Kaempferol-Enhanced Migration and Differentiation of C2C12 Myoblasts via ITG1B/FAK/Paxillin and IGF1R/AKT/mTOR Signaling Pathways.

SCOPE: Kaempferol (KMP), a bioactive flavonoid compound found in fruits and vegetables, contributes to human health in many ways but little is known about its relationship with muscle mass. The effect of KMP on C2C12 myoblast differentiation and the mechanisms that might underlie that effect are studied. METHODS AND RESULTS: This study finds that KMP (1, 10 µM) increases the migration and differentiation of C2C12 myoblasts in vitro. Studying the possible mechanism underlying its effect on migration, the study finds that KMP activates Integrin Subunit Beta 1 (ITGB1) in C2C12 myoblasts, increasing p-FAK (Tyr398) and its downstream cell division cycle 42 (CDC42), a protein previously associated with cell migration. Regarding differentiation, KMP upregulates the expression of myosin heavy chain (MHC) and activates IGF1/AKT/mTOR/P70S6K. Interestingly, pretreatment with an AKT inhibitor (LY294002) and siRNA knockdown of IGF1R leads to a decrease in cell differentiation, suggesting that IGF1/AKT activation is required for KMP to induce C2C12 myoblast differentiation. CONCLUSION: Together, the findings suggest that KMP enhances the migration and differentiation of C2C12 myoblasts through the ITG1B/FAK/paxillin and IGF1R/AKT/mTOR pathways. Thus, KMP supplementation might potentially be used to prevent or delay age-related loss of muscle mass and help maintain muscle health.

EGFR-mediated hyperacetylation of tubulin induced docetaxel resistance by downregulation of HDAC6 and upregulation of MCAK and PLK1 in prostate cancer cells.

Docetaxel-based chemotherapy has generally been considered as one of the effective treatments for castration-resistant prostate cancer (PCa). However, clinical treatment with docetaxel often encounters a number of undesirable effects, including drug resistance. Tubulin isoforms have been previously examined for their resistance to docetaxel in many cancers, but their real mechanisms remained unclear. In this study, a series of docetaxel-resistant PC/DX cell sublines were established by chronically exposing PC3 to progressively increased concentrations of docetaxel. Western blotting results showed significantly higher expression of acetyl-tubulin, α-tubulin, ß-tubulin, γ-tubulin, and ßIII-tubulin in PC/DX25 than in parental PC3 cells. PC/DX25 with greater resistance to docetaxel had higher levels of acetyl-tubulin and mitotic centromere-associated kinesin (MCAK) than PC3 cells. This study found that docetaxel induced the expression of acetyl-tubulin and MCAK in PC3 cells at a dose- and time-dependent manner. Both mRNA and protein levels of histone deacetylase 6 (HDAC6) were significantly decreased in PC/DX25 compared with PC3 cells. PC3 increased the resistance to docetaxel by HDAC6 knockdown and Tubastatin A (HDAC6 inhibitor). Conversely, PC/DX25 reversed the sensitivity to docetaxel by MCAK knockdown. Notably, flow cytometry analysis revealed that MCAK knockdown induced significantly sub G1 fraction in PC/DX cells. Overexpression of polo-like kinase-1 increased the cell survival rate and resistance to docetaxel in PC3 cells. Moreover, epidermal growth factor receptor (EGFR) activation induced the upregulation of acetyl-tubulin in docetaxel-resistant PCa cells. These findings demonstrated that the EGFR-mediated upregulated expression of acetyl-tubulin played an important role in docetaxel-resistant PCa.

Caffeic acid phenethyl ester suppresses EGFR/FAK/Akt signaling, migration, and tumor growth of prostate cancer cells.

BACKGROUND: Epidermal growth factor receptor (EGFR) is upregulated in prostate cancer (PCa). However, suppression of EGFR did not improve the patient outcome, possibly due to the activation of PI3K/Akt signaling in PCa. Compounds able to suppress both PI3K/Akt and EGFR signaling may be effective for treating advanced PCa. PURPOSE: We examined if caffeic acid phenethyl ester (CAPE) simultaneously suppresses the EGFR and Akt signaling, migration and tumor growth in PCa cells. METHODS: Wound healing assay, transwell migration assay and xenograft mice model were used to determine the effects of CAPE on migration and proliferation of PCa cells. Western blot, immunoprecipitation, and immunohistochemistry staining were performed to determine the effects of CAPE on EGFR and Akt signaling. RESULTS: CAPE treatment decreased the gene expression of HRAS, RAF1, AKT2, GSK3A, and EGF and the protein expression of phospho-EGFR (Y845, Y1069, Y1148, Y1173), phospho-FAK, Akt, and ERK1/2 in PCa cells. CAPE treatment inhibited the EGF-induced migration of PCa cells. Combined treatment of CAPE with EGFR inhibitor gefitinib showed additive inhibition on migration and proliferation of PCa cells. Injection of CAPE (15 mg/kg/3 days) for 14 days suppressed the tumor growth of prostate xenografts in nude mice as well as suppressed the levels of Ki67, phospho-EGFR Y845, MMP-9, phospho-Akt S473, phospho-Akt T308, Ras, and Raf-1 in prostate xenografts. CONCLUSIONS: Our study suggested that CAPE can simultaneously suppress the EGFR and Akt signaling in PCa cells and is a potential therapeutic agent for advanced PCa.

Gamma Frequency Inhibits the Secretion and Aggregation of Amyloid-ß and Decreases the Phosphorylation of mTOR and Tau Proteins in vitro.

BACKGROUND: Alzheimer's disease (AD) was the main cause of dementia in an aging society; unfortunately, there is no effective treatment for AD now. Meditation has been reported to thicken the cerebral cortex, and gamma wave at a frequency of 40 hertz (Hz) was recorded during the meditation process from the brain. Previous study showed that non-invasive scintillation gamma frequency oscillation increased the space in recognition and memory of auditory cortex hippocampal gyrus in AD mice model. However, the AD-related molecular change by exposure of 40 Hz gamma frequency in brain cells was still unclear. OBJECTIVE: We investigated the AD-related molecular change by exposure of 40 Hz gamma frequency in SH-SY5Y cells. METHODS: We designed the light and sound generators at 40 Hz gamma frequency for this study. SH-SY5Y cells were exposed to sound or light of 40 Hz gamma frequency, respectively. The concentrations of amyloid-ß40 (Aß40) and amyloid-ß42 (Aß42) were quantified by enzyme-linked immunosorbent assay. The protein levels were examined by Western blotting. The aggregation of Aß42 was examined by thioflavin T assay. RESULTS: Our results showed that the secretion of Aß, phosphorylation of AKT, mTOR, and tau, and aggregation of Aß42 were significantly inhibited by 40 Hz gamma frequency in SH-SY5Y cells. The phosphorylation of 4E-BP1, downstream of mTOR, was induced by 40 Hz gamma frequency in SH-SY5Y cells. CONCLUSION: Our study showed 40 Hz gamma frequency involved in the inhibition of secretion and aggregation of Aß and inhibition of p-Tau protein expression through the mTOR/4E-BP1/Tau signaling pathway.

The Promotion of Migration and Myogenic Differentiation in Skeletal Muscle Cells by Quercetin and Underlying Mechanisms.

Aging and muscle disorders frequently cause a decrease in myoblast migration and differentiation, leading to losses in skeletal muscle function and regeneration. Several studies have reported that natural flavonoids can stimulate muscle development. Quercetin, one such flavonoid found in many vegetables and fruits, has been used to promote muscle development. In this study, we investigated the effect of quercetin on migration and differentiation, two processes critical to muscle regeneration. We found that quercetin induced the migration and differentiation of mouse C2C12 cells. These results indicated quercetin could induce myogenic differentiation at the early stage through activated p-IGF-1R. The molecular mechanisms of quercetin include the promotion of myogenic differentiation via activated transcription factors STAT3 and the AKT signaling pathway. In addition, we demonstrated that AKT activation is required for quercetin induction of myogenic differentiation to occur. In addition, quercetin was found to promote myoblast migration by regulating the ITGB1 signaling pathway and activating phosphorylation of FAK and paxillin. In conclusion, quercetin can potentially be used to induce migration and differentiation and thus improve muscle regeneration.

Application of Micro-Western Array for Identifying Different Serum Protein Expression Profile among Healthy Control, Alzheimer's Disease Patients and Patients' Adult Children.

(1) Background: Alzheimer's disease (AD) is the most common form of dementia. Increased levels of inflammatory proteins have been observed in brain and plasma samples of AD patients; however, it is not clear if other serum proteins correlate to the development or disease progression of AD. (2) Methods: Micro-Western Array (MWA) is a high-throughput antibody-based proteomics system which allows detection of the expression levels of 24-96 different proteins within 6-30 samples simultaneously. We applied MWA to explore potential serum protein biomarkers correlated to the development and progression of AD by examining the difference in serum protein profile of 31 healthy control (HC), 30 patients with AD and 30 patients' adult children (ACS). (3) Results: Compared to HC, AD and ACS express similar pattern of serum proteins, including higher protein levels of ABCA1, ABCG1, SREBP1 and LXRß but lower protein levels of ApoD, ApoE, ApoH, c_Myc, COX2 and Hippo-YAP signaling proteins. AD patients had higher serum levels of ABCG1, ApoD, ApoH, COX2, LXRα and YAP, but lower levels of ABCA1, ApoE, c_Myc, LATS1, MST1, MST2, Nanog, NFκB_p50, PPARγ and SREBP2, as compared to ACS. Pearson's correlation analysis revealed that the protein expression level of ApoE, c_Myc, LATS1, MST2, NFκB p50, PPARγ and SREBP1 was negatively correlated to age, while that of ApoE, c_Myc, LATS1, MST1, MST2, Nanog, NFκB p50 and PPARγ was positively correlated to age. (4) Conclusions: We identified a group of serum proteins which may correlate to disease progression of AD and can be potential diagnostic serum protein biomarkers.

Plasma biomarkers and their correlation in adult children of parents with Alzheimer's disease.

Family history (FH) of late-onset Alzheimer's disease (AD) is associated with changes in several cerebrospinal fluid (CSF) biomarkers in cognitively normal individuals. However, potential changes in plasma biomarkers remain unknown. This study aimed to evaluate potential plasma biomarkers and their correlation in cognitively normal adult children (AC) and to compare this data with their AD parents and unrelated non-demented controls (NC). Participants with dementia due to AD, their AC and NC were recruited. Plasma samples were assessed for amyloid beta (Aß)1-42, Aß1-40, total tau (T-tau) and phosphorylated tau (P-tau). Kruskal-Wallis test was used for the comparison of this data between the three groups. Spearman rank correlation was used for evaluation of the correlations between Aß1-40 and Aß1-42, and T-tau and P-tau in the AD and AC groups. A total of 99 subjects completed the assessment (30 had AD; 38 were AC group; and 31 were NC). Compared with the NC group, there were significantly higher levels of Aß1-40, P-tau, and P-tau/T-tau ratio, and lower levels of Aß1-42 and Aß1-42/Aß1-40 ratio in the AD and AC groups. The correlation between the level of Aß1-42 and Aß1-40 and level of T-tau and P-tau was only observed in the AC but not in the AD group. AC of AD parents demonstrate some indicators of AD like their parents. Disruption to the correlation between Aß and tau in AD may be a biomarker for the development of AD in AC, which should be examined in a longitudinal cohort.

Chlorogenic Acid Inhibition of Esophageal Squamous Cell Carcinoma Metastasis via EGFR/p-Akt/Snail Signaling Pathways.

BACKGROUND/AIM: Chlorogenic acid (CGA) is a polyphenol compound found in a variety of foods, including coffee, tea, cherries, and apples. It has been found by a number of studies to affect the viability of human cancer cells. No study has investigated its effect on esophageal squamous cell carcinoma (ESCC) metastasis or the molecular mechanism underlying its effect on this disease. MATERIALS AND METHODS: We first used the Taiwanese ESCC cell line CE81T/VGH to create CE81T-M4 cells. Treatment of higher motility cells with chlorogenic acid for 24 h led to inhibition of cell migration and invasion as shown by scratch migration and transwell assays. RESULTS: Western blotting showed that chlorogenic acid halted the activation of EGFR/p-Akt/Snail pathway and suppressed the expression of MMP-2 and MMP-9. Knockdown of either EGFR or Akt inhibited Snail, MMP2, and MMP9 activity as well as cell migration and invasion. CONCLUSION: Chlorogenic acid inhibited cancer cell motility via the EGFR/p-Akt/Snail pathway and could potentially be used to develop an antimetastatic agent for ESCC in the future.

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.

Impact of the CYP2D6 single nucleotide polymorphism on the concentration of and therapeutic response to donepezil in mild-to-moderate Alzheimer's disease.

BACKGROUND/PURPOSE: Donepezil was approved for the treatment of Alzheimer's disease (AD) but causes variable therapeutic responses. Thus, identifying specific genetic polymorphisms, which can predict a therapeutic response to donepezil, would enable a development of personalized strategy to treatment for patients with AD. The research aimed to exam the impact of the cytochrome P450 2D6 (CYP2D6) single nucleotide polymorphism (SNP) rs1080985 on the concentration of and therapeutic response to donepezil in AD. METHODS: In total, 40 newly diagnosed AD patients who had a clinical dementia rating (CDR) of 0.5-2 and who were on donepezil were enrolled and followed up. Plasma concentrations of donepezil were determined after 6 months of donepezil treatment. Cognitive and functional statuses were evaluated annually during follow-up. The response to therapy was defined based on the change in CDR. RESULTS: At a mean of 21.8 ± 5.7 months of follow-up, 10 of 40 patients (25.0%) were nonresponders to donepezil treatment. Patients who were homozygous for the G allele exhibited a higher concentration of donepezil and concentration-to-dose ratio than those with other genotypes. Furthermore, a significantly higher proportion of patients with the G/G genotype were responders than nonresponders (90.0% vs 50.0%, P = 0.015, effect size of V: 0.457) to donepezil treatment. Conversely, patients carrying the C allele had a significantly high risk of poor responses to donepezil treatment (odds ratio: 9.00, 95% confidence interval: 1.611-50.275). CONCLUSION: The CYP2D6 SNP rs1080985 might be a useful pharmacogenetic marker of the long-term therapeutic response to donepezil in patients with AD.

Purified Astaxanthin from Haematococcus pluvialis Promotes Tissue Regeneration by Reducing Oxidative Stress and the Secretion of Collagen In Vitro and In Vivo.

Intracellular reactive apoptosis and reactive oxygen species (ROS) play a crucial role in ultraviolet- (UV-) induced inflammation and aging reaction in human dermal tissues. This study determines the mechanism by which Haematococcus pluvialis extracts (HPE) and purified astaxanthin (HPA) to promote skin regeneration in the injured tissue in vitro and in vivo. The results show that HPE and HPA decrease the DNA damage and promote the secretion of collagen from the human normal fibroblast cell line (Hs68) in a dose-dependent manner. UV irradiation and HPA reduce oxidative stress damage due to phorbol-12-myristate-13-acetate (PMA). When skin cells are injured by free radicals, cells undergo a programmed cellular death. Cellular apoptotic death is determined using annexin V-fluorescein isothiocyanate (FITC)/propidium iodide (PI) double staining to verify that there is no cell membrane asymmetry and that the nuclear membrane is broken. Inflammatory symptoms and apoptotic injuries to experimental rats in a group that is treated with HPA treated are decreased in a dose-dependent manner after UVB exposure (300 mJ/cm2) for 15 min in vivo, compared to the vehicle control group. These positive results show that HPA repairs UVB-triggered skin tissue injury and aging by conducting electrons out of cells to maintain a low level of oxidative stress so that collagen is synthesized in vitro and in vivo.

IL-10 from plasmacytoid dendritic cells promotes angiogenesis in the early stage of endometriosis.

An elevated level of IL-10 has been considered a critical factor for the development of endometriosis; however, its detailed mechanism and causal relationship remain unclear. This study explored the cellular source and angiogenic activity of local IL-10 during the early stage of endometriosis. Using a surgical murine model, we found that localised treatment with exogenous recombinant IL-10 on the day of surgery significantly enhanced endometriotic lesion growth and angiogenesis, whereas blocking local IL-10 activity using mAbs significantly suppressed those effects. Adoptive transfer of Il10+/+ plasmacytoid dendritic cells into mice significantly enhanced lesion development, whereas Il10-/- plasmacytoid dendritic cells significantly inhibited lesion development. Furthermore, in vitro angiogenesis analyses demonstrated that the IL-10 and IL-10 receptor pathway stimulated the migratory and tube formation ability of HUVECs as well as ectopic endometrial mesenchymal stem cells through, at least in part, a VEGF-dependent pathway. We also found that recombinant IL-10 directly stimulated angiogenesis, based on a Matrigel plug assay as well as a zebrafish model. Pathological results from human endometrioma tissues showed the increased infiltration of CD123+ plasmacytoid dendritic cells and higher percentages of cells that express the IL-10 receptor and CD31 as compared with the corresponding normal counterparts. Taken together, these results show that IL-10 secreted from local plasmacytoid dendritic cells promotes endometriosis development through pathological angiogenesis during the early disease stage. This study provides a scientific basis for a potential therapeutic strategy targeting the IL-10-IL-10 receptor pathway in the endometriotic milieu. © 2019 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland.

CD44 Promotes Migration and Invasion of Docetaxel-Resistant Prostate Cancer Cells Likely via Induction of Hippo-Yap Signaling.

Patients receiving docetaxel developed a drug resistance within a few months. We generated docetaxel-resistant PC/DX25 and DU/DX50 CRPC cells from PC-3 and DU-145 PCa cells, respectively. We investigated the mechanism behind why PC/DX25 and DU/DX50 cells exhibited higher migration and invasion ability. Transwell assays were used to measure the migration and invasion of PCa cell. Fluorescence activated cell sorter (FACS) analysis was used to determine the population of cancer stem cell (CSC)-like cell. Micro-Western Array (MWA) was used to study the changes of the protein profile. FACS analysis revealed that PC/DX25 cells and DU/DX50 cells contain higher CD44+ population. MWA and Western blotting assay revealed that protein expression of CD44, YAP, CYR61, CTGF, phospho-ERK1/2 T202/Y204, ERK and vimentin was elevated in PC/DX25 cells. Knockdown of CD44 or YAP suppressed migration and invasion of PC/DX25 and DU/DX50 cells. Knockdown of CD44 decreased expression of YAP, CTGF and CYR61 but increased phosphorylation of S127 on YAP. CD44 knockdown also suppressed protein level of AKT, phospho-AKT T308, phospho-ERK1/2 T202/Y204 and vimentin. CD44 promotes migration and invasion of docetaxel-resistant PCa cells probably via induction of Hippo-Yap signaling pathway and CD44/YAP pathway may be a therapeutic target for docetaxel-resistant PCa.

Preliminary evidence of polymorphisms of cell cycle regulatory genes and their roles in urinary tract urothelial cancer susceptibility and prognosis in a Taiwan population.

OBJECTIVE: Our aims were to investigate the correlations between polymorphisms in the cell cycle regulatory genes (aurora kinase A [AURKA], survivin, p21, and p53) and UC risk and prognosis. PATIENTS AND METHODS: This study recruited 185 patients with UTUC, 46 patients with bladder cancer, and 188 hospital controls. Demographic data and possible confounding factors were collected using a standardized questionnaire. Genotyping was determined by real-time polymerase chain reaction and TaqMan probe methods. RESULTS: Compared to the UTUC group, the control group had significantly more alcohol drinkers and coffee drinkers but not cigarette smokers. Statistical analyses showed that UTUC risk correlated with AURKA rs2273535 (adjusted odds ratio = 2.51, 95% CI = 1.07-6.15) and with survivin rs9904341 (adjusted odds ratio = 1.82, 95% CI = 1.03-3.26) polymorphisms but not with p21 and p53 polymorphisms. The bladder cancer risk showed no significant correlations with the 4 polymorphisms. In both the UTUC and UC groups, disease-free survival and overall survival were significantly longer in patients with the rs1801270CC genotype compared to those with CA+AA genotypes. Poor prognosis did not correlate with polymorphisms in AURKA, survivin, and p53 genes. CONCLUSION: These results suggest that cell cycle regulatory gene polymorphisms rs2273535 and rs9904341 may contribute to UTUC susceptibility; gene polymorphism rs1801270 may contribute to prognosis of UTUC and prognosis of UC. The p53 polymorphisms may not correlate with UTUC susceptibility and prognosis of UTUC or UC. Further well-designed, large-scale studies are needed to clarify these findings.

Differential resistance to platinum-based drugs and 5-fluorouracil in p22phox-overexpressing oral squamous cell carcinoma: Implications of alternative treatment strategies.

BACKGROUND: We have previously shown that p22phox confers resistance to cisplatin in oral squamous cell carcinoma (OSCC). Whether p22phox has clinical correlation with cisplatin resistance and affects the efficacy of other platinum or nonplatinum drugs is unknown. METHODS: The p22phox expression in tissues and apoptotic markers in cell lines was detected by immunoblotting. The cytotoxicity of chemotherapy drugs was determined by methylthiazol tetrazolium assay. In vivo chemoresistance of p22phox-overexpressing tumors was confirmed by the xenograft mouse model. RESULTS: The p22phox was upregulated in tumors of patients with OSCC refractory to cisplatin treatment. The p22phox overexpression markedly increased the resistance to cisplatin and carboplatin, but not oxaliplatin and 5-fluorouracil (5-FU), in OSCC cells by differentially inhibiting the drug-induced apoptosis. Furthermore, p22phox-dependent resistance to cisplatin, but not 5-FU, was demonstrated in mouse xenograft tumors. CONCLUSION: The p22phox expression may not only be a prognostic biomarker for prediction of chemotherapy outcomes, but the indication for alternative treatment strategies in oral cancer.

Inhibition of the EGFR/STAT3/CEBPD Axis Reverses Cisplatin Cross-resistance with Paclitaxel in the Urothelial Carcinoma of the Urinary Bladder.

PURPOSE: Cisplatin (CDDP) is frequently used in combination chemotherapy with paclitaxel for treating urothelial carcinoma of the urinary bladder (UCUB). CDDP cross-resistance has been suggested to develop with paclitaxel, thus hindering successful UCUB treatment. Therefore, elucidating the mechanisms underlying CDDP-induced anticancer drug resistance is imperative and may provide an insight in developing novel therapeutic strategy. EXPERIMENTAL DESIGN: Loss-of-function assays were performed to elucidate the role of the EGFR and STAT3 in CDDP-induced CCAAT/enhancer-binding protein delta (CEBPD) expression in UCUB cells. Reporter and in vivo DNA-binding assays were employed to determine whether CEBPD directly regulates ATP binding cassette subfamily B member 1 (ABCB1) and ATP binding cassette subfamily C member 2 (ABCC2) activation. Finally, a xenograft animal assay was used to examine the abilities of gefitinib and S3I-201 (a STAT3 inhibitor) to reverse CDDP and paclitaxel sensitivity. RESULTS: CEBPD expression was maintained in postoperative chemotherapy patients, and this expression was induced by CDDP even in CDDP-resistant UCUB cells. Upon CDDP treatment, CEBPD activated ABCB1 and ABCC2. Furthermore, the EGFR/STAT3 pathway contributed to CDDP-induced CEBPD expression in UCUB cells. Gefitinib and S3I-201 treatment significantly reduced the expression of CEBPD and enhanced the sensitivity of CDDP-resistant UCUB cells to CDDP and paclitaxel. CONCLUSIONS: Our results revealed the risk of CEBPD activation in CDDP-resistant UCUB cells and suggested a therapeutic strategy for patients with UCUB or UCUB resisted to CDDP and paclitaxel by combination with either gefitinib or S3I-201. Clin Cancer Res; 23(2); 503-13. ©2016 AACR.

MiR-193b Mediates CEBPD-Induced Cisplatin Sensitization Through Targeting ETS1 and Cyclin D1 in Human Urothelial Carcinoma Cells.

Transcription factor CCAAT/enhancer-binding protein delta (CEBPD) plays multiple roles in tumor progression. Studies have demonstrated that cisplatin (CDDP) induced CEBPD expression and had led to chemotherapeutic drug resistance. However, the underlying molecular mechanisms of CDDP-regulated CEBPD expression and its relevant roles in CDDP responses remain elusive. MicroRNAs (miRNAs) are small non-coding RNAs that negatively regulate gene expression in a sequence-specific manner. Abnormal miRNAs expression is associated with tumor progression. In current study, a large-scale PCR-based miRNA screening was performed to identify CEBPD-associated miRNAs in urothelial carcinoma cell line NTUB1. Eleven miRNAs were selected with more than twofold changes. MiR-193b-3p, a known tumor suppressor, down-regulated proto-oncogenes Cyclin D1, and ETS1 expression and led to cell cycle arrest, cell invasion, and migration inhibition. The expression of miR-193b-3p was associated with the DNA binding ability of CEBPD in CDDP response. CEBPD knocking-down approach provided a strong evidence of the positive correlation between CEBPD and miR-193b-3p. CDDP-induced CEBPD trans-activated miR-193b-3p expression and it directly targeted the 3'-UTR of Cyclin D1 and ETS1 mRNA, and silenced the protein expression. In addition, miR-193b-3p also inhibited cell migration activity, arrested cell at G1 phase, and sensitized NTUB1 to CDDP treatment. In conclusion, this study indicates that CEBPD exhibits an anti-tumorigenic function through transcriptionally activating miR-193b-3p expression upon CDDP treatment. This study provides a new direction for managing human urothelial carcinoma. J. Cell. Biochem. 118: 1563-1573, 2017. © 2016 Wiley Periodicals, Inc.

Enriched Astaxanthin Extract from Haematococcus pluvialis Augments Growth Factor Secretions to Increase Cell Proliferation and Induces MMP1 Degradation to Enhance Collagen Production in Human Dermal Fibroblasts.

Among many antioxidants that are used for the repairing of oxidative stress induced skin damages, we identified the enriched astaxanthin extract (EAE) from Haematococcus pluvialis as a viable ingredient. EAE was extracted from the red microalgae through supercritical fluid carbon dioxide extraction. To compare the effectiveness, EAE wastreated on human dermal fibroblasts with other components, phorbol 12-myristate 13-acetate (PMA), and doxycycline. With sirius red staining and quantitative real-time polymerase chain reaction (qRT-PCR), we found that PMA decreased the collagen concentration and production while overall the addition of doxycycline and EAE increased the collagen concentration in a trial experiments. EAE increased collagen contents through inhibited MMP1 and MMP3 mRNA expression and induced TIMP1, the antagonists of MMPs protein, gene expression. As for when tested for various proteins through western blotting, it was seen that the addition of EAE increased the expression of certain proteins that promote cell proliferation. Testing those previous solutions using growth factor assay, it was noticeable that EAE had a positive impact on cell proliferation and vascular endothelial growth factor (VEGF) than doxycycline, indicating that it was a better alternative treatment for collagen production. To sum up, the data confirmed the possible applications as medical cosmetology agentsand food supplements.

Histone deacetylase inhibitor trichostatin A resensitizes gemcitabine resistant urothelial carcinoma cells via suppression of TG-interacting factor.

Gemcitabine and cisplatin (GC) has been widely used for advanced and metastatic urothelial carcinoma (UC). However, resistance to this remedy has been noticed. We have demonstrated that increase of TG-interacting factor (TGIF) in specimens is associated with worse prognosis of upper tract UC (UTUC) patients. The roles of TGIF in the gemcitabine resistance of UC were explored. Specimens of 23 locally advanced/advanced stage UTUC patients who received GC systemic chemotherapy after radical nephroureterectomy were collected to evaluate the alterations of TGIF in the resistance to the remedy by using immunohistochemistry. In vitro characterizations of mechanisms mediating TGIF in gemcitabine resistance were conducted by analyzing NTUB1 cells and their gemcitabine-resistant subline, NGR cells. Our results show that increased TGIF is significantly associated with chemo-resistance, poor progression-free survival, and higher cancer-related deaths of UTUC patients. Higher increases of TGIF, p-AKT(Ser473) and invasive ability were demonstrated in NGR cells. Overexpression of TGIF in NTUB1 cells upregulated p-AKT(Ser473) activation, enhanced migration ability, and attenuated cellular sensitivity to gemcitabine. Knockdown of TGIF in NGR cells downregulated p-AKT(Ser473) activation, declined migration ability, and enhanced cellular sensitivity to gemcitabine. In addition, histone deacetylases inhibitor trichostatin A (TSA) inhibited TGIF, p-AKT(Ser473) expression and migration ability. Synergistic effects of gemcitabine and TSA on NGR cells were also demonstrated. Collectively, TGIF contributes to the gemcitabine resistance of UC via AKT activation. Combined treatment with gemcitabine and TSA might be a promising therapeutic remedy to improve the gemcitabine resistance of UC.

Autophagy is deficient and inversely correlated with COX-2 expression in nasal polyps: a novel insight into the inflammation mechanism.

BACKGROUND: Nasal polyposis is characterised by persistent inflammation of the upper airways. Autophagy has been implicated in many chronic inflammatory diseases. Whether autophagy plays a role in nasal polyp (NP) inflammation is completely unknown and deserves investigation. METHODS: LC3 and COX-2 expression, the common autophagy and inflammation indicators, respectively, was analysed by immunoblotting in fresh tissues of NP and control nasal mucosa (NM). Primary cultures of NP-derived fibroblasts (NPDFs) and NMDFs were established for in vitro studies. Autophagy was induced by amino acid starvation and LC3 ectopic overexpression or inhibited by 3-methyladenine in the fibroblasts. Inflammation was induced by IL1-ß and TNF-α. LC3 and COX-2 expression was confirmed in NP specimens by immunohistochemistry. RESULTS: LC3 expression was decreased while COX-2 expression was significantly increased in fresh NP tissues compared with the NM control. In NMDFs and NPDFs, autophagy induction by starvation and LC3 overexpression downregulated COX-2 expression. Conversely, autophagy inhibition by 3-methyladenine enhanced COX-2 expression. However, IL1-ß and TNF-α had no effect on autophagy. Immunohistochemical studies on the NP specimens showed that most displayed low LC3 expression, whereas COX-2 was highly expressed in >50% of the specimens. Examination of two consecutive NP sections from the same tissue blocks revealed a negative correlation between LC3 and COX-2 expression. CONCLUSION: Autophagy is deficient in NP tissues and COX-2 is negatively regulated by autophagy in NP-derived fibroblasts. Since COX-2 is essential for the production of pro-inflammatory mediators, this study might help interpret persistent mucosal inflammation in NP. Attenuation of inflammation by restoring autophagy might be a therapeutic strategy for treating NP.