Covalent assembly-based two-photon fluorescent probes for in situ visualizing nitroreductase activities: From cancer cells to human cancer tissues.

Nitroreductase (NTR) is widely regarded as a biomarker whose enzymatic activity correlates with the degree of hypoxia in solid malignant tumors. Herein, we utilized 2-dimethylamino-7-hydroxynaphthalene as fluorophore linked diverse nitroaromatic groups to obtain four NTR-activatable two-photon fluorescent probes based on covalent assembly strategy. With the help of computer docking simulation and in vitro assay, the sulfonate-based probe XN3 was proved to be able to identify NTR activity with best performances in rapid response, outstanding specificity, and sensitivity in comparison with the other three probes. Furthermore, XN3 could detect the degree of hypoxia by monitoring NTR activity in kinds of cancer cells with remarkable signal-to-noise ratios. In cancer tissue sections of the breast and liver in mice, XN3 had the ability to differentiate between healthy and tumorous tissues, and possessed excellent fluorescence stability, high tissue penetration and low tissue autofluorescence. Finally, XN3 was successfully utilized for in situ visualizing NTR activities in human transverse colon and rectal cancer tissues, respectively. The findings suggested that XN3 could directly identify the boundary between cancer and normal tissues by monitoring NTR activities, which provides a new method for imaging diagnosis and intraoperative navigation of tumor tissue.

A near-infrared fluorescent probe with two-photon excitation for in situ imaging of NQO1 in human colorectum cancer tissue.

Colorectum cancer has become one of the most fatal cancer diseases, in which NAD(P)H: quinone oxidoreductase 1 (NQO1) plays a role in intracellular free radical reduction and detoxification and has been linked to colorectum cancer and chemotherapy resistance. Therefore, rational design of optical probe for NQO1 detection is urgent for the early diagnosis of colorectum cancer. Herein, we have developed a novel two-photon fluorescent probe, WHFD, which is capable of selectively detecting of intracellular NQO1 with two-photon (TP) absorption (800 nm) and near-infrared emission (620 nm). Combination with a substantial Stokes shift (175 nm) and biocompatibility, we have assessed its suitability for in vivo imaging of endogenous NQO1 activities from HepG2 tumor-bearing live animals with high tissue penetration up to 300 µm. Particularly, we for the first time used the probe to image NQO1 activities from human colorectum cancer samples by using TP microscopy, and proving our probe possesses reliable diagnostic performance to directly in situ imaging of cancer biomarker and can clearly distinguish the boundary between human colorectum cancer tissue and their surrounding normal tissue, which shows great potential for the intraoperative navigation.

LINC00460 contributes to colorectal cancer cell invasion.

RESEARCH BACKGROUND: Colorectal cancer (CRC) is one of the most prevalent malignant tumors in the world. Research on long noncoding RNAs (LncRNAs) may illuminate tumorigenesis and progression of CRC. METHODS: We screened long non-coding RNA LINC00460 as a new candidate, which promoted the development of CRC in two independent datasets (GSE39582 and GSE21510) from the Gene Expression Omnibus (GEO). In 98 CRC tissues, expression levels of LINC00460 were significantly increased in cancerous tissues compared to paired adjacent normal tissues (P < 0.001). In addition, in the most common CRC cell lines. LINC00460 expression was up-regulated compared to normal human intestinal epithelial cell line NCM460. siRNA was transfected into CRC cell lines. LINC00460 knockdown reduced cell invasion ability and did not affect cell proliferation. The association between LINC00460 expression and clinical pathological features and prognosis were also analyzed. RESULTS: This increased expression was found to significantly correlate with lymph node metastasis (P = 0.002), distant metastasis (P = 0.045) and TNM stage (P < 0.001); but not related to age, gender, location of tumor, and histological grade. The overall survival (OS) in CRC patients with overexpression of LINC00460 was inferior to that with low expression (P = 0.0167). Multivariate Cox regression analyses indicated that LINC00460 expression, as well as TNM stage was an independent prognostic risk factor for patients with CRC. CONCLUSION: These results showed that a higher expression level of LINC00460 might play an oncogenic role in colorectal cancer invasion and metastasis. It also proved that LINC00460 might be used as a potential diagnostic and prognostic biomarker in CRC patients.

Activating IL-6/STAT3 Enhances Protein Stability of Proteasome 20S α+ß in Colorectal Cancer by miR-1254.

A widely recognized feature of colorectal cancer (CRC) is an increase in cytokine levels, which result in an inflammatory environment in the tumor. Interleukin-6 (IL-6) is a robust protumor cytokine. Several studies suggest that IL-6 plays a role in the development of tumors. Most intracellular protein breakdown occurs in eukaryotes via the ubiquitin-proteasome pathway; this mechanism may also be involved in cancer pathogenesis. The tumor tissues and paracancerous tissues were collected from 90 patients with colorectal cancer. The expressions of pSTAT3, proteasome 20S α+ß, miR-1254, and PSMD1 in tissues were detected by immunohistochemistry, ELISA, and qRT-PCR, and the effects of pSTAT3 and proteasome 20s α+ß expressions on the survival of patients were studied. HCT116 and HCT116-R cells were cultured and added IL-6, AG490, STAT3 plasmid, or overexpression/knockdown of miR-1254 in cells. Immunofluorescence, western blot, qRT-PCR, double luciferase gene reporter assay, and flow cytometry were used to detect the expression of pSTAT3, STAT3, proteasome 20s α+ß, miR-1254, and PSMD1 and cell cycle. The nude mouse xenograft model was constructed and divided into 3 groups: PBS group, IL-6 treatment group, and IL-6+miR-1254 mimic group. After 28 days, the tumor tissues were collected, and the expressions of miR-1254, pSTST3, proteasome 20s α+ß, and PSMD1 in the tissues were detected by qRT-PCR and immunohistochemistry, respectively. Our study discovered that the level of proteasome 20S α+ß had a strong connection with pSTAT3 in CRC patients. They were also linked to the development and clinical outcome of CRC. In addition, we found that IL-6 dramatically increased the expression of proteasome 20S α+ß and pSTAT3; however, it did not affect the proteasome 20S α+ß mRNA synthesis. Circulating proteasome concentration correlated with tumor tissue proteasome 20S α+ß. STAT3 could occupy the miR-1254 promoter to inhibit transcription, and it could suppressed miR-1254 which targeted PSMD10, promoting proteasome 20S α+ß protein stability. This is a prospective target for developing a new colorectal cancer therapy strategy.

Two-Photon Small-Molecule Fluorogenic Probes for Visualizing Endogenous Nitroreductase Activities from Tumor Tissues of a Cancer Patient.

Nitroreductase (NTR), a common enzymatic biomarker of hypoxia, is widely used to evaluate tumor microenvironments. To date, numerous optical probes have been reported for NTRs detection. Approaches capable of concisely guiding the probe design of NTRs suitable for deep-tissue imaging, however, are still lacking. As such, direct optical imaging of endogenous NTR activities from tumors derived from cancer patients is thus far not possible. Herein, aided by computational calculations, the authors have successfully developed a series of two-photon (TP) small-molecule fluorogenic probes capable of sensitively detecting general NTR activities from various biological samples; by optimizing the distance between the recognition moiety and the reactive site of NTRs from different sources, the authors have discovered and experimentally proven that X4 displays the best performance in both sensitivity and selectivity. Furthermore, X4 shows excellent TP excited fluorescence properties capable of directly monitoring/imaging endogenous NTR activities from live mammalian cells, growing zebrafish, and tumor-bearing mice. Finally, with an outstanding TP tissue-penetrating imaging property, X4 is used, for the first time, to successfully detect endogenous NTR activities from the liver lysates and cardia tissues of a cancer patient. The work may provide a universal strategy to design novel TP small-molecule enzymatic probes in future clinical applications.

Small-molecule fluorescent probes based on covalent assembly strategy for chemoselective bioimaging.

Fluorescent probes have been widely studied and applied in environment and health analysis, where among them small molecular "covalent assembly" probes are a novel type of reaction probes with many advantages, including no background interference, remarkable colorimetric change, rapid response, high sensitivity, and strong fluorescent signal. During the past decade, significant contributions have been made globally to both the application and mechanism of covalent assembly probes. In this review, we summarize the recent development of covalent assembly probes, classifying them based on different analytes, such as anions, metal ions, small biological molecules, reactive oxidative spices (ROS), reactive nitrogen species (RNS), nerve agent mimics, and enzymes, and introduce their detection mechanism in detail. Furthermore, the perspective on the next generation of covalent-assembly probes toward biomolecules imaging is presented.

Overexpression of the long non-coding RNA BLACAT1 promotes cell proliferation and invasion in colorectal cancer.

BACKGROUND: Recent evidence demonstrates that the long non-coding RNA (lncRNA) BLACAT1 is associated with the progression and development of various cancers; however, its effect on tumorigenesis of colorectal cancer (CRC) is still poorly understood. The aim of the present study was to investigate the expression and function of BLACAT1 in CRC. METHODS: Expression data from the GEO and GEPIA databases and results obtained from clinical samples/patients were used to determine the correlation between BLACAT1 expression, and CRC metastasis and overall survival (OS). Furthermore, we knocked down BLACAT1 using short interfering RNA (siRNA) and observed its biological functions using quantitative reverse transcription-polymerase chain reaction (qRT-PCR), cell counting kit-8 (CCK-8) assay, tumor cell clone formation, and Matrigel invasion assays in the HCT116 cell line. RESULTS: BLACAT1 level was higher in CRC tissues and cell lines than in normal colon mucosal tissues and cell lines. Correlation of data from the GEO and GEPIA databases with several clinical parameters revealed that CRC patients with high BLACAT1 expression showed poor OS. Multivariate analysis indicated that high BLACAT1 expression is an independent risk factor in patients with CRC. Furthermore, siRNA-mediated knockdown of BLACAT1 suppressed proliferation and invasion of CRC cells in vitro. This in turn was associated with reduced expression of cyclin D1, CDK6, and vimentin, and enhanced expression of E-cadherin. CONCLUSIONS: BLACAT1 may play an important role in the progression and development of CRC, and may serve as a potential therapeutic target for patients with CRC.

Upregulated plasmacytoma variant translocation 1 promotes cell proliferation, invasion and metastasis in colorectal cancer.

Emerging evidence indicates that the long non-coding RNA (lncRNA) plasmacytoma variant translocation 1 (PVT1) is associated with tumourigenesis in various types of cancer. However, its specific effects on the proliferation, invasion and metastasis of colorectal cancer (CRC) are still poorly understood. The present study aimed to investigate PVT1 expression in CRC and explore its role in CRC pathogenesis. The reverse transcriptase­quantitative polymerase chain reaction (RT­qPCR) technique was used to assess PVT1 expression in CRC cell lines. Gene Expression Omnibus (GEO) database analysis and measurement of clinical samples was used to analyse the correlation between PVT1 expression, CRC metastasis and overall survival (OS). In addition, knockdown of PVT1 expression was performed using short interfering RNA (siRNA) and RT­qPCR, western blotting, CCK­8 assays, tumour cell clone­formation and Matrigel invasion assays were used to observe its biological functions in HCT116 cells. The present study demonstrated that the expression of PVT1 in CRC cell lines was higher than that in normal colon mucosal cell lines. Using GEO database analysis and the measurement of clinical samples, it was revealed that CRC patients with high PVT1 expression demonstrated poor OS. Multivariate analysis indicated that high PVT1 expression is an independent risk factor for patients with CRC. In addition, PVT1 knockdown suppressed the proliferation, invasion and metastasis of CRC cells in vitro, which were associated with decreasing vimentin, cyclin D1 and cyclin­dependent kinase 4 expression and enhanced E­cadherin expression. The results of the present study suggest that PVT1 may serve a critical role in CRC progression and metastasis and may serve as a potential prognostic biomarker for CRC.

Human mutL homolog 1 expression characteristic and prognostic effect on patients with sporadic colorectal cancer.

The aim of the present study was to analyze the relationship between aberrant human mutL homolog 1 (hMLH1) expression and clinicopathological parameters of patients with sporadic colorectal cancer, and to explore the prognostic effect of aberrant hMLH1 expression in these patients. The relationship was measured by chi-square test and Fisher's exact test. Survival analysis was performed with Kaplan-Meier analysis and Cox regression model to measure 5-year disease-free survival (DFS) and 5-year overall survival (OS) rates. Totally 17.13% of the patients with sporadic colorectal cancer showed aberrant nuclear staining for hMLH1 expression. Aberrant hMLH1 expression was related with tumor pathologic types, tumor location and TNM staging (P<0.05) in the patients with sporadic colorectal cancer. Cox regression analysis indicated important prognostic factors were age (RR: 1.021, 95% CI: 1.003-1.039, P=0.023), mucinous adenocarcinoma (RR: 2.603, 95% CI: 1.705-3.974, P<0.0001), TNM staging (RR: 2.071, 95% CI: 1.170-3.666, P=0.012), lymphangion invasion (RR: 2.013, 95% CI: 1.227-3.303, P=0.006) and aberrant hMLH1 expression (RR: 0.414, 95% CI: 0.216-0.791, P=0.008). Consequently, hMLH1 expression level is related with some clinicopathologic features. Aberrant hMLH1 expression plays a significant part in prognosis for patients with sporadic colorectal cancer and it will promisingly become an independent prognostic factor.