Results and Discussion

6.1.1 Effects of PUFAs on HCV RNA replication

To study the effects of PUFAs on HCV replication, the HCV sub-genomic replicon cells (Ava5) containing HCV subgenomic RNA were employed (19). Cells were treated with fatty acids, including polyunsaturated, monounsaturated and saturated fatty acids, for 24 hrs. As shown in Fig. 9 (lanes 2 - 4), several PUFAs, including arachidonic acid (AA), docosahexaenoic acid (DHA), and eicosapentaenoic acid (EPA) were able to exert potent anti-HCV activities at 100 µM. At the same concentration, α-linolenic acid, γ-linolenic, and linoleic acid only reduced HCV RNA levels slightly (Fig. 9, lanes 5 - 7). In contrast, saturated fatty acids including oleic acid, myristic acid, palmitic acid, and steric acid slightly enhanced HCV RNA levels (Fig. 9, lanes 8 - 11). The RNA levels of GADPH, a house-keeping gene, were not affected by drug treatments.

6.1.2 Arachidonic acid reduced HCV RNA level dose-dependently

To further confirm the results, we chose to analyze the effect of AA for its anti-HCV activity in more details. Ava5 cells were treated with AA at various concentrations for 24 hrs. In Fig. 10A, it is clear that AA was able to suppress HCV RNA levels in a dose-dependent manner. When cells were treated with 100 µM of AA, there was only 7.5% of HCV RNA left compared to untreated cells while treatment with 500 IU/mL of IFN-α reduced HCV RNA level to 13.4 % of the control (Fig. 10B). The EC50 (effective

concentration required to inhibit 50% of HCV RNA level) of AA is approximate 4 µM which is physiologically relevant. The plasma concentration for AA varied from 5.8 to 49.3 µM (34). As a comparison, the anti-HCV activity of IFN-α was measured in parallel and the EC50 of IFN-α was around 3.1 IU/ml (Fig. 9B).

There was no cellular toxicity for cells treated with AA at this range as revealed by MTS assay (Fig. 10C) and the IC50 (concentration required to inhibit 50% of cell viability) was measured to be around 350 µM and 380 after 24 and 72 hrs, respectively, of drug treatment. The cellular morphology did not change after treatment with AA for 24 hrs at 100 µM (not show). Thus, AA might exert its anti-HCV effects through a specific pathway but not because of its cellular toxicity.

6.1.3 Synergistic antiviral activity of AA combined with IFN-α

Whether AA and IFN-α combination exert synergistic, additive, or antagonistic effects was assessed by an isobologram method (31, 104). In general, representation of an isobologram to measure drug-drug interaction is shown in Fig. 11A. It was proposed that synergy, additivity, and antagonism would be represented by concave, linear, and convex isoeffective curves (isoboles), respectively. The anti-HCV effects of AA and IFN-α in combination were evaluated. Ava5 cells were treated with these two drugs in combination in a checkerboard titration manner. HCV subgenomic RNA levels in cells were then measured. Dose-response inhibition of HCV RNA replication was evaluated for varying AA concentrations (0, 0.78, 3.13, 12.5, 50, 200 µM) in the presence of various doses of

IFN-α (0, 0.78, 3.13, 12.5, 50, 200 IU/mL) (Table 1). The data in Table 4 were used to generate isoboles of 50% and 90% inhibition of HCV replication (Fig. 11B). AA and IFN-α exerted strong synergistic anti-HCV activities as revealed by the curvy concave plots of 50% and 90% isoboles.

Current IFN-based therapy for treating HCV infection is not satisfactory and development of more effective drugs has not been very fruitful over the past few years. In fact, many HCV patients often seek other complement and alternative medicine (CAM) and some may even avoid or abandon standard IFN-based therapy and seek other types of therapy (170, 177). Many forms of CAM have shown scientific evidence as cytoprotective agents. However, few were known to possess specific antiviral activity.

PUFAs such as AA, DHA, and EPA were all recognized as essential nutrients in human diet. The metabolites of PUFAs also play numerous important roles in normal physiological conditions and progression of diseases (59). In this study, AA was found to be able to inhibit HCV replication at physiologically relevant concentration. Further research is needed to evaluate the therapeutic role of PUFAs in the clinical management for HCV-infected patients. It may be possible to that these fatty acids can be both as adjunctive or complementary treatment to benefit HCV patients through dietary control.

In this study, we also found that antiviral activity of IFN-α can be accentuated by AA and probably also by other PUFAs. Thus, further studies are warranted if management of AA or other PUFAs through dietary control could increase the effectiveness of current IFN-based treatment as antiviral therapy.

Polyunsaturated fatty acids (PUFAs) are important for many physiologic functions (64, 98, 187). AA, the precursor of eicosanoids, can be catalyzed by at least three types of enzymes in cells, cyclooxygenases (COXs), lipoxygenases (LOXs), and P450 epoxygenase (CYPs), to generate numerous metabolites that can mediate diverse physiological and pathological responses such as blood pressure, inflammation, phagocyte activation, pain, and fever (25, 63, 151, 187). The mechanism of action of PUFAs in inhibition of HCV replication is not clear. Nevertheless, this study provides a potentially favorable observation of drug-food interactions. A human trial is mandatory to understand the clinical value of PUFAs in HCV therapy. It is also important to elucidate of the exact anti-HCV mechanism caused by the PUFAs identified this study. Such understanding may lead to the development of agents with potent activity against HCV or related viruses.

Chapter 7: