Nitric oxide in liver cancer

NITRIC OXIDE IN LIVER CANCER BEATRICE JOANNE GOH HWEY NEI (B.Sc Hons) A THESIS SUBMITTED FOR THE DEGREE OF DOCTOR OF PHILOSOPHY DEPARTMENT OF BIOCHEMISTRY NATIONAL UNIVERSITY OF SINGAPORE 2009 Acknowledgements I would like to express my deepest appreciation and heartfelt thanks to Dr Tang Bor Luen and Dr Theresa Tan for their invaluable help when I was most in need. Without their time and efforts, this thesis would not have seen the light of day. To Prof Khoo Hoon Eng, I would also like to extend my thanks and gratitude. Lessons learnt went far beyond lectures and lab. To Dr Hon Wei Min, your guidance and patience while I learnt the basic techniques is remembered with gratitude. My thanks to Ms Ng Foong Har and her ‘fei zais’ that were faithful companions during those late nights. I would also like to thank Ms Tan Bee Tee, her assistance during the project was invaluable. To Dr Yau Yin Hoe and Dr Sun Wentian, special thanks for many insightful discussions with no topic too taboo to explore, sometimes to my horror. Also to the members of STUN, particularly Mr Wu Feiyi and Mr Ng Weijun, for keeping morale high, 20% per hour to be exact. My thanks and appreciation for the friendship from Chan Mann Yin, Gregory Tan, Sun Guang Wen, Neeyor Bose, Joyce Sun, Chen Kang. Tan Weiqi and Bian Haosheng. You guys know. My thanks and love to my family: mother, father, sister, dog and newly acquired husband for your love, understanding and unfailing patience. - ii – Contents Chapter 1: Introduction 1.1 Liver Cancer 1.1.1 Etiology and Prevalence 1.1.2 Diagnosis 1.1.3 Current Treatments 1.2 Nitric Oxide 1.2.1 Reactions of Nitric Oxide 12 15 1.2.1.1 Direct Reactions of NO 15 1.2.1.2 Indirect Reactions of NO 17 1.2.2 Biosynthesis of Nitric Oxide 1.3 Nitric Oxide Synthase 19 20 1.3.1 Structure of NOS 22 1.3.2 Regulation of NOS 24 1.3.2.1 Transcriptional Regulation 25 1.3.2.2 Post-translational Regulation 25 1.4 Nitric Oxide and Diseases 27 1.4.1 Diseases Associated with Overproduction of NO 28 1.4.1.1 Septic Shock 28 1.4.1.2 Inflammation 29 1.4.1.3 Neurodegenerative Diseases 30 1.4.2 Diseases Associated with deficiency of NO 30 1.4.2.1 Atherosclerosis 30 1.4.2.2 Hypertension 31 1.5 Nitric Oxide and Cancer 32 1.6 Aims of Study 33 Chapter 2: General Materials and Methods 2.1 Materials 36 2.1.1 Human Samples 36 2.1.2 HCC Animal Model 37 2.2 Methods 40 2.2.1 Immunohistochemistry 40 2.2.2 RNA Extraction 41 2.2.3 Reverse Transcription 43 2.2.4 Conventional PCR 44 2.2.4.1 PCR Reaction 44 2.2.4.2 Agarose Gel Electrophoresis 45 2.2.4.3 Gel Clean-up of PCR products 45 2.2.5 Real Time PCR 46 2.2.5.1 Standard Curve 48 2.2.5.2 Real Time PCR 49 2.2.6 Extraction and Quantitation of Proteins from Tissues 52 2.2.7 Immunoblot/ Western Blot 52 2.2.7.1 SDS-PAGE 52 2.2.7.2 Transblot 54 2.2.7.3 Checking Gel for Blotting Efficiency 54 2.2.7.4 Immunodetection 55 2.2.8 NOS Activity Assay 55 2.2.9 Plasma Nitrate/Nitrite Assay 57 2.2.10 Statistical Analysis 58 Chapter 3: Gene Expression Studies in Liver Cirrhosis and HCC 3.1 Introduction 59 3.2 Specific Materials and Methods 60 3.2.1 Patient Samples 60 3.2.2 Microarray 61 3.2.2.1 RNA Extraction and Purification 61 3.2.2.2 Reverse Transcription 61 3.2.2.3 Preparation of Dye Pack 62 3.2.2.4 Coupling of Cy Dye to cDNA 63 3.2.2.5 Hybridization 64 3.2.2.6 Scanning 64 3.2.2.7 Data Analysis 65 3.2.2 Real Time PCR 65 3.2.4 Hepatocyte Growth Factor ELISA 65 3.3 Results and Discussion 66 3.3.1 Microarray Results 66 3.3.2 Real Time PCR Results 71 Chapter 4: NO in Liver Cirrhosis and HCC 4.1 Introduction to Liver Cirrhosis 81 4.2 Specific Materials and Methods 83 4.3 Results and Discussion 84 Chapter 5: NO in the progression of HCC: an animal study 5.1 Introduction 99 5.2 Specific Materials and Methods 101 5.2.1 Development of a HCC Model 101 5.3 Results and Discussion 101 5.3.1 Development of a HCC Model 101 5.3.2 Role of NO in HCC Animal Model 107 Chapter 6: NO in the Intermediate Stages of HCC: an animal model study 6.1 Introduction 117 6.2 Specific Materials and Methods 121 6.2.1 NOS Selective Inhibition 122 6.2.2 NO donors 123 6.3 Results and Discussion 125 Chapter 7: Conclusions and Future Directions 151 References 158 Appendix A 172 List of Tables Table 1.1: Liver function test parameters Table 1.2: Some chemical properties of NO Table 1.3: Table listing the three known isoforms of NOS Table 1.4: List of NOS isoforms and their general characteristics Table 1.5: Some regulatory interactions of the three isoforms of NOS Table 2.1: Primary antibodies used in Immunohistochemistry Table 2.2: Components of RNA loading buffer Table 2.3 Components of the Reverse Transcription reaction mix Table 2.4: Components of the PCR reaction mix Table 2.5: List of genes examined and quantitated by real-time PCR Table 2.6: Primers used in the generation of Standards as well as for real-time PCR reactions. Table 2.7: Preperation of SDS-PAGE gels Table 3.1: Components of the 25x dNTP mix for use in RT-reaction Table 3.2: Components of the RT reaction Table 3.3: List of reagents in the probe preparation Table 3.4: Cross-labeling of Cy3 and Cy5 to eliminate dye bias Table 3.5: List of some genes found to be differentially expressed in the liver diseases investigated Table 6.1: List of NOS selective inhibitors and NO donors given in drinking water to normal (or control) rats and 10 weeks DEN-treated (or HCC) rats Table 6.2: AST and ALT levels Table 7.1: Summary of our findings. List of Figures Figure 1.1: Pie chart showing the different types of primary liver cancer Figure 1.2: Roles of NO Figure 1.3: Reactions catalysed by NOS Figure 1.4: Structural similarities between the NOS isoforms Figure 2.1: Development of a rodent HCC model Figure 2.2: Representative disassociation curve Figure 2.3: Representative standard curve obtained for each real-time PCR run Figure 3.1: Heat map of transcripts in the Operon Array tested against various liver diseases Figure 3.2: Real-time PCR results of various genes found to have dfferential expression in preliminary microarray experiments Figure 3.3: Real-time PCR results of HGFA, HGF and the receptor c-met Figure 3.4: HGF levels in patient plasma samples Figure 3.5: Real-time PCR results of genes showing differential expression between HCC and HCC + HBV samples in the Stanford DNA array Figure 4.1: Liver function test on patient samples Figure 4.2: Total NOx- from patient samples Figure 4.3: Real-time PCR results of iNOS and Enos Figure 4.4: Protein expression of iNOS and eNOS in various liver diseases Figure 4.6: Protein levels of caveolin-1, Rac2 and sGC Figure 4.7: Immunohistochemistry of iNOS, eNOS and caveolin-1 in patient lives sections Figure 5.1: AST and ALT levels of control and DEN-treated animals over time Figure 5.2: Liver weight expressed as a percentage against body weight in the HCC animal model Figure 5.3: Livers of the animals during the development and progression of hCC by 0.01% DEN (v/v) over – 16 weeks Figure 5.4: Immunohistochemistry of GST-π on liver sections of control and DEN-treated animals over time Figure 5.5: Plasma levels of NOx- in DEN-treated animals expressed as a percentage of control animals at the same time point Figure 5.6: Real-time PCR analysis of iNOS and eNOS expression in the HCC animal model Figure 5.7: NOS activity assay of DEN-treated rats over time Figure 5.8: iNOS and eNOS protein expression of DEN-treated rats over time Figure 5.9: Caveolin-1, Rac2 and sGC protein expression of DEN-treated rats over time, relative to controls Figure 6.1: Percentage of liver weight to body weight of HCC rats with various treatments against their respective controls Figure 6.2: Livers excised from controls and HCC rats with different treatments Figure 6.3: Immunohistochemistry of GST- π on sections of liver from controls and HCC rats with various treatments Figure 6.4: Total NOx- in control and HCC animals Figure 6.5: iNOS mRNA and protein levels in control and HCC rats with various treatments Figure 6.6: eNOS mRNA and protein levels in control and HCC rats with various treatments Figure 6.7: NOS activity of HCC as a percentage of their respective controls. Figure 6.8: Western blot results of caveolin-1, Rac2 and sGC protein levels in DEN treated animals expressed as a percentage against controls. Figure 7.1: Overview of the development of HCC. Summary Nitric oxide (NO) has been implicated in the pathogenesis of liver cancer or hepatocellular carcinoma (HCC). However, the exact role(s) of NO and the expression pattern of its enzymes, nitric oxide synthases (NOS), remain unclear. Thus, this study was designed to investigate the expression and regulation of NOS in both human and animal HCC samples. An animal model of HCC was established and this allowed the tracking of NOS expression during the progression of HCC. NOS selective inhibitors and NO donors were administered to determine the possible role(s) of NO in HCC in an animal model. Microarray analysis was initially used to examine several liver diseases from patient samples, viz, liver cirrhosis, primary HCC, HCC with hepatitis B infection and secondary HCC. These were compared to normal liver samples. Expression patterns were distinct for each liver disease examined. Gene expression changes in selected transcripts were confirmed with quantitative real time PCR. Of these transcripts, NOS was furthered studied in detail. Nitrates and nitrites, a measurable form of NO, alongside NOS expression and activity were found to be increased in liver cirrhosis and HCC. Rac2 and caveolin-1, endogenous post-translational regulatory factors of inducible NOS (iNOS) and endothelial NOS (eNOS) were also examined. Both were found to be highly expressed in the disease states. This suggests the regulatory factors are likely to contribute to NOS regulation. However, the expression of soluble guanylate cyclase (sGC), a target of NO, was found to be unchanged. An animal model for HCC was established with the administration of diethlynitrosamine (DEN) in drinking water. This treatment resulted in the development of cirrhosis in the liver prior to the development of visible foci, similar to the progress of HCC in humans. DEN was given to male Sprague-Dawley rats ad libitum for a period of 4, 6, 8, 10, 12, 14 and 16 weeks. Apart from visible foci formations, glutathione S-transferase-π (GST-π), a commonly used marker for neoplasms, staining was carried out to confirm the development of neoplastic lesions. With time, DEN-treated rats were found to have increased NO and NOS, Rac2 and caveolin-1 expression. However, eNOS and iNOS, the two isoforms of NOS studied, showed differential expression activities which were most distinct at week 10. To further investigate the role of NO in the progression of HCC, NOS selective inhibitors and NO donors were administrated to rats after 10 weeks of DEN treatment. Again the levels of NO and NOS expression and activities were studied. NOS selective inhibitors, N-nitro-L-arginine (L-NNA) and N-1-iminoethly-L-lysine (L-NIL), were selected for their high inhibitory effects against eNOS and iNOS respectively. NO donors, isosorbide dinitrate (ISDN) and molsidomine were selected as these are currently used in prescription drugs for humans. Interestingly, NOS inhibitors exacerbated HCC progression, which implies a protective role for eNOS. NO donor treatments on the other hand, showed decreased visible foci, cirrhotic lesions and GST-π positive staining. This implies NO plays a protective role in HCC progression in our animal model for HCC. 10 NM_004822 E01408 Y10479 NM_001166 Netrin-1 Manganese superoxide dismutase E2F3 API1 NM_000227 M61916 J03133 LAMA3 LAMB1 TSFP1 AF087438 PPP2R1B M31165 M90813 TSG-6 Cyclin D2 AF174487 BOK M26760 ALDH-2 U43746 U29680 BRCA2 BCL2-related protein A1 U14680 AB017167 NM_005526 BRCA SLIT1 HSF1 M21616 PDGFRB Thymidine kinase Ubiquitin Specific Protease HSPF2 U77088 U20657 NM_005528 X06234 U75898 M81933 NM_000843 U17760 U72355 Z23115 U16296 Calgranulin A Heat shock 27kD protein Cdc25A GRM6 LAMB3 Hsp27 ERETATA-binding Bcl-xL TIAM1 2.52 Netrin 2.52 Manganese superoxide dismutase 2.46 E2F-3 transcription factor 2.43 Apoptosis inhibitor (API1) Laminin, alpha (nicein (150kD), 2.42 kalinin (165kD) 2.41 Laminin B1 2.39 Transcription factor SP1 Protein phosphatase subunit A 2.39 isoform beta Tumor necrosis factor, alpha-induced 2.38 protein 2.36 D-type cyclin (CCND2) BCL-2-related ovarian killer protein; 2.32 BOK Aldehyde dehydrogenase 2, 2.23 mitochondrial Breast cancer susceptibility (BRCA2) 2.21 gene 2.20 BCL2-related protein A1; Bfl-1; GRS Breast and ovarian cancer 2.19 susceptibility 2.16 Slit-1 2.15 Heat shock transcription factor Platelet-derived growth factor 2.14 receptor, beta 2.14 Thymidine kinase (TK2) 2.12 Ubiquitin Specific Protease 2.12 Heat shock 40kD protein 2; HSPF2 S100 Calcium-binding protein A8 2.11 (calgranulin A) 2.11 2.10 2.10 2.10 Heat shock 27kD protein Cell division cycle 25A; Cdc25a Glutamate receptor, metabotropic Laminin S B3 chain; LAMB3 Hsp27 ERE-TATA-binding protein 2.08 (HET); Scaffold attachment factor B 2.05 BCL2-like 2.04 T-lymphoma invasion and metastasis 230 inducing Forkhead (Drosophila) homolog U02368 FKHR 2.04 (rhabdomyosarcoma) Y11416 P73 2.03 Tumor protein P73 JNK1; Mitogen-activated protein L26318 JNK1 2.03 kinase Table A5: Primary HCC compared with normal, genes up regulated by at least fold in the Operon array. Gene Bank ID M65217 E01408 J03358 U75898 M59828 Short Gene Name HSF2 Manganese superoxide dismutase FER Heat shock 27kD protein HSP70-1 Fold Diffrence Long Gene Name Heat shock transcription factor 69.77 (HSTF2) 55.56 Manganese superoxide dismutase Fer (fps/fes related) tyrosine kinase 54.55 (phosphoprotein NCP94) 42.25 Heat shock 27kD protein 40.54 MHC class III HSP70-1 AF088982 HSP40-3 NM_004419 DUSP5 39.47 Heat shock protein hsp40-3 36.59 Dual specificity phosphatase X89986 29.41 NBK apoptotic inducer protein BH3 interacting domain death 26.79 agonist Bik NM_001196 BID DNA polymerase D29013 beta M13228 N-myc M29870 Rac1 M34353 AF028832 ROS1 Hsp89-alphadelta-N L35253 MAPK 14 M15400 Rb NM_003244 TGIF 25.42 DNA polymerase beta 24.39 N-myc oncogene Ras-related C3 botulinum toxin substrate (rho family, small GTP 23.08 binding protein Rac1) Transmembrane tyrosine-specific 17.86 protein kinase (ROS1) 12.66 Hsp89-alpha-delta-N 9.32 Mitogen-activated protein kinase 14 Retinoblastoma susceptibility gene, 9.09 Rb TG-interacting factor (TALE family 8.11 homeobox) 231 U19727 Tau 6.85 M36542 OCTF2 6.32 M63167 Rac-alpha NM_001752 CAT 6.13 6.12 NM_007031 HSF2 Immunoglobulin heavy chain U00568 variable region 5.92 L09159 M13975 Rho Protein Kinase C Beta II AB023420 APG-2 X02811 PDGFB M15990 C-yes-1 Microtubule-associated protein (MAP4) Octamer-binding transcription factor V-akt murine thymoma viral oncogene homolog 1; rac protein kinase alpha Catalase Heat shock transcription factor binding protein Immunoglobulin heavy chain 5.86 variable region Ras homolog gene family, member 5.55 A; Rho 5.52 Protein kinase C beta-II Heat shock protein apg-2; 5.50 HS24/p52; hsp70 5.17 Platelet-derived growth factor beta V-yes-1 Yamaguchi sarcoma viral 4.68 oncogene homolog 1; C-yes-1 X79981 VE-cadherin NM_000426 LAMA2 4.17 VE-cadherin 4.11 Laminin, alpha precursor NM_001752 CAT Zinc finger transcriptional M92843 regulator 4.08 Catalase L09159 U43747 Rho Frataxin 3.83 3.69 M23452 3.64 L14283 MIP1-ALPHA Protein Kinase C Zeta M64240 MAX U33841 ATM D14497 M31732 Map3K8 BCL 4.04 Zinc finger protein homologous to Zfp-36 in mouse Ras homolog gene family, member A; Rho Frataxin; Friedreich ataxia Macrophage inflammatory protein; Small inducible cytokine A3 (homologous to mouse Mip-1a) 3.61 Protein kinase C zeta Helix-loop-helix zipper protein 3.55 (max) 3.55 Ataxia telangiectasia gene Mitogen-activated protein kinase 3.47 kinase kinase 3.47 B-cell CLL/lymphoma 232 AF010309 U96876 Pig3 Insulin induced protein M34353 ROS1 Hepatic nuclear M57732 factor 1-alpha AF075601 HSJ2 X01394 TNF Death Associated AF015956 protein V00568 C-myc GABA(A) NM_000808 receptor alpha-3 Z23115 Bcl-xL M95712 D13316 AF003595 B-raf Respiratory factor-2 subunit gamma 8-oxoguanine DNA glyosylase 3.38 Quinone oxidoreductase homolog 3.33 Insulin induced protein Transmembrane tyrosine-specific 3.20 protein kinase (ROS1) Transcription factor 1, hepatic; LFB1, hepatic nuclear factor (HNF1), 3.17 albumin proximal factor 3.11 Heat shock J2 protein; HSJ2 3.06 Tumor necrosis factor 3.02 Death associated protein (DAXX) 3.02 C-myc oncogene Gamma-aminobutyric acid (GABA) 3.00 A receptor, alpha 2.95 BCL2-like V-raf murine sarcoma viral 2.82 oncogene homolog B1; B-raf GA-binding protein transcription 2.80 factor, beta subunit (47kD) 2.76 8-oxoguanine DNA glycosylase NM_001715 Blk 2.70 B lymphoid tyrosine kinase AB017169 SLIT3 2.69 Slit-3 M96577 E2F1 X59798 PRAD1 L33075 IQGAP1 M65217 U29680 HSF2 Protein Kinase CL Mitochondrial transcription factor P53 BCL2-related protein A1 2.60 E2F transcription factor Cyclin D1; PRAD1; parathyroid 2.41 adenomatosis Ras GTPase-activating-like protein; 2.34 IQGAP1 Heat shock transcription factor 2.32 (HSTF2) X01394 TNF M55284 M62810 M14694 2.29 Protein kinase C-L 2.27 Mitochondrial transcription factor 2.26 P53 cellular tumor antigen BCL2-related protein A1; Bfl-1; 2.24 GRS 2.20 Tumor necrosis factor 233 X52104 U24166 p68 EB1 AB023421 APG-1 GATA-binding protein U66075 DEAD/H (Asp-Glu-Ala-Asp/His) box polypeptide (RNA helicase, 2.16 68kD); p68 2.14 EB1 Heat shock protein apg-1 (HSP110 2.06 family) 2.04 GATA-binding protein M93255 Fli-1 2.00 Friend leukemia virus integration Table A6: Primary HCC compared with normal, genes down regulated by at least fold in the Operon array. GeneBank ID AA702737 W72473 Gene Symbol PPP6C PIK3CA N93214 R94360 AA406372 AA700222 AA088857 AA633931 H09613 AA165409 KIAA0318 PRLR ZFX SCAM-1 LIPH ZFP28 CTPS SEMA3D AA702013 SLC22A1 AA417012 AA181978 AA425821 N78927 ZNF336 COQ4 RER1 MYL2 AI654715 W86853 R35941 H68922 AA478752 W46773 PEX16 TIGD2 FGF14 ITGA1 DKK3 TRUB2 AA779415 ZNF11B Fold Long Gene Name Difference 40.07 protein phosphatase 6, catalytic subunit 36.79 phosphoinositide-3-kinase, catalytic, alpha polypeptide 20.75 RIM binding protein 20.65 prolactin receptor 14.60 zinc finger protein, X-linked 14.46 vinexin beta (SH3-containing adaptor molecule-1) 13.59 lipase, member H 13.54 zinc finger protein 28 homolog (mouse) 13.36 CTP synthase 12.89 sema domain, immunoglobulin domain (Ig), short basic domain, secreted, (semaphorin) 3D 12.24 solute carrier family 22 (organic cation transporter), member 11.62 zinc finger protein 336 11.17 coenzyme Q4 homolog (yeast) 11.03 RER1 homolog (S. cerevisiae) 10.76 myosin, light polypeptide 2, regulatory, cardiac, slow 10.74 peroxisomal biogenesis factor 16 10.69 tigger transposable element derived 10.39 **fibroblast growth factor 14 9.90 integrin, alpha 9.14 dickkopf homolog (Xenopus laevis) 9.07 TruB pseudouridine (psi) synthase homolog (E. coli) 8.63 zinc finger protein 11b (KOX 2) 234 AI362062 AA973337 R68463 AA455428 AA035657 AI922402 AA600184 N91246 AA879437 AI241258 AA284589 NOVA1 PCF11 JAM2 SIAH1 LUM GARP OVOS2 TRNT1 RGS5 TINAG TFAP4 8.61 8.56 8.25 8.10 8.08 8.06 7.98 7.91 7.80 7.78 7.68 neuro-oncological ventral antigen pre-mRNA cleavage complex II protein Pcf11 junctional adhesion molecule seven in absentia homolog (Drosophila) lumican glycoprotein A repetitions predominant ovostatin tRNA nucleotidyl transferase, CCA-adding, regulator of G-protein signalling tubulointerstitial nephritis antigen transcription factor AP-4 (activating enhancer binding protein 4) glutathione S-transferase M4 serine/threonine kinase 38 like thyroid hormone receptor, beta (erythroblastic leukemia viral (v-erb-a) oncogene homolog 2, avian) MCF.2 cell line derived transforming sequence thymosin, beta 10 semenogelin II SHB (Src homology domain containing) adaptor protein B CD2-associated protein aldehyde dehydrogenase family, member A1 (succinate-semialdehyde dehydrogenase) AA486570 AA255900 AA022600 GSTM4 STK38L THRB 7.60 7.23 7.21 H05800 AA486085 AI675504 AA427595 MCF2 TMSB10 SEMG2 SHB 7.01 6.89 6.55 6.48 AA173625 H06675 CD2AP ALDH5A1 6.30 6.28 AA708440 AA176453 MYADM NDUFS6 6.21 myeloid-associated differentiation marker 6.16 NADH dehydrogenase (ubiquinone) Fe-S protein 6, 13kDa (NADH-coenzyme Q reductase) AA443688 AI097227 R11979 GCH1 NEURL2 STAU2 AA609640 AA911690 AA468451 AA070357 AA054714 T99311 AA431126 FBXO15 ZCCHC2 ALOX15B TKT KPNA1 MTB CITED1 5.96 GTP cyclohydrolase (dopa-responsive dystonia) 5.88 neuralized-like (Drosophila) 5.87 **staufen, RNA binding protein, homolog (Drosophila) 5.84 F-box protein 15 5.56 zinc finger, CCHC domain containing 5.56 arachidonate 15-lipoxygenase, second type 5.51 transketolase (Wernicke-Korsakoff syndrome) 5.50 karyopherin alpha (importin alpha 5) 5.49 more than blood homolog 5.48 **Cbp/p300-interacting transactivator, with Glu/Asp-rich carboxy-terminal domain, AI796687 SOLH 5.35 small optic lobes homolog (Drosophila) 235 AI004671 R22977 H46487 38231.00 MSN MGAT3 5.26 **strand-exchange protein 5.16 moesin 5.07 mannosyl (beta-1,4-)-glycoprotein beta-1,4-Nacetylglucosaminyltransferase AA719257 AI955961 N73301 W91980 FBS1 VPS41 SRP46 TMPIT 5.04 5.03 4.93 4.82 N71001 AA427490 EIF4EL3 KCNH2 AA485027 AA446565 AA775453 AI023541 AA146826 NAPSB RBM25 CGI-105 CA9 RUNX1 4.64 4.64 4.60 4.60 4.58 AA969508 HEYL H28209 AA778392 AA631098 AA513640 AA443181 AA443982 FABP4 BENE SCLY SCGB2A2 RAB5B PPP1CA AA489700 AA838691 R74467 AA937899 AA431226 CD37 EPHX1 RHOB ZG16 PRPF18 AA464525 AA490462 AI053446 AA284113 AI273242 W37752 IL1R1 AEBP1 IQGAP3 NOTCH3 KIAA0861 CLN3 4.45 hairy/enhancer-of-split related with YRPW motiflike 4.43 fatty acid binding protein 4, adipocyte 4.36 BENE protein 4.36 selenocysteine lyase 4.36 secretoglobin, family 2A, member 4.34 RAB5B, member RAS oncogene family 4.24 protein phosphatase 1, catalytic subunit, alpha isoform 4.13 CD37 antigen 4.13 epoxide hydrolase 1, microsomal (xenobiotic) 4.09 ras homolog gene family, member B 4.03 zymogen granule protein 16 4.03 **PRP18 pre-mRNA processing factor 18 homolog (yeast) 3.90 interleukin receptor, type I 3.84 AE binding protein 3.76 IQ motif containing GTPase activating protein 3.74 Notch homolog (Drosophila) 3.63 Rho family guanine-nucleotide exchange factor 3.62 **ceroid-lipofuscinosis, neuronal 3, juvenile (Batten, Spielmeyer-Vogt disease) fibrosin vacuolar protein sorting 41 (yeast) Splicing factor, arginine/serine-rich, 46kD transmembrane protein induced by tumor necrosis factor alpha 4.73 eukaryotic translation initiation factor 4E-like 4.64 potassium voltage-gated channel, subfamily H (eag-related), member napsin B aspartic peptidase pseudogene RNA binding motif protein 25 CGI-105 protein carbonic anhydrase IX runt-related transcription factor (acute myeloid leukemia 1; aml1 oncogene) 236 AA044151 FABP3 3.55 fatty acid binding protein 3, muscle and heart (mammary-derived growth inhibitor) W46976 H25413 R20106 AA876054 AA719131 LXN PQLC2 WRNIP1 HFE MGAT1 3.55 3.49 3.47 3.44 3.42 latexin PQ loop repeat containing Werner helicase interacting protein hemochromatosis mannosyl (alpha-1,3-)-glycoprotein beta-1,2-Nacetylglucosaminyltransferase AA478606 AA446222 AA100787 AA459392 T61547 AA862529 MDS006 MYO18A GLS RPC155 FBXL15 SUMO3 3.42 3.41 3.33 3.33 3.30 3.29 AA970851 T51539 N75028 AA974248 LRRC19 MSTP9 PSPH SORCS2 3.29 3.28 3.28 3.26 AA132502 AA894437 R12840 TTLL2 BTBD9 FOS 3.20 3.19 3.14 AA873341 AI361560 R26732 W74418 AA430331 W72140 AA010974 AA705306 AA644587 AA487864 PBEF1 HOXC9 PMP22 K6IRS3 SEC31L2 PFKL C7orf9 MLR2 LOC117584 U1SNRNPBP 3.12 3.12 3.11 3.11 3.09 3.08 3.07 3.04 3.00 2.99 x 006 protein myosin XVIIIA glutaminase polymerase (RNA) III (DNA directed) (155kD) F-box and leucine-rich repeat protein 15 **SMT3 suppressor of mif two homolog (yeast) leucine rich repeat containing 19 macrophage stimulating, pseudogene phosphoserine phosphatase sortilin-related VPS10 domain containing receptor tubulin tyrosine ligase-like family, member BTB (POZ) domain containing v-fos FBJ murine osteosarcoma viral oncogene homolog **pre-B-cell colony enhancing factor homeo box C9 peripheral myelin protein 22 keratin irs3 SEC31-like (S. cerevisiae) phosphofructokinase, liver chromosome open reading frame ligand-dependent corepressor fring U11/U12 snRNP 35K AA425545 AA488895 AA626240 AA704995 AA443624 AA188768 RP42 STYX RICS GLYAT ZDHHC8 WRNIP1 2.99 2.98 2.98 2.98 2.97 2.97 RP42 homolog serine/threonine/tyrosine interacting protein Rho GTPase-activating protein glycine-N-acyltransferase zinc finger, DHHC domain containing Werner helicase interacting protein 237 AA921815 SLC22A14 AA137072 AA937342 H05654 AA406362 AA953069 AA664237 AA421099 AA114250 R73909 AA947914 AA001835 AA844818 AA448569 AI652142 AA973391 AA489386 PTPRS SPATS2 CROC4 PTGER3 TPK1 SYNPO SLC41A3 ARMCX2 PSG11 GRIA3 ZNF262 AMY2B SRPX ACACA CAS1 QKI AI668612 AA088748 AA425934 AA278169 PLA2R1 CIDE-3 S100A1 THAP1 AI358848 AA434420 AI003528 MARS PTPN9 AP1G2 AI522311 H73241 AA670382 AA278426 T68274 AA447797 AI055825 ZNF237 VIPR1 MAP1B ZNF469 C8B PLAT FCER2 N95433 AI023726 AA969969 AI094626 AA431988 SNX9 ZNF335 PAOX OSBPL6 FAAH 2.89 solute carrier family 22 (organic cation transporter), member 14 2.87 protein tyrosine phosphatase, receptor type, S 2.87 spermatogenesis associated, serine-rich 2.86 transcriptional activator of the c-fos promoter 2.84 prostaglandin E receptor (subtype EP3) 2.77 thiamin pyrophosphokinase 2.72 synaptopodin 2.71 solute carrier family 41, member 2.71 armadillo repeat containing, X-linked 2.67 pregnancy specific beta-1-glycoprotein 11 2.66 glutamate receptor, ionotrophic, AMPA 2.66 zinc finger protein 262 2.66 amylase, alpha 2B; pancreatic 2.65 sushi-repeat-containing protein, X-linked 2.62 acetyl-Coenzyme A carboxylase alpha 2.61 O-acetyltransferase 2.58 quaking homolog, KH domain RNA binding (mouse) 2.57 phospholipase A2 receptor 1, 180kDa 2.57 cell death activator CIDE-3 2.54 S100 calcium binding protein A1 2.52 THAP domain containing, apoptosis associated protein 2.50 methionine-tRNA synthetase 2.49 protein tyrosine phosphatase, non-receptor type 2.48 adaptor-related protein complex 1, gamma subunit 2.47 zinc finger protein 237 2.45 vasoactive intestinal peptide receptor 2.45 microtubule-associated protein 1B 2.44 zinc finger protein 469 2.43 complement component 8, beta polypeptide 2.43 plasminogen activator, tissue 2.42 Fc fragment of IgE, low affinity II, receptor for (CD23A) 2.42 sorting nexin 2.40 zinc finger protein 335 2.40 polyamine oxidase (exo-N4-amino) 2.39 oxysterol binding protein-like 2.37 fatty acid amide hydrolase 238 AA450055 AA487527 AA458982 T57877 ING1 MPEG1 SCNN1A 2.36 2.24 2.23 2.20 AA393596 PPP2R5E 2.14 R25016 TNFRSF7 2.14 AA463188 T68859 NEK6 SERPINF2 2.13 2.12 W92764 AA406285 TNFAIP6 DRAP1 2.09 2.08 AI668571 ATP6V1B1 2.05 AA043416 CES1 2.02 inhibitor of growth family, member macrophage expressed gene sodium channel, nonvoltage-gated alpha Similar to Zinc finger protein 20 (Zinc finger protein KOX13) (DKFZp572P0920) (LOC342970), mRNA protein phosphatase 2, regulatory subunit B (B56), epsilon isoform tumor necrosis factor receptor superfamily, member NIMA (never in mitosis gene a)-related kinase serine (or cysteine) proteinase inhibitor, clade F (alpha-2 antiplasmin, pigment epithelium derived factor), member tumor necrosis factor, alpha-induced protein DR1-associated protein (negative cofactor alpha) ATPase, H+ transporting, lysosomal 56/58kDa, V1 subunit B, isoform (Renal tubular acidosis with deafness) carboxylesterase (monocyte/macrophage serine esterase 1) Table A7: Primary HCC compared with normal, genes up regulated by at least fold in the Stanford array. Gene Bank ID R24333 Gene Symbol FLJ20516 Fold difference Long Gene Name 8.42 timeless-interacting protein likely ortholog of rat vacuole membrane 7.94 protein fusion (involved in t(12;16) in malignant 7.80 liposarcoma) AA485373 VMP1 AA025165 FUS AA644092 AI049502 AA448395 NME1 TUSC3 HSPE1 non-metastatic cells 1, protein (NM23A) 6.06 expressed in 5.90 tumor suppressor candidate 5.76 **heat shock 10kDa protein (chaperonin 10) AW004605 POLR2K polymerase (RNA) II (DNA directed) 5.35 polypeptide K, 7.0kDa AA487521 AA100145 GOT2 STX16 glutamic-oxaloacetic transaminase 2, 4.94 mitochondrial (aspartate aminotransferase 2) 4.83 syntaxin 16 239 AA460685 N66278 AA468759 AA489583 AA134861 BIRC5 JUND SPEC1 PEMT IRS2 4.83 4.76 4.66 4.60 4.58 AA455786 MCM3 MCM3 minichromosome maintenance 4.42 deficient (S. cerevisiae) AA454935 NRF1 AA625632 baculoviral IAP repeat-containing (survivin) jun D proto-oncogene **small protein effector of Cdc42 phosphatidylethanolamine N-methyltransferase insulin receptor substrate 4.34 nuclear respiratory factor Similar to similarity to monoubiquitin/carboxy-extension protein 4.21 fusion (LOC149329), mRNA AA452531 T66154 AA668811 QKI LOC401152 H3F3A **quaking homolog, KH domain RNA binding 4.13 (mouse) 4.01 HCV F-transactivated protein 3.97 H3 histone, family 3A AA598863 EIF3S8 eukaryotic translation initiation factor 3, 3.94 subunit 8, 110kDa AA664241 AA983905 AI015641 NACA WDR4 SDC1 nascent-polypeptide-associated complex alpha 3.92 polypeptide 3.88 WD repeat domain 3.84 syndecan H85355 ATP2A2 ATPase, Ca++ transporting, cardiac muscle, 3.75 slow twitch AA490256 AI366195 R63318 N21329 AA284265 GNAI3 SRP68 ZNF444 TFRC e(y)2 3.73 3.72 3.67 3.65 3.64 AA477226 AA708342 LASS2 ENO1 guanine nucleotide binding protein (G protein), alpha inhibiting activity polypeptide signal recognition particle 68kDa zinc finger protein 444 transferrin receptor (p90, CD71) e(y)2 protein LAG1 longevity assurance homolog (S. 3.61 cerevisiae) 3.54 enolase 1, (alpha) AA541802 AA291772 AI333932 AA676836 AI359981 B3GNT5 TETRAN ZFP106 SMPDL3A SOX3 3.51 3.51 3.49 3.47 3.39 AA629897 LAMR1 **UDP-GlcNAc:betaGal beta-1,3-Nacetylglucosaminyltransferase tetracycline transporter-like protein zinc finger protein 106 homolog (mouse) sphingomyelin phosphodiesterase, acid-like 3A SRY (sex determining region Y)-box laminin receptor (ribosomal protein SA, 3.39 67kDa) 240 LAG1 longevity assurance homolog (S. 3.38 cerevisiae) AA699770 LASS2 W81684 TCEB1 AI346026 PLEKHA1 AA452095 SMC4L1 R26186 PPP1CB AA448164 AA115186 AA664067 AA427468 KBTBD2 GTF2I HSBP1 CLDN4 AA485965 W96163 N53133 SUCLG1 TOB1 STRBP **SMC4 structural maintenance of 3.27 chromosomes 4-like (yeast) protein phosphatase 1, catalytic subunit, beta 3.24 isoform kelch repeat and BTB (POZ) domain 3.23 containing 3.22 **general transcription factor II, i 3.21 heat shock factor binding protein 3.19 claudin succinate-CoA ligase, GDP-forming, alpha 3.19 subunit 3.19 transducer of ERBB2, 3.18 spermatid perinuclear RNA binding protein AA100289 AA159620 T72088 T63046 AA129397 T40899 AA292964 AA188647 AA535703 H05489 N24059 AA044993 PSME2 EVI2B AGXT2 ITM2B DAZ4 TAS2R14 CKS2 TRIM33 REG4 DCK MDN1 CTGF 3.18 3.18 3.18 3.16 3.15 3.14 3.12 3.12 3.11 3.09 3.07 3.06 AA150505 T56221 C1QR1 MT1F complement component 1, q subcomponent, 3.06 receptor 3.06 metallothionein 1F (functional) AI985214 TFPI tissue factor pathway inhibitor (lipoprotein3.01 associated coagulation inhibitor) AA465237 PSMA3 proteasome (prosome, macropain) subunit, 3.00 alpha type, AW006596 AA495935 CS MGST1 2.98 citrate synthase 2.97 microsomal glutathione S-transferase transcription elongation factor B (SIII), 3.29 polypeptide (15kDa, elongin C) pleckstrin homology domain containing, family A (phosphoinositide binding specific) 3.28 member proteasome (prosome, macropain) activator subunit (PA28 beta) ecotropic viral integration site 2B alanine-glyoxylate aminotransferase integral membrane protein 2B deleted in azoospermia taste receptor, type 2, member 14 CDC28 protein kinase regulatory subunit tripartite motif-containing 33 regenerating islet-derived family, member deoxycytidine kinase MDN1, midasin homolog (yeast) connective tissue growth factor 241 AA608514 H3F3B 2.97 H3 histone, family 3B (H3.3B) AA922326 AA454585 NDUFV2 SFRS2 NADH dehydrogenase (ubiquinone) 2.95 flavoprotein 2, 24kDa 2.95 splicing factor, arginine/serine-rich AA455544 PSK-1 type I transmembrane receptor (seizure-related 2.95 protein) H26841 R38685 UBE2D3 MI-ER1 ubiquitin-conjugating enzyme E2D (UBC4/5 2.95 homolog, yeast) 2.91 **mesoderm induction early response R27585 R87699 AA418670 T57556 AA126913 AI343840 H37989 R83223 PSMA1 ADAM11 JUND HINT1 GMDS TERF2 OK/SW-cl.56 RIN2 2.91 2.89 2.89 2.87 2.86 2.85 2.85 2.82 proteasome (prosome, macropain) subunit, alpha type, a disintegrin and metalloproteinase domain 11 jun D proto-oncogene histidine triad nucleotide binding protein GDP-mannose 4,6-dehydratase telomeric repeat binding factor beta 5-tubulin Ras and Rab interactor AA488782 AA452257 AA961071 AA196465 AA169355 AA661722 AA775509 ROCK1 CKLFSF5 RPN1 SLN TTC17 FLJ23471 PERP 2.81 2.81 2.76 2.75 2.74 2.72 2.72 Rho-associated, coiled-coil containing protein kinase chemokine-like factor super family ribophorin I sarcolipin **tetratricopeptide repeat domain 17 MICAL-like PERP, TP53 apoptosis effector AW074796 COMTD1 catechol-O-methyltransferase domain 2.72 containing AA489245 AA040169 N50654 MAPK8IP3 ACTA2 CP AA159669 N64706 VMP1 ZNF526 **mitogen-activated protein kinase 2.72 interacting protein 2.70 actin, alpha 2, smooth muscle, aorta 2.70 ceruloplasmin (ferroxidase) likely ortholog of rat vacuole membrane 2.68 protein 2.66 **zinc finger protein 526 AA043679 RAB7 2.65 RAB7, member RAS oncogene family T63998 AA598486 GNA11 SAE1 guanine nucleotide binding protein (G protein), 2.65 alpha 11 (Gq class) 2.64 SUMO-1 activating enzyme subunit 242 AA994038 AA599102 CLDN12 Similar to ataxin binding protein isoform gamma; hexaribonucleotide binding protein 2.57 (LOC339162), mRNA 2.56 claudin 12 N62348 AI732296 COL4A3BP ANXA2 collagen, type IV, alpha (Goodpasture 2.54 antigen) binding protein 2.53 annexin A2 T47814 PSME1 proteasome (prosome, macropain) activator 2.52 subunit (PA28 alpha) N93865 EPB41 H25560 DGAT2 erythrocyte membrane protein band 4.1 2.50 (elliptocytosis 1, RH-linked) diacylglycerol O-acyltransferase homolog 2.49 (mouse) AI017237 SLC39A4 **solute carrier family 39 (zinc transporter), 2.46 member AA983252 AA598754 AA708905 W80692 AA455942 STAT2 PEG10 WASF2 SEPHS1 WWP1 2.45 2.42 2.40 2.39 2.39 AA434024 R56055 AA081106 H89996 N50859 LSS TUB SLC38A2 CTCF TNIP2 2.38 2.38 2.36 2.34 2.33 AA458661 W73892 AA916752 AA953747 HADH2 RBM5 VPS35 PLS3 2.32 2.32 2.31 2.30 R36515 AA699707 H29484 T53219 AA434408 AA788767 GJA5 FNBP1 SSB FABP1 CCNI GGCX 2.30 2.29 2.29 2.29 2.29 2.29 signal transducer and activator of transcription 2, 113kDa **paternally expressed 10 WAS protein family, member selenophosphate synthetase WW domain-containing protein lanosterol synthase (2,3-oxidosqualenelanosterol cyclase) **tubby homolog (mouse) solute carrier family 38, member CCCTC-binding factor (zinc finger protein) TNFAIP3 interacting protein hydroxyacyl-Coenzyme A dehydrogenase, type II RNA binding motif protein vacuolar protein sorting 35 (yeast) plastin (T isoform) gap junction protein, alpha 5, 40kDa (connexin 40) formin binding protein Sjogren syndrome antigen B (autoantigen La) fatty acid binding protein 1, liver cyclin I gamma-glutamyl carboxylase 243 T47454 AA416685 AA864681 AI820509 TFPI UNC13B MAP2K7 PB1 2.28 2.26 2.21 2.21 tissue factor pathway inhibitor (lipoproteinassociated coagulation inhibitor) unc-13 homolog B (C. elegans) mitogen-activated protein kinase kinase polybromo AW006512 AA481248 AA193380 AA679454 AI636025 AI246829 N66948 AA599574 H50238 AI061063 PSME3 CGGBP1 AOF2 STAR ZBTB11 SDCCAG1 LOC56902 LIPG LIPE MLH3 2.21 2.21 2.20 2.19 2.19 2.18 2.18 2.17 2.16 2.16 proteasome (prosome, macropain) activator subunit (PA28 gamma; Ki) CGG triplet repeat binding protein amine oxidase (flavin containing) domain steroidogenic acute regulator zinc finger and BTB domain containing 11 serologically defined colon cancer antigen putatative 28 kDa protein lipase, endothelial lipase, hormone-sensitive **mutL homolog (E. coli) AA045463 AA190871 SPARC ALPI R00496 AA609218 AA664009 GALNT2 DNAI1 SCP2 secreted protein, acidic, cysteine-rich 2.16 (osteonectin) 2.16 alkaline phosphatase, intestinal UDP-N-acetyl-alpha-Dgalactosamine:polypeptide Nacetylgalactosaminyltransferase (GalNAc2.15 T2) 2.15 dynein, axonemal, intermediate polypeptide 2.14 sterol carrier protein AA070618 AI024999 H37817 H51065 PSMB6 VCP USP42 LEPR 2.14 2.13 2.12 2.11 AA609598 AA490721 AA872402 AI935290 YWHAZ SFRS9 EIF4B CSRP1 2.11 2.10 2.10 2.10 T49984 AA991590 W85854 DKFZP586A0522 APOC1 EBPL 2.10 DKFZP586A0522 protein 2.10 apolipoprotein C-I 2.09 emopamil binding protein-like AA449362 CHD2 chromodomain helicase DNA binding protein 2.09 proteasome (prosome, macropain) subunit, beta type, valosin-containing protein ubiquitin specific protease 42 leptin receptor tyrosine 3-monooxygenase/tryptophan 5monooxygenase activation protein, zeta polypeptide splicing factor, arginine/serine-rich eukaryotic translation initiation factor 4B cysteine and glycine-rich protein 244 AI452900 JPH2 AI300352 R52964 AA172012 AA190558 GPD1 PPHLN1 GNPDA2 NUP133 2.08 junctophilin glycerol-3-phosphate dehydrogenase 2.07 (soluble) 2.06 periphilin 2.05 glucosamine-6-phosphate deaminase 2.05 nucleoporin 133kDa N92443 UBTF **upstream binding transcription factor, RNA 2.05 polymerase I AA599741 AA626327 AA583574 SLICK TTC15 S100A7 N59089 AA504272 AA046090 HNRPM FABP4 sodium- and chloride-activated ATP-sensitive 2.05 potassium channel 2.05 tetratricopeptide repeat domain 15 2.04 S100 calcium binding protein A7 (psoriasin 1) **Human HepG2 partial cDNA, clone 2.04 hmd5d04m5. 2.02 heterogeneous nuclear ribonucleoprotein M 2.01 fatty acid binding protein 4, adipocyte AI083844 AA435999 NFIX LACTB2 nuclear factor I/X (CCAAT-binding 2.01 transcription factor) 2.01 lactamase, beta Table A8: Primary HCC compared with normal, genes down regulated by at least fold in the Stanford array. 245 [...]... calmodulin is dependent on intracellular calcium concentrations This couples NO synthesis to physiological stimuli This is not so for inducible NOS or iNOS where calmodulin is bound tightly regardless of intracellular calcium levels Calmodulin binding however does not affect the binding affinity of NOS for arginine, implying that arginine has a discrete active site (Su et al., 1995) 1.3 Nitric oxide. .. homology, indicating that NOS is highly conserved (Nathan et al., 1994b) NOS incorporates several domains in a single polypeptide: the P450-like haem domain that binds a tetrahydrobiopterin (BH4) cofactor and is the site of oxidation of arginine to nitric oxide and citrulline; a P450-reductase domain that has binding sites for FMN, FAD, and NADPH and is responsible for providing electrons to the haem domain,... formation In contrast, the systemic and liver specific NO donors, both showed dramatic improvement in reducing neoplasm formation This could be used as a platform for examining the therapeutic potential of NO donors in the treatment of HCC 11 Chapter 1: Introduction 1.1 Liver Cancer The liver, or hēpar in Greek, is one of the largest glandular organs in vertebrates including humans In humans, the liver. .. Enzyme also found in bone 0-3 Enzyme used to determine cholestatic damage if ALP levels are increased 12-15 Clotting assay; increased clotting time indicative of seconds abnormal levels of clotting factors, including vitamin K, which are specifically synthesized in the liver 300-700 Enzyme not exclusive to the liver, found in heart and U/L muscle, but elevated levels indicative of liver damage when... threatening Hippocrates later added the suffix –oma, Greek for swelling, resulting in carcinoma, often used now to describe cancers deriving from cells of the epithelium or endothelium 12 Primary liver cancer, twice as common in men as in women, has two main cellular origins Cholangiocarcinoma, arises from bile duct cells, while hepatocellular carcinoma (HCC) arises from hepatocytes, the main cell... L-citrulline Figure 1.3: Reaction catalyzed by NOS Substrates include L-arginine, oxygen and NADPH, forming L-citrulline and NO 5 other co-factors are involved and each has a specific binding site on the enzyme The availability of these co-factors also plays a part in determining the activity of the enzyme This reaction is part of amino acid metabolism where L-citrulline can be converted back to L-arginine,... Calmodulin binding site FMN binding site b) Reductase Domain Oxygenase Domain CaM FMN FAD NADPH Figure 1.4: Structural similarities between the NOS isoforms a) The various domains found on the isoforms of NOS b) A dimer formation of NOS The oxygenase domain is linked via a helical peptide to the reductase domain Binding of calmodulin acts as a ‘switch’, enabling electron flow from FAD to the haem group in. .. connecting peptide between the haem and flavin domains that binds calmodulin in a Ca2+-dependent manner for eNOS and nNOS forms and essentially Ca2+-independent manner in iNOS (Nathan et al., 1994b) Figure 1.4 illustrates the many domains of NOS and its dimer formation 33 Oxygenase domain Reductase domain a) nNOS iNOS eNOS FAD NH2NH2- FAD NADPH FAD NH2- -COOH NADPH NADPH -COOH -COOH ARG/Heme/BH4 binding... bilirubin or direct bilirubin is measured to determine bile duct obstruction 24-53 Hepatocyte specific enzyme that is released into the blood stream upon liver damage; increased levels indicative of liver damage 77-157 An enzyme found in liver, red blood cells and muscle; increased levels may be indicative of liver damage 132-312 Enzyme found in bile ducts; excessive levels in blood indicative of bile duct... Lcitrulline, via the formation of an intermediate: N ω-hydroxy- L-arginine This involves a total transfer of 5 electrons, 3 substrates, namely L-arginine, molecular oxygen and NADPH, and 5 prosthetic groups (Bruckdorfer, 2005) All this is catalysed by the nitric oxide synthase (NOS) A simplified diagram of this reaction is shown in figure 1.3 NADPH ½NADPH O2 L-arginine + NO⋅ O2 N-hydroxy-L-arginine +H2O . 12 Chapter 1: Introduction 1.1 Liver Cancer The liver, or hēpar in Greek, is one of the largest glandular organs in vertebrates including humans. In humans, the liver contains more than 10%. Albumin 4.0-4.8 Protein specifically synthesized in the liver; low levels indicative of impaired liver function /liver damage. Total Bilirubin 5-30 umol/L Bilirubin is a product in the clearing. Hepatocellular Carcinoma 84% Cholangiocarcinoma 14% Fibrolamellar Carcinoma 1% Angiosarcoma 1% Primary liver cancer, twice as common in men as in women, has two main cellular origins. Cholangiocarcinoma,