Neutrophil gelatinase-associated lipocalin (NGAL), also known as lipocalin 2, is a 25-kDa lipocalin initially purified from neutrophil granules. It is thought to play a role in regulating cellular growth since its expression is highly upregulated in a variety of proliferative cells such as cancer cells. However, experimental evidence showing a clear causal relationship between NGAL expression and the proliferation of tumor cells is lacking. Here, we found NGAL expression in highly and poorly metastatic colon cancer cell lines of the same genetic origin correlated inversely with the metastatic potential of these cells, which suggests NGAL participates in the metastatic process. To explore the role NGAL plays in tumor growth and metastasis, the KM12SM human colon cancer cell line, which is highly metastatic while showing decreased NGAL expression, was genetically manipulated to overexpress NGAL. The effects of this on tumor growth and liver metastasis were then analyzed using experimental animal models established by injecting BALB/c nude mice with tumor cells subcutaneously or intrasplenically. Ectopic expression of NGAL in the colon cancer cells had little effect on the growth and viability of the tumor cells both in vitro and in vivo. However, NGAL expression not only suppressed the ability of the colon carcinoma cells to invade Matrigel in vitro, it also substantially inhibited liver metastasis in an experimental animal model. Collectively, these results indicate that NGAL may be a candidate metastasis suppressor in colon cancer cells. ' 2005 Wiley-Liss, Inc.
Mesenchymal stem cells (MSC) are ideal materials for stem cell-based therapy. As MSCs reside in hypoxic microenvironments (low oxygen tension of 1% to 7%), several studies have focused on the beneficial effects of hypoxic preconditioning on MSC survival; however, the mechanisms underlying such effects remain unclear. This study aimed to uncover the potential mechanism involving 78-kDa glucose-regulated protein (GRP78) to explain the enhanced MSC bioactivity and survival in hindlimb ischemia. Under hypoxia (2% O2), the expression of GRP78 was significantly increased via hypoxia-inducible factor (HIF)-1α. Hypoxia-induced GRP78 promoted the proliferation and migration potential of MSCs through the HIF-1α-GRP78-Akt signal axis. In a murine hind-limb ischemia model, hypoxic preconditioning enhanced the survival and proliferation of transplanted MSCs through suppression of the cell death signal pathway and augmentation of angiogenic cytokine secretion. These effects were regulated by GRP78. Our findings indicate that hypoxic preconditioning promotes survival, proliferation, and angiogenic cytokine secretion of MSCs via the HIF-1α-GRP78-Akt signal pathway, suggesting that hypoxia-preconditioned MSCs might provide a therapeutic strategy for MSC-based therapies and that GRP78 represents a potential target for the development of functional MSCs.
Although mesenchymal stem cells (MSCs) are a promising cell source for regenerative medicine, ischemia-induced endoplasmic reticulum (ER) stress induces low MSC engraftment and limits their therapeutic efficacy. To overcome this, we investigated the protective effect of tauroursodeoxycholic acid (TUDCA), a bile acid, on ER stress in MSCs in vitro and in vivo. In ER stress conditions, TUDCA treatment of MSCs reduced the activation of ER stress-associated proteins, including GRP78, PERK, eIF2α, ATF4, IRE1α, JNK, p38, and CHOP. In particular, TUDCA inhibited the dissociation between GRP78 and PERK, resulting in reduced ER stress-mediated cell death. Next, to explore the ER stress protective mechanism induced by TUDCA treatment, TUDCA-mediated cellular prion protein (PrPC) activation was assessed. TUDCA treatment increased PrPC expression, which was regulated by Akt phosphorylation. Manganese-dependent superoxide dismutase (MnSOD) expression also increased significantly in response to signaling through the TUDCA-Akt axis. In a murine hindlimb ischemia model, TUDCA-treated MSC transplantation augmented the blood perfusion ratio, vessel formation, and transplanted cell survival more than untreated MSC transplantation did. Augmented functional recovery following MSC transplantation was blocked by PrPC downregulation. This study is the first to demonstrate that TUDCA protects MSCs against ER stress via Akt-dependent PrPC and Akt-MnSOD pathway.
Apolipoprotein(a) (apo(a)) contains tandemly repeated kringle domains that are closely related to plasminogen kringle 4, followed by a single kringle 5-like domain and an inactive protease-like domain. Recently, the anti-angiogenic activities of apo(a) have been demonstrated both in vitro and in vivo. However, its effects on tumor angiogenesis and the underlying mechanisms involved have not been fully elucidated. To evaluate the anti-angiogenic and anti-tumor activities of the apo(a) kringle domains and to elucidate their mechanism of action, we expressed the last three kringle domains of apo(a), KIV-9, KIV-10, and KV, in Escherichia coli. The resultant recombinant protein, termed rhLK68, exhibited a dose-dependent inhibition of basic fibroblast growth factor-stimulated human umbilical vein endothelial cell proliferation and migration in vitro and inhibited the neovascularization in chick chorioallantoic membranes in vivo. The ability of rhLK68 to abrogate the activation of extracellular signal-regulated kinases appears to be responsible for rhLK68-mediated anti-angiogenesis. Furthermore, systemic administration of rhLK68 suppressed human lung (A549) and colon (HCT-15) tumor growth in nude mice. Immunohistochemical examination and in situ hybridization analysis of the tumors showed a significant decrease in the number of blood vessels and the reduced expression of vascular endothelial growth factor, basic fibroblast growth factor, and angiogenin, indicating that suppression of angiogenesis may have played a significant role in the inhibition of tumor growth. Collectively, these results suggest that a truncated apo(a), rhLK68, is a potent antiangiogenic and anti-tumor molecule.
Chronic kidney disease (CKD) is caused by dysfunctional kidneys, which result in complications like cardiovascular diseases. Chronic kidney disease‐induced pathophysiological conditions decrease efficacy of autologous mesenchymal stem/stromal cell (MSC)‐based therapy by reducing MSC functionality. To enhance therapeutic potential in patients with CKD, we isolated exosomes derived from melatonin‐treated healthy MSCs (MT exosomes) and assessed the biological functions of MT exosome–treated MSCs isolated from patients with CKD (CKD‐MSCs). Treatment with melatonin increased the expression of cellular prion protein (PrPC) in exosomes isolated from MSCs through the upregulation of miR‐4516. Treatment with MT exosomes protected mitochondrial function, cellular senescence, and proliferative potential of CKD‐MSCs. MT exosomes significantly increased the level of angiogenesis‐associated proteins in CKD‐MSCs. In a murine hindlimb ischemia model with CKD, MT exosome–treated CKD‐MSCs improved functional recovery and vessel repair. These findings elucidate the regenerative potential of MT exosome–treated CKD‐MSCs via the miR‐4516‐PrPC signaling axis. This study suggests that the treatment of CKD‐MSCs with MT exosomes might be a powerful strategy for developing autologous MSC‐based therapeutics for patients with CKD. Furthermore, miR‐4516 and PrPC could be key molecules for enhancing the regenerative potential of MSCs in ischemic diseases.
Mesenchymal stem cells (MSCs) are ‘adult' multipotent cells that promote regeneration of injured tissues in vivo. However, differences in oxygenation levels between normoxic culture conditions (21% oxygen) and both the MSC niche (2–8% oxygen) and ischemic injury-induced oxidative stress conditions in vivo have resulted in low efficacy of MSC therapies in both pre-clinical and clinical studies. To address this issue, we examined the effectiveness of hypoxia preconditioning (2% oxygen) for enhancing the bioactivity and tissue-regenerative potential of adipose-derived MSCs. Hypoxia preconditioning enhanced the proliferative potential of MSCs by promoting the expression of normal cellular prion protein (PrPC). In particular, hypoxia preconditioning-mediated MSC proliferation was regulated by PrPC-dependent JAK2 and STAT3 activation. In addition, hypoxia preconditioning-induced PrPC regulated superoxide dismutase and catalase activity, and inhibited oxidative stress-induced apoptosis via inactivation of cleaved caspase-3. In a murine hindlimb ischemia model, hypoxia preconditioning enhanced the survival and proliferation of transplanted MSCs, ultimately resulting in improved functional recovery of the ischemic tissue, including the ratio of blood flow perfusion, limb salvage, and neovascularization. These results suggest that Hypo-MSC offer a therapeutic strategy for accelerated neovasculogenesis in ischemic diseases, and that PrPC comprises a potential target for MSC-based therapies.
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