa staining at 24 hr after scratched. Gelatin Zymography To understand the effect of these genes on MMP activities, gelatin zymography was performed according to procedures described as before. Briefly, the treated cultured medium containing 5 µg of protein was separated on a 10% SDS-PAGE gel that was copolymerized with 1 mg/ml of gelatin. After removing SDS with 2.5% Triton-X100 solution, the gel was incubated with Zymogen developing buffer (Bio-Rad Laboratories, Hercules, CA) and then stained with 0.5% Coomassie Blue R-250. The enzyme-digested regions, observed as clear bands in destained gel against a blue background, indicated the presence of MMP-2 (72 kda) and MMP-9 (92 kda). The visualized bands were also quantitated. Collecting the tissues of HCC patients HCC specimens were collected from tumor bank at Chang-Gung Memorial Hospital. For each patient, one cancer or one normal tissue biopsy will be surgically resected. The diseased tissue were dissected and frozen immediately in liquid nitrogen until used for molecular assay. 20

22 Figures SK Transwell Pulmonary SKM SKR Fig. 2 The in vivo mouse model used in this proposal. Fig. 3 The metastatic ability of SK, SK-M, and SK-R cell lines. Fig. 3A is the schematic process of in vitro transwell invasion assay. (B) The traversed cell staining with DAPI. (C) The invaded cell on the bottom of up chamber was trypsinized and counted using hemocytometer. 21

23 SK cells SK-R Cells mrna extraction mrna mrna RT-PCR labeling cdna (Cy5) cdna (Cy3) Hybridize to micrroarray slide Laser excitation Fig. 4 The flowchart of cdna microarray. 22

24 (A) Abbreviation Name R2 / P Fold of induction MCG#1 Metastasis candidate gene # ANPEP Alanyl aminopeptidase N EPHA2 EphA VCAM1 Vascular cell adhesion molecule FN1 Fibronectin (B) Abbreviation Gene name SKM1/SK Fold SKR3/SK Fold MCG#1 Metastasis candidate gene # IL-6 Interleukin ANPEP Alanyl aminopeptidase N CXCL1 Chemokine (C-X-C motif) ligand Table 3 Microarray array data. (A) Thirty-five up-regulated genes (SK-R/SK > 1.7) and thirty-six down-regulated genes were identified (SK-R/SK < -0.6). (B) Sixty-eight up-regulated (SK-R3/SK > 1.7) and forty down- regulated (SK-R3/SK < 0.6) genes were identified. 23

25 A Fold induction B 0 Mcg1 SK SKM1 SKR2#11 SKR3#2-2 SKR3# S C Mcg1 Fig. 5 The mrna expression level of Mcg1 (A, B) was determined by Q-PCR and RT-PCR. Panel C shows the protein expression level of Mcg1 detected by western blot. A Q-PCR : Mcg1 mrna B SK Mcg#1 Mcg#2 TNT 25 Fold induction SK Mcg#1 Mcg#2 Fig. 6 The Mcg1 stable over-expressed cell lines. The Mcg1 mrna and protein expression level were detected by Q-PCR (A) and western blot (B) respectively. Mcg#1~2 were the clones with highly Mcg1 level. 24

26 A 0 hr 24 hr SK SKM1 B 0 hr 24 hr SKR2 SK_C2 SKR3 Mcg#1 Fig. 7 The mobility of SK derived cell lines. Cells were incubated for 24 hrs after scraped, washed with PBS and were photographed by phase-contrast microscopy. A B Cell number ( X 10 3 ) Cell number ( X10 2 ) SK SKR3#2-2 SKR3# SK_C2#1 Mcg#1 N7+N12 Fig. 8 The invasive ability of SK derived cell lines detected by using transwell assay. (A) SK-R3 cell lines were the more invasive cells compared to SK parental cell. (B) Overexpression of Mcg1 in SK promotes the cell invasion. SK_C2#1 was the control cell transfected with empty vector. 25

27 A nm OD SK SKM1 SKR2#11 SKR3# B Time (hs) nm OD SK_C2#1 N7 Mcg#1 N12 Mcg#2 N26 Mcg# Time (hrs) Fig. 9 The proliferation activity of SK derived cell lines detected by MTT assay. There was no significant different between SK, SK-M, and SK-R cells or between Mcg#1~3 and SK_C2#1 cell. A SK R3#1 R3#2 B SK M1 SKR3#1 SKR3#2 Serum Soup MMP9 MMP9 MMP2 Cell lysate Fig. 10 The MMP2 and MMP9 enzyme activities were detected by gelatin zymography. MMP9 activity was highly expressed in SKM1 cell (B) and slightly increased in SK-R3 cell lines (A and B). MMP2 activity could not been detected (B). 26

28 A C SK SKM1 SKR2SKR3#1SKR3#2SK_P B D 2 1 SK SKM1 SKR2SKR3#1SKR3#2SK_P SK SKM1 SKR2 SKR3#1SKR3#2SK_P R3#2 Mcg#1 0 SK SKM1 R2 R3#1 R3#2 Mcg#1 Fig. 11 The mrna expression level of adhesion molecules in SK derived cell lines and Mcg#1~4 stable line were analyzed by Q-PCR. (A) β-catenin, (B) E-cadherin, (C) Fibronectin, (D) Vimentin. PT1 PT2 PT3 N T N T N T TNT Mcg1 Fig. 12 The protein expression patterns of Mcg1 in human HCC tissues. PT1~3, patient number; N, adjacent benign tissue; T, tumor tussue. 27

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