Mechanism And Effect Of HLJYT On The Inhibition Of Pancreatic Cancer Through Inhibiting The Differentiation Of Tumor Microenvironment M2 Macrophages

Pancreatic cancer is a highly malignant tumor,and effective therapeutics are urgently needed.Chronic inflammation has been proved to play an pivotal role in the progression of pancreatic cancer.On the basis of the classic formual Huang-Lian-Jie-Du-Tang(Coptidis Rhizoma,Scutellaria,Phellodendron,and Gardeniae Fructus),we developed a formual termed as Huang-Lian-Jie-Yi-Tang(HLJYT),which consists of Coptidis Rhizoma,Scutellaria,Phellodendron,Gardeniae Fructus and Bupleurum.HLJYT has been proved to be effective in clinical applications.Our clinical observation showed that the combination of chemotherapy and HLJYT had markedly improved performances of pancreatic cancer patients.In the present study,we examined the effects of HLJYT on pancreatic cancer cell proliferation both in vitro and in vivo,,and also investigated the mechanisms involved in the anti-cancer of HLJYT via microarrays and bioinformatic analyses.Part ?:Effect of HLJYT on the proliferation and migration of pancreatic cancer cells in vitro.Objeetives:Aceording to the theory of traditional Chinese medieine,most patients with pancreatic cancer have "damp heat" as the main syndrome.Clinical.application shows that HLJYT with clearing and detoxifying effects can improve the clinical symptoms of patients with pancreatic cancer.This section aims to investigate the effects of HLJYT on the proliferation and migration of PANC-1 pancreatic cancer cells in vitro.Methods:Medicated sera of HLJYT was prepared for in vitro studies.Methyl thiazolyl tetrazolium(MTT)and wound healing assays were performed to evaluate cell growth and migration.Results:HLJYT inhibited migration of PANC-1 cells,but had no effect on cell proliferation in vitro.Conclusions:This part of the study clarifled that HLJYT had no direct inhibitory effect on cell proliferation,but could inhibit the migration ability of pancreatic cancer cells.Part ?:Effect of HLJYT on tumor growth in vivo by using orthotopic xenograft models.Objectives:This section aims to explore whether HLJYT can affect the growth of pancreatic cancer in vivo by using orthotopof ic xenograft models.Methods:Decoction of HLJYT was prepared for animal experiments.The mouse model of chronic pancreatitis was established by tail vein injection of dibutyltin dichloride(DBTC).The orthotopic xenograft tumor model of pancreatic cancer was established by using PANC-1 pancreatic cancer cells.Luminex technology was used to detect the average fluorescence intensity of orthotopic xenografts.Immunohistochemistry was used to detect the expressions of Ki-67,CD68 and CD163 in pancreatic cancer xenografts.Results:HLJYT inhibited tumor growth in orthotopic xenograft models.This antitumor effect was more significant in pancreatitis models.HLJYT decreased Ki-67 level and repressed infiltration of macrophages in orthotopic xenograft models.Conclusions:This part of the study confirmed that HLJYT could reduce the infiltration of tumor-associated macrophages(TAMs)in tumor tissues,suggesting that HLJYT possibly exerted anti-tumor effects through immune microenvironment recombinant of pancreatic cancer.Part ?:Effect of HLJYT on M2 macrophage differentiation and angiogenesis in tumor microenvironmentObjectives:Studies have shown that tumor-associated macrophages(TAMs)are more similar to M2 macrophages,and cytokines,chemokines,growth factors released are involved in tumor angiogenesis and tumor cell invasion and metastasis.This section aims to investigate the effect of HLJYT on M2 macrophage differentiation and angiogenesis in tumor microenvironment.Methods:Co-culture system of macrophages and PANC-1 cells was established by transwell chambers.The distributions of macrophage markers were analyzed by flow cytometry.The differential expressed genes were detected by microarrays and analyzed by bioinformatics methods.Angiogenesis assay was used to detect the effect of MCM(macrophage conditioned medium)on angiogenesis.Results:Medicated sera of HLJYT inhibited the polarization of MO macrophages towards M2 phenotype(TAM)in vitro.The downstream molecular events responsible for HLJYT-mediated repression on TAM differentiation were detected by microarrays and bioinformatics analyses.Pathways relating to macrophage differentiation,including MAPK,Notch,WNT and JAK/STAT,were identified.Angiogenesis experiments showed that macrophage conditioned medium(MCM)promoted tumor angiogenesis.HLJYT decreased the proportion of CD34 positive cells and microvessel density(MVD)in orthotopic xenograft models.Conclusions:This part of the study demonstrated that HLJYT inhibited M2 phenotype macrophage polarization,leading to remodeling of tumor inflammatory microenvironment,repressed angiogenesis...