A genetic dimorphism incorporates either alanine (Ala) or valine (Val) in the mitochondrial targeting sequence (MTS) of manganese superoxide dismutase (MnSOD). The Ala-MTS confers a 40% higher MnSOD activity than the Val-MTS after import into isolated mitochondria in vitro. The present study aimed to characterize functional consequences in whole cells. HuH7 human hepatoma cells were transfected with vectors encoding for the human Ala- or Val-MnSOD variants fused to a Myc-His-tag. The Ala-variant resulted in four-fold higher levels of the mature exogenous protein and MnSOD activity than the Val-variant. Studies with a proteasome inhibitor indicated that precursor proteins are either imported into the mitochondria or degraded by the proteasome. Despite identical levels 8 h after transfection, mRNA levels at 36 h were two-fold higher for the Ala-encoding mRNA than the Val-mRNA. Decreasing the mitochondrial membrane potential decreased both MnSOD mitochondrial import and its mRNA levels. Much larger differences in the activity of the human Val- and Ala-MnSOD variants are observed in whole cells rather than after import experiments performed in vitro. First, the slowly imported Val-MnSOD is degraded by the proteasome in cells. Second, the slower mitochondrial import of the Val-variant may be associated with decreased mRNA stability, possibly due to impaired cotranslational import.

Resistance to apoptosis is a recurrent theme in colon cancer. We have shown previously that the 7-transmembrane spanning receptor OX1R for orexins promotes robust apoptosis in the human colon cancer cell line HT29 through an entirely novel mechanism involving phosphorylation of tyrosine-based motifs in OX1R. Here, we investigated the status of OX1R in a large series of human colorectal tumors and hepatic metastases. All primary colorectal tumors regardless of their localization and Duke's stages and all hepatic metastases tested expressed OX1R mRNA and/or protein. In sharp contrast, adjacent normal colonocytes or hepatocytes as well as control normal tissues were negative. Next, we showed that nine human colon cancer cell lines established from primary tumors or metastases expressed OX1R mRNA and underwent important apoptosis on orexin-A challenge. Most interestingly, orexin-A also promoted robust apoptosis in cells that are resistant to the most commonly used drug in colon cancer chemotherapy, 5-fluorouracil. When human colon cancer cells were xenografted in nude mice, orexin-A administered at day 0 strongly slowed the tumor growth and even reversed the development of established tumors when administered 7 days after cell inoculation. Orexin-A also acts by promoting tumor apoptosis in vivo because caspase-3 is activated in tumors on orexin treatment of nude mice. These findings support that OX1R is an Achilles heel of colon cancers, even after metastasis or chemoresistance. They suggest that OX1R agonists might be novel candidates for colon cancer therapy. Cancer Res; 71(9);

This study shows that leptin induced a rapid phosphorylation of p42/44 mitogen-activated protein kinase, an enhancement of both NF-B DNA binding and transcriptional activities, and a concentration-dependent increase of HT-29 cell proliferation. These effects are consistent with the presence of leptin receptors on cell membranes. The leptin induction of cell growth was associated with an increase of cell population in S and G 2 /M phase compared with control cells found in G 0 /G 1 phase of the cell cycle. Moreover, cyclin D1 immunoreactivity was enhanced in leptin-treated HT-29 cells and this increase was essentially associated with cell population in G 0 /G 1 phase. On the other hand, we observed that sodium butyrate inhibited cell proliferation by blocking HT-29 cells in G 0 /G 1 phase of the cell cycle. Interestingly, at physiological concentration, leptin prevented sodium butyrate-induced morphological nucleus changes, DNA laddering and suppressed butyrate-induced cell cycle arrest. This anti-apoptotic effect of leptin was associated with HT-29 cell proliferation and activation NF-B pathways. However, the phosphorylation of p42/44 MAP kinase in response to leptin was reduced in butyrate-treated cells. These data demonstrated that leptin is a potent mitogenic factor for intestinal epithelial cells through the MAP kinase and NF-B pathways. They also showed, for the first time, that leptin promotes colon cancer HT-29 cell survival upon butyrate challenge by counteracting the apoptotic programs initiated by this short chain fatty acid probably through the NF-B pathways. Although further studies are required to unravel the precise mechanism, these data may have significance in the pathogenesis of colorectal cancer and ulcerative colitis diseases.Leptin, the protein product encoded by the ob gene, is a 16-kDa circulating hormone produced primarily by adipose tissue and is a multifunctional hormone that regulates body weight homeostasis, neuroendocrine function, fertility, immune function, and angiogenesis (1-4). The biological actions of leptin on target tissues are carried out through interaction with its specific receptors, Ob-R. The leptin receptor (Ob-R) 1 is a member of the gp130 family of cytokine receptors (5), which occurs in several receptor variants (Ob-Ra through Ob-Rf) that are generated by alternative splicing of the db leptin receptor gene. These isoforms share the same extracellular domain but differ in the length of the transmembrane/cytoplasmic (6, 7). The long Ob-Rb subtype (Ob-R L ) appears as the functional, signal-transducing isoform, responsible for the action of leptin (8). The long isoform, Ob-Rb, can activate the signal transducers and activators of transcription (STAT) pathways, whereas both Ob-Rb and the short isoform (Ob-Ra) can transduce signals through insulin receptor substrates and through mitogenactivated protein kinase-(MAP kinase) dependent pathways (for review, see Ref. 9). Although, it is currently believed that leptin action is largely mediated by the central nervous system, t...