Comparison of Helicobacter bilis-Associated Protein Expression in Huh7 Cells Harbouring HCV Replicon and in Replicon-Cured Cells

Abstract

Hepatocellular carcinoma (HCC) is one of the leading causes of cancer-related deaths worldwide. Hepatitis B or C infections are the main causes of HCC with hepatitis C being the major risk factor for liver cancer in the developed countries. Recently, complications with bacteria of the genus Helicobacter have been associated with HCV-induced HCC. To further understand the mechanisms leading to the development of HCC in the presence of HCV and/or Helicobacter spp., investigation of the differential protein expression in Huh7 cells harbouring HCV-replicon, and replicon cured-Huh7 cells cocultured with H. bilis was done employing two-dimensional gel electrophoresis and mass spectrometry. In the transfected-Huh7 cells exposed to sublethal inoculum densities of H. bilis, 53 different proteins were identified comprising of 28 upregulated and 16 downregulated proteins including 9 potential protein isoforms; in the cured Huh7 cells, 45 different proteins were identified including 33 upregulated, 8 downregulated and, 9 potential protein isoforms. H. bilis affected the modulation of proteins involved in different pathways of Huh7-derived cells physiology including proteins involved in the progression from dysplasia to neoplasm. The result also indicated that the response of the Huh7-derived cells to the presence of H. bilis depended on whether or not HCV replicon was present.

Figure 1

Growth of transfected- and cured Huh7 cells in cocultures with H. bilis at different inoculum densities. The initial number of Huh7 cells determined by the improved Neuber cytometer after staining with trypan blue was ca. 10^5.5 cells/ml. The number of viable Huh7 cells counted after 48 h in cocultures with H. bilis are shown in the figure. The values represent the mean count of Huh7 cells from three independent experiments.

Figure 2

2D-PAGE gels showing spots of soluble proteins of transfected and cured Huh7 cells in the pI 4–7 range. Gels (a) and (b) correspond to proteins of transfected Huh7 cells, gels (c) and (d) correspond to proteins of cured Huh7 cells. Cells were incubated in the presence of an initial H. bilis inoculum of approximately 10³ cfu/mL. Protein spots in gels were visualised by silver staining. The spot intensities from triplicate gels of three independent experiments were determined using the Z3 computational software. Spots corresponding to proteins differentially expressed are indicated on the figure.

Figure 3

Relative expression of HCC-related genes in transfected- & cured Huh7 cells in response to H. bilis. Values are presented as log₂ of the relative expression and represent the mean relative expression derived from biological triplicates. *P < 0.05.