sirolimus and dorsomorphin

Kaempferol (KEM) has been observed to stimulate Krt-14 protein which subsequently contributes to matrix maturation and mineralization in rat primary osteoblast cells. Incorporation of Krt-14 siRNA results in reduced mRNA and protein expression after 48h post transfection and remained low for 9days. At day 9 Krt-14 siRNA significantly reduced mineralization without concomitant change in the cell number. ColI and OCN gene expression was reduced in Krt-14 siRNA-treated osteoblast cells. Soluble osteocalcin and collagen levels were markedly decreased in conditioned medium as well as in acid-salt soluble cell-ECM layer treated with Krt-14 siRNA compared to control siRNA treated cells corroborated at the ultrastructral level by AFM. Further, knockdown of Krt-14 and inhibitors against AMPK and mTOR, repressed the activation of mTOR and mineralization attenuated by KEM confirmed the role of Krt-14 in mineralization. These findings strongly suggest that Krt-14 regulates osteoblast mineralization by organizing osteoblast derived ECM.

Topics: Animals; Calcification, Physiologic; Cell Count; Cells, Cultured; Collagen; Extracellular Matrix; Gene Expression; Gene Knockdown Techniques; Kaempferols; Keratin-14; Osteoblasts; Osteocalcin; Pyrazoles; Pyrimidines; Rats; RNA, Small Interfering; Sirolimus; TOR Serine-Threonine Kinases

pH homeostasis is essential for development, proliferation and apoptosis of cells. Once the pH balances are broken, cell functions and survival will be affected. Nevertheless, moderate acidosis could result in adaptive responses for cell survival and increase tolerance to harmful stress. Here we found that acidic preconditioning (APC) could significantly increase the survival rate of Daphnia pulex, a freshwater invertebrate, during severe hypoxic insult. Meanwhile, the acidic treatment significantly increased the gene expression of hypoxia inducible factor (HIF). Both echinomycin, an inhibitor of HIF, and compound C, an inhibitor of AMP-activated protein kinase (AMPK), could effectively eliminate the acid-induced hypoxic tolerance and the enhanced transcription of HIF. Temsirolimus, an inhibitor of mammalian Target of Rapamycin (mTOR), though effectively abolished the increased transcription of HIF, improved the APC-mediated protection. This result suggests that the involvement of the HIF and AMPK and mTOR could signal the pathways in APC-induced protection against hypoxic insult.

Topics: Acids; Adaptation, Physiological; AMP-Activated Protein Kinases; Animals; Daphnia; Hypoxia; Hypoxia-Inducible Factor 1; Ischemic Preconditioning; Pyrazoles; Pyrimidines; RNA, Messenger; Sirolimus; TOR Serine-Threonine Kinases

Most cancer cells exhibit increased glycolysis for generation of their energy supply. This specificity could be used to preferentially kill these cells. In this study, we identified the signaling pathway initiated by glycolysis inhibition that results in sensitization to death receptor (DR)-induced apoptosis. We showed, in several human cancer cell lines (such as Jurkat, HeLa, U937), that glucose removal or the use of nonmetabolizable form of glucose (2-deoxyglucose) dramatically enhances apoptosis induced by Fas or by tumor necrosis factor-related apoptosis-inducing ligand. This sensitization is controlled through the adenosine monophosphate (AMP)-activated protein kinase (AMPK), which is the central energy-sensing system of the cell. We established the fact that AMPK is activated upon glycolysis block resulting in mammalian target of rapamycin (mTOR) inhibition leading to Mcl-1 decrease, but no other Bcl-2 anti-apoptotic members. Interestingly, we determined that, upon glycolysis inhibition, the AMPK-mTOR pathway controlled Mcl-1 levels neither through transcriptional nor through posttranslational mechanism but rather by controlling its translation. Therefore, our results show a novel mechanism for the sensitization to DR-induced apoptosis linking glucose metabolism to Mcl-1 downexpression. In addition, this study provides a rationale for the combined use of DR ligands with AMPK activators or mTOR inhibitors in the treatment of human cancers.

Topics: Aminoimidazole Carboxamide; AMP-Activated Protein Kinases; Antibodies; Apoptosis; Blotting, Western; Deoxyglucose; Enzyme Activation; fas Receptor; Glucose; Glycolysis; HeLa Cells; Humans; Intracellular Signaling Peptides and Proteins; Jurkat Cells; Models, Biological; Myeloid Cell Leukemia Sequence 1 Protein; Protein Biosynthesis; Protein Serine-Threonine Kinases; Proto-Oncogene Proteins c-bcl-2; Pyrazoles; Pyrimidines; Receptors, Death Domain; Recombinant Proteins; Reverse Transcriptase Polymerase Chain Reaction; Ribonucleotides; RNA Interference; Sirolimus; TNF-Related Apoptosis-Inducing Ligand; TOR Serine-Threonine Kinases; U937 Cells