Study On The Therapeutic Effect And Mechanism Of Attenuated Salmonella Typhimurium VNP2000920009 On Acute Leukemia

Acute leukemia due to rapid onset,drug resistance,easy relapse,more difficult to treat after relapse,high mortality,is the most common disease type of leukemia death.With the improvement of drug and risk classification management in recent decades,the treatment of pediatric acute leukemia has made great progress.However,the prognosis of adults,especially elderly patients with acute leukemia,is poor,and many patients relapse into treatment failure and eventually die.To date,chemotherapy is still the main treatment strategy for leukemia.Bone marrow transplantation(BMT)or hematopoietic stem cell transplantation(HSCT)is usually considered when the chemotherapy fails or leukemia relapses.However,chemotherapy has strong side effects and is prone to drug resistance relapse,which often leads to treatment failure and death.BMT or HSCT also have problems such as difficulty in finding donors,high surgical cost,and unbearable pain during surgery,and there is still a certain risk of recurrence.Therefore,it is of great significance to search new therapeutic drugs with less side effects,reliable efficacy and economy.Clinical reports of spontaneous remission after severe bacterial infection in many patients with acute leukemia,combined with current highly concerned bacterial therapy for cancer,suggest that bacterial therapy may be a new direction for the treatment of acute leukemia.Among a variety of bacteria used in tumor therapy research,Salmonella has been widely used in various solid tumor therapy due to its good tumor targeting and anti-tumor effect.However,the treatment of blood tumors by salmonella has not been reported.Genetically engineered attenuated Salmonella typhimurium VNP20009 produces a 50,000-fold reduction in endotoxin toxicity,and its tolerance and safety have been demonstrated in phase I clinical trials,making it an ideal bacterial strain for the treatment of acute leukemia.This study is the first to evaluated the therapeutic effect of VNP20009 and explore its action mechanism on acute leukemia using mouse acute lymphocyte leukemia(ALL)L1210cell or human acute myeloid leukemia(AML)cells HL-60 subcutaneous transplantation tumor models,and a systemic AML mouse model driven by human AML fusion protein MLL-AF9,respectively.The main contents are as follows:1.Bacteria-cell co-culture experiment results in vitro showed that attenuated Salmonella typhimurium VNP20009 inhibited the proliferation and induced apoptosis of various acute leukemia cells with dose and time effects.2.The experimental results showed that VNP20009 could inhibit the growth of subcutaneous xenograft tumors of acute leukemia.Results from Hematoxylin&Eosin(H&E)staining and(Terminal deoxynucleotidyl transferase(Td T)d UTP Nick-End Labeling,TUNEL)immunofluorescence for paraffin sections of tumor tissues showed that VNP20009 could significantly induce apoptosis of tumor cells,which lead to the degradation of DNA fragments,the fragmentation of cell nuclei and the disintegration of cell membrane,and extensive tissue necrosis in tumoral central region.Western Blot analysis showed that VNP20009 up-regulated the expression levels of proapoptotic proteins Bax,Cleaved-caspase-3 and Cleaved-PARP,and induced apoptosis of acute leukemia cells.3.Treatment results of AML mice driven by MLL-AF9 showed that VNP20009 significantly inhibited the proliferation of AML cells,reduced and restored the counts of white blood cells(WBCs)and its five subsets in peripheral blood of AML mice to near normal physiological values,and prolonged the survival of the AML-carrying mice.4.The detection results of serum cytokines&chemokines and flow cytometry showed that VNP20009 could kill and inhibit the proliferation of leukemia cells by up-regulating the anti-tumor factor interferon-γ(IFN-γ),tumor necrosis factor-α(TNF-α)and leukemia inhibitory factor(LIF)secretion and down-regulating the secretion of granulocyte-macrophage colony-stimulating factor(GM-CSF).Moreover,by up-regulating the secretion of chemokines C-X-C motif ligand-10(CXCL-10)and C-C motif ligand-2(CCL-2)to recruit and activate the proliferations of natural killer cells and CD4~+dominated T lymphocytes,the proliferation of leukemia cells can be killed and inhibited.VNP20009 can further stimulate the polarization of T lymphocytes to CD4~+IFN-γ~+and CD8~+IFN-γ~+effector T lymphocytes,which release IFN-γto kill leukemia cells directly and further maintain and enhance the body’s anti-tumor immunity to inhibit the proliferation of leukemia cells.Conclusion:Attenuated Salmonella typhimurium VNP20009 could significantly inhibit the proliferation of leukemia cells and promote their apoptosis,playing a role in the treatment and delay of leukemia.On the one hand,after entering the body,VNP20009 could induce the secretion of various cytokines and chemokines and the proliferation of various immune cells to enhance the body’s anti-tumor immunity,inhibit and kill leukemia cells,and prolong the survival period of MLL-AF9-induced AML mice.On the other hand,increased expressions of pro-apoptotic proteins Bax,Cleaved-caspase-3 and Cleaved-PARP in transplanted tumor tissues suggested that acute leukemia cell apoptosis might be induced by mitochondrial apoptosis after VNP20009 infection.This study provides a new strategy and theoretical basis for the treatment of acute leukemia.