Examination of the activity of camel milk casein against hepatitis C virus (genotype-4a) and its apoptotic potential in hepatoma and hela cell lines

Abstract

Background: Hepatitis C is a global health concern that represents a major cause of liver disease and socioeconomic burden. Currently, there is no vaccine that protects against this infection or drug that treats it effectively. The current treatment for hepatitis C virus (HCV) infection does not produce a sustained virologic response. Therefore,discovery and identification of a new drug for HCV treatment is a high priority.Camel milk is a traditional medicine that could improve the control of HCV.

Objectives: To assess the potential effect of casein purified from camel milk on HCV cellular infectivity in a tissue culture model.

Materials and methods: Casein was purified from defatted camel milk to electrophoretic homogeneity. PBMCs and HepG2 and HeLa cell lines were used. Three kinds of experiments were conducted. HCV was directly interacted with casein and then mixed with different cell types, casein was incubated with the cells and then exposed to HCV, and the HCV pre-infected cells were treated with casein at different concentrations and time intervals. Non-infected cells were used to assess cytotoxicity and the apoptosis effect of casein.

Results: Direct interaction of casein (with or without α-lactalbumin) with neither the virus nor the cells prevented HCV cell entry. However, casein with α-lactalbumin induced a cytotoxic effect in HepG2 and HeLa cell lines but not in human naïve leukocytes. At all concentrations tested, casein with α-lactalbumin could induce apoptosis in both infected and non-infected HepG2 cells.

Conclusions: Camel milk casein (with or without α-lactalbumin) did not demonstrate any anti-HCV activity. However, the cellular apoptotic cascade was initiated in HepG2 and HeLa cells treated with casein (with α-lactalbumin) but not in naïve leukocytes.

Keywords: Apoptosis; Blocking; Camel; Casein; Cell entry; Hepatitis C virus.

Figure 1

(A) 12% SDS-Urea PAGE of Casein Purified from Camel Milk. Four bands: 3 with molecular weights of 27.7, 24.9, 22.0 kDa, and α-lactalbumin with a molecular weight of 14.4 kDa. Lane 1 is repurified casein without α-lactalbumin, and lane 2 is purified casein with α-lactalbumin. (B) The agarose gel picture reveals the effect of camel casein on HepG2 and HeLa cells (II and III, respectively). The apoptotic DNA nucleosomes were run in 1.8% agarose gel after 8 days of casein (with α-lactalbumin) treatment. Lane 1 was the negative control (untreated cells). Lane 2 was the positive control (cells treated with the apoptosis inducer H2O2). Lane 3 was cells treated with 0.5 mg/mL casein, and lane 4 was cells treated with 2.0 mg/mL casein. DNA fragmentation was visualized with ethidium bromide as described in Materials and Methods.

Figure 2

Camel Casein Activity against HCV. Lane 1 is the DNA ladder, lanes 2 and 5 are negative control samples for HepG2 cells, and lanes 3 and 6 are positive control samples for HepG2 cells. Casein with and without α-lactalbumin at a concentration of 2.0 mg/mL was tested for its ability to block HCV entry into HepG2 cells (lanes 4 and 7, respectively). Lanes 8 and 9 are the negative and positive control samples for PBMCs, respectively, and casein at a concentration of 2.0 mg/mL was tested for its ability to block HCV entry into PBMCs (lane 10). Amplified products were resolved in 3% agarose gel, followed by ethidium bromide staining.

Figure 3

(I) Typical Pictures of HCV-Infected (A) PBMCs and (B) HepG2 cells under a light microscope (40x). Pictures A1 and B1 are the negative control. Pictures A2 and B2 are HCV-infected cells treated with camel casein for 4 days. Pictures A3 and B3 are HCV-infected cells treated with camel casein for 8 days. The arrows indicate the foci. (II) A panel indicates naïve human PBMCs and cells treated with casein (containing α-lactalbumin) at concentrations of 0.5 and 2.0 mg/mL (a, b, and c, respectively) for 8 days. Both naïve and treated cells were healthy. The panel below demonstrates the symptoms of apoptosis in HepG2 cells, which were visualized using the EB/AO method. Untreated HepG2 cells (a), and HepG2 cells were treated with 2.0 mg/mL camel casein for 4 days (b) and 8 days (c). (N) indicates necrotic cells; (A) indicates bright orange apoptotic cells. Normal green cells indicate non-apoptotic cells. (III) A panel showing the pictures of (A1-3) HepG2 cells, (B1-3) HeLa cells, and (C1-3) PBMCs under a light microscope (×20). Pictures A1, B1, and C1 show untreated cells. Pictures A2, B2, and C2 show cells treated with 0.5 mg/mL camel casein, and pictures A3, B3, and C3 show cells treated with 2.0 mg/mL casein. Camel casein did not exert any effect on PBMCs but exhibited typical apoptotic activity against HepG2 and HeLa cells.

Figure 4

Viability of PBMCs (I) and HepG2 Cells (II) Treated with Camel Casein Containing α-Lactalbumin for 4 and 8 Days. Cell Viability was Examined by the MTT Assay, and the Data Presented are the mean ± standard Deviation of the Assays Conducted in Triplicate.