Research On The Antitumor Effect And The Related Mechanisms By Pneumolysin Mutant ?A146Ply

Backgroud: Increasing evidences show that microbiome-based therapy has unique advantages in metabolic,infectious and malignant diseases.However,there are certain concerns about the safety of using live microorganisms as therapeutic agents,which may cause infection or even fatal infection.The inherent heterogeneity of microbial agents makes it difficult to guarantee its effect.Exploring more safe and effective microbiome-based therapy is a hot and difficult point in the field of microbial drugs.Microbe-derived bioactive components can exert effective therapeutic effects and may represent a more controlled,reproducible and reliable therapeutic strategy.It is of great clinical value to indentify single bioactive component with therapeutic potential and explore its related mechanisms.?A146Ply is a mutant of pneumolysin(Ply)without hemolysis activity.Unexpectedly,our group found that the combination of ?A146Ply and berbamine(BBM)significantly inhibited the progression of triple negative breast cancer(TNBC).?A146Ply alone significantly inhibited the migration and invasion ability of TNBC cells.In mouse model ?A146Ply inhibited the metastasis of TNBC cells.These findings suggest that?A146Ply has therapeutic potential against TNBC,but the related molecular mechanisms remain unclear.Objective: This study aims to explore the related mechanisms by which ?A146Ply inhibits the migration and invasion ability of TNBC cells,which may provide a new option for the treatment of TNBC.Methods: Scratch and Transwell assay were used to evaluate the effect of ?A146Ply on the migration and invasion ability of human TNBC cells MDA-MB-231 and mouse TNBC cells py8119 and 4T1.Western blot assay was used to detect the expression of transforming growth factor ?1(TGF-?1),mammalian target of rapamycin(m TOR)signaling,autophagy and epithelial-mesenchymal transition(EMT)-associated proteins.Enzyme-linked immunosorbent assay(ELISA)was used to detect the level of TGF-?1 in the culture supernatant of TNBC cells.Flow cytometry assay was used to detect the expression of toll-like receptor 4(TLR4)and mannose receptor(MR)on the surface of TNBC cells.Immunofluorescence assay was used to detect the bound of ?A146Ply with TLR4 and MR.si RNA was transfected to knock down the expression of TLR4 and MR on TNBC cells.Results:1.?A146Ply inhibited the migration and invasion of MDA-MB-231,py8119,and 4T1 cells: In MDA-MB-231,py8119,and 4T1 cells,after?A146Ply treatment the width of the scratch was greater in ?A146Ply group than in the negative control group.Futhermore,transwell assay showed that the migrating and invading TNBC cells were significantly less in ?A146Ply group than in the negative control group(p < 0.001).2.?A146Ply inhibited EMT of MDA-MB-231,py8119,and 4T1 cells:In MDA-MB-231 cells,after ?A146Ply treatment the expression of epithelial marker E-cadherin increased,while the expression of mesenchymal markers N-cadherin,vimentin,and ZEB1 decreased at 48 h stimulation.In py8119 and 4T1 cells,after ?A146Ply treatment the expression of E-cadherin increased,while the expression of N-cadherin,vimentin,and SNAIL decreased at 48 h stimulation.3.?A146Ply inhibited EMT of MDA-MB-231 and py8119 cells through inhibiting TGF-?1 expression: After ?A146Ply treatment,TGF-?1level in the culture supernatant of MDA-MB-231 significantly decreased(p< 0.01).With the addition of TGF-?1 protein,?A146Ply failed to up-regulate E-cadherin and down-regulate N-cadherin,vimentin expression,and it failed to inhibit the migration and invasion ability of MDA-MB-231 cells.After ?A146Ply treatment,TGF-?1 expression in py8119 cell was decreased.With the addition of TGF-?1 protein,?A146Ply failed to down-regulate vimentin and SNAIL expression,and its effect on the migration and invasion ability of py8119 cells was dampened.4.?A146Ply inhibited TGF-?1 expression of MDA-MB-231 and py8119 cells through inhibiting autophagy: After ?A146Ply treatment,p62 levels in MDA-MB-231,MDA-MB-453,py8119,and 4T1 cells increased.With the addition of rapamycin,an autophagy inducer,?A146Ply failed to inhibit TGF-?1 secretion,up-regualte E-cadherin,down-regulate N-cadherin and vimentin expression,and inhibit the migration and invasion of MDA-MB-231 cells.With the addition of rapamycin,?A146Ply failed to inhibit TGF-?1 expression,up-regualte E-cadherin,and down-regulate N-cadherin and vimentin expression,and its effect on the migration and invasion ability of py8119 cells was dampened.5.?A146Ply inhibited autophagy of MDA-MB-231 and py8119 cells through activating m TOR signaling: After ?A146Ply treatment,p-Akt,p-ERK,and p-m TOR levels in MDA-MB-231,MDA-MB-453,py8119,and 4T1 cells were increased.With the addition of MK2206,a small molecule inhibitor of Akt,and U0126,a small molecule inhibitor of ERK,?A146Ply failed to increase p-m TOR and p62 levels in MDA-MB-231 and py8119 cells.6.?A146Ply activated m TOR signaling through activating TLR4 and MR receptors: MDA-MB-231,MDA-MB-453,py8119,and 4T1 cells expressed both TLR4 and MR,and ?A146Ply directly bond to TLR4 and MR receptors on the surface of MDA-MB-231 and 4T1 cells.After TLR4 and MR knock down with si RNA,?A146Ply failed to phosphorylate m TOR and inhibit the migration and invasion of MDA-MB-231 cells.After TLR4 and MR blockade with TLR4 antibody and mannose,?A146Ply failed to inhibit the migration and invasion of MDA-MB-231 cells.Conclusion: The work in this part demonstrated that ?A146Ply inhibited the migration and invasion of TNBC cells through sequentially activating TLR4 and MR receptor,activating m TOR signaling,inhibiting autophagy,inhibiting TGF-?1 expression,and inhibiting EMT.Our work provides a new theoretical and experimental basis for MR downstream signaling pathway and a new option for TNBC therapy.Backgroud: The work in the first part has demonstreated that?A146Ply exerts anti-TNBC effect through activating TLR4 and MR receptor.Therefore,we speculated that ?A146Ply can directly act on other tumor cells expressing TLR4 or MR.Acute myeloid leukemia(AML)is the most common type of leukemia with high mortality in adults.Previous studies have shown that TLR4 and MR receptors were highly expressed on myeloid cells,suggesting that ?A146Ply may act directly on AML cells through TLR4 and MR receptor.Objective: In this part we aim to explore the effect of ?A146Ply on AML cells and the related mechanisms,which will provide a new option for the treatment of AML.Methods: Flow cytometry assay was used to detect TLR4 and MR expression on the surface of human AML cells KG–1 and mouse AML cells C1498 and the effect of ?A146Ply on apoptosis and the cell cycle of KG-1and C1498 cells.Western blot analysis was used to detect the expression of apoptosis,autophagy and m TOR signaling-associated proteins.ELISA was used to detect tumor necrosis factor ?(TNF-?)levels in the culture supernatant of KG-1 and C1498 cells.Immunofluorescence assay was used to detect microtubule-associated protein 1 light chain 3(LC3)puncta in KG-1 and C1498 cells.Cell counting kit 8(CCK8)was used to detect cell proliferation.Transwell assay was used to evaluate the effect of ?A146Ply on the migration and invasion of KG-1 cells.Results:1.?A146Ply induced apoptosis of KG-1 and C1498 cells: TLR4 and MR were expressed on the surface of KG-1 and C1498 cells,and?A146Ply treatment did not affect TLR4 and MR expression.After?A146Ply treatment,the percentages of apoptotic KG-1 and C1498 cells significantly increased(p < 0.001,p < 0.001).2.?A146Ply inhibited autophagy of KG-1 and C1498 cells: After?A146Ply treatment,anti-apoptotic protein B cell lymphoma 2(BCL-2)expression in KG-1 and C1498 cells increased,and TNF-? levels in the culture supernatant of KG-1 and C1498 cells did not increase.p62 levels in?A146Ply-treated KG-1 and C1498 cells increased,and LC3 puncta decreased.3.?A146Ply inhibited autophagy of KG-1 and C1498 cells through activating m TOR signaling: p-Akt,p-ERK,and p-m TOR leves in?A146Ply-treated KG-1 and C1498 cells increased.With the pretreatment of MK2206 and U0126 ?A146Ply failed to phosphorylate m TOR and accumulate p62 levels in KG-1 and C1498 cells.4.?A146Ply induced apoptosis of KG-1 and C1498 cells through inhibiting autophagy: With the pretreatment of MK2206 and U0126,?A146Ply failed to increase intracellular cleaved caspase 3(CC3)levels and induce apoptosis of KG-1 and C1498 cells.5.The combination of ?A146Ply and other autophagy inhibitors synergistically exerts anti-leukemia effect: The combination of ?A146Ply and chlorquine(CQ)synergistically inhibited proliferation and induced apoptosis of KG-1 and C1498 cells.The combination of ?A146Ply and berbamine(BBM)or CQ synergistically inhibited proliferation and induced apoptosis of THP-1,Raw264.7,and K562 cells.6.The combination of ?A146Ply and CQ synergistically inhibited autophagy of KG-1 and C1498 cells: In KG-1 and C1498 cells,p-Akt,p-ERK,LC3,and p62 levels were higher in the combination group than in the single ?A146Ply or CQ group.Conclusion: The work in this part demonstrated that ?A146Ply induced apoptosis of KG-1 and C1498 cells through activating m TOR signaling and inhibiting autophagy.The combination of ?A146Ply and CQ synergistically inhibited autophagy of KG-1 and C1498 cells,inhibited their proliferation,and induced their apoptosis.Furthermore,the combination of ?A146Ply and CQ or other autophagy inhibitor BBM synergistically inhibited proliferation and induced apoptosis of THP-1,Raw264.7,and K562 cells.This work provides a new option for the adjuvant therapy of AML.Taken together,our work demonstrated the molecular mechanisms by which ?A146Ply inhibited the migration and invasion of TNBC cells and induced the apoptosis of AML cells,which will provide new theoretical and experimental basis for MR downstream signaling pathway and valuable experimental basis for the development of ?A146Ply as an anti-tumor agent.