**3. Hepatocellular carcinoma**

204 Hepatocellular Carcinoma – Basic Research

This is also developed from the lining of blood vessel, however, with relatively a slow speed. Blood-filled channels and spaces can be delineated under microscope. This is highly invasive type of cancer. It is commonly found in children. In patients suffering from

Malignant cells migrated from any tissues other than liver may invade hepatic tissue and develop neoplastic tumor in liver. This is described as secondary liver cancer. Spread of cancerous cells from outside the liver to the liver through blood flow or through the lymphatic system, the anchorage of the cells in liver, angiogenesis (formation of new blood vessels for supply of food and oxygen for new cells), and cellular proliferation leading to solid growth of mass are the possible sequences of secondary liver cancer. It is also called as

Cancer in general is a multistage complex process by which uncontrolled proliferation of cells occurs. To understand the underlying mechanism in the process of development of the disease and its progress, it is important to develop the strategy to combat the dreadful disease. The need for the development of various in vivo cancer models has been in demand. Experimental liver cancer in animals is thus developed for studying the progress of the disease scientifically minutely in vivo and to develop therapeutic and other combating strategies to fight against it. There are various in vivo animal models already available for the purpose. Generally, virus-induced, radiation-induced, neoplastic cell-transplanted and chemical-induced liver cancer animal models have been

Hepatocarcinogenesis is the development of liver cancer due to the exposure of carcinogen (a chemical that produces cancer). Many hepatocarcinogens such as aflatoxins, acetylaminofluorene, diethylnitrosamine have been successfully used to develop hepatocarcinogenesis in animals. Experimentally, hepatocarcinogenesis is developed using different carcinogens and also in different animal species. Several genetic and epigenetic changes such as chromosomal deletions, rearrangements, aneuploidy, gene amplification, and mutations, formation of DNA adducts, DNA strand-break, modulation of DNA methylation, and modulation of cell signaling pathways, due to direct or indirect effect of carcinogen exposure lead to neoplastic transformation of hepatocytes in experimental animals. Hepatocarcinogenesis is a multistage complex process, which is preceded by early appearance of morphologically and genetically altered hepatic focal lesions, also known as preneoplastic lesions. Initially monoclonal populations of hepatocytes evolve primarily due to carcinogenic insult. These aberrant monoclonal populations of regenerative hepatocytes (focal lesions) develop hyperplastic nodules to dysplastic nodules, leading to hepatocellular

hemangiosarcoma, the rapture of tumor leads to bleeding to death.

**1.3.1.5 Hemangiosarcoma** 

**1.3.2 Secondary liver cancer** 

**1.3.3 Experimental liver cancer in animals** 

metastatic cancer.

widely studied.

carcinoma.

**2. Hepatocarcinogenesis** 

The most common primary malignant tumor of liver is hepatocellular carcinoma. This primary liver cancer is also called hepatoma. As described above, liver has different types of cells such as hepatocytes, billiary cells, blood cells, Kupffer cells, ito cells, perisinusoidal cells etc. However, about more than 80% of liver tissue consists of hepatocytes. The majority of primary liver cancer (>90%) arises from hepatocytes and is called hepatocellular carcinoma. During hepatocarcinogenesis, initial carcinogen insult results in initiated cells from normal liver parenchyma cells or hepatocytes by genetic alteration following an interaction generally with DNA. Subsequent tumor promotion by chronic exposure of carcinogen or a tumor promoter such as phenobarbital develop clonally selected expansions of initiated cell populations called hepatic altered foci by fixing the mutations for further genetic changes. Additional accumulations of genetic changes within these foci produce hyperplastic nodules that ultimately lead to the development of hepatocellular carcinoma.

#### **3.1 Stages of hepatocellular carcinoma**

Transformation of the initiated hepatocytes into hepatocellular carcinoma is a multistage complex process. Based on the various morphological (such as appearance, size, shape, growth) and biochemical (such as variation in staining patterns, and altered enzyme expression patterns) changes of these hepatocytes, leading to hepatocellular carcinoma, various stages of development of the disease, namely *initiation*, *promotion* and *progression,* have been described to understand the progress of the disease in a more defined way and to develop better therapeutic strategies.

### **3.1.1 Initiation**

Exposure of genotoxic agents such as aflatoxins, 2-acetylaminofluorene, diethylnitrosamine, ionizing radiation etc. alters DNA sequence, causing mutations in the hepatocytes that

Fig. 2. Glycogen-stored early preneoplastic focal lesion (shown by yellow arrows) in rat hepatic tissue with Periodic Acid Schiff reaction. (Mukherjee et al. 2005)

Chemically Induced Hepatocellular Carcinoma and Stages of Development with Biochemical and Genetic Modulation: A Special Reference to Insulin-Like-Growth Factor II and Raf Gene Signaling 207

look grey or whitish gray or greenish (because of the presence of bile in them). Eventually by a slow process through lots of biochemical and genetic alterations those hepatic altered foci or hepatic nodules develop increasingly malignant cellular characteristics and are transformed into neoplasia without any further external stimulus or intervention (Farber & Sarma 1987).

**4. Sequential changes in hepatocellular lineages leading to hepatocellular** 

components (Bannasch et al., 1998). They named it amphiphilic cell lineage.

Fig. 4. Mixed cell focal lesion (shown by green arrows) and basophilic lesion (shown by blue

arrows) in diethylnitrosamine-treated rat liver. (Mukherjee et al. 2007)

Etiology of hepatocellular carcinoma has probably been studied and analyzed in the best defined manner. Various groups of scientists have studied minutely and described the process of development of hepatocellular carcinoma during hepatocarcinogenesis. Other than initiation, promotion and progression models, Bannasch and his coworkers (Bannasch, 1995) have established and described the involvement of defined cellular lineages in the process of development of liver cancer. Predominant sequential cellular changes during the development of hepatocellular carcinoma commence with glycogenotic clear and acidophilic (due to proliferation of smooth endoplasmic reticulum) cell focal lesions and progress through intermediate phenotype of mixed cell population to glycogen poor basophilic (ribosome rich) cell phenotypes (Figure 4). The group has described few other cellular lineages. In the tigroid basophilic lineage, initially the cells have abundant highly ordered stacks of the rough endoplasmic reticulum and thereby they have uniqueness. The scientist group further reported that the lineage is common to the animals treated with a low dose treatment of hepatocarcinogen (Gournay et al., 2002). Another type of cellular lineage has been found to involve in the development of hepatocellular carcinoma. Rats when treated with nongenotoxic peroxisome proliferators or woodchucks chronically infected with woodchuck hepatitis virus showed foci with glycogen-poor cytoplasm containing abundantly granular acidophilic (mitochondria and peroxisome proliferators) and basophilic (ribosome)

**carcinoma** 

develop potential to begin the transformation of normal cells to cancer cells. The mutations generally activate proto-oncogenes and/or inactivate tumor suppressor genes to develop hepatocellular carcinoma in the carcinogenic mechanism. The chemicals that cause the process (initiation) are called "initiators". Initiation is an irreversible process for a small population of cells. It occurs with a single/ brief exposure to a carcinogen. Electrophilic moieties generated by genotoxic agents generally bind with DNA to form DNA adduct, hamper cellular DNA repair mechanism, and develop permanent DNA lesions. Thus, the normal cell becomes an initiated cell. The initiated cells can develop focal lesions (Figure 2), one or more of which can act as sites of origin for the subsequent development of malignant neoplasia (Farber & Sarma 1987).
