Langchong He

Coating and fusing cell membranes onto a silica surface and their chromatographic characteristics

SummaryA new cell membrane stationary phase (CMSP) consisting of porous silica coated with active cell membranes is presented for affinity chromatography. By immersing, silica into a suspension of cell membranes, the whole surface of silica was covered by the cell membranes due to the irreversible adsorption of silanol groups (Si−OH) on the silica surface and the self-fusion of the cell membranes. CMSP can be used directly as a chromatographic packing material without any additional chemical modification. The surface characteristics, enzymatic activity, and chromatographic behavior of CMSP were investigated. The results obtained from scanning electron microscope, surface energy spectrometer, enzyme assay, and liquid chromatography showed that the surface characteristics of CMSP were very different from that of normal and reversed stationary phases. CMSP was found to have the characteristics of both cell membrane activity and chromatographic separation. Moreover, CMSP, as a chiral stationary phase, could be used for the enantiomeric separation of (±) Bay-K8644. The capacity factor of some calcium antagonists on CMSP was found to have a good correlation with their pharmacological actions. It is concluded that CMSP may be used not only as a kind of packing material in bio-affinity chromatography, but also as a tool for studying the interactions between a drug and its receptor.

Enzymatic activity and chromatographic characteristics of the cell membrane immobilized on silica surface

A new cell membrane preparation. silica carrier cell membrane (CCM), has been presented, in which cell membrane was immobilized on silica which acted as carrier by the absorption. The surface characteristics of the silica CCM have been tested by a scanning electron microscope and surface energy spectrometer. Comparison has been made of the changeable rule of enzymatic activity of rabbit red cell membrane in three states: the silica CCM, the suspension and the precipitation cell membranes, with temperature and time. The result showed that the silica CCM still keeps the same enzymatic activity as the other two states of cell membranes. The interactions between calcium antagonists and rabbi red and cardiac muscle cell membranes, and the stereoscopic interactions between dihydropridines and rabbit cerebellar cell membrane have been investigated by the chromatographic system of the silica CCM as a stationary phase. From the foregoing results, the chromatographic parameters obtained from the system would be expected to indicate the special interaction and enantioselectivity between drugs and membrane receptorsin vivo.

A new A431/cell membrane chromatography and online high performance liquid chromatography/mass spectrometry method for screening epidermal growth factor receptor antagonists from Radix sophorae flavescentis

The intracellular kinase domains of epidermal growth factor receptor (EGFR) in some tumor cells such as human epidermal squamous cells (A(431) cells) are an important target for drug discovery. We have developed a new A(431)/cell membrane chromatography (A(431)/CMC)-online-high performance liquid chromatography/mass spectrometry (HPLC/MS) method for screening EGFR antagonists from medicinal herbs such as traditional Chinese medicines (TCMs). In this study, A(431) cells with high EGFR expression levels were used to prepare cell membrane stationary phase (CMSP) in an A(431)/CMC model. The retention fractions eluted from the CMSP column were enriched onto an ODS pre-column and then switched into an HPLC/MS system by combining a 10 port columns switching valve. The screening results found that oxymatrine and matrine from Radix sophorae flavescentis (RSF) were the targeted components which could act on EGFR in similar manner of gefitinib as a control drug. There was a good relationship of their inhibiting effects on EGFR secretion and A(431) cell growth in vitro. This new A(431)/CMC-online-HPLC/MS method can be applied for screening EGFR antagonists from TCMs such as RSF. It will be a useful method for drug discovery with natural medicinal herbs as a leading compound resource.

A vascular smooth muscle/cell membrane chromatography–offline-gas chromatography/mass spectrometry method for recognition, separation and identification of active components from traditional Chinese medicines

We describe an analytical method of vascular smooth muscle cell membrane chromatography (VSM/CMC) combined with gas chromatography/mass spectrometry (GC/MS) for recognition, separation and identification of active components from traditional Chinese medicines (TCMs). VSM cells by means of primary culture with rat thoracic aortas were used for preparation of the stationary phase in the CMC model. Retention components by the VSM-CMC model were collected and then analyzed by GC/MS under the optimized conditions in offline conditions. After investigating the suitability and reliability of the VSM/CMC-offline-GC/MS method using nifedipine and nitrendipine as standard compounds, this method was applied in screening active components from the extracts of TCMs such as Radix Angelicae Dahuricae (RAD), Rhizomza Seu Radix Notopterygii (RSRN), Radix Glehniae (RG) and Fructus Cnidii (FC). Retention components from the extracts in the VSM-CMC model were imperatorin and osthole identified by the GC/MS method. In vitro pharmacological trials indicated that imperatorin and osthole could concentration dependently relax the rat thoracic artery pre-contracted by KCl (P<0.05). The maximum relaxation effects (R(max)) were 63+/-5% and 40+/-6% for imperatorin and osthole, respectively. The VSM/CMC-offline-GC/MS method is an effective screening system that can rapidly detect and enrich target components from a complex sample and then accurately identify them.

Graphene oxide/poly-l-lysine assembled layer for adhesion and electrochemical impedance detection of leukemia K562 cancercells

A novel biocompatible film assembled by combining of graphene oxide (GO) and poly-L-lysine (PLL) for adhesion and electrochemical impedance detection of leukemia K562 cells was proposed. The biocompatible film showed an improved immobilization capacity for living cells and a good biocompatibility for preserving the activity of the immobilized living cells. The immobilized K562 cells on the biocompatible film-modified electrode can be directly monitored with electrochemical impedance spectroscopy in the presence of [Fe(CN)₆]³⁻/⁴⁻ as redox probes. A highly sensitive electrochemical impedance method for the detection of leukemia K562 cancer cells was developed. Under the optimized conditions, the increased electron-transfer resistance with a good correlation to the logarithmic value of concentration of K562 cells ranging from 10² to 10⁷ cells mL⁻¹, and with the detection limit of 30 cells mL⁻¹ (S/N=3). Additionally, the proposed method was used to describe the viability of cells and to evaluate the effectiveness of antitumor drug Nilotinib on K562 cells. The obtained results of Nilotinib cytotoxicity are well agreed with those from WST-1 assays. Furthermore, the work demonstrates that a highly biocompatible film of PLL/GO assembled is also expected to be an appropriate matrix for the electrochemical investigation of adhesion, proliferation, apoptosis of other relevant mammalian cells which is not limited to adherent cells, and the study of cell-based biosensors.

Houttuynia cordata, a novel and selective COX-2 inhibitor with anti-inflammatory activity.

Abstract Ethnopharmacological relevance Houttuynia cordata Thunb. (Saururaceae; HC) has been long used in traditional oriental medicine for the treatment of inflammation diseases. Modern research has implicated inducible cyclooxygenase-2 (COX-2) as a key regulator of the inflammatory process. Aim of the study In the present study, we aimed to investigate the effect of HC on COX-2. We examined the effects of HC on lipopolysaccharide (LPS)-induced prostaglandin (PG) E2 production, an indirect indicator of COX-2 activity, and COX-2 gene and protein expression in mouse peritoneal macrophages. Materials and methods LPS-induced mouse peritoneal macrophages were employed as an in vitro model system. LPS-induced PGE2 production was assessed by enzyme-linked immunosorbant assay and COX-2 protein expression was assessed by Western blot assay. Results The results showed that HC was able to inhibit the release of LPS-induced PGE2 from mouse peritoneal macrophages (IC50 value: 44.8μg/mL). Moreover, the inhibitory activity of HC essential oil elicited a dose-dependent inhibition of COX-2 enzyme activity (IC50 value: 30.9μg/mL). HC was also found to cause reduction in LPS-induced COX-2 mRNA and protein expression, but did not affect COX-1 expression. The non-steroidal anti-inflammatory drug (NSAID) and specific COX-2 inhibitor NS398 functioned similarly in LPS-induced mouse peritoneal macrophages. Conclusion Taken together, our data suggest HC mediates inhibition of COX-2 enzyme activity and can affect related gene and protein expression. HC works by a mechanism of action similar to that of NSAIDs. These results add a novel aspect to the biological profile of HC.

A novel angiogenesis inhibitor impairs lovo cell survival via targeting against human VEGFR and its signaling pathway of phosphorylation.

Colorectal cancer represents the fourth commonest malignancy, and constitutes a major cause of significant morbidity and mortality among other diseases. However, the chemical therapy is still under development. Angiogenesis plays an important role in colon cancer development. We developed HMQ18–22 (a novel analog of taspine) with the aim to target angiogenesis. We found that HMQ18–22 significantly reduced angiogenesis of chicken chorioallantoic membrane (CAM) and mouse colon tissue, and inhibited cell migration and tube formation as well. Then, we verified the interaction between HMQ18–22 and VEGFR2 by AlphaScreen P-VEGFR assay, screened the targets on angiogenesis by VEGF Phospho Antibody Array, validated the target by western blot and RNAi in lovo cells. We found HMQ18–22 could decrease phosphorylation of VEGFR2(Tyr1214), VEGFR1(Tyr1333), Akt(Tyr326), protein kinase Cα (PKCα) (Tyr657) and phospholipase-Cγ-1 (PLCγ-1) (Tyr771). Most importantly, HMQ18–22 inhibited proliferation of lovo cell and tumor growth in a human colon tumor xenografted model of athymic mice. Compared with normal lovo cells proliferation, the inhibition on proliferation of knockdown cells (VEGFR2, VEGFR1, Akt, PKCα and PLCγ-1) by HMQ18–22 decreased. These results suggested that HMQ18–22 is a novel angiogenesis inhibitor and can be a useful therapeutic candidate for colon cancer intervention.

Synthesis and preliminary biological evaluation of novel taspine derivatives as anticancer agents.

Antiangiogenic therapy might represent a new promising anticancer therapeutic strategy. Taspine can significantly inhibit cell proliferation of human umbilical vein endothelial cells (HUVECs) induced by vascular endothelial growth factor-165, which is crucial for angiogenesis. In this study, a series of novel taspine derivatives were synthesized and screened for in vitro anticancer and antiangiogenesis activities. The majority of the derivatives demonstrated a moderate degree of cytotoxicity against human cancer cell lines. One of them (14) exhibited much better antiproliferative activity against CACO-2 (IC(50)=52.5microM) and ECV304 (IC(50)=2.67microM) cells than taspine did. Some of them were also effective in antiproliferative assays against HUVECs. The in silico estimate of solubility of title compounds were higher than that of taspine.

Suppression of tumor-induced angiogenesis by taspine isolated from Radix et Rhizoma Leonticis and its mechanism of action in vitro

The present study was to demonstrate the effect of taspine isolated from Radix et Rhizoma Leonticis on tumor angiogenesis and its mechanism of action. The anti-angiogenic effect in vivo was evaluated on chicken chorioallantoic membrane (CAM) neovascularisation model and CAM transplantation tumor model. Taspine exerted inhibitory influence on CAM angiogenesis and the growth and microvessel density (MVD) of CAM transplantation tumor at concentrations of 0.5-2μg/egg. The mechanism was demonstrated through detecting vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF) protein secretion by enzyme-linked immunosorbent assay (ELISA), as well as mRNA expression of VEGF, Flt-1 and Flk-1/KDR by reverse transcription-polymerase chain reaction (RT-PCR). The results showed that taspine down-regulated the VEGF and bFGF secretion in human non-small cell lung cancer cell (A549 cell) and human umbilical vein endothelial cell (HUVEC), and the VEGF and Flk-1/KDR mRNA expression in HUVEC. Additionally, the effect of taspine on HUVEC migration was detected with the method of cell scrape. The result indicated that taspine inhibited HUVEC migration in a dose-dependent manner. These findings suggest that taspine might be a promising candidate as angiogenesis inhibitors.

Applications of HPLC/MS in the analysis of traditional Chinese medicines

In China, traditional Chinese medicines (TCMs) have been used in clinical applications for thousands of years. The successful hyphenation of high-Performance liquid chromatography (HPLC) and mass spectrometry (MS) has been applied widely in TCMs and biological samples analysis. Undoubtedly, HPLC/MS technique has facilitated the understanding of the treatment mechanism of TCMs. We reviewed more than 350 published papers within the last 5 years on HPLC/MS in the analysis of TCMs. The present review focused on the applications of HPLC/MS in the component analysis, metabolites analysis, and pharmacokinetics of TCMs etc. 50% of the literature is related to the component analysis of TCMs, which show that this field is the most populär type of research. In the metabolites analysis, HPLC coupled with electrospray ionization quadrupole time-of-flight tandem mass spectrometry has been demonstrated to be the powerful tool for the characterization of structural features and fragmentation behavior patterns. This paper presented a brief overview of the applications of HPLC/MS in the analysis of TCMs. HPLC/MS in the fingerprint analysis is reviewed elsewhere.