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.
Highlights
Phosphatase and tensin homolog (PTEN) has been identified as a new Rho-associated coiled-coil containing protein kinase (ROCK) substrate.[6]
We found that AT-101 induces apoptosis in both human leukemia cell lines and primary human leukemia cells, and inhibits tumor growth of U937 xenograft mouse model via RhoA/ROCK1/PTEN signaling, Akt inactivation, Mcl-1 downregulation, Bcl-2-associated death promoter (Bad) dephosphorylation, and Bcl2-associated X protein (Bax) translocation
We examined the effects of AT-101 on apoptosis and mitochondrial injury in U937 leukemia cells
Summary
Phosphatase and tensin homolog (PTEN) has been identified as a new ROCK substrate.[6]. We found that AT-101 induces apoptosis in both human leukemia cell lines and primary human leukemia cells, and inhibits tumor growth of U937 xenograft mouse model via RhoA/ROCK1/PTEN signaling, Akt inactivation, Mcl-1 downregulation, Bcl-2-associated death promoter (Bad) dephosphorylation, and Bax translocation. These findings identify a novel mechanistic basis for AT-101 in leukemia treatment
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