**2.3 Angiogenesis biomarkers in hepatocarcinoma**

 In the past decades, several biomarkers, such as VEGF, angiogenin, and MVD, have been selected for elucidating angiogenic potential of hepatocarcinoma. **Table 3** summarized the potential of these biomarkers for hepatocarcinoma angiogenesis and angiogenesis-related tumor biological actions. Among these biomarkers, VEGF is concerned especially because of its clinic significance. For example, a hospitalbased clinic samples analyses (including 7 cases with liver low-grade dysplastic nodule [DN], 8 cases with liver high-grade DN, 11 cases with early hepatocarcinoma, 17 cases with small hepatocarcinoma, and 21 cases with advanced hepatocarcinoma) by Park et al*.* [26] showed that the amount of VEGF increased gradually from low-grade DN to early hepatocarcinoma. Furthermore, this increasing expression of VEGF is significantly associated with neoangiogenesis (marked by MVD with CD34 staining) and cancer cell proliferation. Collectively, we can conclude that increasing VEGF expression and MVD are positively associated with tumor vascularization and the following tumor progression and poor survival of tumor cases. Furthermore, increasing evidence has exhibited that serum levels of VEGF are not only parallel with the amount in tumor tissues but also can predict therapy response of patients with hepatocarcinoma [29–32]. Thus, VEGF may be useful for improving therapeutic strategies of hepatocarcinoma based on the angiogenesis thesis.



**Abbreviations:** *LGDs, patients with low-grade dysplasia; HGD, patients with high-grade dysplasia; eHCCs, patients with early hepatocellular carcinoma; HCC, hepatocellular carcinoma; sHCCs, patients with small HCC; aHCCs, patients with advanced HCC; HCCs, patients with HCC; HCs, healthy controls; LCs, patients with liver cirrhosis; VEGF, vascular endothelial growth factor; MVD, microvessel density; OS, overall survival; RFS, tumor reoccurrencefree survival; TACE, transarterial chemoembolization; bFGF, basic fibroblast growth factor; EPCs, bone marrowderived endothelial progenitor cells; TEMs, TIE2-expressing monocytes/macrophages; Ang-2, angiopoietin-2; G-CSF, granulocyte colony-stimulating factor; HGF, hepatocyte growth factor; IL-8, interleukin-8.* 

#### **Table 3.**

*The potential of biomarkers for hepatocarcinoma angiogenesis and angiogenesis-related tumor biological actions.* 

### **3. DNA damage and DRR in hepatocarcinoma**

#### **3.1 DNA damage induced by risk factors for hepatocarcinoma**

Multiple risk factors, including HBV and HCV infection, AFB1 exposure, ethanol consumption, and obesity, have been reported to correlate with hepatocarcinogenesis (**Figure 2**) [4]. These risk factors can induce multiple types of DNA damage, such as DNA single-stand break (SSB), double-strand break (DSB), base damage, DNA-adduct formation, oxidation damage, gene mutation, chromosomal aberration, and genomic instability [4]. Results from epidemiological and experimental studies show that viral-DNA damage relationship is characterized by:

**Figure 2.** 

*Risk factors-induced DNA damage and damage repair response during hepatocarcinoma.* **Abbreviations***: HBV, hepatitis B virus; HCV, hepatitis C virus; AFB1, aflatoxin B1; AFBO, AFB1-8,9-epoxide; IL, interleukin; TGF, transforming growth factor.* 

(1) the integration of viral gene (such as HBx gene) into the genome of liver cells and resulting genomic instability of host cells [21, 24, 40, 41]; (2) TP53 mutation conducted by HBx integration resulting in abnormal cell response, including DNA repair, cell proliferation and cycle, and apoptosis potential [22]; (3) HCV core interfering the formation of Mre11/Rad50/Nbs1 (MRN) complex through the bind with Nbs1 [5]; (4) the inhibition of such DNA repair proteins as Ataxia telangiectasia mutated kinase (ATM) [42, 43]; and (5) inducing dysregulation of signal pathways, including Wnt/β-catenin pathway, sex steroid pathway, p38MAPK pathway, PI3K/Akt pathway, transforming growth factor (TGF) pathway, NF-B pathway, and so on [11].

 For AFB1-induced DNA damage, adducts formation and gene mutations are concerned especially [44]. AFB1 is a known I-type chemical hepatocarcinogen produced mainly by *A. parasiticus* and *A. flavus* and a suspected risk factor for hepatocarcinoma in some dependent areas such as Sub-Saharan area, the southeast region of Asia, and the coast of southeast China. Results from prospective epidemiological and animal studies have exhibited that AFB1-induced DNA damage plays a vital role in the process of hepatocarcinoma caused by AFB1 exposure [40, 45]. Studies of AFB1 metabolism have further proved that cytochrome P450 (CYP) enzymes

 in hepatocytes can facilitate AFB1 into its epoxy compound, also termed as AFB1- 8,9-epoxide (AFBe). AFBe can covalently bind to genomic DNA and ultimately induce multiple types of DNA damage [46, 47]. Increasing evidence exhibits that AFB1 can multiplicatively interact with HBV and/or HCV infection during hepatocarcinogenesis, and that, this multiplicative interaction may be associated with more noticeable DNA damage induced by both AFB1 exposure and HBV/ HCV infection [23]. Epidemiological studies based on the case-control design with a large sample have proved that patients with chronic virus hepatitis (including B and C type) will feature increasing hepatocarcinoma risk under the conditions of high AFB1 exposure [46]. Furthermore, patients with high AFB1 often companies with chronic virus infection and faces higher frequency of gene mutation like TP53 and ras [47]. Interestingly, the mutation at the codon 249 of TP53 gene, namely G:C > T:A mutation resulting in the change of arginine to serine, has been identified as a relatively specifically change and named AFB1-induced hot-spot mutation [44]. This mutation may lead to the dysfunction of TP53 protein and abnormal cell actions like promoting cell growth, inhibiting cell apoptosis, and inhibiting transcription mediated by TP53 [40].

 Other risk factors like alcohol intake also cause malignant transformation of hepatocytes. Chronic ethanol intake will significantly increase hepatocarcinoma risk (about five times) if more than 80 g/day × 10 years. Actually, less than this amount of uptake also increases cancer risk in spite of nonsignificance [48]. Although mechanisms are not still clear, increasing data have shown that chronic hepatic injury, abnormal regeneration, and cirrhosis may act some role in hepatocarcinogenesis [4]. Pathological and molecular biological studies display that acetaldehyde, an important metabolic product of ethanol, can bind to DNA and form DNA adducts. The DNA adduct formation caused could trigger replication errors and/or mutations in tumor suppressor genes and/or oncogene [4]. Additionally, oxidative DNA damage is more noticeable in tissues with hepatocarcinoma than peri-tumor tissues [40, 46]. However, it is unclear whether acetaldehyde-DNA adducts and oxidative damages are true carcinogens and how they trigger hepatocarcinogenesis [4, 49]. Therefore, future studies on DNA damage are needed to better validate these risk factors and detailed molecular mechanisms.

#### **3.2 DRR in hepatocarcinoma**

 DNA damage will trigger DRR pathways, a kind of prompt signal event which can harmonize whether cells obtain cycle arrest for DNA repair or induce death for eliminating cells with severe DNA damage and genomic instability [4]. In human, cells develop several types of surveillance mechanisms consisting of SSB repair (SSBR), DSB repair (DSBR), base excision repair (BER), base mismatch repair (MMR), and nucleotide excision repair (NER) (**Figure 2**) [4, 40]. Among these DNA repair pathways, BER, MMR, and NER can repair base damage such as base mismatches, AFB1-DNA adducts, DNA pyrimidine dimers, and DNA damage induced by irradiation and anticancer drugs. SSBR can repair SSB that is a severe DNA damage, if not repaired quickly, will disrupt genic transcription and replication and ultimately results in lethal DNA damage [40]. DSBR pathway involves in homologous recombination (HR), single-strand annealing (SSA), and nonhomologous end joining (NHEJ). HR pathway can repair DSBs through an accurate repair method using the undamaged homologous chromosome or sister-chromatid as DNA repair temple; whereas NHEJ and SSA pathways are nonhomologous repair methods and usually lead to essential mutagenesis, so far




**Abbreviations:** *hOGG1, human oxoguanine glycosylase 1; XRCC1, X-ray repair cross complementing 1; BER, base excision repair; SSBR, single-strand break repair; HBV, hepatitis B virus; XRCC3, X-ray repair cross complementing 3; AFB1, aflatoxin B1; DSBR, double-strand break repair; WBC, white blood cell; TP53M, hot-spot mutation at codon 249 of TP53 gene; DNA-PKcs, DNA-activated protein kinase catalytic subunit; XRCC4, X-ray repair cross complementing 4; XRCC5, X-ray repair cross complementing 5; XRCC6, X-ray repair cross complementing 6; XRCC7, X-ray repair cross complementing 7; XPC, xeroderma pigmentosum, complementation group C; XPD, xeroderma pigmentosum, complementation group D; NER, nucleotide excision repair; PARP-1, poly(ADP-ribose) polymerase 1; ATM, Ataxia telangiectasia mutated kinase.* 

#### **Table 4.**

*The association between abnormal DRR potential and hepatocarcinogenesis.* 

as to induce chromosomal aberrations, abnormal cell cycle, and/or uncontrolled cell proliferation [50]. During DRR pathways, DNA repair genes play a central role [4]. Dysregulation of DRR caused by DNA repair genic mutations or low DNA repair capacity will increase hepatocarcinoma risk. **Table 4** summarized the effects of abnormal DRR in hepatocarcinogenesis. This evidence shows that dysregulation of DRR resulting from mutations in DNA repair genes and corresponding dysfunctions may promote hepatocarcinogenesis through the following pathways: (1) increasing individuals' susceptibility to risk factors such as hepatitis virus infection and AFB1 exposure [40, 60, 101]; (2) increasing individuals' susceptibility to cancer [45]; (3) increasing amount of carcinogens-DNA adducts in liver tissues [40]; (4) increasing amount of adducts (such as AFB1-DNA and AFB1-albumin adducts) in the peripheral WBCs and affecting immune reaction [61]; (5) increasing the frequency of tumor suppressor genes or oncogenes like Ras and TP53M [40, 47, 52, 61, 79]; and (6) interacting with risk factors during hepatocarcinogenesis [23]. Thus, the potential of DRR pathways should play an important function for hepatocarcinogenesis.
