ATO Increases ROS Production and Apoptosis of Cells by Enhancing Calpain-Mediated Degradation of the Cancer Survival Protein TG2

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  Figure 1. Tissue transglutaminase impairs the capability of ATRA + ATO-differentiated NB4 cells for endogenous ROS production. The production of endogenous ROS was determined for each cell line using a DCFDA-based method and reported in Relative Fluorescence Units (RFU). The graphs represent mean ± SD values normalised to 100 µg protein of total cell lysate content. (A) NB4 TG2 WT cells were treated with ATRA 1 µM, ATO 0.5 µM, ATRA 1 µM + ATO 0.5 µM, ATO 2.0 µM, and ATRA 1 µM + ATO 2.0 µM for five days. Endogenous ROS values were measured on each day in triplicate (n = 5). (B) NB4 TG2 KO cells were treated with ATRA 1 µM, ATO 0.5 µM, ATRA 1 µM + ATO 0.5 µM, ATO 2.0 µM, and ATRA 1 µM + ATO 2.0 µM for five days. Endogenous ROS values were measured on each day in triplicate (n = 5).

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  Figure 2. Transglutaminase 2 attenuates ATO-induced cell death. NB4 cells were treated, harvested, and labelled with FITC/PE-conjugated Annexin-V/PI for 15 min. Cells were analysed on a BD FACSAria™ III. Apoptotic features were evaluated by the size and granularity of the NB4 cells, followed by gating out the FITC/PI-positive cell population. (A) NB4 TG2 WT cells were treated with ATRA 1 µM, ATO 0.5 µM, ATRA 1 µM+ATO 0.5 µM, ATO 2.0 µM, and ATRA 1 µM + ATO 2.0 µM for five days. Dead cell ratios were measured each day in triplicate (n = 5). (B) NB4 TG2 KO cells were treated with ATRA 1 µM, ATO 0.5 µM, ATRA 1 µM + ATO 0.5 µM, ATO 2.0 µM, and ATRA 1 µM + ATO 2.0 µM for five days. Dead cell ratios were measured each day in triplicate (n = 5).

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  Figure 3. Endogenous ROS production is regulated via the transcription factor NRF2, influencing the expression of antioxidant genes. NB4 TG2 WT and TG2 KO cells were incubated with ATRA 1 µM, ATO 0.5 µM, ATRA 1 µM + ATO 0.5 µM, ATO 2.0 µM, and ATRA 1 µM + ATO 2.0 µM for five days. Relative mRNA expressions of (A,B) NRF2, (C,D) GST-Ω, and (E,F) PRDX5 were measured on the indicated days using real-time Q-PCR and were normalised to GAPDH (means ± SD, n = 3). Representative Western blots/densitometry analysis showed protein expression levels upon various treatments over 5 days (n = 3). Statistical significance was determined via two-way analysis of variance (ANOVA; Bonferroni post hoc test; ATRA vs. ATRA + ATO * p < 0.05).

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  Figure 4. ATO-induced ROS production elevates calpain expression/activation, resulting in increased proteolysis of transglutaminase 2. NB4 WT cells were incubated with ATRA 1 µM, ATRA 1 µM + ATO 0.5 µM, and ATRA 1 µM + ATO 2.0 µM for five days. (A) Representative Western blots/densitometry analysis showing calpain total protein expression levels upon treatment over 5 days (n = 3). (B) The graph represents relative normalised calpain activity RFU values on 100 µg protein content of the total, Annexin-V positive/negative sorted cell lysates. Samples’ activity values were determined with a Synergy Multireader fluorometer (400/505 nm). (C) Representative Western blots/densitometry analysis showing calpain protein expression levels in total and Annexin-V sorted cell lysates (n = 3). (D) Untreated, ATRA differentiated, and ATRA + ATO 2.0 μM treated NB4 WT cells were treated with 1 μM or 2 μM calpain inhibitor (C.I.) for 5 days. Flow cytometric analysis shows the cell viability changes upon the treatments. (E) Representative Western blots/densitometry analysis showing TG2 protein expression levels in total cell lysates upon calpain inhibitory treatments (n = 3). Statistical significance was…

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  Figure 5. Transglutaminase 2 levels are reduced by ATO treatment. (A) Relative mRNA expressions of TG2 were measured in NB4 TG2 cells on the indicated days using real-time Q-PCR and normalised to GAPDH. (B) Representative Western blots/densitometry analysis showing TG2 protein expression levels in total cell lysates upon ATRA and ATRA + ATO treatments (n = 3). (C) MCF-7 cells were treated with 1 µg/mL doxorubicin (DOX), ATO 2.0 µM, or with a combination. As a positive control, ATRA-treated NB4 WT cells were used. Relative mRNA expressions of TG2 were measured using real-time Q-PCR and normalised to GAPDH. (D) Representative Western blots/densitometry analysis showing TG2 protein expression levels in total MCF-7 and NB4 WT cell lysates (n = 3). (E) Relative mRNA expressions of TG2 were measured in monocytes on the indicated days using real-time Q-PCR and normalised to GAPDH. (F) Representative Western blots and (G) densitometry analysis showing TG2 protein expression levels in total cell lysates upon ATO 2.0 µM, calpain inhibitor 2.0 µM, MCSF, MSCF + ATO 2.0 µM, MSCF + ATO 2.0 µM + calpain inhibitor 2.0 µM, and MSCF + calpain inhibitor 2.0 µM treatments for 3 and 5 days (n = 3). Statistical…