ケッカン シンセイ ソガイ チリョウ ニヨッテ カッセイカ スル アクセイ シュヨウカ ノンコーディング RNA ノ カイメイ

The most well-studied sequences in the human genome are those of protein-coding genes. However, the coding exons of these genes account for only１．５％ of the genome. In recent years, FANTOM and ENCODE projects reveal that more than ８０％ of the human genome is transcribed into RNAs that do not encode protein. It has become increasingly apparent that the non-protein-coding RNAs （ncRNAs） are of crucial functional importance : for normal development and physiology, and for disease. In particularly cancer, it is increasing evidence that not only miRNAs but also other ncRNAs, such as large intergenic non-coding RNAs （lincRNAs） and long non-coding RNAs （lncRNAs）, function as oncogenic ncRNAs and tumor-suppressive ncRNAs

NADPH oxidase-derived reactive oxygen species are essential for differentiation of a mouse macrophage cell line (RAW264.7) into osteoclasts

Reactive oxygen species (ROS) derived from NADPH oxidase (Nox) homologues have been suggested to regulate osteoclast differentiation. However, no bone abnormalities have been documented in Nox1 deficient, Nox2 deficient, or Nox3 mutant mice. During receptor activator of nuclear factor-κB ligand (RANKL)-stimulated differentiation of a mouse macrophage cell line (RAW264.7) into osteoclasts, mRNA levels of Nox enzymes (Nox1-4) and their adaptor proteins were monitored by real-time reverse transcriptase PCR. RAW264.7 cells constitutively expressed abundant Nox2 mRNA and small amounts of Nox1 and Nox3 transcripts. RANKL markedly attenuated Nox2 mRNA expression in association with reciprocal up-regulation of Nox1 and Nox3 transcripts. Introduction of small interference RNA targeting p67phox or p22phox into RAW264.7 cells effectively downregulated ROS generation and significantly suppressed the RANKL-stimulated differentiation, which was assessed by appearance of tartrate resistant acid phosphatase (TRAP)- positive, multinucleated cells having an ability to form resorption pits on calcium phosphate thin film-coated disks, and by expression of osteoclast marker genes (TRAP, cathepsin K, Atp6i, ClC-7, and NFATc1). Our results suggest that RANKL may stimulate switching between Nox homologues during osteoclast differentiation, and Nox-derived ROS may be crucial for RANKL-induced osteoclast differentiation

UCP3 ト Hax-1 ノ ソウゴ サヨウ ニヨル ミトコンドリア ノ カルシウム ノ ウド ノ チョウセツ

Mitochondrial Ca２＋ plays an important role in the regulations of various cellular functions. Uncoupling protein ３ （UCP３） is primarily expressed in the inner membrane of skeletal muscle mitochondria. Recently, it has been reported that UCP３ is associated with Ca２＋ uptake into mitochondria. However, the mechanisms by which UCP３ regulates mitochondrial Ca２＋ uptake are not well understood. Here we report that UCP３interacts with HS‐１associated protein X‐１ （Hax‐１）, an anti-apoptotic protein that is localized in mitochondria, which is involved in cellular responses to Ca２＋. The hydrophilic sequences within the loop２, matrix-localized hydrophilic domain of mouse UCP３are necessary for binding to Hax‐１of the C-terminal domain in adjacent to mitochondrial innermembrane. Interestingly, interaction of these proteins occurs the calciumdependent manner. Moreover, NMR spectrum of the C-terminal domain of Hax‐１was dramatically changed by removal of Ca２＋, suggesting that the C-terminal domain of Hax‐１ underwent a Ca２＋-induced conformation change. In the Ca２＋-free states, C-terminal Hax‐１ didn’t change the structure, suggesting that Ca２＋ binding may induce the change of protein structure of Hax‐１ C-terminus. These studies identify a novel UCP３‐Hax‐１complex regulates the influx of Ca２＋ into mitochondria. Thus, the efficacy of UCP３‐Hax‐１in mitochondrial calcium regulation may provide a novel therapeutic approach against mitochondrial dysfunction-related disease

Chronic inhibition of tumor cell-derived VEGF enhances the malignant phenotype of colorectal cancer cells

Abstract Background Vascular endothelial growth factor-a (VEGF)-targeted therapies have become an important treatment for a number of human malignancies. The VEGF inhibitors are actually effective in several types of cancers, however, the benefits are transiently, and the vast majority of patients who initially respond to the therapies will develop resistance. One of possible mechanisms for the acquired resistance may be the direct effect(s) of VEGF inhibitors on tumor cells expressing VEGF receptors (VEGFR). Thus, we investigated here the direct effect of chronic VEGF inhibition on phenotype changes in human colorectal cancer (CRC) cells. Methods To chronically inhibit cancer cell-derived VEGF, human CRC cell lines (HCT116 and RKO) were chronically exposed (2 months) to an anti-VEGF monoclonal antibody (mAb) or were disrupted the Vegf gene (VEGF-KO). Effects of VEGF family members were blocked by treatment with a VEGF receptor tyrosine kinase inhibitor (VEGFR-TKI). Hypoxia-induced apoptosis under VEGF inhibited conditions was measured by TUNEL assay. Spheroid formation ability was assessed using a 3-D spheroid cell culture system. Results Chronic inhibition of secreted/extracellular VEGF by an anti-VEGF mAb redundantly increased VEGF family member (PlGF, VEGFR1 and VEGFR2), induced a resistance to hypoxia-induced apoptosis, and increased spheroid formation ability. This apoptotic resistance was partially abrogated by a VEGFR-TKI, which blocked the compensate pathway consisted of VEGF family members, or by knockdown of Vegf mRNA, which inhibited intracellular function(s) of all Vegf gene products. Interestingly, chronic and complete depletion of all Vegf gene products by Vegf gene knockout further augmented these phenotypes in the compensate pathway-independent manner. These accelerated phenotypes were significantly suppressed by knockdown of hypoxia-inducible factor-1α that was up-regulated in the VEGF-KO cell lines. Conclusions Our findings suggest that chronic inhibition of tumor cell-derived VEGF accelerates tumor cell malignant phenotypes.http://deepblue.lib.umich.edu/bitstream/2027.42/112625/1/12885_2012_Article_3866.pd

Helicobacter pylori lipopolysaccharide from type I, but not type II strains, stimulates apoptosis of cultured gastric mucosal cells

The cag pathogenicity island (cag PAI) genes are a major determinant of virulence of Helicobacter pylori (Hp). Lipopolysaccharide (LPS) purified from the cag PAI-positive (type I) strains induced apoptosis of primary cultures of guinea pig gastric mucosal cells. Lipid A catalyzed this apoptosis. These cells expressed the Toll-like receptor 4 (TLR4) mRNA and its protein, and type I Hp LPS phosphorylated transforming growth factor β-activated kinase 1 (TAK1) and TAK1-binding protein 1 (TAB1) in association with up-regulation of the TLR4 expressions, suggesting that type I Hp LPS evoked distinct TLR4 signaling. In contrast, Hp LPS from type II strains with complete or partial deletion of the cag PAI genes did not phosphorylate TAK1 and TAB1 and failed to induce apoptosis. Accelerated apoptosis of gastric epithelial cells is one of the important events relevant to chronic, atrophic gastritis caused by Hp infection. The difference in proapoptotic action of LPS between the type I and II strains may support an important role of the cag PAI genes in the pathogenesis of gastric lesions caused by Hp infection

Antiangiogenic agent sunitinib induces epithelial to mesenchymal transition and accelerates motility of colorectal cancer cells

Although vascular endothelial growth factor receptor (VEGF-R)-targeted antiangiogenic agents are important treatment for a number of human malignancies, there is accumulating evidence that the therapies may promote disease progression, such as invasion and metastasis. How tumors become to promote their evasiveness remains fully uncertain. One of possiblemechanisms for the adaptationmay be a direct effect of VEGF-R inhibitors on tumor cells expressing VEGF-R. To elucidate a direct effect of VEGF-R-targeting drug (sunitinib), we established a human colorectal cancer cell model adapted to sunitinib. The sunitinib-conditioned cells showed a significant increase in cellular motility and migration activities, compared to the vehicle-treated control cells. Consistent with the phenotype, the sunitinib-conditioned cells decreased the expression levels of E-cadherin (an epithelial marker), while significantly increased the levels of Slug and Zeb1 (mesenchymal markers). Expression profiles of VEGF-R in the sunitinib-conditioned cells showed that only neuropilin-1 (NRP1) expression was significantly increased among all VEGF-R tested. Blockade of NRP1 using its antagonist clearly repressed the migration activationin sunitinib-conditioned cells, but not in the control cells. These results suggest that inhibition of VEGF-R on colorectal cancer cells can drive the epithelial-mesenchymal transition, leading to activation of cell motility in an NRP1-dependent manner

Gene expression profiling in peripheral blood leukocytes as a new approach for assessment of human stress response

Stress is the coordinated physiological processes to maintain a dynamic equilibrium under stressful conditions. The equilibrium is threatened by certain physiological and psychological stressors. Stressors trigger physiological, behavioural, and metabolic responses that are aimed at reinstating homeostasis. The hypothalamus-pituitary-adrenal (HPA) axis and the sympathetic nervous system play an essential role in the stress response. Excessive, prolonged, or inadequate response that is termed as “allostasis” or “allostatic load” leads to pathological outcomes. Dysregulation of the HPA axis activity is involved in the pathogenesis of stress related disorders including major depression. The complex brain-immune-endocrine network regulates the HPA axis, and hereditary predisposition as well as environmental factors such as traumatic experiences in early life also modifies the capacity of an individual to cope. Therefore, it is difficult to correctly assess the complex stress response. We have developed a microarray carrying 1,467 cDNAs that were selected to specifically measure stress response in peripheral blood leukocytes. Using this tool, we have succeeded to objectively assess individual response to acute psychological stress and to detect unique expression profiles in patients with depression. Gene expression profile in peripheral blood leukocytes may be a potentially useful for the detection of disease-associated, abnormal stress responses

DIRECT EFFECTS OF VEGF/VEGF-R TARGETING AGENTS ON COLON CANCER CELLS

Anti-angiogenic therapies targeting vascular endothelial growth factor (VEGF) and its receptor (VEGF-R) are important treatments for a number of human malignancies, including colorectal cancers. However, there is increasing evidence that VEGF/VEGF-R inhibitors promote the adaptive and evasive resistance of tumor cells to the therapies. The mechanism by which the cancer cells become resistant remains unclear. One potential mechanism is that VEGF/VEGF-R blockers directly act on tumor cells independently of anti-angiogenic effects. In this study, the direct effects of an anti-VEGF antibody (bevacizumab) and a VEGF-R tyrosine kinase inhibitor (sunitinib) on the evasive adaptation of colon cancer cells were compared. HCT116 and RKO human colon cancer cell lines were chronically exposed (3 months) to bevacizumab or sunitinib in vitro to establish bevacizumab- and sunitinib-adapted cells, respectively. Transwell migration and invasion assays, western blotting, reverse transcription-quantitative polymerase chain reaction, co-immunoprecipitation analysis, cell survival assays and ELISAs were conducted to analyze the adapted cells. Compared with the control vehicle-treated cells, the two cell models exhibited increased migration and invasion activities to different degrees and through different mechanisms. The bevacizumab-adapted cells, but not in the sunitinib-adapted cells, exhibited redundantly increased expression levels of VEGF/VEGF-R family members, including VEGF-A, placental growth factor, VEGF-C, VEGF-R1 and VEGF-R3. In addition, the phosphorylation levels of VEGF-R1 and VEGF-R3 were increased in the bevacizumab-adapted cells compared with the control cells. Thus, the inhibition of VEGF-R1 and VEGF-R3 decreased the evasive activities of the cells, suggesting that they remained dependent on redundant VEGF/VEGF-R signaling. By contrast, the sunitinib-adapted cells exhibited increased neuropilin-1 (NRP1) expression levels compared with the control cells. In the sunitinib-adapted cells, NRP1 interacted with phosphorylated cMet, and the cMet activation was dependent on NRP1. Thus, NRP1 or cMet blockade suppressed the evasive activation of the sunitinib-adapted cells. These results suggest that the sunitinib-adapted cells switched from a VEGF-R-dependent pathway to an alternative NRP1/cMet-dependent one. The findings of the present study indicate that VEGF/VEGF-R inhibitors directly act on colon cancer cells and activate their evasive adaptation via different mechanisms

Regorafenib induces adaptive resistance of colorectal cancer cells via inhibition of vascular endothelial growth factor receptor

Recently, inhibition of tumor angiogenesis has become an important anti-cancer therapy. Tumor angiogenesis is regulated by multiple signaling pathways, including VEGF and VEGF receptor (VEGF-R), FGF and FGF receptor (FGF-R), and PDGF and PDGF receptor (PDGF-R) pathways. Thus, the antiangiogenic agents, such as regorafenib, simultaneously target those receptors on vascular endothelial cells. In addition to endothelial cells, cancer cells express the three receptors, suggesting that the antiangiogenic inhibitors affect tumor cells. In fact, we previously demonstrated that regorafenib directly acted on human colorectal cancer cells and accelerated their apoptosis resistance and migration capability. Thus, we here elucidated how regorafenib induced the malignant phenotypes in colorectal cancer cells. To identify the responsible receptor among the regorafenibtargeting proangiogenic receptors, we examined the effects of a potent selective inhibitor for VEGF-R, FGF-R or PDGF-R on apoptosis resistance and migration capability. We clarified that blockade of VEGF-R, but not FGF-R and PDGF-R, induced the malignant phenotypes. We confirmed that blocking of VEGF ligands derived from colorectal cancer cells also induced the phenotypes. These results suggest that regorafenib progressed the malignancy via prevention of autocrine and paracrine VEGF signaling in colorectal cancer cells

DIRECT EFFECTS OF VEGF/VEGF-R TARGETING AGENTS ON COLON CANCER CELLS

Anti-angiogenic therapies targeting vascular endothelial growth factor (VEGF) and its receptor (VEGF-R) are important treatments for a number of human malignancies, including colorectal cancers. However, there is increasing evidence that VEGF/VEGF-R inhibitors promote the adaptive and evasive resistance of tumor cells to the therapies. The mechanism by which the cancer cells become resistant remains unclear. One potential mechanism is that VEGF/VEGF-R blockers directly act on tumor cells independently of anti-angiogenic effects. In this study, the direct effects of an anti-VEGF antibody (bevacizumab) and a VEGF-R tyrosine kinase inhibitor (sunitinib) on the evasive adaptation of colon cancer cells were compared. HCT116 and RKO human colon cancer cell lines were chronically exposed (3 months) to bevacizumab or sunitinib in vitro to establish bevacizumab- and sunitinib-adapted cells, respectively. Transwell migration and invasion assays, western blotting, reverse transcription-quantitative polymerase chain reaction, co-immunoprecipitation analysis, cell survival assays and ELISAs were conducted to analyze the adapted cells. Compared with the control vehicle-treated cells, the two cell models exhibited increased migration and invasion activities to different degrees and through different mechanisms. The bevacizumab-adapted cells, but not in the sunitinib-adapted cells, exhibited redundantly increased expression levels of VEGF/VEGF-R family members, including VEGF-A, placental growth factor, VEGF-C, VEGF-R1 and VEGF-R3. In addition, the phosphorylation levels of VEGF-R1 and VEGF-R3 were increased in the bevacizumab-adapted cells compared with the control cells. Thus, the inhibition of VEGF-R1 and VEGF-R3 decreased the evasive activities of the cells, suggesting that they remained dependent on redundant VEGF/VEGF-R signaling. By contrast, the sunitinib-adapted cells exhibited increased neuropilin-1 (NRP1) expression levels compared with the control cells. In the sunitinib-adapted cells, NRP1 interacted with phosphorylated cMet, and the cMet activation was dependent on NRP1. Thus, NRP1 or cMet blockade suppressed the evasive activation of the sunitinib-adapted cells. These results suggest that the sunitinib-adapted cells switched from a VEGF-R-dependent pathway to an alternative NRP1/cMet-dependent one. The findings of the present study indicate that VEGF/VEGF-R inhibitors directly act on colon cancer cells and activate their evasive adaptation via different mechanisms