MiR-1180 from bone marrow-derived mesenchymal stem cells induces glycolysis and chemoresistance in ovarian cancer cells by upregulating the Wnt signaling pathway / 浙江大学学报（英文版）（B辑：生物医学和生物技术）

BACKGROUND@#Bone marrow-derived mesenchymal stem cells (BM-MSCs) play an important role in cancer development and progression. However, the mechanism by which they enhance the chemoresistance of ovarian cancer is unknown.@*METHODS@#Conditioned media of BM-MSCs (BM-MSC-CM) were analyzed using a technique based on microRNA arrays. The most highly expressed microRNAs were selected for testing their effects on glycolysis and chemoresistance in SKOV3 and COC1 ovarian cancer cells. The targeted gene and related signaling pathway were investigated using in silico analysis and in vitro cancer cell models. Kaplan-Merier survival analysis was performed on a population of 59 patients enrolled to analyze the clinical significance of microRNA findings in the prognosis of ovarian cancer.@*RESULTS@#MiR-1180 was the most abundant microRNA detected in BM-MSC-CM, which simultaneously induces glycolysis and chemoresistance (against cisplatin) in ovarian cancer cells. The secreted frizzled-related protein 1 (SFRP1) gene was identified as a major target of miR-1180. The overexpression of miR-1180 led to the activation of Wnt signaling and its downstream components, namely Wnt5a, β-catenin, c-Myc, and CyclinD1, which are responsible for glycolysis-induced chemoresistance. The miR-1180 level was inversely correlated with SFRP1 mRNA expression in ovarian cancer tissue. The overexpressed miR-1180 was associated with a poor prognosis for the long-term (96-month) survival of ovarian cancer patients.@*CONCLUSIONS@#BM-MSCs enhance the chemoresistance of ovarian cancer by releasing miR-1180. The released miR-1180 activates the Wnt signaling pathway in cancer cells by targeting SFRP1. The enhanced Wnt signaling upregulates the glycolytic level (i.e. Warburg effect), which reinforces the chemoresistance property of ovarian cancer cells.

Interacciones moleculares de un flavonoide prenilado con transportadores de antimicóticos dependientes de ATP / Molecular interactions of a prenylated flavonoid with ATP-depending antimycotic transporters

Uno de los principales mecanismos para la resistencia de Candida albicans a azoles es la sobreexpresión de transportadores que extruyen los fármacos antimicóticos y disminuyen su concentración intracelular. Anteriormente nuestros laboratorios habían informado que el flavonoide prenilado 2', 4'-dihidroxi - 5'-(1'''-dimetilalil)-6-prenil pinocembrina (6PP) inhibe en forma competitiva los transportadores cdr, dependientes del ATP, además de ser antimicrobiano per se. En este trabajo realizamos estudios bioinformáticos para analizar los posibles sitios de interacción molecular estereoespecífica del 6PP, fluconazol y adenosín trifosfato (ATP) con los transportadores cdr1 y cdr2. Se confeccionaron los modelos tridimensionales de los ligandos y de los transportadores a través de estudios de homología de datos respecto de otras macromoléculas. Se calculó la variación de energía libre de Gibbs ( G) asociada a 45 modelos de interacción molecular y se estudiaron cdr1 y cdr2 en formacomparativa, dada su similitud estructural. Se seleccionaron los modelos más probables desde el punto de vista termodinámico. El 6PP y el fluconazol podrían competir de diversas maneras por un sitio estereoespecífico que atraviesa la membrana de la levadura. Este sitio, numerado como 1, fue estudiado exhaustivamente, es común a los transportadores cdr1 y cdr2 y aporta selectivamente distintosaminoácidos. Entre estos últimos, los de mayor participación en los modelos de interacción fueron, en cdr1 y cdr2, respectivamente: PHE 754,756 y TYR 747,749 . Se seleccionaron los 9 mejores complejos formados por cada ligando, de acuerdo a su energía de afinidad decreciente (en KJ/mol en mejor modelo de cdr2: 6PP = -9,3; fluconazol = -6,8). Además el flavonoide prenilado competiría con el ATP en el dominio citosólico y con menor probabilidad lo imitaría el fluconazol ( G en KJ/mol en mejor modelo de cdr2: 6P P= -8,2, ATP = -7,6; fluconazol = -6,7)...

Chi-square analysis of the reduction of ATP levels in L-02 hepatocytes by hexavalent chromium

This study explored the reduction of adenosine triphosphate (ATP) levels in L-02 hepatocytes by hexavalent chromium (Cr(VI)) using chi-square analysis. Cells were treated with 2, 4, 8, 16, or 32 μM Cr(VI) for 12, 24, or 36 h. Methyl thiazolyl tetrazolium (MTT) experiments and measurements of intracellular ATP levels were performed by spectrophotometry or bioluminescence assays following Cr(VI) treatment. The chi-square test was used to determine the difference between cell survival rate and ATP levels. For the chi-square analysis, the results of the MTT or ATP experiments were transformed into a relative ratio with respect to the control (%). The relative ATP levels increased at 12 h, decreased at 24 h, and increased slightly again at 36 h following 4, 8, 16, 32 μM Cr(VI) treatment, corresponding to a "V-shaped" curve. Furthermore, the results of the chi-square analysis demonstrated a significant difference of the ATP level in the 32-μM Cr(VI) group (P < 0.05). The results suggest that the chi-square test can be applied to analyze the interference effects of Cr(VI) on ATP levels in L-02 hepatocytes. The decreased ATP levels at 24 h indicated disruption of mitochondrial energy metabolism and the slight increase of ATP levels at 36 h indicated partial recovery of mitochondrial function or activated glycolysis in L-02 hepatocytes.

Innovative In Vitro Chemo-Hormonal Drug Therapy for Refractory Thyroid Carcinomas

More than 95% of the thyroid carcinomas are well differentiated types showing favorable prognosis. However, only a few therapeutic options are available to treat the patients with undifferentiated thyroid carcinomas, especially with refractory thyroid carcinomas that are not amenable to surgery or radioiodine ablation. We investigated the anticancer effects of 20 chemotherapy and hormonal therapy drugs on 8 thyroid carcinoma cell lines. In vitro chemosensitivity was tested using the adenosine-triphosphate-based chemotherapy response assay (ATP-CRA). The tumor inhibition rate (TIR; or cell death rate) or half maximal inhibitory concentration (IC50) was analyzed to interpret the results. Of the 12 chemotherapy drugs, etoposide (178.9 index value in follicular carcinoma cell line) and vincristine (211.7 in Hurthle cell carcinoma cell line) were the most active drugs showing the highest chemosensitivity, and of the 8 additional drugs, trichostatin A (0.03 microg/mL IC50 in follicular carcinoma cell line) showed favorable outcome having the anticancer effect. In our study, the result of etoposide and vincristine show evidence as active anticancer drugs in thyroid carcinoma cell lines and trichostatin A seems be the next promising drug. These drugs may become an innovative therapy for refractory thyroid carcinomas in near future.

MsbA ATP-binding cassette (ABC) transporter of E. coli: Structure and possible flippase mechanism.

ATP-binding cassette (ABC) transporters utilize the energy present in cellular ATP to drive the translocation of structurally diverse set of solutes across the membrane barriers of eubacteria, archaebacteria and eukaryotes. In bacteria, these transporters are considered to be important virulence factors because they play role in nutrient uptake and in the secretion of toxins. The advances in structural determination and functional analysis of bacterial transporters have greatly increased our understanding of the mechanism of transport of these ABC transporters. Although progress in the field of structural biology has been made with the prokaryotic family members, it is likely that eukaryotic transporters will utilize the same mechanisms for translocation process. In this review, we summarize the function of the known MsbA ABC transporters in E. coli and mechanistic insights from structural and possible flippase mechanism studies.

Improved method for estimation of inorganic phosphate: implications for its application in enzyme assays.

The conventional method of Fiske and Subba Row for the estimation of inorganic phosphate (Pi) is although rapid, but suffers from the disadvantage that the color is unstable and hence the optical density (OD) measurements have to be carried out within a short time span of 8-12 min. This poses a restriction on the number of samples, which can be handled in a batch. Although, modified procedures involving use of alternate reducing agents/or increasing the concentration of H2SO4 in conventional method have been subsequently developed, but the problem of color stability could not be solved. In addition, the use of higher concentrations H2SO4 has rendered the methods unsuitable in enzyme assays, especially if the acid labile phosphate containing substrates have been used. In the present study, attempts have been made to suitably modify the method to improve the stability of the color and sensitivity and also for its applicability in enzyme assays, especially when acid labile phosphate containing substrates such as ATP is used. We used the higher concentrations (0.625, 0.8 and 1.0 N) of H2SO4 rather than 0.5 N used in the conventional assay procedures. Under these conditions, the reagent blanks do not develop color for up to 24 h, whereas the intensity of the molybdenum blue color in the standard and/or experimental tubes increased with time reaching optimum value at 24 h. Simultaneously, the absorption maximum shifts from 660 nm to 820 nm. The highest concentration of H2SO4 (1.0 N) is found to be the most effective in the process of color development. The sensitivity of the method is from 1.7 to 2.1 times higher, as compared to the conventional Fiske and Subba Row method for the measurements carried out at the end of 15 min at 820 nm and with the highest concentration of H2SO4 (1.0 N); the sensitivity increased 4.8-fold at the end of 24 h. Presence of glucose and sucrose (1-10 mM), NaCl and KCI (5-100 mM), MgCl2 (1-10 mM) and BSA (10 to 500 microg per assay tube) do not interfere either with color development or with OD measurements. The extent of ATP hydrolysis is 1.6 to 3.4% for up to 1 hi, depending upon the concentration of H2SO4 used. Only negligible hydrolysis of G6P is observed under these conditions. These results suggest that the presently modified method is suitable for Pi analysis in the enzyme assays, in the presence of labile phosphate containing substrates.

ATP y metabolismo cardiaco: acerca del tratamiento metabólico del infarto agudo del miocardio / ATP and cardiac metbolism: about metabolic treatment of acute cardiac infarction

Investigaciones sobre el ATP han revelado principios fundamentales de biología molecular cuyos descubridores fueron galardonados con el premio Nobel de química en 1997. En este artículo se revisan avances recientes sobre síntesis e hidrólisis del ATP y su relación con el metabolismo cardiaco. La síntesis de ATP se debe a la enzima F0 F1 sintetasa (o H+-ATP sintetasa) acoplada con la fuerza electromotriz de un gradiente de protones (H+) generado por fotosíntesis o por fosforilación oxidativa. Al pasar por FO, la corriente de H+ produce rotación interna de la subunidad ß y catálisis rotativa de las tres subunidades ß de F1. La rotación de la subunidad ç ha sido demostrada directamante. La hidrólisis de ATP está acoplada con la función de bombas iónicas (ATPasas) dependientes del aporte continuo de sangre oxigenada al cerebro y corazón. Al disminuir el aporte de O2, se produce zonas heterogéneas de metabolismo anaeróbico con acumulación de radicales libres y formación de productos tóxicos. A últimas fechas, hay un renovado interés para el tratamiento metabólico del infarto agudo del miocardio

Photoaffinity labeling and photoaffinity crosslinking of enzymes

Photoaffinity labeling is a special type of chemical modification, where the label is activated by the action of light. This article presents the general principles and limitations of this technique, its application to the study of Micrococcus luteus ATPase and the use of photoaffinity crosslinking to probe the structure of this enzyme

Chemical modification of allosteric properties of enzymes

Chemical modification is usually employed to study enzyme active sites. Valuable information can also be obtained, however, when this technique is used to probe allosteric sites. This approach is discussed in this article, and it is exemplified in chemical modification studies of the allosteric enzyme phosphofructokinase