ARD1 stabilizes NRF2 through direct interaction and promotes colon cancer progression.

AIMS: Aberrant overactivation/overexpression of NRF2 is implicated as a driving event in tumor progression, which has been attributed to its mutation or inactivation of the inhibitory protein, KEAP1. However, alternative mechanisms responsible for sustained activation of NRF2 are less understood. MAIN METHODS: Human colon cancer cell lines and tissues obtained from colorectal cancer (CRC) patients were used. To examine the expression levels of ARD1 and NRF2, Western blot and immunofluorescence analyses were performed. To investigate the potential relevance of NRF2 and ARD1 to human CRC, NRF2 and ARD1 were individually silenced in human colon cancer cells (HCT-116) by transfection with their specific small interfering RNA (siRNA). To determine the functional role of ARD1 in NRF2 regulation, in situ proximate ligation, co-immunoprecipitation, nano-LC-ESI MS/MS, and in vitro acetylation assays were performed. KEY FINDINGS: ARD1 knockdown in human colon cancer cell lines significantly reduced the protein levels of NRF2 without affecting its mRNA expression; however, silencing of NRF2 did not alter ARD1 protein expression. In addition, these two proteins were co-localized and physically interacted with each other both in human colon cancer cells (HCT-116) and human colon tumor tissues. Mechanistically, ARD1 overexpression increased the acetylation levels of NRF2. Moreover, an in vitro acetylation assay and mass spectrometric analysis demonstrated that ARD1 could directly acetylate NRF2. Ectopic expression of mutant forms of ARD1 with defective acetyltransferase activity reduced the stability of NRF2. SIGNIFICANCE: In conclusion, ARD1 may potentiate the oncogenic function of NRF2 in human colon cancer by stabilizing this transcription factor.

Discriminating Potential Genetic Markers for Complete Response and Non-Complete Response Patients to Neoadjuvant Chemotherapy with Locally Advanced Rectal Cancer.

Background: Neoadjuvant chemoradiotherapy (nCRT) prior to surgery is considered standard therapy for locally advanced rectal cancer. Unfortunately, most patients with rectal cancer are resistant to radiotherapy. This might be a genetic cause. The role of certain rectal cancer-causing genes has not been completely elucidated. This study aims to investigate the genes responsible for locally advanced rectal cancer patients not reacting to radiotherapy. Methods: Whole exome sequencing of the DNA samples was performed on the samples. Bioinformatic analysis on the subjects was established. Individual genetic information was screened to identify differently expressed genes that more frequently appeared in non-complete response (NCR) compared to complete response (CR) patients after nCRT. All variations were verified by Sanger sequencing. Results: Genotyping information and pathway analyses of the samples indicated genes such as FLCN, CALML5, and ANTXR1 to be commonly mutated in CR group, whereas genes such as GALNTL14, CNKSR1, ACD, and CUL3 were more commonly mutated in the NCR group. Chi-square test revealed some significant variants (<0.05) such as rs3744124 (FLCN), rs28365986 (ANTXR1), rs10904516 (CALML5), rs3738952 (CUL3), rs13394 and rs2293013 (PIH1D1), rs2274531 (GPA33), rs4963048 (BRSK2), rs17883366 (IL3RA), rs2297575 (PSMD5), rs2288101 (GALNT14), and rs11954652 (DCTN4). Conclusion: Identifying an array of genes that separate NCRs from CRs would lead to finding genetic biomarkers for early detection of rectal cancer patients that are resistant to nCRT. A further investigation to validate the significance of genetic biomarkers to segregate NCRs from CRs should be performed with a larger CRC dataset. Protein expression levels, as well as transcriptomic analysis, would also help us understand the mechanism of how these genes could play a role in preventing radiation therapy to patients. This would be essential to prevent redundant radiation therapy.

Examining the Effectiveness of the Discharge Plan Model on the South Korean Patients with Cancer Completed Cancer Treatment and Are Returning to the Community: A Pilot Study.

This study aimed to examine the effectiveness of a discharge plan model for South Korean patients with cancer who had completed treatment and were returning to the community. Overall, 23 patients with cancer were recruited at the National Cancer Center in Goyang-si. The effectiveness of the discharge plan was examined using four methods: Social Needs Screening Toolkit (2018), early screening for discharge plan, current life situation v.2.0, and a questionnaire regarding problems after discharge from the hospital. Subsequently, the results were analyzed using descriptive statistical analysis methods with the Stata 14.0 program. The largest age group of study participants was between 45 and 64 years. No participants responded to urgent needs, whereas nine (39.13%) participants needed support for their social needs. According to the in-depth evaluation of participants, more than 80% of the respondents answered that patients with cancer needed no help in self-management, daily living activities, or mental health. The satisfaction survey results showed that the degree to which the "discharge plan" was helpful for health management at home after discharge was 4.41 of 5, and the degree to which it helped return to daily life was 3.86 of 5.

JNK-mediated Ser27 phosphorylation and stabilization of SIRT1 promote growth and progression of colon cancer through deacetylation-dependent activation of Snail.

Sirtuin 1 (SIRT1), an NAD+ -dependent histone/protein deacetylase, has multifaceted functions in various biological events such as inflammation, aging, and energy metabolism. The role of SIRT1 in carcinogenesis, however, is still under debate. Recent studies have indicated that aberrant overexpression of SIRT1 is correlated with metastasis and poor prognosis in several types of malignancy, including colorectal cancer. In the present study, we found that both SIRT1 and SIRT1 phosphorylated on serine 27 were coordinately upregulated in colon cancer patients' tissues and human colon cancer cell lines. This prompted us to investigate a role of phospho-SIRT1 in the context of colon cancer progression. A phosphorylation-defective mutant form of SIRT1, in which serine 27 was substituted by alanine (SIRT1-S27A), exhibited lower protein stability compared to that of wild-type SIRT1. Notably, human colon cancer (HCT-116) cells harboring the SIRT1-S27A mutation showed decreased cell proliferation and reduced capability to form xenograft tumor in athymic nude mice, which was accompanied by diminished transcriptional activity of Snail. HCT-116 cells carrying SIRT1-S27A were less capable of deacetylating the Snail protein, with a concomitant decrease in the levels of interleukin (IL)-6 and IL-8 mRNA transcripts. Taken together, these observations suggest that SIRT1 stabilized through phosphorylation on serine 27 exerts oncogenic effects at least partly through deacetylation-dependent activation of Snail and subsequent transcription of IL-6 and IL-8 in human colon cancer cells.

Effect of mistletoe extract on tumor response in neoadjuvant chemoradiotherapy for rectal cancer: a cohort study.

BACKGROUND: Mistletoe extract, used as a complementary chemotherapeutic agent for cancer patients, has anticancer effects against various malignancies. The aim of the present study was to evaluate the effect of mistletoe extract (Abnoba Viscum Q®) on tumor responses to neoadjuvant chemoradiotherapy (NCRT) for locally advanced rectal cancer. METHODS: This study included patients with rectal cancer who underwent NCRT between January 2018 and July 2020. In the mistletoe group (MG), the patients were administered Abnoba Viscum Q® subcutaneously during chemoradiotherapy-maintained just before surgery. Patient demographics, clinical outcomes, histopathological outcomes, and terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick-end labeling (TUNEL) assay results were compared between the MG and non-mistletoe group (NMG). Two rectal cancer cell lines (SNU-503 and SNU-503R80Gy) were treated with Abnoba Viscum Q® to assess its mechanistic effects in vivo. RESULTS: Overall, the study included 52 patients (MG: n = 15; NMG: n = 37). Baseline demographics between the two groups were similar, except carbohydrate antigen 19-9 levels and tumor location from the anal verge. There was no difference in the clinical stage between the two groups. A better tumor response in the MG, relative to the NMG, was observed with respect to tumor regression grade (TRG), T stage, and overall tumor-node-metastasis stage. Tumor response was significantly better in the MG than in the NMG in terms of pathologic complete response rate (53.3% vs. 21.6%, P = 0.044), good TRG response (66.7% vs. 32.4%, P = 0.024), T downstaging (86.7% vs. 43.2%, P = 0.004), and overall downstaging (86.7% vs. 56.8%, P = 0.040). The toxicities during NCRT were minimal in both groups. More apoptotic cells were noted in MG samples than in the NMG samples on TUNEL staining. Cleaved caspase-3 level following treatment with Abnoba Viscum Q® was higher in SNU-503R80Gy cells than in SNU-503 cells. CONCLUSION: Patients treated with chemoradiation combined with mistletoe extract showed better outcomes than patients not treated with mistletoe extract in terms of tumor responses. This diversity in treatment may improve the efficacy of NCRT, leading to better oncologic outcomes. Prospective and randomized studies with long-term follow-up are warranted to confirm and extend these results.

Hemodynamic effects of carbetocin administered as an intravenous bolus or infusion during cesarean delivery.

BACKGROUND: Postpartum hemorrhage is the leading cause of maternal mortality. Oxytocin being the most popular uterotonic agent, has been routinely administered after both vaginal delivery and cesarean section. Carbetocin is a newer uterotonic agent and provides the benefit of a longer duration of action without additional administration post-delivery. METHODS: We recruited 34 women undergoing elective cesarean section under spinal anesthesia. All patient was received spinal anesthesia using 0.5% hyperbaric Marcaine 8-10 mg in conjugation with fentanyl 20 µg in the left lateral decubitus position. Hartmann's solution 10-15 ml/kg was administered before carbetocin. The operation started as soon as sensory block at level T4-T6 was confirmed. A non-invasive hemodynamic monitoring cuff (Finometer®) was attached to the patient's finger soon after the induction of spinal anesthesia. Using the Finometer, we recorded the heart rate and mean arterial pressure at every 15 s, starting from 15 s before the administration of carbetocin to 5 min after. After the removal of the placenta, the bolus group was administered intravenous bolus injection of carbetocin 100 µg and the infusion group was administered carbetocin 100 µg diluted in 50 ml normal saline, over 5 min using an infusion pump. RESULTS: The demographic data showed no significant difference between the two groups. Furthermore, there were no significant hemodynamic differences between the two groups. CONCLUSIONS: The method of administration of carbetocin does not influence its hemodynamic effects.

Chemotherapeutic effects of MEK kinase inhibitor and BRAF kinase inhibitor on KRAS-mutated human colon cancer cell lines with different microsatellite instability.

We analyzed responsiveness of KRAS-mutated CRC cell lines with distinctive MSI status against mitogen-activated protein kinase (MEK) inhibitor (selumetinib; AZD) and/or B-raf proto-oncogene (BRAF) kinase inhibitor (vemurafenib; PLX). The viability of MSI-high (MSI-H) KRAS-mutated LS174T cells treated with AZD or PLX was 24.5 ± 0.9% or 71.4 ± 3.6%, respectively, and the viability of microsatellite stable (MSS) KRAS-mutated SW480 cells for AZD or PLX was 57.4 ± 3.1% or 43.1 ± 1.8%, respectively. These observations imply that the therapeutic efficacy of MEK kinase inhibitors or BRAF kinase inhibitors against KRAS-mutated colon cancer cells may differ between MSI-H and MSS. However, a combination of both inhibitors synergistically inhibits the proliferation of KRAS-mutated colon cancer cells regardless of MSI status. The underlying synergistic cytotoxic efficacy of AZD/PLX combination on KRAS-mutated colon cancer cells with different MSI status was further substantiated by markedly decreased phosphorylation of ERK in both LS74T and SW480 cell lines upon AZD and PLX treatment. Based on these collective data, we propose that MSI status should be considered when MEK kinase inhibitor or BRAF kinase inhibitor is treated for KRAS-mutated colon cancer, and that combination of both inhibitors synergistically inhibit proliferation of KRAS-mutated colon cancer cells independent of MSI status.

The effect of interleukin-8 in the early stage after anterior cruciate ligament reconstruction with remnant preservation.

PURPOSE: We studied the effect of interleukin-8 (IL-8) as the factor for angiogenesis in the joint fluid of remnant-preserved anterior cruciate ligament reconstruction (RP-ACLR). MATERIALS AND METHODS: We measured 12 cytokines in joint fluid by multiplex assay and assessed the relationship between IL-8 and vascular endothelial growth factor (VEGF) concentrations. The signal intensity and mean sagittal diameter via postoperative magnetic resonance imaging (MRI) scans were evaluated and the stress X-ray image was analyzed at 3, 6, and 12 months after operation. RESULTS: The IL-8 concentration was highest 3 months postoperatively in those patients who underwent RP-ACLR. Clinical data also showed that the signal intensity and stress radiography of the knee graft were significantly better at the early postoperative stage. DISCUSSION: Our results show that IL-8 plays an important role in angiogenesis within 3 months after RP-ACLR. This effect yields better recovery after operation. RP-ACLR patients with high knee stability in clinical data were identical to those with high expression of IL-8 in experimental data. Therefore, IL-8 has been shown to help revascularization and ligamentization of the grafted tendon. These results indicate that IL-8 in RP-ACLR is an important factor for angiogenesis after operation. Unfortunately, the relationship of IL-8 and VEGF in vivo has not been studied. CONCLUSION: Our results showed that the IL-8 concentration was very high within 3 months after RP-ACLR operation. The increase in concentration of IL-8 over time was consistent with the increase in VEGF concentration. In the IL-8 clinical setting, MRI analysis showed that ACL synovialization and tension were better in patients who underwent the remnant preservation method. In addition, it was shown that RP-ACLR may be advantageous for early anterior stability within 1 year post operation and beneficial for tendon graft in the early stage post operation. Taken together, our findings suggest that IL-8 may contribute to angiogenesis which is helpful for revascularization and ligamentization of the graft tendon in the early stages of RP-ACLR.

Proteomic Classification and Identification of Proteins Related to Tissue Healing of Platelet-Rich Plasma.

BACKGROUND: Platelet-rich plasma (PRP) is a plasma component of autologous blood containing a high concentration of platelets. PRP is used to promote healing of damaged tissues. However, there are not many studies on the composition and expression patterns of active proteins in PRP. The purpose of this study was to identify unknown factors that contribute to tissue healing by proteomic analysis of proteins in PRP. METHODS: Three men in their 30s with no basal disease participated in this study. All identified proteins were classified for tissue healing-related functions on the basis of the gene ontology analysis of adhesion molecule with Ig-like domain 2 (AmiGO2). PRP was prepared by using the ACP kit and GPS III kit. RESULTS: We identified a total of 125 proteins related to wound healing, along with three proteins for angiogenesis involved in wound healing, two proteins for fibroblast migration, four proteins for collagen biosynthesis process, two proteins for glycosaminoglycan biosynthesis process, and 13 proteins for glycosaminoglycan binding. So, in addition to the growth factors that have been already known to be involved in tissue healing, 25 new proteins were identified. CONCLUSIONS: We identified the unknown proteins associated with tissue healing in PRP. Our findings may serve as a foundation for the establishment of basic medical evidence for PRP applications.

Electroacupuncture for treatment-resistant insomnia: study protocol for a randomised, controlled, assessor-blinded, pilot clinical trial.

INTRODUCTION: A considerable number of insomnia patients experience sleep disturbance even with long-term use of hypnotic medication. Previous studies have indicated that electroacupuncture (EA) could be an efficacious treatment for managing insomnia. However, few trials have been conducted to evaluate the effectiveness and safety of EA for treatment-resistant insomnia. This pilot study aims to explore the feasibility and preliminary effectiveness and safety of EA as an adjunct treatment for treatment-resistant insomnia. METHODS AND ANALYSIS: This is a multicentre, randomised, usual care controlled and assessor-blinded pilot study protocol. Fifty patients presenting with sleep problems who have been taking hypnotic medication for more than 3 months will be randomly allocated to either an EA group or a usual care group at a 1:1 ratio. The EA group will undergo 12 EA treatment sessions twice a week for 6 weeks whereas the usual care group will not receive EA treatment. All the participants will receive a brochure containing educational information on sleep hygiene. The primary outcome will be the measured mean change of the total score of the Insomnia Severity Index from the baseline to week 7. The secondary outcome regarding sleep quality will be measured using the Pittsburgh Sleep Quality Index, a sleep diary and actigraphy. Moreover, we will assess the quality of life, the direct and indirect cost of treating insomnia for economic evaluation. After 4 weeks, the subjects will visit the research sites for a follow-up assessment. ETHICS AND DISSEMINATION: Ethical approval of this study protocol was established by the institutional review boards of the each involved study site. All potential subjects will be provided written informed consent. The results of this study will be accessible in peer-reviewed publications and be presented at academic conference. TRIAL REGISTRATION NUMBER: KCT0003235.

Exogenous 8-hydroxydeoxyguanosine ameliorates liver fibrosis through the inhibition of Rac1-NADPH oxidase signaling.

BACKGROUND AND AIM: Exogenous 8-hydroxydeoxyguanosine (8-OHdG) was suggested as an inhibitor of Rac1 and NADPH oxidase (NOX). The aim of this study was to evaluate the effects of the exogenous 8-OHdG on hepatic fibrogenesis in vitro and in vivo model of liver fibrosis. METHODS: Adult Sprague-Dawley rats were allocated to sham-operated rats (n = 7), rats that underwent bile duct ligation (BDL) (n = 6), and BDL rats treated with 8-OHdG (60 mg/kg/day by gavage, n = 6). All rats were sacrificed on day 21. Double immunofluorescence staining between either NOX1 or NOX2 and α-smooth muscle actin (SMA) in liver was performed. Hepatic fibrotic contents were assessed by hydroxyproline assay and quantified by Sirius red staining. In vitro, hepatic stellate cell (HSC) line LX-2 and HHSteC cells were stimulated by angiotensin II (10 µM). The reactive oxygen species (ROS) production was measured by confocal microscopy. The expressions of NOX1, NOX2, α-SMA, transforming growth factor (TGF)-ß1, and collagen Iα were analyzed by quantitative real-time polymerase chain reaction or immunoblotting. RESULTS: The 8-OHdG treatment in BDL rats reduced the NOX1 and NOX2 protein expression, which overlapped with α-SMA compared with BDL rats. The 8-OHdG treatment in BDL rats significantly decreased the mRNA expression of NOX1, NOX2, α-SMA, TGF-ß1, and collagen Iα, and fibrotic contents. Increases of ROS production, Rac1 activation, NOX1, NOX2, and fibronectin expression induced by angiotensin II in HSCs were attenuated by 8-OHdG. CONCLUSIONS: Rac1 activation and NOX-derived ROS are implicated to liver fibrosis. The 8-OHdG ameliorates liver fibrosis through the inhibition of Rac1 activation and NOX-derived ROS.

Leukocyte-Poor Platelet-Rich Plasma-Derived Growth Factors Enhance Human Fibroblast Proliferation In Vitro.

BACKGROUND: Leukocyte-poor platelet-rich plasma (LP-PRP) from peripheral blood is currently used as a concentrated source of growth factors to stimulate repair at sites of soft tissue injury. Fibroblasts are primary mediators of wound healing. Thus, we aimed to assess the positive effect of LP-PRP on human fibroblast proliferation in vitro. METHODS: LP-PRP was prepared from 49 donors. The fibroblasts were seeded, and at 24 hours after seeding, 1 × 107/10 µL LP-PRP was added once to each well. The cells were harvested 10 times during study period at our planned points, and we examined cell proliferation using the water-soluble tetrazolium salt-1 assay. We collected the supernatants and measured the amount of growth factors such as platelet-derived growth factor (PDGF)-AB/BB, insulin-like growth factor-1 (IGF-1), transforming growth factor-ß1 (TGF-ß1), and vascular endothelial growth factor (VEGF), which are known to be involved in wound healing processes, by multiplex assay. RESULTS: Human fibroblasts treated with LP-PRP showed a significant increase in proliferation when compared to untreated controls (p < 0.001 at days 4, 6, and 8). Multiplex cytokine assays revealed various secretion patterns. PDGF-AB/BB appeared at early time points and peaked before fibroblast proliferation. IGF-1 and TGF-ß1 secretion gradually increased and peaked on days 4 and 6 post-treatment. The early VEGF concentration was lower than the concentration of other growth factors but increased along with cell proliferation. CONCLUSIONS: Platelets in LP-PRP release growth factors such as PDGF, IGF-1, TGF-ß1 and VEGF, and these growth factors have a promoting effect for human fibroblast proliferation, one of the important mediators of wound healing. These results suggest that growth factors derived from LP-PRP enhance the proliferation of human fibroblast.

Enhanced Tendon-to-Bone Healing of Chronic Rotator Cuff Tears by Bone Marrow Aspirate Concentrate in a Rabbit Model.

BACKGROUND: To evaluate the influence of bone marrow aspirate concentrate (BMAC) on tendon-to-bone healing in a rabbit rotator cuff model and to characterize the composition of growth factors in BMAC. METHODS: In this in vivo study, 40 rabbits were allocated into five groups: control (C), repair + saline (RS), repair + platelet-rich plasma (PRP; RP), repair + BMAC (RB) and repair + PRP + BMAC (RPB). A tear model was created by supraspinatus tendon transection at the footprint. Six weeks after transection, the torn tendon was repaired along with BMAC or PRP administration. Six weeks after repair, shoulder samples were harvested for biomechanical and histological testing. Ten rabbits were used for processing PRP and BMAC, followed by analysis of blood cell composition and the levels of growth factors in vitro. RESULTS: The ultimate load-to-failure was significantly higher in RPB group compared to RS group (p = 0.025). BMAC-treated groups showed higher values of biomechanical properties than RS group. The histology of BMAC-treated samples showed better collagen fiber continuity and orientation than RS group. BMAC contained significantly higher levels of the several growth factors than PRP. CONCLUSIONS: Locally administered BMAC enhanced tendon-to-bone healing and has potential for clinical applications.

Age-Related Changes in Nuclear Factor Erythroid 2-Related Factor 2 and Reactive Oxygen Species and Mitochondrial Structure in the Tongues of Fischer 344 Rats.

OBJECTIVES: Previously the authors reported age-related changes in the activities of anti-oxidative enzyme activities and protein expressions in the tongues of rats. Because more information is required about relations between aging and oxidative stress and anti-oxidative enzyme efficiency, the authors investigated differences between the expression of master regulator of anti-oxidative enzymes (nuclear factor erythroid 2-related factor 2 [Nrf2]), levels of reactive oxygen species (ROS), and mitochondrial structures in the tongues of young and aged Fischer 344 rats. METHODS: Age-dependent changes in Nrf2 protein and ROS were determined by Western blotting and using chemical kits, respectively. Tongue specimens were examined by electron microscopy. The study was conducted using rats aged 7 months (young, n=8) or 22 months (old, n=8). RESULTS: Nrf2 protein levels in the tongues of aged rats were lower than in young rats. ROS levels were higher in older rats and mitochondrial structural deficits were observed their tongues. Three young rats showed moderate mitochondrial degeneration, whereas profound degeneration with mitochondrial cristae disruption, swelling, rupture, or intramitochondrial vacuole formation was observed in all 8 old rats. Notably, mitochondrial rupture was observed in 5 old rats. CONCLUSION: Antioxidant defense systems of old rats were compromised by Nrf2 deficiency, which could lead to the deleterious accumulation and release of ROS and probably mitochondrial structural deficits in aged tongue tissues.

Age-Related Changes in Antioxidative Enzyme Capacity in Tongue of Fischer 344 Rats.

OBJECTIVES: Antioxidative enzyme efficiency changes in some organs with age. However, no study has been conducted on age-related antioxidant enzyme changes in tongue. In the present study, the authors investigated the activities of four antioxidative enzymes and their protein expressions in the tongues of young and old Fischer 344 rats. METHODS: Age-dependent changes in the enzyme activities of total superoxide dismutase (SOD), Mn-SOD, Cu/Zn-SOD, catalase (CAT), and glutathione peroxidase (GPx) were determined using chemical kits, and the protein expressions levels of these enzymes by Western blotting. The study was conducted using rats aged 7 months (the young group, n=8) and 22 months (the old group, n=8). RESULTS: Total SOD, Cu/Zn-SOD, and GPx activities in the tongues of old rats were lower than in young rats, and similarly, corresponding protein expressions were downregulated in old rats. On the other hand, although the protein expressions of Mn-SOD and CAT were lower in old rats, their enzyme activities were not. CONCLUSION: The results of this study provide a possible mechanism for the tongue aging process, as in old Fischer 344 rats the antioxidant defense system was diminished with respect to enzyme activity levels and protein abundances.

Anatomic Double-Bundle Anterior Cruciate Ligament Reconstruction Using an Outside-in Technique: Two- to Six-Year Clinical and Radiological Follow-up.

PURPOSE: We evaluated the clinical and radiological outcomes of double-bundle anterior cruciate ligament (ACL) reconstruction using an outside-in technique with a follow-up of two- to six-years, especially in terms of the sports activity level and radiological degeneration. MATERIALS AND METHODS: Sixty-seven patients who were available for a minimum two-year follow-up after double-bundle ACL reconstruction using an outside-in technique were retrospectively evaluated. The mean follow-up period was 43.7 months. The knee function and stability were evaluated before the operation, one year after the operation (short-term follow-up), and more than two years after the operation (last follow-up). RESULTS: Regarding the knee function, the Lysholm score, International Knee Documentation Committee (IKDC) evaluation, and hop test showed significant improvement. Regarding the stability, the Lachman test, pivot shift test, KT-2000 arthrometer data, and anterior drawer radiographs using Telos showed significant improvement. Regarding the sports activity level, the patients who returned to pre-injury level activity was 68.7% according to the Tegner activity score and 76.1% according to the Cincinnati sports activity scale score. The incidence of aggravated degeneration or development of greater than IKDC grade A degeneration after surgery was 10.4%. CONCLUSIONS: Double-bundle ACL reconstruction using an outside-in technique showed favorable clinical and radiological outcomes with respect to the knee function and stability, joint degeneraion, and, especially, return to pre-injury sports activity.

The dipeptide H-Trp-Arg-OH (WR) Is a PPARα agonist and reduces hepatic lipid accumulation in lipid-loaded H4IIE cells.

Dipeptides absorbed by the intestinal epithelium are delivered to circulation, but their metabolic roles are not yet clearly understood. We investigated the biological activities of a dietary dipeptide, H-Trp-Arg-OH (WR), on the regulation of peroxisome proliferator-activated receptor (PPAR) α activity. Reporter gene assays revealed that WR dose-dependently induced PPARα transactivation. Surface plasmon resonance experiments demonstrated that WR interacts directly with the PPARα ligand binding domain, and time-resolved fluorescence energy transfer analyses revealed recruitment of a co-activator peptide, fluorescein-PGC1α, to PPARα, confirming the direct binding of WR to PPARα and occurrence of conformational changes. WR induced cellular fatty acid uptake and the expression of PPARα response genes in fatty acid oxidation, thus reducing intracellular triglyceride accumulation in lipid-loaded hepatocytes. In conclusion, the dietary dipeptide WR activates PPARα and reduces hepatic lipid accumulation in lipid-loaded hepatocytes.

Proteomic identification of 14-3-3ϵ as a linker protein between pERK1/2 inhibition and BIM upregulation in human osteosarcoma cells.

Despite advancements in multimodality chemotherapy, conventional cytotoxic treatments still remain ineffective for a subset of patients with aggressive metastatic or multifocal osteosarcoma. It has been shown that pERK1/2 inhibition enhances chemosensitivity to doxorubicin and promotes osteosarcoma cell death in vivo and in vitro. One of the pro-apoptotic mechanisms is upregulation of Bim by pERK1/2 inhibitors. To this end, we examined proteomic changes of 143B human osteosarcoma cells with and without treatment of PD98059, pERK1/2 inhibitor. Specifically, we identified 14-3-3ϵ protein as a potential mediator of Bim expression in response to inhibition of pERK1/2. We hypothesized that 14-3-3ϵ mediates upregulation of Bim expression after pERK1/2 inhibition. We examined the expression of Bim after silencing 14-3-3ϵ using siRNA. The 14-3-3ϵ gene silencing resulted in downregulation of Bim expression after PD98059 treatment. These data indicate that 14-3-3ϵ is required for Bim expression and that it has an anti-cancer effect under pERK1/2 inhibition in 143B cells. By playing an essential role upstream of Bim, 14-3-3ϵ may potentially be a coadjuvant factor synergizing the effect of pERK1/2 inhibitors in addition to conventional cytotoxic agents for more effective osteosarcoma treatments.

CXCL10 promotes osteolytic bone metastasis by enhancing cancer outgrowth and osteoclastogenesis.

Amplification of the chemokines CXCL10 and RANKL has been suggested to promote osteoclast differentiation and osteolytic bone metastasis, but a function for endogenous CXCL10 in these processes is not well established. In this study, we show that endogenous CXCL10 is critical to recruit cancer cells to bone, support osteoclast differentiation and promote for the formation of osteolytic bone metastases. Neutralizing CXCL10 antibody reduced migration of cancer cells expressing the CXCL10 receptor CXCR3, and loss of CXCR3 or CXCL10 decreased bone tumor burden in vivo. Bone colonization augmented host production of CXCL10, which was required for cancer growth and subsequent osteolysis. Direct interactions between cancer cells and macrophages further stimulated CXCL10 production from macrophages. Growth of bone metastases required CXCL10-stimulated adhesion of cancer cells to type I collagen as well as RANKL-mediated osteoclast formation. Together, our findings show that CXCL10 facilitates trafficking of CXCR3-expressing cancer cells to bone, which augments its own production and promotes osteoclastic differentiation. CXCL10 therefore may represent a therapeutic target for osteolytic bone metastasis.

Ski inhibits TGF-ß/phospho-Smad3 signaling and accelerates hypertrophic differentiation in chondrocytes.

Since transforming growing factor-ß (TGF-ß)/Smad signaling inhibits chondrocyte maturation, endogenous negative regulators of TGF-ß signaling are likely also important regulators of the chondrocyte differentiation process. One such negative regulator, Ski, is an oncoprotein that is known to inhibit TGF-ß/Smad3 signaling via its interaction with phospho-Smad3 and recruitment of histone deacetylases (HDACs) to the DNA binding complex. Based on this, we hypothesized that Ski inhibits TGF-ß signaling and accelerates maturation in chondrocytes via recruitment of HDACs to transcriptional complexes containing Smads. We tested this hypothesis in chick upper sternal chondrocytes (USCs), where gain and loss of Ski expression experiments were performed. Over-expression of Ski not only reversed the inhibitory effect of TGF-ß on the expression of hypertrophic marker genes such as type X collagen (colX) and osteocalcin, it induced these genes basally as well. Conversely, knockdown of Ski by RNA interference led to a reduction of colX and osteocalcin expression under basal conditions. Furthermore, Ski blocked TGF-ß induction of cyclinD1 and caused a basal up-regulation of Runx2, consistent with the observed acceleration of hypertrophy. Regarding mechanism, not only does Ski associate with phospho-Smad2 and 3, but its association with phospho-Smad3 is required for recruitment of HDAC4 and 5. Implicating this recruitment of HDACs in the phenotypic effects of Ski in chondrocytes, the HDAC inhibitor SAHA reversed the up-regulation of colX and osteocalcin in Ski over-expressing cells. These results suggest that inhibition of TGF-ß signaling by Ski, which involves its association with phospho-Smad3 and recruitment of HDAC4 and 5, leads to accelerated chondrocyte differentiation.