Benefits of Combined All-Trans Retinoic Acid and Arsenic Trioxide Treatment of Acute Promyelocytic Leukemia Cells and Further Enhancement by Inhibition of Atypically Expressed Transglutaminase 2

Abstract

Randomized trials in acute promyelocytic leukemia patients have shown that treatment with a combination of all-trans retinoic acid (ATRA) and arsenic trioxide (ATO) is superior in efficacy to monotherapy, with significantly decreased mortality. So far, there are little data available to explain the success of the ATRA and ATO combination treatment in molecular terms. We showed that ATRA-and ATO-treated cells had the same capacity for superoxide production, which was reduced by two-thirds in the combined treatment. Secreted inflammatory biomarkers (monocyte chemoattractant protein-1 [MCP-1], interleukin-1 beta [IL-1β] and tumor necrosis factor-α [TNF-α]) were significantly decreased and were further reduced in a transglutaminase 2 (TG2) expression-dependent manner. The amount of secreted TNF-α in the supernatant of NB4 TG2 knockout cells was close to 50 times lower than in ATRA-treated differentiated wild-type NB4 cells. The irreversible inhibitor of TG2 NC9 not only decreased reactive oxygen species production 28-fold, but decreased the concentration of MCP-1, IL-1β and TNF-α 8-, 15-and 61-fold, respectively in the combined ATRA + ATO-treated wild-type NB4 cell culture. We propose that atypical expression of TG2 leads to the generation of inflammation, which thereby serves as a potential target for the prevention of differentiation syndrome.(ATRA) both activates the PML-RARα chimera protein and initiates its proteolysis, resulting in the differentiation of APL leukemic cells [1][2][3].In the 2000s, arsenic trioxide (As 2 O 3 /ATO) was approved by the Food and Drug Administration (US FDA) to treat APL. Clinical data have shown that ATO, either as a single agent or combined with ATRA, can improve the outcomes in newly diagnosed and relapsed APL, compared to ATRA treatment alone [4][5][6].When combined with ATRA and chemotherapy, ATO can induce complete remission of APL patients, with a five-year overall survival rate up to 90% [5]. At the molecular level, ATO exhibits cytotoxic effects in a concentration-dependent manner, while at lower concentrations (< 0.5 µM), ATO can induce partial differentiation and at higher concentrations (> 0.5 µM), it initiates apoptosis of APL cells. Some of the signal transduction pathways and transactivations of transcription factors involved are arsenic-induced and redox-sensitive. These include the mitogen-activated protein kinases (MAPKs), extracellular signal-regulated kinases 1/2 (ERK1/2), stress-activated protein kinase/Jun-N terminal protein kinases (SAPK/JNK), the apoptosis signal-regulating kinase 1/thioredoxin (ASK1/TRX) system, the AKT-mTOR pathway, transcription factor AP-1 and the nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) [7]. Although both ATO and ATRA prime the PML-RARα oncoprotein for proteolysis, only ATO is effective as a monotherapy, through the elimination of residual leukemia-initiating cells. The major limitation to ATO treatment is coagulopathy, which is one of the major causes of early death in APL, driven by procoagulant and fibrino...

Dysregulation of Transglutaminase type 2 through GATA3 defines aggressiveness and Doxorubicin sensitivity in breast cancer

“…The latter feature of NC9 is particularly relevant, if we consider that the TGH isoform of the enzyme is still capable of hydrolysing GTP, albeit with low binding efficiency [12]. NC9 has been shown to irreversibly block both functions, especially in cancer types [45,[47][48][49]55,56]. Our data demonstrated that the choice to use NC9 in combined treatments proved effective, driving MDA-MB-231 cells to early apoptosis and MCF-7 and T47D cells to late apoptosis, limiting the progression of cell cycle but inducing accumulation of cells in different phases, depending on the type of cell, while also decreasing NF-ĸB in MDA-MB-231 cells.…”

“…Experiments with NC9 were performed on BrCa cells to verify whether the action of Doxo depends on an accumulation of TG2 as in other types of cancers [45][46][47][48][49]. In this approach NC9 was added at 30 μM concentration (K i value) for 16 h, after 24 h of treatment with Doxo for a total exposure time of 40 h. In this way we studied the effects of this drug combination on the expression of NF-ĸB in MCF-7 and MDA-MB-231 cells.…”

Transglutaminase 2 as a Marker for Inflammation and Therapeutic Target in Sepsis

“…Cystamine inhibits LPS-induced liver injury TG2 is mainly expressed and activated in midzonal F4/80/CD80+ M1 macrophages in the livers of septic mice ⇧ Promoting [22] Cystamine inhibits neuroinflammation in amyotrophic lateral sclerosis TG2 catalyzes the oligomerization of superoxide dismutase 1 and induces TNF-α, IL-1β, and nitric oxide in microglial cells ⇧ Promoting [107] Cystamine ameliorates TNBS-induced colitis in a rat model of inflammatory bowel disease TG2 activity is associated with the production of mucosal TNF-α and serological IL-6 ⇧ Promoting [108] Cystamine rescues defective CFTR-induced cystic fibrosis TG2 catalyzes the crosslinking of beclin 1, leading to the sequestration of phosphatidylinositol-3-kinase complex III, accumulation of p62, and aggresome formation ⇧ Promoting [109] NC9 NC9 reduces pro-inflammatory cytokine production in combined ATRA and ATOtreated APL cells TG2 leads to inflammation, which is probably due to reactive oxygen species production ⇧ Promoting [116] ⇧, promoting role in inflammation or sepsis; ⇩, protective role in inflammation or sepsis; APL, acute promyelocytic leukemia; ATO, arsenic trioxide; ATRA, all-trans retinoic acid; CFTR, cystic fibrosis transmembrane conductance regulator; DC, dendritic cell; EC, endothelial cell; IFN-γ, interferon-γ; IL-1β, interleukin-1β; IL-17, interleukin-17; iNOS, inducible nitric oxide synthase; KO, knockout; LPS, lipopolysaccharide; MPTP, 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine; MSU, monosodium urate; NFκΒ, nuclear factorκΒ; PMN, polymorphonuclear leukocyte; TGF-β, transforming growth factor-β; TNBS, 2,4,6-trinitrobenzene sulfonic acid; UV, ultraviolet.…”

“…These novel irreversible inhibitors of TG2, such as NC9 and VA4 [ 114 ], which specifically react with Cys-277 in the TG2 transamidation site and inhibit both transamidation and GTP-binding activities, are potent anticancer agents that inhibit the survival of cancer stem cells [ 115 ]. Very recently, an in vitro study showed that NC9 effectively reduced the levels of pro-inflammatory cytokines such as monocyte chemotactic protein 1, IL-1β, and TNF-α, in combined all-trans retinoic acid and arsenic trioxide-treated acute promyelocytic leukemia cells [ 116 ]. Evidence for genetic and pharmacological inhibition of TG2 during inflammation and sepsis is summarized in Table 1 .…”

Acute Promyelocytic Leukemia

“…Differentiation syndrome, which develops in approximately 5–25% of APL patients, is a life-threatening condition triggered by a release of inflammatory cytokines and chemokines by blastic cells; these differentiate in response to ATRA and/or ATO therapy. An original article of this special issue demonstrates that atypical expression of the enzyme transglutaminase 2 (TG2) leads to the generation of inflammation, and could serve as a potential target for the prevention of differentiation syndrome [ 7 ].…”