Pyruvate Kinase Differentially Alters Metabolic Signatures during Head and Neck Carcinogenesis.

During glycolysis, the muscle isoform of pyruvate kinase PKM2 produces ATP in exchange for dephosphorylation of phosphoenolpyruvate (PEP) into pyruvate. PKM2 has been considered as a tumor-promoting factor in most cancers, whereas the regulatory role of PKM2 during head and neck carcinogenesis remained to be delineated. PKM2 mRNA and protein expression was examined in head and neck tumorous specimens. The role of PKM2 in controlling cellular malignancy was determined in shRNA-mediated PKM2-deficient head and neck squamous cell carcinoma (HNSC) cells. In agreement with the results in other cancers, PKM2 expression is enriched in both mouse and human HNSC tissues. Nevertheless, PKM2 mRNA expression reversely correlated with tumor stage, and greater recurrence-free survival rates are evident in the PKM2high HNSC population, arguing that PKM2 may be tumor-suppressive. Multifaceted analyses showed a greater in vivo xenografic tumor growth and an enhanced cisplatin resistance in response to PKM2 loss, whereas PKM2 silencing led to reduced cell motility. At the molecular level, metabolic shifts towards mitochondrial metabolism and activation of oncogenic Protein kinase B (PKB/Akt) and extracellular signal-regulated kinase (ERK) signals were detected in PKM2-silencing HNSC cells. In sum, our findings demonstrated that PKM2 differentially modulated head and neck tumorigenicity via metabolic reprogramming.

Assessing hand hygiene knowledge, attitude, behavior and adherence among nursing assistants: A cross-sectional study.

OBJECTIVE: This study explored hand hygiene knowledge, attitude, behaviors and adherence amongst nursing assistants. METHODS: This cross-sectional study was conducted with structured questionnaires and direct observation. Nursing assistants were recruited from two long-term care facilities in eastern Taiwan from July to September, 2021. RESULTS: The nursing assistants had high levels of hand hygiene knowledge, attitude, and behavior, however, the direct observational revealed hand hygiene adherence at 58.6% with 17.99 seconds duration on average. Comparing with alcohol-based hand rub, the nursing assistants performed very low adherence rate with soap and water wash, and the use of paper towel was the least followed skill applying to soap and water wash. CONCLUSIONS: The study finds that hand wash with soap and water has lower adherence than the alcohol-based hand rub. Future innovations in hand hygiene, such as accessible and easy-to-use hand washing agents and easy-to-remember hand cleansing techniques will be valuable.

Determination of Pyruvate Metabolic Fates Modulates Head and Neck Tumorigenesis.

Even with increasing evidence for roles of glycolytic enzymes in controlling cancerous characteristics, the best target of candidate metabolic enzymes for lessening malignancy remains under debate. Pyruvate is a main glycolytic metabolite that could be mainly converted into either lactate by Lactate Dehydrogenase A (LDHA) or acetyl-CoA by Pyruvate Dehydrogenase E1 component α subunit (PDHA1) catalytic complex. In tumor cells, accumulating lactate is produced whereas the conversion of pyruvate into mitochondrial acetyl-CoA is less active compared with their normal counterparts. This reciprocal molecular association makes pyruvate metabolism a potential choice of anti-cancer target. Cellular and molecular changes were herein assayed in Head and Neck Squamous Cell Carcinoma (HNSCC) cells in response to LDHA and PDHA1 loss in vitro, in vivo and in clinic. By using various human cancer databases and clinical samples, LDHA and PDHA1 levels exhibit reversed prognostic roles. In vitro analysis demonstrated that decreased cell growth and motility accompanied by an increased sensitivity to chemotherapeutic agents was found in cells with LDHA loss whereas PDHA1-silencing exhibited opposite phenotypes. At the molecular level, it was found that oncogenic Protein kinase B (PKB/Akt) and Extracellular signal-regulated kinase (ERK) singling pathways contribute to pyruvate metabolism mediated HNSCC cell growth. Furthermore, LDHA/PDHA1 changes in HNSCC cells resulted in a broad metabolic reprogramming while intracellular molecules including polyunsaturated fatty acids and nitrogen metabolism related metabolites underlie the malignant changes. Collectively, our findings reveal the significance of pyruvate metabolic fates in modulating HNSCC tumorigenesis and highlight the impact of metabolic plasticity in HNSCC cells.

Porcine Neonatal Pancreatic Cell Clusters Maintain Their Multipotency in Culture and After Transplantation.

Ductal epithelium is primarily detected in porcine neonatal pancreatic cell clusters (NPCCs) bearing grafts, suggesting that transplants might exhibit progenitor-like phenotypes. Here we found that soon after NPCC isolation, PDX1+/insulin- and SOX9+ pancreatic progenitor-like cells dramatically increased while dual-hormonal progenitor-like cells were routinely observed in NPCC culture. After transplantation (Tx), insulin+ cells increased and PDX1+ and SOX9+ cells gradually decreased in both non-diabetic (NDM) and streptozotocin-induced diabetic (DM) grafts over 2 months. Strikingly, a significantly higher percentage of insulin+ cells were detected in 9-day and 16-day, but not in 23-day, 30-day and 60-day grafts implying that hyperglycemia could only facilitate NPCC-derived ß cells early post-Tx. A higher percentage of NPCC-derived ß cells in early DM grafts was determined via an enhanced neogenic differentiation based on the detection of insulin+ cells budding out from PDX1+/SOX9+ epithelium. Interestingly, a drop in SOX9+ progenitor-like cells was detected 16 days post-Tx in DM grafts whilst PDX1+ cells do not show a significant difference until 60 days post-Tx between DM and NDM grafts, demonstrating that distinct progenitor-like populations fuel new ß cells post-Tx. In conclusion, PDX1+/SOX9+ cells could be quickly activated after NPCC isolation, maintain their multipotency in culture and differentiate into new ß cell post-Tx.

miRTarBase update 2018: a resource for experimentally validated microRNA-target interactions.

MicroRNAs (miRNAs) are small non-coding RNAs of ∼ 22 nucleotides that are involved in negative regulation of mRNA at the post-transcriptional level. Previously, we developed miRTarBase which provides information about experimentally validated miRNA-target interactions (MTIs). Here, we describe an updated database containing 422 517 curated MTIs from 4076 miRNAs and 23 054 target genes collected from over 8500 articles. The number of MTIs curated by strong evidence has increased ∼1.4-fold since the last update in 2016. In this updated version, target sites validated by reporter assay that are available in the literature can be downloaded. The target site sequence can extract new features for analysis via a machine learning approach which can help to evaluate the performance of miRNA-target prediction tools. Furthermore, different ways of browsing enhance user browsing specific MTIs. With these improvements, miRTarBase serves as more comprehensively annotated, experimentally validated miRNA-target interactions databases in the field of miRNA related research. miRTarBase is available at http://miRTarBase.mbc.nctu.edu.tw/.

Cell survival and death program modulated by LMP1: implication in antitumor immunity.

The genome of Epstein-Barr virus (EBV) encodes proteins essential for malignant transformation, for example, latent membrane protein 1(LMP1). Whereas, LMP1 up-regulates anti-apoptotic proteins to support viral replication, it also potentiates apoptosis, suggesting that a viral protein contributes to the survival of the virus, and it also elicit host defense leading to the destruction of the infected cells. The antitumor immunity is exerted by infiltrated CD8+ T cells elaborating cytotoxic effectors, like Fas ligand (FasL, CD95L or CD178). As a nuclear factor-kappaB (NF-kappaB)-dependent molecule, Fas is induced by LMP1, and LMP1 enhances Fas-mediated apoptosis, according to our finding of stimulus-dependent apoptosis regulation by LMP1. Data has shown that FasL-mediated cytotoxicity has significant therapeutic effect on EBV-associated nasopharyngeal carcinoma (NPC). Recent reports suggest that mutations affecting the Fas-mediated apoptotic pathway reduce individuals' susceptibility to cancers, but cytokine-targeting therapy which precisely regulates the Fas level on tumor cells could still contribute to enhancement of antitumor immunity in cancer patients.

[Expression of chemokine receptor CXCR4 in nasopharyngeal carcinoma cells].

BACKGROUND & OBJECTIVE: It was reported that chemokine receptor CXCR4 and its ligand stromal cell-derived factor 1(SDF-1) were involved in the proliferation, differentiation, and metastasis of tumor. This study was designed to observe the expression of CXCR4 in NPC cells with different differentiation grade and proliferative ability to primitively clarify the relationship between CXCR4 and the malignity of NPC cells. METHODS: After treated with all-trans-retinoic acid(RA) and telomerase antisense oligodeoxynucleotide (ASODN) respectively, the expression of CXCR4 mRNA and CXCR4 protein in NPC CNE1 and CNE2Z cells were determined by in situ hybridization and immunohistochemistry, respectively; the distribution of cell cycle was examined with flow cytometry and the proliferation of cells was identified by MTT method. RESULTS: CXCR4 mRNA and CXCR4 protein were strongly expressed in both CNE1 and CNE2Z cells, and their expression in CNE2Z cells was stronger than that in CNE1 cells. After treated with 1x10(-5) mol/L and 1x10(-4) mol/L RA, CNE1 cells were arrested in G1 phase and CNE2Z cells in S phase, while the CXCR4 mRNA expression was significantly decreased in both CNE1 and CNE2Z cells compared with control group cells (P< 0.01). The effect of 1x10(-4) mol/L RA was more powerful than that of 1x10(-5) mol/L RA. After treated with ASODN, the proliferation of CNE1 and CNE2Z cells was inhibited, and the expression of CXCR4 protein was decreased compared with the control (P< 0.01). CONCLUSION: CXCR4 is highly expressed in NPC cells,and its expression was associated with differentiation grade and proliferation ability of NPC cells.

[Effects of two LMP1 variants on resistance of CNE1 cell strain to TGF-beta1].

BACKGROUND & OBJECTIVE: We had proved that different latent membrane protein 1 (LMP1) variants of Epstein-Barr virus (EBV) had different effects upon growth characteristics of a human well-differentiated nasopharyngeal carcinoma (NPC) cell strain CNE1. This study was designed to investigate the possible effects of different LMP1 variants on resistance of CNE1 to TGFbeta1 and the related mechanism(s) so as to further elucidate the intrinsic mechanisms of their growth-promoting effects upon CNE1. METHODS: The plasmids including J124 (served as a control plasmid),124-B95-8 (carried LMP1 gene cloned from B95-8 lymphocytes,B95-8-LMP1), and J124- CAO-5 (carried LMP1 gene cloned from NPC tissues,CAO-LMP1) were introduced into CNE1 by liposomal transfection. The transfected cell strains were named CNE1-V,CNE1-B,and CNE1-C,respectively. Gene and protein expression of LMP1 in CNE1 were identified by reverse transcription-polymerase chain reaction (RT-PCR) and Western blot analysis,respectively. Then growth inhibition assay using MTT colorimetric method and flow cytometry were conducted to investigate different effects of the two LMP1 variants on resistance of CNE1 to TGFbeta1. Meanwhile,TGFbetaRI,TGFbetaRII, and cyclin D1 were detected by Western blot analysis and p15 mRNA was examined by semi-quantitative RT-PCR. RESULTS: Two transfected cell strains (CNE1-B and CNE1-C) stably expressing different LMP1 variants were established successfully. When the treating concentration of TGFbeta1 was 5 ng/ml, the growth inhibitory rates of CNE1, CNE1-V,CNE1-B,and CNE1-C were 31.8%, 27.9%, 10.94%, and -4.26%, respectively, and the proliferation index (PI) were (24.55+/-2.55)%, (25.43+/-2.18)%, (46.78+/-2.56)%, and (54.70+/-3.84)%, respectively. CAO-LMP1 induced complete TGFbeta1 resistance in CNE1, whereas B95-8-LMP1 induced partial resistance. Neither of the two LMP1 variants had effects on TGFbetaRI and TGFbetaRII protein expression in CEN1,whereas both of them induced cyclin D1 expression significantly. CAO-LMP1 induced higher level of cyclin D1 than B95-8-LMP1 did (P< 0.05). B95-8-LMP1 had no significant effect on p15 mRNA expression (P >0.05), whereas CAO-LMP1 down-regulated p15 mRNA level obviously (P< 0.05). CONCLUSION: B95-8-LMP1 could induce partial resistance of CNE1 to TGFbeta1 and the main mechanism was correlated with up-regulation of cyclin D1 protein, whereas CAO-LMP1 could induce complete resistance to TGFbeta1 and the mechanisms were correlated with up-regulation of cyclin D1 protein as well as down-regulation of p15 mRNA.

[Effects of telomerase sense and antisense oligodeoxynucleotides on growth and differentiation of nasopharyngeal carcinoma cells].

BACKGROUND & OBJECTIVE: It was reported that telomere and telomerase were associated with the development of cancers. Telomerase antisense oligodeoxynucleotides(ASODN) directly act on telomerase, or induce tumor cells to differentiate, result in telomerase activity decreases, and cells growth inhibited. However, whether the inhibition of telomerase activity by ASODN could induce tumor cells to differentiate is still unknown. Telomerase RNA acts as a template for synthesis of telomere, so telomerase sense oligodeoxynucleotides(SODN) can be hybridized to the telomere DNA. Whether this reaction could affect the growth of tumor cells is worth being studied. This research was just to investigate the effects of telomerase SODN and ASODN on the growth and differentiation of nasopharyngeal carcinoma(NPC) cells. METHODS: After transfecting telomerase SODN and ASODN into NPC CNE1 and CNE2Z cells by lipofectin, the proliferation of the cells was identified by MTT method, and the expression of keratin was detected by immunohistochemistry. Meanwhile, the morphological changes of the cells were observed. RESULTS: SODN inhibited the growth of CNE1 and CNE2Z cells as well as ASODN in dose- and time-dependent manner. After being treated with 1.5 mumol/L, 3.0 mumol/L, 4.5 mumol/L, and 6.0 mumol/L SODN, the inhibition rates in CNE1 cells for 6 h were 16.28%, 19.38%, 22.48%, and 23.26%, respectively; for 48 h were 26.26%, 38.89%, 39.90%, and 38.89%, respectively; the inhibition rates in CNE2Z cells for 6 h were 7.69%, 8.24%, 18.13%, and 20.32%, respectively, and for 48 h were 28.84%, 28.88%, 32.89%, and 31.54%, respectively. The keratin expressions in cells of SODN and ASODN groups were significantly increased and the cells tended to be mature. CONCLUSION: The telomerase SODN and ASODN could both inhibit the growth of NPC cells and induce cells to differentiate.