Cancer Letters

Cancer Letters

Volume 364, Issue 1, 1 August 2015, Pages 79-88
Cancer Letters

Original Articles
Nelfinavir, an HIV protease inhibitor, induces apoptosis and cell cycle arrest in human cervical cancer cells via the ROS-dependent mitochondrial pathway

https://doi.org/10.1016/j.canlet.2015.04.027Get rights and content

Highlights

Abstract

HIV protease inhibitors (HIV-PIs) are a class of antiretroviral drugs designed to target the viral protease. Strikingly, these drugs have also been reported to possess antitumor effect. In this study, we evaluated the activity of one HIV-PI, Nelfinavir, against human cervical cancer cells. We found that Nelfinavir inhibited the growth of cervical cancer cell lines at the lowest micromolar concentrations clinically attainable. Nelfinavir promoted apoptosis and arrested the cell cycle at G1 phase. Apoptosis is attributed to the promotion of mitochondrial reactive oxygen species (ROS) production, which results in the translocation of mitochondrial apoptosis inducing factor (AIF) to the nucleus. We further showed that Nelfinavir increased mitochondrial ROS production by decreasing manganese superoxide dismutase (MnSOD) protein levels. Taken together, our results suggest that Nelfinavir can be repositioned as a cervical cancer therapeutic.

Introduction

Cervical cancer is the third most common type of cancer in women worldwide and a leading cause of cancer-related death for women in developing countries [1]. Conventional therapies for cervical cancer include surgery, radiotherapy and chemotherapy [2]. Surgery is the first option for patients with early-stage cervical cancer, while radiotherapy and chemotherapy have proven to be effective treatments for patients in the advanced stages. The most commonly used chemotherapy drugs in advanced cervical cancer is platinum-based chemotherapy [3]; however, the effect of these drugs is limited, and their side effects are serious [4], [5]. Therefore, development of new chemotherapeutic agents is required.

HIV protease inhibitors (HIV-PIs) are a class of small-molecule drugs that were rationally designed to target the viral aspartyl protease. They are peptidomimetic drugs designed to mimic the peptide bond targeted by viral protease, but not by any other mammalian endopeptidase [6], which means they have a good specificity of action with tolerable side effects. Currently, nine FDA-approved HIV-PIs, Nelfinavir, Indinavir, Ritonavir, Amprenavir, Saquinavir, Lopinavir, Tipranavir, Atazanavir and Darunavir, are available. These drugs, given in combination with reverse transcriptase inhibitors, are the mainstays of the current therapeutic regimens for HIV-infected patients. In recent years, accumulating evidence supports that HIV-PIs can be promising antineoplastic agents. Early support for this came from a report of a patient with regression of Kaposi's sarcoma (KS) following therapy with HIV-PIs. Such antitumor mechanism was attributed to HIV load reduction or CD4 T cell gain [7]. However, several recent studies support that HIV-PIs have direct antitumor activities that are independent of their antiviral activity, including leukemia [8], lung cancer [9], breast cancer [10], glioblastoma [11], multiple myeloma [12], melanoma [13] and ovarian cancer [14]. Studies also show that treatment of HIV-infected women with Saquinavir, an HIV-PI, can reduce the onset of uterine cervical intraepithelial neoplasia (CIN) and halt its progression to cervical carcinoma [15], but the direct effect and mechanism of HIV-PIs on cervical carcinoma remains unknown.

In this study, we aimed to identify an HIV-PI-based anti-HIV drug with the most potent inhibitory activity on the growth of cervical cancer cells and investigate its direct effect on cervical cancer cells and elucidate the underlying mechanisms of action.

Section snippets

Cell viability assays

Cell viability was determined by XTT array as previously described [16]. Briefly, the cervical cancer cell lines (Hela, SiHa and CaSki) were seeded in triplicate in 96-well plates at 104 cells per well and cultured in the presence of HIV-PIs as indicated for 3 days, the medium was exchanged, and the cells were incubated for 4 h with 1 mg/mL XTT reagent and PMS. Absorbance was measured at 450 nm in the microplate reader.

BrdU/7-AAD staining

For assessment of in vitro proliferation, the APC BrdU flow kit (BD

Effect of HIV-PIs on the growth of cervical cancer cell lines

We first systematically investigated the effectiveness of 9 HIV-PIs (Nelfinavir, Indinavir, Ritonavir, Amprenavir, Saquinavir, Lopinavir, Tipranavir, Atazanavir and Darunavir) on the growth of cervical cancer cell line HeLa. Cell viability was assessed using XTT assay after treating HeLa cells with different concentrations (1–1000 µM) of HIV-PIs for 72 h. We found that exposure of HeLa cells to the 9 HIV-PIs showed very different activity against HeLa cells. Nelfinavir was the most active drug

Discussion

The direct antitumor activities of HIV-PIs have been explored in various cancer models, but the direct effect and mechanism of HIV-PIs on cervical carcinoma remains unknown. In this study, we showed a direct effect and a new mechanism on the antitumor activity of the HIV-PI Nelfinavir in human cervical cancer cell lines. Among the HIV-PIs we tested, Nelfinavir was the most active drug for inhibiting the growth of three human cervical cancer cell lines, SiHa, HeLa, and CaSki, with IC50 between

Funding

This work was supported by the China Scholarship Council (No. 201307610018) and an intramural fund of the New York Blood Center (NYB000068).

Authors' contributions

Conception and design: S. Jiang, B. Zhu, T. Xiang.

Development of methodology: T. Xiang, P. Pham.

Acquisition of data (provided animals, acquired and managed patients, provided facilities, etc.): T. Xiang, P. Pham.

Analysis and interpretation of data (e.g., statistical analysis, biostatistics, computational analysis): T. Xiang, L. Du, P. Pham.

Writing, review, and/or revision of the manuscript: T. Xiang, B. Zhu, S. Jiang.

Study supervision: S. Jiang, L. Du, B. Zhu.

Conflict of interest

No potential conflicts of interest are disclosed by the authors.

Acknowledgments

The authors thank Dr. Titia de Lange of the Laboratory for Cell Biology and Genetics, The Rockefeller University, for the HeLa cells. The following reagents were obtained through the NIH AIDS Reagent Program, Division of AIDS, NIAID, NIH: Nelfinavir, Indinavir, Ritonavir, Amprenavir, Saquinavir, Lopinavir, Tipranavir, Atazanavir and Darunavir, as well as SiHa and CaSki cells.

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