RESUMO
A new type of fluorescent silver nanoclusters consisting of one silver bound to several strands of DNA, called multi-DNA-AgNCs, have been constructed using a bifunctional oligonucleotide with the recognition sequence 5'-CTAC[combining low line]G[combining low line]T[combining low line]G[combining low line]CT-3' as a stabilizing agent. The target oligonucleotide causes the multi-DNA-AgNCs to reassemble into smaller sized Ag clusters with quenched emission properties, while BSA induces the reassembly of the multi-DNA-AgNCs to give large particles with an enhanced emission. This demonstrates that the multi-DNA-AgNCs can specifically detect this recognition sequence. Furthermore, the multi-DNA-AgNCs show different fluorescence responses toward the total protein of normal cells (WRL-68), HepG-2 cells and HepG-2 cells incubated with 5-fluorourcil (5-Fu). The results show that the total protein of the HepG-2 cells, in which HIF is highly expressed, significantly decreases the fluorescence emission. Consequently, the multi-DNA-AgNCs can be used as a fluorescence probe for the detection of cancer cells, which have a high expression of HIF.
RESUMO
Pathologic assessment of colorectal adenomas, a complex task with significant interobserver variability, typically defines the scheduling of surveillance colonoscopies after removal of adenomas. We have characterized the activity levels of pro-matrix metalloproteinase-2, active matrix metalloproteinase-2, and matrix metalloproteinase-9 in colorectal adenomas and carcinomas as potential markers of pathologic progression during colorectal tumorigenesis. Endogenous fully activated matrix metalloproteinase-2, in particular, has been studied less frequently in adenomas due to difficulties in detection. For this report, tissues (n = 119) from 51 individuals were extracted and assayed on gelatin zymograms with digital standardization to nanogram quantities of purified active controls. Resulting data were assessed by graphical and multinomial logit regression analyses to test whether matrix metalloproteinase-2 or matrix metalloproteinase-9 activities could discriminate among 4 different types of colorectal tissue (normal mucosa, adenomas with or without high-grade dysplasia, and invasive carcinomas). Active matrix metalloproteinase-2 successfully discriminated among these tissue categories. Median activity for active matrix metalloproteinase-2 increased in a stepwise fashion with pathologic progression from normal mucosa to adenoma without high-grade dysplasia to adenoma with high-grade dysplasia to cancer. Although pro-matrix metalloproteinase-2 and pro-matrix metalloproteinase-9 activities could discriminate to some extent among tissue categories, those effects did not contribute additional information. Active matrix metalloproteinase-2 activity correlated significantly with histopathologic assessment of colorectal tissues. The ability of active matrix metalloproteinase-2 to distinguish adenomas with high-grade dysplasia from adenomas without high-grade dysplasia may be particularly useful in predicting future colorectal cancer risk for an individual, thus optimizing scheduling of surveillance colonoscopies.
Assuntos
Adenoma/patologia , Colo/citologia , Neoplasias Colorretais/patologia , Mucosa Intestinal/citologia , Metaloproteinase 2 da Matriz/metabolismo , Adenoma/enzimologia , Polipose Adenomatosa do Colo/enzimologia , Polipose Adenomatosa do Colo/patologia , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Colo/enzimologia , Neoplasias Colorretais/enzimologia , Diagnóstico Diferencial , Progressão da Doença , Ativação Enzimática , Feminino , Humanos , Mucosa Intestinal/enzimologia , Masculino , Metaloproteinase 9 da Matriz/metabolismo , Pessoa de Meia-Idade , Adulto JovemRESUMO
Fully active MMP-2 is expressed at such low levels in human tissues that studies often fail to confirm its value as a cancer marker despite strong associations with malignancy. Our study utilized careful extraction, accurate activity measurements, standardization to purified controls and a new statistical metric to determine whether active MMP-2 is an effective indicator of colorectal cancer compared to pro-MMP-2 or pro-MMP-9. MMP-2 and MMP-9 activities were analyzed in matched normal and cancer samples from 269 patients by gelatin zymography, computer-assisted image analysis, serial dilutions of strong samples and standardization to controls. An index of effect size was designed for comparative evaluation of active MMP-2, pro-MMP-2 and pro-MMP-9 activities. For each gelatinase, mean activity and protein levels/mg soluble protein in normal mucosa and colorectal cancer were calculated for the first time with respect to commercial standards. Active MMP-2 activity, detected in 99% of colorectal cancers, was higher in 95% of cancers (on average 10-fold) than in normal mucosa. Levels of pro-MMP-2 and pro-MMP-9, but not active MMP-9, activities were also significantly higher in cancers versus normal. However, active MMP-2 activity provided the most effective test for the presence of cancer (p<0.0.0001) with an effect size statistically significantly larger than for either pro-MMP-2 or pro-MMP-9. Receiver operating characteristic (ROC) curves demonstrated that a cut-off for active MMP-2 of >44 SDU activity/mg soluble protein (>180 pg/mg), which is three times mean normal levels, would permit detection of colorectal cancer with an estimated sensitivity of 84% and estimated specificity of 93%.
Assuntos
Biomarcadores Tumorais/metabolismo , Neoplasias Colorretais/enzimologia , Precursores Enzimáticos/metabolismo , Gelatinases/metabolismo , Metaloproteinase 2 da Matriz/metabolismo , Adulto , Idoso , Neoplasias Colorretais/patologia , Feminino , Humanos , Immunoblotting , Masculino , Metaloproteinase 9 da Matriz/metabolismo , Pessoa de Meia-Idade , Estadiamento de Neoplasias , Prognóstico , Curva ROCRESUMO
Cathepsin D protein patterns were analyzed in 59 colorectal tumors by Western blotting, glycosylation and immunohistochemical assays. Measurement of protein content by laser densitometry of tumor/normal pairs on Western blots revealed loss of cathepsin D protein in more than 50% of colorectal tumors. Independent loading controls and statistical estimates of reproducibility on duplicate assays confirmed frequent decreases in cathepsin D. For cases having a tumor/normal ratio (T/N) <1, the average T/N was 0.50+/-0.19, equivalent to the loss of one cathepsin D allele. However, 2-fold increases in cathepsin D protein levels were also observed in approximately 1/3 of tumors, supporting the concept that colorectal cancers develop via divergent molecular pathways and that cathepsin D may function differently in different cancers. Although normal cathepsin D expression was detected in some earlier stage tumors, protein levels became increasingly bimodal with progression such that cathepsin D levels were increased in 1/3 but decreased in 2/3 of stage III and IV cancers. Other laboratories have reported both significant loss and gain of chromosome 11 (site of the cathepsin D gene) in different colorectal tumors, providing a possible mechanism for our observations on cathepsin D. However, differential regulation of cathepsin D expression by mutant versus wild-type p53 may also contribute to variable cathepsin D levels in colorectal cancers. Immunohistochemical studies demonstrated a shift from a predominantly punctate distribution of cathepsin D protein in normal mucosa to a more diffuse cytoplasmic distribution in tumor tissues. Mutant forms of cathepsin D were not detected in tumors either as changes in electrophoretic mobility or altered glycosylation but minor changes in protein sequence could not be ruled out. Loss of cathepsin D protein may provide an advantage to colorectal tumors related to a loss of cathepsin D function in proapototic or antiangiogenic pathways while increased cathepsin D may promote cancer cell proliferation or invasion.