Apoptosis Of Acute Myeloid Leukemia Cells Induced By LukS-PV Targeting C5aR

Leukemia is the result of multi-gene,multi-factor and multi-step action.Its common cellular events are blocked apoptosis,impaired differentiation and uncontrolled proliferation.Inducing apoptosis and/or differentiation is an important strategy for the treatment of leukemia.Bacterial toxins,which are specific and cytotoxic to target cells,have become new concerns in the development of anti-cancer drugs.Some toxins have been shown to have anti-tumor activities,such as botulinum neurotoxin A,which has function against prostate cancer and breast cancer,Diphtheria toxin,which has function against adrenocortical carcinoma and glioma,Pseudomonas aeruginosa exotoxin A,which has function against melanoma and pancreatic cancer,etc.It shows that bacterial toxins have potential advantages and good application prospects in tumor therapy.Panton-Valetine leukocidin(PVL)is a two-component toxin secreted by Staphylococcus aureus.It is composed of LukS-PV and LukF-PV subunits.The two subunits can cause perforation dissolution of target cells.Single component LukS-PV has no perforation toxicity,but can specifically bind with target cells and cause a series of signal pathway changes.In the early stage,our group used different concentrations of LukS-PV to act on human acute myeloid leukemia(AML)cells THP-1 and HL-60 respectively.It was found that LukS-PV has anti leukemia activity of inducing leukemia cell apoptosis in a dose and time-dependent manner.Spaan et al reported that complement C5a receptor(C5aR)is the binding site of PVL.The expression of C5aR is tissue and organ specific.It is highly expressed in myeloid cells,but not in lymphocytes and erythrocytes.Recently,it was found that C5aR is highly expressed in hepatocellular carcinoma and non-small cell lung cancer,but not in normal liver and lung tissues.Whether LukS-PV induced leukemia cell apoptosis depends on the expression of C5aR in the target cell membrane and the specific mechanism are unclear.Objective:This study will select cell lines with different expression of C5aR,study the role of C5aR in the apoptosis of acute myeloid leukemia cells induced by LukS-PV through knockdown and overexpression intervention test,further explore the molecular mechanism of apoptosis of myeloid leukemia cells induced by LukS-PV,and study whether C5aR can be used as a molecular marker of LukS-PV targeted therapy.To provide experimental basis for exploring new targets and methods of leukemia treatment.Methods:(1)The apoptosis of leukemia cells induced by LukS-PV and its mechanism were studied by flow cytometry and western blot.1.0 ?mol/L LukS-PV acted on leukemia cell THP-1.After 24 hours,annexin V/PI fluorescence double staining flow cytometry was used to detect the apoptosis rate,JC-1 fluorescence probe was used to detect the mitochondrial membrane potential,colorimetry was used to detect the activity of caspase3,and western blot was used to detect the expression levels of mitochondrial apoptosis related proteins Bak,Bax and Bcl-2.(2)The correlation between apoptosis of leukemia cells induced by LukS-PV and C5aR expression was studied by using patient primary and cell lines.20 hospitalized patients with AML and 20 healthy people were selected as the research objects,and bone marrow primary cells were isolated and cultured.Meanwhile,leukemia cell lines such as THP-1,HL-60 and Jurkat were cultured in vitro.The expression of C5aR mRNA and protein in primary cells and leukemia cell lines were detected by RT-PCR and western blot.Then LukS-PV was used to act on patients' primary cells.The apoptosis rate was detected by flow cytometry to evaluate the correlation between apoptosis induced by LukS-PV and C5aR expression.(3)The localization of LukS-PV and C5aR was observed by laser confocal scanning microscope.Staphylococcus aureus DNA was extracted by boiling method as PCR template,and LukS-PV gene product was amplified.After gel recovery,it was connected with plasmid pMD-18T,and then transformed into E.coli DH5? competent cells.The plasmid was then extracted and identified accurately by PCR and sequencing.After double enzyme digestion,it was connected to pet28a-N6H-GFP vector and transformed into E.coli DH5? competent cells again and introduced into BL21 host bacterial to prepare green fluorescent labeled LukS-PV.The expression products of LukS-PV GFP were analyzed by SDS-PAGE electrophoresis,and the smears of bacterial solution before and after IPTG induction were observed under fluorescence microscope.THP-1 cells were cultured in vitro.After standing and washing,green fluorescent labeled LukS-PV and red fluorescent labeled C5aR antibody were added.After paraformaldehyde was fixed,blocking solution was added.After re-fixation,nuclei were stained by DAPI.The binding of LukS-PV to C5aR was observed under laser confocal scanning microscope.(4)The effect of C5aR expression on apoptosis induced by LukS-PV was studied by knocking down C5aR in acute myeloid leukemia cells(THP-1)with high expression of C5aR.After THP-1 cells were cultured in vitro to logarithmic growth stage,they were transferred to the lentivirus vector knockdown C5aR and hU6-MCS-Ubiquitin-EGFP-IRES-puromycin control lentivirus vector respectively.RT-PCR and western blot were used to observe the knockdown effect of C5aR,and then 1.00 ?M LukS-PV cells were added.After 24 hours,AnnexinV-PE/7-AAD fluorescence double staining flow cytometry,JC-1 fluorescence probe mitochondrial membrane potential,Rhod-2 AM fluorescence probe intracellular calcium content,caspase3 activity,RT-PCR and western blot were used to detect change of gene and protein of mitochondrial apoptosis related Bak,Bax,Bcl-2,Bcl-x and the phosphorylation of p38 protein and reflect the experimental indexes related to apoptosis in each group.(5)To study the effect of C5aR expression on apoptosis induced by LukS-PV,we overexpressed C5aR in acute lymphoblastic leukemia cells(Jurkat)without C5aR expression by lentivirus vector.Jurkat cells were cultured in vitro to logarithmic growth stage,and transferred into lentivirus vector overexpressing C5aR and Ubi-MCS-3FLAG-SV40-EGFP-IRES-puromycin control lentivirus vector pretreatment cells.RT-PCR and western blot were used to observe the overexpression effect of C5aR,and then 1.00?M LukS-PV cells were added.After 24 hours,AnnexinV-PE/7-AAD fluorescence double staining flow cytometry,JC-1 fluorescence probe mitochondrial membrane potential,Rhod-2 AM fluorescence probe intracellular calcium content,caspase3 activity,RT-PCR and western blot were used to detect change of gene and protein of mitochondrial apoptosis related Bak,Bax,Bcl-2,Bcl-x and the phosphorylation of p38 protein and reflect the experimental indexes related to apoptosis in each group.Results:(1)After LukS-PV acted on THP-1,the apoptosis rate increased from 11.2%±1.15%to 46.6%±2.11%,the proportion of cells with decreased mitochondrial membrane potential increased from 0.82%±1.35%to 83.2%±2.16%,the activity of caspase3 increased about twice,the expression of pro-apoptotic proteins Bak and Bax increased,and the expression of anti-apoptotic protein Bcl-2 decreased.These results suggest that LukS-PV can induce leukemia cell apoptosis through mitochondrial pathway.(2)RT-PCR showed that the expression of C5aR mRNA in bone marrow primary cells of AML patients was higher than that of healthy control group,p<0.05.The apoptosis rate of AML primary cells induced by LukS-PV was positively correlated with the expression level of C5aR,R2=0.729.Meanwhile,RT-PCR showed that the expression of C5aR mRNA on the surface of THP-1 cells was the highest,while Jurkat cells did not express C5aR.(3)Laser confocal scanning microscopy showed that green fluorescent labeled LukS-PV and red fluorescent labeled C5aR antibody were co-located on the surface of THP-1 cells,suggesting that LukS-PV can bind to C5aR.With the extension of culture time,no green fluorescence of LukS-PV was observed in the cytoplasm.It is speculated that LukS-PV does not enter the cytoplasm,which may play a biological effect mediated by C5aR.(4)After THP-1 cells were transferred into C5aR knockdown lentivirus vector,the gene and protein expression levels of C5aR?were significantly lower than those in the control group.After adding LukS-PV to THP-1 cells,compared with the group without C5aR knockdown treatment,the apoptosis rate of cells in C5aR knockdown group decreased significantly,from 28.6%±1.76%to 13.69%±1.75%,the proportion of cells with elevated mitochondrial membrane potential increased from 59.87%±3.01%to 85%±2.1%,the concentration of calcium ions in cytoplasm decreased from 61.8%±3.23%to 25.6%±1.89%,the activity of caspase3 decreased by about 1/3.The gene and protein expression levels of Bak,Bax and p38 phosphorylation decreased significantly,and the gene and protein expression levels of Bcl-2,Bcl-x increased significantly.(5)After Jurkat cells were transferred into lentivirus vector overexpressing C5aR,the gene and protein expression levels of C5aR were significantly higher than those in the control group.Compared with the group without C5aR overexpression,the apoptosis rate of cells in C5aR overexpression group increased significantly from 10.58%±1.25%to 25.7%±1.77%,the proportion of cells with elevated mitochondrial membrane potential decreased from 85.4%±1.51%to 59.53%±4.11%,and the concentration of calcium ion in cytoplasm increased from 16.9%±2.14%to 34.1%±3.55%,the activity of caspase3 increased about twice.The gene and protein expression levels of Bak,Bax and p38 phosphorylation.increased significantly,and the gene and protein expression levels of Bcl-2,Bcl-x decreased significantly.Conclusion:(1)LukS-PV can induce apoptosis of acute myeloid leukemia cells through p38 mitochondria/endoplasmic reticulum pathway apoptosis signal pathway.(2)LukS-PV induces apoptosis of acute myeloid leukemia cells through C5aR,and its effect is positively correlated with the expression of C5aR.(3)LukS-PV is co-located with C5aR in the cell membrane.(4)C5aR must be involved in the apoptosis of acute myeloid leukemia cells induced by LukS-PV.(5)C5aR may become a potential target for the treatment of acute leukemia and can be used as a molecular marker of LukS-PV targeted therapy.Significance:This study proves for the first time that LukS-PV induces apoptosis of acute myeloid leukemia cells through C5aR.LukS-PV may have potential therapeutic effect on tumors with high expression of C5aR and is expected to become a new molecular targeted drug.