Abstract
BackgroundCryptotanshinone (CPT), a fat-soluble phenanthraquinone from Salvia miltiorrhiza Bunge, has been demonstrated to inhibit phosphorylation of p70 S6 kinase 1 (S6K1) and eukaryotic initiation factor 4E binding protein 1 (4E-BP1), a couple of direct downstream effectors of the mammalian target of rapamycin complex 1 (mTORC1), resulting in cancer cell arrested in G0 phase and subsequent inhibition of proliferation. However, its concrete molecular mechanism about how CPT inhibits mTORC1 signaling pathway is unclear.Methodsone solution was used to check cell viability and western blotting for determining expression of the indicated proteins. Molecular docking was performed to assess the binding of CPT with mTOR. The co-immunoprecipitation assay was to analyze whether CPT could disrupt the mTORC1 and TSC1/TSC2 complex. Recombinant adenoviral dominant-negative AMPKα was used to downregulate expression of AMPKα and lentiviral AMPK and TSC2 to silence the AMPK and TSC2 in Rh30 cells.ResultsPrimarily, Rh30 cells expressing rapamycin-resistant mutant mTOR are also sensitive to CPT, while the molecular docking result for CPT binding to mTOR is negative, suggesting that CPT inhibition of mTORC1 is different from rapamycin. Then the related proteins of PTEN-PI3K pathway was proved not to be affected, but the phosphorylation of adenosine monophosphate-activated protein kinase (AMPK) was activated by a concentration- and time- dependent manner, meaning that it may be associated with AMPK. Further results indicated that compound C, inhibitor of AMPK, could clearly reversed CPT inhibitory effect on Rh30 cells, and dominant-negative AMPK in cancer cells conferred resistance to CPT inhibition of 4E-BP1 and phosphorylation of S6K1, as well as sh-AMPK. Furthermore, compared with AMPK-positive MEF cells, AMPK-negative MEF cells are less sensitive to CPT by the findings that 4E-BP1 and phosphorylation of S6K1 express comparatively more. Additionally, phosphorylation of tuberous sclerosis complex 2 (TSC2) was activated under the treatment of CPT, and down-expression of TSC2 by shRNA slightly recovered expression of 4E-BP1 and phosphorylation of S6K1, while co-immunoprecipitation of TSC2 did not alter expression of TSC1 by CPT.ConclusionCPT inhibiting mTORC1 pathway was mostly due to activation of AMPK-TSC2 axis rather than specific binding to mTORC1. CPT is a potent anticancer agent targeting AMPK.
Highlights
Cryptotanshinone (CPT), a fat-soluble phenanthraquinone from Salvia miltiorrhiza Bunge, has been demonstrated to inhibit phosphorylation of p70 S6 kinase 1 (S6K1) and eukaryotic initiation factor 4E binding protein 1 (4E-BP1), a couple of direct downstream effectors of the mammalian target of rapamycin complex 1, resulting in cancer cell arrested in G0 phase and subsequent inhibition of proliferation
The mTOR, a 289-KD serine/threonine protein kinase, plays a core role in multiple signaling pathways involving the mediation of cell growth, proliferation, apoptosis and autophagy, being considered a hotspot target for cancer therapy [1, 2]. mTOR has a pair of multiprotein complex forms including mTOR complex 1 and 2 [3, 4]. mammalian target of rapamycin complex 1 (mTORC1) activation results in phosphorylation of 4EBP1 and S6K1, regulating cell proliferation [3, 5], while mTORC2 phosphorylates Akt at Ser473 and probably promotes Akt phosphorylation by regulating integrin related kinase [3, 6]. mTORC2 controls actin cytoskeleton and cell migration via regulating phosphorylation of PKC [3, 6]
Activation of the mTOR signaling is firstly initiated by a variety of cellular signals comprising mitogenic growth factors, hormones, nutrients, cellular energy, stress conditions and so on, initiative signals are transmitted by many important node molecules via multiple pathways to regulate mTOR in final [2]
Summary
Cryptotanshinone (CPT), a fat-soluble phenanthraquinone from Salvia miltiorrhiza Bunge, has been demonstrated to inhibit phosphorylation of p70 S6 kinase 1 (S6K1) and eukaryotic initiation factor 4E binding protein 1 (4E-BP1), a couple of direct downstream effectors of the mammalian target of rapamycin complex 1 (mTORC1), resulting in cancer cell arrested in G0 phase and subsequent inhibition of proliferation. The mTOR, a 289-KD serine/threonine protein kinase, plays a core role in multiple signaling pathways involving the mediation of cell growth, proliferation, apoptosis and autophagy, being considered a hotspot target for cancer therapy [1, 2]. Activation of the mTOR signaling is firstly initiated by a variety of cellular signals comprising mitogenic growth factors, hormones, nutrients, cellular energy, stress conditions and so on, initiative signals are transmitted by many important node molecules via multiple pathways to regulate mTOR in final [2]. A key pathway activating mTOR is the PI3K/Akt, which critically mediates cell survival and proliferation and is started by mitogenic stimuli from the binding of growth factors with receptors in the cell membrane [7,8,9]. Activation of AMPK in turn phosphorylates TSC2 obviously, which subsequently suppresses mTORC1 activity [10, 11]
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
