Antimyeloma In Vitro Activity Of 15-Deoxy-D12,14-Prostaglandin J2 Is Associated With Endoplasmic Reticulum Stress In a Reactive Oxygen Species-Dependent Fashion
Authors: Demasi Ana Paula , Napimoga Marcelo Henrique , Martinez Elizabeth Ferreira , Santos Duarte Adriana Silva , Pericole Fernando V , Saad Sara T O , Araujo Ney Soares , Araujo Vera Cavalcanti 
Published: October 2019

Abstract
Multiple myeloma (MM) is an incurable malignancy characterized by the accumulation of terminally differentiated plasma cells in the bone marrow, usually accompanied by continuous monoclonal immunoglobulin production. This production requires proper folding and assembly of immunoglobulin chains, conducted by disulfide bonds formation in the endoplasmic reticulum (ER). Disulfide bonds are formed via electron transfer from thiol groups in a protein relay system, from protein disulfide isomerase (PDI) to ER oxidoreductase1 (ERO1) and finally to molecular oxygen, generating one molecule of hydrogen peroxide (H2O2) as secondary product for each disulfide bond formed. The high ER protein-folding demand usually placed on MM cells predisposes them to accumulation of unfolded proteins, which results in ER stress and also oxidative stress, both capable to induce cell death. Cellular defense systems for these specific forms of stress, known as the unfolded protein response (UPR) and oxidative stress response, have been explored as therapeutic targets for MM.
15-Deoxy-D12,14-prostaglandin J2 (15d-PGJ2) is a member of J2 series of PGs, characterized by the presence of an electrophilic carbonyl group in the cyclopentenone ring, which confers them biological properties that are different from those of other components of the PG family. These include anti-inflammatory, anti-proliferative and pro-apoptotic activities, depending on the concentration and cell type. Starting from the observation that15d-PGJ2 is emerging as the most potent antineoplastic agent of the J2 series of PGs, in the present study we investigated its effect on MM cells.
We found that 15d-PGJ2 displayed superior cytotoxicity than dexamethasone in MM cell lines. Using ﬂow cytometry, we demonstrated intracellular reactive oxygen species (ROS) formation in these cells after 15d-PGJ2 treatment, which was associated with an augmented ratio of oxidized to reduced glutathione (GSH), indicating that the cells were under oxidative stress. Due to its electrophilic property, it has been suggested that 15d-PGJ2 may directly react with GSH and/or other thiol compounds. Such reactions would deplete essential cellular antioxidants thereby leading to oxidative stress in MM cells.

Results
from qPCR analysis showed that 15d-PGJ2 induces transcription of UPR genes, including GRP78, GRP94, EDEM1 and also PRDX4. GRP78/BiP and GRP94 are ER-resident chaperones, members of the heat shock protein (HSP) 70 family and HSP 90 family, respectively. These chaperones were described to bind to immunoglobulin chains assisting their assembly. The ER degradation-enhancer mannosidase-like protein 1 (Edem1) is involved in the degradation of misfolded proteins to prevent their aggregation and cytotoxicity. PRDX4 encodes the ER-localized peroxiredoxin (Prx) IV, an enzyme not yet recognized as an UPR component, but recently demonstrated to use the H2O2 produced by ERO1 during disulfide bond formation to generate another pair of disulfide, with the additional benefit of reducing H2O2 to water. Increased transcription of these genes indicates an attempt to preserve ER-processing function and MM cells survival in response to accumulated misfolded proteins. In this regard, 15d-PGJ2-induced oxidative stress, by provoking oxidative modification of proteins, could have intensified the formation of misfolded proteins, thereby activating UPR signaling. In agreement with 15d-PGJ2-mediated accumulation of misfolded proteins, which are prevented from progressing further along the secretory pathway, our results from ELISA have shown a massive decrease in the concentration of light chain immunoglobulin secreted by MM cells in the presence of 15d-PGJ2, but not dexamethasone.
These data suggest that 15d-PGJ2 favors ROS accumulation and protein misfolding, especially threatening the survival of cells that have a high protein-folding load and/or are susceptible to oxidative stress. In this sense, multiple myeloma and related disorders may benefit from 15d-PGJ2 therapy.

Disclosures:
Demasi: FAPESP: Research Funding. Napimoga:FAPESP: Research Funding.