Mining Of The Secondary Metabolites From Microorganisms And Study On The Roles Of PLEK2 In Colorectal Cancer

This dissertation consists of two parts:one is mining of the secondary metabolites from microorganisms,the other one is study on the roles of PLEK2 in the proliferation and metastasis of colorectal cancer cells.Natural products from microorganisms are a prolific source of drugs.Many antibiotics,antifungal agents,anticancer drugs in clinic are derived from microbial natural products.However,there is an urgent need to develop new drugs with the emergence of multi-drug resistance.Mining structurally diverse bioactive metabolites from rich microbial sources can provide lead compounds for drug research and development.Strategies to mine natural products from microbes mainly include investigation of unexplored microbial sources,alteration of culture conditions and genome mining.In the first part of this dissertation,secondary metabolites from Streptomyces sp.XZQH130E,Amycolatopsis alba DSM 44262,Amycolatopsis mediterranei S699?rifA,Lysobacter capsici DSM 19286 and Lysobacter antibioticus DSM 2044 were systematically investigated.After Medium screening,small molecule elicitation or genetic manipulation,we carried out large-scale fermentation of strains.The fermentation products were isolated with the methods of sephadex LH-20,MPLC,TLC and HPLC.The compounds were elucidated by UV,IR,MS,NMR and X-ray single crystal diffraction.A total of 58 pure compounds were identified,including 24 new ones,and bioactivities of several compounds were studied.In order to exploit the diversity of post-PKS modifications in the biosynthesis of ansatrienins,we carried out systematical isolation of the fermentation products of mutant strain XZQH130E?astC.Eleven ansatrienins(1-11)without the N-cyclohexanoyl D-alanyl side chain but with diverse post-PKS modifications were obtained,including 7 new ones(1-7).And 13 other compounds(12-24)were obtained,such as cyclic dipeptide(12-16),phenylthiazoline(17),indole(18),alkaloids(19-20)and so on,including 3 new ones.The results showed that deleting the side chain facilitates the structure diversity of ansatrienins and is critical in determining their structure-activity relationship.Compounds 1-7 exhibited no evident activities against tested microbes and human cancer cell lines.Compound 3 showed modest inhibitory activity against the secretion of the type three secretion system(T3SS)SPI-1 effectors SipA/B/C/D.Two known ansatrienins without hydroxylation in benzene ring,namely trienomycin G(25)and trienomycin A(26)were isolated from mutant strain XZQH130E?astF2.The results confirmed that hydrolase gene astF2 is responsible for the hydroxylation of C-19 in ansatrienins,laying the foundation for the biosynthesis of ansatrienins.Amycolatopsis alba DSM 44262,which belongs to rare actinomycetes,was a potential ansamycin-producer based on bioinformatic analysis of the whole genome sequence.After medium screening,we isolated 5 ansacarbamitocins(27-31)and 2 new abscisic acid-type sesquiterpenes(32-33)from fermentation products on 80 L waksman agar media.Compounds 27-33 showed no evident activity against tested pathogenic bacteria.Amycolatopsis mediterranei S699 is a well-known rifamycins-producer.In the past years,no other types of secondary metabolites were isolated from the strain.In our study,ten new pentangular polyphenols,namely amexanthomycins A-J(40-49)were isolated from S699?rifA on YMG agar media.The mutant strain was constructed by deleting the polyketide synthase genes responsible for the biosynthesis of rifamycins.Compounds 40-42 showed inhibitory activity against human DNA topoisomerases ??.Bioinformatic information analysis showed that DSM 44262 and S699 contains multiple biosynthetic gene clusters(BGCs).In order to exploit more secondary metabolites,genetic manipulation and small molecule elicitation were applied in DSM 44262?a6bmP and S699?rifA?PKS,which was knocked out ansacarbamitocins and amexanthomycins,respectively.A total of 15 mutant strains were constructed by overexpressing positive regulator or exchanging promoter,while no secondary metabolites were overexpressed in screened medium by HPLC detection.Five elicitors(LaCl3·7H2O,ScCl3-6H2O,?-butyrolactones,N-acetylglucosamine(GlcNAc)and sodium butyrate)were applied in the two strains,and 25 mM GlcNAc was capable of inducing secondary metabolite production in DSM 44262?abm9 on MM agar media.Six compounds(34-39)were isolated in our subsequent large-scale fermentation,including a new carbamothioic S-acid derivative(34)and 5 known kigamicins(35-39).Lysobacter capsica DSM 19286 and Lysobacter antibioticus DSM 2044 belongs to gram-negative bacteria.After the screen of culture medium and liquid fermentation,7 known cyclic dipeptides(50-55,15)and 3 known phenazines(56-58)were isolated and elucidated,respectively.The second part of this dissertation is to explore the role and mechanism of PLEK2 in the proliferation and metastasis of colorectal cancer(CRC)cells.CRC is the third most common cancer worldwide,the incidence and mortality of CRC in China is rising rapidly.The pathogenesis of CRC is complex,involving a variety of genetic and epigenetic alterations,and the molecular mechanism is not clear.Searching for new molecules that contribute to colorectal carcinogenesis and exploring the roles and mechanisms of these molecules in tumor progression are of great significance to promote the precise treatment of CRC.Pleckstrin 2(PLEK2)is a member of the pleckstrin family that was first found in platelets and leukocytes,and it was widely expressed in other types of cells.The function of PLEK2 is mainly limited to actin cytoskeleton reorganization and actin-dependent spreading.Gene Expression Omnibus(GEO)data sets analysis and immunohistochemistry(IHC)staining of PLEK2 in human CRC or adjacent normal samples revealed that PLEK2 was upregulated in CRC.Knockdown of PLEK2 in CRC cell lines inhibited cell proliferation,induced cell cycle arrest at G0/G1 phase and apoptosis.PLEK2 knockdown also inhibited CRC cell migration and invasion by wound-healing and transwell assay.Mice injected with shPLEK2 cells inhibited the tumorigenesis compared to the shNC group.Mass spectrometry data analysis in proteomics and RNA-seq analysis showed that TS was downregulated and p21 was upregulated significantly both in mRNA and protein level.However,overexpressing TS or knocking down p21 did not rescue cell biological behavior inhibited by knockdown of PLEK2.These results showed that PLEK2 might act as an oncogene and mediate CRC cell proliferation and metastasis.PLEK2 could be a novel therapeutic target for antitumor drugs.