Translationally controlled tumor protein (TCTP) is a growth-related protein under transcriptional as well as translational control. We screened a rat skeletal muscle cDNA library using yeast two-hybrid system and found that TCTP interacts with the third large cytoplasmic domain of ␣1 as well as ␣2 isoforms of Na,K-ATPase, believed involved in the regulation of Na,K-ATPase activity. Interaction between TCTP and Na,K-ATPase was confirmed by coimmunoprecipitation in yeast and mammalian cells. We also showed, using 86 Rb ؉ uptake assay, that overexpression of TCTP inhibited Na,K-ATPase activity in HeLa cells. Northern and Western blotting studies of HeLa cells transiently transfected with GFPtagged TCTP showed that overexpression of TCTP did not change mRNA and protein levels of Na,K-ATPase. Recombinant TCTP protein purified from an Escherichia coli expression system inhibited purified HeLa cell plasma membrane Na,K-ATPase in a dose-dependent manner. Using deletion analysis, we also found that the C-terminal 102-172-amino-acid region of rat TCTP that contains the TCTP homology region 2 is essential for its association with, and inhibition of, Na,K-ATPase. Na,K-ATPase, a multimembrane-spanning enzyme, is essential for maintaining transmembrane gradients of Na ϩ and K ϩ ions and thus for cell homeostasis (1). These ionic gradients serve to control essential cellular processes such as cell volume, membrane potential, and nutrient transport (2). In addition, Na,K-ATPase is involved in cell proliferation and differentiation, heart and vascular muscle contraction, and neurotransmitter and hormone secretion (3). Thus dysfunction of this enzyme can profoundly affect cell function. Na,K-ATPase is composed of a catalytic 110-kDa ␣ subunit and a glycosylated 40 -60-kDa  subunit. The ␣ subunit contains binding sites for cations, ATP, and cardiac glycosides. It has been suggested that there might exist a diffusible cytoplasmic regulator of Na,K-ATPase activity, possibly modulated by protein kinases and hormones (4, 5). The third large cytoplasmic domain (CD3) 1 of Na,K-ATPase was proposed to be one of the domains involved in the regulation of its activity by insulin, thereby playing an important role in the catalytic function and regulation of this enzyme (6). Interactions between the Nterminal region of the Na,K-ATPase ␣ subunit with phosphoinositide-3 kinase (7), cytoplasmic domain 2 (CD2) and CD3 with ankyrin (8, 9), CD3 with cofilin (10), and purified Na,KATPase with actin (11) and adducin (12) have also been demonstrated.We looked for other cytoplasmic agents that might interact with the CD3 of Na,K-ATPase ␣ subunit and regulate its activity and found that translationally controlled tumor protein (TCTP) acts as a cytoplasmic repressor of Na,K-ATPase. TCTP is a growth-related protein, under tight transcriptional as well as translational control (13,14). It occurs as a 23-kDa protein in humans and has a 21-kDa homologue in mice but shows no significant homology with any other family of proteins. Based on structural studies o...
Hepatocellular carcinoma (HCC) is a highly malignant human cancer that has increasing mortality rates worldwide. Because CD133+ cells control tumor maintenance and progression, compounds that target CD133+ cancer cells could be effective in combating HCC. We found that the administration of chromenopyrimidinone (CPO) significantly decreased spheroid formation and the number of CD133+ cells in mixed HCC cell populations. CPO not only significantly inhibited cell proliferation in HCC cells exhibiting different CD133 expression levels, but also effectively induced apoptosis and increased the expression of LC3-II in HCC cells. CPO also exhibits in vivo therapeutic efficiency in HCC. Specifically, CPO suppressed the expression of CD133 by altering the subcellular localization of CD133 from the membrane to lysosomes in CD133+ HCC cells. Moreover, CPO treatment induced point mutations in the ADRB1, APOB, EGR2, and UBE2C genes and inhibited the expression of these proteins in HCC and the expression of UBE2C is particularly controlled by CD133 expression among those four proteins in HCC. Our results suggested that CPO may suppress stemness and malignancies in vivo and in vitro by decreasing CD133 and UBE2C expression in CD133+ HCC. Our study provides evidence that CPO could act as a novel therapeutic agent for the effective treatment of CD133+ HCC.
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