Dihydroartemisinin (DHA) induces ferroptosis and causes cell cycle arrest in head and neck carcinoma cells

Highlights

• Dihydroartemisinin (DHA) induces ferroptosis in head and neck carcinoma cells.

• FOXM1 is implicated in DHA-mediated cell cycle arrest.

• DHA holds promise as a treatment strategy for head and neck cancer.

Abstract

Head and neck cancer is the sixth most common cancer worldwide. Dihydroartemisinin (DHA), a semi-synthetic derivative of artemisinin, exhibits a wide range of biological roles including a highly efficient and specific anti-tumor activity. Here, we aimed to examine the effect of DHA on head and neck carcinoma cells and elucidate the potential mechanisms. We used five head and neck carcinoma cell lines and two non-tumorigenic normal epithelial cell lines to achieve our goals. Cells were exposed to DHA and subjected to cellular activity assays including viability, cell cycle analysis, cell death, and angiogenic phenotype. Our results show that DHA causes cell cycle arrest which is mediated through Forkhead box protein M1 (FOXM1). We also demonstrate that DHA induces ferroptosis and apoptosis in head and neck carcinoma cells. Lastly, our results show that DHA alters the angiogenic phenotype of cancer cells by reducing the expression of angiogenic factors and the ability of cancer cells to support endothelial cell tubule formation. Our study suggests that DHA specifically causes head and neck cancer cell death through contribution from both ferroptosis and apoptosis. DHA may represent an effective strategy in head and neck cancer treatment.

Introduction

Head and neck squamous cell carcinoma (HNSCC) is the sixth most common cancer worldwide with an incidence of over 500,000 cases each year [1], [2], [3]. HNSCC also accounts for approximately 350,000 annual deaths globally [1]. Despite advances in surgery, chemotherapy, and radiotherapy, the five-year survival rate has remained essentially the same. Currently, the treatment for early-stage HNSCC includes either surgery or radiotherapy [4]. At advanced stages, treatment may be multimodal with surgery and chemotherapy [5]. However, high risk of relapse and metastasis following treatment warrants the identification and development of new and efficient treatment options for patients with HNSCC.

Dihydroartemisinin (DHA) is a derivative and active metabolite of the artemisinin (ART). Both were first successfully isolated from Chinese medicinal herb, Artemisia annua. DHA is one of the first-line antimalarial therapies recommended by the World Health Organization. The anti-malarial mechanism of these endoperoxide-containing sesquiterpenes involves reaction with Fe²⁺ ions to generate radicals. In addition to potent anti-malarial activity, DHA has been shown to possess antiviral and antibacterial activities [6], [7]. Since ARTs have an established safety record for the treatment of malaria, there has been considerable interest in re-purposing this class of compounds for other indications such as cancer. Numerous large scale epidemiological and pharmacological studies have also shown that ARTs and DHA have efficient and selective anti-tumor activities [8], [9], [10], [11]. However, the underlying mechanisms are not fully known.

Antitumor activity of DHA and ARTs has been attributed to apoptosis induction [8], [12], [13], [14], cell cycle arrest [12], [15], [16], reduced cell proliferation [13], [14], [16], [17], alteration of tumor-associated genes [18], and inhibition of tumor angiogenesis [19], [20]. In HNSCC, studies evaluating the effect of DHA and possible underlying mechanisms are scarce. Just recently, DHA was shown to strongly inhibit cell proliferation in CNE-2 HNSCC cell line [21]. Reduced cell numbers together with cell cycle arrest was attributed to apoptosis although this was not directly tested. Similarly, Jia and colleagues showed that DHA inhibited STAT3 activation and inhibited HNSCC growth possibly through induction of apoptosis [22].

Insight into how DHA may specifically target tumor cells came from two recent studies. Ooko and colleagues showed that mRNA expression of iron-related genes correlated with the response to artemisinin derivatives [23]. Treatment of 60 cell lines of the National Cancer Institute with artemisinin derivatives caused significant changes in the expression of iron-related genes suggesting ferroptosis-inducing activity of artemisinin. The second study conducted by Ba et al. showed that DHA caused cellular iron depletion in time- and concentration-dependent manner. It was suggested that DHA disrupted iron homeostasis in cancer cells [24]. Cancer cells do exhibit an enhanced dependence on iron relative to normal cells. This phenomenon has been termed iron addiction [25].

DHA-mediated changes to iron homeostasis in cancer cells may involve direct disruption of basic iron-dependent cellular processes such as DNA synthesis or may directly lead to cell death by utilizing iron in a specific form of programmed cell death called ferroptosis. This form of cell death is defined by the iron-dependent accumulation of lipid reactive oxygen species and depletion of plasma membrane polyunsaturated fatty acids. The metal ion can cause cleavage of DHA and ARTs endoperoxide bridge via the Fenton reaction, which leads to the generation of reactive oxygen species (ROS).

In the present study, we have utilized a panel of HNSCC lines to understand the mechanisms underlying the antitumor activity of DHA. Based on the known mode of action of DHA and dependence of cancer cell on iron, we hypothesize that DHA causes HNSCC cell death by inducing ferroptosis. Here we show that DHA does specifically target HNSCC cells and reduces viability. DHA also caused cell cycle arrest and induced iron-dependent cell death.