Altered lipid profile, oxidative status and hepatitis B virus interactions in human hepatocellular carcinoma

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Abstract

Altered membrane integrity in hepatocellular carcinoma (HCC) tissue was indicated by an elevation in cholesterol and significant decrease in phosphatidylcholine (PC). The resultant decreased phosphatidylcholine/phosphatidylethanolamine (PC/PE) and increased cholesterol/phospholipid ratios are associated with decreased fluidity in the carcinoma tissue. The lower PC was associated with a decrease in the quantitative levels of the saturated (C16:0, C18:0), ω6 (C18:2, C20:4) and ω3 (C22:5, C22:6) fatty acids (FAs), resulting in reduced long-chain polyunsaturated fatty acids (LCPUFAs), total PUFA and an increase in ω6/ω3 FA ratio. In PE, the saturated and ω3 (C22:5, C22:6) FAs were reduced while the total ω6 FA level was not affected, leading to an increased ω6/ω3 FA ratio. Increased levels of C18:1ω9, C20:2ω6 and reduction of 22:6ω3 in PC and PE suggest a dysfunctional delta-6 desaturase. The reduced PC/PE ratio resulted in a decreased C20:4ω6 (PC/PE) ratio, implying a shift towards synthesis of the 2-series eicosanoids. Lipid peroxidation was reduced in both hepatitis B negative (HBV) and positive (HBV+) HCC tissues. Glutathione (GSH) was decreased in HCC while HBV had no effect, suggesting an impairment of the GSH redox cycle. In contrast HBV infection enhanced GSH in the surrounding tissue possibly to counter oxidative stress as indicated by the increased level of conjugated dienes. Apart from the reduced LCPUFA, the low level of lipid peroxidation in the carcinoma tissue was associated with increased superoxide dismutase and glutathione peroxidase activity. The disruption of the redox balance, resulting in increased cellular antioxidant capacity, could create an environment for resistance to oxidative stress in the carcinoma tissue. Alterations in membrane cholesterol, phospholipids, FA parameters, C20:4ω6 membrane distribution and low lipid peroxidation are likely to be important determinants underlying the selective growth advantage of HCC cells.

Introduction

Hepatocellular carcinoma (HCC) is one of the most prevalent human cancers, ranking the fifth most common worldwide [1]. Geographical distribution of this disease varies greatly, i.e. in parts of Asia and Africa the prevalence is more than 100 per 100 000 population while in Europe and North America it is estimated to be 2–4 per 100 000 population [2]. The main risk factors associated with HCC are hepatitis B (HBV) and C (HCV) viral infections, which account for approximately 80% of reported cases worldwide [1], [3]. Other factors that play a role in HCC development, either alone or in conjunction with viral infection include aflatoxin B1 (AFB1) exposure, cigarette smoking, alcohol consumption, p53 gene mutations, repeated cycles of necrosis and regeneration, and chronic inflammation [4]. Experimental and clinical observations suggest that persistent proliferation of genetically altered liver cells plays a key role in the progression of chronic hepatitis into cancer [5]. The development of HCC has also been associated with disorders in plasma lipid and lipoprotein metabolism [6].

Cancer development is associated with alterations in lipid metabolism, affecting cellular function and growth [7]. The development of hepatocyte nodules in rat liver is associated with changes in lipid parameters and oxidative status. These alterations involve increased cholesterol and phosphatidylethanolamine (PE), resulting in increased cholesterol/phospholipid and decreased phosphatidylcholine/phosphatidylethanolamine (PC/PE) ratios. Changes in the phospholipid levels resulted in a decrease in the polyunsaturated fatty acids (PUFAs), especially the long-chain PUFA (LCPUFA), which was associated with low oxidative status [8], [9]. The low levels of PUFA may result from either increased requirement during cell proliferation or impaired functioning of the delta-6 desaturase enzyme, a rate-limiting enzyme in the PUFA metabolic pathway [10], [11]. LCPUFA may play a role in the control of cell proliferation by inhibiting cell growth and stimulating apoptosis either directly [12] or via increased lipid peroxidation (LPO) [7]. Besides their effect on LPO, antitumour effects of the ω3 LCPUFA, such as C20:5ω3 and C22:6ω3, are mainly attributed to the suppression of cell proliferation and induction of apoptosis via various processes, including regulating the level of C20:4ω6 [13], [14]. The ω6 fatty acid (FA) 20:4ω6 is known to stimulate cell proliferation via overexpression of cyclo-oxygenase-2 (COX-2) [15] and subsequent increased formation of PGE2 while on the other hand it may also enhance apoptosis via ceramide [16]. Therefore, regulation of membrane phospholipid content, 20:4ω6 metabolism as well as cellular oxidative status seems to play an important role in determining cell survival. An imbalance in cellular redox state has been observed in various cancer cells compared with normal cells [17], which may be related to oncogenic stimulation and cancer cell survival. Cancer cells exhibit low oxidative status presumably due to low PUFA levels and alterations in the redox status, including the glutathione (GSH)/GSSG ratio and activity of antioxidant enzymes such as superoxide dismutase (SOD), glutathione peroxidase (GPx) and catalase (CAT) [12], [17].

It would appear that the regulatory mechanisms related to lipid metabolism ensuring normal liver homeostasis are disrupted and these changes play a role in the survival and growth of malignant cells [8], [12]. In this regard, the control of cell proliferation and apoptosis has been recognised as a key event in cancer development [18] and the use of dietary constituents such as FAs has been recognised to be an important tool in cancer therapy [19], [20]. The present study investigated the lipid composition and different oxidative parameters in human HCC as compared to those in matching surrounding tissue.

Section snippets

Ethical considerations

Ethical approval for the study was granted by the Ethics Committees of the University of the Witwatersrand and the Medical Research Council of South Africa. Consent for necropsy and removal of cancerous and normal liver tissue was obtained from close relatives of the patient and consent for resection of the tumour from the patient prior to the surgical procedure.

HCC and surrounding non-tumorous liver tissue

HCC and surrounding non-tumorous liver tissue were obtained from 13 patients either at necropsy or at the time of surgical resection

Phospholipid and cholesterol parameters (Table 1)

No significant difference in the phospholipid and cholesterol levels (Table 1) was observed between the HBV and HBV+ tissue samples. The PC concentration was significantly (P<0.05) decreased in the carcinoma tissue, while no significant effect was observed in the PE concentration, resulting in a significant (P<0.05) decrease in the PC/PE ratio when compared with the surrounding tissue. The cholesterol concentration was marginally (0.05<P<0.1) increased in the carcinoma tissue and with the

Discussion

Alterations in lipid profiles and oxidative status in malignant tissue are of importance due to the effect on membrane integrity, fluidity and regulation of cellular processes related to growth and cell survival [12], [35]. Regarding the lipid profile of the HCC tissue, the present study indicated perturbations in the membrane phospholipid distribution and content as demonstrated by a decreased PC/PE ratio. This is suggestive of increased membrane PE content relative to PC, which could be an

Acknowledgements

The authors wish to thank Mrs Amelia Damons and Mr John Mokotary for technical assistance. This study was funded by the Cancer Association of South Africa (CANSA) and a Technology and Human Resources for Industry Programme grant (THRIP) by the South African Department of Trade and Industry.

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