Inhibition of antiviral drug cidofovir on proliferation of human papillomavirus-infected cervical cancer cells

Abstract

In order to evaluate the potential application value of cidofovir (CDV) in the prevention of human papillomavirus (HPV) infection and treatment of cervical cancer, the inhibitory effect of CDV on the proliferation of HPV 18-positive HeLa cells in cervical cancer was preliminarily investigated, using cisplatin (DDP) as a positive control. An MTT assay was used to analyze the effects of CDV and DDP on HeLa cell proliferation. In addition, clone formation assay and Giemsa staining were used to examine the extent of HeLa cell apoptosis caused by CDV and DDP. Flow cytometry was also used to detect the shape and size of apoptotic cells following propidium iodide staining, while western blot analysis identified the expression levels of of E6 and p53 proteins in HeLa cells. A cell climbing immunofluorescence technique was used to locate the subcellular position of p53 in HeLa cells. The results demonstrated that CDV and DDP inhibited the proliferation of HeLa cells in a concentration- and time-dependent manner. Flow cytometry showed that CDV and DDP treatments resulted in cell arrest in the S-phase, and triggered programmed cell death. Furthermore, western blot analysis revealed that CDV and DDP inhibited E6 protein expression and activated p53 expression in HeLa cells. Finally, the immunofluorescence results indicated that CDV and DDP inhibited the nuclear export of p53 by E6 protein, which is required for degradation of endogenous p53 by MDM2 and human papilloma virus E6. In conclusion, CDV and DDP inhibited HeLa cell proliferation in a concentration- and time-dependent manner, reduced the expression of E6 protein, and reinstated p53 protein activity. Thus, CDV regulates cell cycle arrest and apoptosis, and may be a potential cervical cancer therapeutic strategy.

Keywords: HeLa cells; apoptosis; cervical cancer; cidofovir; cisplatin; human papillomavirus.

Figure 1.

(A) Growth of HeLa cells treated with different concentrations of (A) cidofovir and (B) cisplatin, for 24, 48 and 72 h (magnification, ×200).

Figure 2.

Cell viability of HeLa cells treated with (A) CDV and (B) DDP for 24, 48 or 72 h, as detected by MTT assay. Data were analyzed by one-way analysis of variance. Values are expressed as the mean ± standard deviation in each group (n=3). *P<0.05 compared to the control group. CDV, cidofovir; DDP, cisplatin.

Figure 3.

Colony formation of HeLa cells, observed using Giemsa staining. CDV, cidofovir; DDP, cisplatin.

Figure 4.

Detection of apoptosis of HeLa cells using Giemsa staining (magnification, ×200). The arrows show apoptotic cell nuclei. CDV, cidofovir; DDP, cisplatin.

Figure 5.

HeLa cells stained by DAPI (magnification, ×1,000). The arrows show apoptotic cell nuclei. CDV, cidofovir; DDP, cisplatin.

Figure 6.

Cell cycle progression and apoptosis of HeLa cells. Histograms of (A) cell cycle distribution, and (B) apoptosis percentage. Values are expressed as the mean ± standard deviation in each group (n=3). (C) Apoptosis of HeLa cells detected by flow cytometry. *P<0.05 vs. the control group. CDV, cidofovir; DDP, cisplatin.

Figure 7.

(A) Western blots and (B) quantified western blotting results, showing the expression levels of E6 and p53 proteins treated with CDV and DDP. Values are expressed as the mean ± standard deviation for three wells in each group. *P<0.05 vs. control group. CDV, cidofovir; DDP, cisplatin.

Figure 8.

Immunofluorescence microscopy detection of p53 expression in HeLa cells treated by CDV and DDP (magnification, ×1,000). Nuclei were shown in the DAPI-stained cell (blue) and p53 protein was shown as green fluorescence. CDV, cidofovir; DDP, cisplatin.