The HIV-protease inhibitor saquinavir reduces proliferation, invasion and clonogenicity in cervical cancer cell lines

Abstract

Innovative therapies in cervical cancer (CC) remain a priority. Recent data indicate that human immunodeficiency virus (HIV)-protease inhibitors used in highly active antiretroviral therapy can exert direct antitumor activities also in HIV-free preclinical and clinical models. The aim of the present study was to evaluate the antineoplastic effects of various HIV-protease inhibitors (indinavir, ritonavir and saquinavir) on primary and established CC cell lines. Two CC cell lines established in our laboratory and four commercially available CC cell lines were treated with indinavir, ritonavir and saquinavir at different concentrations and for different times. Proliferation, clonogenicity and radiosensitivity were evaluated by crystal violet staining. Proteasomal activities were assessed using a cell-based assay and immunoblotting. Cell cycle was analyzed by propidium iodide staining and flow cytometric analysis. Invasion was tested with Matrigel chambers. A t-test for paired samples was used for statistical analysis. In all cell lines, saquinavir was more effective than ritonavir in reducing cell proliferation and inhibiting proteasomal activities (P≤0.05). Conversely, indinavir exerted a negligible effect. The saquinavir concentrations required to modulate the proteasome activities were higher than those observed to be effective in inhibiting cell proliferation. In HeLa cells, saquinavir was strongly effective in inhibiting cell invasion and clonogenicity (P≤0.05) at concentrations much lower than those required to perturb proteasomal activities. Saquinavir did not contribute to increase the sensitivity of HeLa cells to X-rays. In conclusion, the present results demonstrate that saquinavir is able to significantly reduce cell proliferation, cell invasion and clonogenicity in a proteasome-independent manner in in vitro models of CC, and suggest that saquinavir could be a promising CC therapeutic agent.

Keywords: HIV-protease inhibitors; cell lines; cervical cancer; proteasomal activities; saquinavir.

Figure 1.

(A) Expression of HPV16 E6 and E7 mRNA in CC1 and CaSki cell lines, and expression of HPV18 E6 and E7 mRNA in CC2 and HeLa cell lines. The HT3 cell line served as HPV-negative control. (B) Immunoblotting for intracellular levels of ubiquitinated proteins and ubiquitin, which was performed with whole HeLa cell extracts following treatment with 40, 60 and 80 µM Saq. CTRL, control; Saq, saquinavir; DMSO, dimethyl sulfoxide; HPV, human papillomavirus; mRNA, messenger RNA.

Figure 2.

Dose-response curve for HeLa cells treated with saquinavir at different concentrations for 96 h. Means and standard deviations were represented by points and error bars, respectively. IC₅₀, inhibitory concentration.

Figure 3.

HeLa (A) cell invasion (magnification, ×10) and (B) clonogenicity upon treatment with 10 and 19 µM Saq for 96 h. CTRL, control; Saq, saquinavir; DMSO, dimethyl sulfoxide.

Figure 4.

(A) Growth inhibitory effects induced on HeLa cells treated for 24, 48, 72 and 96 h with 10 and 19 µM Saq. (B) Graphic representation of HeLa cells sensitivity to X-rays following the treatment with 10 and 19 µM Saq for 96 h. Following irradiation, cells were treated with Saq for an additional 6 days. Means and standard deviations were represented by points and error bars, respectively. CTRL, control; Saq, saquinavir; DMSO, dimethyl sulfoxide.