Original ArticlesDihydroartemisinin (DHA) induces ferroptosis and causes cell cycle arrest in head and neck carcinoma cells
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.
Section snippets
Cell lines and reagents
Human larynx carcinoma HEP-2, nasopharyngeal carcinoma cell lines (5-8F, CNE-1, CNE-2, CNE-2Z), non-cancerous immortalized nasopharyngeal epithelial cell line NP-69, human hepatocyte cell line HL-7702, and human umbilical vein endothelial cells (HUVECs) were purchased from the Cell Bank of Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences (Shanghai, China). HUVECs were maintained in DMEM medium. Other cell lines were cultured in RPMI 1640 medium (Gibco, Thermo Fisher)
DHA specifically reduces viability of HNSCC cell lines
We first tested the effect of DHA on cell viability utilizing a panel of head and neck cancer cell lines (5-8F, CNE-1, CNE-2Z, CNE-2, HEP-2) and two normal epithelial cell lines (nasopharyngeal epithelial cell line NP-69 and human liver cell line HL-7702) by MTT assay. As shown in Fig. 1A–C, DHA reduced cell viability in a dose-dependent manner in all five HNSCC cell lines. DHA also exerted its pharmacological effect in a time-dependent manner in cancer cells. Surprisingly, DHA had no effect on
Discussion
Novel therapeutic agents that specifically and efficiently target cancer cells are crucial for developing promising treatment modalities for HNSCC [35]. DHA is an effective treatment drug for falciparum malaria; it shows remarkable safety, is well tolerated, and is widely used in the clinic. Studies are now emerging that show DHA also exhibits strong antitumor activity in different human cancer cells [36], [37], [38]. However, less than a handful of studies have examined the effect of DHA on
Conflict of interest
The authors disclose no potential conflicts of interest.
Acknowledgements
The work was supported by National Natural Science Foundation of China (81572443 and 81503107), Zhejiang Province Natural Science Funding of China (LY14H130003 and LY16H310011), Zhejiang Key Health Science and Technology Project (WKJ2013-2-021), and the Opening project from Zhejiang Provincial Extremely Key Discipline of Pharmaceutical Science (2014-KFKT-06).
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These authors contribute equally to this work.