br Materials and methods br Results br Discussion

Materials and methods
Results
Discussion The data in the present study showed that ABZ induces SIRT3 downregulation and SIRT3 overexpression attenuates the microtubule-destabilizing effect of ABZ in U937 cells. In agreement, previous studies have revealed that SIRT3 overexpression inhibits the disassembly of microtubule polymers and mitotic arrest elicited by vinblastine [15]. Noticeably, brRESV-induced SIRT3 downregulation does not cause G2/M brompheniramine maleate arrest in U937 cells. These results allow us to deduce that SIRT3 downregulation could not affect microtubule organization but plays a role in promoting the microtubule-destabilizing effect of ABZ. ABZ-induced SIRT3 degradation largely causes an elevated generation of ROS from mitochondria, while abolishment of ROS generation by NAC does not affect ABZ-induced cell cycle arrest and tubulin depolymerization. These indicate that ABZ-induced mitochondrial ROS generation is not involved in its microtubule-destabilizing effect. Conversely, Harkcom et al. [15] proposed that suppression of ROS generation increases microtubule polymer stability in vinblastine-treated breast cancer MCF-7 cells. Evidently, microtubule-destabilizing agent-induced ROS generation affects microtubule stability in a cell-type dependent manner. Disruption of microtubule organization has been suggested to affect mitochondrial biogenesis and dynamic [22], [41]. Therefore, the possibility that ABZ-induced microtubule depolymerization elicits SIRT3 degradation could not be excluded. However, Signorile et al. [43] reported that an alteration in mitochondrial cAMP/protein kinase A signaling induced by oxidative stress elicits SIRT3 degradation in cardiac myoblast cells. Some studies have shown that the interference of paclitaxel with the mitochondrial signaling cascade occurs upstream of any alteration to microtubule organization [24]. Together with the functional role of SIRT3 in attenuating ABZ-induced microtubule depolymerization, these suggest that ABZ-induced SIRT3 degradation may be attributed to its direct effect on mitochondria. Nonetheless, it is worth noting that the mechanistic pathways responsible for ABZ-induced SIRT3 downregulation and SIRT3-modulated microtubule stability in leukemia cells remain to be studied further. On the other hand, our data revealed that an increase in mitochondrial ROS generation is closely related to SIRT3 degradation in ABZ-treated cells. Mitochondrial SOD2 is an important antioxidant enzyme for eliminating superoxide in mitochondria [53]. Some studies have reported that SIRT3 directly deacetylates SOD2 which promotes the SOD2 activity on mitochondrial ROS-scavenging [6], [37]. Thus, it is conceivable that ABZ-induced SIRT3 downregulation reduces SOD2 activity by which increases the production of mitochondrial ROS. Our data also reveal that ABZ-induced SIRT3 degradation increases mitochondrial ROS generation, subsequently eliciting p38 MAPK-mediated TTP degradation. The results of our immunoprecipitation assay demonstrated that phosphorylation of TTP by p38 MAPK is crucial for its binding with PKM2, indicating the connection of p38 MAPK activation with PKM2-mediated TTP degradation in ABZ-treated cells. This is in line with previous studies showing that the inactivation of p38 MAPK alleviated PKM2-mediated TTP degradation [20]. Nonetheless, Liu et al. [30] suggested that the p38 MAPK-elicited upregulation of PP2Acα reduces the level of phospho-TTP and thus promotes TTP degradation in hydroquinone-treated U937 cells. Sun et al. [47] proposed that p38 MAPK-mediated TTP phosphorylation increases the binding of TTP with 14-3-3 and dampens the degradation of TTP in lipopolysaccharide-treated mouse alveolar macrophage cells. In contrast, Lee et al. [31] reported that p38 MAPK-mediated TTP phosphorylation causes TTP degradation in casein kinase 2 inhibitor-treated colon cancer cells. Thus, it appears that the precise role of p38 MAPK in modulating the stability of TTP is stimulus- and/or cellular context-dependent. In line with previous studies showing the TNF-α mRNA decay activity of TTP [2], [20], [30], [46], [47], our data reveal that TTP degradation increases TNF-α mRNA stability and protein expression in ABZ-treated cells. In support of this finding, TTP overexpression mitigates ABZ-induced TNF-α upregulation. Collectively, these results indicate that ABZ-induced TNF-α upregulation is mediated through the SIRT3/ROS/p38 MAPK/TTP axis.