title: RhoA/ROCK/PTEN signaling is involved in AT-101-mediated apoptosis in human leukemia cells in vitro and in vivo.
abstract: R-(-)-gossypol acetic acid (AT-101) is a natural cottonseed product that exhibits anticancer activity. However, the molecular mechanism behind the antileukemic activity of AT-101 has not been well characterized. In this study, we investigated how AT-101 induces apoptosis in human leukemia cells. Exposure to AT-101 significantly increased apoptosis in both human leukemia cell lines and primary human leukemia cells. This increase was accompanied by the activation of caspases, cytochrome c release, Bcl2-associated X protein (Bax) translocation, myeloid cell leukemia-1 (Mcl-1) downregulation, Bcl-2-associated death promoter (Bad) dephosphorylation, Akt inactivation, and RhoA/Rho-associated coiled-coil containing protein kinase 1/phosphatase and tensin homolog (RhoA/ROCK1/PTEN) activation. RhoA, rather than caspase-3 cleavage, mediated the cleavage/activation of ROCK1 that AT-101 induced. Inhibiting RhoA and ROCK1 activation by C3 exoenzyme (C3) and Y27632, respectively, attenuated the ROCK1 cleavage/activation, PTEN activity, Akt inactivation, Mcl-1 downregulation, Bad dephosphorylation, and apoptosis mediated by AT-101. Knocking down ROCK1 expression using a ROCK1-specific siRNA also significantly abrogated AT-101-mediated apoptosis. Constitutively active Akt prevented the AT-101-induced Mcl-1 downregulation, Bad dephosphorylation, and apoptosis. Conversely, AT-101 lethality was potentiated by the phosphatidylinositol 3-kinase inhibitor LY294002. In vivo, the tumor growth inhibition caused by AT-101 was also associated with RhoA/ROCK1/PTEN activation and Akt inactivation in a mouse leukemia xenograft model. Collectively, these findings suggest that AT-101 may preferentially induce apoptosis in leukemia cells by interrupting the RhoA/ROCK1/PTEN pathway, leading to Akt inactivation, Mcl-1 downregulation, Bad dephosphorylation, and Bax translocation, which culminate in mitochondrial injury and apoptosis.
authors.affiliation: Department of Pharmacognosy, College of Pharmacy, Third Military Medical University, Chongqing, China.
authors.forename: G
authors.initials: G
authors.lastname: Li
authors.affiliation: Department of Pharmacognosy, College of Pharmacy, Third Military Medical University, Chongqing, China.
authors.forename: L
authors.initials: L
authors.lastname: Liu
authors.affiliation: Department of Pharmacognosy, College of Pharmacy, Third Military Medical University, Chongqing, China.
authors.forename: C
authors.initials: C
authors.lastname: Shan
authors.affiliation: Department of Pharmacognosy, College of Pharmacy, Third Military Medical University, Chongqing, China.
authors.forename: Q
authors.initials: Q
authors.lastname: Cheng
authors.affiliation: Graduate Center for Toxicology, College of Medicine, University of Kentucky, Lexington, KY, USA.
authors.forename: A
authors.initials: A
authors.lastname: Budhraja
authors.affiliation: Department of Pharmacognosy, College of Pharmacy, Third Military Medical University, Chongqing, China.
authors.forename: T
authors.initials: T
authors.lastname: Zhou
authors.affiliation: State Key Laboratory of Silkworm Genome Biology, Southwest University, Chongqing, China.
authors.forename: H
authors.initials: H
authors.lastname: Cui
authors.affiliation: Department of Pharmacognosy, College of Pharmacy, Third Military Medical University, Chongqing, China.
authors.forename: N
authors.initials: N
authors.lastname: Gao
mesh_terms: D000818:Animals; D017209:Apoptosis; D053148:Caspase 3 / Q000378:metabolism; D045744:Cell Line, Tumor; D006072:Gossypol / Q000008:administration & dosage / Q000031:analogs & derivatives*; D006801:Humans; D007938:Leukemia / Q000188:drug therapy* / Q000201:enzymology* / Q000235:genetics / Q000503:physiopathology; D051379:Mice; D016688:Mice, Inbred NOD; D016513:Mice, SCID; D051059:PTEN Phosphohydrolase / Q000235:genetics / Q000378:metabolism*; D051057:Proto-Oncogene Proteins c-akt / Q000378:metabolism; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000378:metabolism; D015398:Signal Transduction; D051028:bcl-2-Associated X Protein / Q000378:metabolism; D054460:rho-Associated Kinases / Q000235:genetics / Q000378:metabolism*; D020742:rhoA GTP-Binding Protein / Q000235:genetics / Q000378:metabolism*
chemical_list: D019253:Proto-Oncogene Proteins c-bcl-2; D051028:bcl-2-Associated X Protein; D051057:Proto-Oncogene Proteins c-akt; D054460:rho-Associated Kinases; D051059:PTEN Phosphohydrolase; D053148:Caspase 3; D020742:rhoA GTP-Binding Protein; D006072:Gossypol; C028178:gossypol acetic acid