Xianjing Hu

Structural Characterization and Biological Activities of a Novel Polysaccharide from Cultured Cordyceps militaris and Its Sulfated Derivative

A novel polysaccharide (CMPA90-1; compound 1) was isolated from the cultured fruiting bodies of Cordyceps militaris. The chemical structure of compound 1 was elucidated by acid hydrolysis, periodate oxidation, Smith degradation, and methylation analysis, along with Fourier transform infrared spectroscopy, high-performance anion-exchange chromatography coupled with pulsed amperometric detection, gas chromatography-mass spectrometry, and one-dimensional [(1)H and (13)C nuclear magnetic resonance (NMR)] and two-dimensional NMR (heteronuclear single-quantum coherence and heteronuclear multiple-bond correlation). Sulfation of compound 1 by the chlorosulfonic acid-pyridine (CSA-Pyr) method led to synthesis of its sulfated analogue (CMPA90-M1; compound 2). The ultrastructures of both compounds 1 and 2 were further characterized by scanning electron microscopy and atomic force microscopy. The results of antioxidant assays showed that compounds 1 and 2 exhibited free-radical-scavenging effects, ferrous-ion-chelating ability, and reducing power. Also, in the cytotoxicity assay, compounds 1 and 2 showed inhibitory activity against A549 cells, with IC50 values of 39.08 and 17.33 μg/mL, respectively.

Antitumor Effect of a Polypeptide Fraction from Arca subcrenata in Vitro and in Vivo

Arca subcrenata Lischke is a marine traditional Chinese medicine. The study investigated the antitumor effects of P2, a polypeptide fraction from A. subcrenata, and its toxicity in vitro and in vivo. The results showed that P2 could inhibit the proliferation of seven tumor cell lines, especially in HeLa and HT-29 cell lines. The IC50 values were 11.43 μg/mL for HeLa and 13.00 μg/mL for HT-29 treated by P2 for 48 h. P2 had little cytotoxicity on normal liver cells (L-02). The maximum tolerated dose (MTD) of P2 on KM mice was 1000 mg/kg by i.p. or i.v. The tumor growth inhibitory ratios of P2 were 26.4%, 41.4% and 46.4% for H-22, and 34.0%, 45.8% and 60.1% for S-180 tumor-bearing mice. The results demonstrated that P2 might be a potential antitumor agent with high efficiency in dose-dependent and time-dependent manners and low toxicity.

The Inhibitory Effect of a Novel Polypeptide Fraction from Arca subcrenata on Cancer-Related Inflammation in Human Cervical Cancer HeLa Cells

Inflammation is known to be closely associated with the development of cancer. The study was launched in human cervical cancer HeLa cells to investigate the antitumor and anti-inflammatory effects of P2, a marine polypeptide fraction from an important fishery resource Arca subcrenata. The basic research showed that P2 could suppress the production of nitric oxide in LPS-induced RAW264.7 macrophage cells as well as the secretion of inflammatory cytokines IL-6 and TNF-α in human cervical cancer HeLa cells. For the molecular mechanisms, P2 was shown to downregulate the gene expression of proinflammatory cytokines IL-6 and IL-8 and to inhibit the COX-2 and iNOS-related pathways in HeLa cells. In consequence, P2 might inhibit tumor development by blocking the interaction between tumor microenvironment and proinflammatory mediators. All findings indicate that P2 possesses the potential to be developed as a novel agent for cancer therapy.

Purification and characterization of an antibacterial and anti-inflammatory polypeptide from Arca subcrenata

A polypeptide coded as PGC was isolated from Arca subcrenata muscle using ion exchange, Sephadex G-50 gel chromatography and RP-HPLC. PGC was identified to be a homogeneous compound by Native-PAGE and the purity was more than 98.9% measured by HPLC. The isoelectric point of PGC was determined to be 9.76 by IEF-PAGE. The molecular weight was determined to be 15,973.0Da by ESI-MS/MS. The conformational structure of PGC was characterized by UV-vis, FT-IR and CD spectroscopy. N terminal amino acid sequence of PGC was shown as PSVYDAAAQLTADVKKDLRDSWKVIGGDKKGNGVA by Edman degradation. The results demonstrated that there is a high degree of homology between PGC and the subunit from hemoglobin, and proposed that PGC is the depolymerized polypeptide of Hemoglobin I (HbI) from A. subcrenata. The evaluation of biological activities showed that the diameters of the inhibitory ring of PGC on Escherichia coli and Staphylococcus aureus were 14.5±0.44mm and 16.5±1.15mm, respectively. The IC50 of inhibition rate for PGC on NO production was 9.60±0.71μg/mL. Therefore, PGC might be developed as one of potential antibacterial and anti-inflammatory agents.

GZD856, a novel potent PDGFRα/β inhibitor, suppresses the growth and migration of lung cancer cells in vitro and in vivo

Platelet-derived growth factor receptors (PDGFRα/β) play critical roles in the autocrine-stimulated growth and recruitment of cancer-associated fibroblasts (CAFs) of human lung cancer cells. We have identified GZD856 as a new PDGFR inhibitor that potently inhibits PDGFRα/β kinase activity and blocks this signaling pathway in lung cancer cells both in vitro and in vivo. GZD856 strongly suppresses the proliferation of PDGFRα-amplified H1703 (PDGFRβ(-)) human lung cancer cells and demonstrates significant in vivo antitumor efficacy in a xenograft mouse model. Although GZD856 displays only limited in vitro antiproliferative efficiency against PDGFRα(-)/PDGFRβ(+) A549 lung cancer cells, it efficiently inhibits the in vivo growth and metastasis of A549 cancer cells in xenograft and orthotopic models, respectively. The promising in vivo antitumor activity of GZD856 in A549 models may result from its suppression of PDGFR-related microenvironment factors, such as recruitment of CAFs and collagen content in stromal cells. GZD856 may be considered as a promising new candidate for anti-lung cancer drug development.

Polypeptide Fraction from Arca subcrenata Induces Apoptosis and G2/M Phase Arrest in HeLa Cells via ROS-Mediated MAPKs Pathways

Arca subcrenata is documented in the literature of marine Traditional Chinese Medicine. Polypeptide fraction from A. subcrenata, coded as P2, was demonstrated to possess significant antitumor activity in our previous study. However, the underlying mechanism remains undefined. The present study was carried out to investigate the underlying antitumor mechanism of P2 in human cervical cancer HeLa cells by MTT, FCM, LSCM, and western blot assays. The results revealed that P2 significantly induced apoptosis of HeLa cells in a concentration- and time-dependent manner. High level of ROS was provoked by P2, which was in turn responsible for induction of apoptosis through activation of intrinsic mitochondrial pathway and JNK1/2, p38 MAPK pathways, as well as inhibition of ERK1/2 pathway, as evidenced by the abrogation of P2s effect on HeLa cells preincubated with the ROS scavenger NAC. P2 also was observed to display significant effect on G2/M phase arrest by downregulating the expression of cyclin B1/cdc2 complex and upregulating the expression of p21. These findings demonstrate that P2 induces apoptosis and G2/M phase arrest in HeLa cells through ROS-mediated MAPKs pathways, suggesting that P2 would be worth investigating as a promising agent within the scope of marine drugs for treatment of cervical cancer.

Induction of apoptosis by Cordyceps militaris fraction in human chronic myeloid leukemia K562 cells involved with mitochondrial dysfunction

Background: Cordyceps militaris is widely used for various ethno medical conditions including cancer and inflammation complications in traditional Chinese medicine. Objective: To investigate the in vitro antitumor activity of Cordyceps militaris fraction (CMF) and the molecular mechanism underlying the apoptosis it induces in human chronic myeloid leukemia K562 cells. Materials and Methods: CMF was prepared according to our previous report. Cell viability was assessed by MTT assay. The rate of apoptosis, distribution of cell cycle and loss of mitochondrial membrane potential were measured by flow cytometry. Caspase activities were analyzed by Western blot and oxygen consumption rate was recorded using the Oxytherm system. Results: The results demonstrated that CMF triggered growth inhibition in K562 cells with only minor toxicity on a normal human cell line and inhibited the proliferation of K562 cells in a dose- and time-dependent manner with IC50 value of 34.1 ± 2.0 μg/ml after 48 h incubation. This most likely resulted from cell cycle arrest at the S phase and the induction of apoptosis. In addition, CMF induced activation of caspase-3 and subsequent cleavage of poly ADP-ribose polymerase (PARP). The caspase signals may originate from mitochondrial dysfunction, which was supported by the finding of decreased mitochondria transmembrance potential and the lower oxygen consumption rate. Conclusion: CMF possessed the in vitro antitumor effect on K562 cells and CMF-induced apoptosis might be involved by the mitochondrial dysfunction and valuable to research and develop as a potential antitumor agency.

A novel polysaccharide isolated from Litchi chinensis by using a simulated gastric medium and its immunomodulatory activity

A novel polysaccharide (LCPA50-S1) with immunomodulatory activity was extracted with simulated gastric medium from Litchi chinensis, and purified by DEAE-52 cellulose column, Sephadex G-50 column and Sephacryl S-300 HR chromatography. The structural characteristics of LCPA50-S1 were expounded through complete acid hydrolysis, partial acid hydrolysis, methylation and instrumental analysis. The results demonstrated that LCPA50-S1 is a heteropolysaccharide with a molecular weight of 1.58 × 10(5) Da. The backbone was composed of (1→4)-linked β-D-glucopyranosyl residues, (1→6)-linked β-D-galactopyranosyl, (1→3,6)-linked β-D-galactopyranosyl residues, (1→4,6)-linked α-D-glucopyranosyl residues and branched at O-6. The branches were consisted of (1→2)-linked α-L-rhamnopyranosyl residues, (1→4)-linked β-D-glucopyranosyl residues, and (1→6)-linked β-D-galactopyranosyl, terminated with (1→)-linked α-L-arabinopyranosyl residues and (1→)-linked β-D-galactopyranosyl residues, respectively. The immunoregulatory activity of LCPA50-S1 was evaluated through determination the effect of LCPA50-S1 on nitric oxide (NO) production of RAW264.7 macrophages and spleen lymphocyte proliferation as well as its cytokines secretion level. The results demonstrated that LCPA50-S1 increased NO and TNF-α production in RAW264.7 macrophages significantly, enhanced the proliferation as well as the interleukin-2 (IL-2) production of splenocytes. The data indicated that LCPA50-S1 had the potential to be explored as a novel natural immunomodulator for application in functional foods and medicine.

Cordyceps militaris fraction inhibits the invasion and metastasis of lung cancer cells through the protein kinase B/glycogen synthase kinase 3β/β‑catenin signaling pathway

Cordyceps militaris is widely used as a traditional Chinese medicine health supplement, and is also used in the development of anticancer agents. In our previous studies, it was revealed that C. militaris fraction (CMF) possessed an antitumor effect against K562 cells in vitro, induced apoptosis and caused cell cycle arrest in the S phase. The published results also demonstrated that CMF-induced apoptosis was involved in mitochondrial dysfunction. The aim of the present study was to investigate the anti-invasion and anti-metastasis effects of CMF in NCI-H1299 and Lewis lung cancer (LLC) cell lines, which have high metastatic potential. MTT and clone formation assays were initially used to investigate the inhibitory effect of CMF on the viability of NCI-H1299 and LLC cells. The results of cell adhesion, wound healing, migration and Matrigel invasion assays in vitro indicated that NCI-H1299 cells (treated with 1, 3, 10 or 30 µg/ml CMF) and LLC cells (treated with 0.1, 0.3, 1 or 3 µg/ml CMF) demonstrated a concentration-dependent reduction in cell migration and invasion compared with the control. In vivo experiments demonstrated that the oral administration of CMF (65, 130 or 260 mg/kg) decreased the tumor growth and decreased the lung and liver metastasis in an LLC xenograft model, compared with untreated mice. Furthermore, western blot analysis was used to investigate the mechanism of the effect of CMF on the migration of NCI-H1299 cells and metastasis in the xenograft model. The results revealed that CMF may promote glycogen synthase kinase 3β (GSK-3β)-mediated degradation of β-catenin inhibited the phosphorylation of upstream protein kinase B (Akt), which resulted in the attenuation of the expression of matrix metalloproteinase (MMP)-2 and MMP-9. These results suggested that CMF may possess potential for the treatment of lung cancer metastasis via the Akt/GSK-3β/β-catenin pathway.

Region-selective biosynthesis of artemisinic acid glycosides by crown galls of Panax quinquefolium and their in vitro antitumor activities

Background: The biosynthesis of artemisinin derivatives is one of the interesting subjects. Artemisinic acid (AA) has been widely studied as a supposed intermediate in the biosynthetic pathway leading to artemisinin in Artemisia annua. Objective: To investigate the bioconversion of AA by transgenic crown galls of Panax quinquefolium. Materials and Methods: AA was administered into crown galls of P. quinquefolium and co-cultured for 2 days. The methanol extract was separated by column chromatography, and the structures of two biosynthesis products were elucidated by physicochemical and spectroscopic methods. Co-culture time curves on conversion were also established. In addition, the effects of AA on the growth and ginsenosides production of crown galls of P. quinquefolium were investigated. Furthermore, the in vitro antitumor activities of AA and two glycosides against HepG2 cell line were evaluated by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT) assay. Results: Glycosylation of AA by crown galls of P. quinquefolium was observed, and two region-selectively glycosylated products were obtained (AA-1, AA-2), involving one new compound (AA-2). Their structures were elucidated to be AA β-D-glucopyranosyl ester (AA-1) and AA β-D-glucopyranosyl-(2 → 1)-β-D-glucopyranosyl ester (AA-2). The maximum yield of AA-1 was 19.3% on the 1st co-culture day while that of AA-2 was 59.1% on the 2nd day. MTT assay showed that the activity of monosaccharide glycoside (AA-1) was better than that of disaccharide glycoside (AA-2). Conclusion: Two AA glycosides involved one new compound with potential antitumor activity were obtained by region-selective biosynthesis with crown galls of P. quinquefolium.