Piperlongumine

Chitosan, fucoidan, and 2′,7′-dichlorofluorescein diacetate (DCF-DA) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Piperlongumine was supplied from Cayman Chemical (Ann Arbor, MI, USA). hDFB human dermal fibroblast cells and PC-3 human prostate cancer cells were obtained from ATCC (Manassas, VA, USA). The AnnexinV-FITC/PI staining kit was purchased from BD Life Sciences (San Jose, CA, USA). Tween 80 was purchased from Duksan Pure Chemicals (Ansan-si, Gyunggido, Korea).

Sodium arsenite (Na₂AsO₂, CAS:7784-46-5), apocynin, 5,5-dimethyl-1-pyrroline-1-oxide (DMPO), and Annexin V/propidium iodide (PI) were purchased from Sigma (St Louis, MO). Both 5-(and -6)-chloromethyl-2,7-dichlorodihydrofluorescein diacetate, acetyl ester (DCFDA) and dihydroethidium (DHE) were purchased from Molecular Probes (Eugene, OR). Curcumin, luteolin, genistein, and butein were purchased from Sigma-Aldrich (St. Louis, MO). Shikonin, wogonin, plumbagin, bis(2-hydroxybenzal) acetone, phenethyl isothiocyanate, rutin, and piperlongumine were purchased from Cayman Chemical (Ann Arbor, MI). Plasmid DNA encoding human catalase and SOD2 and catalase shRNA were purchased from Origene (Rockville, MD). Antibody against SOD2 was purchased from Millipore (Billerica, MA). Antibodies against catalase (#14097), AKT(#9272), pAKT^Ser473(#9271), PI3K(#4257), p-PI3K(#4228), C-PARP(#5625), C-Caspase-3(#9664), Bcl-2(#3498), and wortmannin were purchased from Cell Signaling Tech (Danvers, MA).

Piperlongumine was purchased from Cayman Chemical (Ann Arbor, Michigan, USA). The apoptosis assay kit was purchased from eBioscience and the cell proliferation ELISA kit (BrdU) was purchased from Roche Diagnostics. Propidium iodide (PI), Ribonuclease A (RNase A), and dimethyl sulfoxide (DMSO) were purchased from Sigma-Aldrich (Shanghai, China). The cell counting kit-8 (CCK-8) was purchased from Dojindo Laboratories (Japan). Antibodies against p-STAT3, STAT3, PSMB6 (β1), PSMB8 (β5i), CDK2, cyclin E2, cyclin A, Bax, Bcl-2, c-myc, p21, p27, survivin, p65, p-p65, IL-1β, Histone-H3, caspase-8, cleaved caspase-9, and cleaved caspase-3 were purchased from Cell Signaling Technology (Beverly, MA, USA). PSMB5 (β5) and PSMB9 (β1i) were purchased from Abcam (Cambridge, MA). p38, p-p38, IL-18, ERK, p-ERK, and GAPDH were purchased from Santa Cruz Biotechnology, Inc. (USA). PSMA5 was purchased from Bioworld Technology. CD138 was purchased from Proteintech. Recombinant human macrophage colony-stimulating factor (M-CSF) and human soluble receptor activator of NF-κB ligand (sRANK Ligand) were purchased from PeproTech (Rocky Hill, NJ). STAT3-CA (constitutively active, A661C, N663C) plasmid was purchased from Addgene. The Cys712 of STAT3-CA was mutated to Ala (STAT3-CA-C712A) using the Fast Mutagenesis System.

For experiments that involved the TrxR inhibitor auranofin (Cayman Chemical Company), solid auranofin was dissolved in sterile-filtered DMSO to make a 32 mM stock solution the day of the experiment. The auranofin stock solution was then added to culture medium comprised of DMEM supplemented with 10% FBS to reach final auranofin concentrations of 32, 16, 8, 4, and 1 μM in a total volume of 2 mL culture medium. Pure dimethyl sulfoxide (DMSO) was used as a control. Cells were then incubated at 37 °C for a period of 90 min. After incubation, the signal in each fluorescent channel was acquired in three different regions of each well. For experiments that involved the cancer therapeutic piperlongumine, solid piperlongumine (Cayman Chemical Company) was dissolved in DMSO to make 30 mM stock solution the day of the experiment. Before addition of the piperlongumine to the cell culture, images in each fluorescent channel were acquired for each well. After these images were acquired, the piperlongumine stock solution was then added to the media to reach a final piperlongumine concentration of 10 μM in a total volume 2.5 mL; as a control, an equivalent volume of DMSO was added to a separate cell sample. Images were acquired in each fluorescent channel in each well every 2 h for a period of 10 h.