**4.1 Arginine metabolism**

Arginine content is well-known to affect transplanted tumor in mice. Enhanced in vivo tumor growth is observed when mice were fed with diet rich in arginine. On the other hand, depletion of arginine from their diet inhibits the growth of metastatic tumor (Gonzalez & Byus, 1991). It is later proved that arginine is essential for the survival of cancer cells. Cancer cells are dependent on exogenous arginine for growth because most of them cannot synthesize their own and become auxotropic for arginine (Dillon et al., 2004). There are various explanations for the acquisition of arginine auxotropic phenotype in various cancer cells, but generally it is associated with the downregulation of argininosuccinate synthase (ASS) (Dillon et al., 2004). Arginine auxotrophy is also a common phenomenon in HCC cells due to their lackage of ASS (Ensor et al., 2002).

In somatic cells, deficiency of arginine puts cell cycle on hold, and cells enter the quiescence G0 phase. They can tolerate the depletion of arginine for weeks and return to normal condition once the arginine content is resumed. On the other hand, arginine deficiency is not sustainable in cancer cells (Delage et al., 2010). Defect in cell cycle checkpoint drives continuous cell proliferation even with insufficient arginine, but arginine is necessary for metabolic and enzymatic pathways in malignant cells. In essence, cancer cells with shortage of ASS rely heavily on exogenous arginine. If the uptake of arginine is disrupted, or the stability of arginine is lowered, cell death will occur due to a loss of gross balance (Delage et al., 2010). This physiological difference between normal and cancer cell makes the arginine metabolic pathway a potent target in treatment to distinguish HCC cells from normal cells.

Reducing arginine stability is one of the strategies against malignant cells, and argininedegrading enzyme is the major group of enzymes that can serve the purpose in depleting internal arginine. Arginase belongs to such group of enzyme which is responsible for arginine degradation in the urea cycle, and its anticancer effect is well documented (Bach et al., 1963). In addition to arginase, the enzyme arginine deiminas is proved to efficiently deplete cellular arginine in vitro and in vivo (Cheng et al., 2007). Recombinant argininedegrading enzymes were developed, and their anticancer effect was investigated in HCC. Satisfactory result was obtained using recombinant arginase and arginine deiminas to combat ASS-deficient tumors (Izzo et al., 2004). Studies are conducted to improve the efficacy of these arginine-degrading enzymes. Modification such as pygelation can increase the half-life of the enzyme and prolong its activity. Phase III trial deploying a pegylated form of recombinant ADI is undertaken in HCC patients who have failed prior systemic treatment. It is also reported that a modified recombinant human arginase is able to inhibit ASS-positive HCC, and inhibit tumor cell growth (Cheng et al., 2007).

Novel Therapeutic Targets for Hepatocellular Carcinoma Treatment 41

associated with increased cyclin-dependent kinase 4 activity, and both were believed to contribute to HCC development (Pascale et al., 2005). HSP90 is involved in the folding and activity of many bona fide oncoproteins in tumor cells, maintaining their dysregulated expression and mutational status. Subsequently, tumor cells become sustainable for cell growth and survival, and equip with crucial aberrations required for metastasis (Whitesell

HSP90 is proved to be an efficacious therapeutic target in HCC. Inhibition of a broadspectrum tumorigenic mechanism is resulted when HSP90 is targeted in vitro and in vivo. Independent of the etiological background, all HCC cell lines responded to HSP90 inhibition similarly with increased cell cycle arrest and apoptosis (Breinig et al., 2009). It might due to the fact that HSP90 inhibition triggered a simultaneous degradation of various hepatocarcinogenesis driving factors. In vivo studies showed that inhibitor of HSP90 is tumor-cell specific, and is able to efficiently reduce HCC tumor growth. Newly developed HSP90 inhibitor showed a lack of significant hepatotoxicity and is more tolerated, which become more practical in therapeutic treatment (Breinig et al., 2009). HSP90 inhibition further prevents tumor growth by disruption of tumor angiogenesis, as demonstrated by blocking PDGFR-β expression in vascular smooth muscle cells and VEGF2 expression on endothelial cells (Lang et al., 2009). Moreover, the combinatory use of HSP90 inhibitor and other anticancer agents is proved to be beneficial. Blockage of HSP90 is able to enhance the antitumor effect of mTOR inhibitor rapamycin by blocking the alternative AKT signaling induced by rapamycin (Lang et al., 2009). Inhibition of HSP90 can be invaluable clinically during HCC treatment, either by targeting HSP90 alone or in combination with other

The ubiquitin-proteasome system plays a crucial role in maintaining cellular homeostasis such as regulation of the cell cycle, apoptosis, receptor signaling and endocytosis. Aberration in different ubiquitin-proteasome systems is recognized as the fundamental cause of various human diseases including cancer. The dysregulation of NF-κB in HCC is one of the oncogenic events induced upon defect in ubiquitin-proteasome system. It is also observed proteins expressed by HBV (Hu et al., 1999) and HCV (Munakata et al., 2005) are reported to cause the alteration of different ubiquitin-proteasome systems, contributing to viral replication, hepatotumorigenesis and impairment of host immunity. These findings demonstrate the importance of an intact ubiquitin-proteasome system in preventing HCC development (Chen, 2005). In the United States, a proteasome inhibitor bortezomib is used clinically to manage late-stage multiple myeloma (Chauhan et al., 2008). Studying the mechanisms of various ubiquitin-proteasome systems not only enable a better understanding of cancers, but also help to explore new strategies to cancer management.

In human HCC, a small proteasome regulatory subunit called gankyrin is frequently overexpressed at both mRNA and protein levels. A study showed gankyrin expression levels were highly upregulated in hepatoma cell lines, and its level was higher in HCC samples compared to normal livers, and premalignant or cirrhotic livers (Higashitsuji et al., 2000). Gankyrin is found to interact with retinoblastoma, increasing its phosphorylation level so as

& Lindquist, 2005).

anticancer agent.

**5.2.1 Gankyrin** 

**5.2 Ubiquitin-proteasome system** 

#### **4.2 GLUT1-mediated anaerobic glycolysis**

Due to the high proliferation rate and cell motility rate, the energy requirement of malignant cells is immense. It is suggested that malignant cells have their metabolic rate accelerated in order to accommodate the excess energy consumption, and glucose is the basic unit necessary. Like most cancers, HCC has high glucose requirement and is observed with an increase of glucose metabolism. While glucose metabolism in eukaryotic cells has multiple levels of control, transport of glucose across the cell membrane is the first rate limiting step. Indeed, accelerated glucose metabolism in cancer cells has been associated with increased expression of glucose transporter proteins. In HCC, increase uptake of glucose is mediated by glucose transporter GLUT1. GLUT1 expression is elevated in hypoxic conditions, and this elevates the rate of anaerobic glycolysis which is a metabolic event frequently observed in HCC (Amann et al., 2009). Level of GLUT1 determines the rate of anaerobic glycolysis, affects glucose uptake and utilization, and plays a role in metastasis, chemoresistance and immunity evasion.

Increased GLUT1 expression is observed in all HCC cell lines compared with primary hepatocytes, and this increase could be found in a subset of HCC patients (Amann et al., 2009). It is demonstrated that suppression of GLUT1 expression by siRNA significantly impaired the tumorigenicity of HCC cells. Inhibition of GLUT1 could decelerate anaerobic glcolysis, implied by the reduction of both glucose uptake and lactate secretion (Amann et al., 2009). RNAi-mediated targeting of GLUT1 is a potent way to combat cancer cells as shown in the study of gastric cancer and laryngeal cancer. GLUT1 is possibly a druggable target as it is shown that the ATP-binding site is important for the conformation and transporter affinity (Liu et al., 2001). Several substances have demonstrated the ability to inhibit GLUT1 and cause cancer cell death (Martin et al., 2003). To increase the GLUT1 targeting specificity, derivatives of the GLUT1 inhibitors were generated and showed promising anticancer effect (Morris et al., 1991).

Glucose analogues or glucose conjugates also serve to inhibit anaerobic glycolysis. Glucose analog such as 2-Deoxyglucose reduced the proliferation rate of many hepatoma cells (Ingram et al., 2006), and showed enhanced anticancer effects in combination with conventional chemotherapeutic drugs such as adriamycin or paclitaxel in xenografted mice (Maschek et al., 2004). The use of ketogenic diets is an alternative strategy to target the anaerobic metabolism, which is based on a high fat and low carbohydrate diet and mimic the metabolic state of fasting (Zhou et al., 2007). As a result, a reduction of carbohydrate intake occurs which allows ketones as an alternative fuel for normal tissue. All in all, disruption of critical metabolic pathways in HCC cells or targeting the main components of the pathways might become alternative therapeutic strategies.
