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  • br Material and methods br Results br Discussion

    2024-06-05


    Material and methods
    Results
    Discussion The present study demonstrates for the first time that Mino selectively induces apoptosis in Jurkat 2-D08 via an H2O2 signaling-dependent mechanism. Structurally, Mino is composed of a C10 and C12 phenol group, C12a hydroxyl group, and C1 and C11 aldehyde group in the DCBA naphthacene ring system (lower peripheral region of tetracycline molecule, Fig. 9). These enol and keto groups can participate in redox reactions with molecular oxygen (O2) leading to production of superoxide anion (O2) and H2O2 (Fig. 9, step 1). Therefore, Mino might be capable of generating H2O2. This capability might explain the cytotoxic effect not only of Mino (this work) but also of other tetracycline analogues (Song et al., 2014). However, whether tetracycline analogues (e.g., doxycycline, tetracycline, terramycin (Nelson and Levy, 2011)) can eliminate leukemia cells via an H2O2-induced oxidative stress mechanism requires further investigation. Whatever the mechanism of generation, we demonstrated that H2O2 is a critical molecule in Mino-induced apoptosis. Three main observations support this view. First, Jurkat cells treated with Mino induced a sudden and continued increase in DCF fluorescence in a time-independent fashion, indicating excessive generation of H2O2 (or ROS), but scarce fluorescence was detected in untreated cells. Second, the antioxidant NAC significantly reduced DCF fluorescence. Last, DJ-1 Cys106 (i.e., the cysteine thiol residue) is oxidized into Cys106-sulfonate (SO3) (step 2), as detected by monoclonal antibodies. Taken together, these results suggest that Mino can produce H2O2, at least under the present experimental conditions, and oxidized DJ-1 protein. The oxidized form of the DJ-1 protein may represent a diagnostic biochemical biomarker of intracellular oxidative stress. Therefore, our results comply with the notion that an elevation in the levels of ROS/H2O2 by anticancer agents (e.g., doxorubicin, (Mendivil-Perez et al., 2015)), oxidant compounds (e.g., ascorbate at pharmacological concentrations (Doskey et al., 2016)) or drugs (e.g., Mino, this work) might efficiently destroy leukemia cells (Irwin et al., 2013). The present work shows for the first time that Mino induced activation of a molecular signaling pathway in Jurkat cells. Indeed, Mino-treated cells displayed up-regulation (activation) of the transcription factors NF-κB, P53 and c-JUN assessed by Western blotting, and pharmacological inhibitors PDTC, PFT-α and SP600125 significantly reduced DNA fragmentation, as evaluated by flow cytometry. H2O2-induced apoptosis in Jurkat cells is known to require the activation of NF-κB (Dumont et al., 1999). However, the mechanism by which H2O2 induces NF-κB, P53 and c-JUN activation has yet to be determined. One possible explanation is that H2O2 indirectly activates NF-κB through several kinases (step 3) such as the spleen tyrosine kinase (Syk), Src homology 2-containing inositol phosphatase-1 (SHIP), and mitogen-activated protein kinase/ERK kinase −1 (MEKK1) [17]. Once NF-κB is activated (step 4), it translocates to the nucleus and transcribes several anti-apoptotic and pro-apoptotic genes (e.g., https://www.bu.edu/nf-kb/gene-resources/target-genes/), among them TP53 (step 5). These observations comply with the notion that NF-κB is involved in apoptosis (Siomek, 2012). Interestingly, P53 in turn reinforces the NF-κB-induced apoptotic signal by transcribing BAX (Miyashita and Reed, 1995) (step 6) and PUMA (Nakano and Vousden, 2001) (step 7) proteins. In accordance with these observations, we found that BAX and PUMA were up-regulated in Jurkat cells exposed to Mino/OS stimuli compared to their expression in untreated cells. However, how exactly BAX/PUMA proteins operate in the mitochondria are still unknown (Luna-Vargas and Chipuk, 2016). Taken together, our data suggest that NF-κB and P53 are involved in H2O2 signaling (Redza-Dutordoir and Averill-Bates, 2016). Alternatively, H2O2 indirectly induces c-JUN activation through successive activation of apoptosis signal-regulating kinase 1 (ASK1), MKK4 kinase, and JNK kinase (Jimenez-Del-Rio and Velez-Pardo, 2012) (step 8). This last kinase in turn activates c-JUN (step 9). Remarkably, c-JUN has been shown to transactivate the PUMA gene (Lu et al., 2014) (step 7). These data indicate that cells under oxidative stress were undergoing apoptosis by at least 2 independent but complementary pathways that converge onto PUMA protein activation and mitochondrial damage (step 10).