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  • It is well known that the cell cycle consists of


    It is well known that the Bortezomib (PS-341) consists of four phases, i.e., the G0/G1, S, G2/M and M phases, which are regulated by each cell cycle checkpoint. Various anticancer drugs, such as cisplatin and paclitaxel, have been recently applied to induce cancer cell death by interfering with cell cycle checkpoints, especially G2/M checkpoints [[32], [33], [34]]. In our study, the results fully demonstrate that GLA can induce G2/M phase arrest via regulating the G2/M checkpoints, including cyclin B1, CDC2 and P21 in vitro. In addition, given cell cycle arrest is closely associated with the apoptosis of OS cells, we further detect that GLA can induce substantial apoptosis in two distinct OS cells by flow cytometric assays. Meanwhile, we note that GLA induce apoptosis in OS cells by increasing the expression of cleaved caspase 3 and cleaved PARP. Therefore, these results demonstrate that GLA can inhibit the growth of OS cells via inducing G2/M phase arrest and cellular apoptosis in vitro. We further observe that GLA also induce significant ROS production in vitro. As reported, ROS can serve dual functions in cancer cells. Hole PS et al. reported that low levels of ROS promote the proliferation and growth of cancer cells under normal physiological conditions [35,36]. In contrast, upregulated ROS can markedly induce apoptosis and suppress the growth of cancer cells under nonphysiological conditions, as reported by Ryter et al. [37]. In our study, we observe that GLA significantly inhibit cell viability and induce cellular apoptosis by upregulating ROS production, which can be reversed by two ROS scavengers, NAC and GSH. These results suggest that ROS substantially contribute to the anticancer effect of GLA in OS cells. GSSG/GSH is an important cellular anticancer system that contributes to maintaining low levels of ROS [14,38,39]. The imbalance of GSSG/GSH system reduces ROS scavenge ability. In our study, we indeed observe a significant increase in GSSG and a significant decrease in GSH after GLA treatment in vitro. Furthermore, BSO, GSH synthesis blocker, can enhance the effect of ROS production and apoptosis induced by GLA. These findings further demonstrate the GSSG/GSH system plays pivotal role in GLA-induced ROS production and anticancer effect. As GR can maintain the balance of the GSSG/GSH system by catalyzing the conversation of GSSG to GSH. In our study, we observe a significant inhibition of GR activity in GLA-treated OS cells, which demonstrate the inhibition of GR contribute to the imbalance of GSSG/GSH. Meanwhile, we further conclude that GR inhibition is mainly caused by the reduction of mRNA and protein levels of GR in GLA-treated OS cells. Next, we for the first time demonstrate that GLA markedly suppress both constitutive and IL-6-inducible activation of STAT3 (at tyrosine 705 residue) and JAK2 which is the upstream of STAT3 in OS cells. In view of STAT3 is closely associated with the proliferation and growth of OS, we aim to elucidate the relationship between ROS-mediated oxidative stress and STAT3 inactivation. Interestingly, we observe that the inhibition of STAT3 is abolished by ROS scavengers, NAC and GSH, in GLA-treated OS cells. It has been reported that the kinase activity of JAK2 can be suppressed by ROS-mediated oxidative stress, which oxidizes the cysteine residues in the catalytic domain. Previous studies demonstrate that there are many cysteine residues located at the C-terminal region of the STAT3 Tyr705 residue, and oxidative stress can also directly oxidize these cysteines to suppress the phosphorylation of STAT3 [40,41]. In our study, we also note that GLA-induced ROS production and cellular apoptosis can be partially reversed by STAT3 overexpression. Thus, these findings demonstrate that GLA can abrogate STAT3 activation via ROS-mediated oxidative stress and then inhibit the growth of OS cells in vitro. Finally, we investigate the anticancer effect of GLA in a xenograft mouse model. Our study for the first time demonstrate that GLA can significantly inhibit the growth of OS by abrogating GR expression and STAT3 activation in vivo, which correlates well with its observed in vitro effect. In addition, no obvious major organ toxicity was observed in the GLA-treated OS mouse model.