Lactoferrin inhibits apoptosis through insulin-like growth factor I in primary rat osteoblasts.
Ontology highlight
ABSTRACT:
SUBMITTER: Hou JM
PROVIDER: S-EPMC4813718 | biostudies-other | 2014 Apr
REPOSITORIES: biostudies-other
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Project description:Cadmium (Cd) is a common environmental pollutant that can damage many organs and the fetus. We previously reported that Cd induced apoptosis in primary rat osteoblasts (OBs). OB apoptosis induced by Cd will eventually lead to osteoporosis. In this study, a novel pharmacotherapeutic approach was investigated involving the regulation of autophagy to prevent Cd osteoporosis. The results showed that Cd treatment induced apoptosis in OBs, as demonstrated by the ratio of Bax/Bcl-2, activation of poly (ADP-ribose) polymerase (PARP) and nuclear condensation. In addition, cells treated with Cd were observed to undergo autophagic cell death by monitoring the induction of the beclin 1, autophagy gene 5 (Atg5) and the expression of microtubule-associated protein 1 light chain 3 (LC3). The results indicated that promotion of apoptotic cell death by Cd is accompanied by induction of autophagy in OBs. Interestingly, Cd-mediated apoptotic cell death was suppressed by pretreatment with the autophagy activator rapamycin (RAP) and potentiated by the autophagy inhibitor chloroquine (CQ) or small interfering RNA against beclin 1. These findings suggest that the autophagic response plays a protective role that impedes eventual cell death. Activation of autophagy could therefore be an adjunctive strategy for treatment of Cd-induced osteoporosis.
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Project description:Colorectal cancer is a common cause of death with few available therapeutic strategies, and the preventative complexes in adjunctive therapy are urgently needed. Increasing evidences have shown that natural ingredients, including lactoferrin, oleic acid, docosahexaenoic acid (DHA) and linolenic acid, possess anti-inflammatory and anti-tumor activities. However, investigations and comparisons of their combinations in colorectal tumor model have not been reported, and the mechanism is still unrevealed. In the study, we examined the viability, migration, invasion and apoptosis of HT29 cells to choose the proper doses of these components and to select the effective combination in vitro. BALB/c nude mice bearing colorectal tumor were used to explore the role of selected combination in inhibiting tumor development in vivo. Additionally, metabonomic detection was performed to screen out the specific changed metabolitesand related pathway. The results demonstrated that lactoferrin at 6.25 μM, oleic acid at 0.18 mM, DHA at 0.18 mM, and linolenic acid at 0.15 mM significantly inhibited the viabilities of HT29 cells (p < 0.05). The combination of lactoferrin (6.25 μM) + linolenic acid (0.15 mM) exhibited the strongest activity in inhibiting the migration and invasion of HT29 cells in vivo and suppressing tumor development in vitro (p < 0.05). Furthermore, the lactoferrin + linolenic acid combination activated p-AMPK and p-JNK, thereby inducing apoptosis of HT29 cells (p < 0.05). The present study was the first to show that lactoferrin + linolenic acid combination inhibited HT29 tumor formation by activating AMPK/JNK related pathway.
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Project description:Insulin-like growth factor (IGF)-1 is a key factor in bone homeostasis and could be involved in bone tissue sclerosis as observed in osteoarthritis (OA). Here, we compare the key signaling pathways triggered in response to IGF-1 stimulation between normal and OA osteoblasts (Obs). Primary Obs were prepared from the subchondral bone of tibial plateaus of OA patients undergoing knee replacement or from normal individuals at autopsy. Phenotypic characterization of Obs was evaluated with alkaline phosphatase and osteocalcin release. The effect of IGF-1 on cell proliferation, alkaline phosphatase and collagen synthesis was evaluated in the presence or not of 50 ng/ml IGF-1, whereas signaling was studied with proteins separated by SDS-PAGE before western blot analysis. We also used immunoprecipitation followed by western blot analysis to detect interactions between key IGF-1 signaling elements. IGF-1 receptor (IGF-1R), Shc, Grb2, insulin receptor substrate (IRS)-1, and p42/44 mitogen-activated protein kinase (MAPK) levels were similar in normal and OA Obs in the presence or absence of IGF-1. After IGF-1 stimulation, the phosphorylation of IGF-1R in normal and OA Obs was similar; however, the phosphorylation of IRS-1 was reduced in OA Ob. In addition, the PI3K pathway was activated similarly in normal and OA Obs while that for p42/44 MAPK was higher in OA Obs compared to normal. p42/44 MAPK can be triggered via an IRS-1/Syp or Grb2/Shc interaction. Interestingly, Syp was poorly phosphorylated under basal conditions in normal Obs and was rapidly phosphorylated upon IGF-1 stimulation, yet Syp showed a poor interaction with IRS-1. In contrast, Syp was highly phosphorylated in OA Obs and its interaction with IRS-1 was very strong initially, yet rapidly dropped with IGF-1 treatments. The interaction of Grb2 with IRS-1 progressively increased in response to IGF-1 in OA Obs whereas this was absent in normal Ob. IGF-1 stimulation altered alkaline phosphatase in Ob, an effect reduced in the presence of PD98059, an inhibitor of p42/44 MAPK signaling, whereas neither IGF-1 nor PD98059 had any significant effect on collagen synthesis. In contrast, cell proliferation was higher in OA Obs compared to normal under basal conditions, and IGF-1 stimulated more cell proliferation in OA Obs than in normal Ob, an effect totally dependent on p42/44 MAPK activiy. The altered response of OA Obs to IGF-1 may be due to abnormal IGF-1 signaling in these cells. This is mostly linked with abnormal IRS-1/Syp and IRS-1/Grb2 interaction in these cells.
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Project description:The induced pluripotent stem cell (iPSC) is a promising cell source for tissue regeneration. However, the therapeutic value of iPSC technology is limited due to the complexity of induction protocols and potential risks of teratoma formation. A trans-differentiation approach employing natural factors may allow better control over reprogramming and improved safety. We report here a novel approach to drive trans-differentiation of human fibroblasts into functional osteoblasts using insulin-like growth factor binding protein 7 (IGFBP7). We initially determined that media conditioned by human osteoblasts can induce reprogramming of human fibroblasts to functional osteoblasts. Proteomic analysis identified IGFBP7 as being significantly elevated in media conditioned with osteoblasts compared with those with fibroblasts. Recombinant IGFBP7 induced a phenotypic switch from fibroblasts to osteoblasts. The switch was associated with senescence and dependent on autocrine IL-6 signaling. Our study supports a novel strategy for regenerating bone by using IGFBP7 to trans-differentiate fibroblasts to osteoblasts.
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Project description:In this study, we determined the molecular mechanisms whereby forkhead transcription factor Foxo1, a key downstream signaling molecule of insulin-like growth factor 1 (IGF1)/insulin actions, regulates Runx2 activity and expression of the mouse osteocalcin gene 2 (Bglap2) in osteoblasts in vitro. We showed that Foxo1 inhibited Runx2-dependent transcriptional activity and osteocalcin mRNA expression and Bglap2 promoter activity in MC-4 preosteoblasts. Co-immunoprecipitation assay showed that Foxo1 physically interacted with Runx2 via its C-terminal region in osteoblasts or when co-expressed in COS-7 cells. Electrophoretic mobility shift assay demonstrated that Foxo1 suppressed Runx2 binding to its cognate site within the Bglap2 promoter. IGF1 and insulin prevented Foxo1 from inhibiting Runx2 activity by promoting Foxo1 phosphorylation and nuclear exclusion. In contrast, a neutralizing anti-IGF1 antibody decreased Runx2 activity and osteocalcin expression in osteoblasts. Chromatin immunoprecipitation assay revealed that IGF1 increased Runx2 interaction with a chromatin fragment of the proximal Bglap2 promoter in a PI3K/AKT-dependent manner. Conversely, knockdown of Foxo1 increased Runx2 interaction with the promoter. This study establishes that Foxo1 is a novel negative regulator of osteoblast-specific transcription factor Runx2 and modulates IGF1/insulin-dependent regulation of osteocalcin expression in osteoblasts.
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Project description:PurposeRegorafenib, a multikinase inhibitor targeting the Ras/Raf/MEK/ERK pathway, has recently been approved for the treatment of metastatic colorectal cancer. However, the mechanisms of action of regorafenib in colorectal cancer cells have been unclear. We investigated how regorafenib suppresses colorectal cancer cell growth and potentiates effects of other chemotherapeutic drugs.Experimental designWe determined whether and how regorafenib induces the expression of PUMA, a p53 target and a critical mediator of apoptosis in colorectal cancer cells. We also investigated whether PUMA is necessary for the killing and chemosensitization effects of regorafenib in colorectal cancer cells. Furthermore, xenograft tumors were used to test if PUMA mediates the in vivo antitumor, antiangiogenic, and chemosensitization effects of regorafenib.ResultsWe found that regorafenib treatment induces PUMA in colorectal cancer cells irrespective of p53 status through the NF-κB pathway following ERK inhibition and glycogen synthase kinase 3β activation. Upregulation of PUMA is correlated with apoptosis induction in different colorectal cancer cell lines. PUMA is necessary for regorafenib-induced apoptosis in colorectal cancer cells. Chemosensitization by regorafenib is mediated by enhanced PUMA induction through different pathways. Furthermore, deficiency in PUMA abrogates the in vivo antitumor, antiangiogenic, and chemosensitization effects of regorafenib.ConclusionsOur results demonstrate a key role of PUMA in mediating the anticancer effects of regorafenib in colorectal cancer cells. They suggest that PUMA induction can be used as an indicator of regorafenib sensitivity, and also provide a rationale for manipulating the apoptotic machinery to improve the therapeutic efficacy of regorafenib and other targeted drugs.
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Project description:Estradiol (E2) is believed to modulate physiological functions relevant to osteoblast biology through the actions of estrogen receptors (ERs) that in turn regulate the expression of target genes. The molecular effects of estrogen action in bone remain to be fully elucidated. This study reports a genome-wide molecular and computational analysis of the interaction between ER and regulatory elements on the DNA of target genes in human primary osteoblasts. Of approximately 54,000 gene probes surveyed in this study, a total of 375 genes were up-regulated and 418 genes were down-regulated on exposure to E2, with only 46 of these being direct target genes after 24 h, as determined by concomitant cycloheximide treatment. Computational analysis discovered several pathways where E2 co-regulates multiple functionally linked components. Examination of the genomic sequence of IGF binding protein 4 located ER response elements within the first intron. Using by chromatin immunoprecipitation, we show a site- and cell-specific recruitment of transcription factors to this newly identified regulatory region. Transient transfection studies revealed that this intronic region acts as a functional promoter in human osteoblasts. Taken together, this analysis provides a comprehensive gene transcription profile and identifies several genes of potential physiological importance in controlling estrogen-mediated signaling in primary osteoblasts.
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