Induction of Hepatocellular Carcinoma and treatment

Different lobes in LIVER

1)Right lobe2)Left lobe3)Caudate lobe4)Quadrate lobe

Functions of liver

– Storage of Nutrients– Breakdown of erythrocytes– Bile Secretion– Synthesis of plasma Proteins– Synthesis of cholesterol

Types of cells present in Liver

Hepatic cells Non-hepatic cells

Stromal cells,epithelial ,endothelial,Liver progeniator cells

•Hepatocellular carcinoma (HCC) is the most common

primary malignancy of the liver.

• Approximately 500,000 new patients are diagnosed with

HCC each year

• The highest incidence of HCC is found in Asia and sub-

Saharan Africa.

•Hepatocellular Carcinoma is a primary cancer meaning it

originated in the Liver

• It is commonly associated with Cirrhosis and Hepatitis.

Introduction

(1) cirrhosis(2) chronic HBV infection(3) chronic HCV infection(4)Exposure to the food-borne

mycotoxin ,aflatoxin B1

The four major risk factors for HCC include

Intracellular pathways involved in HCC formation and development

•DEN

•DEN +Phenobarbital

•DEN+ Ccl4

•Choline deficient diet

•Thioacetamide (organosulfur compound)

•Aflotoxins (Aspergillusflavus and Aspergillus parasiticus)

cassava, chili peppers, corn, cotton

seed, millet, peanuts, rice, sorghum,sunflower seeds, tree

nuts ,wheat

Inducer of HCC in Mice

DEN is hyroxylated mediated by cytochrome p450 α-hydroxylnitrosamine

ethyldiazonium

causes DNA damage

Time to develop

% of mice with HCC

metastasis

Remarks

DEN Single Dose

45-104 Weeks

80-100% in Male

/ Poorly reproducible

Short term Dose

40-60 Weeks 100%in male /

Long term Dose

20-35 weeks 30% in female YES H-ras mutation

+ PB 20-40 weeks / B-catanin activation

CCl4 104 weeks 50-90% in male & female

YES

Aflatoxins

Choline deficient diet

Theoactamide

92-110 weeks

50-54 weeks

50-80 weeks

50-90% in male & female

100%

70-100%

YES

Different strains of Mice and Rats used in induction of HCC

MICE Rats C57BL/6J F344/N Balbc mice Wistar albino B6C3F1 Sprague-Dawley C3H/ HE Fischer 344

C57BL/6J C3H Sprague Dawley

B6C3F1

•Morphology of liver

•Enzyme levels in serum

•Detection of ROS•IHC staining for glutathione S-transferase, placental form (GST-P),

•Detection of level of Pro-inflammatory cytokines

Steps to detect HCC after induction

Morphology of Liver due to Hepatocellular carcinoma induced by DEN

Morphology of mice liver at 16 weeks (I) and 28 weeks (a) Control, (b) diethylnitrosamine (DEN)-exposed,

16 weeks

28 weeks

Alpha-fetoprotein (AFP) is a glycoprotein with a molecular weigh of approximately

70,000 daltons. AFP is normally produced during fetal and neonatal development by the

liver, yolksac, and in small concentrations by the gastrointestinal tract. After birth, serum

AFP concentrations decrease rapidly, and by the second year of life and thereafter only

trace amounts are normally detected in serum.

Elevation of serum AFP to high values occurs in several malignant diseases, most

notably in hepatocellular carcinoma.

Alpha-fetoprotein (AFP)

AST is a cellular enzyme present in many tissues such as heart, skeletal muscles , kidney,

brain, liver, pancreas or erythrocytes. It exists in two isoforms, cytoplasmic and

mitochondrial. The cytoplasmic isoenzyme is released into the blood during the moderate

cell damage. The determination of AST activity in serum is used mainly to assess the liver

damage.

Aspartate transaminase catalyzes the interconversion of aspartate and α-ketoglutarate to oxaloacetate and glutamate.Aspartate (Asp) + α-ketoglutarate ↔ oxaloacetate + glutamate (Glu)

PRINCIPALThe determination is based on the absorbance of hydrazones of 2-oxoglutarate and pyruvate in an alkaline medium.

Aspartate aminotransferase (AST)

An increase in pyruvate concentration corresponds with the levels of AST and ALT activities. The pyruvate concentration is determined spectrophotometrically in the form of hydrazone, which is produced by reaction with 2,4-dinitrophenylhydrazine in an alkaline medium. The pyruvate hydrazone absorbs at 510 nm more than 2-oxoglutarate hydrazone.

ALT is a cytoplasmic enzyme. It is primarily localized in hepatocytes. ALT catalyzes the transfer of an amino group from L-alanine to α-ketoglutarate, the products of this reversible transamination reaction being pyruvate and L-glutmate.It is released into the blood during the cell damage. The determination of ALT activity in serum is used mainly to assess the liver damage.

Alanine aminotransferase (ALT)

The pyruvate concentration is determined spectrophotometrically in the form of hydrazone, which is produced by reaction with 2,4-dinitrophenylhydrazine in an alkaline medium. The pyruvate hydrazone absorbs at 510 nm more than 2-oxoglutarate hydrazone.

PRINCIPALThe determination is based on the absorbance of hydrazones of 2-oxoglutarate and pyruvate in an alkaline medium.

Interleukin-6: At a threshold of 7.9 pg/mL,

interleukin-6 (IL-6) has a sensitivity of 0.83, a specificity of 0.83, and an

AUC of 0.810 [62]. Higher C reactive protein and IL 6 levels correlated

well with larger tumour sizes, poorer Child Pugh functions, shorter survival

times and more predictable outcomes in patients with HCC receiving loco

regional therapy

Vascular endothelial growth factor

Vascular endothelial growth factor (VEGF), especiallyVEGF A, was found to be

elevated in HCC, particularly inadvanced tumour stages and metastasis [65] .

High serumlevels of VEGF indicate poor HCC prognosis [66].

Alpha-l-fucosidase

Alpha l fucosidase (AFU) is a lysosomal enzyme. Itserum levels have been

shown to increase in patientwith cirrhosis and HCC . At a threshold of

2.3005µ mol/L per minute, AFU yielded a sensitivity andspecificity of 90%

and 97.5%, respectively

Transforming growth factor beta-1

Transforming growth factor beta 1 TGF beta 1) is a cytokine with multiple

biological functions. It has a role in cell growth and extracellular matrix

formation . With a threshold of 64.33 ng/mL, TGF beta 1 has a sensitivity of

78.3% and a specificity of 29.5% for the diagnosis of HCC.

Desγ-carboxyprothrombin

Des γ carboxyprothrombin (DCP) is produced in HCC cell lines, and it is found at

significantly higher concentrations than normal in 50% to 60% of all HCC

patients and in 15% to 30% of early HCC cases. DCP can be used together with

AFP L3 to diagnose HCC . At a 125 mAU/mL threshold, DCP has high sensitivity

(89%), specificity (95%) and AUC (0.797)in the prediction of HCC.

PRINCIPLE

The assay of SOD is based on the inhibition of the formation of NADH-

phenazine methosulphate-nitroblue tetrazolium formazon. The colour formed

at the end of the reaction can be extracted into butanol and measured at

560nm

ASSAY FOR SUPEROXIDE DISMUTASE (SOD)

Superoxide dismutases (SOD) are enzymes that alternately catalyze

the dismutation (or partitioning) of the superoxide (O2−) radical into either

ordinary molecular oxygen(O2) or  (H2O2) .Decrease in the level of this

enzyme indicates generation of ROS

PRINCIPLE

The UV absorption of hydrogen peroxide can be measured at

240nm, whose absorbance decreases when degraded by the enzyme

catalase. From the decrease in absorbance, the enzyme activity can be

calculated.

This enzyme plays important role in protecting the cell from oxidative

damage by  ROS Likewise one catalase molecule can convert approximately 5

million molecules of hydrogen peroxide to water and oxygen each second.

Assay for Catalase 

LIPID PEROXIDATION ASSAY 

Lipid peroxidation, is used as an indicator of oxidative stress in cells and tissues. Lipid peroxides are unstable and decompose to form a complex series of compounds including reactive carbonyl compounds. Polyunsaturated fatty acid peroxides generate malondialdehyde (MDA) and 4-hydroxyalkenals (HAE) upon decomposition, and the measurement of MDA and HAE has been used as an indicator of lipid peroxidation

Principle This assay is based on the reaction of a chromogenic reagent, N-methyl-2-

phenylindole (R1), with MDA and 4-hydroxyalkenals at 45°C. One molecule of either MDA or 4-hydroxyalkenal reacts with 2 molecules of reagent R1 to yield a stable chromophore with maximal absorbance at 586 nm.