An activity-based functional test for identifying homologous recombination deficiencies across cancer types in real time.

Homologous recombination (HR)-mediated DNA repair is a prerequisite for maintaining genome stability. Cancer cells displaying HR deficiency (HRD) are selectively eliminated by poly(ADP-ribose) polymerase inhibitors (PARPis). To date, sequencing of HR-associated genes and analyzing genome instability have been used as clinical predictions for PARPi therapy. However, these genetic tests cannot reflect dynamic changes in the HR status. Here, we have developed a virus- and activity-based functional assay to quantify real-time HR activity directly. Instead of focusing on a few HR-associated genes, our functional assay detects endpoint HR activity and establishes an activity threshold for identifying HRD across cancer types, validated by PARPi sensitivity and BRCA status. Notably, this fluorescence-based assay can be applied to primary ovarian cancer cells from patients to reflect their level of HRD, which is associated with survival benefits. Thus, our work provides a functional test to predict the response of primary cancer cells to PARPis.

Hyperphosphorylation-Mimetic TDP-43 Drives Amyloid Formation and Possesses Neuronal Toxicity at the Oligomeric Stage.

TDP-43 proteinopathies cover a range of neurodegenerative diseases, including frontotemporal lobar degeneration (FTLD) and amyotrophic lateral sclerosis (ALS). Hyperphosphorylated TDP-43 was found within the inclusion bodies in disease lesions; however, the role of hyperphosphorylation and the toxic species are still ambiguous. To characterize the hyperphosphorylation effect of TDP-43, here, we employed five serine mutations implicated in the diseases at serine locations 379, 403, 404, 409, and 410 in the C-terminus to aspartate (S5D) and to alanine (S5A). We systematically characterized the conformation, liquid-liquid phase separation, oligomerization, and fibrillization of TDP-43 variants. Results revealed that the recombinant TDP-43 variants readily formed structurally similar spherical oligomers, as evidenced by circular dichroism spectroscopy, fluorescence spectroscopy, the TDP-43 oligomer-specific antibody assay, dynamic light scattering, and transmission electron microscopy. After incubation, only the phosphor-mimic S5D TDP-43 formed thioflavin-positive amyloid fibrils, whereas wild-type and S5A TDP-43 formed amorphous aggregates. We also examined membrane disruption, the cytotoxicity of human neuroblastoma, and the synaptic loss of primary neurons induced by oligomers and large aggregates of TDP-43. The results showed that all oligomeric TDP-43 variants were toxic regardless of hyperphosphorylation, but the fibrils and amorphous aggregates were not. Overall, our results demonstrated the hyperphosphorylation effect on fibril formation and the toxicity attributed from TDP-43 oligomers. This study facilitates the understanding and therapeutic development for TDP-43 proteinopathies.

Aspalathus linearis suppresses cell survival and proliferation of enzalutamide-resistant prostate cancer cells via inhibition of c-Myc and stability of androgen receptor.

Enzalutamide, a nonsteroidal antiandrogen, significantly prolonged the survival of patients with metastatic castration-resistant prostate cancer (CRPC). However, patients receiving enzalutamide frequently develop drug resistance. Rooibos (Aspalathus linearis) is a shrub-like leguminous fynbos plant endemic to the Cedarberg Mountains area in South Africa. We evaluated the possibility of using a pharmaceutical-grade green rooibos extract (GRT, containing 12.78% aspalathin) to suppress the proliferation and survival of enzalutamide-resistant prostate cancer (PCa) cells. Treatment with GRT dose-dependently suppressed the proliferation, survival, and colony formation of enzalutamide-resistant C4-2 MDV3100r cells and PC-3 cells. Non-cancerous human cells were more resistant to GRT treatment. GRT suppressed the expression of proteins involved in phosphoinositide 3-kinase (PI3K)-Akt signaling, androgen receptor (AR), phospho-AR (Ser81), cyclin-dependent kinase 1 (Cdk1), c-Myc and Bcl-2 but increased the expression of apoptotic proteins. Overexpression of c-Myc antagonized the suppressive effects of GRT, while knockdown of c-Myc increased the sensitivity of PCa cells to GRT treatment. Expression level of c-Myc correlated to resistance of PCa cells to GRT treatment. Additionally, immunofluorescence microscopy demonstrated that GRT reduced the abundance of AR proteins both in nucleus and cytoplasm. Treatment with cycloheximide revealed that GRT reduced the stability of AR. GRT suppressed protein expression of AR and AR's downstream target prostate specific antigen (PSA) in C4-2 MDV3100r cells. Interestingly, we observed that AR proteins accumulate in nucleus and PSA expression is activated in the AR-positive enzalutamide-resistant PCa cells even in the absence of androgen. Our results suggested that GRT treatment suppressed the cell proliferation and survival of enzalutamide-resistant PCa cells via inhibition of c-Myc, induction of apoptosis, as well as the suppression of expression, signaling and stability of AR. GRT is a potential adjuvant therapeutic agent for enzalutamide-resistant PCa.

Combination treatment of docetaxel with caffeic acid phenethyl ester suppresses the survival and the proliferation of docetaxel-resistant prostate cancer cells via induction of apoptosis and metabolism interference.

BACKGROUND: Docetaxel has been approved by USFDA as a first-line treatment for castration-resistant prostate cancer (CRPC) patients. Patients receiving androgen deprivation therapy along with docetaxel result in superior survival, lower serum prostate specific antigen (PSA) level, and better quality of life. However, a significant proportion of these patients ultimately develop resistance to docetaxel within months. Caffeic acid phenethyl ester (CAPE), one of the main bioactive components extracted from the propolis, has been reported to be effective for repressing the tumor growth, the migration and invasion of prostate cancer (PCa) cells, as well as the downstream signaling and stability of androgen receptor (AR). We hence determined if combination treatment of docetaxel with CAPE can suppress the proliferation and the survival of docetaxel-resistant PCa cells. METHODS: We established docetaxel-resistant PC/DX25 and DU/DX50 CRPC cell lines from PC-3 and DU-145 human PCa cells, respectively. Proliferation assay, MTT assay, flow cytometry with Annexin V staining, Comet Assay, and nude mice xenograft model were applied to determine the effects of combination treatment on cell proliferation and survival of the docetaxel-resistant PCa cells. Micro-Western Array (MWA) and qRT-PCR were used to investigate the molecular mechanism lying underneath. RESULTS: Combination treatment effectively suppressed the proliferation, survival and tumor growth of docetaxel-resistant PCa cells both in vitro and in nude mice. Comet assay and flow cytometry indicated that combination treatment induced apoptosis in docetaxel-resistant PCa cells. MWA and Western blotting assay revealed that combination treatment suppressed protein expression of Bcl-2, AKT2, c-Myc, apoptosis and caspase activation inhibitor (AVEN), pyruvate kinase M2 (PKM2) but increased protein expression of Bax, caspase 3, cytochrome c, glucose-6-phosphate dehydrogenase (G6PD) and acylglycerol kinase (AGK). Overexpression of Bcl-2 in the docetaxel-resistant PCa cells enhanced cell proliferation of docetaxel-resistant PCa cells under combination treatment. Analysis with qRT-PCR suggested that combination treatment decreased cholesterol biosynthesis genes DHCR24 (24-dehydrocholesterol reductase) and LSS (lanosterol synthase) but increased genes involved in glycolysis and TCA cycle. CONCLUSIONS: Combination treatment of docetaxel with CAPE effectively suppressed the proliferation and survival of docetaxel-resistant PCa cells via inhibition of Bcl-2 and c-Myc as well as induction of metabolism interference. Combination treatment can be beneficial for patients with docetaxel-resistant PCa.

Betulinic acid induces autophagy-dependent apoptosis via Bmi-1/ROS/AMPK-mTOR-ULK1 axis in human bladder cancer cells.

Betulinic acid (BA), a pentacyclic triterpenoid isolated from tree bark, exhibits antitumor effects against solid malignancies and triggers autophagy and/or apoptosis in human cancer cells. Nonetheless, the relationship between autophagy and apoptosis and the potential modulatory actions of BA on autophagy-dependent bladder cancer cell death remain unclear. The present study showed that BA exposure significantly suppressed viability, proliferation, and migration of EJ and T24 human bladder cancer cells. These effects reflected caspase 3-mediated apoptosis and could be attenuated or abolished by inhibiting ROS production with N-acetyl-L-cysteine, inhibiting autophagy with chloroquine, or silencing ATG7 with targeted siRNA. BA-induced autophagy was evidenced by epifluorescence imaging of lentivirus-induced expression of mCherry-GFP-LC3B and increased expression of two autophagy-related proteins, LC3B-II and TEM. Moreover, enhanced AMPK phosphorylation and decreased mTOR and ULK-1 phosphorylation suggested BA activates autophagy via the AMPK/mTOR/ULK1 pathway. Accordingly, exposure to dorsomorphin (Compound C), an AMPK inhibitor, and AICAR, an AMPK activator, respectively inhibited and stimulated BA-induced autophagy in EJ and T24 cells. The effects of Bmi-1 overexpression in vitro and decreased Bmi-1 expression in BA-treated T24 cell xenografts in nude mice suggested that downregulation of Bmi-1 is the underlying mechanism in BA-mediated, autophagy-dependent apoptosis.

ROR2 suppresses metastasis of prostate cancer via regulation of miR-199a-5p-PIAS3-AKT2 signaling axis.

Bones are the most common metastatic sites for prostate cancer (PCa). Receptor tyrosine kinase-like orphan receptor 2 (ROR2), a noncanonical Wnt receptor, plays crucial roles in skeletal morphogenesis, osteoblast differentiation, and bone formation. The role of ROR2 in PCa metastasis is unclear. We analyzed online datasets from Oncomine as well as using IHC staining on tissue array to determine the relationship between ROR2 expression level and disease outcome of PCa. To investigate how ROR2 regulates migration and invasion of PCa cells, we performed transwell assay and orthotopic xenograft model in nude mice. We then applied the Micro-Western Array (MWA), a high-throughput western blotting platform to analyze the downstream signaling pathways being regulated by ROR2. Compared with nonmalignant PZ-HPV-7 and RWPE-1 cells, PCa cell lines express lower level of ROR2 protein. Constitutive expression of ROR2 in PC-3, DU-145, or C4-2B PCa cells significantly suppressed the cell migration, invasion, and epithelial-mesenchymal transition (EMT) proteins. MWA, western blotting, and microRNA analysis showed that elevation of ROR2 suppressed the expression of miR-199a-5p, which in turn increased the expression of PIAS3. The upregulation of PIAS3 then decreased AKT2 and the phosphorylation of AKT, resulting in the inhibition of migration and invasion of PCa cells both in vitro and in orthotopic xenograft mice model. IHC staining of tissue array and Oncomine datasets analysis indicated that the gene and protein level of ROR2 is much lower in metastatic prostate tumors as compared with primary tumors or adjacent normal prostate tissues. Low level of ROR2 correlated to poor survival and high recurrent frequency in PCa patients. In conclusion, we discovered that ROR2 suppresses PCa metastasis via regulation of PIAS3-PI3K-AKT2 signaling axis.

Aspalathin-rich green Aspalathus linearis extract suppresses migration and invasion of human castration-resistant prostate cancer cells via inhibition of YAP signaling.

BACKGROUND: More than 80% of advanced prostate cancer (PCa) cases have bone metastasis, with a 5-year survival rate of 25%. Previously, we reported that GRT, a standardized, pharmaceutical-grade aspalathin-rich extract (12.78 g aspalathin/100 g extract), prepared from green rooibos produced from the leaves and fine stems of Aspalathus linearis, inhibits the proliferation of PCa cells, meriting this investigation to determine if GRT can suppress the migration and invasion of castration-resistant prostate cancer (CRPC) cells. PURPOSE: In the present study, we investigated whether GRT extract can interfere with the migration and invasion of human CRPC cells. METHODS: Transwell assays were used to explore the effects of GRT on the migration and invasion of CRPC cells. Micro-Western Array (MWA) and Western blot analysis were carried out to unravel the underlying molecular mechanism(s). RESULTS: Treatment with 25-100 µg/ml GRT suppressed the migration and invasion of LNCaP C4-2B and 22Rv1 CRPC cells. MWA and Western blot analysis indicated that GRT treatment suppressed the protein level of yes-associated protein (YAP), macrophage stimulating 1 protein (MST1), phospho-MST1/phospho-MST2 T183/T180, and paxillin, but increased the abundance of E-cadherin. Over-expression of YAP rescued the suppressive effects of GRT on migration and invasion of CRPC cells. Treatment with the major flavonoid of GRT - the C-glucosyl dihydrochalcone, aspalathin - at a concentration of 75-100 µg/ml also reduced the migration and invasion of CRPC cells, and the inhibition was partially rescued by YAP over-expression. CONCLUSIONS: GRT treatment suppresses the migration and invasion of CRPC cells via inhibition of YAP signaling and paxillin.

Rooibos suppresses proliferation of castration-resistant prostate cancer cells via inhibition of Akt signaling.

BACKGROUND: Androgen ablation therapy is the primary treatment for metastatic prostate cancer (PCa). However, the majority of PCa patients receiving the androgen deprivation therapy develop recurrent castration-resistant prostate cancer (CRPC) within two years. Chemotherapies show little effect on prolonging survival of CRPC patients and new treatments are needed. Previous studies reported that the extracts from rooibos (Aspalathus linearis) exhibit chemopreventive properties in some cancer models, including skin, liver and oesophagus cancers in animals. We therefore investigate if extracts from rooibos can suppress the proliferation of CRPC cells. PURPOSE: We investigated whether an aspalathin-rich green rooibos extract (GRT™; 12.78 g aspalathin/100 g extract) demonstrates anti-cancer activity against CRPC cells. METHODS: High performance liquid chromatography (HPLC) was used to profile the major flavonoids in GRT. Hoechst-dye proliferation assay, 3,4,5-dimethylthiazol-2-yl)-2-5-diphenyltetrazolium bromide (MTT) viability assay and flow cytometry assay were used to explore the effects of GRT on the proliferation and cell cycle progression of CRPC cells. Comet assay was used to survey whether GRT induces apoptosis in CRPC cells. LNCaP 104-R1 xenograft nude mice model was used to determine the inhibitory effect of GRT on CRPC tumors in vivo. Micro-Western Array (MWA) and Western blot analysis were carried out to unravel the underlying molecular mechanism. RESULTS: GRT contained aspalathin as the most abundant flavonoid. GRT suppressed the proliferation and survival of LNCaP 104-R1, LNCaP FGC and PC-3 PCa cells. Flow cytometry analysis showed that GRT decreased the population of PCa cells in S phase but increased the cell population in G2/M phase. Comet assay confirmed that GRT induced apoptosis in LNCaP 104-R1 cells. Gavage of 400 mg/kg GRT suppressed LNCaP 104-R1 xenografts in castrated nude mice. MWA and Western blot analysis indicated that GRT treatment suppressed Akt1, phospho-Akt Ser473, Cdc2, Bcl-2, TRAF4 and Aven, but increased activated Caspase 3, cytochrome c, and p27Kip1. Overexpression of Akt rescued the suppressive effects of GRT on CRPC cells. Co-treatment of GRT with Bcl-2 inhibitor ABT-737, PI3K inhibitor LY294002 and Akt inhibitor GSK 690693 exhibited additive inhibitory effect on proliferation of CRPC cells. CONCLUSIONS: GRT suppresses the proliferation of CRPC cells via inhibition of Akt signaling.

The effect of increased frequency of hemodialysis on serum cystatin C and ß2-microglobulin concentrations: A secondary analysis of the frequent hemodialysis network (FHN) trial.

INTRODUCTION: Small molecular weight toxin clearance is the main method of assessment of hemodialysis efficiency. Middle molecules including cystatin C (CysC) and Beta-2 microglobulin (ß2-M) are understudied. We hypothesized that lowering of predialysis CysC and ß2-M serum concentrations would be affected by switching to more frequent hemodialysis. METHODS: Predialysis CysC and ß2-M serum concentrations were measured from serum samples of the Frequent Hemodialysis Network (FHN) Daily and Nocturnal Trials. The differences between predialysis concentrations at baseline (while on conventional thrice weekly dialysis) and those after 12-months of study (on more frequent dialysis) were compared separately by trial (Nocturnal, Daily). We tested the associations between predialysis serum CysC and ß2-M concentrations and outcomes. FINDINGS: Forty-nine percent and 52% of the patients from the FHN Daily and Nocturnal Trials respectively were included in this ancillary study. Predialysis serum CysC concentrations remained unchanged after intensifying hemodialysis dose by either modality. There was significant lowering of the serum ß2-M concentrations in the frequent Daily Trial hemodialysis group at 12 months in all patients and in patients without residual renal function at baseline (-3.8 ± 12.62 µg/mL, P = 0.004; -5.9 ± 12.99 µg/mL, P = 0.02, respectively). There were no significant differences between the baseline and the 12-months predialysis ß2-M serum concentrations in the two control groups (Daily 3× and Nocturnal 3× groups). No association between the changes in the two biomarkers between baseline and 12-months and in changes in left ventricular mass, physical-health composite scores, hospitalization rate, and death were found. The numbers of hospitalizations and deaths were small. DISCUSSION: ß2-M may be a better biomarker of dialysis dose than CysC. Reduction in the concentration of potentially toxic long-lived proteins of the size of ß-2M is one potential long-term benefit of more intensive dialysis that may be explored.

KDM8/JMJD5 as a dual coactivator of AR and PKM2 integrates AR/EZH2 network and tumor metabolism in CRPC.

During the evolution into castration or therapy resistance, prostate cancer cells reprogram the androgen responses to cope with the diminishing level of androgens, and undergo metabolic adaption to the nutritionally deprived and hypoxia conditions. AR (androgen receptor) and PKM2 (pyruvate kinase M2) have key roles in these processes. We report in this study, KDM8/JMJD5, a histone lysine demethylase/dioxygnase, exhibits a novel property as a dual coactivator of AR and PKM2 and as such, it is a potent inducer of castration and therapy resistance. Previously, we showed that KDM8 is involved in the regulation of cell cycle and tumor metabolism in breast cancer cells. Its role in prostate cancer has not been explored. Here, we show that KDM8's oncogenic properties in prostate cancer come from its direct interaction (1) with AR to affect androgen response and (2) with PKM2 to regulate tumor metabolism. The interaction with AR leads to the elevated expression of androgen response genes in androgen-deprived conditions. They include ANCCA/ATAD2 and EZH2, which are directly targeted by KDM8 and involved in sustaining the survival of the cells under hormone-deprived conditions. Notably, in enzalutamide-resistant cells, the expressions of both KDM8 and EZH2 are further elevated, so are neuroendocrine markers. Consequently, EZH2 inhibitors or KDM8 knockdown both resensitize the cells toward enzalutamide. In the cytosol, KDM8 associates with PKM2, the gatekeeper of pyruvate flux and translocates PKM2 into the nucleus, where the KDM8/PKM2 complex serves as a coactivator of HIF-1α to upregulate glycolytic genes. Using shRNA knockdown, we validate KDM8's functions as a regulator for both androgen-responsive and metabolic genes. KDM8 thus presents itself as an ideal therapeutic target for metabolic adaptation and castration-resistance of prostate cancer cells.

Activation of liver X receptor suppresses angiogenesis via induction of ApoD.

Liver X receptors (LXRs) are important sensors and regulators for cholesterol, fatty acid, and glucose. LXRs play essential roles in the development and progression of cardiovascular diseases. We examined the effects of T0901317, a potent LXR agonist, on angiogenesis of human umbilical vein endothelial cells (HUVECs). Treatment with T0901317 inhibited the tube formation and migration of HUVECs and reduced the in vivo angiogenesis, as determined by chorioallantoic membrane assay. T0901317 stimulated gene and protein expression of LXR target gene apolipoprotein D (ApoD). Overexpression of ApoD suppressed the tube formation of HUVECs. ApoD interacted with scavenger receptor class B member 1 (SR-B1), while knockdown of SR-B1 blocked suppressive effects of T0901317 on HUVEC migration. T0901317 treatment or overexpression of ApoD lessened expression of proteins regulating angiogenesis, including phospho-eNOS S1177, phospho-Akt T308, phospho-Akt S473, eNOS, mammalian target of rapamycin, VEGF-A, VEGF-C, IL-8, RhoB, matrix metalloproteinase (MMP)-8, -9, and monocyte chemoattractant protein 1. Our study suggested that activation of LXR interferes with angiogenesis through induction of LXR target gene ApoD, which in turn suppresses PI3K-Akt-eNOS signaling, an essential pathway regulating angiogenesis. ApoD may be a potential therapeutic target for tumor angiogenesis.-Lai, C.-J., Cheng, H.-C., Lin, C.-Y., Huang, S.-H., Chen, T.-H., Chung, C.-J., Chang, C.-H., Wang, H.-D., Chuu, C.-P. Activation of liver X receptor suppresses angiogenesis via induction of ApoD.

A cross-sectional study measuring vanadium and chromium levels in paediatric patients with CKD.

OBJECTIVES: Although many secondary effects of high levels of vanadium (V) and chromium (Cr) overlap with symptoms seen in paediatric patients with chronic kidney disease (CKD), their plasma V and Cr levels are understudied. DESIGN: Ancillary cross-sectional study to a prospective, longitudinal, randomised controlled trial. SETTING: Children's Hospital of Western Ontario, London Health Sciences Centre, London, Ontario, Canada. PARTICIPANTS: 36 children and adolescents 4-18 years of age with CKD. INTERVENTIONS: 1-6 trace element measurements per patient. Cystatin C (CysC) estimated glomerular filtration rate (eGFR) was calculated using the Filler formula. Plasma V and Cr levels were measured using high-resolution sector field inductively coupled mass spectrometry. Anthropomorphic data and blood parameters were collected from our electronic chart programme. Water Cr and V data were obtained from the Ontario Water (Stream) Quality Monitoring Network. PRIMARY AND SECONDARY OUTCOME MEASURES: Primary outcomes: plasma Cr and V. SECONDARY OUTCOMES: age, season, CysC, CysC eGFR, and Cr and V levels in environmental water. RESULTS: The median (IQR) eGFR was 51 mL/min/1.73 m2 (35, 75). The median V level was 0.12 µg/L (0.09, 0.18), which was significantly greater than the 97.5th percentile of the reference interval of 0.088 µg/L; 32 patients had at least one set of V levels above the published reference interval. The median Cr level was 0.43 µg/L (0.36, 0.54), which was also significantly greater than the established reference interval; 34 had at least one set of Cr levels above the published reference interval. V and Cr levels were moderately correlated. Only some patients had high environmental exposure. CONCLUSIONS: Our study suggests that paediatric patients with CKD have elevated plasma levels of V and Cr. This may be the result of both environmental exposure and a low eGFR. It may be necessary to monitor V and Cr levels in patients with an eGFR <30 mL/min/1.73 m2. TRIAL REGISTRATION NUMBER: NCT02126293; HC#172241.

High prevalence of elevated molybdenum levels in pediatric CKD patients. A cross-sectional and longitudinal study
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AIMS: Many of the secondary effects of high levels of molybdenum (Mo) overlap with symptoms commonly seen in pediatric patients with chronic kidney disease (CKD). We measured plasma Mo levels and examined the relationship between Mo levels and kidney function. MATERIALS AND METHODS: The study was carried out at the London Health Sciences Centre in London, Ontario, Canada with 36 children and adolescents 4 - 18 years of age with CKD. There were 1 - 6 trace element measurements (Mo and copper (Cu)) per patient. We studied the proportion of patients with abnormal trace element levels and the relationship between trace element levels and estimated glomerular filtration rate (eGFR), calculated using the Filler formula. Plasma Mo and Cu levels were measured using High Resolution Sector Field Inductively Coupled Mass Spectrometry. Anthropomorphic data and blood parameters were collected from our electronic chart program. RESULTS: Median eGFR was 51 mL/min/1.73m2 (35, 75). Median Mo level was 2.00 µg/L (1.40, 2.88). 20 patients had at least one set of Mo levels above the published reference interval in either unit, and the results of 46% of the tests were above the interval. There was a strong negative correlation between the Mo levels and the eGFR (Spearman's r = -0.627, p < 0.0001). CONCLUSIONS: Our study suggests that pediatric patients with CKD have elevated plasma levels of Mo, which may cause secondary effects commonly associated with CKD. The elevated Mo levels in our center's catchment area may cause an accumulation of this trace element in patients with impaired renal function.
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Duchesnea indica extract suppresses the migration of human lung adenocarcinoma cells by inhibiting epithelial-mesenchymal transition.

Epithelial-mesenchymal transition (EMT) is a process through which epithelial cells are transformed into mesenchymal cells; EMT diminishes cell polarity and cell-cell adhesion in cancer cells, leading to enhanced migratory and invasive properties. In this experiment, zymography, cell invasion, and migration assays were performed. Results indicated that Duchesnea indica extracts (DIE) inhibited highly metastatic A549 and H1299 cells by reducing the secretions of matrix metalloproteinase-2 and urokinase-type plasminogen activator. Cell adhesion assay also demonstrated that DIE reduced the cell adhesion properties. Western blot analysis showed that DIE down-regulated the expression of N-cadherin, fibronectin, and vimentin, which are mesenchymal markers, and enhanced that of E-cadherin, which is an epithelial marker. In vivo study showed that tumor growth was significantly reduced in BALB/c nude mouse xenograft model administered with oral gavage of DIE. Therefore, DIE could be exhibits potential as a phytochemical-based platform for prevention and treatment of lung cancer. © 2016 Wiley Periodicals, Inc. Environ Toxicol 32: 2053-2063, 2017.

Spot urine protein to creatinine ratio.

In a recent article in Pediatric Nephrology, EM Yang and colleagues (Pediatr Nephrol 2017: doi: 10.1007/s00467-016-3587-6 ) published a retrospective cross-sectional study involving a cohort of 442 children with an mean estimated glomerular filtration rate of >60 mL/min/1.73 m2. The authors measured 24-h urine protein excretion (24-h UProt) alongside the morning spot urine protein to creatinine ratio (Prot/Cr) in this group of patients. While the Prot/Cr may be the only feasible way to routinely estimate the daily protein excretion of a young child, inter-individual variability in childrens' urinary creatinine excretion (UCr) may heavily influence the result. The authors sought to determine which equation was the most accurate in predicting UCr. Not only did they discover that the adult Cockcroft-Gault equation worked best, they also found that multiplying the Prot/Cr by the estimated UCr significantly improved the accuracy of the 24-h UProt estimate. In this editorial we discuss both the strengths and limitations of the study by EM Yang and colleagues. We also highlight the importance of adhering to internationally agreed upon reporting guidelines such as the STrengthening the Reporting of OBservational studies in Epidemiology (STROBE) statement.

Can the new CKD-EPI BTP-B2M formula be applied in children?

Although measuring creatinine to determine kidney function is currently the clinical standard, new markers such as beta-trace protein (BTP) and beta-2-microglobulin (B2M) are being investigated in an effort to measure glomerular filtration rate more accurately. In their recent publication, Inker et al. (Am J Kidney Dis 2015; 67:40-48) explored the use of these two relatively new markers in combination with some commonly available clinical characteristics in a large cohort of adults with chronic kidney disease. Their research led them to develop three formulae using BTP, B2M, and a combination of the two. The combined formula is particularly attractive as it removes all gender bias, which applies to both serum creatinine and cystatin C. Using data from a cohort of 127 pediatric patients from our center, we sought to determine whether these formulae would be equally as effective in children as in adults. Unfortunately, we found that the formulae cannot be applied to the pediatric population.

Minimum mycophenolic acid levels are associated with donor-specific antibody formation.

Although de novo DSA are associated with inferior graft survival, there are no effective strategies to prevent their formation. Underexposure to MPA (prodrug: MMF) also contributes to rejection rates early after transplantation, but the effect of this phenomenon on the formation of DSA long-term post-transplantation is unknown. Data are expressed as mean (standard deviation). All available data from 32 renal transplant recipients (age at transplantation 7.5 [4.5] yr) on tacrolimus and MPA immunosuppression with an average follow-up of 9.4 (s.d. 4.6) yr were analyzed. DSA were measured using the Luminex assay (>500 MFI was considered DSA-positive). Tacrolimus and MPA levels were measured with the Abbot Tacro II and EMIT assay, respectively. Among 1964 MPA and 3462 tacrolimus trough levels, the average MPA trough level was 3.2 (1.5) mg/L and the average tacrolimus level was 6.7 (2.8) ng/mL. At last follow-up, only 5/32 patients had undetectable DSA, with 5/32 having no class I antibodies and 6/32 having no class II antibodies. DSA formation was associated with a lower minimum MPA trough level (0.27 [0.23] vs. 0.47 [0.18] mg) and cystatin C eGFR (48 [21] vs. 70 [23] mL/min/1.73 m(2)) for class I DSA formers. The average eGFR of patients without class I DSA was 70 (23) mL/min/1.73 m(2), whereas the average eGFR of patients with class I DSA was 48 (21) mL/min/1.73 m(2) (p = 0.0071). MPA trough levels <1.3 mg/L long-term post-transplantation are associated with the formation of DSA. The association between the formation of DSA and minimum MPA exposure may support a strategy for preventing the formation of DSA.

What is the intrapatient variability of mycophenolic acid trough levels?

TDM of MPA, the active compound of MMF, is rarely used despite its substantial intra- and interpatient variability. Little is known about the utility of long-term MPA TDM. Data are expressed as mean (one standard deviation). All available data from 27 renal transplant recipients (mean age at transplantation: 7.7 [5.0] yr) with an average follow-up of 9.3 (4.6) yr were analyzed. MPA levels were measured using the EMIT. GFR was measured using cystatin C and eGFR was calculated using the Filler formula. Intrapatient CV of the trough level was calculated as the ratio of the mean divided by one standard deviation. Mean cystatin C eGFR was 56.9 (24.4) mL/min/1.73 m(2) . There was a weak but significant correlation between the MPA trough level and the AUC (Spearman r = 0.6592, p < 0.0001). A total of 1964 MPA trough levels (73 [45]/patient) were measured, as compared to 3462 Tac trough levels (144 [71]/patient). The average MPA trough level was 3.01 (1.26) mg/L and the average trough Tac level was 7.3 (1.8) ng/mL. Intrapatient CV was statistically higher (p = 0.00093) for MPA at 0.68 (0.29) when compared to Tac with a CV of 0.46 (0.12). CV did not correlate with eGFR. Intrapatient MPA trough level CV is significantly higher than for Tac, while CV for both MPA and Tac was high. MPA trough level monitoring may be a feasible monitoring option to improve patient exposure and possibly outcomes.

Caffeic Acid phenethyl ester is a potential therapeutic agent for oral cancer.

Head and neck cancers, which affect 650,000 people and cause 350,000 deaths per year, is the sixth leading cancer by cancer incidence and eighth by cancer-related death worldwide. Oral cancer is the most common type of head and neck cancer. More than 90% of oral cancers are oral and oropharyngeal squamous cell carcinoma (OSCC). The overall five-year survival rate of OSCC patients is approximately 63%, which is due to the low response rate to current therapeutic drugs. In this review we discuss the possibility of using caffeic acid phenethyl ester (CAPE) as an alternative treatment for oral cancer. CAPE is a strong antioxidant extracted from honeybee hive propolis. Recent studies indicate that CAPE treatment can effectively suppress the proliferation, survival, and metastasis of oral cancer cells. CAPE treatment inhibits Akt signaling, cell cycle regulatory proteins, NF-κB function, as well as activity of matrix metalloproteinase (MMPs), epidermal growth factor receptor (EGFR), and Cyclooxygenase-2 (COX-2). Therefore, CAPE treatment induces cell cycle arrest and apoptosis in oral cancer cells. According to the evidence that aberrations in the EGFR/phosphoinositide 3-kinase (PI3K)/protein kinase B (Akt) signaling, NF-κB function, COX-2 activity, and MMPs activity are frequently found in oral cancers, and that the phosphorylation of Akt, EGFR, and COX-2 correlates to oral cancer patient survival and clinical progression, we believe that CAPE treatment will be useful for treatment of advanced oral cancer patients.

The potential of lactulose and melibiose, two novel trehalase-indigestible and autophagy-inducing disaccharides, for polyQ-mediated neurodegenerative disease treatment.

The unique property of trehalose encourages its pharmaceutical application in aggregation-mediated neurodegenerative disorders, including Alzheimer's, Parkinson's, and many polyglutamine (polyQ)-mediated diseases. However, trehalose is digested into glucose by trehalase and which reduced its efficacy in the disease target tissues. Therefore, searching trehalase-indigestible analogs of trehalose is a potential strategy to enhance therapeutic effect. In this study, two trehalase-indigestible trehalose analogs, lactulose and melibiose, were selected through compound topology and functional group analyses. Hydrogen-bonding network analyses suggest that the elimination of the hydrogen bond between the linker ether and aspartate 321 (D321) of human trehalase is the key for lactulose and melibiose to avoid the hydrolyzation. Using polyQ-mediated spinocerebellar ataxia type 17 (SCA17) cell and slice cultures, we found the aggregation was significantly prohibited by trehalose, lactulose, and melibiose, which may through up-regulating of autophagy. These findings suggest the therapeutic applications of trehalase-indigestible trehalose analogs in aggregation-associated neurodegenerative diseases.