Hsc 3

The human OSCC cell lines (HSC3 and its subclone HSC3-M3 and HSC2) and human skin-derived squamous cell carcinoma cell lines (HSC1 and HSC5) [40 (link)] were obtained from Health Science Research Resources Bank (Osaka, Japan). HSC3, HSC3-M3 and HSC2 were maintained in RPMI1640. HSC1 and HSC5 were maintained in Dulbecco’s minimum essential medium (DMEM) and Iscove’s Modified Dulbecco’s Medium (IMDM), respectively. Each medium was supplemented with 10% FCS, 50 μg/ml streptomycin and 50 U/ml penicillin (10% FCS-RPMI, 10% FCS-DMEM and 10% FCS-IMDM). To test Ni²⁺ ion stimulation, 2 × 10⁵ cells were plated in a 24-well dish on the day before the experiment. The cells were washed twice with 10% FCS-RPMI and further cultured in the presence or absence of 1 mM Ni²⁺ ions.

Fe(Salen) nanoparticle reagent was purchased from Tokyo Chemical Industry Co. Ltd. Fe(Salen) was sonicated for 30 min and used in normal saline suspension as previously described18 (link). The average size of nanoparticles was about 200 nm. Cetuximab (Erbitax^®) was purchased from Merck Serono (Tokyo, Japan). Rabbit squamous cell carcinoma (VX2) cells were purchased from American Type Culture Collection (ATCC) (Virginia, U.S.A.). VX2 cells were transfected with an EGFP-encoding vector as previously described23 (link). Human oral squamous cell carcinoma cell lines, OSC-19 and HSC-3, were purchased from the Health Science Research Resources Bank (Japan Health Sciences Foundation, Tokyo, Japan). In all cases, early passage cultures were stored and used for experiments. These cell lines were cultured in RPMI-1640 with L-glutamine and phenol red medium containing 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin.

All human oral cancer cell lines (Ca9-22, CAL 27, HSC-3, OC-2, and SCC-9) and a normal oral cell line (HGF-1) were used from Health Science Research Resources Bank (HSRRB) (Osaka, Japan) and American Type Culture Collection (ATCC; Manassas, VA, USA) except for OECM1 [57 (link)], a generous gift from Dr. Wan-Chi Tsai (Kaohsiung Medical University, Taiwan). Cells were cultured in 5% CO₂ at 37 °C with humidity and maintained by regular formula (Gibco, Grand Island, NY, USA) with 10% fetal bovine serum as previously described [49 (link)].
Manoalide (CAYMAN CHEMICAL, Ann Arbor, MI, USA) was dissolved in dimethyl sulfoxide (DMSO) for treatment. A ROS scavenger N-acetylcysteine (NAC) [58 (link)] (Sigma-Aldrich; St. Louis, MO, USA) was dissolved in double distilled water. The mitochondrial superoxide inhibitor MitoTEMPO [59 (link)] (Cayman Chemical, Ann Arbor, MI, USA), panapoptosis inhibitor Z-VAD-FMK [60 (link)], Cas 8 inhibitor Z-IETD-FMK, and Cas 9 inhibitor Z-LEHD-FMK (Selleckchem.com; Houston, TX, USA) was dissolved in DMSO. All experiments contain the same concentration of DMSO.

The human oral squamous cell carcinoma cell lines HSC-3 and OSC-19, which are high metastatic potential lines, were purchased from the Health Science Research Resources Bank (Japan Health Sciences Foundation, Tokyo, Japan)^{9 (link),46 (link)}. The human oral squamous cell carcinoma cell lines SAT and HSC-4 were acquired from the Japanese Collection of Research Bioresources (Osaka, Japan). The SAT cell line exhibits non-metastatic characteristics, whereas HSC-4 is characterized by a low rate of metastasis^{10 (link),24 (link)}. The human glioblastoma cell line LN229 was purchased from American Type Culture Collection (ATCC) (Virginia, USA). These cell lines were cultured in Dulbecco’s modified Eagle’s medium (DMEM [High Glucose], WAKO, Osaka, Japan) supplemented with L-glutamine and phenol red or in sodium pyruvate medium containing 10% fetal bovine serum (FBS), 1% penicillin‒streptomycin and 1% L-glutamine. Human gingival fibroblasts (HGnF cells) and human oral keratinocytes were purchased from ScienCell Research Laboratories (CA, USA).

Human oral cancer cell lines from gingival carcinoma (Ca9.22) and tongue carcinoma (SCC9 and HSC-3) were respectively ordered from Health Science Research Resources Bank (HSRRB) (Osaka, Japan) and American Type Culture Collection (ATCC; Manassas, VA, USA). A normal human gingival fibroblast cell line (HGF-1) was ordered from ATCC. All tested cells were maintained in DMEM: F-12/3:2 ratio and supplemented with 10% FBS, 2 mM glutamine, and antibiotics (100 units/mL penicillin and 100 μg/mL streptomycin) at 37 °C in a humidified atmosphere of 5% CO₂.