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BackgroundThe percutaneous screw reconstruction technique, known as the “Tripod Technique,” has demonstrated favorable clinical outcomes in the management of metastatic periacetabular lesions, as evidenced by our prior investigations and corroborated by independent studies. Nevertheless, there is a steep learning curve in handling this technique, with possible complications such as intraarticular screw placement.MethodsPreoperative pelvic CT scans were acquired before surgery and utilized for the guiding frame design. A convolutional neural network model was trained with annotated data to identify the starting point and trajectory of each potential screw. A model boundary intersection detection technology was used to determine the optimal diameter and length of each screw. A non-rigid registration technology was matched with a prefabricated model of the body surface to design personalized anchoring skin pads. Finally, a polylactic acid-based guiding frame for intraoperative was custom-made with a 3D printer.Results12 patients underwent a guiding frame-assisted Tripod procedure for treatment of periacetabular metastatic lesions. An intraoperative CT scan was performed in all cases to confirm screw trajectories. Among 36 screws that were implanted, 26 screws were implanted as designed. The remaining ten screws drifted, but all remained within the intra-osseous conduit without any complications. The mean surgical time was 1.22 h with the guiding frame compared with 2.3 h without the guiding frame. Following the surgical procedure, a noteworthy enhancement in pain management, as evidenced by a reduction in scores on the visual analog scale (p < 0.01), and an improvement in functional status, as assessed through the Eastern Cooperative Oncology Group score (p < 0.01), were observed when compared to the patient’s pre-operative condition.ConclusionThis proof-of-concept investigation demonstrates that the amalgamation of AI-assisted surgical planning and additive manufacturing can improve surgical accuracy and shorten surgical duration. While access to this technology is currently constrained during its early stages of development, it is anticipated that these limitations will diminish as the potential of AI and additive manufacturing in facilitating complex orthopedic procedures becomes more evident, leading to a surge in interest and adoption of this approach.

ObjectivePost-traumatic osteoarthritis (PTOA) is a degenerative joint disease initiated by injury. Early phase (0-7 days) treatments often include rest (unloading) and anti-inflammatory medications, but how those early interventions impact PTOA progression is unknown. We hypothesized that early unloading and anti-inflammatory treatment would diminish joint inflammation and slow PTOA progression.DesignMice were injured with non-invasive ACL rupture followed by hindlimb unloading (HLU) or normal cage activity (ground control: GC) for 7 days, after which all mice were allowed normal cage activity. HLU and GC mice were treated with daily celecoxib (CXB; 10 mg/kg IP) or vehicle. Protease activity was evaluated using in vivo fluorescence imaging, osteophyte formation and epiphyseal trabecular bone were quantified using micro-computed tomography, and synovitis and articular cartilage were evaluated using whole-joint histology at 7, 14, 21, and 28 days post-injury.ResultsHLU significantly reduced protease activity (-22-30% compared to GC) and synovitis (-24-50% relative to GC) at day 7 post-injury (during unloading), but these differences were not maintained at later timepoints. Similarly, trabecular bone volume was partially preserved in HLU mice at during unloading (-14-15% BV/TV for HLU mice, -21-22% for GC mice relative to uninjured), but these differences were not maintained during reloading. Osteophyte volume was reduced by both HLU and CXB, but there was not an additive effect of these treatments (HLU: -46%, CXB: -30%, HLU + CXB: -35% relative to vehicle GC at day 28).ConclusionsThese data suggest that early unloading following joint injury can reduce inflammation and potentially slow PTOA progression.

Vietnamese American males have high smoking rates. This study explored social support mechanisms provided by lay health workers (LHWs) and family members through a smoking cessation intervention. Eight focus groups (N = 54) were conducted in Vietnamese stratified by intervention arms (Tobacco [experimental] and healthy living [control]) with 18 smokers, 18 family members, and 18 LHWs. Smokers reported feeling more accountable for their health behaviors, and smoking changes were reinforced by family members, peers, and LHWs through conversations facilitated during and outside the program. Culturally appropriate interventions with multiple social support mechanisms may reduce smoking in minority populations.

Osteosarcoma is a highly aggressive malignancy for which treatment has remained essentially unchanged for years. Our previous studies found that the F-box protein SKP2 is overexpressed in osteosarcoma, acting as a proto-oncogene; p27Kip1 (p27) is an inhibitor of cyclin-dependent kinases and a downstream substrate of SKP2-mediated ubiquitination. Overexpression of SKP2 and underexpression of p27 are common characteristics of cancer cells. The SCFSKP2 E3 ligase ubiquitinates Thr187-phosphorylated p27 for proteasome degradation, which can be abolished by a Thr187Ala knock-in (p27T187A KI) mutation. RB1 and TP53 are two major tumor suppressors commonly coinactivated in osteosarcoma. We generated a mouse model with a double knockout (DKO) of Rb1 and Trp53 within cells of the osteoblastic lineage, which developed osteosarcoma with full penetrance. When p27T187A KI mice were crossed on to the DKO background, p27T187A protein was found to accumulate in osteosarcoma tumor tissues. Furthermore, p27T187A promoted apoptosis in DKO tumors, slowed disease progression, and significantly prolonged overall survival. RNA sequencing analysis also linked the SCFSKP2 -p27T187A axis to potentially reduced cancer stemness. Given that RB1 and TP53 loss or coinactivation is common in human osteosarcoma, our study suggests that inhibiting the SKP2-p27 axis may represent a desirable therapeutic strategy for this cancer.

Synovial sarcoma (SS) is an aggressive soft-tissue cancer with a poor prognosis and a propensity for local recurrence and distant metastasis. In this study, we investigated whether S phase kinase-associated protein (Skp2) plays an oncogenic role in tumor initiation, progression, and metastasis of SS. Our study revealed that Skp2 is frequently overexpressed in SS specimens and SS18-SSX transgenic mouse tumors, as well as correlated with clinical stages. Next, we identified that genetic depletion of Skp2 reduced mesenchymal and stemness markers, and inhibited the invasive and proliferative capacities of SS cell lines. Furthermore, Skp2 depletion markedly suppressed the growth of SS xenografts tumors. Treatment of SS cell lines with the skp2 inhibitor flavokawain A (FKA) reduced Skp2 expression in a dose-dependent manner and resulted in cell cycle arrest and apoptosis. FKA also suppressed the invasion and tumor-initiating properties in SS, similar to the effects of Skp2 knockdown. In addition, a combination of FKA and conventional chemotherapy showed a synergistic therapeutic efficacy. Taken together, our results suggest that Skp2 plays an essential role in the biology of SS by promoting the mesenchymal state and cancer stemness. Given that chemotherapy resistance is often associated with cancer stemness, strategies of combining Skp2 inhibitors with conventional chemotherapy in SS may be desirable.

IntroductionAmericans have low levels of knowledge of and adherence to recommendations for healthy eating of fruits and vegetables and for physical activity (HEPA). We conducted a cluster randomized controlled trial of a lay health worker intervention to increase HEPA among Vietnamese Americans.MethodsWe randomized 64 lay health workers to 2 intervention arms. Each lay health worker recruited 10 participants aged 50 to 74. From 2008 to 2013, using flip charts, lay health workers led 2 educational sessions on HEPA (intervention) or colorectal cancer (comparison). We assessed HEPA knowledge and self-reported behaviors by preintervention and postintervention surveys 6 months apart.ResultsOf the 640 participants, 50.0% were female, 38.4% had lived in the United States for 10 years or fewer, and 71.4% reported limited English proficiency. Knowledge of the recommended intake of fruits and vegetables (≥5 servings daily) increased from 2.6% to 60.5% in the intervention group (n = 311) and from 2.9% to 6.7% in the comparison group (n = 316) (intervention vs comparison change, P < .001). Knowledge of the physical activity recommendation (≥150 minutes weekly) increased from 2.6% to 62.4% among intervention participants and from 1.0% to 2.5% among comparison participants (P < .001). Consumption of 5 or more daily servings of fruits and vegetables increased more in the intervention group (8.4% to 62.1%) than in the comparison group (5.1% to 12.7%) (P < .001). Participants reporting 150 minutes or more of physical activity weekly increased from 28.9% to 54.0% in the intervention group and from 38.0% to 46.8% in the comparison group (intervention vs comparison change, P = .001).ConclusionA lay health worker intervention increased both healthy eating and physical activity knowledge and self-reported behaviors among older Vietnamese Americans.

BackgroundFlavokawain B (FKB) has been identified from kava root extracts as a potent apoptosis inducer for inhibiting the growth of various cancer cell lines, including prostate cancer. However, the molecular targets of FKB in prostate cancer cells remain unknown.MethodsAn in vitro NEDD8 Initiation Conjugation Assay was used to evaluate the neddylation inhibitory activity of FKB. Molecular docking and a cellular thermal shift assay were performed to assess the direct interaction between FKB and the NEDD8 activating enzyme (NAE) complex. Protein neddylation, ubiqutination, stability and expression in cells were assessed with immunoprecipitation and Western blotting methods using specific antibodies. Deletion and site specific mutants and siRNAs were used to evaluate deep mechanisms by which FKB induces Skp2 degradation. Cell growth inhibition and apoptosis induction were measured by MTT, ELISA and Western blotting methods.ResultsFKB inhibits NEDD8 conjugations to both Cullin1 and Ubc12 in prostate cancer cell lines and Ubc12 neddylation in an in vitro assay. Molecular docking study and a cellular thermal shift assay reveal that FKB interacts with the regulatory subunit (i.e. APP-BP1) of the NAE. In addition, FKB causes Skp2 degradation in an ubiquitin and proteasome dependent manner. Overexpression of dominant-negative cullin1 (1-452), K720R mutant (the neddylation site) Cullin1 or the F-box deleted Skp2 that losses its binding to the Skp1/Cullin1 complex causes the resistance to FKB-induced Skp2 degradation, whereas siRNA knock-down of Cdh1, a known E3 ligase of Skp2 for targeted degradation, didn't attenuate the effect of FKB on Skp2 degradation. These results suggest that degradation of Skp2 by FKB is involved in a functional Cullin1. Furthermore, proteasome inhibitors Bortezomib and MG132 transcriptionally down-regulate the expression of Skp2, and their combinations with FKB result in enhanced inhibitory effects on the growth of prostate cancer cell lines via synergistic down-regulation of Skp2 and up-regulation of p27/Kip1 and p21/WAF1 protein expression. FKB also selectively inhibits the growth of RB deficient cells with high expression of Skp2.ConclusionThese findings provide a rationale for further investigating combination of FKB and Bortezomib for treatment of RB deficient, castration-resistant prostate cancer.

Overdispersion models have been extensively studied for correlated normal and binomial data but much less so for correlated multinomial data. In this work, we describe a multinomial overdispersion model that leads to the specification of the first two moments of the outcome and allows the estimation of the global parameters using generalized estimating equations (GEE). We introduce a Global Blinding Index as a target parameter and illustrate the application of the GEE method to its estimation from (1) a clinical trial with clustering by practitioner and (2) a meta-analysis on psychiatric disorders. We examine the impact of a small number of clusters, high variability in cluster sizes, and the magnitude of the intraclass correlation on the performance of the GEE estimators of the Global Blinding Index using the data simulated from different models. We compare these estimators with the inverse-variance weighted estimators and a maximum-likelihood estimator, derived under the Dirichlet-multinomial model. Our results indicate that the performance of the GEE estimators was satisfactory even in situations with a small number of clusters, whereas the inverse-variance weighted estimators performed poorly, especially for larger values of the intraclass correlation coefficient. Our findings and illustrations may be instrumental for practitioners who analyze clustered multinomial data from clinical trials and/or meta-analysis.

BackgroundThe Kidney Donor Profile Index (KDPI) provides a numerical estimate of deceased donor kidney quality. The KDPI uses 10 donor factors but it does not consider histopathologic findings. We examined whether the KDPI and its component donor factors correlate with the degree of histopathologic changes seen in implantation renal allograft biopsies.MethodsAll deceased donor kidney transplants at our institution from July 1, 2016 to March 15, 2017 that had an implantation biopsy were included. The biopsies were graded based on the Banff criteria for interstitial fibrosis, tubular atrophy, arterial intimal fibrosis, and arteriolar hyalinosis, as well as percent glomerulosclerosis. Linear and logistic regression were used to assess the correlation between histopathologic findings and KDPI and the ability of these variables to predict 30-day serum creatinine (SCr) and delayed graft function (DGF).ResultsOne hundred thirty-four recipients from 107 donors were included. All histopathologic features examined correlated significantly with KDPI, with arteriolar hyalinosis correlating most strongly. Arteriolar hyalinosis was also associated with the most component donor factors of the KDPI. Histopathologic findings alone or in combination with KDPI predicted 30-day SCr but not DGF. Using the KDPI in combination with degree of interstitial fibrosis and tubular atrophy was the best predictor of 30-day SCr.ConclusionHistopathologic changes seen in implantation renal allograft biopsies correlate with KDPI and predict 30-day SCr. Using a combination of donor histopathologic findings and KDPI may be the best predictor of short-term graft function.

Osteosarcoma (OS), the most common primary cancer of bone, exhibits a high propensity for local invasion and distant metastasis. This study sought to elucidate the role of S phase kinase-associated protein (Skp2) in osteosarcoma invasion and metastasis and to explore flavokawain A (FKA), a natural chalcone from kava extract, as a potential Skp2 targeting agent for preventing osteosarcoma progression. Skp2 was found to be overexpressed in multiple osteosarcoma cell lines, including 5 standard and 8 primary patient-derived cell lines. Patients whose tumors expressed high levels of Skp2 sustained a significantly worse metastasis-free (p = 0.0095) and overall survival (p = 0.0013) than those with low Skp2. Skp2 knockdown markedly reduced in vitro cellular invasion and in vivo lung metastasis in an orthotopic mouse model of osteosarcoma. Similar to Skp2 knockdown, treatment with FKA also reduced Skp2 expression in osteosarcoma cell lines and blocked the invasion of osteosarcoma cells in vitro and lung metastasis in vivo. Together, our findings suggest that Skp2 is a promising therapeutic target in osteosarcoma, and that FKA may be an effective Skp2-targeted therapy to reduce osteosarcoma metastasis.

HER2/neu positive breast tumors predict a high mortality and comprise 25%-30% of breast cancer. We have shown that Flavokawain A (FKA) preferentially reduces the viabilities of HER2-overexpressing breast cancer cell lines (i.e., SKBR3 and MCF7/HER2) versus those with less HER2 expression (i.e., MCF7 and MDA-MB-468). FKA at cytotoxic concentrations to breast cancer cell lines also has a minimal effect on the growth of non-malignant breast epithelial MCF10A cells. FKA induces G2M arrest in cell cycle progression of HER2-overexpressing breast cancer cell lines through inhibition of Cdc2 and Cdc25C phosphorylation and downregulation of expression of Myt1 and Wee1 leading to increased Cdc2 kinase activities. In addition, FKA induces apoptosis in SKBR3 cells by increasing the protein expression of Bim and BAX and decreasing expression of Bcl₂, BclX/L, XIAP, and survivin. FKA also downregulates the protein expression of HER-2 and inhibits AKT phosphorylation. Herceptin plus FKA treatment leads to an enhanced growth inhibitory effect on HER-2 overexpressing breast cancer cell lines through downregulation of Myt1, Wee1, Skp2, survivin, and XIAP. Our results suggest FKA as a promising and novel apoptosis inducer and G2 blocking agent that, in combination with Herceptin, enhances for the treatment of HER2-overexpressing breast cancer.

Purpose: The article unpacks potentials of and resistance towards facilitating meetings between refugee and non-refugee youth in global citizenship education. Design/methodology/approach: The analyses are based on participant observation in a school-based intervention in three locations, developed on the principles of design-based research [DBR]. Findings: The article exposes both how meetings between students could be deeply educational and how teachers prevent meaningful interaction between students out of concern for refugee students. Research limitations/implications: More research is needed on how students care for themselves and others in transformational learning contexts. Practical implications: Privileged teachers’ concern for retraumatising students can veil unconscious protection of the privileged self against students’ trauma and should therefore be subject to critical reflection.

Synovial sarcoma (SS) is an aggressive soft tissue sarcoma with poor prognosis due to late recurrence and metastasis. Metastasis is an important prognostic factor of SS. This study aimed to identify the core genes and mechanisms associated with SS metastasis. Microarray data for GSE40021 and GSE40018 were obtained from the Gene Expression Omnibus database. 186 differentially expressed genes (DEGs) were identified. The biological functions and signalling pathways closely associated with SS metastasis included extracellular matrix (ECM) organization and ECM‐receptor interaction. Gene set enrichment analysis showed that the terms cell cycle, DNA replication, homologous recombination and mismatch repair were significantly enriched in the metastasis group. Weighted gene co‐expression network analysis identified the most relevant module and 133 hub genes, and 31 crossover genes were identified by combining DEGs. Subsequently, four characteristic genes, EXO1, NCAPG, POLQ and UHRF1, were identified as potential biomarkers associated with SS metastasis using the least absolute shrinkage and selection operator algorithm and validation dataset verification analysis. Immunohistochemistry results from our cohort of 49 patients revealed visible differences in the expression of characteristic genes between the non‐metastatic and metastatic groups. Survival analysis indicated that high expression of characteristic genes predicted poor prognosis. Our data revealed that primary SS samples from patients who developed metastasis showed activated homologous recombination and mismatch repair compared to samples from patients without metastasis. Furthermore, EXO1, NCAPG, POLQ and UHRF1 were identified as potential candidate metastasis‐associated genes. This study provides further research insights and helps explore the mechanisms of SS metastasis. [ABSTRACT FROM AUTHOR]

Based largely on a number of short-term administration studies, growing evidence suggests that central oxytocin is important in the regulation of energy balance. The goal of the current work is to determine whether long-term third ventricular (3V) infusion of oxytocin into the central nervous system (CNS) is effective for obesity prevention and/or treatment in rat models. We found that chronic 3V oxytocin infusion between 21 and 26 days by osmotic minipumps both reduced weight gain associated with the progression of high-fat diet (HFD)-induced obesity and elicited a sustained reduction of fat mass with no decrease of lean mass in rats with established diet-induced obesity. We further demonstrated that these chronic oxytocin effects result from 1) maintenance of energy expenditure at preintervention levels despite ongoing weight loss, 2) a reduction in respiratory quotient, consistent with increased fat oxidation, and 3) an enhanced satiety response to cholecystokinin-8 and associated decrease of meal size. These weight-reducing effects persisted for approximately 10 days after termination of 3V oxytocin administration and occurred independently of whether sucrose was added to the HFD. We conclude that long-term 3V administration of oxytocin to rats can both prevent and treat diet-induced obesity.

ObjectiveThe aim of the study was to determine the differential expression patterns of the wingless-type (Wnt) pathway inhibitors Dkk3 (Dickkopf 3), SFRP1 (secreted frizzled-related protein 1), and SFRP4 in normal müllerian tissue and endometrial endometrioid adenocarcinoma specimens.MethodsMessenger RNA (mRNA) and protein levels of the Wnt pathway inhibitors Dkk3, SFRP1, and SFRP4 were evaluated by real-time reverse transcription-polymerase chain reaction and Western blot analysis. A total of 87 human tissue specimens were obtained from 60 women who participated in Gynecologic Oncology Group protocol 210. Twenty-seven normal müllerian tissues, 32 early-stage, and 28 advanced-stage endometrial endometrioid cancer specimens were analyzed.ResultsMedian age for this cohort was 60 years, with median body mass index of 32 kg/m. There was a difference in Dkk3 protein expression between normal müllerian tissues and primary endometrial endometrioid adenocarcinoma samples (P = 0.05). There was down-regulation of Dkk3, SFRP1, and SFRP4 mRNA expression in patients with high-grade disease (P = 0.08, 0.06, and 0.05, respectfully). Furthermore, a decrease in SFRP1 and SFPR4 mRNA expression was noted in patients with a diagnosis of locoregional and distant disease recurrence. Lastly, a trend toward decreased progression-free survival in patients with low Dkk3, SFRP1, and SFRP4 mRNA expression levels was noted.ConclusionsWnt pathway inhibitor (Dkk3, sFRP1, and/or sFRP4) expression was down-regulated in patients with high-grade disease and was associated with locoregional and distant disease recurrence. Despite sample size (power) limitations, these results support previous preclinical studies and may suggest a therapeutic role for Wnt signaling in endometrial cancer.

Abstract Local control for the treatment of primary bone tumors is generally delayed following neoadjuvant chemotherapy. This was born out of the historical need to manufacture custom implants when performing limb-salvage resection. There is increasing reason to reconsider the timing of local control in the setting of primary bone tumors. In this report, we describe two cases in which upfront surgery was utilized and review rationale, prior literature, and potential benefits of this approach.

Increasing evidence has indicated that Wnt signaling plays complex roles in castration resistant prostate cancer (CRPC). Although not all data were consistent, β-catenin nuclear localization and its co-localization with androgen receptor (AR) were more frequently observed in CRPC compared to hormone naïve prostate cancer. This direct interaction between AR and β-catenin seemed to elicit a specific expression of a set of target genes in low androgen conditions in CRPC. Paracrine Wnt signaling also was shown to aid resistance to chemotherapy and androgen deprivation therapy. Results from the next generation sequencing studies (i.e. RNA-seq and whole exosome sequcing) of CRPC specimens have identified the Wnt pathway as one of the top signaling pathways with significant genomic alterations in CRPC, whereas, Wnt pathway alterations were virtually absent in hormone naïve primary prostate cancer. Furthermore, Wnt signaling has been suggested to play an important role in cancer stem cell functions in prostate cancer recurrence and resistance to androgen deprivation therapy. Therefore, in this review we have summarized existing knowledge regarding potential roles of Wnt signaling in CRPC and underline Wnt signaling as a potential therapeutic target for CRPC. Further understanding of Wnt signaling in castration resistance may eventually contribute new insights into possible treatment options for this incurable disease.

PurposeTo identify all available reconstruction methods for a total sacrectomy. Secondarily, we aimed to evaluate outcomes based on different interventions.MethodsWe searched PubMed to identify sacral resections for tumors requiring internal fixation for stabilization. Demographic information, fixation techniques and postoperative outcomes were abstracted.ResultsTwenty-three publications (43 patients) met inclusion criteria from an initial search of 856 (κ 0.93). Mean age was 37 years and follow-up was 33 months. Fixation methods included a combination of spinopelvic fixation (SPF), posterior pelvic ring fixation (PPRF), and/or anterior spinal column fixation (ASCF). For the purposes of analysis, patients were segregated based on whether they received ASCF. Postoperative complications including wound/instrument infections, GI or vascular complications were reported at a higher rate in the non-ASCF group (1.63 complications/patient vs. 0.7 complications/patient). Instrument failure was seen in 5 (16.1 %) out of the 31 patients with reported outcomes. Specifically, 1 out of 8 patients (12.5 %) with ASCF compared with 4 out of 23 patients (17.4 %) without ASCF had hardware failure. At final follow-up, 35 of 39 patients were ambulating.ConclusionWhile surgical treatment of primary sacral tumors remains a challenge, there have been advances in reconstruction techniques following total sacrectomy. SPF has shifted from intrapelvic rod and hook constructs to pedicle and iliac screw-rod systems for improved rigidity. PPRF and ASCF have adapted for deficiencies in the posterior ring and anterior column. A trend toward a lower rate of hardware failure emerged in the group utilizing anterior spinal column support. Despite a more involved reconstruction with ASCF, surgical complications such as infection rates and blood loss were lower compared to the group without ASCF. While we cannot definitively say one system is superior to the other, based on the data gleaned from this systematic review, it is our opinion that incorporation of ASCF in reconstructing the spinopelvic junction may lead to improved outcomes. However, most importantly, we recommend that the treating surgeon operate on patients requiring a total sacrectomy based on his or her level of comfort, as these cases can be extremely challenging even among experts.

Flavokawain A (FKA), a major chalcone in the Kava plant, has recently demonstrated promising anti-cancer activities. A systematic evaluation of FKA's safety profile has not been reported before. In this study, male FVB/N mice were fed with an AIN-76A diet or AIN-76A diet supplemented with 0.6% (6 g/kg food) FKA or 0.6% commercial kava root extract (KRE) for three weeks. Dietary feeding of FKA did not affect food consumption and body weight. Histopathological examination of liver, kidney, colon, lung, heart, spleen, and thymus revealed no signs of FKA-induced toxicity. Biochemical serum analysis and histological examination confirmed normal organ function in FKA-treated mice. The cytotoxicity profile showed FKA had minimal side effects on bone marrow and small intestinal epithelial cells compared with Adriamycin. In addition, oral feeding of FKA increased activities of both glutathione S-transferase and quinone reductase in the liver, lung, prostate and bladder tissues of mice. In comparison, dietary feeding of 0.6% KRE increased liver/body weight ratio and decreased spleen, thymus, and testis/body weight ratios, as well as induced nodular proliferation in liver tissues. Therefore, dietary feeding FKA showed no adverse effects on major organ function and homeostasis in mice, suggesting the potential of FKA for chemoprevention study of human cancers.

Abstract Background Flavokawain B (FKB) has been identified from kava root extracts as a potent apoptosis inducer for inhibiting the growth of various cancer cell lines, including prostate cancer. However, the molecular targets of FKB in prostate cancer cells remain unknown. Methods An in vitro NEDD8 Initiation Conjugation Assay was used to evaluate the neddylation inhibitory activity of FKB. Molecular docking and a cellular thermal shift assay were performed to assess the direct interaction between FKB and the NEDD8 activating enzyme (NAE) complex. Protein neddylation, ubiqutination, stability and expression in cells were assessed with immunoprecipitation and Western blotting methods using specific antibodies. Deletion and site specific mutants and siRNAs were used to evaluate deep mechanisms by which FKB induces Skp2 degradation. Cell growth inhibition and apoptosis induction were measured by MTT, ELISA and Western blotting methods. Results FKB inhibits NEDD8 conjugations to both Cullin1 and Ubc12 in prostate cancer cell lines and Ubc12 neddylation in an in vitro assay. Molecular docking study and a cellular thermal shift assay reveal that FKB interacts with the regulatory subunit (i.e. APP-BP1) of the NAE. In addition, FKB causes Skp2 degradation in an ubiquitin and proteasome dependent manner. Overexpression of dominant-negative cullin1 (1–452), K720R mutant (the neddylation site) Cullin1 or the F-box deleted Skp2 that losses its binding to the Skp1/Cullin1 complex causes the resistance to FKB-induced Skp2 degradation, whereas siRNA knock-down of Cdh1, a known E3 ligase of Skp2 for targeted degradation, didn’t attenuate the effect of FKB on Skp2 degradation. These results suggest that degradation of Skp2 by FKB is involved in a functional Cullin1. Furthermore, proteasome inhibitors Bortezomib and MG132 transcriptionally down-regulate the expression of Skp2, and their combinations with FKB result in enhanced inhibitory effects on the growth of prostate cancer cell lines via synergistic down-regulation of Skp2 and up-regulation of p27/Kip1 and p21/WAF1 protein expression. FKB also selectively inhibits the growth of RB deficient cells with high expression of Skp2. Conclusion These findings provide a rationale for further investigating combination of FKB and Bortezomib for treatment of RB deficient, castration-resistant prostate cancer.

Background. Six cell surface receptors, human epidermal growth factor receptor-2 (Her-2), platelet-derived growth factor receptor-β (PDGFR-β), insulin-like growth factor-1 receptor (IGF-1R), insulin receptor (IR), c-Met, and vascular endothelial growth factor receptor-3 (VEGFR-3), previously demonstrated variable expression across varying patient-derived and standard osteosarcoma (OS) cell lines. The current study sought to validate previous expression patterns and evaluate whether these receptors offer prognostic and/or therapeutic value. Methods. Patient-derived OS cell lines (n = 52) were labeled with antibodies to Her-2, PDGFR-β, IGF-1R, IR, c-Met, and VEGFR-3. Expression was characterized using flow cytometry. The difference in geometric mean fluorescent intensity (geoMFIdiff = geoMFIpositive − geoMFInegative) was calculated for each receptor across all cell lines. Receptor expression was categorized as low (Q1), intermediate (Q2, Q3), or high (Q4). The event-free survival (EFS) and overall survival for the six cell surface receptors were estimated by the Kaplan–Meier method. Differences in hazard for EFS event and overall survival event for patients in each of the three expression levels in each of the six cell surface receptors were assessed using the log-rank test. Results. All 6 receptors were variably expressed in the majority of cell lines. IR and PDGFR-β expressions were found to be significant predictors for EFS amongst patients with nonmetastatic disease (p=0.02 and 0.01, respectively). The hazard ratio for EFS was significantly higher between high IR and intermediate IR expression (HR = 2.66, p=0.02), as well as between high PDGFR-β and intermediate PDGFR-β expression (HR = 5.68, p=0.002). Her-2, c-Met, IGF-1R, and VEGFR-3 were not found to be significant predictors for either EFS or overall survival. Conclusion. The six cell surface receptors demonstrated variable expression across the majority of patient-derived OS cell lines tested. Limited prognostic value was offered by IR and PDGFR-β expression within nonmetastatic patients. The remaining receptors do not provide clear prognostic utility. Nevertheless, their consistent, albeit variable, surface expression across a large panel of patient-derived OS cell lines maintains their potential use as future therapeutic targets.

Abstract Background Osteosarcoma (OS) is the most common primary bone malignancy with a high propensity for local invasion and distant metastasis. Limited by the severe toxicity of conventional agents, the therapeutic bottleneck of osteosarcoma still remains unconquered. Flavokawain B (FKB), a kava extract, has been reported to have significant anti-tumor effects on several carcinoma cell lines both in vitro and in vivo. Its efficacy and low toxicity profile make FKB a promising agent for use as a novel chemotherapeutic agent. Results In the current study, we investigated the anti-proliferative and apoptotic effects of FKB against human osteosarcomas. Exposure of OS cells to FKB resulted in apoptosis, evidenced by loss of cell viability, morphological changes and the externalization of phosphatidylserine. Apoptosis induced by FKB resulted in activation of Caspase-3/7, -8 and −9 in OS cell lines, 143B and Saos-2. FKB also down-regulated inhibitory apoptotic markers, including Bcl-2 and Survivin and led to concomitant increases in apoptotic proteins, Bax, Puma and Fas. Therefore, the induction of apoptosis by FKB involved both extrinsic and intrinsic pathways. FKB also caused G2/M phase cell cycle arrest, which was observed through reductions in the levels of cyclin B1, cdc2 and cdc25c and increases in Myt1 levels. Furthermore, migration and invasion ability was decreased by FKB in a dose-dependent manner. The cytotoxicity profile showed FKB had significant lower side effects on bone marrow cells and small intestinal epithelial cells compared with Adriamycin. Conclusions Taken together, our evidence of apoptosis and cell cycle arrest by FKB treatment with less toxicity than the standard treatments provides an innovative argument for the use of FKB as a chemotherapeutic and chemopreventive compound. In vivo experiments utilizing FKB to reduce tumorigenesis and metastatic potential will be crucial to further justify clinical application.

Osteosarcoma (OS) is the most common primary bone malignancy with a high propensity for local invasion and distant metastasis. Despite current multidisciplinary treatments, there has not been a drastic change in overall prognosis within the past 2 decades. Dickkopf-3 protein (Dkk-3/REIC) has been known to inhibit canonical Wnt/β-catenin pathway, and its expression has been shown to be downregulated in OS cell lines. Using in vivo and in vitro studies, we demonstrated that Dkk-3-transfected 143B cells inhibited tumorigenesis and metastasis in an orthotopic xenograft model of OS. Inoculation of Dkk-3-transfected 143B cell lines into nude mice showed significant decreased tumor growth and less metastatic pulmonary nodules (88.7%) compared to the control vector. In vitro experiments examining cellular motility and viability demonstrated less anchorage-independent growth and decreased cellular motility for Dkk-3-transfected 143B and SaOS2 cell lines compared to the control vector. Downstream expressions of Met, MAPK, ALK, and S1004A were also downregulated in Dkk-3-transfected SaOS2 cells, suggesting the ability of Dkk-3 to inhibit tumorigenic potential of OS. Together, these data suggest that Dkk-3 has a negative impact on the progression of osteosarcoma. Reexpressing Dkk-3 in Dkk-3-deficient OS tumors may prove to be of benefit as a preventive or therapeutic strategy.

Synovial sarcoma (SS) is an aggressive soft-tissue cancer with a poor prognosis and a propensity for local recurrence and distant metastasis. In this study, we investigated whether S phase kinase-associated protein (Skp2) plays an oncogenic role in tumor initiation, progression, and metastasis of SS. Our study revealed that Skp2 is frequently overexpressed in SS specimens and SS18-SSX transgenic mouse tumors, as well as correlated with clinical stages. Next, we identified that genetic depletion of Skp2 reduced mesenchymal and stemness markers, and inhibited the invasive and proliferative capacities of SS cell lines. Furthermore, Skp2 depletion markedly suppressed the growth of SS xenografts tumors. Treatment of SS cell lines with the skp2 inhibitor flavokawain A (FKA) reduced Skp2 expression in a dose-dependent manner and resulted in cell cycle arrest and apoptosis. FKA also suppressed the invasion and tumor-initiating properties in SS, similar to the effects of Skp2 knockdown. In addition, a combination of FKA and conventional chemotherapy showed a synergistic therapeutic efficacy. Taken together, our results suggest that Skp2 plays an essential role in the biology of SS by promoting the mesenchymal state and cancer stemness. Given that chemotherapy resistance is often associated with cancer stemness, strategies of combining Skp2 inhibitors with conventional chemotherapy in SS may be desirable.

Abstract Background Aberrations in the Wnt pathway have been reported to be involved in the metastasis of prostate cancer (PCa) to bone. We investigated the effect and underlying mechanism of a naturally-occurring Wnt inhibitor, WIF1, on the growth and cellular invasiveness of a bone metastatic PCa cell line, PC3. Results The WIF1 gene promoter was hypermethylated and its expression down-regulated in the majority (7 of 8) of PCa cell lines. Restoration of WIF1 expression in PC-3 cells resulted in a decreased cell motility and invasiveness via up-regulation of epithelial markers (E-cadherin, Keratin-8 and-18), down-regulation of mesenchymal markers (N-cadherin, Fibronectin and Vimentin) and decreased activity of MMP-2 and -9. PC3 cells transfected with WIF1 consistently demonstrated reduced expression of Epithelial-to-Mesenchymal Transition (EMT) transcription factors, Slug and Twist, and a change in morphology from mesenchymal to epithelial. Moreover, WIF1 expression significantly reduced tumor growth by approximately 63% in a xenograft mouse model. This was accompanied by an increased expression of E-cadherin and Keratin-18 and a decreased expression of vimentin in tumor tissues. Conclusion These data suggest that WIF1 regulates tumor invasion through EMT process and thus, may play an important role in controlling metastatic disease in PCa patients. Blocking Wnt signaling in PCa by WIF1 may represent a novel strategy in the future to reduce metastatic disease burden in PCa patients.

BackgroundMethotrexate (MTX) uptake is mediated by the reduced folate carrier (RFC). Defective drug uptake in association with decreased RFC expression is a common mechanism of MTX resistance in many tumor types. Heavy promoter methylation was previously identified as a basis for the complete silencing of RFC in MDA-MB-231 breast cancer cells, its role and prevalence in RFC transcription regulation are, however, not widely studied.MethodsIn the current study, RFC promoter methylation was assessed using methylation specific PCR in a panel of malignant cell lines (n = 8), including MDA-MB-231, and M805, a MTX resistant cell line directly established from the specimen of a patient with malignant fibrohistocytoma, whom received multiple doses of MTX. A quantitative approach of real-time PCR for measuring the extent of RFC promoter methylation was developed, and was validated by direct bisulfite genomic sequencing. RFC mRNA levels were determined by quantitative real-time RT-PCR and were related to the extent of promoter methylation in these cell lines.ResultsA partial promoter methylation and RFC mRNA down-regulation were observed in M805. Using the quantitative approach, a reverse correlation (correlation coefficient = -0.59, p < 0.05) was identified between the promoter methylation and RFC mRNA levels in this a panel of malignant cell lines.ConclusionThis study further suggests that promoter methylation is a potential basis for MTX resistance. The quantitative correlation identified in this study implies that promoter methylation is possibly a mechanism involved in the fine regulation of RFC transcription.

Previous reports document expression of low-density lipoprotein receptor-related protein 5 (LRP5) in osteosarcoma (OS) tissue. Expression of this Wnt receptor correlated with metastatic disease and poor disease-free survival. Forced expression of dominant-negative LRP5 (dnLRP5), which lacks the membrane binding domain of the native protein and therefore functions as a soluble receptor-sponge for Wnt ligands, reduced in vitro cellular invasion and in vivo xenograft tumor growth for osteosarcoma cell lines. Here, we use a genetically engineered mouse model of osteosarcomagenesis with and without expression of dnLRP5 to assess to what degree tumorigenesis is affected and whether Wnt/β-catenin signaling is circumvented or maintained. Each cohort of mice developed osteosarcoma at a similar ultimate prevalence, but after a slightly increased latency in those also expressing dnLRP5. On histology, there was no difference between groups, despite previous reports that the dnLRP5 osteosarcoma cells specifically undergo a mesenchymal-to-epithelial transition in vitro. Finally, immunohistochemistry showed the presence of cytosolic and nuclear β-catenin and nuclear Cyclin D1, markers consistent with preserved Wnt/β-catenin signaling despite constitutive blockade of the cell surface receipt of Wnt signaling ligand. These data suggest that canonical Wnt signaling plays a role in OS progression and that while blockade of singular nodes in signaling pathways can have dramatic effects on individual cell lines, real tumors readily evade such focused attacks.

Flavokawain A (FKA), a major chalcone in the Kava plant, has recently demonstrated promising anti-cancer activities. A systematic evaluation of FKA's safety profile has not been reported before. In this study, male FVB/N mice were fed with an AIN-76A diet or AIN-76A diet supplemented with 0.6% (6 g/kg food) FKA or 0.6% commercial kava root extract (KRE) for three weeks. Dietary feeding of FKA did not affect food consumption and body weight. Histopathological examination of liver, kidney, colon, lung, heart, spleen, and thymus revealed no signs of FKA-induced toxicity. Biochemical serum analysis and histological examination confirmed normal organ function in FKA-treated mice. The cytotoxicity profile showed FKA had minimal side effects on bone marrow and small intestinal epithelial cells compared with Adriamycin. In addition, oral feeding of FKA increased activities of both glutathione S-transferase and quinone reductase in the liver, lung, prostate and bladder tissues of mice. In comparison, dietary feeding of 0.6% KRE increased liver/body weight ratio and decreased spleen, thymus, and testis/body weight ratios, as well as induced nodular proliferation in liver tissues. Therefore, dietary feeding FKA showed no adverse effects on major organ function and homeostasis in mice, suggesting the potential of FKA for chemoprevention study of human cancers.

Myung Joo Kang1, Jae Yoon Eum1, Mi Sook Jeong2, Sang Han Park1, Ki Young Moon1, Mean Hyung Kang1, Min Soo Kim1, Sun Eun Choi1, Min Won Lee1, Do Ik Lee1, Hyoweon Bang2, Chung Soo Lee2, Seong Soo Joo3, Kapsok Li2, Mi-Kyung Lee2, Seong Jun Seo2, Young Wook Choi11College of Pharmacy, ChungAng University, Heuksuk-dong, Dongjak-gu, Seoul, 2College of Medicine, Chung-Ang University, Heuksukdong, Dongjak-gu, Seoul, 3Division of Marine Molecular Biotechnology, Gangneung-Wonju National University, Gangneung, South KoreaBackground: The aim of the present study was to enhance a topical delivery of hirsutenone (HST), a naturally occuring immunomodulator, employing Tat peptide-admixed elastic liposomes (EL/T).Methods: HST-loaded EL, consisting of phosphatidylcholine and Tween 80 (85:15 w/w%), were prepared using thin film hydration method. By adding Tat peptide to EL (0.16 w/w%), EL/T were formulated. The in vitro skin permeation of HST was examined using a Franz diffusion cell mounted with depilated mouse skin. Lesions for atopic dermatitis (AD) were induced by a topical application of diphenylcyclopropenone to NC/Nga mice. Therapeutic improvements of AD were evaluated by clinical skin severity scores. Immunological analyses on inducible nitric oxide synthase and cyclooxygenase-2 levels in the skin and interleukin (IL)-4, IL-13, immunoglobulin E, and eosinophil levels in the blood were also performed.Results: EL systems were superior to conventional cream, revealing greater flux values in a permeation study. The addition of Tat peptide further increased the skin permeation of HST. In an efficacy study with AD-induced NC/Nga mice, an HST-containing EL/T formulation brought a significant improvement in both skin severity score and immune-related responses for the levels of nitric oxide synthase, cyclooxygenase-2, IL-4, IL-13, immunoglobulin E, and eosinophils.Conclusion: A novel EL/T formulation was successfully developed for topical delivery of HST to treat AD.Keywords: hirsutenone, elastic liposomes, atopic dermatitis, NC/Nga mice, Tat peptide

Fucoidans from brown macroalgae have beneficial biomedical properties but their use as pharma products requires homogenous oligomeric products. In this study, the action of five recombinant microbial fucoidan degrading enzymes were evaluated on fucoidans from brown macroalgae: Sargassum mcclurei, Fucus evanescens, Fucus vesiculosus, Turbinaria ornata, Saccharina cichorioides, and Undaria pinnatifida. The enzymes included three endo-fucoidanases (EC 3.2.1.-GH 107), FcnA2, Fda1, and Fda2, and two unclassified endo-fucoglucuronomannan lyases, FdlA and FdlB. The oligosaccharide product profiles were assessed by carbohydrate-polyacrylamide gel electrophoresis and size exclusion chromatography. The recombinant enzymes FcnA2, Fda1, and Fda2 were unstable but were stabilised by truncation of the C-terminal end (removing up to 40% of the enzyme sequence). All five enzymes catalysed degradation of fucoidans containing α(1→4)-linked l-fucosyls. Fda2 also degraded S. cichorioides and U. pinnatifida fucoidans that have α(1→3)-linked l-fucosyls in their backbone. In the stabilised form, Fda1 also cleaved α(1→3) bonds. For the first time, we also show that several enzymes catalyse degradation of S. mcclurei galactofucan-fucoidan, known to contain α(1→4) and α(1→3) linked l-fucosyls and galactosyl-β(1→3) bonds in the backbone. These data enhance our understanding of fucoidan degrading enzymes and their substrate preferences and may assist development of enzyme-assisted production of defined fuco-oligosaccharides from fucoidan substrates.

HER2/neu positive breast tumors predict a high mortality and comprise 25%–30% of breast cancer. We have shown that Flavokawain A (FKA) preferentially reduces the viabilities of HER2-overexpressing breast cancer cell lines (i.e., SKBR3 and MCF7/HER2) versus those with less HER2 expression (i.e., MCF7 and MDA-MB-468). FKA at cytotoxic concentrations to breast cancer cell lines also has a minimal effect on the growth of non-malignant breast epithelial MCF10A cells. FKA induces G2M arrest in cell cycle progression of HER2-overexpressing breast cancer cell lines through inhibition of Cdc2 and Cdc25C phosphorylation and downregulation of expression of Myt1 and Wee1 leading to increased Cdc2 kinase activities. In addition, FKA induces apoptosis in SKBR3 cells by increasing the protein expression of Bim and BAX and decreasing expression of Bcl2, BclX/L, XIAP, and survivin. FKA also downregulates the protein expression of HER-2 and inhibits AKT phosphorylation. Herceptin plus FKA treatment leads to an enhanced growth inhibitory effect on HER-2 overexpressing breast cancer cell lines through downregulation of Myt1, Wee1, Skp2, survivin, and XIAP. Our results suggest FKA as a promising and novel apoptosis inducer and G2 blocking agent that, in combination with Herceptin, enhances for the treatment of HER2-overexpressing breast cancer.