PRESENTED BY: DHARTI SHAH DEPARTMENT OF NUTRITION AND FOOD SCIENCES TEXAS WOMAN’S UNIVERSITY DENTON, TEXAS The Role of Isoprenoids in Liver Cancer: A Review

SIGNIFICANCE OF THE STUDY Most rapidly increasing cancer type Increased incidence in developed countries Tumor suppressive effects of isoprenoids have been shown in many cancers A comprehensive review would provide a good insight on the use of isoprenoids in liver cancer

METHODS Literature search was carried out using research databases Key words: o Mevalonate pathway o Liver cancer  HBV, HCV, Aflatoxins o Molecular mechanisms in liver cancer o Isoprenoids  Farnesol  Geraniol  d-limonene, Perillyl alcohol  geranylgeraniol

INTRODUCTION Cancer - second leading cause of death in U.S. Estimated new cases and deaths in U.S. (all cancers) o 2008 – 1,437,920; 565,650 Liver cancer – 5 th most common cancer o 2007 – 19,160; 16,780 o 2008 – 21,370; 18,410 5 year survival rate is < 3% ACS Lencioni. et. al., Ital. J Gastroenterol, 1994http://

LIVER CANCER Primary liver cancers (PLC) o Hepatocellular carcinoma (HCC) – hepatocytes, most common type o Cholangiocarcinoma (CCA) – cells of intrahepatic bile ducts Global incidence o > 80% of all cases in developing countries o Increased incidence seen in developed countries Srivatanakul et. al., Asian Pacific J Cancer Prev.,2004

INCIDENCE IN UNITED STATES El-Serag et. al. confirm an increase in the number of HCC cases from o Study period divided into 7 3-year periods o Age groups (20-49, 50-64, 65-74, or ≥ 75 yrs) Results o 2-fold increase in incidence of HCC between  Highest incidence seen among Asian migrants and Blacks  25% increase during the last 3 year period – Whites affected the most o Contributing factors  HBV - Asian countries (Southeast Asia)  HCV – Black and White people  Needle sharing, drug use, blood transfusions El-Serag et. al., Intern. Med., 2003

ETIOLOGY OF LIVER CANCER Major contributing factors o Hepatitis B (HBV) o Hepatitis C (HCV) o Aflatoxin-B1 Minor contributing factors o Chronic alcoholism Cirrhosis HCC o Obesity Hepatic steatosis oxidative stress o Diabetes o Anabolic steroids o Toxins – arsenic, vinyl chloride ACS ncer_25.asp?sitearea ncer_25.asp?sitearea

HEPATITIS B X protein (HBX) HBV DNA fuses with cellular DNA – gene expression Feitelson, Hum. Pathol., 2004 Tumor suppressive activity

HEPATITIS C (HCV) Core protein HCV core protein upregulates many oncogenic genes, including c-myc Hepatic steatosis – an independent risk factor in HCC oxidation Moriya et. al., J. Gen. Virol., 1997; Moriya et. al., Nat. Med., 1998; Ohata et. al., Cancer, 2003

Aflatoxin-B 1 Viral agents and environmental carcinogens - cancer Aflatoxin exposure ---- G to T transversion in codon 249 of p53 HBV + aflatoxin interaction may be synergistic o Liver injury as a result of HBV is a critical factor  Increased expression of cytochrome P450’s that metabolize carcinogens Wild et. al., Environ. Health Persp., 1993; Eaton and Gallagher, Annu. Rev. Pharmacol. Toxicol, 1994; Hussain et. al., Oncogene, 1994

MOLECULAR EVENTS IN CANCER

CARCINOGENESIS Murukami, et.al., Cancer Res., 1993 Malignancy ProtooncogenesTumor suppressive genesGrowth factors

OVERVIEW OF LIVER CANCER Hepatocytes altered phenotypically Feitelson, Hum. Pathol., 2004; Anthony, J. Clin. Path., 1973; Thorgeirsson et. al., Nat. Genet., 2002

Protooncogenes and growth factors c-myc transcription factor cell growth o Early event in hepatocarcinogenesis o Hypomethylation of c-myc o c-myc transgenic mice  Dysplastic changes – 2 months, well developed HCC – months TGF-α active liver regeneration o Tumor progression o TGF-α transgenic mice  Multifocal, well differentiated HCC – months Bhave et.al., Carcinogenesis, 1988; Sandgren et.al.,Oncogene,1989

Co-expression (c-myc-TGF-α) Dysplastic changes in hepatocytes Apoptosis of healthy liver cells Murukami et.al.,Cancer Res.,1993; Factor et.al.,Hepatology,2001 phosphorylated forms of ras and phosphoinositol-3 kinase IĸB kinase (phosphorylated) Nuclear factor κB Apoptosis

C-myc-TGF-β1 and Hedgehog pathway Overexpression of TGF-β1 or c-myc/TGF-β1 o sensitivity of cells to TGFβ1  Overexpression of c-myc and loss of TGF-β II receptors  HBV X and HCV core protein interfere with function of TGF-β1 Hedgehog pathway o Cell differentiation o expression of two target genes  PTCH1 and Gli1 Alexandrow et.al.,Cancer Res.,1995; Fynn et.al.,Crit.Rev.Oncog.,1993; Tsuchihara et.al.,Virology,1999; Huang et.al.,Carcinogenesis,2006

TNF-α Nuclear factor-κB (NF-κB) Proapoptotic genes DNA damage Arsura and Cavin,Cancer Lett.,2005 Anti-apoptotic genes Carcinogens, viruses, growth factors NF-κB α-fetoprotein (AFP) Cell survival

NF-κB’s role in liver cancer Rel/NF-κB (cytoplasm) transcription factors o Role studied in HBV positive Hep 3B cells TGF-α Chiao et.al.,Cancer,2002 releases Rel/NF-κB IκBα (phosphorylated) Rel/NF-κB (cytoplasm)Rel/NF-κB (nucleus) cell survival IĸBα

c-Jun c-Jun component of activator protein 1 (AP-1) o Cell survival, cell cycle progression o After initiation stage of tumor development Oncogenic activity of c-Jun o N-terminal phosphorylation o Interaction with Ras o Suppression of p53 o TNF-α induced NF-κB activity Johnson et. al., Mol. Cell Biol., 1996; Eferl et. al., J. Cell Biol., 1999; Eferl et.al., Cell, 2003; Schreiber et. al., Genes Dev., 1999

Angiogenesis Multistep and a well coordinated process Vascular endothelium specific angiogenic factors Vascular endothelial growth factors (VEGF) Angiopoietin familyEphrin family Endothelium Angiogenesis Rac Hypoxia inducible factor 1α NF-κB Interleukin-6,8 Yancopoulas et. al., Nature, 2000; Arsura and Cavin, Cancer lett, 2005; Lee et.al., Clin. Cancer Res., 2006

MEVALONATE PATHWAY IN NORMAL CELLS Acetyl CoA HMG CoA Mevalonate Geranyl-PP Farnesyl-PP Cholesterol Protein prenylation Ras, lamin B Cell growth FTase HMG CoA reductase Brown and Goldstein,Nature,1990; Goldstein et.al.,Cell,2006 Farnesol Isopentenyl-5-PPDimethylallyl-PP Isopentenyl-PP isomerase

SREBP pathway

MEVALONATE PATHWAY IN NORMAL CELLS Acetyl CoA HMG CoA Mevalonate Geranyl-PP Farnesyl-PP Cholesterol Protein prenylation Ras, lamin B Cell growth FTase HMG CoA reductase Brown and Goldstein,Nature,1990; Goldstein et.al.,Cell,2006 Farnesol Isopentenyl-5-PPDimethylallyl-PP Isopentenyl-PP isomerase

MEVALONATE PATHWAY IN CANCER CELLS Acetyl CoA HMG CoA Mevalonate Geranyl-PP Farnesyl-PP Cholesterol Protein prenylation Ras, lamin B Cell growth FTase HMG CoA reductase Brown and Goldstein,Nature,1990; Goldstein et.al.,Cell,2006 Farnesol Isopentenyl-5-PPDimethylallyl-PP Isopentenyl-PP isomerase

DYSREGULATED PATHWAY Reductase lacks active site or no conformational change occurs Hypomethylation of reductase gene Mutation in SCAP protein o Transition at one of the codons of SCAP Increase in active form of HMG CoA reductase o Altered enzyme kinase - phosphatase system Siperstein and Fagan,Cancer Res.,1964; Vasudevan et.al.,FASEB J.,1994; Goldstein et.al.,Cell,2006; Kawata et.al.,Cancer Res.,1990

Reductase regulation in Hep G2 cells Acetyl CoA HMG CoA Mevalonate Geranyl-PP Farnesyl-PP Cholesterol HMG CoA reductase Farnesol Isopentenyl-5-PP Compactin U18666A Enzyme activity Enzyme mRNA content Cohen and Griffioen, Biochem. J.,1998

ISOPRENOIDS AND CANCER Secondary metabolites of mevalonate metabolism in plants Five carbon isoprene units ISOPRENOIDS PureMixed Monoterpenes – d-imonene (orange peel oil), Perillyl alcohol (lavender, cherries), Geraniol (berries) Diterpenes – G eranylgeraniol Sesquiterpenes – F arnesol Isoflavones Tocotrienols (vegetable oils, whole grains) Blocking and suppressing agents Elson and Yu, J. Nutr.,1994; Elson et. al., Proc. Soc.Exp.Biol.Med., 1999

Mechanism of action Isoprenoids o Cell cycle arrest o Apoptosis o Degradation of HMG-CoA reductase o Anti-angiogenic o Inhibition of protein isoprenylation

CELL CYCLE ARREST Perillyl alcohol (POH), d-limonene, geraniol Mo and Elson, Nutritional Oncology, 2006; Clark, Oncology, 2006; Packham and Cleaveland, Mol. Cell Biol., 1994 G1 S G2 M Cyclin/cdk complex c-myc Mitosis DNA replication

APOPTOSIS Geranylgeraniol (GGOH), Perillyl alcohol (POH) Proapoptotic proteins (Bad, Bax) Anti-apoptotic proteins (Bcl-2, Bcl-XL) Caspase activity Apoptosis Masuda et.al., Leuk. Res., 2000

HMG CoA reductase inhibitors Acetyl CoA HMG CoA Mevalonate Geranyl-PP Farnesyl-PP Cholesterol Protein prenylation Ras, lamin B Cell growth FTase HMG CoA reductase Crowell et.al., J. Biol. Chem., 1991; Hohl and Lewis, J. Biol. Chem., 1995; Keller et.al., Arch. Biochem. Biophys., 1996 Farnesol Translation Degradation Translation Farnesol d-limonene Perillyl alcohol Geranylgeraniol Geranylgeranyl-PP

Monoterpenes Perillyl alcohol (POH) mannose-6-phosphate/insulin growth factor II receptor TGF-β type I, II and III receptors in tumor cells Mills et.al., Cancer Res., 1995 Male Fischer rats diethylnitrosamine (DEN) 1 month – induced liver tumors Control (n=11) POH treated (n=10) 1% w/w POH – 1 st week 2% w/w POH – 19 weeks (Powdered diet) Mean tumor mass0.8g 7.0g Apoptotic index 5-fold – large tumors 10-fold – small tumors LowHigh

ANTI-ANGIOGENIC ROLE Anti-angiogenic function – Perillyl alcohol (POH) Rac – changes in cell morphology POH inhibits Rac interaction with membranes p21 activated kinase – cell migration Angiogenic factors o VEGF – cell migration and cell survival POH decreases release of VEGF, increases Ang2 o Ang2 – neovascularization Capillary formation Blood vessel formation Loutari et.al., J. Pharmacol. Exp. Ther., 2004; Connolly et.al., Mol. Biol. Cell, 2002

Monoterpenes d-limonene Mechanism - Apoptosis and decreased cell proliferation Kaji et. al., Int. J. Cancer, 2001 Sprague-Dawley ratsN-nitrosomorpholine (NNM) 8 weeks Group 1 (n=20) Chow pellets Group 2 (n=20) Chow pellets + 1% d-limonene Group 3 (n=60) Chow pellets + 2% d-limonene GST-P positive foci and lesions Apoptotic index Membrane associated p21 ras No significant effects

Monoterpenes Geraniol o Results Tumor volumes in experimental rats was 20% that of control rats o Mechanism  Apoptosis Yu et.al., J. Nutr., 1995 Morris 7777 hepatomasMale buffalo rats Control (n=6) AIN-76A Experimental (AIN-76A + geraniol 350μmol/d) Diet – 14 days before and 42 days after tumor transplant

Diterpenes – Geranylgeraniol (GGOH) HuH-7 cells GGOH (1-50μmol/L) CADICAD Nucleus DNA fragmentation Caspase-8 Caspase-3 Cytochrome-c Caspase-9 cleaves Bcl-XL Bid + Bax - COOH BH3 domain apaf1 Takeda et. al., Jpn. J. Cancer Res., 2001; Enari et. al., Nature, 1998; Wang et. al., Genes &Dev., 1996; Luo et. al., Cell, 1998

Sesquiterpenes Farnesol o Mechanism - FOH – inhibited HMG CoA reductase; GOH induced apoptosis Ong et.al., Carcinogenesis, 2006 Male Wistar ratsInitiated – DEN, 2-AAF Corn oil( n=12) 0.25 ml/100g FOH (n=12) 25mg/100g GOH (n=12) 25mg/100g Incidence + number of nodules Mean area of PNL’s % liver section Occupied by PNL’s Apoptosis

MIXED ISOPRENOIDS Tocotrienols In vivoIn vitro C3H/He – spontaneous liver tumor Experimental (n=14) T3 mixture (2.25mg/d) γ, α, δ Control (n=17) 7.6 tumors/mouse1.4 tumors/mouse Hep G2 cells DMSO γ, δ isoforms (3 days) Reduced cell viability IC50 conc. – γ – 27.4µM δ – 9.6 µM apoptosis 100 µM – 4 hours conc. of δ isoforms 72 hours ControlExperimental S-phase arrest Cell proliferation Anti-carcinogenic effect

SYNTHETIC ISOPRENOIDS Contain a farnesol or geraniol molecule o More potent  Geranylgaranoic acid (GGA)  4,5 didehydro GGA Mo and Elson, Exp. Biol. Med., 2004

SYNTHETIC ISOPRENOIDS HuH-7 cells Geranylgeranoic acid 10 µM Interleukin-1-β converting enzymeCysteine protease precursor 32 APOPTOSIS TGF-α + EGF Shidoji et. al., Biochem. Biophys. Res. Commun., 1997; Enari et. al., Nature, 1996; Nakamura et. al., Biochem. Biophys. Res. Commun., 1996

SUMMARY Isoprenoids in liver cancer o Liver tumor regression achieved chiefly by apoptosis o Decrease cell proliferation and tumor growth  Inhibition of protein isoprenylation  Degradation of reductase

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