Inhibitory Effect Of Mesenchymal Stem Cells Secreting STRAIL Protein On B-cell Acute Lymphoblastic Leukemia

Objective:B-cell acute lymphoblastic leukemia(B-ALL)is a malignant neoplastic disease in which B-lymphoblastic lymphocytes grow abnormally in the bone marrow.It has been shown that tumor necrosis factor-related apoptosis-inducing ligand(TRAIL)can induce apoptosis in leukemic cells without significant cytotoxicity to normal cells.However,TRAIL treatment also has three major limitations:its short in vivo half-life,its poor tumor-targeting efficacy,and resistance to TRAIL monotherapy.Therefore,an approach needs to be found to address these issues.The emergence of mesenchymal stem cells(MSCs)provides a useful tool for this purpose.Based on MSCs as carriers it is possible to improve the stability of TRAIL,prolong its half-life in the blood,specifically deliver TRAIL to the target site and overcome resistance to TRAIL.Based on the above background and theory,this study was conducted to genetically engineer MSCs to continuously express and secrete soluble TRAIL(s TRAIL)protein within the tumor microenvironment.We investigated the inhibitory effect of MSCs secreting s TRAIL protein on B-lymphocytic acute leukemia.Methods:We examined whether our purchased B-ALL cells expressed TRAIL receptor-related m RNA by Q-PCR.And examined the effect of recombinant TRAIL(rh TRAIL)on the proliferation of B-ALL cells.The lentiviral expression vectors p LV-3Flag-TRAIL-Green Puro and p LV-3Flag-N-Green Puro(empty vector control)were constructed by Q-PCR using PCR,enzymatic digestion and ligation,and then verified by sequencing for correctness.The lentiviral expression vector p LV-3Flag-TRAIL-Green Puro was transfected into 293T cells,and the culture supernatant was collected and concentrated by PEG8000.Human umbilical cord tissue-derived MSCs were infected with the concentrated lentivirus and puromycin screened for stable expression of TRAIL.the expression of TRAIL in the constructed MSCs was detected by western blot.the secretion level of TRAIL in the constructed MSCs was detected by ELISA.The stemness and chemotaxis of MSCs to Nalm-6 cells and Sup-b15 cells after lentiviral infection were examined by inducing MSCs to differentiate into lipid,osteogenic and transwell.CCK-8 assayed the inhibitory effect of TRAIL secreted by MSCs on the proliferation of Nalm-6 and Sup-b15 cells in vitro,and western blot assayed the expression of leukemia proliferation-associated proteins PI3K,AKT,P-RAF-1,P-MEK and P-ERK by western blot.Meanwhile,the effect of MSC-s TRAIL on the apoptosis of Nalm-6 and Sup-b15 cells was analyzed by flow cytometric fluorescence activated cell sorting(FACS).The expression of Bcl-2,Bax,cleaved caspsae3,cleaved caspase8,cleaved caspase9,cleaved PARP and autophagy-related protein LC3A/B was determined by Western blot.Changes in the expression of TRAIL receptor-related genes TNFRSF10A,TNFRSF10B,TNFRSF10C and TNFRSF10D in Nalm-6 and Sup-b15 cells were detected by Q-PCR.For in vivo mice experiments,SCID mice were random Ly divided into five groups with tail vein inoculation,namely the untreatment group,the PBS group,the MSC-EV group,the MSC-s TRAIL group and the rh TRAIL group.the PBS group,the MSC-EV group,the MSC-s TRAIL group and rh TRAIL group were injected twice with 2×10~6Nalm-6 cells per injection to establish B-ALL model.On day 35 after inoculation with Nalm-6 cells,mice in the untreatment group,PBS group,MSC-EV group,MSC-s TRAIL group and rh TRAIL group were each injected with 200μL PBS,200μL PBS,4×10~6MSC-EV cells,4×10~6MSC-s TRAIL cells and 5mg/kg rh TRAIL.The mice were observed every other day and weighed.Mice were inoculated with Nalm-6 cells for about 60 days,and bone marrow of each group of mice was taken for Richter-Gimza staining.The amount of TRAIL in the bone marrow of mice was also determined by ELISA.HE staining was performed to observe the pathological changes in the heart,liver,spleen,lung and kidney of mice examined by tissue,and the effect of MSC-s TRAIL was evaluated according to the results.Results:Our purchased B-ALL cells express TRAIL receptor-associated m RNA.rh TRAIL inhibits the proliferation of B-ALL cells in a time-dose dependent manner.The TRAIL-expressing plasmid p LV-3Flag-TRAIL-Green Puro was successfully constructed and sequenced to prove its genetic correctness.After transfection of plasmid p LV-3Flag-TRAIL-Green Puro into 293T cells,the lentiviral particles p LV-3Flag-TRAIL-Green Puro were concentrated and obtained.MSCs infected with lentiviral p LV-3Flag-TRAIL-Green Puro expressed the target protein TRAIL efficiently,and it was found that the lentiviral p LV-3Flag-TRAIL-Green Puro expressed the target protein TRAIL efficiently after infection.The ability of MSCs to differentiate and converge to B-ALL cells was not significantly altered before and after infection.CCK8 results showed that TRAIL secreted by MSC-s TRAIL inhibited the proliferation of Nalm-6 and Sup-b15 cells.And the expression of proliferation-related proteins PI3K,AKT,P-MEK,P-ERK1/2,and P-RAF-1 was found to be decreased in Nalm-6 cells at 72h by western blot assay.Meanwhile,FACS assay showed that s TRAIL secreted by MSC-s TRAIL promoted apoptosis in Nalm-6 and Sup-b15cells.Western blot results showed decreased expression of anti-apoptosis-related proteinsBcl-2/Baxdecreased,and pro-apoptosis-related proteins cleaved caspsae3,cleaved caspase8,cleaved caspase9,cleaved PARP expression increased in MSC-s TRAIL-CM group and rh TRAIL group.And MSC-s TRAIL down-regulated the expression of autophagy-related proteins LC3A/B.Its suggests that MSC-s TRAIL may have some connection with autophagy in B-lymphocyte acute leukemia.Meanwhile,Q-PCR assay revealed that rh TRAIL decreased the expression levels of TNFRSF10A and TNFRSF10B m RNA in Nalm-6 cells compared with the control group;rh TRAIL increased the expression levels of TNFRSF10BD m RNA in Sup-b15 cells.MSC-s TRAIL group compared with rh TRAIL group.TNFRSF10A expression was increased in both Nalm-6 cells.The MSC-s TRAIL group increased the expression levels of TNFRSF10A and TNFRSF10B m RNA in Sup-b15 cells compared with the control group.This indicates that MSC-s TRAIL increased the sensitivity of Nalm-6 and Sup-b15 cells to TRAIL compared with rh TRAIL.In vivo experiment,the Richter-Gimza bone marrow smear staining of SCID mice in the untreatment group,PBS group,MSC-EV group,MSC-s TRAIL group and rh TRAIL group a large number of morphological cells similar to Nalm-6 cells could be observed in the PBS group.Whereas similar Nalm-6 cells were not found in the untreatment group and some similar Nalm-6 cells were found in the MSC-EV group,most of similar Nalm-6 cells were reduced in the bone marrow smears of mice in the MSC-s TRAIL and rh TRAIL groups.Thereby,it was shown that MSC-s TRAIL and rh TRAIL could inhibit the proliferation and growth of Nalm-6 cells in large numbers.And the TRAIL content in bone marrow of mice in the MSC-s TRAIL group was significantly increased by the analysis of the results of ELISA assay.Weighing of mouse tissue and organ weights revealed that the spleen and kidney weight/body weight ratios of leukemic mice in the MSC-s TRAIL and rh TRAIL groups were reduced compared to the PBS group(P<0.05).HE staining of pathological sections showed that the spleen of leukemic mice in the PBS and MSC-EV groups had sparse cells in the white marrow structure and significantly damaged cells;the medullary discharge lines of the kidney were also disordered in texture.the MSC-s TRAIL and rh TRAIL groups showed no significant changes in the spleen,the white marrow structure was normal,and the kidney medullary discharge was also clear in texture.It indicates that inoculation of MSC-s TRAIL and rh TRAIL can restore the spleen and kidney damage in leukemic mice.Conclusion:MSC-s TRAIL significantly inhibited the proliferation of Nalm-6 and Sup-b15 cells by downregulating the leukemia proliferation-associated proteins PI3K,AKT,P-RAF-1,P-MEK and P-ERK.and the inhibitory effect of MSC-s TRAIL was stronger than that of rh TRAIL.MSC-s TRAIL promoted Nalm-6 and Sup-b15 apoptosis by downregulating the anti-apoptosis-related protein Bcl-2/Bax and upregulating the pro-apoptosis-related proteins cleaved caspsae3,cleaved caspase8,cleaved caspase9,and cleaved PARP.And MSC-s TRAIL has a stronger promoting effect than rh TRAIL.Compared with rh TRAIL,Nalm-6 and sup-b15 cells were more sensitive to MSC-s TRAIL.Compared with rh TRAIL,MSC-s TRAIL increased TRAIL levels in B-ALL mice.MSC-s TRAIL exerted a therapeutic effect on spleen and kidney injury.The related study of MSC-s TRAIL in B-ALL may provide new ideas for finding leukemia treatment strategies.