The anti-diabetes drug metformin has been shown to have anti-neoplastic effects in several tumor models through its effects on energy metabolism and protein synthesis. signaling seems to be promising from a therapeutic point of view in vitro, more research is needed when implementing this combination strategy in vivo. < 0.05 vs. control; (C) Protein expression ... In line with this, cyclin D1 protein expression was drastically decreased after treatment with 5 mM metformin, especially in the rapidly proliferating PC3 Bay 65-1942 and DU145 cell lines (Figure 1C). Additionally, metformin activated its downstream signaling components AMPK and Acetyl-CoA carboxylase (ACC) in a dose-dependent manner in all PCa cell lines (Figure 1C). 2.2. Metformin Increases Radiosensitivity of PCa Cells Independent of Adenosine Monophosphate (AMP)-Activated Protein Kinase (AMPK) Activation Metformin (5 mM) increased radiosensitivity of DU145 and 22Rv1 cells with a dose-enhancement factor (DEF) of 1.6 0.15 (< 0.05) and 1.36 0.08 (< 0.05) respectively. In contrast, the radiosensitivity of PC3 cells was not affected by metformin (Figure 2A). To evaluate the role of AMPK in the metformin-induced radiosensitization effect in the DU145 and 22Rv1 cells, AMPK was silenced by means of silencing RNA (siRNA). Downregulation of (phospho)AMPK did not affect Rabbit polyclonal to GnT V the intrinsic radiosensitivity of either cell line nor did it change the metformin-induced radiosensitization (Figure 2B). Figure 2 Effect of metformin (MF) on radiosensitivity of PCa cells. (A) Clonogenic survival after 72-h treatment with Bay 65-1942 metformin (5 mM) prior to/during ionizing radiation (IR); (B) Clonogenic survival of DU145 and 22Rv1 cells transfected with AMPK Bay 65-1942 silencing RNA … 2.3. Metformin Regulates Hedgehog Signaling in an AMPK-Dependent Manner Next, we investigated if there was a link between metformin and Hh signaling in PCa cells. Indeed, metformin (5 mM) significantly decreased glioma-associated oncogene homolog 1 (and gene expression after 72-h metformin treatment. Means SEM of two independent experiments. * < 0.05 vs. control; (B) PTCH1, GLI1 and GLI2 protein expression after ... 2.4. Combination of Metformin and GANT61 (GLI-ANTagonist 61) Synergistically Decreases PCa Cell Growth The link between AMPK and GLI1 led to the question as to whether the combination of metformin with Hh inhibitors could enhance the cytotoxic effect of the individual drugs. We have previously shown that the GLI1/2 inhibitor GANT61 significantly decreased cell survival of PC3 and Bay 65-1942 22Rv1 cells [19]. Indeed, combining metformin and GANT61 significantly decreased cell Bay 65-1942 growth of all PCa cell lines, resulting in an almost complete blockage of cell growth in PC3 and 22Rv1 cells (Figure 4A). Additionally, we confirmed decreased gene expression in all cells treated with the drug combination (Figure S2). Cell cycle analyses revealed that the drug combination in the PC3 cells led to a G2/M-arrest after only 24 h, which persisted until 72 h of treatment (Figure 4B). This corresponds to the dramatic decrease in cell growth already observed after 24 h of treatment. The drug combination also significantly increased the sub-G1 population which peaked at 48 h (Figure 4C). In the DU145 cells, no significant cell cycle effects were observed after 24C72 h of either treatment (Figure 4B), whereas the combination treatment did significantly increase apoptosis after 72 h compared to either single agent (Figure 4C). In the 22Rv1 cells, GANT61 induced a G1-arrest after only 24 h. Metformin alone did not have a significant effect.
Integrins contribute to lymphopoiesis, whereas Toll-like receptors (TLRs) facilitate the myeloid
Integrins contribute to lymphopoiesis, whereas Toll-like receptors (TLRs) facilitate the myeloid replenishment during inflammation. were cell-intrinsic and could be recapitulated on bone marrow stromal cell culture. Furthermore, defective lymphopoiesis correlated strongly with failure of hematopoietic progenitors to form close contact with stromal cell niche and was not the result of the defect in the assembly of antigen receptor or interleukin-7 signaling. These findings define gp96 as the only known molecular chaperone to specifically regulate T- and B-cell development. Introduction Integrins are a family of 24 heterodimers in vertebrates formed noncovalently by 18 and 8 Rabbit polyclonal to SERPINB9 integrins, of which 17 integrins are expressed in the hematopoietic system.1,2 Known best for their adhesion properties, integrins also orchestrate signals between extracellular matrix and intracellular cytoskeletons in regulating diverse functions of cells, including proliferation and differentiation. However, despite the expression of integrins on hematopoietic stem cells (HSCs) and the role of integrins in HSC homing to the bone marrow (BM) niche, their function in hematopoiesis remains controversial. For example, although 4 integrin has been implicated in both T and B lymphopoiesis from fetal HSCs,3,4 it appears to play a less significant role in adult hematopoiesis.5,6 Furthermore, combined deletion of both 1 and 7 integrins, which are the only known partners of 4 integrin, causes no defect in either lymphopoiesis or myelopoiesis.7 Genetic 2 integrin deficiency causes myeloid hyperplasia, including profound granulocytosis and splenomegaly, but no significant problems in hematopoiesis.8 Clearly, both 4 and 2 integrins are involved in homing of HSCs in the BM and recruitment of leukocytes to sites of inflammation.5,9,10 Although Bay 65-1942 pan-integrin deficient system is now available,11 no resolution of the roles of integrin in hematopoiesis has emerged. Toll-like receptors (TLRs) are pattern recognition receptors that play important roles in sensing pathogen-associated molecular patterns from microbes, which are critical Bay 65-1942 for host immune response.12 More than 10 TLRs have been described in vertebrates, recognizing a spectrum of microbial moieties, such as endotoxin, flagellin, dsRNA, and DNA. In the steady state, TLRs do not contribute significantly to hematopoiesis, although TLRs on HSCs have been implicated in the replenishment/recruitment of myeloid cells in response to inflammation.13,14 TLRs and integrins do not share significant structural homology. Nevertheless, the folding and proper expression of many TLR and integrin family members are dependent on gp96, the heat shock protein 90 (HSP90) paralogue in the endoplasmic reticulum (ER). Deletion of gp96 leads to posttranslational loss of multiple TLRs (TLR1, TLR2, TLR4, TLR5, TLR6, TLR7, and TLR9) and several integrins Bay 65-1942 (2, 4, and V integrins),15C17 although no study has probed the entire hematopoietic system-specific integrins for their dependence on gp96. As a major ER luminal protein whose expression can be further induced by accumulation of misfolded proteins, gp96 is also thought to participate in the ER-unfolded protein response (UPR)18 and ER-associated protein degradation,19 and has been implicated to play a major housekeeping function to maintain protein homeostasis in the secretory pathway.20 The discovery that gp96 seems to selectively fold TLRs and integrins15C17 was unexpected, which raises the intriguing possibility that gp96 is evolved to play more specialized function in the multicellular organism. In this study, we used tamoxifen (TAM)Cinducible gp96 knockout (floxed mice were crossed to R26R-creERT2 mice (kindly provided by James Y. H. Li, University of Connecticut Health Center [UCHC]) and further backcrossed to C57BL/6 background for 6 to 10 generations. Control mice were mice. All mice were maintained by the Center for Laboratory Animal Care of UCHC (Farmington, CT) on an Institutional Animal Care and Use CommitteeCapproved animal care protocol. Cell lines and gp96 mutant 70Z/3 pre-B cells were a gift from Brian Seed (Harvard University),15 which were cultured in RPMI medium (Sigma-Aldrich) supplemented with 10% heat-inactivated fetal calf serum (Atlas Biologicals), 55M 2-mercaptoethanol (Invitrogen), and penicillin-streptomycin (Invitrogen). OP9 and OP9-DL1 cells were cultured in -minimum essential medium containing l-glutamine and ribonucleotides (Invitrogen) supplemented with 20% fetal calf serum, 1mM.