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DHA-induced activation of AMPK contributes to inhibition of mTORC1. (A) Rh1 cells were treated with DHA (5 M) for indicated time, followed by Western blotting with indicated antibodies. (B) Rh1 cells were pretreated with or without Compound C (CC) (5 μM) for 2 h, and then exposed to with or without DHA (5 μM) for 24 h, followed by Western blotting using indicated antibodies. (C) Rh30 cells were infected with recombinant adenovirus expressing myc-tagged dominant negative (DN) AMPK (Ad-AMPK-DN) or GFP (Ad-GFP) for 24 h, and then treated with DHA (0−30 M) for another 24 h, followed by Western blotting with indicated antibodies. (D) Rh30 cells, infected with lentiviral shRNA to human AMPKα1 or GFP, were treated with DHA (0−30 M) for 24 h, followed by Western blotting with indicated antibodies.
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Dihydroartemisinin (DHA), an anti-malarial drug, has been shown to possess potent anticancer activity, partly by inhibiting the mammalian target of rapamycin (mTOR) complex 1 (mTORC1) signaling. However, how DHA inhibits mTORC1 is still unknown. Here, using rhabdomyosarcoma (RMS) as a model, we found that DHA reduced cell proliferation and viabilit...
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... Rh1 cells were treated with DHA (5 M) for 0-12 h, the phosphorylation level of AMPKα increased in a time-dependent manner. The phosphorylation of AMPKα was modestly induced at 8 h and robustly induced at 12 h, which matched well with the inhibition pattern on mTORC1 ( Figure 7A). The results suggest that DHA-induced mTORC1 inhibition may be associated with activation of AMPK. ...
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... validate whether DHA-induced activation of AMPK contributes to inhibition of mTORC1, Rh1 cells were pre-treated with or without compound C (a selective inhibitor of AMPK) for 2 h and then exposed to DHA (5 M) for 24 h. As predicted, pretreatment with compound C remarkably attenuated DHA-induced p-AMPKα ( Figure 7B). Of interest, the inhibition of AMPK profoundly prevented DHA from inhibiting the phosphorylation of S6K1 and S6 ( Figure 7B). ...
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... predicted, pretreatment with compound C remarkably attenuated DHA-induced p-AMPKα ( Figure 7B). Of interest, the inhibition of AMPK profoundly prevented DHA from inhibiting the phosphorylation of S6K1 and S6 ( Figure 7B). Similar results were observed in Rh30 cells (Supplementary Figure S2). ...
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... corroborate the above finding, Rh30 cells were infected with a recombinant adenovirus expressing DN AMPKα (Ad-AMPK-DN), kinase-dead AMPKα (Ad-AMPK-KD) [37,38], or GFP (Ad-GFP, control) for 24 h, and then treated with DHA for another 24 h. As anticipated, ectopic expression of AMPK-DN or AMPK-KD, but not GFP, attenuated DHA-induced phosphorylation of ACC (S79), a substrate of AMPK ( Figure 7C; Supplementary Figure S3), suggesting that both Ad-AMPK-DN and Ad-AMPK-KD were working well in the cells. Importantly, expression of AMPK-DN or AMPK-KD did render high resistance to the inhibitory effect of DHA on mTORC1 in the cells (Figure 7C; Supplementary Figure S3). ...
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... anticipated, ectopic expression of AMPK-DN or AMPK-KD, but not GFP, attenuated DHA-induced phosphorylation of ACC (S79), a substrate of AMPK ( Figure 7C; Supplementary Figure S3), suggesting that both Ad-AMPK-DN and Ad-AMPK-KD were working well in the cells. Importantly, expression of AMPK-DN or AMPK-KD did render high resistance to the inhibitory effect of DHA on mTORC1 in the cells (Figure 7C; Supplementary Figure S3). Furthermore, similar results were observed in Rh30 cells when AMPKα1 was knocked down with lentiviral shRNA to AMPKα1 ( Figure 7D). ...
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... expression of AMPK-DN or AMPK-KD did render high resistance to the inhibitory effect of DHA on mTORC1 in the cells (Figure 7C; Supplementary Figure S3). Furthermore, similar results were observed in Rh30 cells when AMPKα1 was knocked down with lentiviral shRNA to AMPKα1 ( Figure 7D). Together, our results indicate that activation of AMPK plays a critical role in DHA-induced inhibition of mTORC1. ...
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... Western blotting analysis revealed that treatment with DHA for 8 h remarkably inhibited p-S6 (S235/236) and p-4E-BP1 (T70) but did not apparently affect p-Akt (S473) in the tumors ( Figure 9D), in line with our in vitro results ( Figure 1B). Interestingly, DHA treatment also time-dependently induced p-AMPK (T172) in vivo ( Figure 9D), also consistent with the in vitro data (Figure 7). The results underline that artesunate or DHA has a great potential for RMS therapy. ...
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... AMPK can phosphorylate TSC2 at multiple sites (including s1387), and promote TSC formation and activation. Thus antagonizing Rheb, thereby inhibiting Rheb-mediated mTORC1 (Najafov et al. 2021;Luo et al. 2021). Second, AMPK phosphorylates serines at positions 722 and 792 of the raptor and directly inhibits mTORC1 activity (Jang et al. 2021). ...
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... Some studies have demonstrated that DHA acts as an antineoplastic agent by a variety of mechanisms, depending on the cancer type. For instance, DHA inhibits cell proliferation and induces apoptosis of rhabdomyosarcoma tumors by suppressing mTORC1 pathway, via triggering AMPK signaling [16,17]. Chen et al. reported that DHA can sensitize a panel of cancer cells to ferroptosis by iron-dependent reactive oxygen species (ROS) generation [18]. ...
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Experimental studies on the pathogenetic process of paclitaxel-induced neuropathic pain (PINP) have been initially carried out, but PINP still has no effective therapy. Recently reported studies have highlighted the involvement of glutamate receptors and neuroinflammation in peripheral and central nociceptive transmission in PINP. Artesunate is a first-line antimalarial drug with established efficacy in alleviating pain in a variety of pathologies. The current work assessed whether artesunate inhibits PINP by modulating metabotropic glutamate receptor 5 (mGluR5) and neuroinflammation in mice. The anti-hyperalgesic effect of artesunate was verified by assessing mechanical frequency and thermal latency in the paw withdrawal test as well as spontaneous pain. The expression levels of mGluR5, pain-related receptors and neuroinflammatory markers in dorsal root ganglion (DRG) were examined. In addition, treatment with CHPG and 2-methyl-6-(phenyl ethynyl) pyridine (MPEP) (mGluR5 agonist and antagonist, respectively) was performed to determine mGluR5’s role in the anti-hyperalgesic properties of artesunate. We demonstrated artesunate prevented PINP in a dose-dependent manner, while exerting a clear anti-hyperalgesic effect on already existing PINP. Artesunate normalized paclitaxel-related expression changes in DRG mGluR5, NR1, and GluA2, as well as six paclitaxel related neuroinflammation markers. Intrathecal application of MPEP treated PINP by reversing NR1 and GluA2 expression changes but had no effects on chemokines and inflammatory factors. Furthermore, artesunate treatment reversed acute pain following CHPG application. In conclusion, this study revealed that artesunate alleviates paclitaxel-induced hyperalgesia and spontaneous pain by decreasing DRG mGluR5 expression and neuroinflammation in the mouse model of PINP.
