Integrin α5 mediates intrinsic cisplatin resistance in three-dimensional nasopharyngeal carcinoma spheroids via the inhibition of phosphorylated ERK /caspase-3 induced apoptosis.

Nasopharyngeal carcinoma (NPC) originates in the nasopharynx epithelium. Although concurrent chemoradiation therapy followed by chemotherapy is considered as an effective treatment, there is substantial drug resistance in locally advanced NPC patients. One major contributor to the chemoresistance includes aberrant expression of cell adhesion molecules, such as integrin α and ß subunits, giving rise to cell adhesion-mediated drug resistance. Thus, the aim of this study was to investigate the effect of integrin α5 on the development of intrinsic cisplatin resistance in NPC and the associated underlying mechanisms using in vitro three-dimensional (3D) spheroid models, as well as induced cisplatin-resistant NPC (NPCcisR). We demonstrated that established 3D highly- (5-8F) and lowly- (6-10B) metastatic NPC spheroids overexpressed integrin α5 and aggravated their resistance to cisplatin. Besides, enhanced integrin α5 resulted in substantially reduced growth, corresponding to G0/G1 and G2/M cell cycle arrest. In addition, 5-8FcisR and 6-10BcisR cells in 3D forms synergistically strengthened endurance of their spheroids to cisplatin treatment as observed by increased resistance index (RI) and decreased apoptosis. Mechanistically, the aberrantly expressed integrin α5 decreased drug susceptibility in NPC spheroids by inactivating ERK and inhibition of caspase-3 inducing apoptosis. Furthermore, the effect of integrin α5 inducing intrinsic resistance was verified via treatment with ATN-161, a peptide inhibitor for integrin α5ß1. The results showed dramatic reduction in integrin α5 expression, reversal of ERK phosphorylation and caspase-3 cleavage, together with elevated cisplatin sensitivity, indicating regulation of innate drug resistance via integrin α5. Taken together, our findings suggest that integrin α5 could act as a promising target to enhance the chemotherapeutic sensitivity in NPC.

Reversal of cisplatin sensitization and abrogation of cisplatin-enriched cancer stem cells in 5-8F nasopharyngeal carcinoma cell line through a suppression of Wnt/ß-catenin-signaling pathway.

Nasopharyngeal carcinoma (NPC) is one of the rare cancers in western countries but predominant in Southeast Asian countries including Thailand. One major cause for failure of NPC chemotherapeutic treatments is reportedly correlated with the elevation of cancer stem cell (CSC) fractions. Thus, this present study aims to investigate the effect of cisplatin (CDDP) treatment on the enrichment of cancer stem-like cells (CSCs) and its associated signaling pathway in EBV-negative NPC cells. Cisplatin-pretreated 5-8F NPC cells (5-8F CDDP) were first generated by treating the cells with 0.5 µM cisplatin for 48 h. After the instant treatment, 5-8F CDDP showed increased IC50 values, demonstrating a decrease in CDDP sensitization. Besides, the proportion of NPC cells with cancer stem-like phenotypes comprising side population (SP), key stemness-related gene expressions including SOX2, ALDH1, CD24 was significantly enhanced. Additionally, 5-8F CDDP displayed the upregulation of ß-catenin gene, suggesting its association with the CSC-initiating mechanism. Furthermore, a tankyrase inhibitor for Wnt/ß-catenin pathway, XAV939, substantially reduced CSCs and retrieved the cisplatin sensitivity in 5-8F CDDP. This confirms that the Wnt/ß-catenin signaling is accountable for rising of the CSC population in EBV-negative NPC. Finally, the combined treatment of CDDP and XAV939 exhibited lower 5-8F CDDP cell viability compared to the treatment of CDDP alone, suggesting the reversal of cisplatin sensitization. In conclusion, the enhancement of CSCs in 5-8F NPC cells caused by the instant cisplatin treatment is initially mediated through the upregulation of ß-catenin and activation of Wnt/ß-catenin signaling pathway. As a result, a primary chemotherapeutic treatment with closely monitoring the targeted Wnt/ß-catenin signaling pathway could potentially prevent the development of CSCs and improve the treatment efficiency in NPC.

Lapatinib sensitivity in nasopharyngeal carcinoma is modulated by SIRT2-mediated FOXO3 deacetylation.

BACKGROUND: Chemoresistance is an obstacle to the successful treatment of nasopharyngeal carcinoma (NPC). Lapatinib is a targeted tyrosine kinase inhibitor therapeutic drug also used to treat NPC, but high doses are often required to achieve a result. To investigate the mechanism for the development of Lapatinib resistance, we characterised a number of NPC cell lines to determine the role of FOXO3 and sirtuins in regulating NPC resistance. METHODS: Sulforhodamine B (SRB) assays, Clonogenic assays, Protein extraction, quantification and western blotting, RT qPCR, Co-immunoprecipitation assay. RESULTS: To explore novel treatment strategies, we first characterized the Lapatinib-sensitivity of a panel of NPC cell lines by SRB and clonogenic cytotoxic assays and found that the metastatic NPC (C666-1 and 5-8F) cells are highly resistant whereas the poorly metastatic lines (6-10B, TW01 and HK-1) are sensitive to Lapatinib. Western blot analysis of the Lapatinib-sensitive 6-10B and resistant 5-8F NPC cells showed that the expression of phosphorylated/inactive FOXO3 (P-FOXO3;T32), its target FOXM1 and its regulator SIRT2 correlate negatively with Lapatinib response and sensitivity, suggesting that SIRT2 mediates FOXO3 deacetylation to promote Lapatinib resistance. In agreement, clonogenic cytotoxic assays using wild-type and foxo1/3/4-/- mouse embryonic fibroblasts (MEFs) showed that FOXO1/3/4-deletion significantly attenuates Lapatinib-induced cytotoxicity, confirming that FOXO proteins are essential for mediating Lapatinib response. SRB cell viability assays using chemical SIRT inhibitors (i.e. sirtinol, Ex527, AGK2 and AK1) revealed that all SIRT inhibitors can reduce NPC cell viability, but only the SIRT2-specific inhibitors AK1 and AGK2 further enhance the Lapatinib cytotoxicity. Consistently, clonogenic assays demonstrated that the SIRT2 inhibitors AK1 and AGK2 as well as SIRT2-knockdown increase Lapatinib cytotoxicity further in both the sensitive and resistant NPC cells. Co-immunoprecipitation studies showed that besides Lapatinib treatment, SIRT2-pharmaceutical inhibition and silencing also led to an increase in FOXO3 acetylation. Importantly, SIRT2 inhibition and depletion further enhanced Lapatinib-mediated FOXO3-acetylation in NPC cells. CONCLUSION: Collectively, our results suggest the involvement of SIRT2-mediated FOXO3 deacetylation in Lapatinib response and sensitivity, and that SIRT2 can specifically antagonise the cytotoxicity of Lapatinib through mediating FOXO3 deacetylation in both sensitive and resistant NPC cells. The present findings also propose that SIRT2 can be an important biomarker for metastatic and Lapatinib resistant NPC and that targeting the SIRT2-FOXO3 axis may provide novel strategies for treating NPC and for overcoming chemoresistance.

Meta-analysis of gene expression profiles identifies differential biomarkers for hepatocellular carcinoma and cholangiocarcinoma.

Hepatocellular carcinoma (HCC) and cholangiocarcinoma (CCA) are the members of hepatobiliary diseases. Both types of cancer often exert high levels of similarity in terms of phenotypic characteristics, thus leading to difficulties in HCC and CCA differential diagnoses. In this study, a transcriptome meta-analysis was performed on HCC and CCA microarray data to identify differential transcriptome networks and potential biomarkers for HCC and CCA. Raw data from nine gene expression profiling datasets, consisting of 1,185 samples in total, were methodologically compiled and analyzed. To evaluate differentially expressed (DE) genes in HCC and CCA, the levels of gene expression were compared between cancer and its normal counterparts (i.e., HCC versus normal liver and CCA versus normal bile duct) using t test (P < 0.05) and k-fold validation. A total of 226 DE genes were specific to HCC, 249 DE genes specific to CCA, and 41 DE genes in both HCC and CCA. Gene ontology and pathway enrichment analyses revealed different patterns between functional transcriptome networks of HCC and CCA. Cell cycle and glycolysis/gluconeogenesis pathways were exclusively dysregulated in HCC whereas complement and coagulation cascades as well as glycine, serine, and threonine metabolism were prodominantly differentially expressed in CCA. Our meta-analysis revealed distinct dysregulation in transcriptome networks between HCC and CCA. Certain genes in these networks were discussed in the context of HCC and CCA transition, unique characteristics of HCC and CCA, and their potentials as HCC and CCA differential biomarkers.

Inactivation of Clostridium difficile spores by microwave irradiation.

Spores are a potent agent for Clostridium difficile transmission. Therefore, factors inhibiting spores have been of continued interest. In the present study, we investigated the influence of microwave irradiation in addition to conductive heating for C. difficile spore inactivation in aqueous suspension. The spores of 15 C. difficile isolates from different host origins were exposed to conductive heating and microwave irradiation. The complete inhibition of spore viability at 10(7) CFU/ml was encountered following microwave treatment at 800 W for 60 s, but was not observed in the conductive-heated spores at the same time-temperature exposure. The distinct patterns of ultrastructural alterations following microwave and conductive heat treatment were observed and the degree of damages by microwave was in the exposure time-dependent manner. Microwave would therefore be a simple and time-efficient tool to inactivate C. difficile spores, thus reducing the risk of C. difficile transmission.

Multidrug resistance in Clostridium perfringens isolated from diarrheal neonatal piglets in Thailand.

Clostridium perfringens causes diarrhea in neonatal piglets, thereby affecting commercial swine farming. The objective of this study was to determine the prevalence and characterize antimicrobial resistance in C. perfringens isolated from diarrheal neonatal piglets in Thailand. A total of 260 rectal swab samples were collected from 13 farms and were subjected to C. perfringens isolation. A total of 148 samples were PCR-positive for C. perfringens toxin genes, from which 122 were recovered. All isolates were cpb2-encoding C. perfringens type A and enterotoxin gene negative. Most of the isolates were susceptible to ampicillin, bacitracin, chlorotetracycline, doxycycline, and oxytetracycline with MIC50 values ranging from 0.32 to 8 µg/ml. The high resistance rates were observed for ceftiofur, enrofloxacin, erythromycin, lincomycin, and tylosin. Among resistant isolates, 82% were resistant to more than one type of antibiotics. The distinct pattern of multiple drug resistance in C. perfringens was observed in different regions, potentially reflecting the farm specific usage of these agents.

Transcriptome meta-analysis reveals dysregulated pathways in nasopharyngeal carcinoma.

Nasopharyngeal carcinoma (NPC) is a malignant cancer arising from the epithelial surface of the nasopharynx that mostly appears in advanced stages of the disease, leading to a poor prognosis. To date, a number of mRNA profiling investigations on NPC have been reported in order to identify suitable biomarkers for early detection. However, the results may be specific to each study with distinct sample types. In this study, an integrative meta-analysis of NPC transcriptome data was performed to determine dysregulated pathways, potentially leading to identification of molecular markers. Ten independent NPC gene expression profiling microarray datasets, including 135 samples from NPC cell lines, primary cell lines, and tissues were assimilated into a meta-analysis and cross-validation to identify a cohort of genes that were significantly dysregulated in NPC. Bioinformatics analyses of these genes revealed the significant pathways and individual players involving in cellular metabolism, cell cycle regulation, DNA repair, as well as ErbB pathway. Altogether, we propose that dysregulation of these molecular pathways in NPC might play a role in the NPC pathogenesis, providing clues, which could eventually translate into diagnostic and therapeutic approaches.

Index insurance for pro-poor conservation of hornbills in Thailand.

This study explores the potential of index insurance as a mechanism to finance community-based biodiversity conservation in areas where a strong correlation exists between natural disaster risk, keystone species populations, and the well-being of the local population. We illustrate this potential using the case of hornbill conservation in the Budo-Sungai Padi rainforests of southern Thailand, using 16-y hornbill reproduction data and 5-y household expenditures data reflecting local economic well-being. We show that severe windstorms cause both lower household expenditures and critical nest tree losses that directly constrain nesting capacity and so reduce the number of hornbill chicks recruited in the following breeding season. Forest residents' coping strategies further disturb hornbills and their forest habitats, compounding windstorms' adverse effects on hornbills' recruitment in the following year. The strong statistical relationship between wind speed and both hornbill nest tree losses and household expenditures opens up an opportunity to design wind-based index insurance contracts that could both enhance hornbill conservation and support disaster-affected households in the region. We demonstrate how such contracts could be written and operationalized and then use simulations to show the significant promise of unique insurance-based approaches to address weather-related risk that threatens both biodiversity and poor populations.

The complete catalog of antimicrobial resistance secondary active transporters in Clostridioides difficile: evolution and drug resistance perspective.

Secondary active transporters shuttle substrates across eukaryotic and prokaryotic membranes, utilizing different electrochemical gradients. They are recognized as one of the antimicrobial efflux pumps among pathogens. While primary active transporters within the genome of C. difficile 630 have been completely cataloged, the systematical study of secondary active transporters remains incomplete. Here, we not only identify secondary active transporters but also disclose their evolution and role in drug resistance in C. difficile 630. Our analysis reveals that C. difficile 630 carries 147 secondary active transporters belonging to 27 (super)families. Notably, 50 (34%) of them potentially contribute to antimicrobial resistance (AMR). AMR-secondary active transporters are structurally classified into five (super)families: the p-aminobenzoyl-glutamate transporter (AbgT), drug/metabolite transporter (DMT) superfamily, major facilitator (MFS) superfamily, multidrug and toxic compound extrusion (MATE) family, and resistance-nodulation-division (RND) family. Surprisingly, complete RND genes found in C. difficile 630 are likely an evolutionary leftover from the common ancestor with the diderm. Through protein structure comparisons, we have potentially identified six novel AMR-secondary active transporters from DMT, MATE, and MFS (super)families. Pangenome analysis revealed that half of the AMR-secondary transporters are accessory genes, which indicates an important role in adaptive AMR function rather than innate physiological homeostasis. Gene expression profile firmly supports their ability to respond to a wide spectrum of antibiotics. Our findings highlight the evolution of AMR-secondary active transporters and their integral role in antibiotic responses. This marks AMR-secondary active transporters as interesting therapeutic targets to synergize with other antibiotic activity.

Identification of a microbial sub-community from the feral chicken gut that reduces Salmonella colonization and improves gut health in a gnotobiotic chicken model.

A complex microbial community in the gut may prevent the colonization of enteric pathogens such as Salmonella. Some individual or a combination of species in the gut may confer colonization resistance against Salmonella. To gain a better understanding of the colonization resistance against Salmonella enterica, we isolated a library of 1,300 bacterial strains from feral chicken gut microbiota which represented a total of 51 species. Using a co-culture assay, we screened the representative species from this library and identified 30 species that inhibited Salmonella enterica subspecies enterica serovar Typhimurium in vitro. To improve the Salmonella inhibition capacity, from a pool of fast-growing species, we formulated 66 bacterial blends, each of which composed of 10 species. Bacterial blends were more efficient in inhibiting Salmonella as compared to individual species. The blend that showed maximum inhibition (Mix10) also inhibited other serotypes of Salmonella frequently found in poultry. The in vivo effect of Mix10 was examined in a gnotobiotic and conventional chicken model. The Mix10 consortium significantly reduced Salmonella load at day 2 post-infection in gnotobiotic chicken model and decreased intestinal tissue damage and inflammation in both models. Cell-free supernatant of Mix10 did not show Salmonella inhibition, indicating that Mix10 inhibits Salmonella through either nutritional competition, competitive exclusion, or through reinforcement of host immunity. Out of 10 species, 3 species in Mix10 did not colonize, while 3 species constituted more than 70% of the community. Two of these species were previously uncultured bacteria. Our approach could be used as a high-throughput screening system to identify additional bacterial sub-communities that confer colonization resistance against enteric pathogens and its effect on the host.IMPORTANCESalmonella colonization in chicken and human infections originating from Salmonella-contaminated poultry is a significant problem. Poultry has been identified as the most common food linked to enteric pathogen outbreaks in the United States. Since multi-drug-resistant Salmonella often colonize chicken and cause human infections, methods to control Salmonella colonization in poultry are needed. The method we describe here could form the basis of developing gut microbiota-derived bacterial blends as a microbial ecosystem therapeutic against Salmonella.

Therapeutic Implications of Ceritinib in Cholangiocarcinoma beyond ALK Expression and Mutation.

Cholangiocarcinoma (CCA) is a difficult-to-treat cancer, with limited therapeutic options and surgery being the only curative treatment. Standard chemotherapy involves gemcitabine-based therapies combined with cisplatin, oxaliplatin, capecitabine, or 5-FU with a dismal prognosis for most patients. Receptor tyrosine kinases (RTKs) are aberrantly expressed in CCAs encompassing potential therapeutic opportunity. Hence, 112 RTK inhibitors were screened in KKU-M213 cells, and ceritinib, an approved targeted therapy for ALK-fusion gene driven cancers, was the most potent candidate. Ceritinib's cytotoxicity in CCA was assessed using MTT and clonogenic assays, along with immunofluorescence, western blot, and qRT-PCR techniques to analyze gene expression and signaling changes. Furthermore, the drug interaction relationship between ceritinib and cisplatin was determined using a ZIP synergy score. Additionally, spheroid and xenograft models were employed to investigate the efficacy of ceritinib in vivo. Our study revealed that ceritinib effectively killed CCA cells at clinically relevant plasma concentrations, irrespective of ALK expression or mutation status. Ceritinib modulated multiple signaling pathways leading to the inhibition of the PI3K/Akt/mTOR pathway and activated both apoptosis and autophagy. Additionally, ceritinib and cisplatin synergistically reduced CCA cell viability. Our data show ceritinib as an effective treatment of CCA, which could be potentially explored in the other cancer types without ALK mutations.

Asiatic Acid Inhibits Nasopharyngeal Carcinoma Cell Viability and Migration via Suppressing STAT3 and Claudin-1.

Nasopharyngeal carcinoma (NPC) is a prevalent cancer in Southeast Asia, but effective treatment options remain limited, and chemotherapy has a high resistance rate. Asiatic acid (AA), a triterpenoid found in Centella asiatica, has shown anticancer activity in various cancers. Therefore, this study aims to investigate the anticancer effects and mechanisms of AA in NPC cell lines. The effects of AA on NPC cytotoxicity, apoptosis, and migration were determined in TW-01 and SUNE5-8F NPC cell lines. Western blot analysis was performed to evaluate the protein expression levels affected by AA. The role of AA in proliferation and migration was investigated in STAT3 and claudin-1 knockdown cells. AA inhibited NPC cell viability and migration and induced cell death by increasing cleaved caspase-3 expression. Moreover, AA inhibited STAT3 phosphorylation and reduced claudin-1 expression in NPC cells. Although knockdown of STAT3 or claudin-1 slightly reduced cell viability, it did not enhance the anti-proliferative effect of AA. However, knockdown of STAT3 or claudin-1 increased the anti-migratory effect of AA in NPC cells. These results suggest that AA can be a promising candidate for drug development against NPC.

Targeting FGFRs Using PD173074 as a Novel Therapeutic Strategy in Cholangiocarcinoma.

Cholangiocarcinoma (CCA) is an architecturally complex tumour with high heterogeneity. Discovery at later stages makes treatment challenging. However, the lack of early detection methodologies and the asymptomatic nature of CCA make early diagnosis more difficult. Recent studies revealed the fusions in Fibroblast Growth Factor Receptors (FGFRs), a sub-family of RTKs, as promising targets for targeted therapy for CCA. Particularly, FGFR2 fusions have been of particular interest, as translocations have been found in approximately 13% of CCA patients. Pursuing this, Pemigatinib, a small-molecule inhibitor of FGFR, became the first targeted therapy drug to be granted accelerated approval by the FDA for treating CCA patients harbouring FGFR2 fusions who have failed first-line chemotherapy. However, despite the availability of Pemigatinib, a very limited group of patients benefit from this treatment. Moreover, as the underlying mechanism of FGFR signalling is poorly elucidated in CCA, therapeutic inhibitors designed to inhibit this pathway are prone to primary and acquired resistance, as witnessed amongst other Tyrosine Kinase Inhibitors (TKIs). While acknowledging the limited cohort that benefits from FGFR inhibitors, and the poorly elucidated mechanism of the FGFR pathway, we sought to characterise the potential of FGFR inhibitors in CCA patients without FGFR2 fusions. Here we demonstrate aberrant FGFR expression in CCA samples using bioinformatics and further confirm phosphorylated-FGFR expression in paraffinised CCA tissues using immunohistochemistry. Our results highlight p-FGFR as a biomarker to guide FGFR-targeted therapies. Furthermore, CCA cell lines with FGFR expression were sensitive to a selective pan-FGFR inhibitor, PD173074, suggesting that this drug can be used to suppress CCA cells irrespective of the FGFR2 fusions. Finally, the correlation analysis utilising publicly available cohorts suggested the possibility of crosstalk amongst the FGFR and EGFR family of receptors as they are significantly co-expressed. Accordingly, dual inhibition of FGFRs and EGFR by PD173074 and EGFR inhibitor erlotinib was synergistic in CCA. Hence, the findings from this study provide support for further clinical investigation of PD173074, as well as other FGFR inhibitors, to benefit a larger cohort of patients. Altogether, this study shows for the first time the potential of FGFRs and the importance of dual inhibition as a novel therapeutic strategy in CCA.

Upregulated LAMA3 modulates proliferation, adhesion, migration and epithelial­to­mesenchymal transition of cholangiocarcinoma cells.

A poor outcome for cholangiocarcinoma (CCA) patients is still a clinical challenge. CCA is typically recognized by the desmoplastic nature, which accounts for its malignancy. Among various extracellular matrix proteins, laminin is the most potent inducer for CCA migration. Herein, we accessed the expression profiles of laminin gene family and explored the significance of the key laminin subunit on CCA aggressiveness. Of all 11 laminin genes, LAMA3, LAMA5, LAMB3 and LAMC2 were concordantly upregulated based on the analysis of multiple public transcriptomic datasets and also overexpressed in Thai CCA cell lines and patient tissues in which LAMA3A upregulated in the highest frequency (97%) of the cases. Differential expression genes (DEGs) analysis of low and high laminin signature groups revealed LAMA3 as the sole common DEG in all investigated datasets. Restratifying CCA samples according to LAMA3 expression indicated the association of LAMA3 in the focal adhesion pathway. Silencing LAMA3 revealed that it plays important roles in CCA cell proliferation, adhesion, migration and epithelial-to-mesenchymal transition. Taken together, this research signifies the roles of dysregulated ECM homeostasis in CCA malignancy and highlights, for the first time, the potential usage of LAMA3 as the diagnostic biomarker and the therapeutic target to tackle the CCA stromal.

CP-673451, a Selective Platelet-Derived Growth Factor Receptor Tyrosine Kinase Inhibitor, Induces Apoptosis in Opisthorchis viverrini-Associated Cholangiocarcinoma via Nrf2 Suppression and Enhanced ROS.

Platelet-derived growth factors (PDGFs) and PDGF receptors (PDGFRs) play essential roles in promoting cholangiocarcinoma (CCA) cell survival by mediating paracrine crosstalk between tumor and cancer-associated fibroblasts (CAFs), indicating the potential of PDGFR as a target for CCA treatment. Clinical trials evaluating PDGFR inhibitors for CCA treatment have shown limited efficacy. Furthermore, little is known about the role of PDGF/PDGFR expression and the mechanism underlying PDGFR inhibitors in CCA related to Opisthorchis viverrini (OV). Therefore, we examined the effect of PDGFR inhibitors in OV-related CCA cells and investigated the molecular mechanism involved. We found that the PDGF and PDGFR mRNAs were overexpressed in CCA tissues compared to resection margins. Notably, PDGFR-α showed high expression in CCA cells, while PDGFR-ß was predominantly expressed in CAFs. The selective inhibitor CP-673451 induced CCA cell death by suppressing the PI3K/Akt/Nrf2 pathway, leading to a decreased expression of Nrf2-targeted antioxidant genes. Consequently, this led to an increase in ROS levels and the promotion of CCA apoptosis. CP-673451 is a promising PDGFR-targeted drug for CCA and supports the further clinical investigation of CP-673451 for CCA treatment, particularly in the context of OV-related cases.

Preclinical evidence for anaplastic lymphoma kinase inhibitors as novel therapeutic treatments for cholangiocarcinoma.

Introduction: Bile duct cancer (cholangiocarcinoma, CCA) has a poor prognosis for patients, and despite recent advances in targeted therapies for other cancer types, it is still treated with standard chemotherapy. Anaplastic lymphoma kinase (ALK) has been shown to be a primary driver of disease progression in lung cancer, and ALK inhibitors are effective therapeutics in aberrant ALK-expressing tumors. Aberrant ALK expression has been documented in CCA, but the use of ALK inhibitors has not been investigated. Using CCA cell lines and close-to-patient primary cholangiocarcinoma cells, we investigated the potential for ALK inhibitors in CCA. Methods: ALK, cMET, and ROS1 expression was determined in CCA patient tissue by immunohistochemistry and digital droplet polymerase chain reaction, and that in cell lines was determined by immunoblot and immunofluorescence. The effect on cell viability and mechanism of action of ALK, cMet, and ROS1 inhibitors was determined in CCA cell lines. To determine whether ceritinib could affect primary CCA cells, tissue was taken from four patients with biliary tract cancer, without ALK rearrangement, mutation, or overexpression, and grown in three-dimensional tumor growth assays in the presence or absence of humanized mesenchymal cells. Results: ALK and cMet but not ROS were both upregulated in CCA tissues and cell lines. Cell survival was inhibited by crizotinib, a c-met/ALK/ROS inhibitor. To determine the mechanism of this effect, we tested c-Met-specific and ALK/ROS-specific inhibitors, capmatinib and ceritinib, respectively. Whereas capmatinib did not affect cell survival, ceritinib dose-dependently inhibited survival in all cell lines, with IC50 ranging from 1 to 9 µM and co-treatments with gemcitabine and cisplatin further sensitized cells, with IC50 ranging from IC50 0.60 to 2.32 µM. Ceritinib did not inhibit cMet phosphorylation but did inhibit ALK phosphorylation. ALK was not mutated in any of these cell lines. Only ceritinib inhibited 3D growth of all four patient samples below mean peak serum concentration, in the presence and absence of mesenchymal cells, whereas crizotinib and capmatinib failed to do this. Ceritinib appeared to exert its effect more through autophagy than apoptosis. Discussion: These results indicate that ceritinib or other ALK/ROS inhibitors could be therapeutically useful in cholangiocarcinoma even in the absence of aberrant ALK/ROS1 expression.

Clostridium difficile transcriptome analysis using pig ligated loop model reveals modulation of pathways not modulated in vitro.

A pig ligated loop model was used to analyze the in vivo transcriptome response of Clostridium difficile. Bacterial RNA from the loops was retrieved at different times and was used for microarray analysis. Several virulence-associated genes and genes involved in sporulation cascade were differentially expressed (DE). In concordance with observed upregulation of toxin genes in microarray, enzyme-linked immunosorbent assay estimation of total toxin showed high amounts of toxin in the loops. Several genes that were absent in primary annotation of C. difficile 630 but annotated in a secondary annotation were found to be DE. Pathway comparison of DE genes in vitro and in vivo showed that when several pathways were expressed in all conditions, several of the C. difficile pathways were uniquely expressed only in vivo. The pathways observed to be modulated only in this study could be targets of new therapeutic agents against C. difficile infection.

Transcriptomic profiling revealed FZD10 as a novel biomarker for nasopharyngeal carcinoma recurrence.

Background: Nasopharyngeal carcinoma (NPC) is a type of cancers that develops in the nasopharynx, the very upper part of the throat behind the nose. NPC is typically diagnosed in later stages of the disease and has a high rate of recurrence due to the location of the tumor growth site. In this study, we compared the gene expression profiles of NPC tissues from patients with and without recurrence to identify potential molecular biomarkers of NPC recurrence. Methods: Microarrays were used to analyze the expression of genes in 15 NPC tissues taken at the time of diagnosis and at the site of recurrence following therapeutic treatment. Pathway enrichment analysis was used to examine the biological interactions between the major differentially expressed genes. The target identified was then validated using immunohistochemistry on 86 NPC tissue samples. Results: Our data showed that the Wnt signaling pathway was enhanced in NPC tissues with recurrence. FZD10, a component of the Wnt signaling pathway, was significantly expressed in NPC tissues, and was significantly associated with NPC recurrence. Conclusion: Our study provides new insights into the pathogenesis of NPC and identifies FZD10 as a potential molecular biomarker for NPC recurrence. FZD10 may be a promising candidate for NPC recurrence and a potential therapeutic target.

Distinct cholangiocarcinoma cell migration in 2D monolayer and 3D spheroid culture based on galectin-3 expression and localization.

Introduction: Cholangiocarcinoma (CCA) is difficult to cure due to its ineffective treatment and advanced stage diagnosis. Thoroughly mechanistic understandings of CCA pathogenesis crucially help improving the treatment success rates. Using three-dimensional (3D) cell culture platform offers several advantages over a traditional two-dimensional (2D) culture as it resembles more closely to in vivo tumor. Methods: Here, we aimed to establish the 3D CCA spheroids with lowly (KKU-100) and highly (KKU-213A) metastatic potentials to investigate the CCA migratory process and its EMT-associated galectin-3 in the 3D setting. Results and discussion: Firstly, the growth of lowly metastatic KKU-100 cells was slower than highly metastatic KKU-213A cells in both 2D and 3D systems. Hollow formation was observed exclusively inside the KKU-213A spheroids, not in KKU-100. Additionally, the migration activity of KKU-213A cells was higher than that of KKU-100 cells in both 2D and 3D systems. Besides, altered expression of galectin-3 were observed across all CCA culture conditions with substantial relocalization from inside the 2D cells to the border of spheroids in the 3D system. Notably, the CCA migration was inversely proportional to the galectin-3 expression in the 3D culture, but not in the 2D setting. This suggests the contribution of culture platforms to the alternation of the CCA cell migration process. Conclusions: Thus, our data revealed that 3D culture of CCA cells was phenotypically distinct from 2D culture and pointed to the superiority of using the 3D culture model for examining the CCA cellular mechanisms, providing knowledges that are better correlated with CCA phenotypes in vivo.

Comparative Proteomic Profiling of Cisplatin-resistant Nasopharyngeal Carcinoma Cell Lines: Novel Biomarkers for Improving Chemotherapy of NPC.

BACKGROUND/AIM: Nasopharyngeal carcinoma (NPC) originates in the hidden nasopharynx, causing NPC patients to be diagnosed at a late stage and develop drug resistance. Therefore, the identification of drug-resistance biomarkers is indispensable to improve NPC detection and treatment. Hence, this study aimed to identify novel cisplatinresistance biomarkers using comparative proteomic profiles of cisplatin-resistant (CDDP/NPC) cell lines. MATERIALS AND METHODS: Two cisplatin-resistant NPC cell lines (CDDP/5-8F and CDDP/6-10B) were established by a continuous cisplatin treatment. Then, morphology, proliferation, and migration of all NPC cells were evaluated, followed by the examination of protein profiles using 1D in-gel digestion coupled with mass spectrometry. The potential drugresistance biomarkers were transcriptionally and translationally validated by qPCR and western blotting, respectively. RESULTS: CDDP/5-8F and CDDP/6-10B cells were successfully developed with a resistance index of 8.42 and 2.46, respectively. Furthermore, both CDDP/NPC cells demonstrated relatively altered morphology, retarded growth, and decreased migration. Additionally, the comparative proteomic analysis of CDDP/NPC revealed 92 differentially expressed proteins (DEPs). Specifically, up-regulated DEPs were notably enriched in cellular metabolic processes, while down-regulated DEPs were predominantly enriched in actin filament-based movement, methylation, and programmed cell death. Six up-regulated, namely ALPI, CKB, HMGB1, KHSRP, PDIA4, and STMN1, and three down-regulated proteins, FUBP1, YWHAZ, and PLEC, were validated at the transcriptional level. CKB and FUBP1 were further validated at the translational level and demonstrated corresponding expression levels at both protein and gene levels. CONCLUSION: Our findings suggest novel biomarkers to indicate cisplatin resistance in NPC, expanding the drug resistance knowledge and paving the way for in-depth mechanism studies in NPC.