Anti-tumor Function And Mechanism Of Berberine In Diffuse Large B-cell Lymphoma

Non-Hodgkin lymphoma（NHL）is the most common hematological malignancy,in which diffuse large B-cell lymphoma（DLBCL）accounts for more than forty percent of newly diagnosed NHL in the adult.Although the immunochemotherapy based on rituximab and systematic chemotherapy has significantly improved the prognosis of DLBCL patients,about forty percent of patients presented limited clinical response and became refractory and relapsed.The second-line chemotherapy and salvage therapy could enhance the prognosis of some refractory or relapsed DLBCL（r/r DLBCL）patients.But,because of high heterogeneity,parts of r/r DLBCL patients died from tumor progression and recurrence.Therefore,it is urgent and significant to explore novel biomarkers and signal pathways in DLBCL and find new targeted agents in DLBCL treatment.Cluster of differentiation 47（CD47）is a member of the immunoglobulin superfamily and known as integrin-associated protein（IAP）.CD47 is widely expressed on normal tissue and generally upregulated in tumor tissue.The interaction between overexpressed CD47 on tumor cells and SIRP-α on innate immune cells,especially macrophages,activates a "don’t eat me" signal cascade mediating the inhibition of phagocytosis activity of innate immune cells and inducing immune escape of tumor cells.Thus,targeting CD47 is a novel strategy for tumor immunotherapy.Recently,many preclinical studies showed that blocking CD47-SIRP-α axis by anti-CD47 antibody could inhibit tumor growth.A phase I clinical trial showed that 6 out of 15 r/r DLBCL patients obtained partial remission or complete remission by humanized antiCD47 antibody Hu5F9-G4 combined with rituximab.Because of the small sample size,the clinical efficacy of Hu5F9-G4 needs to be proved in later clinical trials.Although there have been more than 10 years of research on CD47-targeted therapy in DLBCL,the role of CD47 in the development and progression of DLBCL and novel CD47targeted agents are worthy of further investigation.Berberine hydrochloride,also known as berberine,is the major effective component of Chinese herb called coptis chinensis.Berberine is a kind of quaternary amine isoquinoline alkaloid with stable chemical structure and is traditionally used in intestinal infection.Recently,berberine is proved with application prospects in the treatment of diabetes,cardiovascular disease and tumor.Berberine exerts broad antitumor activities in multiple tumors,including colorectal cancer,esophageal cancer,cervical cancer,prostate cancer,liver cancer and leukemia.Induction of cell cycle arrest,apoptosis,senescence,autophagy and epigenetic regulation is accounting for its antitumor mechanisms.Furthermore,it enhances the anti-tumor efficacy of 5-fluorouracil,doxorubicin,tamoxifen and sorafenib.However,most reported studies revealed the anti-tumor activity of berberine in solid tumors,few studies investigated the anti-tumor function of berberine in lymphoma.Moreover,its regulatory activity in the immune escape of tumor cells is unclear.This study researched the role of CD47 in the occurrence and progression of DLBCL and investigated the biological function and involved mechanism of berberine targeting the expression of CD47.Part Ⅰ Expression and Biological Function of CD47 in Diffuse Large B-cell LymphomaIntroduction:Diffuse large B-cell lymphoma（DLBCL）is a kind of invasive non-Hodgkin lymphoma（NHL）with high heterogeneity.Although rituximab has dramatically improved the treatment effect of DLBCL,about 40%of patients presented limited clinical response or relapsed after standard first-line immunochemotherapy.Therefore,it is of vital importance to explore new molecular regulatory mechanisms involved in the occurrence and progression of DLBCL and discover novel potential therapeutic targets and therapeutic strategies in the treatment of DLBCL.Previous studies demonstrated that CD47 was overexpressed in multiple tumor cells.The interaction between CD47 and SIRP-α on macrophages activated an inhibitory "don’t eat me"signal pathway to mediate the immune escape of tumor cells.Abnormal overexpression of CD47 was related with the poor prognosis of breast cancer,thyroid cancer and colorectal cancer.However,the relationship between CD47 and the prognosis of DLBCL patients is still unclear.In this study,the expression of CD47 in DLBCL was detected and the relationship between CD47 and the prognosis of DLBCL patients was explored.Moreover,the biological function of CD47 in the immune escape of DLBCL cells was studied.Material and Methods:1 Obtain of specimen from DLBCL and reactive hyperplasia lymphoid（RHL）patients and immunohistochemistry;2 Obtain of clinical information from DLBCL patients and survival analysis;3 DLBCL cell lines and cell culture;4 Obtain of peripheral blood from healthy donors and separation of peripheral blood mononuclear cells（PBMCs）;5 Flow cytometry;6 RNA isolation,cDNA synthesis and RT-qPCR;7 Cell total protein isolation and western-blot;8 siRNA synthesis and DLBCL cell transfection;9 Lentivirus vector-mediated knockdown of CD47;10 Isolation of CD 14 positive monocytes and induction of macrophages;11 Labeling DLBCL cells with CFSE;12 Co-culture of macrophages with DLBCL cells and detection of phagocytosis ratio;13 Statistical analysis of data.Results:1 The expression of CD47 protein was detected by immunohistochemistry in paraffin sections from 55 newly diagnostic DLBCL patients and 40 RHL patients.Results showed that CD47 was positively stained on cytomembrane.The expression of CD47 was significantly higher in DLBC compared to RHL.CD47 was positively stained in 30 out of 55 DLBCL patients and 11 out of 40 RHL patients（54.55%vs 27.50%,P<0.05）.2 Thirty-eight out of 55 DLBCL patients were followed up.Expression of CD47 was related with the subtype of DLBCL（P=0.007）,but not with age,gender,NCCN IPI score,Ann Abor stage and serum LDH level.The majority of CD47 positive patients were non-GCB.Kaplan-Meier analysis showed that patients with positive expression of CD47 have worse overall survival（OS）.3 The expression of CD68 protein was detected by immunohistochemistry in paraffin sections from 38 followed-up DLBCL patients and the number of CD68 positive macrophages was counted under high power field（HPF）.The number of CD68 positive macrophages per HPF was not related with age,gender,NCCN IPI score,Ann Abor stage,subtype or serum LDH level.Kaplan-Meier analysis showed no relation between OS and the number of CD68 positive macrophages per HPF.4 Thirty-eight DLBCL patients can be divided into 4 subgroups by CD47 and CD68.They are CD47negative/CD68high,CD47negative/CD68low,CD47positive/CD68low and CD47positive/CD68low.Kaplan-Meier analysis showed that patients from CD47positive/CD68low subgroup had relatively worse OS than others.5 The expression of total CD47 protein and cytomembrane CD47 were detected by western-blot and flow cytometry respectively.Results showed that compared with PBMCs,CD47 was overexpressed in DLBCL cell lines,including LY1,LY3,LY8,Val and U2932.6 Three different sequences of CD47 siRNA were transfected into LY1,LY8 and U2932 cells,and western blot showed that CD47 was downregulated by the 3 CD47 siRNAs.The sequence of CD47 siRNA with the best knockdown rate was used to structure the lentivirus.The lentivirus vector-mediated knockdown of CD47 was operated in LY1,LY8 and U2932 cells.Expression of CD47 was significantly decreased in shCD47 cells than shControl cells by western-blot and RT-qPCR.7 CD 14 positive monocytes were isolated from PBMCs by CD 14 positive cell isolation kit.The CD 14 positive monocytes were induced to macrophages by 100ng/ml M-CSF.LY1,LY8 or U2932 cells transfected with shCD47 or shControl were co-cultured with macrophages.Results showed that knockdown of CD47 enhanced the phagocytosis of macrophages.An anti-CD47 antibody called B6H12 or its isotype control IgG was applied in the co-culture system containing macrophages and LY1,LY8 or U2932 cells.Results showed that compared with the isotype control,B6H12 could promote the phagocytosis of macrophages to eliminate DLBCL cells.Conclusions:1 CD47 was overexpressed in DLBCL patients.CD47 level was related with subtype and poor prognosis.The number of CD68 positive macrophages was not related with age,gender,NCCN IPI score,Ann Abor stage,subtype,serum LDH level,as well as,prognosis.DLBCL can be divided into 4 subgroups by the combination of CD47 and CD68,in which patients from CD47positive/CD68low had relatively worse prognosis than the others.Overall,CD47 was a potential biomarker of DLBCL and the combination of CD47 and CD68 has the potential to be a novel prognostic model.2 CD47 was overexpressed in DLBCL cell lines.CD47 knockdown or blocked by antibody could promote the phagocytosis activity of macrophages.Hence,CD47 mediated the immune escape of DLBCL cells from the phagocytosis of macrophages.Part Ⅱ Berberine Exerts Anti-tumor Activity in Diffuse Large B-cell Lymphoma via Targeting CD47Introduction:Berberine possesses broad anti-tumor activity,which can inhibit the proliferation and metastasis of tumor cells by induction of cell senescence,apoptosis and cell cycle arrest and modulation of autophagy of tumor cells.Based on the regulation of tumorintrinsic signal pathway,berberine could induce radiotherapy sensitization and enhance the anti-tumor activity of chemotherapy drugs such as 5-fluorouracil,doxorubicin,tamoxifen and sorafenib.A recent study showed that berberine exerted immunomodulatory activity by regulating the expression of PD-L1 in lung cancer,indicating the application potential of berberine in tumor immunotherapy.However,few studies worked on the anti-tumor activity of berberine in DLBCL,and no study has explored the regulation of berberine on CD47,an innate immune checkpoint protein.To explore the regulation and biological function of berberine on the expression of CD47 in DLBCL,DLBCL cells were treated with berberine.Furthermore,the effects of berberine on the phagocytosis activity of macrophages in the co-culture system and the tumor growth in the tumor-bearing mice model were detected.Furthermore,the additive function of berberine to rituximab and anti-CD47 antibody was explored.Material and Methods:1 DLBCL and mice B cell lymphoma cell culture and drug treatment;2 CCK8 assays to detect the cell viability;3 Flow cytometry;4 RNA isolation,cDNA synthesis and RT-qPCR;5 Protein isolation and western-blot;6 Obtain of peritoneal macrophages of mice;7 Isolation of CD 14 positive monocytes and induction of macrophages;8 Labeling lymphoma cells with CFSE;9 Co-culture of lymphoma cells and macrophages;10 Lentivirus vector mediated overexpression of c-myc;11 Establishment of tumor bearing mice model and berberine oral gavage;12 Frozen section and immunofluorescent staining;13 Statistical analysis of data.Results:1 CCK8 assay showed that berberine inhibited the proliferation of DLBCL cell lines including LY1,LY8 and U2932 in a dose-dependent manner.The half-maximal inhibitory concentration（IC50）of LY1,LY8 and U2932 cells were 46.07μM,40.40μM,42.27μM respectively.2 Western-blot and flow cytometry showed that 30μM berberine could downregulate the expression of CD47 in LY1,LY8 and U2932 cells in a time-dependent manner.In the co-culture system consisting of macrophages and berberine-treated LY1 or U2932 cells,compared to the control group,the phagocytosis ratio was promoted in the berberine group.3 30μM berberine downregulated the expression of CD47 in mice B-cell lymphoma cell line called A20 in a time-dependent manner.Berberine promoted the phagocytosis activity of mice peritoneal macrophages to engulf A20 cells.Immunocompetent BALB/c mice and A20 cells were used to establish the tumorbearing mice model and berberine or sodium carboxymethylcellulose solution oral gavage was operated.Results showed that berberine gavage significantly inhibited tumor growth and final tumor weight.Immunofluorescent staining of the tumor mass sections showed that berberine downregulated the expression of CD47 and promoted the infiltration of F4/80 positive macrophages in the tumor tissue.4 In the regulatory mechanism,berberine inhibited the expression of CD47 mRNA in LY1,LY8 and U2932 cells,indicating that berberine modulated CD47 at the transcriptional level.Meanwhile,we found that berberine inhibited CD47 expression accompanied by down-regulation of c-myc expression in LY1 cells.After treating LY1 cells with 10058-F4,a specific inhibitor c-myc,CD47 expression was significantly reduced.On the contrary,after overexpressing c-myc by lentivirus in LY1 cells,the expression of CD47 was significantly increased.Then c-myc overexpressed LY1 cells were treated with berberine,and the results showed that c-myc overexpression could rescue berberine-induced downregulation of CD47.5 We combined berberine and B6H12 or rituximab in the co-culture system consisting of macrophages and LY1,LY8 or U2932 cells.Results showed that compared to single agents,the combination of berberine and B6H12 or rituximab could enhance the phagocytosis activity of macrophages.Conclusions:Berberine induced CD47 downregulation at the transcriptional level via modulating the expression of c-myc,a transcriptional factor of CD47,and enhance the phagocytosis activity of macrophages to engulf DLBCL cells.In vivo,berberine inhibited tumor growth by inhibiting the expression of CD47 and enhancing the infiltration of macrophages.Berberine enhanced the efficacy of anti-CD47 antibody and anti-CD20 antibody.This study revealed a new anti-tumor mechanism of berberine and provide novel insight into the treatment of DLBCL.