Jie Hou

Synthesis of novel folate conjugated fluorescent nanoparticles for tumor imaging

A novel folate fluorescent nanoconjugate was synthesized and used for detection of cancer cells overexpressing the folate receptor (FR). The folate conjugate (PCMS-NA-FA) was synthesized by conjugating folic acid (FA) and 4-ethylnyl-N-ethyl-1, 8-naphthalimide (NA) to the polychloromethylstyrene (PCMS) functionalized with azido group (PCMS-N₃) through click reaction. The obtained conjugate had clear structure and could form PCMS-NA-FA nanoparticles with particle size around 86 nm in aqueous solution. Ability of PCMS-NA-FA targeting to cancerous cells was investigated by comparing the uptake of the nanoparticles by human adenocarcinoma HeLa cells and by non-FR expressing human lung carcinoma A549 cells. Specificity of the PCMS-NA-FA nanoparticles targeting on FRs was verified with cellular uptake inhibition assay, in which HeLa cells were incubated with both nanoconjugate and free FA. In addition, the specificity was also confirmed by the collocation of the immunofluorescence staining of anti-FR and the cellular uptake of the PCMS-NA-FA nanoparticles. Furthermore, the organ distribution of this folate nanoconjugate was studied on HeLa cell-bearing mice via frozen slicing, and the results showed that the folate nanoparticles were preferentially accumulated in the tumor site rather than other tissues, indicating the desired specificity for tumor targeting and imaging. All these findings suggested that this practical synthetic strategy can potentially facilitate the preparation of multifunctional imaging probe in biology and diagnosis of disease.

Cell surface glycoprotein profiling of cancer cells based on bioorthogonal chemistry.

Bioorthogonal chemistry refers to chemical reactions that can occur within a living system without altering native biochemical processes. Applications of this concept extend to studies on a group of biomolecules that includes glycans, proteins, and lipids. In this study, a strategy for isolating cell surface glycoproteins and based on bioorthogonal chemistry was employed to identify new cancer-related glycoproteins. A novel alkyne reagent containing one disulfide bond was synthesized for the enrichment of glycoproteins metabolized with peracetylated N-azidoacetylmannosamine, which was applied on three different cancer cell lines, and all isolated proteins were analyzed by high-performance liquid chromatography–tandem mass spectrometry. The strategy of purifying cell surface glycoproteins introduced in this article was shown to be reliable, and a total of 56 cell surface glycoproteins were identified. Neuronal cell adhesion molecule was found uniquely expressed in A549 lung adenocarcinoma, and its expression in non-small-cell lung carcinomas was detected by immunohistochemistry. Furthermore, a significant increase of neuronal cell adhesion molecule expression was identified in non-small-cell lung adenocarcinoma compared with adjacent noncancerous tissues, and could be a novel potential target and marker in cancer treatment and detection.

Profiling and comparative analysis of glycoproteins in Hs578BST and Hs578T and investigation of prolyl 4-Hydroxylase alpha polypeptide II expression and influence in breast cancer cells

To identify potential cancer related glycoproteins in breast cancer cells, we enriched N-linked glycoproteins by lentil lectin from the human breast cancer cell line Hs578T and the normal breast cell line Hs578BST for proteomic comparison. Glycoproteins were separated and compared by two-dimensional electrophoresis. Twenty-four glycoproteins were identified that expressed remarkably differently, among which nine were involved in the progress of collagen synthesis. Prolyl 4-hydroxylase alpha polypeptide II (P4HA2) expression and influence in breast cancer was further investigated. Immunohistochemistry revealed that P4HA2 was upregulated in breast tumor cells compared with its adjacent normal tissues. Moreover, overexpression and RNA interference of P4HA2 showed that P4HA2 expression suppressed cell proliferation and migration in Hs578T in vitro.

A novel amino-oligosaccharide isolated from the culture of Streptomyces strain PW638 is a potent inhibitor of α-amylase

A novel amino-oligosaccharide, named SF638-1, was isolated from the culture filtrate of the Streptomyces strain PW638. Its chemical structure was determined by electrospray ionization tandem mass spectrometry (ESI-MS/MS) and two-dimensional nuclear magnetic resonance spectroscopy. The novel compound was a mixed inhibitor of human pancreatic α-amylase, with a K(i) value in the same order of magnitude as that of the α-amylase inhibitor, acarbose. SF638-1 inhibited starch hydrolysis and glucose transfer in vitro, and suppressed postprandial blood glucose elevation in vivo. These results suggest that SF638-1 may be a potent antidiabetic agent.

A novel approach for fluorescent visualization of glycyrrhetic acid on a cell with a quantum dot

Glycyrrhetic acid (GA), a pentacyclic triterpenoid derivative obtained from hydrolysis of glycyrrhizic acid, was found to have synergistic anti-asthmatic effects with the β2-adrenergic receptor (β2AR) agonist via the β2AR-mediated pathway. This study visualized the location of GA on a human cell expressing β2AR via chemical biological approaches. A CdTe/ZnS quantum dot modified with an alkynyl group (QD-AL) was first synthesized, and an azide-terminal GA (ATGA) was also prepared. The QD-AL was used for fluorescence visualization of the distribution of GA on human embryonic kidney 293 cells expressing fusion β2AR (HEK293-β2AR) through the “click reaction” between QD-AL and ATGA. The average size of the QD-AL nanoparticle was about 10 nm, and its fluorescent emission wavelength was 620 nm. The location of GA on the HEK293-β2AR cell membrane can be visualized by the click reaction (between QD-AL and ATGA). The ability of QD-AL targeting to ATGA on the cell membrane of a HEK293-β2AR cell was further investigated using both confocal laser-scanning microscopy and a cellular uptake-inhibition assay. The results reveal that QD-AL can recognize ATGA on the cell membrane through the click reaction, which provides a new approach for visualizing the location of GA on the cell in an indirect way, and it can be applied to explore the synergistic anti-asthmatic mechanism of GA with β2AR agonist through the β2AR mediated pathway.

Enzymatic synthesis of L-tryptophan from D,L-2-amino-Δ2-thiazoline-4-carboxylic acid and indole by Pseudomonas sp. TS1138 L-2-amino-Δ2-thiazoline-4-carboxylic acid hydrolase, S -carbamyl-L-cysteine amidohydrolase and Escherichia coli L-tryptophanase

L-Tryptophan (L-Trp) is an essential amino acid. It is widely used in medical, health and food products, so a low-cost supply is needed. There are 4 methods for L-Trp production: chemical synthesis, extraction, enzymatic synthesis, and fermentation. In this study, we produced a recombinant bacterial strain pET-tnaA of Escherichia coli which has the L-tryptophanase gene. Using the pET-tnaA E. coli and the strain TS1138 of Pseudomonas sp., a one-pot enzymatic synthesis of L-Trp was developed. Pseudomonas sp. TS1138 was added to a solution of D,L-2-amino-Δ2-thiazoline-4-carboxylic acid (DL-ATC) to convert it to L-cysteine (L-Cys). After concentration, E. coli BL21 (DE 3) cells including plasmid pET-tnaA, indole, and pyridoxal 5’-phosphate were added. At the optimum conditions, the conversion rates of DL-ATC and L-Cys were 95.4% and 92.1%, respectively. After purifying using macroporous resin S8 and NKA-II, 10.32 g of L-Trp of 98.3% purity was obtained. This study established methods for one-pot enzymatic synthesis and separation of L-Trp. This method of producing L-Trp is more environmentally sound than methods using chemical synthesis, and it lays the foundations for industrial production of L-Trp from dl-ATC and indole.