一,Research On Anti-colon Cancer Material Base And Mechanism Of Chinese Traditional Herbs 二,Research On Fatty-acid Binding Proteins In Haemonchus Contortus

Part IResearch on anti-colon cancer material base and mechanism of Chinese traditional herbsMalignant tumor is a serious threat to human health and a leading cause of death from disease. In the past dozens of years, the mobility of cancer has increased by some 22%. Colorectal carcinoma is the fourth most common form of cancer occurring worldwide and a major cause of morbidity and mortality in Western countries. In the year 2000, an estimated 900,000 new cases of colorectal cancer were diagnosed worldwide, with an estimated 490,000 deaths. While early stage colorectal cancer is frequently curable with surgery, unresectable metastatic disease is uniformly fatal. At present, treatments for colorectal cancer mainly involve fluorouracil-based chemotherapy and innotecan, and then, more recently, oxaliplatin. However, clinical chemotherapy cases show that these cytotoxic agents can do harm to the health of patients. There are essentially no established treatment options with demonstrated efficacy. Thus, it is urgent to find anti-colon cancer drugs with slender cytotoxity. Recently, natural compounds in Chinese traditional herbs as sources of drug screening raise much attention because of their diversity in structures. At present, many effective anti-cancer drugs are designed and synthesized based on the structure of natural products. Therefore, analysis on the material base of anti-cancer herbs became more and more urgent.According to many references and a lot of clinical cases in China, Oldenlandia (Hedyotis) corymbosa L. is a plant which has definite anti-tumor activity. Moreover, Oldenlandia (Hedyotis) corymbosa L. can also improve the immunity of patients without obvious bad effects on human body. In our previous research, we found that extracts from Oldenlandia (Hedyotis) corymbosa L. could induce apoptosis of several cancer cells, such as Hela, SMMC-7721 and Bcap-37. The extracts also could inhibit the angiogenesis of chick embryo choriollantoic (CAM). Until now, there is no report about the anti-cancer material base of Oldenlandia (Hedyotis) corymbosa L., thus, it is highly likely that new anti-cancer drugs with novel structures could be found in this plant. In this research, we isolated and purified the ingredient compounds of Oldenlandia (Hedyotis) corymbosa L. under a guide of their anti-cancer effects, and consequently identified those with highly anti-cancer effects.Cudrania tricuspidata (Carr.) Bur. is a Chinese traditional medical plant widely used in anti-tumor therapy. Previous researches demonstrated that there are plenty of anti-cancer compounds such as flavonoids in this plant. Isoalvaxanthone (IAX) and Cudratricusxanthone G (CTXG) are xanthones extracted from Cudrania tricuspidata (Carr.) Bur.. Our previous research revealed that both of these two compounds could significantly inhibit in vitro proliferation of colorectal cancer cells HCT-116, hepatocellular carcinoma cells SMMC-7721 and SGC-7901, and also gastric carcinoma cells BGC-823 cells. In this study, we investigated the anti-colon cancer effects of IAX and CTXG and the underlying mechanisms.The paper can be divided into four parts:A. Extraction, isolation and purification of chemical constituents with anti-colon cancer activity in Oldenlandia (Hedyotis) corymbosa L.In this study, we used various chromatography techniques to isolate the ingredients of Oldenlandia (Hedyotis) corymbosa L. We tested the anti-cancer activity of samples from various isolating steps and then extracted the chemical constituents of Oldenlandia (Hedyotis) corymbosa L. step by step from those effective samples. We identified 23 compounds including 3 novel secoiridoid compounds and 20 known compounds using NMR and chromatography technologies.B. Investigation of anti-colon cancer activity of compounds from Oldenlandia (Hedyotis) corymbosa L. and the underlying mechanism.We estimated the anti-colon cancer activity of extracts from Oldenlandia (Hedyotis) corymbosa L. by investigating the effect of samples on cell proliferation, cell cycle, and developed guidance for the isolation of chemical constituents. We also tested the anti-colon cancer activity of the 23 compounds from Oldenlandia (Hedyotis) corymbosa L. and identified 4 compounds with significant effects. Those 4 effective compounds are Quercetin, Hedyotiscone A, P-sitosterol and Syringic acid, with IC₅₀ 104.3μg/ml, 154.2μg/ml, 104.5μg/ml and 112.9μg/ml respectively. We identified Hedyotiscone A as an anti-cancer agent for the first time. We also found that these 4 compounds could significantly induce cell apoptosis. Especially, Hedyotiscone A could inhibit the processing of cell cycle and induce apoptosis in colon cancer cell HCT-8 and RKO. Result of RT-PCR assay revealed that Hedyotiscone A may induce cell apoptosis through Bax signaling pathway.C. Investigation on effects and mechanisms of the anti-colon cancer activity ofIAX.IAX was extracted from medical plant Cudrania tricuspidata (Carr.) Bur. and belongs to a group of small compound termed xanthone. At concentration of 0.1～10μM, IAX could significantly inhibit cell proliferation, achieving the half-maximum effect at～1μM. In addition, IAX exhibits low cytotoxity, with cell viability around 88.9% at 10μM treatment. FACS analysis revealed that IAX could block cell cycle processing at S/G2 control point. Microarray analysis revealed that the effects of IAX refer to genes involved in tumor metastasis, such as Cyclin D1, AKT1, RhoB. Results of wound healing assay and borden chamber assay confirmed that IAX could significantly inhibit cell migration and invasion at 0.1～10μM. We also demonstrated the underlying mechanism of anti-colon cancer effect of IAX by gelatin zymography analysis, GST-Pull Down assay, EMSA assay. Results indicate that IAX may exert its anti-colon cancer effects by inhibiting cell proliferation, migration, invasion through ERK/Rac1/JNK/AP-1 signaling pathways.D. Investigation on effects and mechanisms of the anti-colon cancer activity ofCTXG.Another small xanthone compound extracted from Cudrania tricuspidata (Carr.) Bur., CTXG, has similar structure as IAX. We first identified that CTXG exerted significant inhibitory effects on cell proliferation, migration and invasion at 1～10μM and achieved the half-maximum effect at～1μM. We also investigated the underlying mechanism of its anti-cancer effects. Results reveal that CTXG could exerts its anti-colon cancer effects by inhibiting cell proliferation, migration, invasion through ERK/Racl/JNK/AP-1 signaling pathways.From all the above, we can conclude that:I. Twenty three compounds, including 3 novel compounds, were obtained bybio-function guided isolation from Oldenlandia (Hedyotis) corymbosa L..II. Four compounds out of 23 which exert significant anti-colon cancer effects areQuercetin, Hedyotiscon A, P-sitosterol and syringic acid. III. Quercetin, Hedyotiscon A,β-sitosterol and Syringic acid exert their anti-colon cancer effects by inducing apoptosis. Hedyotiscon A may induce cell apoptosis through Bax signaling pathway.IV. IAX exerts its anti-colon caner effects by inhibiting cell proliferation, migration and invasion through ERK/Rac1/JNK/AP-1 signaling pathway.V. ITXG exerts its anti-colon cancer effects by inhibiting cell proliferation, migrationand invasion through ERK/Rac1/JNK/AP-1 signaling pathway. Part IIResearch on fatty-acid binding proteins in HaemonchuscontortusTwo different classes of small nematode specific lipid binding proteins, the nematode polyprotein allergens/antigens (NPA) and the fatty acid- and retinol-binding (FAR) proteins, are secreted by helminth parasites. Until now, there was no evidence of the expression or secretion of these two families of proteins in H. contortus. In this study, we applied proteomic and bioinformatic tools in an iterative manner to investigate these fatty acid binding proteins in the excretory/secretory products (ESP) of adult H. contortus. Initial examination of the mass spectra of ESP fractions against standard databases returned nine peptides mapping to O. ostertagi NPA and FAR sequences. Searches of the H. contortus EST and genomic contig databases with the O. ostertagi and C. elegans homologues retrieved sequences encoding H. contortus NPA and FAR proteins. The retrieved sequences revealed the diversity of these families in H. contortus, and encouraged further proteomic analysis of ESP fractions. Concurrently, the new sequences were integrated into a customized database and a new search of the mass spectra achieved a 10-fold improvement in coverage of the predicted H. contortus NPAs. The retrieved predicted sequences were also used as search strings for a further search of H. contortus ESTs and genomic contigs and the new sequences were added to the customized database. The final analyses of the mass spectra achieved 49-60% coverage of H. contortus NPAs and 7-47% coverage of H. contortus FARs. Moreover, the structures of the encoding genes were assembled by combining the genomic sequence data with predicted protein sequences confirmed by the peptide evidence. We predict there are at least two hc-npa and six hc-far genes in H. contortus, and life stage expression of the hc-far-1 to -4 genes revealed unique expression patterns for each of these four genes.