Chao Ma

Effects of triterpenes from Ganoderma lucidum on protein expression profile of HeLa cells

To elucidate the cytotoxicity mechanism of Ganoderma triterpenes, a chemoproteomic study using five purified ganoderic acids, ganoderic acid F (GAF), ganoderic acid K (GAK), ganoderic B (GAB), ganoderic acid D (GAD) and ganoderic acid AM1 (GAAM1) was conducted. GAF, GAK, GAB, GAD and GAAM1 treatment for 48 h inhibited the proliferation of HeLa human cervical carcinoma cells with IC(50) values of 19.5+/-0.6 microM, 15.1+/-0.5 microM, 20.3+/-0.4 microM, 17.3+/-0.3 microM, 19.8+/-0.7 microM, respectively. The protein expression profiles of HeLa cells treated with each ganoderic acid at dose of 15 microM for 48 h were checked using two-dimensional electrophoresis (2-DE). The possible target-related proteins of ganoderic acids, i.e. proteins with same change tendency in all five ganoderic acids-treated groups compared with control, were identified using MALDI-TOF MS/MS. Twelve proteins including human interleukin-17E, eukaryotic translation initiation factor 5A (eIF5A), peroxiredoxin 2, ubiquilin 2, Cu/Zn-superoxide dismutase, 14-3-3 beta/alpha, TPM4-ALK fusion oncoprotein type 2, PP2A subunit A PR65-alpha isoform, nucleobindin-1, heterogeneous nuclear ribonucleoprotein K, reticulocalbin 1 and chain A of DJ-1 protein were identified. Ganoderic acids might exert their cytotoxicity by altering proteins involved in cell proliferation and/or cell death, carcinogenosis, oxidative stress, calcium signaling and ER stress.

Interaction of Ganoderma triterpenes with doxorubicin and proteomic characterization of the possible molecular targets of Ganoderma triterpenes

Triterpenes are the main components with cytotoxicity in Ganoderma lucidum, which is used popularly as a complementary treatment for cancer therapy in traditional Chinese medicine. To investigate the possible interaction between chemotherapeutic agents and triterpenes extracted from G. lucidum, the cytotoxicity of doxorubicin (DOX) combined with Ganoderma triterpenes (GTS) or lucidenic acid N (LCN), a purified compound, was examined in HeLa cells. The combinations targeting DOX with GTS or LCN resulted in a synergistic interaction in HeLa cells. Moreover, to identify the molecular targets of GTS, two‐dimensional gel electrophoresis‐based comparative proteomics was carried out and proteins with altered expression levels after GTS treatment in HeLa cells were identified by matrix‐assisted laser desorption/ionization time‐of‐flight tandem mass spectrometry. The results of our proteomic study indicated that the GTS treatment caused regulated expression of 14 proteins, which play important roles in cell proliferation, the cell cycle, apoptosis, and oxidative stress. Flow cytometric analysis confirmed that GTS could induce weak G0–G1 phase arrest and combined use of GTS with DOX could induce apoptosis in cells. Furthermore, GTS enhanced the reactive oxygen species (ROS)‐producing effect of DOX, and a ROS scavenger could affect the synergism between GTS and DOX. In cells with high Ku80 protein expression, the synergism between GTS and DOX was also partly affected. Importantly, in cells with high Ku80 expression that were treated with a ROS scavenger, the synergism between GTS and DOX totally disappeared. These results suggest that the synergism between GTS and DOX might be based on GTS‐induced sensitization of cells to chemotherapeutics through enhanced oxidative stress, DNA damage, and apoptosis. (Cancer Sci 2008; 99: 1461–1470)

Cardio-protection of salvianolic acid B through inhibition of apoptosis network.

Targeting cellular function as a system rather than on the level of the single target significantly increases therapeutic potency. In the present study, we detect the target pathway of salvianolic acid B (SalB) in vivo. Acute myocardial infarction (AMI) was induced in rats followed by the treatment with 10 mg/kg SalB. Hemodynamic detection and pathological stain, 2-dimensional electrophoresis, MALDI-TOF MS/MS, Western blot, pathway identification, apoptosis assay and transmission electron microscope were used to elucidate the effects and mechanism of SalB on cardioprotection. Higher SalB concentration was found in ischemic area compared to no-ischemic area of heart, correlating with improved heart function and histological structure. Thirty-three proteins regulated by SalB in AMI rats were identified by biochemical analysis and were classified as the components of metabolism and apoptosis networks. SalB protected cardiomyocytes from apoptosis, inhibited poly (ADP-ribose) polymerase-1 pathway, and improved the integrity of mitochondrial and nucleus of heart tissue during AMI. Furthermore, the protective effects of SalB against apoptosis were verified in H9c2 cells. Our results provide evidence that SalB regulates multi-targets involved in the apoptosis pathway during AMI and therefore may be a candidate for novel therapeutics of heart diseases.

Structural characterization of a pectic polysaccharide from Nerium indicum flowers.

A polysaccharide fraction, J6, was isolated from the hot-water extract of flowers of oleander Nerium indicum Mill., using ethanol precipitation, cetyltrimethylammonium bromide (CTAB) complexing, anion-exchange chromatography and gel permeation chromatography. J6 was found to contain L-rhamnose, L-arabinose, D-galactose, and D-galacturonic acid, in the ratio of 10.1:49.8:30.1:10.0. Its structure was investigated by methylation analysis, periodate oxidation, Smith degradation, partial acid hydrolysis, electrospray ionization mass spectrometry and NMR spectroscopic methods. It was found that J6 is an RG-I type polysaccharide, which contains a rhamnogalacturonan backbone, with various branches attached to O-4 of L-rhamnose. The branches probably involve (1-->4)-beta-D-galactan, branched L-arabino-(1-->3)(1-->6)-beta-D-galactan, and (1-->5)-alpha-L-arabinan. J6 stimulated NO production of macrophage RAW264.7 cells in a preliminary test.

Differential protein expression in mouse splenic mononuclear cells treated with polysaccharides from spores of Ganoderma lucidum

Polysaccharides were one of the main components of Ganoderma lucidum, a medicinal mushroom well known for its immuno-modulation effects. In the present study, we demonstrated that polysaccharides extracted from Ganoderma lucidum spores (GL-SP) could stimulate splenic mononuclear cells (MNCs) proliferation and cytokine production. To identify the possible cellular targets of GL-SP in MNCs, two-dimensional gel electrophoresis (2-DE)-based comparative proteomics was performed and proteins altered in expressional level after GL-SP treatment were identified by MALDI-TOF MS/MS. Ten proteins with >2-fold increase or decrease expression in GL-SP-treated MNCs compared with control were found and further identified by MALDI-TOF MS/MS analysis and database searching. In the GL-SP-treated MNCs, there were increases in the expression of myosin regulatory light chain 2-A, Rho GDP dissociation inhibitor beta, T-cell-specific GTPase, phosphatidylinositol transfer protein alpha, and decreases in the expression of apoptosis-associated speck-like protein containing a CARD, 14-3-3 tau, beta-actin, tubulin alpha 2, copine I, and gamma-actin. The results of the present study help to provide insight into the immuno-modulating activities of GL-SP.

Insight into potential toxicity mechanisms of melamine: an in silico study.

The toxicity of melamine has attracted much attention since the outbreak of nephrolithiasis among children ingesting melamine-contaminated infant formula in China. However, there is little knowledge about the molecular mechanisms underlying the melamine-induced toxicity. In this paper, a ligand-protein docking method (INVDOCK) was applied to predict the toxicity-related target proteins for melamine and its metabolite, cyanuric acid. As a result, 23 and 35 proteins were finally identified as the potential target proteins for melamine and cyanuric acid, respectively. Through an enrichment analysis, it was found that nephrotoxicity and lung toxicity might be the most significant toxicities induced by melamine and cyanuric acid. Four target proteins (glutathione peroxidase 1, beta-hexosaminidase subunit beta, L-lactate dehydrogenase and lysozyme C) may be related to the molecular basis of the nephrotoxicity induced by melamine except for known kidney crystals formation. After mapping all these toxicity-related target proteins onto cellular pathways, it was indicated that the toxicities of melamine and cyanuric acid might also be caused by breaking down redox balance, perturbing the arginine and proline metabolism and damaging the homeostasis of energy production system. To further explore the mechanisms underlying the toxicities of melamine and cyanuric acid, a biological signal cascades network constructed by some of the toxicity-related target proteins was discussed.

Differential Proteomic Analysis of Platelets Suggested Possible Signal Cascades Network in Platelets Treated with Salvianolic Acid B

Background Salvianolic acid B (SB) is an active component isolated from Danshen, a traditional Chinese medicine widely used for the treatment of cardiovascular disorders. Previous study suggested that SB might inhibit adhesion as well as aggregation of platelets by a mechanism involving the integrin α2β1. But, the signal cascades in platelets after SB binding are still not clear. Methodology/Principal Findings In the present study, a differential proteomic analysis (two-dimensional electrophoresis) was conducted to check the protein expression profiles of rat platelets with or without treatment of SB. Proteins altered in level after SB exposure were identified by MALDI-TOF MS/MS. Treatment of SB caused regulation of 20 proteins such as heat shock-related 70 kDa protein 2 (hsp70), LIM domain protein CLP-36, copine I, peroxiredoxin-2, coronin-1 B and cytoplasmic dynein intermediate chain 2C. The regulation of SB on protein levels was confirmed by Western blotting. The signal cascades network induced by SB after its binding with integrin α2β1 was predicted. To certify the predicted network, binding affinity of SB to integrin α2β1 was checked in vitro and ex vivo in platelets. Furthermore, the effects of SB on protein levels of hsp70, coronin-1B and intracellular levels of Ca(2+) and reactive oxygen species (ROS) were checked with or without pre-treatment of platelets using antibody against integrin α2β1. Electron microscopy study confirmed that SB affected cytoskeleton structure of platelets. Conclusions/Significance Integrin α2β1 might be one of the direct target proteins of SB in platelets. The signal cascades network of SB after binding with integrin α2β1 might include regulation of intracellular Ca(2+) level, cytoskeleton-related proteins such as coronin-1B and cytoskeleton structure of platelets.

Combining genomic and network characteristics for extended capability in predicting synergistic drugs for cancer

The identification of synergistic chemotherapeutic agents from a large pool of candidates is highly challenging. Here, we present a Ranking-system of Anti-Cancer Synergy (RACS) that combines features of targeting networks and transcriptomic profiles, and validate it on three types of cancer. Using data on human β-cell lymphoma from the Dialogue for Reverse Engineering Assessments and Methods consortium we show a probability concordance of 0.78 compared with 0.61 obtained with the previous best algorithm. We confirm 63.6% of our breast cancer predictions through experiment and literature, including four strong synergistic pairs. Further in vivo screening in a zebrafish MCF7 xenograft model confirms one prediction with strong synergy and low toxicity. Validation using A549 lung cancer cells shows similar results. Thus, RACS can significantly improve drug synergy prediction and markedly reduce the experimental prescreening of existing drugs for repurposing to cancer treatment, although the molecular mechanism underlying particular interactions remains unknown.

Calmodulin as a Potential Target by Which Berberine Induces Cell Cycle Arrest in Human Hepatoma Bel7402 Cells

Berberine is an isoquinoline alkaloid that has drawn extensive attention because it possesses various biological activities. Several mechanisms have been proposed to interpret the anticancer activity of berberine. However, these explanations are mostly based on its downstream‐regulated genes or proteins; information on the direct target proteins that mediate the antiproliferative action of berberine remains unclear. In this study, a computational pipeline based on a ligand–protein inverse docking program and mining of the ‘Connectivity MAP’ data was adopted to explore the potential target proteins for berberine. The results showed that four proteins, that is calmodulin, cytochrome P450 3A4, sex hormone‐binding globulin, and carbonic anhydrase II, were suggested to be the potential targets of berberine. The anticalmodulin property of berberine was demonstrated with an in vitro phosphodiesterase activity assay. Flow cytometric analysis found that G1 cell cycle arrest induced by berberine in Bel7402 cells was enhanced by cotreatment with calmodulin inhibitors. Western blotting results indicated that berberine treatment decreased phosphorylation of calmodulin kinase II and blocked subsequent MEK1 activation as well as p27 protein degradation. These results suggested that calmodulin might play crucial roles in berberine‐induced cell cycle arrest in cancer cells.

Proteomic assessment of tanshinone IIA sodium sulfonate on doxorubicin induced nephropathy.

Although doxorubicin (DXR) is an important antineoplastic agent, the serious toxicity mediated by the production of reactive oxygen species has remained a considerable clinical problem. Our hypothesis is that tanshinone II A sodium sulfonate (TSNIIA-SS), which holds significant effects against oxidative stress, protects against DXR-induced nephropathy. Firstly, the antioxidative effects of TSNIIA-SS were confirmed using oxygen radicals absorbance capacities (ORAC) assay in vitro. Then, DXR nephropathy was induced by repeated DXR treatment and verified by kidney index (20.76 ± 3.04 mg/mm versus 14.76 ± 3.04 mg/mm, p < 0.001) and histochemical stain. The mice were randomized into three groups: Control group, DXR group and DXR-TSNIIA-SS group. TSNIIA-SS treatment not only improved DXR lesion identified by histochemical stain, but also regulated the expression of several proteins related with the cytoskeleton, oxidative stress and protein synthesis or degradation detected by two-dimensional electrophoresis (2-DE). These data have provided the evidence that TSNIIA-SS is a protective agent against DXR-induced nephropathy.