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There are no effective treatments for relieving neuronal dysfunction after mild traumatic brain injury (TBI). Here, we evaluated therapeutic efficacy of human embryonic stem cell-derived cerebral organoids (hCOs) in a mild TBI model, in terms of repair of damaged cortical regions, neurogenesis, and improved cognitive function.Male C57BL/6 J mice were randomly divided into sham-operated, mild TBI, and mild TBI with hCO groups. hCOs cultured at 8 weeks were used for transplantation. Mice were sacrificed at 7 and 14 days after transplantation followed by immunofluorescence staining, cytokine profile microarray, and novel object recognition test.8W-hCOs transplantation significantly reduced neuronal cell death, recovered microvessel density, and promoted neurogenesis in the ipsilateral subventricular zone and dentate gyrus of hippocampus after mild TBI. In addition, increased angiogenesis into the engrafted hCOs was observed. Microarray results of hCOs revealed neuronal differentiation potential and higher expression of early brain development proteins associated with neurogenesis, angiogenesis and extracellular matrix remodeling. Ultimately, 8W-hCO transplantation resulted in reconstruction of damaged cortex and improvement in cognitive function after mild TBI.hCO transplantation may be feasible for treating mild TBI-related neuronal dysfunction via reconstruction of damaged cortex and neurogenesis in the hippocampus.

Kallikrein-related peptidase (KLK)6 is associated with inflammatory diseases and neoplastic progression. KLK6 is aberrantly expressed in several solid tumors and regulates cancer development, metastatic progression, and drug resistance. However, the function of KLK6 in the tumor microenvironment remains unclear. This study aimed to determine the role of KLK6 in the tumor microenvironment. Here, we uncovered the mechanism underlying KLK6-mediated cross-talk between cancer cells and macrophages. Compared with wild-type mice, KLK6−/− mice showed less tumor growth and metastasis in the B16F10 melanoma and Lewis lung carcinoma (LLC) xenograft model. Mechanistically, KLK6 promoted the secretion of tumor necrosis factor-alpha (TNF-α) from macrophages via the activation of protease-activated receptor-1 (PAR1) in an autocrine manner. TNF-α secreted from macrophages induced the release of the C-X-C motif chemokine ligand 1 (CXCL1) from melanoma and lung carcinoma cells in a paracrine manner. The introduction of recombinant KLK6 protein in KLK6−/− mice rescued the production of TNF-α and CXCL1, tumor growth, and metastasis. Inhibition of PAR1 activity suppressed these malignant phenotypes rescued by rKLK6 in vitro and in vivo. Our findings suggest that KLK6 functions as an important molecular link between macrophages and cancer cells during malignant progression, thereby providing opportunities for therapeutic intervention.

Retrospective cross-sectional study To evaluate the validity and obtain optimal cutoff values of 3 radiologic measurements for the diagnosis of basilar invagination (BI). Two hundred seventy-six patients (46 patients who underwent atlantoaxial fusion for BI and 230 patients who were treated for minor cervical trauma) seen in a single institution from January 2010 to December 2016 were included in this study. Age, sex, and body mass index were adjusted for the patients. The Ranawat index (RI), modified Ranawat method (MRM), and Redlund-Johnell method (RJM) were used to diagnose BI on plain radiographs. The sensitivity, specificity, positive predictive value, negative predictive value, accuracy, and diagnostic odds ratio of 3 radiologic measurements were compared. We also calculated the optimized cutoff values of 3 radiologic measurements using the receiver operating characteristic curve in our patients. The mean age of the 130 women and 146 men was 58.3 ± 14.5 years. The mean values of RI, MRM, and RJM in the BI group were 12.5 ± 3.3, 23.1 ± 3.8, and 27.3 ± 3.6 in women and 13.6 ± 2.6, 26.8 ± 4.2, and 34.7 ± 5.1 in men. There was a significant difference between the sexes (P .05). The accuracies of RI, MRM, and RJM were 95%, 89.6%, and 92.3% in women and 93%, 68.2%, and 85.4% in men, respectively. The optimized cutoff values of RI, MRM, and RJM were 14, 26, and 32 mm in women and 15, 29, and 38 mm in men. Three radiologic measurements (RI, MRM, and RJM) are reliable for the diagnosis of BI even in the era of cross-sectional images. The validity of these measurements depends on sex and particular radiologic measurement. The optimized cutoff values of RI, MRM, and RJM were 14, 26, and 32 mm in women and 15, 29, and 37 mm in men. These cutoff values showed high validity when compared to the CT and MRI findings.

Strategies for stem cell-based cardiac regeneration and repair are key issues for the ischemic heart disease (IHD) patients with chronic complications related to ischemic necrosis. Cardiac stem cells (CSCs) have demonstrated high therapeutic efficacy for IHD treatment owing to their specific cardiac-lineage commitment. The therapeutic potential of CSCs could be further enhanced by designing a cellular spheroid formulation. The spheroid culture condition of CSCs was optimized to ensure regulated size and minimal core necrosis in the spheroids. The CSC spheroids revealed mRNA profiles of the factors related to cardiac regeneration, angiogenesis, anti-inflammatory, and cardiomyocyte differentiation with a higher expression level than the CSCs. Intramyocardially delivered CSC spheroids in the rat IHD model resulted in a significant increase in retention rate by 1.82-fold (day 3) and 1.98-fold (day 14) compared to CSCs. Endothelial cell differentiation and neovascularization of the engrafted CSC spheroids were noted in the infarcted myocardium. CSC spheroids significantly promoted cardiac regeneration: i.e., decreased infarction and fibrotic area (11.22% and 4.18%) and increased left ventricle thickness (0.62 mm) compared to the untreated group. Cardiac performance was also improved by 2.04-fold and 1.44-fold increase in the ejection fraction and fractional shortening, respectively. Intramyocardial administration of CSC spheroids might serve as an advanced therapeutic modality with enhanced cell engraftment and regenerative abilities for cardiac repair after myocardial infarction.

Radiation therapy is an effective treatment against various types of cancer, but some radiation-resistant cancer cells remain a major therapeutic obstacle; thus, understanding radiation resistance mechanisms is essential for cancer treatment. In this study, we established radiation-resistant colon cancer cell lines and examined the radiation-induced genetic changes associated with radiation resistance. Using RNA-sequencing analysis, collapsin response mediator protein 4 (CRMP4) was identified as the candidate gene associated with radiation sensitivity. When cells were exposed to radiation, intracellular Ca2+ influx, collapse of mitochondrial membrane potential, and cytochrome c release into the cytosol were increased, followed by apoptosis induction. Radiation treatment- or Ca2+ ionophore A23187-induced apoptosis was significantly inhibited in CRMP4-deficient cells, including radiation-resistant or CRMP4-shRNA cell lines. Furthermore, treatment of CRMP4-deficient cells with low levels (10 µM) resulted in higher cell death in the CRMP4-depleted cells compared to CRMP4-expressing control cells. Our results suggest that CRMP4 plays an important role in Ca2+-mediated cell death pathways under radiation exposure and that CRMP4 may be a therapeutical target for colon cancer treatment.

Rho GTPases control a wide range of cellular processes, and their deregulation is associated with promotion of an aggressive and metastatic tumor phenotype in human cancers. Rho guanine nucleotide dissociation inhibitor 1 (RhoGDI1) plays a key role in regulating the activity of Rho GTPases. However, the underlying mechanisms are still unclear. In this study, we show that protein phosphatase 1B (PPM1B) interacts with RhoGDI1 and functions as its phosphatase. Ectopic expression of PPM1B results in dephosphorylation of RhoGDI1 and, thereby, abates the activation of RhoA, Rac1 and CDC42 by epidermal growth factor (EGF). PPM1B overexpression in Hs578T and SKBR3 human breast cancer cells decreases their motility and invasiveness in vitro and cancer metastasis in vivo. In contrast, knockdown of PPM1B in MCF-7 and MDA-MB-468 human breast cancer cells that express endogenous PPM1B enhances EGF-induced RhoGDI1 phosphorylation, activation of Rho GTPases, and cancer cell migration and invasion. Knockdown of RhoA or Rac1 by siRNA reverses the enhanced cell migration seen after PP1MB depletion. Collectively, these results indicate that PPM1B negatively regulates cancer cell motility and invasiveness through dephosphorylating RhoGDI1.

Objective The purpose of this study was to evaluate the morphometric characteristics of ponticulus posticus (PP) and determine the impact of two promising high cervical operations (posterior arch to lateral mass screw fixation (PALMSF) and occipital condyle fixation (OCF)). Patients and methods We evaluated retrospective data from a total of 2628 head and neck 3-dimensional CT-angiographies (3D-CTA) that did not have bony or vascular abnormalities. The PP anomaly cases were confirmed, then we measured vertebral artery groove height (VAGH), arch to vertebral artery distance (AVD) and posterior arch height (PAH) for PALMSF. We also measured the vertebral artery to occipital bone distance (VOD) for OCF. Results In 186 patients (7.1%), 227 PP (complete or near complete) were identified and this anomaly was more common in males and on the left side. The mean VAGH and PAH values ranged from 5.0 to 6.0 mm and from 7.0 to 8.8 mm, respectively. The VAGH value was not statistically different, regardless of the presence of PP, and the feasibility (>4 mm) of a safe PALMSF (range, 87.7–100%) was not influenced by PP anomalies. The mean value of VOD in the anomaly side (range, 6.0–8.0 mm) was much higher than for normal side (range, 4.2–5.7 mm) and the proportion of patients that had a safe OCF (>3.5 mm) was also much higher (range, 90.8–96.9%) than for the normal side. Conclusions A PP anomaly might have some anatomical feasibility and advantage during PALMSF and OCF, due to the relatively sufficient bony (VAGH) and spatial regions (VOD). However, there are gender differences in anatomical dimensions (VAGH, VOD and AVD); therefore, surgery in female patients should be approached more cautiously in the future.

Background We compared left and right vascular anatomy at the L5-S1 disc space and validated the anatomical feasibility of the right oblique approach for L5-S1 oblique lumbar interbody fusion. Methods Axial T2-weighted magnetic resonance imaging studies at the L5-S1 disc level were used to study 274 subjects (164 women and 110 men; average age, 62.97 years). The distance from the center of the L5-S1 disc to the medial wall of the left or right vessel was measured. Using the vessel position, 3 groups were established: medial, middle, and lateral. To describe the morphological configuration, the vessel type and the presence of perivascular adipose tissue (PVAT) around the vessels were identified on both sides. Results The vessels on the left L5-S1 disc space were located 12.47 mm from the midline and most subjects (209 subjects; 76.3%) were included in the medial or middle group. On the right side, the vessels were located more laterally (16.93 mm; P = 0.000) and most subjects (248 subjects; 90.5%) were in the middle or lateral group. On the left side, vessels were mostly veins (260 subjects; 94.9%) and 139 subjects (50.7%) had PVAT. On the right side, the vessels were mostly arteries (213 subjects; 77.7%) and 242 (88.3%) had PVAT. Conclusions The vessels on the right side of the L5-S1 disc were located more laterally, and most vessels on the right side were arteries accompanying PVAT, which might minimize vessel manipulation. These results indicate that the right side of the L5-S1 disc could provide feasible access for oblique lumbar interbody fusion at L5-S1.

The EF‑hand calcium binding protein tescalcin (TESC) is highly expressed in various human and mouse cancer tissues and is therefore considered a potential oncogene. However, the underlying mechanism that governs TESC expression remains unclear. Emerging evidence suggests that TESC expression is under epigenetic regulation. In the present study, the relationship between the epigenetic modification and gene expression of TESC in gastric cancer was investigated. To evaluate the relationship between the methylation and expression of TESC in gastric cancer, the methylation status of CpG sites in the TESC promoter was analyzed using microarray with the Illumina Human Methylation27 BeadChip (HumanMethylation27_270596_v.1.2), gene profiles from the NCBI Dataset that revealed demethylated status were acquired, and real‑time methylation‑specific PCR (MSP) in gastric cancer cells was conducted. In the present study, it was demonstrated that the hypermethylation of TESC led to the downregulation of TESC mRNA/protein expression. In addition, 5‑aza‑2c‑deoxycytidine (5'‑aza‑dC) restored TESC expression in the tested gastric cancer cells except for SNU‑620 cells. ChIP assay further revealed that the methylation of the TESC promoter was associated with methyl‑CpG binding domain protein (MBD)1, histone deacetylase (HDAC)2, and Oct‑1 and that treatment with 5'‑aza‑dC facilitated the dissociation of MBD1, HDAC2, and Oct‑1 from the promoter of TESC. Moreover, silencing of TESC increased MBD1 expression and decreased the H3K4me2/3 level, thereby causing transcriptional repression and suppression of cell survival in NCI‑N87 cells; conversely, overexpression of TESC downregulated MBD1 expression and upregulated the H3K4me2 level associated with active transcription in SNU‑638 cells. These results indicated that the differential expression of TESC via the modification status of the promoter and histone methylation controled cell survival in gastric cancer cells. Overall, the present study provided a novel therapeutic strategy for gastric cancer.

The B cell lymphoma 2 (BCL2) family of proteins constitutes a critical intracellular checkpoint in the intrinsic apoptosis pathway. Among BCL2 members, the anti-apoptotic protein BCL2A1 mediates the resistance to BCL2 inhibitors and may be considered as a target for anti-cancer therapy. Here, we report that prenylated Rab acceptor 1 (RABAC1 or PRA1) inhibits the anti-apoptotic activity of BCL2A1 and induces apoptosis in AGS gastric cancer cells. Protein interaction of BCL2A1 and RABAC1 was verified by an in-vitro glutathione-S-transferase pull-down assay, immunoprecipitation, and confocal microscopy. When apoptosis was induced by cisplatin, the anti-apoptotic activity of BCL2A1 was blocked by RABAC1 expression. RABAC1 caused caspase-3 activation and decreased cell proliferation, clonogenic cell survival, and cell migration and invasion. We suggest RABAC1 as a potential therapeutic target for BCL2A1-related cancer.

Nogo receptor (NgR) has been shown to inhibit the migration and invasion of human glioma cells. However, little is known regarding the regulatory mechanisms of NgR in glioblastoma multiforme (GBM). In this study, we propose a novel mechanism that regulates the maturation process of NgR through an interaction with vimentin. The inhibition of TGFβ1 activity by LY2109761 attenuated the migration/invasion of GBM cells by upregulating cell-surface NgR. Conversely, the treatment of GBM cells with TGFβ1 suppressed NgR maturation. We showed that NgR and vimentin interact, which could be a possible mechanism for the suppression of NgR maturation. The knockdown of vimentin suppressed the migration/invasion of GBM cells through the increased maturation of NgR. Finally, TCGA (The Cancer Genome Atlas) analysis also supported the association of NgR and vimentin. The maturation of NgR is regulated by the interaction of vimentin and NgR, which attenuates the invasive activity of GBM, and might be a potential therapeutic target for brain cancer., Brain cancer: A possible target to stop spread A mechanism that prevents the maturation of a protective cell surface protein during the spread of brain cancer could be a therapeutic target. Aggressive glioblastoma multiforme tumors spread quickly, lowering survival chances. The transforming growth factor-beta 1 (TGFβ1) protein is implicated in the rapid spread of cancer cells through the brain’s white matter fibers. However, cancer spread can be limited by the mature form of a protein receptor called nogo receptor (NgR), which is expressed on white matter cell surfaces. Using human glioblastoma cell cultures, Seung-Hoon Lee and Myung-Shin Lee at Eulji University School of Medicine, Daejeon, South Korea, and co-workers demonstrated how the interaction between NgR and another protein enhances TGFβ1 pathway activity and prevents NgR maturing. When the team inhibited TGFβ1, the interaction was disrupted, allowing NgR maturation and preventing tumor spread.

// Tae Woo Kim 1, 2, * , Seon-Jin Lee 1, 2, * , Jong-Tae Kim 1 , Sun Jung Kim 3 , Jeong-Ki Min 4 , Kwang-Hee Bae 5 , Haiyoung Jung 1 , Bo-Yeon Kim 6 , Jong-Seok Lim 7 , Young Yang 7 , Do-Young Yoon 8 , Yong-Kyung Choe 1 , Hee Gu Lee 1, 2 1 Immunotherapy Convergence Research Center, Korea Research Institute of Bioscience and Biotechnology, Daejeon, Republic of Korea 2 Department of Biomolecular Science, University of Science and Technology (UST), Daejeon, Korea 3 Department of Life Science, Dongguk University-Seoul, Seoul, Republic of Korea 4 Biotherapeutics Translational Research Center, Korea Research Institute of Bioscience and Biotechnology, Daejeon, Republic of Korea 5 Research Center for Metabolic Regulation, Korea Research Institute of Bioscience and Biotechnology, Daejeon, Republic of Korea 6 World Class Institute, Korea Research Institute of Bioscience and Biotechnology, Ochang, Cheongwon, Republic of Korea 7 Department of Biological Sciences, Sookmyung Women’s University, Seoul, Republic of Korea 8 Department of Bioscience and Biotechnology, Konkuk University, Seoul, Republic of Korea * These authors have contributed equally to this work Correspondence to: Hee Gu Lee, email: hglee@kribb.re.kr Yong-Kyung Choe, email: ykccoruk@kribb.re.kr Keywords: kallikrein-related peptidase 6, autophagy, auranofin, cell death, chemoresistance Received: April 14, 2016 Accepted: October 14, 2016 Published: November 15, 2016 ABSTRACT Kallikrein-related peptidase 6 (KLK6) is a biomarker of gastric cancer associated with poor prognosis. Mechanisms by which KLK6 could be exploited for chemotherapeutic use are unclear. We evaluated auranofin (AF), a compound with cytotoxic effects, in KLK6-deficient cells, and we investigated whether KLK6 expression induces autophagy and acquisition of drug resistance in gastric cancer. Using cultured human cells and a mouse xenograft model, we investigated how KLK6 affects antitumor-reagent-induced cell death and autophagy. Expression levels of KLK6, p53, and autophagy marker LC3B were determined in gastric cancer tissues. We analyzed the effects of knockdown/overexpression of KLK6, LC3B, and p53 on AF-induced cell death in cancer cells. Increased KLK6 expression in human gastric cancer tissues and cells inhibited AF-induced cell motility due to increased autophagy and p53 levels. p53 dependent induction of KLK6 expression increased autophagy and drug resistance, whereas KLK6 silencing decreased the autophagy level and increased drug sensitivity. During AF-induced cell death, KLK6 and LC3B colocalized to autophagosomes, associated with p53, and were then trafficked to the cytosol. In the xenograft model of gastric cancer, KLK6 expression decreased AF-induced cell death and KLK6-induced autophagy increased AF resistance. Taken together, the data suggest that the induction of autophagic processes through KLK6 expression may increase acquisition of resistance to AF. Our findings may contribute to a new paradigm for tumor therapeutics.

Unusual entrance of the vertebral artery into the cervical transverse foramen (UE-V2S) is a relatively common clinical anomaly. Because spinal surgeons should understand the anatomical characteristics and clinical implications of this condition, we aimed to provide a description based on a very large number of UE-V2S anomaly cases. We retrospectively analyzed 2207 three-dimensional head-and-neck computed tomographic angiograms (CTAs) that did not have specific vascular abnormalities. After confirming which cases had an unusual vertebral artery (VA) entrance into the transverse foreman (TF), we measured their vertebral artery diameter (VAD), transverse foramen area (TFA) and bilateral pedicle width (PW) from C3 to C7. The shortest horizontal distance from the vertical line in the medial margin of the TF to VA (D-TFVA) was measured in the extra-osseous region to estimate the exact location of the extra-osseous VA, except at the C7 level. An unusual V2 entrance was observed in 11.4 % (252 patients) of all 2207 consecutive patients and 6.5 % (268 courses) of all 4414 courses. The prevalence rankings for the different measures were as follows: unilateral UE-V2S = E5 > E4 > E7 > E3; bilateral UE-V2S = E5 (bilateral) > E4 (Rt) with E5 (Lt) > E4 (bilateral). Generally, the VAD of the anomaly side was statistically smaller than the normal contralateral side, which can induce a smaller TFA value at all sub-axial levels. Cervical pedicle fixation was preferable in the adjacent lower segment of unusual VA entrance level than the normal side in this study due to a broader PW, which was evident on the anomaly side. However, there was no statistical evidence that the side of the C7 entrance of the VA had a narrow PW. The lowest D-TFVA value was −3.8, indicating that we should take care not to exceed 3.8 mm medially into the vertical line of the medial TF wall during dissection when taking an anterior cervical approach. To avoid an unexpected VA injury and to improve the efficiency of even routine cervical operations, spinal surgeons should determine whether the patient has a UE-V2S and have a full understanding of the clinical characteristics of this anomaly.

Interleukin-21 is a common γ-chain cytokine that controls the immune responses of B cells, T cells, and natural killer cells. Targeting IL-21 to strengthen the immune system is promising for the development of vaccines as well as anti-infection and anti-tumor therapies. However, the practical application of IL-21 is limited by the high production cost. In this study, we improved IL-21 production by codon optimization and selection of appropriate signal peptide in CHO-K1 cells. Codon-optimized or non-optimized human IL-21 was stably transfected into CHO-K1 cells. IL-21 expression was 10-fold higher for codon-optimized than non-optimized IL-21. We fused five different signal peptides to codon-optimized mature IL-21 and evaluated their effect on IL-21 production. The best result (a 3-fold increase) was obtained using a signal peptide derived from human azurocidin. Furthermore, codon-optimized IL-21 containing the azurocidin signal peptide promoted IFN-γ secretion and STAT3 phosphorylation in NK-92 cells similar to codon-optimized IL-21 containing original signal peptide. Collectively, these results indicate that codon optimization and azurocidin signal peptides provide an efficient approach for the high-level production of IL-21 as a biopharmaceutical.

The occipital condyle (OC) screw can be a viable alternative option for the occipito-cervical fixation. However, the risk of vertebral artery (VA) injury during the direct OC screw fixation has not been adequately assessed. The purpose of this study was to establish the course of the VA (V3) relative to the nearby osseous structures to estimate the feasibility of OC screw fixation and describe its anatomical relationship depending on patient’s age and sex. A total of 387 three-dimensional computed tomographic angiograms (3D-CTA) were used and compared between two age groups. The vertebral artery diameter and two kinds of bony space were measured. The occipito-C1 arch space (O-C1S) and VA-occipital bone distance (VOD, six entry points) were measured on both sides. The feasibility of direct OC screw fixation can be represented by the VOD value; the minimum feasible value was determined to be 4 mm. Angular measurements (O-C1A and O-C2A) were also taken to assess their relationship to the bony space. The mean value of the O-C1S ranged from 9.0 to 9.9 mm. The mean value of the VOD ranged from 3.2 to 3.5 mm, and the proportion of individuals for which direct OC screw fixation was considered feasible ranged from 32 to 42 % in both age groups and there was no significant difference between two age groups. The VOD value was not affected by laterality or by gender (P > 0.05). The mean kyphosis of the O-C1A (−5° ± 5.2°, range −22° to 8°) was significantly smaller in the young age group compared to the older age group (−7.6° ± 5.3°, range −26° to 2°). The mean lordosis of the O-C2A (12.4° ± 6.4°, range 3°–33°) was significantly higher in the young age group compared to the older age group (10.4° ± 6.4°, range 0°–36°). The direct OC screw fixation was not possible in a considerable number of cases due to the limited space and the position of the VA regardless of age group. Only about one quarter (21–24 %) of all patients was affordable to place the direct OC screw bilaterally. There was more space available to place the direct OC screw when the angle of the upper cervical spine is more kyphotic.

The cervical spine has a linear chain of correlation or reciprocal relationship regionally (within the cervical spine) and globally (head to whole spine). The purpose of this study was to assess the sequential alignment change of the regional and global cervical spine after two-level anterior cervical discectomy and fusion (ACDF) performed on the lower cervical spine. This study included 61 patients (mean age 56 ± 8.6 years; range 35–70 years) who underwent ACDF at C5-6-7 with a plate-cage construct and whose C-spine neutral lateral radiographs showed an identical degree of horizontal gaze (occipital slope) peri-operatively. We compared the change in cervical curvature from the occiput to C7 with the absolute value (slope angle) and relative value (between two different slopes). We also investigated the correlated change in multiple angular parameters according to the change in the occipital slope. The occipital slope was significantly correlated with the value of the C1-slope (r = 0.33) and C2- slope (r = 0.51). The value of the center of the sellar turcica-C7 sagittal vertical axis (St-SVA) was very closely related to the C1-slope (r = −0.83), C2-slope (r = −0.8), C2-7 angle (r = −0.43), and C2-5 angle (r = −0.46). The amount of angular change at the surgical level (C5-7A) was 5.8° (2.9° -> 8.5°), and the sum of the change in the C5-slope and C7-slope was 6° (3.1° + 2.9°). In general, the C2-5 angle decreased about 3°, in proportion to the upward inclination of C5-slope (3.1°), because the C2-slope was fixed. However, patients who showed improvement in cervical alignment (greatly increased C5-7 lordosis or greatly decreased St-SVA after surgery) often had upper cervical slope change (C1-s and C2-s). The ACDF procedure itself can induce regional slope change (C5-s and C7-s) directly at the surgical level and can also influence upper cervical slope change (C1-s and C2s) indirectly. Then the change in the upper cervical spine can induce a change in the St-CVA and spino-cranial angle (SCA).

To retrospectively analyse the synchondrosis from the occipital bone to the whole cervical spine and determine the feasibility and validity of age estimation using computed tomography (CT) images.A total of 231 cervical spine or neck CT images of young children (7 years of age) were examined. Twelve ossification centres were assessed (occiput: n = 2; atlas: n = 2; axis, n = 6; whole sub-axial vertebra: n = 2), and the ossification process was graded as open (O, fully lucent), osseous bridging (B, partially ossified), and fusion (F, totally ossified). After the first analysis was completed, the resulting chronological chart was used to estimate the age of 10 new cases in order to confirm the usefulness of the chart.Infancy was easily estimated using the sub-axial or C2 posterior ossification centres, while the posterior occipital regions provided good estimation of age between 1-2 years. The most difficult period for accurate age estimation was between 2-4 years. However, the C2 anterior (neurocentral ossification) and C1 posterior regions did yield information to help determine the age around 3 years. The anterior occipital region was useful for age estimation between 4-5 years, and the C1-anterior region was potentially useful to help decide among the other parameters. The test for age estimation (TAE) had a very high ICC score (0.973) among the three observers.Segmentalised analysis can enhance the ability to estimate real age, at least by the year. The analysis of the occipital bone made a strong contribution to the usefulness of the chorological chart. An organised chronological chart can provide readily available information for age estimation, and the primary application of the above data (TAE) demonstrated the validity of this approach.

Purpose To evaluate a radiological and clinical difference between the curvilinear type cages compared to the straight type cages for the restoration of lumbopelvic sagittal alignment and its maintenance after transforaminal lumbar interbody fusion (TLIF) procedure. Methods 68 patients who underwent single-level TLIF using either the straight type or curvilinear type cage were retrospectively reviewed. Assessment of the lumbopelvic parameters and the height of disc space was performed before surgery as well as 2 days, 6 and 12 months after surgery. Clinical outcome was assessed using VAS and ODI. Results The curvilinear type cages were positioned more anteriorly than the straight type. Restoration of the segmental lordosis (SL) in the curvilinear group was significantly greater than the straight group and at 12 months of follow-up, the straight group showed greater decrease in the disc height than the curvilinear group. The straight group failed to show improvement of lumbar lordosis (LL), while the curvilinear group showed significant restoration of LL and could maintain it to the 6 months of follow-up. In both groups, pelvic tilt was significantly decreased and it lasted to 6 months in the straight group; whereas in the curvilinear group, it was maintained to the last follow-up of 12 months. There were no significant differences between the two groups in mean VAS and ODI score over the follow-up period. Conclusions This study demonstrates that the curvilinear type cage is superior to the straight type cage in improving the SL and maintaining both the restored lumbopelvic parameters and elevated disc height. These results could be attributable to the anterior position of the curvilinear cage which permits easy restoration of segmental lordosis and less sinking of cages.

To describe the effect of the C1 bursting fracture on the location of the internal carotid artery (ICA) around the atlas. The authors analyzed the morphology of the atlas and the ICA in 15 patients with C1 bursting fracture and compared with control group (77 patients) without any pathology. All patients were evaluated with CT angiography for the anatomical assessment. The laterality of the ICA, the distances of the ICA from the midline, anterior tubercle, and ventral surface of the C1 lateral mass were compared between two groups. The distance between the lateral margin of the longus capitis muscle and the inner edge of the transverse foramen was also measured. Medially located ICA was more common in the C1 bursting fracture group than control group (76.7 vs 42.8 %). There were no significant differences between 2 groups for the distance from the midline, anterior tubercle, and ventral surface of the C1 lateral mass, respectively. The distance of the longus capitis muscle to transverse foramen was 2.52 ± 2.09 and 4.15 ± 3.09 mm in each group, and there was statistically significant difference (p

We studied the applicability of the Acute Physiology and Chronic Health Evaluation II (APACHE II) and Simplified Acute Physiology Score II (SAPS II) in patients admitted to the intensive care unit (ICU) with acute stroke and compared the results with the Glasgow Coma Scale (GCS) and National Institutes of Health Stroke Scale (NIHSS). We also conducted a comparative study of accuracy for predicting hemorrhagic and ischemic stroke mortality. Between January 2011 and December 2012, ischemic or hemorrhagic stroke patients admitted to the ICU were included in the study. APACHE II and SAPS II-predicted mortalities were compared using a calibration curve, the Hosmer-Lemeshow goodness-of-fit test, and the receiver operating characteristic (ROC) curve, and the results were compared with the GCS and NIHSS. Overall 498 patients were included in this study. The observed mortality was 26.3%, whereas APACHE II and SAPS II-predicted mortalities were 35.12% and 35.34%, respectively. The mean GCS and NIHSS scores were 9.43 and 21.63, respectively. The calibration curve was close to the line of perfect prediction. The ROC curve showed a slightly better prediction of mortality for APACHE II in hemorrhagic stroke patients and SAPS II in ischemic stroke patients. The GCS and NIHSS were inferior in predicting mortality in both patient groups. Although both the APACHE II and SAPS II systems can be used to measure performance in the neurosurgical ICU setting, the accuracy of APACHE II in hemorrhagic stroke patients and SAPS II in ischemic stroke patients was superior.

In patients with severe traumatic brain injury (TBI), maintaining systolic blood pressure90 mm Hg, intracranial pressure (ICP)20 mm Hg and cerebral perfusion pressure (CPP)60-70 mm Hg is recommended to improve clinical outcomes. A recommended CPP value for patients treated with decompressive craniectomy (DC) has not been clearly studied. We aimed to determine whether the targeted CPP can be lowered in patients treated with DC.This retrospective analysis included 191 patients who underwent DC for TBI. All patients were monitored for ICP and blood pressure during and after DC. CPP was calculated every 2 hours after DC. Patient outcomes were evaluated 6 months after initial treatment.Mean patient age was 50.8 years (median 52 years), and 79.1% of patients were male. Initial Glasgow Coma Scale score was 6.2 (median 6). Comparing clinical outcome based on postoperative ICP25 mm Hg and25 mm Hg, Extended Glasgow Outcome Scale score was 1.4 (25 mm Hg) and 4.9 (25 mm Hg) (P = 0.000). In patients maintained at ICP25 mm Hg, mortality was increased significantly with CPP between 35 mmHg and 30 mm Hg (χPatients with TBI who underwent DC with postoperative ICP maintained25 mm Hg and CPP35 mm Hg may have similar mortality as patients with CPP60-70 mm Hg who did not undergo DC. For patients with TBI who undergo DC, targeted CPP might be lowered to 35 mm Hg if ICP is maintained25 mm Hg.

Rho GDP dissociation inhibitors (RhoGDIs) play important roles in various cellular processes, including cell migration, adhesion, and proliferation, by regulating the functions of the Rho GTPase family. Dissociation of Rho GTPases from RhoGDIs is necessary for their spatiotemporal activation and is dynamically regulated by several mechanisms, such as phosphorylation, sumoylation, and protein interaction. The expression of RhoGDIs has changed in many human cancers and become associated with the malignant phenotype, including migration, invasion, metastasis, and resistance to anticancer agents. Here, we review how RhoGDIs control the function of Rho GTPases by regulating their spatiotemporal activity and describe the regulatory mechanisms of the dissociation of Rho GTPases from RhoGDIs. We also discuss the role of RhoGDIs in cancer progression and their potential uses for therapeutic intervention.

Previously, we reported that overexpression of Opa (Neisseria gonorrhoeae opacity-associated)-interacting protein 5 (OIP5) caused multi-septa formation and growth defects, both of which are considered cancer-related phenotypes. To evaluate OIP5 as a possible cancer therapeutic target, we examined its expression level in 66 colorectal cancer patients. OIP5 was upregulated about 3.7-fold in tumors and over 2-fold in 58 out of 66 colorectal cancer patients. Knockdown of OIP5 expression by small interfering RNA specific to OIP5 (siOIP5) resulted in growth inhibition of colorectal and gastric cancer cell lines. Growth inhibition of SNU638 by siOIP5 caused an increase in sub-G1 DNA content, as measured by flow cytometry, as well as an apoptotic gene expression profile. These results indicate that knockdown of OIP5 may induce apoptosis in cancer cells. Therefore, we suggest that OIP5 might be a potential cancer therapeutic target, although the mechanisms of OIP5-induced carcinogenesis should be elucidated.

Background The aim of this study is 2-fold: to analyze a clinical case series in which we used laminar screws for cervical posterior instrumentation and to describe the difference between C2 and C7 laminar screws in terms of technique and anatomy. Methods Data were obtained from 25 patients who underwent cervical posterior fixation with intralaminar screws at C2 or C7. C2 intralaminar screw instrumentation was used for 7 patients requiring occipitocervical fixation (basilar invagination [3 patients], C1 unstable bursting fracture [1 patient], C1-C2 instability with occipital assimilation [2 patients], and dystopic os odontoideum [1 patient]), 13 patients with C1-C2 instability, 1 patient with C2-C3 subluxation, and 4 patients undergoing C7 fixation due to pseudoarthrosis or cervical instability after trauma. A total of 34 laminar screws were placed including 1 thoracic laminar screw, and the patients were assessed both clinically and radiographically. Results There were no instances where a screw violated the spinal canal nor any hardware fractures noted during the follow-up period. As for perioperative complications, there were 2 cases of postoperative wound infection, 1 case of dural laceration during dissection, and 2 cases of partial dorsal laminar breach. However, there was no neurologic compromise in any of the cases. The fusion success rate was 100%. Conclusion These preliminary results support the use of intralaminar screws for posterior instrumentation at C2 and C7.

Background Cysteine proteases like cathepsins are widely distributed proteolytic enzymes and form tight equimolar complexes with cystatins at their active sites. Among cystatins, CST1, encoding cystatin SN, is a member of the type 2 salivary cystatin family found in a variety of fluids and secretions, including plasma, tears, and saliva. CST1 was identified as an upregulated gene in gastric cancer tissues compared to noncancerous regions using our Affymetrix GeneChip microarray. Methods The upregulation of CST1 in gastric cancer was analyzed using RT-PCR (n = 15), immnohistochemistry, and clinicopathological (n = 77) analysis. CST1-siRNA was used for the suppression of CST1 gene expression and cathepsin proteolytic activity was assayed. Results CST1 was upregulated in cancerous lesions of gastric cancer tissues compared to noncancerous regions and clinicopathological analysis showed a significant correlation between high expression of CST1 and pTNM stage (p = 0.044). In CST1-siRNA transfected cells, cell proliferation was reduced and the proteolytic activity of cathepsins was increased. Conclusions CST1 might be highly involved in gastric tumorigenesis and regulate the proteolytic activity of cysteine proteases.

NDRG (N-Myc downstream-regulated gene)-2 is a member of the NDRG family. Although it has been suggested that NDRG2 is involved in cellular differentiation and tumor suppression, its intracellular signal and regulatory mechanism are not well known. Here, we show the differential expression of NDRG2 in human colon carcinoma cell lines and tissues by reverse transcription-polymerase chain reaction and immunohistochemical analyses with monoclonal antibody against NDRG2. NDRG2 was strongly expressed in normal colonic mucosa and colonic adenomatous tissues (25 of 25) but not in all invasive cancer tissues [44 of 99 (44%)]. Most distinctive results indicated that the high expression level of NDRG2 has a positive correlation with tumor differentiation and inverse correlation with tumor invasion depth and Dukes' stage of colon adenocarcinoma. To investigate the roles of NDRG2 in tumorigenesis, we used in vitro cell culture system. SW620 colon cancer cell line with a low level of intrinsic NDRG2 protein was transfected with NDRG2-expressing plasmid. TOPflash luciferase reporter assay showed that the transcriptional activity of T-cell factor (TCF)/lymphoid enhancer factor (LEF) was reduced by NDRG2 introduction, but not by the introduction of mutant NDRG2 generated by deletion or site-directed mutagenesis. Intracellular beta-catenin levels were slightly reduced in the NDRG2-transfected SW620 cells and this regulation of beta-catenin stability and TCF/LEF activity were mediated through the modulation of glycogen synthase kinase-3beta activity by NDRG2 function. Our results suggest that NDRG2 might play a pivotal role as a potent tumor suppressor by the attenuation of TCF/beta-catenin signaling for the maintenance of healthy colon tissues.

Recently, the anti-tumor activity of N-myc downstream-regulated gene 2 (NDRG2) was elucidated, but the molecular mechanism of how NDRG2 works as a tumor suppressor is not well known. To determine the function of NDRG2 as a tumor suppressor, we established stable cell lines expressing NDRG2 protein or its mutant forms, and studied their effects on tumor cell growth. Interestingly, constitutive expression of wild-type NDRG2 induced the growth retardation of SW620 colon carcinoma cells. Introduction of NDRG2 into SW620 cells induced the decrease of c-Jun phosphorylation at Ser63, followed by the attenuation of activator protein-1 (AP-1) function as a transcriptional activator. Subsequently, the down-regulation of cyclin D1, which is known as a major target for AP-1 transcription activator, resulted in cell cycle arrest at G1/S phase. Additionally, treatment of NDRG2-siRNA on NDRG2-expressing cells has induced the recovery of c-Jun phosphorylation and cyclin D1 expression. Cell proliferation of those cells was also increased compared with untreated cells. NDRG2 mutants of which the phosphorylation sites at C-terminal region were removed by deletion or site-directed mutagenesis have shown no effect on cyclin D1 expression and could not induce cell growth retardation. In conclusion, NDRG2 modulates intracellular signals to control cell cycle through the regulation of cyclin D1 expression via phosphorylation pathway. © 2008 Wiley-Liss, Inc.

Aims The Wnt/β-catenin pathway plays a critical part in several cell physiology events associated with embryonic development and adult homeostasis, including determination, proliferation, migration, and differentiation. However, the role of Wnt signaling in osteoblastogenesis from mesenchymal stem cells (MSC) remains a controversial matter. Therefore, in the present study, we investigated how ICAT (inhibitor of β-catenin and TCF-4), a negative regulator of the Wnt signaling pathway, influenced differentiation and proliferation of human adipose tissue-derived stromal cells (hASC). Main methods To mediate ICAT overexpression in hASC, we used a lentiviral gene transfer technique. We further determined the role of ICAT by RNAi technique. Key findings ICAT-transduced hASC exhibited lower TCF promoter activity and cellular growth capacity than control cells, but ICAT overexpression did not affect hASC attachment efficiency. ICAT overexpression also increased osteogenic differentiation. Conversely, introduction of an ICAT siRNA oligonucleotide increased TCF promoter activity and cellular proliferation, but it inhibited osteogenic differentiation. Significance Taken together, these findings indicated that ICAT participated in regulating hASC proliferation and differentiation by modulating Wnt signaling.

Background Mac-2 binding protein (Mac-2BP) is a secreted glycoprotein from the culture fluid of several human cancer cells, especially breast, lung, and gastric cells. Mac-2BP plays a role in immune response and cell adhesion activity in patients with various cancer and infectious diseases. In this study, we attempted to identify the regulators of Mac-2BP expression at the transcriptional level. Methods To determine the effect of epidermal growth factor (EGF) to Mac-2BP expression in gastric cancers, we constructed the different lengths of Mac-2BP promoter plasmids and measured the promoter activity and Mac-2BP expression. In addition to investigating the role of signal transducer and activator of transcription3 (STAT3) or human telomerase reverse transcriptase (hTERT) as a regulator of Mac-2BP, we transfected the small interfering RNA (siRNA) specific for STAT3 or hTERT, and Mac-2BP level was observed by a quantitative ELISA. Results EGF treatment could suppress the Mac-2BP transcription in HEK293 or gastric cancer cell lines (SNU-638 or AGS). In 5'-deleted promoter experiment, pGL3-Mac Pro-2377 transfected cells showed a decreased luciferase activity compared to pGL3-Mac Pro-2277. We also identified that (-2,366/-2,356) on Mac-2BP promoter is a putative STAT3 binding site and suppression of STAT3 with STAT3 specific siRNA increased the Mac-2BP level, suggesting the role of STAT3 as a negative regulator, in contrast to hTERT, which is known as a positive regulator. Conclusions EGF signal is critical for the Mac-2BP expression, and more importantly, STAT3 could work as a negative regulator, while hTERT as a positive regulator in Mac-2BP transcription.

We reported previously that N-myc downstream-regulated gene 2 (NDRG2), a member of a new family of differentiation-related genes, is expressed specifically in dendritic cells (DC) differentiated from monocytes, CD34+ progenitor cells, and the myelomonocytic leukemic cell line. In this study, we demonstrate that NDRG2 protein expression is detected, not only in in vitro-differentiated DC but also in primary DC from lymph nodes, thymus, and skin when anti-NDRG2 antibodies are used. As predicted from previous studies investigating the mRNA expression pattern of several types of cell lines, progenitor cells, and DC, NDRG2 protein was expressed strongly in DC. Its expression was detected at significant levels after differentiation from progenitor cells. RNA interference of NDRG2 demonstrated that activated leukocyte cell adhesion molecule (ALCAM) expression is down-regulated specifically in DC differentiated from NDRG2 small interfering RNA (siRNA)-transfected monocytes. This was consistent with our observation that U937 cells transfected with NDRG2 became resistant to the GM-CSF/IL-4-induced ALCAM reduction. Furthermore, DC, which had differentiated from NDRG2 siRNA-transfected monocytes, showed a reduced ability to induce T cell proliferation. Taken together, our results indicate that NDRG2 is able to preserve ALCAM expression during DC differentiation from monocytes under cytokine culture conditions and that its expression helps DC maintain costimulatory signals necessary for T cell stimulation.

// Sang Yoon Park 1, 8 , Seon-Jin Lee 1, 2 , Hee Jun Cho 1 , Tae Woo Kim 1, 2 , Jong-Tae Kim 1 , Jae Wha Kim 3 , Chul-Ho Lee 4 , Bo-Yeon Kim 5 , Young Il Yeom 6 , Jong-Seok Lim 7 , Younghee Lee 8 , Hee Gu Lee 1, 2 1 Genome Structure Research Center, Korea Research Institute of Biosceience and Biotechnology, Daejeon, Korea 2 Department of Biomolecular Science, University of Science and Technology (UST), Daejeon, Korea 3 Biomedical Translational Research Center, Korea Research Institute of Biosceience and Biotechnology, Daejeon, Korea 4 Animal Resource Center, Korea Research Institute of Biosceience and Biotechnology, Daejeon, Korea 5 World Class Institute, Korea Research Institute of Bioscience and Biotechnology, Cheongju, Korea 6 Department of Functional Genomics, Korea Research Institute of Biosceience and Biotechnology, Daejeon, Korea 7 Department of Biological Sciences, and The Research Center for Women’s Diseases, Sookmyung Women’s University, Seoul, Korea 8 Department of Biochemistry, College of Natural Sciences, Chungbuk National University, Cheongju, Korea Correspondence to: Hee Gu Lee, e-mail: hglee@kribb.re.kr Younghee Lee, e-mail: yhl4177@chungbuk.ac.kr Keywords: colon cancer, invasion, metastasis, E-cadherin, dehydropeptidase 1 Received: October 20, 2015 Accepted: January 19, 2016 Published: January 27, 2016 ABSTRACT Dehydropeptidase 1 (DPEP1) is a zinc-dependent metalloproteinase that is expressed aberrantly in several cancers. The role of DPEP1 in cancer remain controversial. In this study, we demonstrate that DPEP1 functions as a positive regulator for colon cancer cell metastasis. The expression of DPEP1 mRNA and proteins were upregulated in colon cancer tissues compared to normal mucosa. Gain-of-function and loss-of-function approaches were used to examine the malignant phenotype of DPEP1-expressing or DPEP1-depleted cells. DPEP1 expression caused a significant increase in colon cancer cell adhesion and invasion in vitro , and metastasis in vivo . In contrast, DPEP1 depletion induced opposite effects. Furthermore, cilastatin, a DPEP1 inhibitor, suppressed the invasion and metastasis of DPEP1-expressing cells. DPEP1 inhibited the leukotriene D4 signaling pathway and increased the expression of E-cadherin. We also show that DPEP1 mediates TGF-β-induced EMT. TGF-β transcriptionally repressed DPEP1 expression. TGF-β treatment decreased E-cadherin expression and promoted cell invasion in DPEP1-expressing colon cancer cell lines, whereas it did not affect these parameters in DPEP1-depleted cell lines. These results suggest that DPEP1 promotes cancer metastasis by regulating E-cadherin plasticity and that it might be a potential therapeutic target for preventing the progression of colon cancer.

The prenylated Rab acceptor 1 (PRA1) is a ubiquitously expressed 21 kDa protein containing two transmembrane domains that possibly induce its localization to the Golgi complex. It binds to prenylated Rab GTPases and VAMP2. In this study, we report that PRA1-overexpressing cells exhibited a significantly retarded growth rate as compared to that of the mock-transfected cells, and the transcriptional activity of TCF, as evaluated by TOPflash luciferase reporter assay, was profoundly reduced in the PRA1-overexpressed cells. These intracellular functions of PRA1 were verified by introducing deletion mutant or site-directed mutants, or small interfering RNA of PRA1. In addition, the translocation of beta-catenin from the cytosol to the nucleus was blocked to a significant degree in the PRA1-cells, and the interaction of PRA1 and beta-catenin was identified by confocal microscopy and immunoprecipitation analysis. Finally, we observed that the inhibition of TCF/beta-catenin signaling by PRA1 is associated with ERK1/2 dephosphorylation. Therefore, our data suggest that the in vivo modulation of PRA1 may be involved in TCF/beta-catenin signaling, as well as cellular proliferation and tumorigenesis.

We searched for genes with expressions specific to human monocyte-derived dendritic cells (DCs) using differential display reverse transcription-polymerase chain reaction, and found that N-myc downstream regulated gene 2 (NDRG2), a member of a new family of differentiation-related genes, was expressed in DCs. While DCs derived from CD34+ progenitor cells also showed strong NDRG2 expression, the corresponding mRNA expression was absent in other cell lines including monocytes, B cells, and NK cells. The inhibition of DC differentiation by dexamethasone or vitamin D3 treatment down-regulated the expression of the NDRG2 gene in DCs. In addition, gene expression was induced in a myelomonocytic leukemia cell line, which is capable of differentiating into DCs in cytokine-conditioned culture. The level of NDRG2 gene expression in DCs was significantly higher than that of other members of the NDRG gene family. Finally, in contrast to the stable NDRG2 expression in CD40-stimulated DCs, the induction of DC maturation by lipopolysaccharide (LPS) resulted in the down-regulation of NDRG2 gene expression. This down-regulation is likely to be due to a modification and subsequent destabilization of NDRG2 mRNA, because co-treating with actinomycin D and LPS significantly blocked this LPS effect. Taken together, our results indicate that NDRG2 is expressed during the differentiation of DCs, and that NDRG2 gene expression is differentially regulated by maturation-inducing stimuli.

Cystatin SN (CST1) is a specific inhibitor belonging to the cystatin superfamily that controls the proteolytic activities of cysteine proteases such as cathepsins. Our previous study showed that high CST1 expression enhances tumor metastasis and invasiveness in colorectal cancer. Recently, auranofin (AF), a gold(I)-containing thioredoxin reductase 1 (TrxR1) inhibitor, has been used clinically to treat rheumatoid arthritis. AF is a proteasome-associated deubiquitinase inhibitor and can act as an anti-tumor agent. In this study, we investigated whether CST1 expression induces autophagy and tumor cell survival. We also investigated the therapeutic effects of AF as an anti-tumor agent in colorectal cancer (CRC) cells. We found that CRC cells expressing high levels of CST1 undergo increased autophagy and exhibit chemotherapeutic resistance to AF-induced cell death, while those expressing low levels of CST1 are sensitive to AF. We also observed that knockdown of CST1 in high-CST1 CRC cells using CST1-specific small interfering RNAs attenuated autophagic activation and restored AF-induced cell mortality. Conversely, the overexpression of CST1 increased autophagy and viability in cells expressing low levels of CST1. Interestingly, high expression of CST1 attenuates AF-induced cell death by inhibiting intracellular reactive oxygen species (ROS) generation, as demonstrated by the fact that the blockage of ROS production reversed AF-induced cell death in CRC cells. In addition, upregulation of CST1 expression increased cellular glutathione reductase (GR) activity, reducing the cellular redox state and inducing autophagy in AF-treated CRC cells. These results suggest that high CST1 expression may be involved in autophagic induction and protects from AF-induced cell death by inhibition of ROS generation through the regulation of GR activity.

Congestive heart failure is mostly resulted in a consequence of the limited myocardial regeneration capacity after acute myocardial infarction. Targeted delivery of proangiogenic factors and/or stem cells to the ischemic myocardium is a promising strategy for enhancing their local and sustained therapeutic effects. Herein, we designed an epicardial delivery system of vascular endothelial growth factor (VEGF) and cardiac stem cells (CSCs) using poly(l-lactic acid) (PLLA) mat applied to the acutely infarcted myocardium. The fibrous VEGF-loaded PLLA mat was fabricated by an electrospinning method using PLLA solution emulsified VEGF. This mat not only allowed for sustained release of VEGF for 4weeks but boosted migration and proliferation of both endothelial cells and CSCs in vitro. Furthermore, sustained release of VEGF showed a positive effect on in vitro capillary-like network formation of endothelial cells compared with bolus treatment of VEGF. PLLA mat provided a permissive 3-dimensional (3D) substratum that led to spontaneous cardiomyogenic differentiation of CSCs in vitro. Notably, sustained stimulation by VEGF-loaded PLLA mat resulted in a substantial increase in the expression of proangiogenic mRNAs of CSCs in vitro. The epicardially implanted VEGF-loaded PLLA mat showed modest effects on angiogenesis and cardiomyogenesis in the acutely infarcted hearts. However, co-implantation of VEGF and CSCs using the PLLA mat showed meaningful therapeutic effects on angiogenesis and cardiomyogenesis compared with controls, leading to reduced cardiac remodeling and enhanced global cardiac function. Collectively, the PLLA mat allowed a smart cargo that enabled the sustained release of VEGF and the delivery of CSCs, thereby synergistically inducing angiogenesis and cardiomyogenesis in acute myocardial infarction.

Tescalcin (TESC) is an EF-hand calcium binding protein that is differentially expressed in several tissues, however it is not reported that the expression and functional roles of TESC in colorectal cancer. Levels of messenger RNA (mRNA) and protein expression of TESC in colorectal cancer tissues were assessed using RT-PCR, real time PCR, immunohistochemistry, and clinicopathologic analyses. Quantitative analysis of TESC levels in serum specimens was performed using sandwich ELISA. Colorectal cancer cells transfected with TESC small interfering RNA and short hairpin RNA were examined in cell proliferation assays, phospho-MAPK array, and mouse xenograft models. Here we demonstrated that TESC is overexpressed in colorectal cancer (CRC), but was not expressed in normal mucosa and premalignant dysplastic lesions. Furthermore, serum TESC levels were elevated in patients with CRC. Knockdown of TESC inhibited the Akt-dependent NF-κB pathway and decreased cell survival in vitro. Depletion of TESC reduced tumor growth in a CRC xenograft model. Thus, TESC is a potential diagnostic marker and oncotarget in colorectal cancer.

// Hee Cheol Kim 3,* , Yong Sung Kim 1,* , Hyun-Woo Oh 1 , Kwoneel Kim 1 , Sang-Seok Oh 2 , Jong-Tae Kim 1 , Bo Yeon Kim 1 , Seon-Jin Lee 1 , Yong-Kyung Choe 1 , Dong Hyeok Kim 2 , Seok-Hyung Kim 4 , Seoung Wan Chae 5 , Kwang Dong Kim 2 , and Hee Gu Lee 1 1 Biomedical Genomics Research Center, Korea Research Institute of Bioscience and Biotechnology, Daejeon, South Korea; 2 Division of Applied Life Science, PMBBRC, Gyeongsang National University, Jinju, South Korea; 3 Department of Surgery, Samsung Medical Center, Sungkyunkwan University School of Medicine, Seoul, South Korea; 4 Department of Pathology, Samsung Medical Center, Sungkyunkwan University School of Medicine, Seoul, South Korea; 5 Department of Pathology, Kangbuk Samsung Hospital, Sungkyunkwan University School of Medicine, Seoul, South Korea * These authors equally contributed to this study. Correspondence: Hee Gu Lee, email: // Kwang Dong Kim, email: // Keywords : CTHRC1, ERK, MMP9, Invasion, Colorectal cancer Received : December 18, 2013 Accepted : January 18, 2014 Published : January 18, 2014 Abstract Collagen triple helix repeat-containing 1 (CTHRC1) is known to be aberrantly upregulated in most human solid tumors, although the functional roles of CTHRC1 in colorectal cancer remain unclear. In this study, we investigated the occurrence of CTHRC1 upregulation and its role in vivo and in vitro. The expression profile and clinical importance of CTHRC1 were examined by reverse transcription-polymerase chain reaction and immunohistochemical analyses in normal and tumor patient samples. CTHRC1 was detectable in normal tissues, but also was highly expressed in tumor specimens. CTHRC1 upregulation was significantly associated with demethylation of the CTHRC1 promoter in colon cancer cell lines and tumor tissues. Clinicopathologic analyses showed that nodal status and expression of CTHRC1 (95% CI 0.999-3.984, p=0.05) were significant prognostic factors for disease-free survival. Promoter CpG methylation and hypermethylation status were measured by bisulfite sequencing and pyrosequencing analysis. Furthermore, we showed that overexpression of CTHRC1 in the SW480 and HT-29 cell lines increased invasiveness, an effect mediated by extracellular signal-regulated kinase (ERK)-dependent upregulation of matrix metalloproteinase 9 (MMP9). Consistent with this, we found that knockdown of CTHRC1 attenuated ERK activation and cancer cell invasivity. These results demonstrate that CTHRC1 expression is elevated in human colon cancer cell lines and clinical specimens, and promotes cancer cell invasivity through ERK-dependent induction of MMP9 expression. Our results further suggest that high levels of CTHRC1 expression are associated with poor clinical outcomes.

NDRG2 is a member of the N-myc downstream regulated gene (NDRG) family, implicated in cell growth and differentiation. Investigation of NDRG2 molecular interactions by yeast two-hybrid screening identified prenylated Rab acceptor-1 (PRA1), involved in vesicle trafficking and protein transport, as binding partner. Binding of NDRG2 (and NDRG1-4) with PRA1 in vitro was confirmed by GST pull-down assay and immunoprecipitation, and colocalization was verified by confocal microscopy in HCT116 cells. Intracellular coexpression showed that NDRG2 and PRA1 synergistically downregulate T-cell factor (TCF) promoter activity and GSK3β phosphorylation. Results suggest that NDRG2 and PRA1 might act synergistically to prevent signaling of TCF/β-catenin.Structured summary of protein interactionsNDRG2a binds to PRA1 by pull down (View interaction)NDRG2a physically interacts with PRA1 by two hybrid (View Interaction: 1, 2)PRA1 physically interacts with NDRG2a by anti tag coimmunoprecipitation (View interaction)NDRG2a, PRA1 and Catenin beta colocalize by cosedimentation (View interaction)NDRG2a and PRA1 colocalize by fluorescence microscopy (View Interaction: 1, 2, 3)

Carcinoembryonic antigen-related cell adhesion molecule 6 (CEACAM6) inhibits anoikis and affects the malignant phenotype of cancer cells. In this study, we analyzed CEACAM6 as a gene that is highly upregulated in colon cancer tissues, and examined the assertion that CEACAM6 might be a suitable candidate tumor marker for the diagnosis of colon cancer.CEACAM6 gene expression in human colon tissues was performed by tissue microarray and analyzed using RT-PCR (each of normal and tumor tissue, n=40) and immunohistochemical and clinicopathological (colon cancer patients, n=143) analyses.CEACAM6 transcriptional and translational levels were significantly upregulated in human tumor tissues compared to non-tumor regions, and clinicopathological analysis revealed a significant correlation between CEACAM6 protein expression and Dukes' stage (p0.001). High expression levels of CEACAM6 were significantly associated with lower overall survival (p0.001) and shorter recurrence-free survival (p0.001). We demonstrated that knockdown of CEACAM6 with CEACAM6-specific small interfering RNA in colorectal cancer cells attenuated invasivity (35%); conversely, the overexpression of CEACAM6 increased invasiveness.CEACAM6 is significantly upregulated in colon cancer tissues and is closely associated with poor prognosis, indicating that CEACAM6 might be used as a tumor biomarker and a potential therapeutic target for colon cancer.

There has been no specific treatment for ischemic colitis. We verified the effects of adipose-derived stem cells (ASCs) on ischemia-induced colitis in a rat model. Forty male Sprague–Dawley rats (10 weeks old; weight, 350 ± 20 g) were divided into two groups: a control group (only fibrinogen and thrombin injected, n = 20) and an ASC group (local implantation of ASCs mixed with thrombin and fibrinogen, n = 20). An ischemic colitis model was established by modifying Nagahata's methods with double-blind randomization. ASCs (1 × 106 cells) were implanted intramurally into the ischemic area using a fibrin glue mixture. The severity of adhesion, degree of ileus, the number and size of the ulcers, Wallace macroscopic and microscopic scores, and microvascular density were measured. The degree of ileus was significantly lower, and significantly fewer and smaller ulcerations were found in the ASC group than those in the control group. Wallace macroscopic and microscopic scores were lower in the ASC group than in the control group (1.90 ± 1.22 versus 3.25 ± 1.83, p

Mesenchymal stem cells (MSCs) have been discovered in a multitude of organs, but their distribution and identity are still uncertain. Furthermore, loose connective tissue (LCT) is dispersed throughout virtually all organs, but its biological role in tissue homeostasis is unclear. Here, we describe a unique organ culture system to explore the omnipresence and in situ identity of MSCs among the LCTs. This culture system included the use of the fibrin hydrogel coupled with dynamic culture conditions, using native LCTs obtained from various organs as starting materials. This culture allowed MSC outgrowth into the hydrogel to be robustly supported, while maintaining the structural integrity of LCTs during in vitro culture. Subcultured outgrown cells fulfilled the minimal requirements for defining MSCs on the basis of clonogenicity, multipotency, and immunophenotypic characteristics. In vitro label-retaining assay demonstrated that the numbers of mobilized and proliferated cells in situ increased in the pericapillary region and expressed both MSCs and pericytes markers, indicating that the in situ identity of MSCs represents a certain population of pericapillary pericytes. Our results indicate that this culture system affords a unique strategy for both isolating MSCs and recapitulating their niche in LCTs.

Abnormal angiogenesis plays a critical role in the pathogenesis of various diseases such as cancer and chronic inflammation. A variety of pro-angiogenic factors, including vascular endothelial growth factor (VEGF), exert their action through endothelial receptor tyrosine kinases (RTKs). The synthetic phenylpropenone derivatives, used in this study were the following: 1,3-diphenyl-propenone (DPhP), 3-phenyl-1-thiophen-2-yl-propenone (PhT2P), 3-phenyl-1-thiophen-3-yl-propenone (PhT3P) and 1-furan-2-yl-3-phenyl-propenone (FPhP). These derivatives were screened for their inhibitory effect on VEGF-induced angiogenesis in vitro using HUVECs and in vivo using chick chorioallantoic membrane (CAM). The order of anti-angiogenic activity was DPhP>FPhP>PhT3P>PhT2P. The most effective compound DPhP, also known as chalcone, showed weak VEGF receptor tyrosine kinase activity compared with the specific inhibitor, SU4312 (3-[[4-(dimethylamino)phenyl]methylene]-1,3-dihydro-2H-indol-2-one). However, DPhP also inhibited several other receptor tyrosine kinases including Tie-2, epithermal growth factor (EGF) receptor, EphB2, fibroblast growth factor (FGF) receptor 3 and insulin-like growth factor-1 (IGF-1) receptor, as revealed by a receptor tyrosine kinase array assay. In addition, the down-stream signaling, including ERK phosphorylation and NF-κB activation, after receptor activation was significantly inhibited by DPhP. Furthermore, in the HT29 human colon cancer cell-inoculated CAM assay, the tumor growth and tumor-induced angiogenesis was significantly inhibited by DPhP (10μg/ml). These results suggest that the simple flavonoid, DPhP (chalcone), has valuable potential as an antiangiogenic and anti-cancer agent, and its action is mediated through the inhibition of multi-target RTKs including VEGF receptor 2.

KLK6 encoding kallikrein-related peptidase 6, a trypsin-like serine protease, has been shown to be upregulated in several cancers, although the tumorigenic role of KLK6 has not been elucidated. In this study, KLK6 was identified as a highly upregulated gene in gastric cancer; therefore, the possibility that KLK6 might be a suitable candidate tumor marker was examined. RT-PCR and immunohistochemical analysis showed overexpression of KLK6 in gastric cancer tissues compared to nontumor regions. Sera from gastric cancer patients had a 1.7-fold increase in KLK6 (373.1 μg/L, P = 0.048) compared to healthy individuals (214.2 μg/L), although there was no significant difference among patients with various tumor stages. Cellular invasiveness decreased by 45% in cells transfected with KLK6-specific small interfering RNA. Exogenous overexpression of KLK6 led to decreased activity of the E-cadherin promoter. This study shows that KLK6 is significantly upregulated and secreted in gastric cancer tissues and sera, suggesting that KLK6 might be used as a potential biomarker and therapeutic target for gastric cancer.

Conventional systems for isolating adipose-derived stem cells (ASC) require enzymatic digestion of adipose tissue (AT), followed by monolayer culture to the enrich the stem cell population. However, these systems are hindered by low cell yields and a lack of reproducibility. The present study was aimed at developing a unique strategy for isolating ASC based on fibrin matrix-supported three-dimensional (3-D) organ culture of native AT. Furthermore, we tried to optimize the fibrin composition by adjusting the fibrinogen and thrombin concentrations to allow rapid outgrowth and proliferation of ASC in the 3-D fibrin matrix. Human cutaneous AT fragments were encapsulated within the fibrin matrix to construct a 3-D environment and cultured under dynamic conditions. During in vitro culture the fibrin matrix provided physical support for the AT and also allowed selective outgrowth of ASC from embedded AT fragments. In situ expanded outgrown cells were recovered from the fibrin matrix by selective fibrinolysis and propagated under monolayer culture conditions. The cultured cells fulfilled the following criteria for ASC: adhesion to culture plastic, multipotent differentiation, correct immunophenotypic profile. Fibrin matrix-supported 3-D organ culture produced ASC that with high competency in terms of growth and differentiation capabilities, and resulted in a larger and more consistent cell yield than obtained with conventional culture systems. The fibrinogen and thrombin concentrations inversely affected spreading, migration, and ASC outgrowth from native AT. Our results indicate that this 3-D organ culture system for AT can be used as an efficient and reproducible method for ASC isolation.

Kallikrein-related peptidase 6 (KLK6) encodes a trypsin-like serine protease that is up-regulated in several cancers, although the putative functions of KLK6 in cancer have not been elucidated. In the current study, overexpression of KLK6 was identified in colon cancer, and the possibility that KLK6 may be a suitable candidate as a tumor marker was examined.Messenger RNA (mRNA) transcript levels and protein up-regulation of KLK6 in colon cancer tissues was examined using reverse transcriptase-polymerase chain reaction, immunohistochemistry, and clinicopathologic analyses. Cell proliferation, invasiveness, and antiapoptotic activity were determined in colon cancer cells that were transfected with small-interfering RNA (siRNA) of KLK6.KLK6 mRNA was up-regulated significantly in tumor tissues compared with nontumor regions. KLK6 protein was strongly expressed in adenocarcinomas but was not expressed in normal mucosa or in premalignant dysplastic lesions. Sera from patients with colon cancer revealed an increase in KLK6 secretion (0.25 μg/mL; P = .031) compared with noncancer cells (0.19 μg/mL). Clinicopathologic and immunohistochemical studies of 143 patients with colon cancer revealed a significant correlation between KLK6 expression and Dukes disease stage (P = .005). High KLK6 expression was associated significantly with shorter overall (P = .001) and recurrence-free survival (P = .001). The rates of proliferation and invasiveness were decreased by 50% in cells that were transfected with KLK6 siRNA. The overexpression of KLK6 led to decreased activity of the E-cadherin promoter.KLK6 was up-regulated significantly in tissues and sera from patients with colon cancer and was associated closely with a poor prognosis, suggesting that KLK6 may be used as a potential biomarker and a therapeutic target for colon cancer.

N-myc downstream-regulated gene 2 (NDRG2) implicated in cellular growth and differentiation was previously reported as it is specifically expressed in primary and in vitro-differentiated dendritic cells (DCs) from monocytes and CD34+ progenitor cells. However, its function has yet to be investigated in DCs. Here, the novel NDRG2 function about modulation of cytokines in DC was observed in this study. The secretion of IL-10 was not found in the monocyte-derived DC cells with high level of NDRG2 expression, but IL-10 was abundantly secreted up to 1 ng/ml in the monocyte-derived macrophages with low level of NDRG2 expression, and further confirmed that the expression of IL-10 was dramatically increased in NDRG2-silenced DCs under presence of LPS, and significantly reduced in the NDRG2-overexpressed U937 cells under stimulation of PMA. The secretion of IL-12p70 was significantly reduced in the siNDRG2 introduced DC cells. The intracellular signaling of IL-10 secretion was markedly inhibited by SB203580, inhibitor of p38 MAPK, in the LPS-activated DCs and phosphorylation of p38 MAPK was decreased in the NDRG2 introduced U937 cells under PMA-stimulation. Taken together, NDRG2 might have a pivotal role as one of intrinsic factors for the modulation of p38 MAPK phosphorylation, and subsequently involve in controlling of IL-10 production.

Apoptosis signal-regulating kinase 1 (ASK1), a member of the MAP kinase kinase kinase, is activated by several death stimuli and is tightly regulated by several mechanisms such as interactions with regulatory proteins and post-translational modifications. Here, we report that dual-specificity phosphatase 13A (DUSP13A) functions as a novel regulator of ASK1. DUSP13A interacts with the N-terminal domain of ASK1 and induces ASK1-mediated apoptosis through the activation of caspase-3. DUSP13A enhances ASK1 kinase activity and thus its downstream factors. Small interfering RNA (siRNA) analyses show that knock-down of DUSP13A in human neuroblastoma SK-N-SH cells reduces ASK1 kinase activity. The phosphatase activity of DUSP13A is not required for the regulation of ASK1. This regulatory action of DSUP13 on ASK1 activity involves competition with Akt1, a negative regulator of ASK1, for binding to ASK1. Taken together, this study provides novel insights into the role of DUSP13A in the precise regulation of ASK1.

The proangiogenic action of human adipose tissue-derived mesenchymal stem cells (hASCs) transplantation has been shown to be mediated by secretory factors. In this study, we determined if human granulocyte chemotactic protein-2(GCP2) or monocyte chemoattractant protein-1(MCP1) is involved in the proangiogenic action of hASCs transplantation in the hindlimb ischemia model. hASCs secrete GCP2 and MCP1, which leads to increased tubule formation. The downregulation of GCP2 or MCP1 decreased MCP1 and GCP2 secretion, respectively, whereas the external addition of GCP2 or MCP1 increased MCP1 and GCP2, respectively. Additionally, the treatment of GCP2 and MCP1 increased VEGF secretion, while the downregulation of GCP2 and MCP1 showed the opposite effect on VEGF secretion. Downregulation of GCP2 and MCP1 expression also inhibited hASCs-induced proangiogenic action, while the overexpression of GCP2 increased it. Finally, the downregulation of MCP1 or VEGF inhibited the GCP2 overexpression-induced increase in blood flow recovery. Taken together, these data indicate that the proangiogenic action of hASCs transplantation is mediated by the interaction between GCP2, MCP1 and VEGF, which are secreted from the transplanted cells.

Mac-2BP is a ligand of the galectin family that has been suggested to affect tumor proliferation and metastasis formation. We as- sessed Mac-2BP expression at the transcriptional and transla- tional levels to evaluate nerve growth factor (NGF)-induced Mac-2BP expression. A time kinetic analysis using reverse tran- scription-polymerase chain reaction showed that NGF-induced Mac-2BP transcript levels were 4-5 times higher than in controls. Mac-2BP enzyme-linked immunosorbent assay and immuno- fluorescence staining showed a 2-3-fold increase in intracellular and secreted Mac-2BP as a result of NGF stimulation. This in- crease was regulated by Akt activation and NF-κB binding. p65 and p50-NF-κB are major transcriptional factors in the Mac-2BP promoter region, and were shown to be regulated in accordance with the Akt activation states. Collectively, these results suggest that NGF induces Mac-2BP expression via the PI3K/Akt/NF-κB pathway. (BMB reports 2008; 41(11): 784-789)

Background Macrophage inhibitory cytokine-1 (MIC-1), a distant member of the transforming growth factor (TGF)-β superfamily, has been reported to be upregulated and secreted from several cancers. We examined MIC-1 expression and secretion in gastric cancers. Methods MIC-1 mRNA and protein levels in cancer tissues and cell lines were analyzed by RT-PCR and Western blot. MIC-1 expression in cancer tissues and its secretion in serum were analyzed using immunohistochemistry and ELISA. Results MIC-1 was significantly upregulated in gastric cancer tissues and cell lines. MIC-1 was secreted from gastric SNU620 cells and its levels in the serum of cancer patients were 10-fold higher than those of healthy controls. In addition, the staining of MIC-1 expression was strongly increased in metastatic gastric cancers. Conclusions MIC-1 was obviously overexpressed in gastric cancers and MIC-1 secretion into blood may be useful for the prediction of gastric cancer progression.