Longistylin A from Cajanus cajan (L.) Millsp. disturbs glycerophospholipid metabolism and cytokinin biosynthesis of Nocardia seriolae.

ETHNOPHARMACOLOGICAL RELEVANCE: Nocardiosis is an uncommon infectious disease that bears certain similarities to tuberculosis, with a continuous increase in its incidence and a poor prognosis. In traditional Chinese medicine, the leaves of Cajanus cajan (L.) Millsp. are employed to treat wounds, malaria, coughs, and abdominal pain. AIM OF THE STUDY: In this study, we investigated the effects and mechanisms of longistylin A (LGA), a natural stilbene isolated from C. cajan, as a potential antibiotic against nocardiosis. MATERIALS AND METHODS: LGA was isolated from the leaves of C. cajan and assessed using a minimum bactericidal concentration (MBC) determination against Nocardia seriolae. Multi-omics analysis encompassing genes, proteins, and metabolites was conducted to investigate the impact of LGA treatment on N. seriolae. Additionally, quantitative analysis of 40 cytokinins in N. seriolae mycelium was performed to assess the specific effects of LGA treatment on cytokinin levels. Cryo-scanning electron microscopy was utilized to examine morphological changes induced by LGA treatment, particularly in the presence of exogenous trans-zeatin-O-glucoside (tZOG). The therapeutic effect of LGA was investigated by feeding N. seriolae-infected largemouth bass. RESULTS: LGA exhibited significant efficacy against N. seriolae, with MBC value of 2.56 µg/mL. Multi-omics analysis revealed that LGA disrupted glycerophospholipid metabolism and hormone biosynthesis by notably reducing the expression of glycerol-3-phosphate dehydrogenase and calmodulin-like protein. Treatment with LGA markedly disrupted 12 distinct cytokinins in N. seriolae mycelium. Additionally, the addition of exogenous tZOG counteracted the inhibitory effects of LGA on filamentous growth, resulting in mycelial elongation and branching. Furthermore, LGA treatment improved the survival rate of largemouth bass infected with N. seriolae. CONCLUSIONS: We found for the first time that LGA from C. cajan exhibited significant efficacy against N. seriolae by interfering with glycerophospholipid metabolism and cytokinin biosynthesis.

Configurational paths to turnover intention among primary public health workers in Liaoning Province, China: a fuzzy-set qualitative comparative analysis.

BACKGROUND: A stable public health workforce plays an indispensable role in the realization of the goal of health for all. However, there is an exodus of public health workers from the Centers for Disease Control and Prevention (CDC). Given the limited evidence on the mechanisms shaping turnover intention (TI) among public health workers, the study aims to investigate the triggering mechanisms of high and low turnover intention by combining job demands, job resources, and personal resources through a set theory perspective based on the Job-Demand-Resources (JD-R) model. METHODS: The cross-sectional study was conducted from September 7 to 18, 2020 at district (county) level CDC in Liaoning Province, China. A total of 584 public health professionals were included. Overcommitment, effort, social respect, occupational identity, job rewards, self-efficacy, and psychological resilience were included in the study as configuration factors. The data were gathered through an online questionnaire and were analyzed using multiple regression and fuzzy-set Qualitative Comparative Analysis (fsQCA). RESULTS: Social respect (B = -0.682, P < 0.001), occupational identity (B = -0.168, P < 0.001), and effort (B = 0.114, P < 0.001) were associated with turnover intention. Five configurations for high turnover intention and five for low turnover intention were obtained through the fsQCA, with occupational identity and effort playing an essential role in all pathways. Moreover, the configurations for low turnover intention are not the antithesis of the configurations for high turnover intention. CONCLUSION: Managers should synthesize the combined effects of factors when implementing interventions and formulating policies. Given the vital role of occupational identity and effort, mechanisms for the rational distribution of work to avoid excessive efforts and measures to promote occupational identity should be implemented to reduce the turnover intentions of primary public health workers and encourage their intention to stay.

An interfacial synergism effect of Pd-g-C3N4 in Pd/g-C3N4 for highly active and selective hydrogenation of 4-nitrophenol.

Herein, we report that Pd nanoparticles (NPs) anchored on graphitic nitride carbon (Pd/g-C3N4) catalysts with various Pd contents (1.55 wt%, 0.14 wt%, 0.04 wt%) are successfully prepared via a simple NaBH4 reduction method, exhibiting excellent catalytic activity and selectivity toward 4-aminophenol (4-AP) in 4-nitrophenol (4-NP) selective hydrogenation. 4-NP is completely converted to 4-AP (yield ∼ 100%) under quite moderate reaction conditions (40 °C, 2.0 MPa H2 and 5 min) over the 1.55 wt% Pd/g-C3N4 catalyst, with a high reaction rate r = 134.4 mol4-NP molPd-1 min-1. The excellent catalytic performance can be attributed to the following reasons: (1) a higher ratio of Pd(0)/Pdn+ provides much more exposed active sites for the potential adsorption and activation of the reactants, which is beneficial for increasing the reaction rate and catalytic activity; (2) Pd NPs are highly dispersed on g-C3N4 due to the strong interaction of Pd-N or Pd-C; (3) the interfacial synergism effect between Pd NPs and g-C3N4 enables the effective adsorption and activation of H2 (4-NP) at Pd (g-C3N4), promoting the catalytic hydrogenation of 4-NP and improving their catalytic properties. In addition, this catalyst has superior reusability.

Dual-energy CT virtual non-calcium: an accurate method for detection of knee osteoarthritis-related edema-like marrow signal intensity.

OBJECTIVES: To evaluate the performance of a dual-energy computed tomography (DECT) virtual non-calcium (VNCa) technique in the detection of edema-like marrow signal intensity (ELMSI) in patients with knee joint osteoarthritis (OA) compared to magnetic resonance imaging (MRI). METHODS: The study received local ethics board approval, and written informed consent was obtained. DECT and MRI were used to examine 28 knees in 24 patients with OA. VNCa images were generated by dual-energy subtraction of calcium. The knee joint was divided into 15 regions for ELMSI grading, performed independently by two musculoskeletal radiologists, with MRI as the reference standard. We also analyzed CT numbers through receiver operating characteristics and calculated cut-off values. RESULTS: For the qualitative analysis, we obtained CT sensitivity (Readers 1, 2 = 83.7%, 89.8%), specificity (Readers 1, 2 = 99.5%, 99.5%), positive predictive value (Readers 1, 2 = 95.3%, 95.7%), and negative predictive value (Readers 1, 2 = 97.9%, 98.7%) for ELMSI. The interobserver agreement was excellent (κ = 0.92). The area under the curve for Reader 1 and Reader 2 was 0.961 (95% CI 0.93, 0.99) and 0.992 (95% CI 0.98, 1.00), respectively. CT numbers obtained from the VNCa images were significantly different between regions with and without ELMSI (p < .001). CONCLUSIONS: VNCa images have good diagnostic performance for the qualitative and quantitative analysis of knee osteoarthritis-related ELMSI.

Pinostrobin from plants and propolis against human coronavirus HCoV-OC43 by modulating host AHR/CYP1A1 pathway and lipid metabolism.

Coronaviruses, as enveloped positive-strand RNA viruses, manipulate host lipid compositions to enable robust viral replication. Temporal modulation of the host lipid metabolism is a potential novel strategy against coronaviruses. Here, the dihydroxyflavone pinostrobin (PSB) was identified through bioassay that inhibited the increment of human coronavirus OC43 (HCoV-OC43) in human ileocecal colorectal adenocarcinoma cells. Lipid metabolomic studies showed that PSB interfered with linoleic acid and arachidonic acid metabolism pathways. PSB significantly decreased the level of 12, 13- epoxyoctadecenoic (12, 13-EpOME) and increased the level of prostaglandin E2. Interestingly, exogenous supplement of 12, 13-EpOME in HCoV-OC43-infected cells significantly stimulated HCoV-OC43 virus replication. Transcriptomic analyses showed that PSB is a negative modulator of aryl hydrocarbon receptor (AHR)/cytochrome P450 (CYP) 1A1signaling pathway and its antiviral effects can be counteracted by supplement of FICZ, a well-known AHR agonist. Integrative analyses of metabolomic and transcriptomic indicated that PSB could affect linoleic acid and arachidonic acid metabolism axis through AHR/CYP1A1 pathway. These results highlight the importance of the AHR/CYP1A1 pathway and lipid metabolism in the anti-coronavirus activity of the bioflavonoid PSB.

[Effects of moxibustion on serum levels of ß-EP, SP and expression of IL-1ß and COX-2 protein in brainstem in rats with migraine].

OBJECTIVE: To observe the effects of moxibustion at "Baihui" (GV 20) and "Dazhui" (GV 14) at different time points on the serum level of ß-endorphin (ß-EP), substance P (SP) and expression of interleukin-1ß (IL-1ß) and cyclooxygenase-2 (COX-2) protein in brainstem in rats with migraine, and to explore the effect and mechanism of moxibustion in preventing and treating migraine. METHODS: Forty male SD rats were randomly divided into a blank group, a model group, a prevention+treatment (PT) group and a treatment group, 10 rats in each group. Except the blank group, the rats in the remaining groups were injected with nitroglycerin subcutaneously to prepare migraine model. The rats in the PT group were treated with moxibustion 7 days before modeling (once a day) and 30 min after modeling, while the rats in the treatment group were treated with moxibustion 30 min after modeling. The "Baihui" (GV 20) and "Dazhui" (GV 14) were taken for 30 minutes each time. The behavioral scores in each group were observed before and after modeling. After intervention, ELISA method was used to detect the serum level of ß-EP and SP; the immunohistochemistry method was used to detect the number of positive cells of IL-1ß in brainstem; the Western blot method was used to detect the expression of COX-2 protein in brainstem. RESULTS: Compared with the blank group, the behavioral scores in the model group were increased 0-30 min, 60-90 min and 90-120 min after modeling (P<0.01); compared with the model group, in the treatment group and the PT group, the behavioral scores were decreased 60-90 min and 90-120 min after modeling (P<0.01). Compared with the blank group, in the model group, the serum level of ß-EP was decreased (P<0.01), while the serum level of SP, the number of positive cells of IL-1ß in brainstem and the expression of COX-2 protein were increased (P<0.01). Compared with the model group, in the PT group and and the treatment group, the serum level of ß-EP was increased (P<0.01), while the serum level of SP, the number of positive cells of IL-1ß and the expression of COX-2 protein in brainstem were decreased (P<0.01, P<0.05). Compared with the treatment group, in the PT group, the serum level of ß-EP was increased and COX-2 protein expression was decreased (P<0.05). CONCLUSION: Moxibustion could effectively relieve migraine. The mechanism may be related to reduce the serum level of SP, IL-1ß and COX-2 protein expression in brainstem, and increase the serum level of ß-EP, and the optimal effect is observed in the PT group.

The oncogenic role of TFAP2A in bladder urothelial carcinoma via a novel long noncoding RNA TPRG1-AS1/DNMT3A/CRTAC1 axis.

BACKGROUND: Overexpression of TFAP2A has been linked to increased lymph node metastasis in basal-squamous bladder cancer. However, its downstream targets in bladder urothelial carcinoma (BLCA), the most malignant cancer of the urinary tract, remain unclear. In the current study, we aim to explore the function and mechanism of TFAP2A in BLCA. METHODS: TFAP2A expression and the prognostic significance in BLCA was analyzed using TCGA and GTEX projects. TFAP2A was knocked-down in BLCA cells to study its impact on glucose uptake, lactate and ATP production, expression of HK2, and the number of vascular meshes formed by HUVEC. The target long noncoding RNAs (lncRNAs) of TFAP2A were predicted by bioinformatics tools, followed by ChIP-qPCR and luciferase assays. The downstream targets of TPRG1-AS1 were analyzed by microarray analysis. Rescue experiments were conducted for validation. RESULTS: TFAP2A upregulation in BLCA predicted dismal survival of patients. Loss of TFAP2A inhibited glycolysis (as evidenced by reduced glucose uptake, lactate, ATP production, and the expression of HK2) and angiogenesis (decreased number of vascular meshes formed by HUVEC). TFAP2A promoted the transcription of TPRG1-AS1. TPRG1-AS1 reversed the inhibitory effect of TFAP2A knockdown on glycolysis and angiogenesis in BLCA cells. TPRG1-AS1 inhibited the transcription of CRTAC1 by recruiting a DNA methyltransferase to the promoter of CRTAC1 and increasing the DNA methylation of its promoter. CRTAC1 inhibited glycolysis and angiogenesis in BLCA cells. TFAP2A silencing curbed tumor growth in vivo via the TPRG1-AS1/CRTAC1 axis. CONCLUSION: TFAP2A reduces CRTAC1 expression by promoting TPRG1-AS1 transcription, thereby expediting BLCA glycolysis and angiogenesis.

Inactivation of a wheat protein kinase gene confers broad-spectrum resistance to rust fungi.

Wheat crops are frequently devastated by pandemic stripe rust caused by Puccinia striiformis f. sp. tritici (Pst). Here, we identify and characterize a wheat receptor-like cytoplasmic kinase gene, TaPsIPK1, that confers susceptibility to this pathogen. PsSpg1, a secreted fungal effector vital for Pst virulence, can bind TaPsIPK1, enhance its kinase activity, and promote its nuclear localization, where it phosphorylates the transcription factor TaCBF1d for gene regulation. The phosphorylation of TaCBF1d switches its transcriptional activity on the downstream genes. CRISPR-Cas9 inactivation of TaPsIPK1 in wheat confers broad-spectrum resistance against Pst without impacting important agronomic traits in two years of field tests. The disruption of TaPsIPK1 leads to immune priming without constitutive activation of defense responses. Taken together, TaPsIPK1 is a susceptibility gene known to be targeted by rust effectors, and it has great potential for developing durable resistance against rust by genetic modifications.

MicroRNA­16­5p/BIMP1/NF­κB axis regulates autophagy to exert a tumor­suppressive effect on bladder cancer.

Bladder cancer (BC) is the second most common urological disease worldwide. Previous studies have reported that microRNA (miR)­16­5p is associated with the development of BC, but whether miR­16­5p regulates BC cell autophagy remains unknown. Thus, the aim of the present study was to investigate this issue. miR­16­5p expression in BC cells was assessed by reverse transcription­quantitative PCR. Cell viability and apoptosis were detected via Cell Counting Kit­8 and flow cytometry assays, respectively. For cell autophagy detection, autophagic flux was detected using a mCherry­green fluorescent protein­microtubule­associated proteins 1A/1B light chain 3B (LC3) puncta formation assay, followed by determination of autophagy­related protein markers. The targeting relationship between miR­16­5p and caspase recruitment domain family member 10 (BIMP1) was confirmed using a dual­luciferase reporter assay, followed by detection of the BIMP1/NF­κB signaling pathway. The results showed that miR­16­5p overexpression inhibited cell viability, whereas miR­16­5p knockdown promoted cell viability in BC. Furthermore, miR­16­5p overexpression induced autophagy, which was accompanied by increased autophagic flux and expression of the autophagy­related proteins LC3­II and beclin 1, as well as decreased p62 expression, whereas miR­16­5p silencing led to an inhibition of autophagy in BC cells. Moreover, autophagy inhibitor 3­methyladenine treatment inhibited cell autophagy and apoptosis in miR­16­5p­overexpressing cells. Mechanistic studies demonstrated that miR­16­5p could inhibit the BIMP1/NF­κB signaling pathway and this inhibition was achieved by directly targeting BIMP1. Furthermore, it was found that blockade of the BIMP1/NF­κB signaling pathway inversed the inhibitory effects of miR­16­5p knockdown on autophagy in BC cells. In vivo experiments further verified the tumor­suppressive effect on BC of the miR­16­5p/BIMP1/NF­κB axis. Therefore, the results of the present study indicated that miR­16­5p promotes autophagy of BC cells via the BIMP1/NF­κB signaling pathway, and an improved understanding of miR­16­5p function may provide therapeutic targets for clinical intervention in this disease.

Rsf­1 regulates malignant melanoma cell viability and chemoresistance via NF­κB/Bcl­2 signaling.

Remodeling and spacing factor 1 (Rsf­1) has been reported as overexpressed in numerous cancers; however, its expression, biological functions and mechanisms in malignant melanoma remain unknown. In the present study, the expression of Rsf­1 was investigated in 50 cases of malignant melanoma samples using immunohistochemistry. The results revealed that Rsf­1 expression was elevated in 38% of specimens. MTT, colony formation, Transwell and flow cytometry assays were performed to investigate the functions of Rsf­1. Knockdown of Rsf­1 in the MV3 and A375 melanoma cell lines decreased the viability, invasion and cell cycle transition of cells. Conversely, overexpression of Rsf­1 in M14 cells with low endogenous Rsf­1 expression induced opposing effects. Further analysis revealed that Rsf­1 knockdown decreased matrix metalloproteinase­2, cyclin E and phosphorylated­IκB expression. Additionally, Rsf­1 depletion reduced cisplatin resistance and significantly increased the cisplatin­associated apoptotic rate, whereas Rsf­1 overexpression exhibited opposing effects. Rsf­1 also maintained the mitochondrial membrane potential following cisplatin treatment. Analysis of apoptosis­associated proteins revealed that Rsf­1 positively regulated B­cell lymphoma 2 (Bcl­2), cellular inhibitor of apoptosis 1 (cIAP1) and cIAP2, and downregulated Bcl­2­associated X protein expression. Nuclear factor κ­light­chain­enhancer of activated B­cells (NF­κB) inhibition reversed the effects of Rsf­1 on Bcl­2. In conclusion, Rsf­1 was overexpressed in malignant melanoma and may contribute to the malignant behaviors of melanoma cells, possibly via the regulation of NF­κB signaling. Therefore, Rsf­1 may be a potential therapeutic target in the treatment of malignant melanoma.

Silencing of CARMA3 inhibits bladder cancer cell migration and invasion via deactivating ß-catenin signaling pathway.

BACKGROUND: Bladder cancer (BC) is the ninth most common cancer and the fourteenth leading death worldwide. CARD-containing MAGUK 3 (CARMA3) protein is a novel scaffold protein known to activate NF-κB pathway and is overexpressed in BC tissues. PURPOSE: The objective of this study was to identify how CARMA3 affects the metastasis of BC cells via the ß-catenin signaling pathway. MATERIALS AND METHODS: In the present study, 5637 and T24 BC cells with stable low expression of CARMA3 were established, and their migratory and invasive capabilities were further evaluated by wound-healing and transwell assay. The activity and expression of ß-catenin were determined by Luciferase assay and immunofluoresence staining. The mRNA and protein expression levels of CARMA3, matrix metallopeptidase (MMP) 9 and MMP2 were detected by quantitative real-time PCR (qRT-PCR) and Western blot analysis. The nude mouse tumor xenograft model was established for in vivo study. RESULTS: By comparison to the control cells, CARMA3-silenced cells acquired a less aggressive phenotype: decreased migration and invasion. More importantly, we confirmed that CARM3 knockdown could inhibit ß-catenin mRNA and protein expression and activity, and reduce the expression and/or activity of matrix metallopeptidase (MMP) 9, MMP2 and C-myc. Also, CARM3 silencing increased E-cadherin expression and attenuated the expression of ß-catenin. Moreover, we demonstrated that ß-catenin overexpression reversed the inhibiting effect of CARMA3 silencing on cell invasion and migration. Furthermore, our study illustrated that knockdown of CARMA3 suppressed BC cells xenograft tumor growth in nude mice. CONCLUSION: We demonstrated that CARMA3 contributes to the malignant phenotype of BC cells at least by activating ß-catenin signaling pathway, and it may serve as a therapeutic target for clinic treatment in BC.

Selective [5,5]-Sigmatropic Rearrangement by Assembly of Aryl Sulfoxides with Allyl Nitriles.

Aromatic [5,5]-sigmatropic rearrangement is an appealing protocol for accessing 1,4-substituted arenes. However, such a protocol has not been well utilized in organic synthesis because of the difficulties in the synthesis of the substrates, selectivity issues, and limited substrate scope. Described herein is a new [5,5]-sigmatropic reaction utilizing readily available aryl sulfoxides and allyl nitriles. This reaction features mild reaction conditions, high chemo- and regioselectivity, excellent functional-group compatibility, and broad substrate scope. Computational studies suggest that the success of the reaction can be attributed to the selective electrophilic assembly of the rearrangement precursors, in which a linear -C=C=N- linkage favors [5,5]-sigmatropic rearrangement over the competitive [3,3]-sigmatropic rearrangement.

Remodeling and spacing factor 1 overexpression is associated with poor prognosis in renal cell carcinoma.

The present study aimed to assess the expression and prognostic significance of remodeling and spacing factor 1 (RSF1; HBXAP) in renal cell carcinoma (RCC). RSF1 expression was analyzed using immunohistochemistry on tissue samples from a consecutive series of 137 patients with RCC who underwent tumor resection between November 2000 and March 2004. The associations between RSF1 expression, clinicopathological factors and patient survival were investigated. Immunohistochemistry revealed that RSF1 was highly expressed in 43.1% (59/137) of the RCC samples. RSF1 expression levels were associated with the T stage of the Tumor-Node-Metastasis grading system. Kaplan-Meier survival analysis indicated that high RSF1 expression in RCC was significantly associated with a poor prognosis. Multivariate analysis revealed that RSF1 expression is an independent prognostic parameter for the duration of overall survival of patients with RCC. The results demonstrated that a high expression level of RSF1 in RCC is associated with advanced tumor stages and a poor prognosis. To the best of our knowledge, the present study provides novel evidence of the biological significance of RSF1 expression in RCC.

Overexpression of Rsf-1 correlates with poor survival and promotes invasion in non-small cell lung cancer.

Rsf-1 (HBXAP) was recently reported to play roles in tumorigenesis and tumor progression. There have been many reports referred to Rsf-1 overexpression in various cancers and associated with the malignant behavior of cancer cells. However, the molecular mechanism of Rsf-1 in non-small cell lung cancer aggressiveness remains ambiguous. In the present study, we found that there was a significant association between Rsf-1 overexpression and poor overall survival (p = 0.028) in lung cancer. Furthermore, knockdown of Rsf-1 expression in H1299 and H460 cells with high endogenous Rsf-1 expression inhibited cell migration and invasion and downregulated MMP2 expression and nuclear levels of NF-κB. NF-κB inhibitor could also block the effect of Rsf-1 in regulation of MMP2 expression. Further experiments demonstrated that Rsf-1 depletion restrained NF-κB reporter luciferase activity and downregulated bcl-2 and p-IκB protein level. In conclusion, we demonstrated that Rsf-1 was overexpressed in lung cancer and associated with poor survival. Rsf-1 regulated cell invasion through MMP2 and NF-κB pathway.

Redox-Neutral α-Arylation of Alkyl Nitriles with Aryl Sulfoxides: A Rapid Electrophilic Rearrangement.

A facile α-arylation of nitriles has been developed by simply introducing Tf2O and DABCO to the mixture of nitriles and aryl sulfoxides. The transformation consists of two sequential steps: (i) Tf2O-initiated electrophilic assembly and and (ii) DABCO-triggered rearrangement. Each step can be tuned independently by changing the temperature and/or base. This adjustability allows the method to accommodate a wide range of substrates. Notable features of this new protocol include remarkable efficiency (20 min, -30 °C), exclusive regioselectivity, and high functional group compatibility, which can be challenging issues in traditional approaches. NMR studies not only identified a unique, highly unstable sulfonium imine complex but also demonstrated the importance of temperature in the formation and manipulation of this key intermediate. Further DFT calculations suggested that an electrophilic assembly, followed by removal of HOTf (by base), and finally [3,3]-sigmatropic rearrangement are three key stages in the reaction. The versatile transformability of the products and easy scalability of this reaction are also exhibited here.

Synthesis of some ester derivatives of 4'-demethoxyepipodophyllotoxin/2'-chloro-4'-demethoxyepipodophyllotoxin as insecticidal agents against oriental armyworm, Mythimna separata Walker.

Podophyllotoxin is a naturally occurring non-alkaloid toxin isolated from the roots and rhizomes of Podophyllum peltatum and P. hexandrum. In continuation of our program aimed at the discovery and development of natural product-based insecticides, two series of ester derivatives of 4'-demethoxyepipodophyllotoxin/2'-chloro-4'-demethoxyepipodophyllotoxin were prepared. The structures of the target compounds were well characterized by 1H NMR, IR, optical rotation and mp. The precise three-dimensional structural information of 8j was further determined by single-crystal X-ray diffraction. Their insecticidal activity was tested against Mythimna separata Walker. These compounds showed delayed insecticidal activity. Among all derivatives, some compounds showed more potent insecticidal activity than toosendanin against M. separata; especially compounds 8k and 9k exhibited the most potent activity with the final mortality rates of 71.4%. Their structure-activity relationships were discussed.

Clinical significance and biological roles of CARMA3 in human bladder carcinoma.

Caspase recruitment domain and membrane-associated guanylate kinase-like domain protein 3 (CARMA3) was reported as an oncoprotein overexpressed in several cancers. The expression pattern of CARMA3 and its clinical significance in human bladder cancer have not been well characterized. In the present study, CARMA3 expression was analyzed in 90 archived bladder cancer specimens using immunohistochemistry, and the correlation between CARMA3 expression and clinicopathological parameters was evaluated. We found that CARMA3 was overexpressed in 35 of 90 (38.8%) bladder cancer specimens. Significant association was observed between CARMA3 overexpression with tumor status (p = 0.081) and tumor grade (p = 0.027). To further explore the biological functions of CARMA3 in bladder cancer, we depleted CARMA3 in T24 and 5637 cell lines using small interfering RNA (siRNA). Using cell counting kit-8 (CCK8) assay and colony formation assay, we were able to show that CARMA3 depletion inhibited cell proliferation and colony number. Further study demonstrated that CARMA3 depletion decreased an expression of nuclear factor kappa B (NF-κB) targets cyclin D1 and Bcl-2 expression, as well as IκB phosphorylation. Luciferase reporter assay showed that CARMA3 depletion could downregulate NF-κB reporter activity. In conclusion, CARMA3 is overexpressed in bladder cancer and regulates malignant cell growth and NF-κB signaling, which makes CARMA3 a candidate therapeutic target for bladder cancer.

Inhibition of cytoplasmic GSK-3ß increases cisplatin resistance through activation of Wnt/ß-catenin signaling in A549/DDP cells.

Cisplatin-based chemotherapy is recommended as the first-line therapy for advanced non-small cell lung cancer (NSCLC). However, acquired cisplatin resistance is ubiquitous in patients with NSCLC, but the molecular mechanism of such resistance remains ambiguous. The present study sought to examine the role of the Wnt/ß-catenin signaling pathway in cisplatin resistance by assessing the phosphorylation and subcellular distribution of GSK-3ß in a human lung adenocarcinoma cell line, A549, and its cisplatin-resistant subline, A549/DDP. Total GSK-3ß, phosphorylated GSK-3ß(ser9) and phosphorylated GSK-3ß(tyr216) in cytoplasmic and nuclear fractions of A549/DDP and A549 cells were examined by western blot analysis. The regulation of cisplatin resistance, apoptosis, ß-catenin and survivin protein expression by inhibition of cytoplasmic GSK-3ß were determined by MTT assay, flow cytometry analysis, immunofluorescence technique and western blot analysis. In the present study, cytoplasmic levels of p-GSK-3ß(ser9) were significantly increased in A549/DDP cells as compared with A549 cells (P<0.01), and these levels were further increased by cisplatin treatment in A549/DDP cells (P<0.01). In contrast, cytoplasmic levels of p-GSK-3ß(ser9) were reduced in A549 cells after treatment with cisplatin (P<0.01). However, cytoplasmic levels of p-GSK-3ß(tyr216) were significantly decreased in A549/DDP cells as compared with A549 cells (P<0.01), and these levels were further decreased by cisplatin treatment in A549/DDP cells (P<0.01). Conversely, cytoplasmic levels of p-GSK-3ß(tyr216) were raised in A549 cells after treatment with cisplatin (P<0.01). Analysis of downstream effectors of the Wnt/ß-catenin signaling pathway revealed upregulation of ß-catenin and survivin expression in A549/DDP cells treated with cisplatin as compared to untreated cells. In A549 cells, cisplatin treatment decreased the expression of ß-catenin and survivin. Furthermore, phosphorylation of GSK-3ß at serine 9 by LiCl and transient interference of GSK-3ß by siRNA increased ß-catenin and survivin protein expression in A549/DDP cells. Low exogenous and endogenous cytoplasmic GSK-3ß expression enhanced the IC50 and inhibited apoptosis. In conclusion, activation of the Wnt/ß-catenin signaling pathway and upregulated survivin expression due to cytoplasmic GSK-3ß inhibition might lead to cisplatin resistance in NSCLC.