Association of MnSOD Ala16Val genotype and activity with hepatocellular carcinoma risk in HCV-infected Egyptian patients

Introduction 

Hepatocellular carcinoma (HCC) is the predominant histologic subtype of liver cancer in adults, comprising approximately 65% of all cases of primary liver cancer [1].

Primary risk factors for HCC are hepatitis B virus (HBV), hepatitis C virus (HCV), dietary aflatoxin exposure and chronic alcohol consumption [2]. Hospital-based studies from Egypt reported that HCC constitutes >95% of liver cancers, HCV infection reportedly being the cause of >75% of these cases [3], [4]. This may be explained by the high prevalence (11.95%) of HCV in Egypt [5].

The fundamental mechanism by which HCV is related to HCC is not definitely known. The indirect role of HCV may be through induction of cirrhosis and chronic inflammation, which lead to continued generation of reactive oxygen species (ROS) including free oxygen radicals and non-radicals. They can cause oxidative DNA damage, mitochondrial abnormalities and accelerated hepatocyte proliferation [6].

On the contrary, some reports suggested a direct role of HCV core and non-structural 3 proteins (NS3) that activate NADPH oxidase of Kupffer and polymorphonuclear cells in the liver, leading to increased generation of ROS and other reactive oxidative species [7], [8].

The cells are protected against oxidative insults by both exogenous dietary antioxidants and endogenous antioxidant enzymes, such as superoxide dismutase (SOD), glutathione peroxidase 1 (GPx1), catalase and peroxiredoxin III (PrxIII) [9]. Superoxide dismutase catalyses the dismutation of superoxide anion into molecular oxygen and hydrogen peroxide, which is subsequently detoxified to water by mitochondrial GPx1 and to O2 by catalase [10].

In human tissues, SOD is present in at least three forms: (i) constitutive cytoplasmic, copper/zinc SOD; (ii) inducible mitochondrial manganese SOD (MnSOD); and (iii) extracellular SOD [11]. Manganese SOD is synthesised with a mitochondrial-targeting sequence (MTS) enabling its import into the mitochondrial matrix [12]. The MTS is then cleaved and the mature protein assembles to form its active tetramer form, which contains one manganese ion per subunit [13]. Manganese SOD has attracted the most attention since it was reported to be inducible by many stimuli, including inflammatory cytokines, growth factors and viral infections [11], [14].

The manganese SOD gene is located on chromosome 6q25 [15]. Several single nucleotide polymorphisms (SNPs) with functional consequences have been identified in the MnSOD gene [16] and have been implicated in neoplasia of different organs as the lung [17], oesophagus [18], colon [19] and liver [20].

The most commonly studied polymorphism in MnSOD gene is a C47T SNP, that is, a substitution of thymine (T) base to cytosine (C) base at nucleotide 47 within exon 2, resulting in a change of the encoded amino acid from valine (GTT) to alanine (GCT) on the 16th residue of 24-amino acid of MTS (Ala16Val) [21].

It has been shown that the 16Ala variant allows efficient targeting of MnSOD to the mitochondria, as evidenced by Sutton et al. [22] who found that it was 30–40% more efficiently localised to the mitochondria than the 16Val variant. This is because 16Ala variant has an α-helix structure that is easily imported and it reaches high levels of mitochondrial concentration and activity, whereas the 16Val variant has a partial β-sheet structure that is partly stuck within the narrow inner membrane import pore and is subsequently degraded by the proteasome. Furthermore, the mRNA that encodes the 16Val variant is more rapidly degraded than the Ala variant [23].

Imbalance between the activities of MnSOD and other antioxidant enzymes increases the risk of HCC, possibly by leading to high H2O2 steady-state levels, which can lead to cancer [24].

Several case–control studies conducted on liver diseases yielded controversial results about this functional polymorphism. This emphasises the importance of replicating similar studies in novel populations, since the knowledge of allele distribution in specific ethnic groups could have important implications for future treatments.

This work aimed to assess whether there is a relationship between the MnSOD Ala16Val genotype and activity on one hand and HCC development in HCV-infected Egyptian patients on the other hand.

Patients and methods 

Laboratory tests 

Laboratory tests included liver function tests, INR, schistosomal antibodytitre, HBsAg and anti-HCV by Enzyme Immunoassay Kits (DiaSorin, Italy). Quantitation of HCV-RNA load was performed using Versant HCV RNA 3.0 branched chain DNA (bDNA) assay (Siemens Healthcare Diagnostics, USA) according to manufacturer’s instructions based on Elbeik et al. [28]. The assay was performed in a semi-automated Bayer System 340 bDNA analyser (Bayer HealthCare LLC Diagnostics Division, USA).

Manganese-SOD activity was measured using a superoxide dismutase assay kit (Cayman Chemical, Ann Arbor, MI, USA). According to the manufacturer’s protocol, 10μl of diluted serum samples (1:5) with 190μl of diluted tetrazolium solution were added to the sample wells; then 10μl of 1mM potassium cyanide solution were added to inhibit both copper/zinc and extracellular SOD enzymes. Twenty microlitres of diluted xanthine oxidase solution were added as quickly as possible, followed by incubation for 20min at room temperature. Absorbance change was read at 450nm using an SLT Spectra microplate reader (SLT, Labinstruments A-5082 Austria). The activity of Mn-SOD enzyme (expressed as units per millilitre) was calculated from a standard curve constructed with known amounts of standards processed with samples. It was defined as the amount of enzyme needed to exhibit 50% dismutation of the superoxide radical.

Genomic DNA was isolated from peripheral leucocytes using QIAamp DNA Mini extraction system (Qiagen, Valencia, CA, USA). The DNA concentrations were determined by spectrophotometric absorbance measurements of the extracts. The Ala16Val polymorphism was analysed using PCR/RFLP [26]. PCR was done by using a primer pair: (forward, 5′-CAG CCC AGC CTG CGT AGA CGG-3′ and reverse, 5′-CTT GGC CAA CGC CTC CTG GTA CTT-3′) (Operon Biotechnologies Inc., AL, USA). The PCR was carried out using a final volume of 20μl, containing 50ng of genomic DNA, 0.4μmol/L of each primer, 200μmol/L of each dNTP, 1.25U of Taq polymerase in 1.5mmol/L MgCl2, 50mmol/L KCl and 20mmol/L Tris–HCl, pH 8.4 (Gibco-BRL System; Cergy-Pontoise, France). Subsequently, 20μl mineral oils were poured on the surface of the mixture to prevent evaporation of the sample during heating. The temperature settings were a cycle of initial denaturation for 5min at 95°C, 30 cycles of 45s at 95°C, 30s at 54°C, 30s at 72°C each and a last cycle of 5min at 72°C were run in a DNA thermal cycler (Perkin Elmer-Cetus, USA). The resulting 267-base-pair PCR product was digested with restriction endonuclease BsaWI (New England Biolabs, Ozyme, Saint-Quentin-en-Yvelines, France) at 56°C for a minimum of 2h, according to the manufacturer’s recommendation. This enzyme only cleaves this product, when a thymine base is present at position 47 nucleotide of the MTS of MnSOD gene, into 183- and 84-base-pair fragments. Complete, partial or lack of digestion patterns were detected using 2.5% agarose gel electrophoresis allowing clear distinction of Val/Val, Ala/Val and Ala/Ala genotypes, respectively (Fig. 1). Thirty percent of the RFLP assays were repeated and no discrepancy was observed [29], [30].

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Fig. 1. PCR/RFLP analysis of C47T SNP in MTS of MnSOD gene. Lane 1: 20-bp low ladder DNA marker; lanes 2 and 3: TT (Val/Val) genotype (two bands; one at 183bp and one at 84bp); lanes 4 and 5: CC (Ala/Ala) genotype (one band at 267bp); lanes 6 and 7: TC (Val/Ala) genotype (three bands, one at 183bp, one at 84bp, and one band at 267bp) and lane 8 shows the DNA-negative control (no bands).

Results 

Table 1 shows the general characteristics of the studied groups. There were no significant differences between them in terms of age and sex which indicates adequate matching between the groups As regards the Ala16Val MnSOD polymorphism, the frequency of the Ala/Ala genotype in patients (36.0%) was significantly higher than in asymptomatic HCV-infected patients (12.5%) (OR=5.63, 95% CI=1.35–23.52, p=0.029) and the healthy controls (18.9%) (OR=2.91, 95% CI=1.11–7.66, p=0.031). Moreover, the frequency of the Ala allele was higher in patients (57.3%) than in asymptomatic HCV-infected patients (35.4%) (OR=2.45, 95% CI=1.25–4.81, p=0.008) and the healthy controls (43.1%) (OR=1.77, 95% CI=1.09–2.89, p=0.021) (Table 2).

Table 1. Characteristics of the studied groups participating in the study.

	Characteristics	HCV-infected HCC patients (n=75)	Asymptomatic HCV-infected patients (n=24)	Healthy controls (n=58)	p valuea	p valueb
	Age (mean±SD years)	53.3±1.85	52.3±3.5	51.9±3.8	0.960	0.813
	Sex n (%)
	Males	56 (74.7)	18 (75.0)	42 (72.4)	0.974	0.770
	Females	19 (25.3)	6 (25.0)	16 (27.6)

a p value with asymptomatic HCV-infected patients as reference. b p value with healthy controls as reference.

Table 2. MnSOD genotype and allele frequencies in the studied groups and its association with HCC risk.

		HCV-infected HCC patients (n=75)	Asymptomatic HCV-infected patients (n=24)	Healthy controls (n=58)	OR (95% CI)a	p valueb	OR (95% CI)c	p valued
	Genotype frequency n (%)
	Val/Val	16 (21.3)	10 (41.7)	19 (32.8)	1.00		1.00
	Val/Ala	32 (42.7)	11 (45.8)	28 (48.3)	1.82 (0.64–5.17)	0.785	1.36(0.59–3.13)	0.519
	Ala/Ala	27 (36.0)	3 (12.5)	11 (18.9)	5.63 (1.35–23.52)	0.029	2.91 (1.11–7.66)	0.031
	Allele frequency n (%)
	Val	64 (42.7%)	31 (64.6%)	66 (56.9%)	1.00		1.00
	Ala	86 (57.3%)	17 (35.4%)	50 (43.1%)	2.45 (1.25–4.81)	0.008	1.77 (1.09–2.89)	0.021

a OR with asymptomatic HCV-infected patients as reference. b p value with asymptomatic HCV-infected patients as reference. c OR with healthy controls group as reference. d p value with healthy controls group as reference.

Table 3 shows highly significant difference in MnSOD activityamong the studied groups (p=0.000).

Table 3. Serum MnSOD activity (U/ml) in the studied groups.

	Serum MnSOD activity	HCV-infected HCC patients	Asymptomatic HCV-infected patients	Healthy controls	p value
	Mean±SD	0.152±0.049	0.032±0.009	0.037±0.019	0.000

By studying serum MnSOD activity among the different genotypes within each studied group, we found a highly significant increase in its activity in HCC patients with Ala/Ala and Val/Ala than in those with Val/Val genotypes (p=0.007). On the contrary, there was a non-significant increase in its activity among different genotypes in asymptomatic HCV-infected patients and healthy controls (p=0.574 and 0.862), respectively (Table 4).

Table 4. Serum MnSOD activity (U/ml) among the different genotypes within each studied group.

		Serum MnSOD activity (U/ml) mean±SD
		HCV-infected HCC patients	Asymptomatic HCV-infected patients	Healthy controls
	Genotype
	Ala/Ala and Val/Ala	0.14±0.05	0.0292±0.00297	0.0297±0.00315
	Val/Val	0.08±0.04	0.0285±0.00295	0.0295±0.00334
	p value	0.007	0.574	0.862

Finally, the bioclinical parameters of different Child–Pugh classes of HCC patients did not differ significantly in relation to Ala/Ala genotype (p=0.913) (Table 5).

Table 5. Relationship between different Child–Pugh classes and MnSOD genotypes in HCC patients.

		Child–Pugh class			p value
		A (n=25; 33.3%)	B (n=27; 36.0%)	C (n=23; 30.7%)
	Genotype frequency n (%)
	Val/Val	6 (24.0)	5 (18.5)	5 (21.6)	0.889
	Val/Ala	10 (40.0)	13 (48.2)	9 (39.1)	0.770
	Ala/Ala	9 (36.0)	9 (33.3)	9 (39.1)	0.913

Discussion 

HCV-associated HCC may occur due to severe alterations of host redox status, as HCV infection might increase ROS generation [32]. Furthermore, it disrupts reduced glutathione export, which is an important endogenous antioxidant [33], [34]. Moreover, pro-inflammatory cytokines, such as tumour necrosis factor (TNF)-α, may increase production of superoxide radicals in the mitochondria [7].

As regards the association between MnSOD Val16Ala polymorphism and cancer risk, it may be explained by high H2O2 concentrations, which reduce the chance of TNF-α-mediated apoptosis of cancer cells [35]. Furthermore, it may activate cytosolic iron-responsive protein 1 that induces the synthesis of the transferrin receptor, and consequently increases the hepatocellular uptake of iron [36]. Iron can react with H2O2 forming the hydroxyl radical that can lead to cancer by either its damaging effect on lipids, proteins and DNA causing somatic DNA mutations [37] or activation of certain oncogenes [38].

The molecular mechanisms through which HCV infection and MnSOD malfunction synergise to stimulate cell proliferation and carcinogenesis are still unknown. It may be due to the cell transformation effect of ROS-induced signal transducer and activator of transcription 3 (Stat3) transcription factor [39].

The current study was conducted on three groups of HCV-infected HCC patients, asymptomatic HCV-infected patients and healthy controls aiming to study the association between Ala16Val MnSOD polymorphism and occurrence of HCC in HCV-infected Egyptian patients. The analysis of the three groups showed that MnSOD Ala/Ala genotype is increased by 5.63- and 2.91-fold in HCC patients in comparison to asymptomatic HCV-infected patients and healthy controls, respectively. This is in accordance with a study by Ezzikouri et al. [40], who found that Ala/Ala genotype has been associated with a 5.09-fold increase in HCC risk among Moroccan patients.

In another study by Nahon et al. [41], they showed that the Ala/Ala genotype was equally represented in controls and in patients with HCV-related cirrhosis, and was not associated by a significant increase in the risk of HCC or death. One of the probable explanations is the presence of other polymorphisms in the transport coding sequence and in other detoxifying genes [42]. Besides, variations in genetic background and environmental or lifestyle differences may modify the impact of the MnSOD polymorphism [43].

On studying serum MnSOD activity, we found a statistically highly significant difference among the three studied groups where it was higher in patients in comparison to the control groups. This is in agreement with the results of Clemente et al. [44], who found that serum MnSOD activity was significantly higher in cirrhotic patients and in cirrhotic patients with HCC compared with healthy subjects. Furthermore, cirrhotic patients who developed HCC during follow-up showed significantly higher values of MnSOD activity than HCC-free patients. Therefore, it was suggested that MnSOD activity may be a malignancy-associated parameter [44].

The current study suggests that a highly significant increase in MnSOD activity (p=0.007) in Ala/Ala and Val/Ala than in Val/Val genotypes entails a higher HCC risk. Our observations support the study conducted by Sutton et al. [23] who found a greater MnSOD activity with the Ala/Ala genotype in a human hepatoma cell line.

By contrast, Bastaki et al. [45] and Martin et al. [41] who evaluated MnSOD activities in erythrocytes of healthy non-smoking volunteers and lysates of the cryopreserved hepatocytes, respectively, found a reduced activity in Ala/Ala and Val/Ala than in Val/Val genotypes. They explained that the transport efficiency and activity of MnSOD might differ in the non-cancerous normal tissue, normal tissue in a cancer specimen and cancer tissue itself. In the present study, the bioclinical parameters of different Child–Pugh classes of HCC patients did not differ significantly in Ala/Ala genotype (p=0.913). This is consistent with the data from the studies by Sutton et al. [37] and Nahon et al. [43] who found that different classes of Child–Pugh score did not differ among the three genotypic groups.

In conclusion, HCC risk is elevated among Egyptian HCV-infected patients with MnSOD Ala/Ala genotype. However, this important finding needs to be corroborated through studying larger groups of patients from various ethnic backgrounds. Such findings may have major implications in the management of patients with HCV.