ABSTRACT
The human UDP-glucuronosyltransferase 1A1 (UGT1A1), one of the most essential conjugative enzymes, is responsible for the metabolism and detoxification of bilirubin and other endogenous substances, as well as many different xenobiotic compounds. Deciphering UGT1A1 relevance to human diseases and characterizing the effects of small molecules on the activities of UGT1A1 requires reliable tools for probing the function of this key enzyme in complex biological matrices. Herein, an easy-to-use assay for highly-selective and sensitive monitoring of UGT1A1 activities in various biological matrices, using liquid chromatography with fluorescence detection (LC-FD), has been developed and validated. The newly developed LC-FD based assay has been confirmed in terms of sensitivity, specificity, precision, quanti-tative linear range and stability. One of its main advantages is lowering the limits of detection and quantification by about 100-fold in comparison to the previous assay that used the same probe substrate, enabling reliable quantification of lower amounts of active enzyme than any other method. The precision test demonstrated that both intra- and inter-day variations for this assay were less than 5.5％. Further-more, the newly developed assay has also been successfully used to screen and characterize the regu-latory effects of small molecules on the expression level of UGT1A1 in living cells. Overall, an easy-to-use LC-FD based assay has been developed for ultra-sensitive UGT1A1 activities measurements in various biological systems, providing an inexpensive and practical approach for exploring the role of UGT1A1 in human diseases, interactions with xenobiotics, and characterization modulatory effects of small mole-cules on this conjugative enzyme.
ABSTRACT
Polyacrylic acid was firstly grafted by N-amino-4-N-methylpiperazine-1,8-naphthlimide (AMN) to prepare a amphiphilic polymer, which was self-assembled in water producing nanoparticles called as PAAMN.Then the morphology, structure and fluorescence properties of PAAMN were investigated by various methods including transmission electron microscopy, dynamic light scattering, ultraviolet-visible spectroscopy (UV-Vis), nuclear magnetic resonance spectroscopy (HNMR) and fluorescence spectroscopy.MTT assay was carried out to assess the cell compatibility of PAAMN.Finally, the fluorescence from PAAMN self and HeLa cells incubated with PAAMN was observed by fluorescence microscope.The results revealed that PAAMN had spherical structure, in which naphthlimide fluorphores were immobilized in the polyacrylic acid matrix with the degree of substitution of 4.1%.Under the physiological pH condition, PAAMN excited at 390 nm could emit strong and stable fluorescence at 534 nm.In the range of pH 4.0-10.0, its excitation and emission wavelengths had no obvious change.The fluorescence intensity of PAAMN increased with the decrease of pH values, but the pH sensitivity of PAAMN was much lower than that of AMN.PAAMN had good cell compatibility.From the pictures of fluorescence imaging, it was found that both PAAMN self and cells-engulfed PAAMN could emit green fluorescence upon excited at 390 nm, indicating the potential of the developed nanoparticle for cell imaging.
ABSTRACT
A novel probe (DNSBN) towards biothiols on the basis of 4-hydroxynaphthalimide as fluorophores and 2, 4-dinitrobenzenesulfonyloxy group as specific recognition site was designed and synthesized.The result of absorption and fluorescence spectral analyses indicated that the probe had high sensitivity and selectivity towards cysteine (Cys), homocysteine (Hcy) and glutathione (GSH), and the detection was not affected by other 17 kinds of natural amino acids.Meanwhile, it was confirmed that DNSBN was a ratiometric probe through the fluorescence titration experiment, and the fluorescent intensity at 555 nm had a high linear relationship with biothiols concentration in the range of 0-20 μmol/L.The detection limits (3σ) of Cys, Hcy and GSH were 25.9, 92.0 and 77.9 nmol/L, respectively.The absorption, emission and mass spectra indicated that biothiols could be engaged in nucleophilic substitution reaction with 2,4-dinitrobenzenesulfonate, which induced the sulfonic esters decomposed.With the departure of receptor unit, the d-PeT progress (donor-excited photoinduced electron transfer) was blocked with an obvious colorimetric and fluorescence change.Finally, HeLa cell imaging experiments verified that DNSBN had good biocompatibility and could be used to detect exogenous biothiols.
ABSTRACT
OBJECTIVE: To study the effect of a novel naphthalimide-polyamine conjugate on apoptosis of human hepatocellular carcinoma HepG2 cell line. METHODS: MTT assay was used to evaluate the cell inhibition rate. The cellular morphous was detected with AO/EB/Hoechst staining, the ROS was detected with DCFH-DA staining, the mitochondrial membrane potential (ΔΨm) was detected with Rh123 staining, the expression of caspase-9 and PARP-1 was detected by immunofluorescence method. The cell cycle was detected by HCS. Apoptosis of HepG2 cells was quantified by flow cytometry using Annexin V/PI stain. RESULTS: Proliferation of HepG2 cells was inhibited significantly by the naphthalimide-polyamine conjugate in a dosage dependant manner. Under HCS, some HepG2 cells underwent a typical apoptotic morphologic change and the expression of ROS and caspase-9 was increased. The expression of PARP-1 and the mitochondrial membrane potential (ΔΨm) was detected decreased. Flow cytometry indicates 64.12% HepG2 cells were induced apoptosis after 48 h incubation with 25 μmol·L-1 naphthalimide-polyamine conjugate. CONCLUSION: The naphthalimide-polyamine conjugate could significantly induce the apoptosis of HepG2 cells by the mitochondrial pathway. The mechanism is concerned with increasing ROS, decreasing the mitochondrial membrane potential, upgrading caspase-9 and decreasing the expression of PARP-1.
ABSTRACT
Background and purpose:Suppression of apoptotic signaling pathways is an important factor in tumor cell resistance. Research on cell apoptosis will open up a new way of reversing drug resistance and tumor treatment. This study examined the effects of a novel naphthalimide derivative 8c on multidrug resistant colon cancer HCT116/L-OHP cells and explored the molecular mechanisms underlying the apoptosis induction. Methods: The anti-proliferative effects of 8c were detected by CCK-8 assays and the effects on apoptosis induction were examined by lfow cytometry. The mRNA expression levels of p53, Bax and Bcl-2 were measured by real-time PCR;The protein expressions of p-p53, Bax, Bcl-2 and Cyt-c were detected by Western blot. Results:8c (IC50=8.16 μmol/L) seemed to be more potent than amonaifde (IC50=28.37 μmol/L) against HCT116/L-OHP cells. 8c induced apoptosis on HCT116/L-OHP cell lines through intrinsic or mitochondria dependent pathway. The protein expression of phosphorylation of p53 at Ser-15 was increased, but the mRNA level of p53 did not increase in HCT116/L-OHP cells. Bax protein and mRNA levels were signiifcantly increased, and Bcl-2 protein and mRNA levels were decreased, suggesting an increase of Bax/Bcl-2 ratios. Meanwhile, 8c induced a substantial release of cytochrome c from the mitochondria into the cytosol in HCT116/L-OHP cells. Conclusion: 8c induced cell death signal by inducing the activation p53 phosphorylation which subsequently activated related protein expressions of apoptotic pathway, which may be an important mechanism of 8c on inhibiting proliferation of HCT116/L-OHP resistant cells. All the results suggested that 8c was a potent compound to be developed as an anti-tumor and anti-resistance agent for clinic application in the future.