Albendazole

Navitoclax was synthesized at AbbVie, Inc. (North Chicago, IL). FDA-approved drug library was purchased from Enzo (Farmingdale, NY). Albendazole, mebendazole, oxibendazole, and oxfendazole were purchased from Sigma (St. Louis, MO). All siRNA pools were purchased from Dharmacon (Lafayette, CO). Noxa antibody was purchased from Abcam. Bim and Mcl-1 antibodies were purchased from Epitomics (Burlingame, CA). Caspase 3 and caspase 9 inhibitors were purchased from SantaCruz Biotechnology (Santa Cruz, CA). All the branched DNA reagents were purchased from Affymetrix (Santa Clara, CA).

Two approaches for cell synchronization were tested: the starvation [42 (link)] and the albendazole-dependent [43 (link)] methods. Both methods provided the same results concerning the mitosomal dynamics. However, the albendazole treatment had a much greater effect on cell synchrony and therefore was used in the study. Trophozoites from the late log phase were incubated in growth medium supplemented with 100 ng/mL of albendazole (Sigma-Aldrich, St. Louis, MO, USA) for 6 h at 37 °C [43 (link)]. After incubation, the albendazole-affected unattached cells were discarded and the unaffected adherent pre-mitotic cells were washed twice with pre-warmed, fresh, drug-free medium and then detached from the tube by cooling on ice for 10 min. The cells were then allowed to proliferate on slides in the drug-free conditions for 9–14 min, fixed, and permeabilized as described below.

An overview of the tested plant extracts and compounds is provided in Table 1. Levamisole, albendazole, trans-cinnamaldehyde, dimethyl sulfoxide (DMSO), RPMI-1640 media and α-linolenic acid were obtained from Sigma-Aldrich (Stellenborsch, Germany). Grape seed extract, consisting of >95% condensed tannins [35 (link)], was purchased from Bulk Powders (Colchester, UK). A chicory (Cichorium intybus) extract (cv. Spadona) enriched in sesquiterpene lactones was prepared as previously described [36 (link)]. Four different seaweed extracts were produced as described by Bonde et al. [24 (link)]. Briefly, Saccharina latissma was sourced from either Grenå, Denmark, or the Faroe Islands. From each source location, extracts were prepared using water and methanol (polar extracts; SW1, SW2), or dichrolormethane and methanol (non-polar extracts; SW3, SW4). The chemical composition of the extracts has previously been reported [24 (link)].

SCC12 and SCC13 cells are the human squamous cell carcinoma line, established from SCCs of the facial epidermis [14 (link)]. Both the cells were maintained in Dulbecco's modified Eagle's medium (DMEM) supplemented with 5% fetal bovine serum (FBS) and antibiotics (Life Technologies Corporation, Grand Island, NY) at 37°C, 5% CO₂ atmosphere. Cells were routinely passaged with 1:5 ratio when they grew to nearly confluent. Normal human epidermal keratinocytes were isolated from skin specimens obtained in accordance with the ethical committee approval process of Chungnam National University Hospital. Specimens were briefly sterilized in 70% ethanol, minced, and then treated with dispase overnight at 4°C. The epidermis was separated and placed in a solution containing 0.05% trypsin and 0.02% EDTA (Life Technologies Corporation) for 15 min at 37°C. After vigorous pipetting, cells were pelleted and resuspended in keratinocyte-serum free medium (K-SFM) supplemented with bovine pituitary extract and recombinant human epidermal growth factor (Life Technologies Corporation). Albendazole and 4-phenylbutyric acid (4-PBA) were purchased from Sigma-Aldrich (St. Louis, MO) and dissolved in dimethyl sulfoxide (DMSO).

Albendazole (ABZ), 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), Tris base, glycine, NaCl, sodium dodecylsulfate (SDS), and bovine serum albumin (BSA) were purchased from Sigma-Aldrich (St. Louis, MO, USA). LightShift^® Chemiluminescent EMSA kit and Trizol were purchased from Thermo Fisher Scientific Inc. (Waltham, MA, USA). FITC Annexin V Apoptosis Detection Kit was purchased from BD Pharmingen™ (BD Biosciences, Becton-Dickinson, Franklin Lakes, NJ, USA). TUNEL (terminal transferase mediated dUTP-fluorescein nick end labeling) assay kit was from Roche Diagnostics GmbH (Mannheim, Germany). Anti-p-STAT3(Tyr705), anti-p-STAT5 (Tyr694/Tyr699), anti-p-JAK1 (Tyr1022/1023), anti-JAK1, anti-p-JAK2 (Tyr1007/1008), anti-JAK2, anti-p-Src (Tyr416), anti-Src, anti-Cleaved caspase3, and anti-Cyclin D1 antibodies were purchased from Cell Signaling Technology (Beverly, MA, USA). Anti-STAT3, anti-PTPε, anti-SHP-1, anti-Procaspase-3, anti-PARP, anti-Bcl-2, anti-Bcl-xL, anti-Survivin, anti-Cyclin D1, anti-COX-2, anti-β-actin antibodies, and SHP-1 siRNA were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA).