Preparation And Preliminary Study On Antitumor Activity Of Prodigiosin From Cockroach Gut Endophytic Serratia Marcescens

Prodigiosin is a kind of red pigment,which has the ability of antibacterial,anti-fungal,anti-malaria,immunosuppression and anticancer.The national cancer institute has reported that prodigiosin have anticancer activity against 57 hunman tumor cells but little toxicity in normal cells.Therefore,prodigiosin may represent a new class of anticancer agents.The Gram-negative bacterium Serratia marcescens(S.marcescens)belongs to Enterobacteriaceae,widely exist in soil,water and air.Most of prodigiosin is isolated from Serratia marcescens and there are other kind of bacterial can product prodigiosin such as Pseudomonas and Actinomycetes.Previously,we have isolated four S.marcescens strains from the gut of periplaneta americana,and one of strain named WA12-1-18 has been proved that it had ability to product prodigiosin steadily.On this basis,we preliminary study the anticancer activity of prodigiosin through cell experiment in vitro and nude mice experiment in vivo.PurposeTo evaluate the anticancer activity of prodigiosin and explore the mechanism of its anticancer effect.Methods(1)The identification of WA12-1-48 isolated from the gut of cockroach in morphology and molecular biology.The morphological characteristics of WA12-1-18 were observed by optical microscope and scanning electron microscope.The homology comparison of 16 s rDNA blast was used to construct N-J phylogenetic tree.(2)The preparation and identification of prodigiosinThe red pigment was producted by solid fermentation and the red pigment monomerwas purified by silica gel column chromatography.The structure of the compound was characterized by HPLC,UV,IR,LC-MS and NMRs.(3)The anticancer effect of prodigiosin in vitroTake Hela,CNE-2,A549 T,MCF-7/ADR cell strains as the experimental object.MTT,flow cytometry,fluorescence staining,Western blot and qRT-PCR were used to detect the anticancer activity of prodigiosin in vitro.(4)The anticancer effect of prodigiosin in vivoAccording to the nude mice bearing Hela tumor,evaluate the anticancer activity of prodigiosin and toxicity effect of heart,liver,spleen,kidney in vivo.Results(1)The identification of WA12-1-48 isolated from the gut of cockroach in morphology and molecular biology.The red strain WA12-1-18 isolated from the gut of cockroach was identified by morphology as Gram-negative Serratia marcescens.Though molecular biological identification,the similarity with the strain from soil sources was 99.58%.The strain WA12-1-18 was identified as Serratia marcescens.(2)The preparation and identification of prodigiosinAfter separation and purification,the red powder was obtained,whose yield was 2.77 g/ L.The pigment was identified as prodigiosin by HPLC,UV,IR,HPLC and NMR.The results of stability test showed that prodigiosin should be stored in acid,the temperature is not more than 60 ℃ and dark place.What’s more,the chelation of individual metal ions is easy to occur.(3)The anticancer effect of prodigiosin in vitroProdigiosin can inhibit the growth of Hela,CNE-2,A549 T and MCF-7 / ADR.It can also change the distribution of tumor cell cycle and induce tumor cell apoptosis.The results of scanning electron microscopy(SEM)and transmission electron microscopy(TEM)showed that prodigiosin could make the cell surface collapse and shrink,caudal foot disappear,vacuole structure and nuclear edge agglomeration.And form partial apoptotic bodies.The results of westernblot and qRT-PCR showed that prodigiosin could inhibit the activation of Hela cell apoptosis gene Bcl-2 and promote the activation of apoptotic gene Bax and induce the activation of downstream caspase-3 and eventually lead to apoptosis of tumor cells.(4)The anticancer effect of prodigiosin in vivoProdigiosin could effectively inhibit the growth of cervical cancer bearing nude mice,and the tumor inhibition rate was 20.60%.The results of HE staining showed that the number of tumor cells in the treated group was significantly reduced,the number of tumor cells was sparse,the nucleus was small and the staining was shallow.Immunohistochemical results showed that prodigiosin could inhibit the expression of Bcl-2 and Bax and had no toxicity on the heart,liver,spleen,lung,kidney and stomach of nude mice.ConclusionWA12-1-18 isolated from the gut of cockroach has the biological properties of stable production of prodigiosin.Prodigiosin texted by cells experiment in vitro and nude mice bearing tumor in vivo have been found to have antitumor activity,which lays a theoretical foundation for the clinical application.