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

Bandiera,Elisabetta; Todeschini,Paola; Romani,Chiara; Zanotti,Laura; Erba,Eugenio; Colmegna,Benedetta; Bignotti,Eliana; Santin,Alessandro Davide; Sartori,Enrico; Odicino,Franco Edoardo; Pecorelli,Sergio; Tassi,Renata Alessandra; Ravaggi,Antonella

doi:10.3892/ol.2016.5008

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

Authors:
- Elisabetta Bandiera
- Paola Todeschini
- Chiara Romani
- Laura Zanotti
- Eugenio Erba
- Benedetta Colmegna
- Eliana Bignotti
- Alessandro Davide Santin
- Enrico Sartori
- Franco Edoardo Odicino
- Sergio Pecorelli
- Renata Alessandra Tassi
- Antonella Ravaggi
View Affiliations

Affiliations: Department of Obstetrics and Gynecology, ‘Angelo Nocivelli’ Institute for Molecular Medicine, University of Brescia, I‑25123 Brescia, Italy, Department of Oncology, Flow Cytometry Unit, IRCCS - ‘Mario Negri’ Institute for Pharmacological Research, I‑20156 Milan, Italy, Department of Obstetrics, Gynecology and Reproductive Sciences, Yale University School of Medicine, New Haven, CT 06510, USA
Published online on: August 16, 2016 https://doi.org/10.3892/ol.2016.5008
Pages: 2493-2500
Copyright: © Bandiera et al. This is an open access article distributed under the terms of Creative Commons Attribution License.

Metrics: Total Views: 0 (Spandidos Publications: | PMC Statistics: )

Metrics: Total PDF Downloads: 0 (Spandidos Publications: | PMC Statistics: )

Cited By (CrossRef): 0 citations Loading Articles...

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.

View Figures

View References

1	Ferlay J, Soerjomataram I, Dikshit R, Eser S, Mathers C, Rebelo M, Parkin DM, Forman D and Bray F: Cancer incidence and mortality worldwide: Sources, methods and major patterns in GLOBOCAN 2012. Int J Cancer. 136:E359–E386. 2015. View Article : Google Scholar : PubMed/NCBI
2	Arbyn M, Castellsagué X, de Sanjosé S, Bruni L, Saraiya M, Bray F and Ferlay J: Worldwide burden of cervical cancer in 2008. Ann Oncol. 22:2675–2686. 2011. View Article : Google Scholar : PubMed/NCBI
3	Lăără E, Day NE and Hakama M: Trends in mortality from cervical cancer in the Nordic countries: Association with organised screening programmes. Lancet. 1:1247–1249. 1987. View Article : Google Scholar : PubMed/NCBI
4	Hakama M and Louhivuori K: A screening programme for cervical cancer that worked. Cancer Surv. 7:403–416. 1988.PubMed/NCBI
5	Bodily J and Laimins LA: Persistence of human papillomavirus infection: Keys to malignant progression. Trends Microbiol. 19:33–39. 2011. View Article : Google Scholar : PubMed/NCBI
6	Münger K, Werness BA, Dyson N, Phelps WC, Harlow E and Howley PM: Complex formation of human papillomavirus E7 proteins with the retinoblastoma tumor suppressor gene product. EMBO J. 8:4099–4105. 1989.PubMed/NCBI
7	Scheffner M, Werness BA, Huibregtse JM, Levine AJ and Howley PM: The E6 oncoprotein encoded by human papillomavirus types 16 and 18 promotes the degradation of p53. Cell. 63:1129–1136. 1990. View Article : Google Scholar : PubMed/NCBI
8	Crook T, Tidy JA and Vousden KH: Degradation of p53 can be targeted by HPV E6 sequences distinct from those required for p53 binding and trans-activation. Cell. 67:547–556. 1991. View Article : Google Scholar : PubMed/NCBI
9	Boyer SN, Wazer DE and Band V: E7 protein of human papilloma virus-16 induces degradation of retinoblastoma protein through the ubiquitin-proteasome pathway. Cancer Res. 56:4620–4624. 1996.PubMed/NCBI
10	DiSaia PJ and Creasman WT: Cervical CancerClinical Gynecologic Oncology. 5th. Mosby; Maryland Heights: pp. 1–106. 1997
11	Deeks SG, Smith M, Holodniy M and Kahn JO: HIV-1 protease inhibitors. A review for clinicians. JAMA. 277:145–153. 1997. View Article : Google Scholar : PubMed/NCBI
12	Sgadari C, Barillari G, Toschi E, Carlei D, Bacigalupo I, Baccarini S, Palladino C, Leone P, Bugarini R, Malavasi L, et al: HIV protease inhibitors are potent anti-angiogenic molecules and promote regression of Kaposi sarcoma. Nat Med. 8:225–232. 2002. View Article : Google Scholar : PubMed/NCBI
13	Monini P, Sgadari C, Toschi E, Barillari G and Ensoli B: Antitumour effects of antiretroviral therapy. Nat Rev Cancer. 4:861–875. 2004. View Article : Google Scholar : PubMed/NCBI
14	Russo SM, Tepper JE, Baldwin AS Jr, Liu R, Adams J, Elliott P and Cusack JC Jr: Enhancement of radiosensitivity by proteasome inhibition: Implications for a role of NF-kappaB. Int J Radiat Oncol Biol Phys. 50:183–193. 2001. View Article : Google Scholar : PubMed/NCBI
15	Pajonk F, Himmelsbach J, Riess K, Sommer A and McBride WH: The human immunodeficiency virus (HIV)-1 protease inhibitor saquinavir inhibits proteasome function and causes apoptosis and radiosensitization in non-HIV-associated human cancer cells. Cancer Res. 62:5230–5235. 2002.PubMed/NCBI
16	Gaedicke S, Firat-Geier E, Constantiniu O, Lucchiari-Hartz M, Freudenberg M, Galanos C and Niedermann G: Antitumor effect of the human immunodeficiency virus protease inhibitor ritonavir: Induction of tumor-cell apoptosis associated with perturbation of proteasomal proteolysis. Cancer Res. 62:6901–6908. 2002.PubMed/NCBI
17	Pati S, Pelser CB, Dufraine J, Bryant JL, Reitz MS Jr and Weichold FF: Antitumorigenic effects of HIV protease inhibitor ritonavir: Inhibition of Kaposi sarcoma. Blood. 99:3771–3779. 2002. View Article : Google Scholar : PubMed/NCBI
18	André P, Groettrup M, Klenerman P, de Giuli R, Booth BL Jr, Cerundolo V, Bonneville M, Jotereau F, Zinkernagel RM and Lotteau V: An inhibitor of HIV-1 protease modulates proteasome activity, antigen presentation, and T cell responses. Proc Natl Acad Sci USA. 95:13120–13124. 1998. View Article : Google Scholar : PubMed/NCBI
19	Schmidtke G, Holzhütter HG, Bogyo M, Kairies N, Groll M, de Giuli R, Emch S and Groettrup M: How an inhibitor of the HIV–I protease modulates proteasome activity. J Biol Chem. 274:35734–35740. 1999. View Article : Google Scholar : PubMed/NCBI
20	Piccinini M, Rinaudo MT, Chiapello N, Ricotti E, Baldovino S, Mostert M and Tovo PA: The human 26S proteasome is a target of antiretroviral agents. AIDS. 16:693–700. 2002. View Article : Google Scholar : PubMed/NCBI
21	Goldberg AL and Rock K: Not just research tools-proteasome inhibitors offer therapeutic promise. Nat Med. 8:338–340. 2002. View Article : Google Scholar : PubMed/NCBI
22	Barillari G, Iovane A, Bacigalupo I, Palladino C, Bellino S, Leone P, Monini P and Ensoli B: Ritonavir or saquinavir impairs the invasion of cervical intraepithelial neoplasia cells via a reduction of MMP expression and activity. AIDS. 26:909–919. 2012. View Article : Google Scholar : PubMed/NCBI
23	Hampson L, Kitchener HC and Hampson IN: Specific HIV protease inhibitors inhibit the ability of HPV16 E6 to degrade p53 and selectively kill E6-dependent cervical carcinoma cells in vitro. Antivir Ther. 11:813–825. 2006.PubMed/NCBI
24	Justesen US: Protease inhibitor plasma concentrations in HIV antiretroviral therapy. Dan Med Bull. 55:165–185. 2008.PubMed/NCBI
25	Monini P, Sgadari C, Grosso MG, Bellino S, Di Biagio A, Toschi E, Bacigalupo I, Sabbatucci M, Cencioni G, Salvi E, et al: Clinical course of classic Kaposi's sarcoma in HIV-negative patients treated with the HIV protease inhibitor indinavir. AIDS. 23:534–538. 2009. View Article : Google Scholar : PubMed/NCBI
26	Flick DA and Gifford GE: Comparison of in vitro cell cytotoxic assays for tumor necrosis factor. J Immunol Methods. 68:167–175. 1984. View Article : Google Scholar : PubMed/NCBI
27	Tanaka K, Mizushima T and Saeki Y: The proteasome: Molecular machinery and pathophysiological roles. Biol Chem. 393:217–234. 2012. View Article : Google Scholar : PubMed/NCBI
28	Bertuzzi A, Gandolfi A, Germani A, Spanò M, Starace G and Vitelli R: Analysis of DNA synthesis rate of cultured cells from flow cytometric data. Cytometry. 5:619–628. 1984. View Article : Google Scholar : PubMed/NCBI
29	Kalantari M, Karlsen F, Kristensen G, Holm R, Hagmar B and Johansson B: Disruption of the E1 and E2 reading frames of HPV 16 in cervical carcinoma is associated with poor prognosis. Int J Gynecol Pathol. 17:146–153. 1998. View Article : Google Scholar : PubMed/NCBI
30	Liu J and Westin SN: Rational selection of biomarker driven therapies for gynecologic cancers: The more we know, the more we know we don't know. Gynecol Oncol. 141:65–71. 2016. View Article : Google Scholar : PubMed/NCBI
31	Lee CG, Gottesman MM, Cardarelli CO, Ramachandra M, Jeang KT, Ambudkar SV, Pastan I and Dey S: HIV-1 protease inhibitors are substrates for the MDR1 multidrug transporter. Biochemistry. 37:3594–3601. 1998. View Article : Google Scholar : PubMed/NCBI
32	Kraus M, Bader J, Overkleeft H and Driessen C: Nelfinavir augments proteasome inhibition by bortezomib in myeloma cells and overcomes bortezomib and carfilzomib resistance. Blood Cancer J. 3:e1032013. View Article : Google Scholar : PubMed/NCBI
33	Kraus M, Malenke E, Gogel J, Müller H, Rückrich T, Overkleeft H, Ovaa H, Koscielniak E, Hartmann JT and Driessen C: Ritonavir induces endoplasmic reticulum stress and sensitizes sarcoma cells toward bortezomib-induced apoptosis. Mol Cancer Ther. 7:1940–1948. 2008. View Article : Google Scholar : PubMed/NCBI
34	Taura M, Kariya R, Kudo E, Goto H, Iwawaki T, Amano M, Suico MA, Kai H, Mitsuya H and Okada S: Comparative analysis of ER stress response into HIV protease inhibitors: Lopinavir but not darunavir induces potent ER stress response via ROS/JNK pathway. Free Radic Biol Med. 65:778–788. 2013. View Article : Google Scholar : PubMed/NCBI
35	Gupta AK, Cerniglia GJ, Mick R, McKenna WG and Muschel RJ: HIV protease inhibitors block Akt signaling and radiosensitize tumor cells both in vitro and in vivo. Cancer Res. 65:8256–8265. 2005. View Article : Google Scholar : PubMed/NCBI
36	Ikezoe T, Saito T, Bandobashi K, Yang Y, Koeffler HP and Taguchi H: HIV-1 protease inhibitor induces growth arrest and apoptosis of human multiple myeloma cells via inactivation of signal transducer and activator of transcription 3 and extracellular signal-regulated kinase 1/2. Mol Cancer Ther. 3:473–479. 2004.PubMed/NCBI
37	Laurent N, de Boüard S, Guillamo JS, Christov C, Zini R, Jouault H, Andre P, Lotteau V and Peschanski M: Effects of the proteasome inhibitor ritonavir on glioma growth in vitro and in vivo. Mol Cancer Ther. 3:129–136. 2004.PubMed/NCBI
38	Berkers CR, Verdoes M, Lichtman E, Fiebiger E, Kessler BM, Anderson KC, Ploegh HL, Ovaa H and Galardy PJ: Activity probe for in vivo profiling of the specificity of proteasome inhibitor bortezomib. Nat Methods. 2:357–362. 2005. View Article : Google Scholar : PubMed/NCBI