Anti-fungal drug itraconazole exerts anti-cancer effects in oral squamous cell carcinoma via suppressing Hedgehog pathway

doi:10.1016/j.lfs.2020.117695

Life Sciences

Volume 254, 1 August 2020, 117695

https://doi.org/10.1016/j.lfs.2020.117695 Get rights and content

Highlights

•
Itraconazole inhibited the proliferation of OSCC in vitro and in vivo.
•
Itraconazole induced apoptosis of OSCC in vitro and in vivo.
•
Itraconazole perturbed invasion and migration of OSCC.
•
Itraconazole suppressed OSCC through Hedgehog pathway.

Abstract

Aims

To investigate the therapeutic potential of itraconazole in oral squamous cell carcinoma (OSCC) and its molecular mechanism.

Materials and methods

The in vitro anti-cancer effects of itraconazole was determined by CCK-8 assay and colony formation assay. Transwell and wound healing assays were used to examine cell invasion and migration. The in vivo therapeutic efficacy of itraconazole was assessed by OSCC patient-derived xenograft (PDX) model. Western blot was performed to explore the anti-cancer mechanism.

Key findings

Itraconazole inhibited cell proliferation and colony formation of OSCC cells in a time and concentration dependent manner; induced cell cycle arrest and apoptosis, as well as inhibited cell invasion and migration. In the OSCC PDX model, itraconazole impeded tumor growth, reduced Ki-67 expression and induced apoptosis. Itraconazole downregulated the protein expression of Hedgehog pathway to inhibit proliferation and migration of oral squamous cell carcinoma cells, which can be revised by recombinant human sonic hedgehog protein (rSHH).

Significance

Itraconazole showed anti-cancer effects on OSCC via inhibiting the Hedgehog pathway.

Introduction

Oral squamous cell carcinoma (OSCC) is the most frequent malignant tumor in the head and neck squamous cell carcinoma (HNSCC), with the rising overall incidence and stagnant 5-year survival rate of 50% around the world [1]. For the past 20 years, the 5-year overall survival of OSCC only increased slightly by 5% [2]. Late diagnosis, aggressive local invasion, and early lymph node metastasis result in high rates of relapse and mortality [3]. The treatment of OSCC remains a clinical challenge, despite improvements in the standard treatments, which are surgery, radiotherapy, and chemotherapy [4]. Cisplatin (DDP) is the first-line chemotherapy for OSCC with satisfactory efficacy but severe side effects [5]. Cetuximab, targeting the epidermal growth factor receptor (EGFR), was approved for the treatment of OSCC and remains the only molecular targeted therapy available for OSCC [6]. Unfortunately, long-term cetuximab treatment usually causes drug resistance over time [7,8]. Novel therapeutic strategies for OSCC are still in high demand.

Itraconazole is a broad-spectrum anti-fungal drug that blocks the synthesis of ergosterol in the fungal cell membrane [9]. The repurposing of approved drugs as novel anti-cancer agents is becoming increasingly attractive as it saves great amount of time and cost during the drug developing process [10]. Itraconazole is a Food and Drug Administration (FDA)-approved drug that had been tested for its safety for use in human and widely assessed as a novel anti-cancer agent [11]. The superior anti-cancer activity of itraconazole has been demonstrated in a variety of cancers [12]. Prior studies demonstrated that itraconazole is a promising antagonist of the Hedgehog (Hh) pathway as well as an anti-angiogenesis agent and verified it in basal cell carcinoma murine model [13,14]. However, the potential use of itraconazole in OSCC has not yet been examined. We herein aimed to investigate whether itraconazole exerts anti-cancer effects against OSCC and its underlying mechanism of action.

Section snippets

Cell culture and reagents

Human OSCC cell lines, UM1, SCC9, SCC15, SCC25 were cultured in Dulbecco's Modified Eagle Medium/Nutrient Mixture F-12 (DMEM/F – 12) (Gibco, New York, USA) supplemented with 10% fetal bovine serum (FBS) (Gibco, New York, USA). Cells were incubated in humidified air with 5% CO₂ in a 37 °C incubator. For in vitro experiments, itraconazole isomer C (2S,4R,2′S) powder [15] (Aidea Pharma, Jiangsu, China) was dissolved in 0.05% dimethyl sulfoxide (DMSO). For in vivo usage, itraconazole isomer C oral

Itraconazole inhibits cell proliferation and induces cell apoptosis in OSCC cells

To investigate the anti-cancer effect of itraconazole on OSCC, four human OSCC cell lines representing distinct oral cancer subtypes were used. As shown in Fig. 1A–D, itraconazole showed appreciable, time-and dose-dependent inhibition on the proliferation of OSCC cells, including UM1, SCC9, SCC15, and SCC25. At the first 24 h, 0.1 μM itraconazole showed no inhibition on these four OSCC cells. While at 72 h, the inhibition rate of 10 μM itraconazole on these four cell lines reached 90.19%,

Discussion

This study provided evidence that itraconazole manifested significant inhibition of OSCC by negative regulation of the Hedgehog pathway. In vitro, itraconazole inhibited cell proliferation, induced cell cycle arrest and apoptosis, and perturbed cell invasion and migration. In vivo, we found that itraconazole impeded tumor growth, triggered necrosis and apoptosis in the OSCC PDX model. To our knowledge, this is the first study demonstrating the feasibility of re-purposing anti-fungal drug

Conclusion

In summary, our study demonstrated that itraconazole inhibits OSCC markedly in vitro and in vivo. The results might provide insight into the application of repurposing itraconazole in further preclinical and clinical studies for OSCC.

The following are the supplementary data related to this article.

. Itraconazole (ITRA) induces cell cycle arrest and cell apoptosis in OSCC UM1 and SCC15 cells.

. Itraconazole (ITRA) inhibits the abilities of invasion and migration of OSCC UM1 and SCC15 cells.

Acknowledgments

This study was supported by the National Natural Science Foundation of China (NSFC81672659, NSFC81472523); the National Science and Technology Major Project for “Major New Drugs Innovation and Development” (2018ZX09734003); and the Natural Science Foundation of Guangdong Province (2020A1515010291).

Declaration of competing interest

The authors declared that there are no conflicts of interest.

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¹: Co-first authors.

View full text

Anti-fungal drug itraconazole exerts anti-cancer effects in oral squamous cell carcinoma via suppressing Hedgehog pathway

Highlights

Abstract

Aims

Materials and methods

Key findings

Significance

Introduction

Section snippets

Cell culture and reagents

Itraconazole inhibits cell proliferation and induces cell apoptosis in OSCC cells

Discussion

Conclusion

Acknowledgments

Declaration of competing interest

Eur. J. Cancer

Cancer Cell

Biochim. Biophys. Acta (BBA)-Mol. Basis Dis.

Oral Oncol.

J. Thorac. Oncol.

Cancer Lett.

Cancer Cell

Exp. Mol. Pathol.

Hum. Pathol.

Cancer Cell

J. Thorac. Cardiovasc. Surg.

Exp. Cell Res.

J. Cancer Res. Clin. Oncol.

Cancer Treat. Rev.

Current aspects on oral squamous cell carcinoma

Open Dent. J.

Oral cancer: prevention, early detection, and treatment

The changing therapeutic landscape of head and neck cancer

Nat. Rev. Clin. Oncol.

Biochemical mechanisms of cisplatin cytotoxicity

Anti Cancer Agents Med. Chem.

Cetuximab-containing combinations in locally advanced and recurrent or metastatic head and neck squamous cell carcinoma

Front. Oncol.

Predicting tumor sensitivity to chemotherapeutic drugs in oral squamous cell carcinoma patients

Sci. Rep.

Anti-epidermal growth factor receptor therapy in head and neck squamous cell carcinoma: focus on potential molecular mechanisms of drug resistance

Oncologist

Role of itraconazole in haematology/oncology

Arch. Dis. Child.