Integrity of a HPV11 infection cell model and identification of (-)-Epigallocatechin-3-gallate as a potential HPV11 inhibitor

Abstract

Background: Condyloma acuminatum (CA) is one of the most common sexually transmitted diseases and induced by low-risk human papillomaviruses (HPVs), mainly HPV type 6 and 11. Here, we report the identification of (-)-Epigallocatechin-3-gallate (EGCG) by an HPV11 infection cell model.

Results: The recombined HPV11.HaCaT cells had stable HPV 11 early genes expression. The introducing of HPV11 genome significantly increased the proliferation of HPV11.HaCaT cells, as well as the proportion of cells in S and G2/M phases. After treated with rhIFN-α 2a, IFN signaling pathway was activated in both HaCaT and HPV11.HaCaT cells, while HPV11 decreased the activation level. In addition, rhIFN-α 2a, could inhibit expression of HPV 11 E6 and E7 mRNA significantly (P<0.05). However, cell growth and cell cycle did not show statistical difference (P>0.05). Nevertheless, EGCG, a major active constituent in tea polyphenol, showed strong anti-HPV11 effect, which inhibited HPV11 E6 and E7 mRNA.

Methods: Gene transfection technique was used to introduce HPV11 genome into HaCaT cells, named HPV11.HaCaT cells. With the established cell model, we explore the anti-HPV11 effect of (-)-Epigallocatechin-3-gallate (EGCG) on cell growth, viability and affection on expression HPV11 E6 and E7 mRNA.

Conclusions: Our data collectively demonstrated that the recombinant HPV11.HaCaT cells were integral and practical to be a cell model to test anti-HPV11 agents and explore the interaction between HPV11 genes and host cells. And EGCG inhibits expression of HPV11 E6 and E7 mRNA in the recombinant HPV11.HaCaT cells.

Keywords: HPV11; IFN pathway; condyloma acuminatum; early genes; model.

Figure 1. HPV11 early genes were identified in HPV11.HaCaT cells, but not HaCaT cells

A. HPV11 early genes were only expressed in HPV11.HaCaT cells, while β-actin were expressed in both cells. B. Immunofluorescence showed E7 protein was expressed in HPV11.HaCaT cells. Green fluorescence indicated E7 protein and blue fluorescence indicated cell nucleus. C. The expression of E6 and E7 genes were increased significantly in suspension culture HPV11.HaCaT cells comparing to normal culture. D–F. In suspension culture HPV11.HaCaT cells, the proportion of cells in G2/M and S phases is significantly increased. G. The expression of involucrin is significantly increased in suspesion cultured of both HPV11.HaCaT and HaCaT cells, comparing with normal culture. Data are means ± SD from three independent experiments. H refers to HaCaT cells and P refers to HPV11.HaCaT cells.

Figure 2. The cell growth curves and FACS analysis of HaCaT and HPV11.HaCaT cells

A. Cell growth curve showed that HPV gene could promote the host cells’ proliferation. From the 3rd day, HPV11. HaCaT proliferated more rapidly than HaCaT (P<0.05). B–D. Cell cycle analysis in HaCaT and HPV11.HaCaT cells. In HPV11.HaCaT cells, there is a significant increase in the proportion of cells in G2/M and S phases (G1: HPV11.HaCaT<HaCaT, P<0.01; G2: HPV>HaCaT, P<0.05; S: HPV>HaCaT, P<0.01).

Figure 3. FACS analysis of HaCaT and HPV. HaCaT with rhIFN-α 2a treatment

A&C. FACS analysis of cell cycles in HaCaT and HPV11.HaCaT cells with 100 U/ml rhIFN-α 2a treatment. B&D. FACS analysis of cell cycles in HaCaT and HPV11.HaCaT cells with 10⁶ U/ml rhIFN-α 2a treatment. No significant difference showed in different concentration of rhIFN-α 2a treatment.

Figure 4. Gene expression difference and cell viability HPV11.HaCaT cells with dose dependent rhIFN-α 2a treatment for 24h

A&B. After treated for 24h, 1000U/ml or higher concentration rhIFN-α 2a could significantly decrease the expression of HPV11 E6 and E7 mRNA (P<0.05). Data are means ± SD from three independent experiments. C. Cell viability did not change significantly with the different concentration of rhIFN-α 2a concentration.

Figure 5. The suppression of JAK-STAT signaling pathway in HPV11.HaCaT Cell line

A. Western blotting analysis of HaCat and HPV11.HaCaT Cells after rhIFN-α 2a treatment (1000U/ml). B. pSTAT1 was significantly decreased in HPV11.HaCaT Cells after IFN-α treatment for 0.5 to 2 hours. C. pSTAT2 was significantly decreased in HPV11.HaCaT Cells after rhIFN-α 2a treatment for 0.5 to 2 hours. Data are means ± SD from three independent experiments.

Figure 6. The anti-HPV effect of EGCG in HPV11.HaCaT cells

A&B. DMSO treatment showed no cytotoxicity for HaCat and HPV11.HaCaT Cells. C&D. EGCG treatment affects cell viability for HaCat and HPV11.HaCaT Cells. But cell survival was more than 80% in both cells in 24h. High concentration (100μmol/L) EGCG showed significant inhibition on cells proliferation in 48h as the cell survival decreasing to 60%. E&F. After treated for 12 or 24 hours, 50μmol/L and 100μmol/L EGCG could significantly decrease the expression of HPV11 E6 and E7 mRNA (P<0.05), while DMSO as solvent didn't show an ability to decrease HPV11 E6 and E7 mRNA expression. Data are means ± SD from three independent experiments.