Factors linked to prognosis in children with provisional tic disorder: a prospective cohort study.

The purpose of the present study was to estimate the factors linked to the prognosis of children with provisional tic disorder (PTD). We conducted a prospective cohort study enrolled children with PTD who were subsequently followed-up at three-month intervals for 1 year post-enrolment. A total of 259 PTD patients were included in the final analysis. At the end of the follow-up period, 77 (30%) of the patients had achieved clinical remission. Result of the LASSO logistic regression analysis revealed that a disease duration >3 months (OR=4.20, 95% CI 1.20-14.73), moderate/severe tic severity (OR=5.57, 95% CI 2.26-13.76), and comorbid behavioral problems (OR=2.78, 95% CI 1.15-6.69) were significant factors linked to remission in the PTD patients. The path analysis model showed that comorbid behavioral problems and recurrence partially mediated the association between tic severity and remission, with a mediating effect of 37%. Conclusions: We have identified several significant factors linked to prognosis in children with PTD, including comorbid behavioral problems and recurrence, which were found to be important mediators. These findings provide new insights for the clinical management of patients with PTD.

Mechanism study of cross presentation of exogenous antigen induced by cholera toxin-like chimeric protein.

Tumor subunit vaccines have great potential in personalized cancer immunotherapy. They are usually administered with adjuvant owing to their low immunogenicity. Cholera toxin (CT) is a biological adjuvant with diverse biological functions and a long history of use. Our earlier study revealed that a CT-like chimeric protein co-delivered with murine granulocyte-macrophage colony stimulating factor (mGM-CSF) and prostate cancer antigen epitope could co-stimulate dendritic cells (DCs) and enhance cross presentation of tumor epitope. To further study the molecular mechanism of CT-like chimeric protein in cross presentation, major histocompatibility complex class I (MHC I)-restricted epitope 257-264 of ovalbumin (OVAT) was used as a model antigen peptide in this study. Recombinant A subunit and pentameric B subunit of CT protein were respectively genetically constructed and purified. Then both assembled into AB5 chimeric protein in vitro. Three different chimeric biomacromolecules containing mGM-CSF and OVAT were constructed according to the different fusion sites and whether the endoplasmic reticulum (ER) retention sequence was included. It was found that A2 domain and B subunit of CT were both available for loading epitopes and retaining GM1 affinity. The binding activity of GM1 was positively correlated with antigen endocytosis. Once internalized, DCs became mature and cross-presented antigen. KDEL helped the whole molecule to be retained in the ER, and this improved the cross presentation of antigen on MHC I molecules. In conclusion, hexameric CT-like chimeric protein with dual effects of GM1 affinity and ER retention sequence were potential in improvement of cross presentation. The results laid a foundation for designing personalized tumor vaccine based on CT-like chimeric protein molecular structure.

Identification of a potential antigen stimulating immune response against Vibrio parahaemolyticus infection in hybrid tilapia (Oreochromis aureus♂ × Oreochromis niloticus♀).

Vibrio parahaemolyticus (V. parahaemolyticus) is a major pathogen that causes substantial losses in the marine fishery. With the emergence of antibiotic resistance, vaccines have become the most effective approach against V. parahaemolyticus infection. Adhesion factors on the cell surface are pivotal in the colonization and pathogenesis of V. parahaemolyticus within the host, highlighting their potential as vaccine candidates. This study aims to assess the immunogenicity and potential of recombinant V. parahaemolyticus MAM7 (rMAM7) as a vaccine candidate. Initially, we cloned and purified the MAM7 protein of V. parahaemolyticus. Moreover, after 4 weeks of vaccination, the fish were challenged with V. parahaemolyticus. rMAM7 demonstrated a certain protective effect. Immunological analysis revealed that rMAM7 immunization-induced antibody production and significantly increased acid phosphatase (ACP) and alkaline phosphatase (AKP) activity in hybrid tilapia. Furthermore, serum bactericidal tests demonstrated a lower bacterial survival rate in the rMAM7 group compared to PBS and rTrxa. qRT-PCR results indicated that rMAM7 significantly upregulated CD4, CD8 and IgM gene expression, suggesting the induction of Th1 and Th2 responses in hybrid tilapia. Overall, these findings highlight the potential application of MAM7 from V. parahaemolyticus in the development of protein vaccines.

The complete change in bile acids and steroids in systematic metabolomics applied to the intrahepatic cholestasis of pregnancy.

Intrahepatic cholestasis of pregnancy (ICP) is a pregnancy-specific hepatobiliary disease, leading to an abnormal increase in total bile acid in the blood of pregnant women. To systematically explore the similarities and differences in metabolites and metabolic pathways among three types of biological samples from ICP women, a study of 18 ICP and 6 healthy (as a normal control) pregnant women was performed to investigate their clinical information and biochemical features. Based on validated LC-MS/MS methods 1-5 for hydrophilic and hydrophobic metabolites (molecular weight <2000 Dalton), an untargeted-metabolomic strategy was applied to 24 pregnant women to determine the metabolites from 22 serum, 15 placental and 22 urine samples. Then 1137 metabolites from serum, 876 metabolites from placental tissue and 311 metabolites from urine with a coefficient of variation <30% in the pooled quality control samples were found. Furthermore, orthogonal partial least squares-discriminate analysis (OPLS-DA), correlation analysis, chemical enrichment analysis and metabolic pathway analysis were carried out by a bioinformatics process. On the OPLS-DA model analysis, the metabolites in urine were better than those in serum or placental tissue to reflect the metabolic changes of ICP disease. Some metabolites were significantly changed in serum (n = 71), placental tissue (n = 46) and urine (n = 36), such as bile acids, triacylglycerols, lysoPCs, and steroids. Primary bile acid biosynthesis was the main metabolic pathway in ICP disease, and taurine and hypotaurine metabolism and sphingolipid metabolism were also found. More specifically, bile acids increased and steroids decreased in the serum, placental and urine samples. For complex metabolic diseases such as ICP disease, untargeted-metabolomic analysis of multiple biological samples could provide a systematic understanding of the changes in metabolic types and pathways.

Sociodemographic and clinical characteristics of children with tic disorders and behavioral problems: A real-world study and development of a prediction model.

BACKGROUND: Tic disorders (TD) are complex neuropsychiatric disorders frequently associated with a variety of comorbid problems, whose negative effects may exceed those of the tics themselves. In this study, we aimed to explore the sociodemographic and clinical characteristics of children with TD and behavioral problems, and develop a prediction model of behavioral problems based on the predictors under real-world conditions. METHODS: A hospital-based cross-sectional study was conducted on children with TD. Behavioral problems were surveyed using the Achenbach Child Behavior Checklist (CBCL). Sociodemographic information was collected from face-to-face interviews using an electronic questionnaire administered during the initial ambulatory visit. Clinical data were collected from medical records, and quality control was performed. The sociodemographic and clinical characteristics of patients with and without behavioral problems were statistically compared, and a nomogram prediction model was developed based on multivariate logistic regression analysis. The discriminatory ability and clinical utility of the nomogram were assessed by concordance index (C-index), receiver operating characteristic (ROC) curve, decision curve analysis (DCA) and clinical impact curve (CIC). RESULTS: A total of 343 TD cases were included in the final analysis, of which 30.32% had behavioral problems. The prediction model showed age 12-16 years, abnormal birth history, parenting pattern of indulgence, parent/close relatives with psychiatric disorders, chronic motor or vocal tic disorder (CTD)/Tourette syndrome (TS) and moderate/severe tic severity were associated with behavioral problems in children with TD. The C-index of the prediction model (nomogram) was 0.763 (95% confidence interval, 0.710 ~ 0.816). The nomogram was feasible for making beneficial clinical decisions, according to the satisfactory results of the DCA and CIC. CONCLUSIONS: A nomogram prediction model for comorbid behavioral problems in children with TD was established. The prediction model demonstrated a good discriminative ability and predictive performance for beneficial clinical decisions. This model further provides a comprehensive understanding of associated sociodemographic and clinical characteristics by visual graphs and allows clinicians to rapidly identify patients with a higher risk of behavioral problems and tailor necessary interventions to improve clinical outcomes.

Cross-sectional investigation of quality of life determinants among children with tic disorders: The roles of family environmental and clinical factors.

Objective: To examine the association between family environmental and clinical factors with the whole range of quality of life (QOL) in children with tic disorders (TD). Methods: A hospital-based cross-sectional study was conducted among children with TD. All participants were given a family environmental survey and scale evaluations with Yale Global Tic Severity Scale (YGTSS), Achenbach Child Behavior Checklist (CBCL) and PedsQL-Generic Core Scale of the Chinese Version (PedsQL). Variable selection and data analysis was done by the least absolute shrinkage and selection operator (LASSO) method and multivariate logistic regression analysis. Results: A total of 363 TD cases were included in the analysis. YGTSS scores, total CBCL score had significant negative correlations with PedsQL scores (P < 0.05). Of the total 15 factors, 8, 6, 11, 7, 5, 10 potential predictors with nonzero coefficients were identified by LASSO regression models of physical functioning, emotional functioning, social functioning, school functioning, social-psychological domain and PedsQL total scale respectively. Results of multivariate logistic regression analysis showed older age (physical functioning, ORs: 1.77, 3.67; total scale: ORs: 1.73, 2.28), no presence of chronic conditions (school functioning, OR: 1.61), moderate/severe tic severity (physical functioning, OR: 0.57; social functioning, OR: 0.44; social-psychological domain, OR: 0.57), co-morbid behavioral problems (physical functioning, OR: 0.52; emotional functioning, OR: 0.31; social functioning, OR: 0.30; school functioning, OR: 0.35; social-psychological domain, OR: 0.34; total scale, OR: 0.30), no fully parental involvement in care (physical functioning, OR: 0.62), higher paternal (physical functioning, ORs: 2.89, 2.07) and maternal education level (social functioning, ORs: 1.74, 2.03), democratic parenting pattern (emotional functioning, OR: 1.89; social functioning, OR: 2.17; social-psychological domain, OR: 2.33; total scale, OR: 2.11) and inharmony family relationship (emotional functioning, OR: 0.47; total scale, OR: 0.50) were the most important determinants to QOL of TD. Conclusions: This study identifies several QOL determinants among children with TD. Clinicians should be encouraged to screen for family environmental and clinical factors in TD patients, and take tailored interventions to help TD children improve their QOL.

A potential delivery system based on cholera toxin: A macromolecule carrier with multiple activities.

Macromolecular drugs are widely thought to be one of the most promising fields, but there are still many problems, especially with regard to drug delivery. Drug delivery systems are focused on loading efficiency without loss of activity, effective cellular internalization, anti-degradation, target ability, etc. New directions for macromolecular drugs delivery systems are not only to retain the activity of drugs, but bring new bioactivity to carry out dual benefits. Cholera toxin (CT) from Vibrio cholerae is one of such delivery systems and plays a potential role in delivering macromolecular drugs. After released from V. cholerae in the intestine, the B subunit of CT binds to the ganglioside GM1 on intestinal cells, and then the toxin gains access into the intestine. CT has potential as a "vaccine adjuvant-delivery system" (VADS) and is able to bring antigens and serve as adjuvants to induce specific immunity. In addition, it has been well used in the field of mucosal drug delivery and neural targeting. However, native CT is toxic, which restricts its practical application. There are several CT-based proteins with reduced virulence and reserved or even enhanced adjuvant activity under research. In this review, we comprehensively summarize the preparation strategy, advantages, applications and corresponding deficiencies of CT-based proteins. CT is focused on a delivery system when delivering macromolecular cargos such as active protein/peptide and antigen/antigen peptide. CT-based drug delivery system deserves further study due to their superiority.

Co-delivery of PSMA antigen epitope and mGM-CSF with a cholera toxin-like chimeric protein suppressed prostate tumor growth via activating dendritic cells and promoting CTL responses.

Subunit vaccines derived from tumor antigens play a role in tumor therapy because of their unique advantages. However, because of the weak immunogenicity of peptides in subunit vaccines, it is difficult to trigger an effective cytotoxic T lymphocyte (CTL) response, which is critical for cancer therapy. A requirement for the activation of CTL cells by exogenous antigens is the stimulation of antigen presenting cells (APC) with the help of adjuvants and cross-presentation to T lymphocytes. Standard nonconjugated adjuvant-peptide mixtures do not ensure co-targeting of the antigen and the adjuvant to the same APC, which limits the effects of adjuvants. In this study, a fusion protein consisting of murine granulocyte-macrophage colony stimulating factor (mGM-CSF) fused with CTA2 (A2 subunit of cholera toxin) was generated and assembled with CTB-PSMA624-632 (prostate specific membrane antigen (PSMA) peptide 624-632 fused to CTB) to obtain a cholera toxin-like protein. The chimeric protein retained the biological activity of mGM-CSF and had stronger GM1 binding activity than (CTB-PSMA624-632)5. C57BL/6J mice immunized with the CT-like chimeric protein exhibited delayed tumor growth following challenge with human PSMA-EGFP-expressing RM-1 cells. Experiment results showed that the CT-like chimeric protein could induce the maturation of DC cells and improve CTL responses. Overall, these results indicate that the nasal administration of a CT-like chimeric protein vaccine results in the development of effective immunity against prostate tumor cells and might be useful for future clinical anti-tumoral applications.

The preparation of bifunctional hybrid nano-flowers and their application in the enzyme-linked immunosorbent assay for Helicobacter pylori detection.

As the infection by Helicobacter pylori (H. pylori, HP) remains for a lifetime and may induce diseases such as gastric cancer, it is vital to detect and diagnose it. A new non-invasive indirect enzyme-linked immunosorbent assay (iELISA) method based on nano-flowers (NFs) is very advantageous for the sensitive detection of HP. Furthermore, the established iELISA method based on the organic-inorganic bifunctional hybrid nano-flowers including rabbit polyclonal antibody of HP labeled with peroxidase from horseradish (R-HP-Ab-HRP@Cu2+ NFs) showed linearity with HP at a concentration of 0-105 CFU mL-1 (R2 = 0.9997). Moreover, the limit of detection (LOD) reached 50 CFU mL-1, and not only was the detection sensitivity 20 times higher than that based on rabbit polyclonal antibody of HP labeled with peroxidase from horseradish (R-HP-Ab-HRP) but also the stability of R-HP-Ab-HRP in NFs was improved. In addition, the OD450 nm value was still linearly related to the concentration of HP at a range of 0-105 CFU mL-1 (R2 = 0.9952) with a LOD of 50 CFU mL-1 in an artificial saliva system. This study provided a sensitive, low-cost and convenient method for the non-invasive detection of HP.

An in vitro and in vivo study of the brain-targeting effects of an epidermal growth factor-functionalized cholera toxin-like chimeric protein.

The development of neuroprotective drugs has proven to be extremely difficult because of the blood-brain barrier. Intranasal administration is thought to transport the drug from the nasal cavity along the olfactory and trigeminal nerves to the brain, thus bypassing the blood-brain barrier. However, macromolecular protein drugs have low delivery efficiency via this route in general. We hypothesized that an innocuous cholera toxin-like chimeric protein could better enhance the efficiency of protein delivery through the intranasal route. To test this hypothesis, we designed an enhanced green fluorescent protein (EGFP) chimera to evaluate the effect of the cholera toxin (CT) as a carrier for drug delivery into the brain. Then, the EGFP was replaced with epidermal growth factor (EGF) in the chimeric protein, and the therapeutic effect of the new chimeric protein was studied in an LPS-induced neuritis mouse model. The results suggest that the CT-like chimeric protein can bypass the blood-brain barrier and enter the brain in approximately 30 min. This EGF chimeric protein can effectively protect the spatial cognitive ability of and confer anti-anxiety protection to mice. The results indicate that cholera toxin-like chimeric proteins are potential tools for effectively delivering macromodecular drugs into the brain through intranasal administration.

Experimental Autoimmune Encephalomyelitis (EAE) Model of Cynomolgus Macaques Induced by Recombinant Human MOG1-125 (rhMOG1-125) Protein and MOG34-56 Peptide.

BACKGROUND: Experimental autoimmune encephalomyelitis (EAE) induced by self-myelin antigen is a widely used in multiple sclerosis (MS) model for preclinical studies of new therapeutics and potential pathogenesis. By comparison with rodent EAE models, EAE models in primates are more similar to MS. Some groups have developed EAE models in primates by using common marmoset (Callithrix jacchus). However, this model has some limitations. EAE in cynomolgus monkey (Macaque fasciculrais) could overcome these limitations. OBJECTIVES: The main objective of this study was to establish an ideal EAE cynomolgus monkey model resembling human MS by immunizing human myelin oligodendrocyte glycoprotein (MOG) protein. METHODS: In this study, six female cynomolgus monkeys were divided into two separate experiment groups and one monkey as the control. EAE was induced in cynomolgus monkey by immunizing with the recombinant human myelin oligodendrocyte glycoprotein extracellular domain (1-125) (rhMOG1-125) and a synthetic peptide, representing peptide 34-56 of human myelin oligodendrocyte glycoprotein (MOG34-56) in complete Freund's adjuvant (CFA) by subcutaneous multipoint immunization in the armpit and inguinal region and in combination with intravenous injection of pertussis toxin, and subsequent booster immunizations with the same dose of antigen in incomplete Freund's adjuvant (IFA) until the animals developed clinically significant EAE (score≥2). The body weight, clinical scores, magnetic resonance imaging (MRI), histopathology, antibody, cytokine profiling and antigen-specific lymphocyte proliferation were evaluated. RESULTS: The results showed that despite the different time intervals between onset and significant neurological deficits, all the cynomolgus monkeys immunized with rhMOG1-125 or MOG34-56 developed clinically significant acute fulminant or mild forms of EAE, with a success rate of 100%. The clinical courses were obvious heterogeneity which closely resembles MS. MOG34-56 immunization was much milder compared with rhMOG1-125 immunized individuals, at least in cynomolgus monkeys. Inflammation and demyelinated lesions were present in the brains and spinal cords. Immune profiling revealed high IgG levels associated with early onset of EAE but not the course of the disease. Significant antigen-specific T lymphocyte responses against immunodominant epitopes of MOG were also detected. CONCLUSION: Our results indicated rhMOG1-125 and MOG34-56 could induce successfully EAE models resembling MS in cynomolgus monkeys. The synergistic action of anti-myelin T cells and Abs during the pathogenesis of EAE could establish a more ideal EAE model.

The Effects and Mechanism of YK-4-279 in Combination with Docetaxel on Prostate Cancer.

Background: Docetaxel is the first-line treatment for castration-resistant prostate cancer (CRPC). The limited survival benefit associated with the quick emergence of resistance and systemic toxicity diminishes its efficacy in high-dose monotherapy. YK-4-279 is a small molecule inhibitor of ETV1 that plays an important role in the progression of prostate cancer. The aim of this study was to evaluate the hypothesis that the combination of docetaxel and YK-4-279 will have a synergistic effect on inhibiting growth and accelerating apoptosis in human prostate cancer cells. Methods: Cell growth assessed using CCK-8 and trypan blue exclusion assays. Cell apoptosis was determined by morphological assessment in cells stained with propidium iodide. Standard scratch migration and Matrigel-coated transwell invasion assays were used to assess cell migration and invasion, respectively. Western blotting was used to investigate the levels of ETV1, AR, PSA, p-STAT3, survivin, Bcl-2, and p-Akt in prostate cancer cells. Results: The combination of low-dose docetaxel and YK-4-279 synergistically inhibited growth and induced apoptosis in human prostate cancer cells. The combination also more efficiently suppressed the migration and invasion of LNCaP and PC-3 cells. The combination of low-dose docetaxel and YK-4-279 caused a stronger decrease in the levels of ETV1, AR, PSA, p-STAT3, survivin, Bcl-2, and p-Akt in LNCaP cells and of p-Akt, Bcl-2, and p-STAT3 in PC-3 cells compared with either drug alone. Conclusions: These data suggest that the combination of docetaxel and YK-4-279 may be an effective approach for inhibiting the growth and metastasis of prostate cancer. This could permit a decrease in the docetaxel dose necessary for patients with CRPC and thereby lower its systemic toxicity.

Purification and characterization of a novel cell-penetrating carrier similar to cholera toxin chimeric protein.

Developing a recombinant vector for noninvasively delivering biological macromolecules into the brain is important. This study constructed and purified a protein complex based on the cholera toxin (CT) molecular structure. Enhanced green fluorescent protein (EGFP)-modified A2 subunits of CT (CTA2) were used as tracer molecules for introduction of transactivator of transcription (TAT) through the A subunit into cells. The protein complex EGFP-CTA2-TAT/(CTB)5 (CTB: B subunit of CT) was obtained using an in vitro recombination method and verified by monosialoganglioside-enzyme-linked immunosorbent assay and high performance liquid chromatography assay. The protein complexes bound more strongly to monosialoganglioside (GM1) than (CTB)5 at low concentrations (0.625-1.25 µg/mL). In vitro assays revealed that the transmembrane function of TAT was also maintained. The GM1-binding activity and cell membrane-penetrating ability suggested that a CT structure-based protein complexes could be used to design a delivery carrier for intranasal administration through GM1 binding. The expression vector introduced in this study provides a feasible expression frame for constructing several new macromolecular protein drugs for effective cell penetration.

Synthesis and Biological Evaluation of Novel Resveratrol-NSAID Derivatives as Anti-inflammatory Agents.

Long-term use of nonsteroidal antiinflammatory drugs (NSAIDs) may cause serious side effects such as gastric mucosal damage. Resveratrol, a naturally dietary polyphenol, exhibited anti-inflammatory activity and a protective effect against gastric mucosa damage induced by NSAIDs. In this regard, we synthesized a series of resveratrol-based NSAIDs derivatives and evaluated their anti-inflammatory activity against nitric oxide (NO) overproduction in lipopolysaccharide (LPS)-stimulated RAW264.7 macrophages. We identified mono-substituted resveratrol-ibuprofen combination 21 as the most potent anti-inflammatory agent, which is more active than a physical mixture of ibuprofen and resveratrol, individual ibuprofen, or individual resveratrol. In addition, compound 21 exerted potent inhibitory effects on the LPS-induced expression of tumor necrosis factor-α (TNF-α) and interleukin-1ß (IL-1ß). Furthermore, compound 21 significantly increased the survival rate in an LPS-induced acute inflammatory model and produced markedly less gastric damage than ibuprofen. It was found that compound 21 may be a potent anti-inflammatory agent for the treatment of inflammation-related diseases.

Terpenoid composition and the anticancer activity of Acanthopanax trifoliatus.

The petroleum ether and ethyl acetate fractions of extract from an edible and medicinal plant Acanthopanax trifoliatus were found to show significant inhibitory effects against SF-268, MCF-7, HepG2 and NCI-H460 cancer cells. Two new ursane-type triterpenoids, acantrifoic acid C (1) and acantrifoic acid D (2), along with five known triterpenoids (3-7) and eight known diterpenoids (8-15) were obtained from these two fractions. To the best of our knowledge, this is the first report concerning the isolation of compounds (5-12, 14, 15) from A. trifoliatus. Among all the isolated compounds, 3, 5 and 8 from the ethyl acetate fraction showed the strongest inhibitory effects against cancer cells, while 12 and 13 from the petroleum ether fraction showed moderate activities. These terpenoid compounds may be responsible for the anticancer activities of A. trifoliatus. Our study provides the first evidence that terpenoids from A. trifoliatus exert anticancer activities and indicates that A. trifoliatus may be a useful edible plant for further development of anticancer health supplement.

Synthesis and biological evaluation of curcumin derivatives containing NSAIDs for their anti-inflammatory activity.

Oral administration of nonsteroidal anti-inflammatory drugs (NSAIDs) was frequently associated with serious adverse effects. Inspired by curcumin-a naturally traditional Chinese medicine, a series of curcumin derivatives containing NSAIDs, used for transdermal application, were synthesized and screened for their anti-inflammatory activities in vitro and in vivo. Compared with curcumin and parent NSAID (salicylic acid and salsalate), topical application of A11 and B13 onto mouse ear edema, prior to TPA treatment markedly suppressed the expression of IL-1ß, IL-6 and TNF-α, respectively. Mechanistically, A11 and B13 blocked the phosphorylation of IκBα and suppressed the activation of p65 and IκBα. It was found that A11 and B13 may be potent anti-inflammatory agents for the treatment of inflammatory diseases.

Combination of 12-O-tetradecanoylphorbol-13-acetate with diethyldithiocarbamate markedly inhibits pancreatic cancer cell growth in 3D culture and in immunodeficient mice.

The aim of the present study was to determine the effects of 12-O-tetradecanoylphorbol-13-acetate (TPA) and diethyldithiocarbamate (DDTC) alone or in combination on human pancreatic cancer cells cultured in vitro and grown as xenograft tumors in nude mice. Pancreatic cancer cells were treated with either DDTC or TPA alone, or in combination and the number of viable cells was then determined by trypan blue ecxlusion assay and the number of apoptotic cells was determined by morphological assessment by staining the cells with propidium idiode and examining them under a fluorescence microscope. Treatment with DDTC or TPA alone inhibited the growth and promoted the apoptosis of pancreatic cancer cells in a concentration-dependent manner. These effects were more prominent following treatment with TPA in combination with DDTC than following treatment with either agent alone in PANC-1 cells in monolayer cultures and in 3 dimensional (3D) cultures. The potent effects of the combination treatment on PANC-1 cells were associated with the inhibition of nuclear factor-κB (NF-κB) activation and the decreased expression of Bcl-2 induced by DDTC, as shown by NF-κB-dependent reporter gene expression assay and western blot analysis. Furthermore, treatment of nude mice with DDTC + TPA strongly inhibited the growth of PANC-1 xenograft tumors. The results of the present study indicate that the administration of TPA and DDTC in combination may be an effective strategy for inhibiting the growth of pancreatic cancer.

Anticancer activity of Acanthopanax trifoliatus (L) Merr extracts is associated with inhibition of NF-kB activity and decreased Erk1/2 and Akt phosphorylation.

Acanthopanax trifoliatus (L) Merr (AT) is commonly used as an herbal medicine and edible plant in some areas of China and other Asian countries. AT is thought to have anticancer effects, but potential mechanisms remain unknown. To assess the anticancer properties of AT, we exposed prostate cancer cells to AT extracts and assessed cell proliferation and signaling pathways. An ethanol extract of AT was suspended in water followed by sequential extraction with petroleum ether, ethyl acetate and n-butanol. PC-3 cells were treated with different concentrations of each extract and cell viability was determined by the MTT and trypan blue exclusion assays. The ethyl acetate extract of the ethanol extract had a stronger inhibitory effect on growth and a stronger stimulatory effect on apoptosis than any of the other extracts. Mechanistic studies demonstrated that the ethyl acetate extract suppressed the transcriptional activity of NF-kB, increased the level of caspase-3, and decreased the levels of phospho-Erk1/2 and phospho-Akt. This is the first report on the anticancer activity of AT in cultured human prostate cancer cells. The results suggest that AT can provide a plant-based medicine for the treatment or prevention of prostate cancer.

Inhibition of IL-6 expression in LNCaP prostate cancer cells by a combination of atorvastatin and celecoxib.

In the present study, we investigated the effect of a combination of atorvastatin and celecoxib on the formation of interleukin (IL)-6, a cytokine that is increased during the progression of LNCaP tumors from androgen dependence to androgen independence. Culturing LNCaP cells in androgen­depleted (AD) medium increased the levels of IL-6 and survivin, and treatment of the cells in AD medium with a combination of atorvastatin and celecoxib strongly inhibited the increase in IL-6 and survivin which is one of the downstream targets of the IL-6 signaling pathway. Addition of recombinant IL-6 partially abrogated the combined effect of atorvastatin and celecoxib on apoptosis in LNCaP cells cultured in AD medium. In SCID mice, we found that the levels of IL-6 and survivin expression were increased when LNCaP tumors became androgen-independent. Treatment of the mice with atorvastatin or celecoxib alone caused decrease in the levels of IL-6 and survivin as LNCaP tumors became androgen-independent, but treatment of the mice with a combination of celecoxib and atorvastatin resulted in a much stronger inhibition in the increase in IL-6 and survivin expression. Our results indicate that decreases in IL-6 and survivin levels by atorvastatin and celecoxib administration are associated with increased apoptosis in LNCaP cells treated with this drug combination. Our in vivo studies indicate that the inhibitory effect of a combination of atorvastatin and celecoxib on the progression of androgen-dependent LNCaP xenograft tumors to androgen independence is associated with inhibition of the increase in IL-6 and survivin that occurs when androgen-dependent LNCaP prostate tumors become androgen-independent.

Effects of pyridine analogs of curcumin on growth, apoptosis and NF-κB activity in prostate cancer PC-3 cells.

Twelve pyridine analogs of curcumin were studied for their effects on growth and apoptosis in human prostate cancer PC-3 cells. The ability of these compounds to inhibit the transcriptional activity of nuclear factor-kappa B (NF-κB) and the level of phosphorylated extracellular signal-regulated kinases (phospho-ERK1/2) in PC-3 cells was also determined. Treatment of PC-3 cells with the pyridine analogs of curcumin resulted in concentration-dependent growth inhibition and apoptosis stimulation. Only pyridine analogs of curcumin with a tetrahydrothiopyrane-4-one linker (FN compounds) exhibited a strong inhibitory effect on growth and a strong stimulatory effect on apoptosis at low concentrations (≤ 1 µM). Mechanistic studies showed that NF-κB transcriptional activity in PC-3 cells was strongly inhibited by treatment with group FN compounds. Treatment of PC-3 cells with 1 µM FN1 resulted in a decrease of activated ERK1/2. Results from the present study indicate that FN compounds warrant further in vivo studies using suitable animal models of prostate cancer.