Tylophorine, a phenanthroindolizidine alkaloid, is the major medicinal 
constituent of herb Tylophora indica. Tylophorine treatment increased the 
accumulation of c-Jun protein, a component of activator protein 1 (AP1), in 
carcinoma cells. An in vitro kinase assay revealed that the resultant c-Jun 
phosphorylation was primarily mediated via activated c-Jun N-terminal protein 
kinase (JNK). Moreover, flow cytometry indicated that ectopically overexpressed 
c-Jun in conjunction with tylophorine significantly increased the number of 
carcinoma cells that were arrested at the G1 phase. The tylophorine-mediated 
downregulation of cyclin A2 protein levels is known to be involved in the 
primary G1 arrest. Chromatin immunoprecipitation and reporter assays revealed 
that tylophorine enhanced the c-Jun downregulation of the cyclin A2 promoter 
activity upon increased binding of c-Jun to the deregulation AP1 site and 
decreased binding to the upregulation activating transcription factor (ATF) site 
in the cyclin A2 promoter, thereby reducing cyclin A2 expression. Further, 
biochemical studies using pharmacological inhibitors and RNA silencing 
approaches demonstrated that tylophorine-mediated elevation of the c-Jun protein 
level occurs primarily via two discrete prolonged signaling pathways: (i) the 
NF-κB/PKCδ_(MKK4)_JNK cascade, which phosphorylates c-Jun and increases its 
stability by slowing its ubiquitination, and (ii) the PI3K_PDK1_PP2A_eEF2 
cascade, which sustains eukaryotic elongation factor 2 (eEF2) activity and thus 
c-Jun protein translation. To the best of our knowledge, this report is the 
first to demonstrate the involvement of c-Jun in the anticancer activity of 
tylophorine and the release of c-Jun translation from a global translational 
blockade via the PI3K_PDK1_eEF2 signaling cascade.