Purpose Darinaparsin (Zio-101) is a novel organic arsenical compound with encouraging clinical activity in relapsed/refractory T-cell lymphoma (TCL) and Hodgkin lymphoma (HL), however little is known regarding its mechanism of action. phosphorylation of ERK (and relevant downstream substrates) primarily by decreasing the inhibitory SHP1 phosphatase and co-immunoprecipitation showed significant ERK/SHP1 interaction. Furthermore, ERK shRNA knockdown or constitutive overexpression of SHP1 resulted in increased apoptosis, while co-treatment with pharmacologic MEK inhibitors resulted in synergistic cell death. Conversely, SHP1 blockade (via pharmacologic inhibition or RNAi) as well as MEK constitutive activation decreased darinaparsin-related cell death. Conclusions Altogether, these data show that darinaparsin is highly active in HL and TCL and its activity is dependent primarily on MAPK mechanisms. and experiments with leukemia showed that darinaparsin was a potent antineoplastic agent (4, 5). Additionally, early-phase clinical trials in patients with hematological malignancies demonstrated that darinaparsin is safer and effective compared with inorganic arsenic trioxide (ATO).(6C8) Moreover, it is known that darinaparsin and ATO inhibit tumor growth by distinct mechanisms (8). The biological mechanism of SB-408124 action of darinaparsin in lymphoma is unknown. Our goals were to investigate the potency of darinaparsin in TCL and HL cells lines and related xenograft SCID mouse models. Furthermore, we intended to identify the associated biologic mechanisms of action. We found that darinaparsin induced dose- and time-dependent apoptosis against TCL and HL cell lines, and demonstrated in vivo therapeutic activity of darinaparsin in TCL and HL tumor xenografts grown in SCID mice. Furthermore, we show SB-408124 that darinaparsin treatment resulted in the activation of MAPK pathway by a unique mechanism involving the inhibitory SHP1 protein tyrosine phosphatase. Methods Cell culture, reagents, and transfections HL cell lines L540, L428, KMH2 and L1236, and TCL cell lines HH, Hut78 Rabbit polyclonal to PABPC3 and Jurkat were grown in RPMI1640 consisting of 10% heat inactivated fetal bovine serum and 200U of penicillin/streptomycin (Mediatech, Manassas, VA) under 5% CO2 and 37C. Darinaparsin was kindly provided by Ziopharm Oncology, Inc (Boston, MA). U0126 and AZD6244 was obtained from Selleck Chem. (Houston, TX). Non-targeting or smart pool ERK2 siRNA were obtained from Thermo Fisher Scientific. For L428, transfection of siRNA or plasmid DNA was performed using Amaxa Nucleofector device and Amaxa cell line Nucleofector kit L reagent, (Lonza, Walkersville, MD). For RNAi experiments, lentiviral based pGPIZ manifestation system (Open Biosystem) was utilized to transduce scrambled non-targeting, ERK2 or SHP1shRNA sequences into lymphoma cells. For induction of ERK activity, Addgene #21193 plasmid construct which express constitutively active form of MEK was used for the transfection experiment in Hut78 using Amaxa Nucleofector device and Amaxa cell line Nucleofector kit R reagent, (Lonza, Walkersville, MD). For SHP1 overexpression experiments, lentiviral supernatants were prepared by transfecting HEK293T cells with pBABE-puro SHP1 WT (Addgene#8575) or pBABE-puro Vector (Addgene#1764) and packaging plasmids pUMVC (Addgene#8449) and PCMV-VSV-G (Addgene#8454), using Fugene 6 reagent (Promega, Inc). High titer lentiviral supernatants were transduced into 0.5106 cells using retronectin (Clontech Inc) coated 12-well plates and puromycin (2g/mL) was used as selection antibiotic. Mass spectrometry for determination of intracellular arsenic concentration Twenty million L428 cells treated with 3M arsenic trioxide (ATO) or 3M Darinaparsin for 1, 3 and 6 hours were harvested, washed with PBS and dehydrated in the SB-408124 oven at 60C overnight followed by digestion in nitric acid at 80C. 0.1 to 50g/L of arsenic standards were prepared from a 1mg/L stock, previously prepared from the 10mg/L standard in 2% nitric acid. Internal standard without arsenic at a concentration of 50g/L. For sample analysis, the internal standard was added to the cell digest and diluted to 4 mL with double distilled and deionized water. Arsenic (As), concentrations were assessed using inductively coupled plasma mass spectroscopy (ICP-MS, X Series II, Thermo Electron). MTT assay For MTT, 104 cells/100L were plated in a 96-well plate and treated with increasing concentrations of Darinaparsin (1C5M) for 24C72 hours. MTT assay was performed using Cell Titer Aqueous Non-Radioactive Cell Proliferation assay, Promega Inc. Madison, WI, as per instructions supplied by the manufacturer. Apoptosis and cell cycle analysis by flow cytometry Annexin-V/propidium iodide (PI)-based estimation of apoptosis by flow cytometry performed using Apoptosis Detection Kit-I (BD Biosciences, San Jose, CA). Briefly, 106 cells per mL of complete RPMI 1640 medium treated with darinaparsin for 24 or 48 hours was harvested, washed with ice cold PBS and stained with Annexin-V-FITC antibody and PI for 15 minutes and the samples were analyzed by flow cytometry within 1 hour. For cell cycle analysis, darinaparsin treated cells were harvested, washed in ice cold PBS, fixed SB-408124 in ethanol and stained with PI staining,.