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

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) 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)

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 209

(Nordlie & Sukalski, 1986).

tumor (B). (Mukherjee at al. 2007)

Reports suggest that mutation in the G6P gene, G727T, leads to hepatocellular carcinoma

Upon exposure of carcinogens, mutagens or other xenobiotics, reactive oxygen species are generated in the cells. Intracellular reactive oxygen species produce different types of DNA

B Fig. 5. No predominant glucose-6-phosphatase expression in tumor area (A) of a basophilic

A

#### **5. Biochemical modulation in hepatocellular carcinoma**

As discussed above, from the beginning of initiation process to the development of neoplasm, various biochemical and genetic changes occur in the affected cells. Some of these changes are well-distinguishable and vary along with the stages of development during various cancer processes including hepatocarcinogenesis. They have been described as preneoplastic or neoplastic markers, depending on the developmental stages (Pérez-Carreón et al., 2006). Liver is the largest organ in our body and it takes major role in metabolism. It has several enzymes which take part in metabolism and detoxification of various chemicals, including drugs. Most of these enzymes were discovered during investigation of drug metabolism in liver and thus they are called hepatic drug metabolizing enzymes. In liver, glutathione and related enzymes such as glutathione peroxidase, glutathione reductase, superoxide dismutase, catalase and many other hepatic drug metabolizing enzymes and isoenzymes, such as glutathione-S-transferases (GSHT), UDP-glucuronyl transferases (UDPGT), cytochrome-c-reductase, cytochrome P-450 content and cytochrome b5, have been identified as possible markers of preneoplastic and neoplastic hepatocytes (Mukherjee et al., 2005; Sarkar et al., 1994). They are categorized as Phase I and Phase II drug metabolizing enzymes/ isoenzymes. Variation in different such enzyme and isoenzyme levels, enzyme expression patterns assessed by histochemistry, lipid peroxidation profile, oxidative stress markers etc., during hepatocarcinogenesis, has been studied and reviewed by several workers (Sarkar et al., 1994,). Conjugation of toxic metabolites with cellular macromolecules such as proteins and nucleic acids, may lead to several health problems including cancer. The enzymes (glutathione S-transferases, arylhydrocarbon hydroxylases, UDP-glucuronic trasferases, cytochrome monooxygenases etc.) and isoenzymes (cytochrome b5, cytochrome C etc.) are mostly involved in the detoxification process in liver. In Phase I hepatic metabolic reaction, oxygenation and hydroxylation reactions and in Phase II metabolic reaction in liver, glucuronidation and transferase reactions are very predominant. Glucuronic acid is transferred from uridine diphosphate glucuronic acid to a drug or phase I metabolite by the enzyme UDPGT (Vessey, 1996). Thus inefficient phase II processes cause increased deposition of phase I toxic metabolites. Another very important class of transferase enzymes is GSHT. They take enormous role in phase II detoxification process in liver. They detoxify electrophilic groups and thus inactivate even the function of carcinogens or mutagens. Satoh and Hatayama (Satoh & Hatayama, 2002) reported that specific different forms of GSHT are expressed during initiation, promotion and neoplastic cell populations. In the different developmental stages of hepatocellular carcinoma, glutathione peroxidase and reduced triphosphopyridine nucleotide (TPNH)-cytochrome-c-reductase activities, cytochrome b5 and P-450 contents, glutathione content and superoxide dismutase and catalase activities were found to vary. Activities of these enzymes or their levels in hepatocellular carcinoma were always lower than those in initiation and promotion stages (Vessey, 1996). These reports suggest that the effects of Phase I hepatic drug metabolism is dwindled in hepatocellular carcinoma. The importance of glucose-6-phosphatase (G6P) in preneoplastic and neoplastic liver cannot be ruled out. The enzyme catalyzes in the final biochemical reactions of both gluconeogenesis and glycogenolysis (Nordlie & Sukalski, 1986; Shieh et al., 2003). This enzyme has an important role in blood glucose homeostasis (Nordlie & Sukalski, 1986). Histochemical demonstrations of G6P exhibited less pronounced activity in some cancer lesions and enhanced activity in the others. G6P-negative hepatocellular carcinoma (Figure 5) was also found to be basophilic (Mukherjee et al., 2007; Hwang et al., 2004).

As discussed above, from the beginning of initiation process to the development of neoplasm, various biochemical and genetic changes occur in the affected cells. Some of these changes are well-distinguishable and vary along with the stages of development during various cancer processes including hepatocarcinogenesis. They have been described as preneoplastic or neoplastic markers, depending on the developmental stages (Pérez-Carreón et al., 2006). Liver is the largest organ in our body and it takes major role in metabolism. It has several enzymes which take part in metabolism and detoxification of various chemicals, including drugs. Most of these enzymes were discovered during investigation of drug metabolism in liver and thus they are called hepatic drug metabolizing enzymes. In liver, glutathione and related enzymes such as glutathione peroxidase, glutathione reductase, superoxide dismutase, catalase and many other hepatic drug metabolizing enzymes and isoenzymes, such as glutathione-S-transferases (GSHT), UDP-glucuronyl transferases (UDPGT), cytochrome-c-reductase, cytochrome P-450 content and cytochrome b5, have been identified as possible markers of preneoplastic and neoplastic hepatocytes (Mukherjee et al., 2005; Sarkar et al., 1994). They are categorized as Phase I and Phase II drug metabolizing enzymes/ isoenzymes. Variation in different such enzyme and isoenzyme levels, enzyme expression patterns assessed by histochemistry, lipid peroxidation profile, oxidative stress markers etc., during hepatocarcinogenesis, has been studied and reviewed by several workers (Sarkar et al., 1994,). Conjugation of toxic metabolites with cellular macromolecules such as proteins and nucleic acids, may lead to several health problems including cancer. The enzymes (glutathione S-transferases, arylhydrocarbon hydroxylases, UDP-glucuronic trasferases, cytochrome monooxygenases etc.) and isoenzymes (cytochrome b5, cytochrome C etc.) are mostly involved in the detoxification process in liver. In Phase I hepatic metabolic reaction, oxygenation and hydroxylation reactions and in Phase II metabolic reaction in liver, glucuronidation and transferase reactions are very predominant. Glucuronic acid is transferred from uridine diphosphate glucuronic acid to a drug or phase I metabolite by the enzyme UDPGT (Vessey, 1996). Thus inefficient phase II processes cause increased deposition of phase I toxic metabolites. Another very important class of transferase enzymes is GSHT. They take enormous role in phase II detoxification process in liver. They detoxify electrophilic groups and thus inactivate even the function of carcinogens or mutagens. Satoh and Hatayama (Satoh & Hatayama, 2002) reported that specific different forms of GSHT are expressed during initiation, promotion and neoplastic cell populations. In the different developmental stages of hepatocellular carcinoma, glutathione peroxidase and reduced triphosphopyridine nucleotide (TPNH)-cytochrome-c-reductase activities, cytochrome b5 and P-450 contents, glutathione content and superoxide dismutase and catalase activities were found to vary. Activities of these enzymes or their levels in hepatocellular carcinoma were always lower than those in initiation and promotion stages (Vessey, 1996). These reports suggest that the effects of Phase I hepatic drug metabolism is dwindled in hepatocellular carcinoma. The importance of glucose-6-phosphatase (G6P) in preneoplastic and neoplastic liver cannot be ruled out. The enzyme catalyzes in the final biochemical reactions of both gluconeogenesis and glycogenolysis (Nordlie & Sukalski, 1986; Shieh et al., 2003). This enzyme has an important role in blood glucose homeostasis (Nordlie & Sukalski, 1986). Histochemical demonstrations of G6P exhibited less pronounced activity in some cancer lesions and enhanced activity in the others. G6P-negative hepatocellular carcinoma (Figure 5) was also found to be basophilic (Mukherjee et al., 2007; Hwang et al., 2004).

**5. Biochemical modulation in hepatocellular carcinoma** 

Reports suggest that mutation in the G6P gene, G727T, leads to hepatocellular carcinoma (Nordlie & Sukalski, 1986).

Upon exposure of carcinogens, mutagens or other xenobiotics, reactive oxygen species are generated in the cells. Intracellular reactive oxygen species produce different types of DNA

Fig. 5. No predominant glucose-6-phosphatase expression in tumor area (A) of a basophilic tumor (B). (Mukherjee at al. 2007)

B

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 211

Fig. 6. A. Hepatic section of an experimental animal showing glycogen-stored early preneoplastic focal lesion with Periodic Acid Schiff reaction. (Mukherjee et al. 2007)

Fig. 6. B. IGF II mRNA-expressed glycogen storage early preneoplastic lesion detected with Digoxigenin-labeled antisense IGF II mRNA by *in situ* hybridization from the consecutive

section. pv – portal vein. bd – bile duct. (Mukherjee et al. 2005)

damage, including chromosomal aberrations, sister chromatid exchanges, and mutations (Dahm*,* 1996), leading to the initiation and/ or promotion and/ or progression of the cancerous process. Oxidative stress resulting from the imbalance of free radicals and the cellular antioxidant defense enzyme systems is reported to induce damage to cellular membranes and nuclear DNA, which results in lipid peroxidation and oxidative DNA damage, respectively (Dahm*,* 1996). Oxidation of the C8 of guanine which gives rise to the formation of modified base 8-hydroxy-2'-deoxyguanosine (8-OHdG) is one of the most abundant types of oxidative DNA damage (Kasai & Nishimura, 1986). It is therefore considered as a sensitive biomarker for cancer development and an important molecular epidemiological assessment of cancer risk due to oxidative stress (Romano et al., 2000). The lipid peroxidation, another important chemical reaction owing to the oxidative stress during hepatocarcinogenesis, is known to influence tumor growth (Gelderblom,2001).

### **6. Signaling of hepatocellular carcinoma: A special reference to the genes, Insulin-like-growth factor II (IGF II) and c-raf.1**

Accumulation of mutations in a variety of genes transforms phenotypes of cancer cells. Mutations are found in several important genes, including p73, p53, rb, APC, DLC-1(deleted in liver cancer), p16, PTEN, IGF-2, BRCA2, SOCS-1, Smad2 and Smad4, ß-catenin, c-myc, and cyclinD1, in hepatocellular carcinoma (Farber & Sarma, 1987). These gene products normally modulate biochemical pathways that regulate cell death and cell proliferation. Deregulation of signaling pathways during the development of hepatocellular carcinoma affects normal cellular processes such as cell cycle and apoptosis. Many growth factors such as insulin-like growth factor I and II (IGF I/II) have ubiquitous role in the development of the disease. Raf/MEK/ ERK/MAP (Mitogen Activated Protein) kinase pathway, Akt pathway, Wnt pathway and Ink4A pathway are some of the predominant pathways involved in the neoplastic conversion of normal cells. However, here our focus is to establish the role of IGF II and c-raf.1 in Raf/MEK/ ERK/MAP kinase pathway during the development of hepatocellular carcinoma in hepatocarcinogenesis.

#### **6.1 Insulin-like growth factor II (IGF II)**

Insulin-like growth factor II (IGF II), a mitogenic polypeptide, has been widely implicated in the pathogenesis of neoplasm of different tissues including the liver of rats and men (Li et al., 1998; Mukherjee et al., 2005). This growth factor is found to express in neonatal life (in the first few days after birth) and then during neoplasia in rodents and men (Li et al., 1998; Mukherjee et al., 2007). IGF II in signaling for cancer cell proliferation is mediated through the Raf growth factor (Das et al., 2010). IGF II activates c-raf through signaling proteins such as Grb2 and Ras. Thus, the pathological implication of the overexpression of these two genes during the development of hepatocellular carcinoma cannot be ignored.

The correlation between IGF II expression and cancer development has been reported in a number of works (Mukherjee et al., 2005, 2007; Li et al., 1998). In a majority of liver carcinoma, IGF-II mRNA expression was reactivated and high levels of IGF-II expression were detected. We also investigated to understand the stage(s) at which IGF II gene activates during carcinogenesis. In our study, IGF II overexpression was observed in the early hepatic altered lesions (Figure 6A, B, C, D) and in hepatocellular carcinoma (Figure 7) (Mukherjee et

damage, including chromosomal aberrations, sister chromatid exchanges, and mutations (Dahm*,* 1996), leading to the initiation and/ or promotion and/ or progression of the cancerous process. Oxidative stress resulting from the imbalance of free radicals and the cellular antioxidant defense enzyme systems is reported to induce damage to cellular membranes and nuclear DNA, which results in lipid peroxidation and oxidative DNA damage, respectively (Dahm*,* 1996). Oxidation of the C8 of guanine which gives rise to the formation of modified base 8-hydroxy-2'-deoxyguanosine (8-OHdG) is one of the most abundant types of oxidative DNA damage (Kasai & Nishimura, 1986). It is therefore considered as a sensitive biomarker for cancer development and an important molecular epidemiological assessment of cancer risk due to oxidative stress (Romano et al., 2000). The lipid peroxidation, another important chemical reaction owing to the oxidative stress during

hepatocarcinogenesis, is known to influence tumor growth (Gelderblom,2001).

**Insulin-like-growth factor II (IGF II) and c-raf.1** 

development of hepatocellular carcinoma in hepatocarcinogenesis.

during the development of hepatocellular carcinoma cannot be ignored.

**6.1 Insulin-like growth factor II (IGF II)** 

**6. Signaling of hepatocellular carcinoma: A special reference to the genes,** 

Accumulation of mutations in a variety of genes transforms phenotypes of cancer cells. Mutations are found in several important genes, including p73, p53, rb, APC, DLC-1(deleted in liver cancer), p16, PTEN, IGF-2, BRCA2, SOCS-1, Smad2 and Smad4, ß-catenin, c-myc, and cyclinD1, in hepatocellular carcinoma (Farber & Sarma, 1987). These gene products normally modulate biochemical pathways that regulate cell death and cell proliferation. Deregulation of signaling pathways during the development of hepatocellular carcinoma affects normal cellular processes such as cell cycle and apoptosis. Many growth factors such as insulin-like growth factor I and II (IGF I/II) have ubiquitous role in the development of the disease. Raf/MEK/ ERK/MAP (Mitogen Activated Protein) kinase pathway, Akt pathway, Wnt pathway and Ink4A pathway are some of the predominant pathways involved in the neoplastic conversion of normal cells. However, here our focus is to establish the role of IGF II and c-raf.1 in Raf/MEK/ ERK/MAP kinase pathway during the

Insulin-like growth factor II (IGF II), a mitogenic polypeptide, has been widely implicated in the pathogenesis of neoplasm of different tissues including the liver of rats and men (Li et al., 1998; Mukherjee et al., 2005). This growth factor is found to express in neonatal life (in the first few days after birth) and then during neoplasia in rodents and men (Li et al., 1998; Mukherjee et al., 2007). IGF II in signaling for cancer cell proliferation is mediated through the Raf growth factor (Das et al., 2010). IGF II activates c-raf through signaling proteins such as Grb2 and Ras. Thus, the pathological implication of the overexpression of these two genes

The correlation between IGF II expression and cancer development has been reported in a number of works (Mukherjee et al., 2005, 2007; Li et al., 1998). In a majority of liver carcinoma, IGF-II mRNA expression was reactivated and high levels of IGF-II expression were detected. We also investigated to understand the stage(s) at which IGF II gene activates during carcinogenesis. In our study, IGF II overexpression was observed in the early hepatic altered lesions (Figure 6A, B, C, D) and in hepatocellular carcinoma (Figure 7) (Mukherjee et

Fig. 6. A. Hepatic section of an experimental animal showing glycogen-stored early preneoplastic focal lesion with Periodic Acid Schiff reaction. (Mukherjee et al. 2007)

Fig. 6. B. IGF II mRNA-expressed glycogen storage early preneoplastic lesion detected with Digoxigenin-labeled antisense IGF II mRNA by *in situ* hybridization from the consecutive section. pv – portal vein. bd – bile duct. (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 213

hepatocellular carcinoma development.

experimental animals. (Mukherjee et al. 2005)

**6.2 The Ras/raf/MEK/ ERK/MAP kinase pathway** 

al., 2005). IGF II gene expressed in the sequence of events leading from glycogen-richacidophilic lesions to glycogen poor basophilic lesions through intermediate type lesions to hepatocellular carcinoma with an expression pattern of "high-low-high" in terms of degree of expression. More precisely, IGF II overexpression was found to be predominant in hepatocellular carcinoma and partially in early preneoplastic lesions. Thus, the gene has an essential role at the initiation stage of carcinogenesis (first few weeks) and during

Fig. 7. IGF II mRNA expression in hepatocytes in tumor area (hepatocellular carcinoma) in

The MAP kinase pathway has probably undergone the most extensive characterization in the process of development of hepatocellular carcinoma. Binding of a growth factor to a tyrosine kinase receptor causes receptor phosphorylation, leading to the formation of a molecular complex with an adaptor protein growth factor receptor bound-2 (Grb2), Grb-2 associated binder 1 and signal relay protein SH-2 domain-containing tyrosine phosphatase-2. This is then localized in the plasma membrane. Other protein such as an exchange factor, Son-of-sevenless (SOS) joins. This complex activates ras while exchange GDP to GTP in the ras/raf/MEK/ERK/MAP kinase pathway. Ras/raf/MEK/ ERK/MAP kinase pathway is known to involve in cell proliferation, dedifferentiation, angiogenesis and cell survival process (Rapp, 1991). Activation of the components of this pathway has been reported to contribute to tumorigenesis, including liver cancer. The GTPase (Guanine neucleotides triphosphate)-Ras and the serine/threonine kinase raf (signaling regulators) regulate the signaling process immediately by activating raf which then phosphorylates the

Fig. 6. C. Consecutive hepatic section after Digoxigenin-labeled sense IGF II mRNA treatment during *in situ* hybridization method. (Mukherjee et al. 2007)

Fig. 6. D. IGF II-expressed one of the early preneoplastic lesions in an animal of initiation group. (Mukherjee et al.2005)

al., 2005). IGF II gene expressed in the sequence of events leading from glycogen-richacidophilic lesions to glycogen poor basophilic lesions through intermediate type lesions to hepatocellular carcinoma with an expression pattern of "high-low-high" in terms of degree of expression. More precisely, IGF II overexpression was found to be predominant in hepatocellular carcinoma and partially in early preneoplastic lesions. Thus, the gene has an essential role at the initiation stage of carcinogenesis (first few weeks) and during hepatocellular carcinoma development.

Fig. 7. IGF II mRNA expression in hepatocytes in tumor area (hepatocellular carcinoma) in experimental animals. (Mukherjee et al. 2005)
