Oral squamous cell carcinoma (OSCC), one type of head and neck cancer, is located at the 10th of human malignant and reached about 40 and 55 % survival rates after clinical treatment [1]. OSCC is typically treated with surgery, radiotherapy, chemoradiation, or combined with these clinical treatments. To date, photodynamic therapy (PDT) was collected as a new efficacy clinical approach in OSCC treatment by specifically visible light excitation due to it holding low-toxic, non-invasive treatment and low-side effects, etc. A previous study showed that human tongue squamous cell carcinoma treated with 5-aminolevulinic acid, one precursor of photosensitizer protoporphyrin XI, reduces cancer cell proliferation by increasing cell apoptosis signaling [2]. Since PDT was applied in clinical cancer treatment by its specificity and sensitivity, the antitumor effects of PDT may result in direct or indirect tumor cell death/damage through activation of non-specific and specific immune responses against the tumor cell progress [[3], [4], [5]].

5, 10, 15, 20-Tetrakis(3-hydroxyphenyl)chlorin (also called temoporfin) is one of the potent second-generation synthetic photosensitizers of PDT for treating with head-neck carcinoma in clinical therapy by specifically light excitation, including oral malignant tissue [6,7]. Moreover, temoporfin (Temo) induced cell death may correlate with cellular reactive oxygen species (ROS) generation [8,9]. In addition, cellular ROS induct several ways of cell death, such as apoptosis, autophagy, and ferroptosis [10]. Ferroptosis is an iron-dependent form of nonapoptotic cell death. System Xc− regulates cystine uptake for glutathione (GSH) synthesis, playing a crucial role in antioxidant defense. Its inhibition or suppression by P53 leads to reduced glutathione peroxidase 4 (GPX4) activity, lipid ROS buildup, and ferroptosiss [11]. GSH is necessary for the normal physiological function of GPX4. Intracellular GSH synthesis is catalyzed by glutamate–cysteine ligase [12]. Ferroptosis has been implicated in a range of different diseases including cancer, inflammation, neurodegeneration, and kidney injury [13]. Numerous studies have shown that the regulation of ferroptosis can influence the effectiveness of cancer therapies and may even reverse resistance to treatment. However, it is still incompletely known that temoporfin-triggered cancer cell mortality by which cell death induction signals, especially in ferroptosis.

Induction of intracellular levels of cellular free radicals leads to elimination of cancer cells which might provide a new concept in clinical therapy [14]. In the antioxidant defense system, the enzymatic components, such as superoxide dismutase (SOD) and catalase (CAT), as well as non-enzymatic mechanisms, such as glutathione (GSH), for against cellular oxidative stress. At the beginning, intracellular O2•− was formed during the action of SOD that converted to the hydrogen peroxide (H2O2). Next, nuclear factor erythroid 2–related factor 2 (Nrf-2) as a nuclear transcription factor accounts for increasing the transcription/translation of catalase, which leads to degradation of H2O2 in the cell [15]. On the other side, GSH played another chelation role in the prevention of O2•−/H2O2 induced damages which formed oxidative GSH (GSSG) by glutathione peroxidases (GPX) and recycled to GSH by glutathione reductase (GR) [16]. This defense system prevents normal or cancer cell death [17]. If the antioxidant defense system fails, high levels of H2O2 have damaging effects on cells caused the lipid peroxidation, DNA damage or mitochondria dysfunction are induced cell death under dysfunctional of antioxidant defense system [18].

It is well known that the promotion of the hypoxia microenvironment contributed to cancer cell proliferation, angiogenesis, and tumor metastasis [19]. Recently, hypoxia has been illustrated it limited therapeutic effects of PDT to treatment with oral carcinoma [20]. Another study indicated that PDT remained maintain ability in induction of cancer cell toxicity in the hypoxia environment [21]. Therefore, from the literature review, we hypothesized that temoporfin would resistance to oral carcinoma development by elevating ROS accumulation for activating cell death programing by oxidative stress possibly. However, temoporfin-induced cell death presented controversial results in different oxygen level in the previous studies. Thus, we also investigate the effects of the temoprofin-induced cell death under normoxic and hypoxic conditions in the present study.