In vitro and in vivo cytotoxic activity of humanized CD19 CAR-T cells against Raji cell line / 中华微生物学和免疫学杂志

Objective@#To investigate the different functions of humanized and murinized CD19 chimeric antigen receptor (CAR)-T cells against Raji cell line in vitro and in vivo.@*Methods@#Peripheral blood samples were collected from eight patients with lymphoma who were going to receive CD19 CAR-T cell therapy and used for the preparation of peripheral blood mononuclear cells (PBMC) as well as humanized and murinized CAR-T cells. Cell proliferation and cytotoxicity were detected with CCK-8 and LDH assays, respectively. A tumor-bearing mouse model was established by injecting BALB/c female nude mice with fluorescent Raji cells. Changes in tumor volume in these mice were observed by in vivo imaging technology. The transfection efficiency and amount of CAR-T cells in the mice were detected with flow cytometry.@*Results@#No statistical difference in transfection efficiency was found between humanized and murinized CAR-T cells, nor in cell proliferation at 24 h of culture in vitro(P=0.104). The proliferation of humanized CAR-T cells showed a significant increase compared with that of murinized CAR-T cells at 48 h of culture (P=0.009). Similarly, the cytotoxicity of the two types of CAR-T cells against Raji cells showed no significant difference at 24 h at any effector/target (E/T) ratio (1∶1 or 4∶1), and that of humanized CAR-T cells was higher than that of murinized CAR-T cells at both E/T ratios at 48 h (E/T ratio=1∶1, P=0.005; E/T ratio=4∶1, P=0.008). Moreover, the cytotoxicity of CAR-T cells was higher than that of PBMC in any case. Tumor volumes in mice were reduced 14 d after humanized or murinized CAR-T cell therapy, while the mice in the PBMC control group suffered tumor progression. Tumor volume began to increase in mice 21 d after murinized CAR-T cell therapy, while no significant change was observed in the mice treated with humanized CAR-T cells. All of the mice died 25 d after murinized CAR-T cell therapy, while the deaths among those under humanized CAR-T cell therapy occurred on 31 d. The proportion of CAR-T cells in mice reached the peak 7 d after receiving humanized or murinized CAR-T cell therapy, while that in the humanized group was significantly higher than that in the murinized group at any time point (P4 d=0.001, P7 d=0.000, P14 d=0.003). Murinized CAR-T cells became undetectable on 21 d, while humanized CAR-T cells on 35 d. The maximum survival time for mice in the PBMC and murinized and humanized CAR-T cell groups was 20 d, 25 d and 53 d, respectively.@*Conclusions@#Compared with murinized CD19 CAR-T cells, humanized CD19 CAR-T cells showed stronger proliferation potential and cytotoxicity and remained in vivo detectable for a longer period of time. This study indicated that humanized CD19 CAR-T cells were superior to murinized CD19 CAR-T cells for the treatment of B cell lymphoma.

In vitro and in vivo cytotoxic activity of humanized CD19 CAR-T cells against Raji cell line / 中华微生物学和免疫学杂志

Objective To investigate the different functions of humanized and murinized CD19 chimeric antigen receptor ( CAR)-T cells against Raji cell line in vitro and in vivo. Methods Peripheral blood samples were collected from eight patients with lymphoma who were going to receive CD19 CAR-T cell therapy and used for the preparation of peripheral blood mononuclear cells ( PBMC) as well as humanized and murinized CAR-T cells. Cell proliferation and cytotoxicity were detected with CCK-8 and LDH assays, respectively. A tumor-bearing mouse model was established by injecting BALB/c female nude mice with flu-orescent Raji cells. Changes in tumor volume in these mice were observed by in vivo imaging technology. The transfection efficiency and amount of CAR-T cells in the mice were detected with flow cytometry. Re-sults No statistical difference in transfection efficiency was found between humanized and murinized CAR-T cells, nor in cell proliferation at 24 h of culture in vitro(P=0. 104). The proliferation of humanized CAR-T cells showed a significant increase compared with that of murinized CAR-T cells at 48 h of culture ( P=0. 009). Similarly, the cytotoxicity of the two types of CAR-T cells against Raji cells showed no significant difference at 24 h at any effector/target (E/T) ratio (1 : 1 or 4 : 1), and that of humanized CAR-T cells was higher than that of murinized CAR-T cells at both E/T ratios at 48 h (E/T ratio=1 : 1, P=0. 005;E/T ratio=4 : 1, P=0. 008). Moreover, the cytotoxicity of CAR-T cells was higher than that of PBMC in any case. Tumor volumes in mice were reduced 14 d after humanized or murinized CAR-T cell therapy, while the mice in the PBMC control group suffered tumor progression. Tumor volume began to increase in mice 21 d after murinized CAR-T cell therapy, while no significant change was observed in the mice treated with hu-manized CAR-T cells. All of the mice died 25 d after murinized CAR-T cell therapy, while the deaths among those under humanized CAR-T cell therapy occurred on 31 d. The proportion of CAR-T cells in mice reached the peak 7 d after receiving humanized or murinized CAR-T cell therapy, while that in the humanized group was significantly higher than that in the murinized group at any time point (P4 d=0. 001, P7 d=0. 000, P14 d=0. 003). Murinized CAR-T cells became undetectable on 21 d, while humanized CAR-T cells on 35 d. The maximum survival time for mice in the PBMC and murinized and humanized CAR-T cell groups was 20 d, 25 d and 53 d, respectively. Conclusions Compared with murinized CD19 CAR-T cells, humanized CD19 CAR-T cells showed stronger proliferation potential and cytotoxicity and remained in vivo detectable for a longer period of time. This study indicated that humanized CD19 CAR-T cells were superior to murinized CD19 CAR-T cells for the treatment of B cell lymphoma.

Effect of the two-step pretargeting radioimmunotherapy of CD45 monoclonal antibody and 188Re-Avidin on lymphoma Raji cell line / 中华放射医学与防护杂志

Objective To study the effect of two-step pretargeting radioimmunotherapy of CD45 McAb and 188Re-Avidin on lymphoma Raji cell line.Methods The CD45 McAb and Avidin were directly labeled with 188Re,and the labeling efficiency and radiochemical purity were measured by the paper chromatography.The specific binding test and competition binding test between 188 Re-CD45 McAb and Raji cells in vitro were also performed.CCK-8 assay was used to determine the inhibition effect on Raji cell proliferation in the pretargeted group,188Re-CD45 McAb,188Re-Avidin and 188ReO4 groups,then the cell survival and proliferation inhibition rate were calculated.Results The specific cell binding rate of 188Re-CD45 McAb with Raji cells was (70.92 ± 1.91) ％,in the competition group,the binging rate of 188Re-CD45 McAb with Raji cells was only (7.96 ± 0.87)％.The Raji cells proliferation was inhibited in all groups with 188Re radiolabel,and the inhibition rate was positively correlated with the radioactivity dose (r=0.907-0.992,P ＜0.05).However,at the same dose,the inhibition effect in the group of two-step pretargeting at each time point were all stronger than those of 188Re-CD45 McAb,188Re-Avidin and 188 ReO4-alone (t =124.76-607.98,P ＜ 0.05).But there was no significantly statistical difference in the inhibitory effect between the groups of 188Re-Avidin and 188ReO4-(P ＞ 0.05).Conclusions It is confirmed that 188Re-CD45 McAb could be specifically bound to Raji cells,and the two-step pretargeting of CD45 McAb and 188Re-Avidin has obvious inhibitory effect on the Raji cell proliferation.

Inhibitory effects of the phosphoinostitide-3'-kinase delta inhibitor CAL-101 on Raji and SUDHL-10 lymphoma cells and its relative mechanism / 中国肿瘤临床

Objective:To detect the inhibitory effects of CAL-101, a selective inhibitor of phosphoinostitide-3'-kinase delta (PI3Kδ), on Burkitt's lymphoma cell line Raji and diffused large B-cell lymphoma cell line SUDHL-10 and elucidate its relative mechanism. Methods:Raji and SUDHL-10 cells were treated with various concentrations of CAL-101. Methyl thiazolyl tetrazolium (MTT) assay was performed to determine the inhibitory effect of CAL-101 on lymphoma cells, and cell apoptosis was measured by Annexin V/PI and DAPI staining. Migration assays were performed with transwell to detect the migration of lymphoma cells derived from the stromal cell line HK. Western blot was used to detect the phosphorylation status of the ERK pathway. MTT and CalcuSyn software analyses were preformed to detect whether or not combining CAL-101 with bortezomib induces synergistic cytoxicity. Results:CAL-101 at con-centrations of 5, 10, 15, and 20μmol/L inhibited cell proliferation in a dose-dependent manner. The proliferation rates of the Raji cells treated with 5, 10, 15, and 20μmol/L for 48 h were 29.17%± 1.23%, 38.15%± 1.51%, 46.46%± 1.78%, and 55.8%± 2.01%, respec-tively, which were significantly higher (P<0.05) than that of the control group (1.15% ± 0.02%). Similar results were found in the SUDHL-10 cells after treatment with CAL-101 (P<0.05). CAL-101 also exerted an apoptotic effect on the lymphoma cells. The apop-totic rates of the Raji cells treated with CAL-101 for 21 h were 22.69%± 3.83%and 49.96%± 7.36%, respectively, which were signifi-cantly higher (P<0.05) than that of the control group (5.23%± 2.04%). Similar results were found in the SUDHL-10 cells (P<0.05). Treatment with 5 and 10 μmol/L CAL-101 dose-dependently inhibited the migration activity of lymphoma cells to stromal cells (P<0.05). Western blot analysis showed that the expression level of ERK phosphorylation protein was significantly downregulated in the cells treated with CAL-101. A synergistic effect between CAL-101 and bortezomib was verified. That is, these two drugs can signifi-cantly inhibit the proliferation of lymphoma cells with CI values less than 1. Conclusion:The PI3Kδ-specific inhibitor CAL-101 sup-pressed the proliferation of Raji and SUDHL-10 cells, induced apoptosis, and inhibited stromal cell-derived migration. This inhibitory effect may be induced by blocking the ERK pathway. Overall, our study indicated that CAL-101 is a novel and potential agent in the therapeutic strategy against aggressive B-cell lymphoma.

MiR-15a and miR-16-1 enhance sensitivity of Raji cells to Ara-C / 第二军医大学学报

Objective To study whether miR-15a and miR-16-1 can enhance the sensitivity of Raji cells to cytarabine (Ara-C). Methods MiR-15a and miR-16-1 oligonucleotides were transfected into Raji cells with Lipofectamine™ 2000, and then the cells were treated with Ara-C. The IC50 values of Ara-C was detected by CCK8 assay. The growth of Raji cells was measured by trypan blue dye exclusion method. The apoptotic cells were observed by Hoechst dyeing; AnnexinV/PI double dyeing and glow cytometry(FCM) were used to examine the cell apoptotic rate. Results After transfection of miR-15a or miR-16-1 into Raji cells, the IC50 values of Ara-C were 10. 41 and 10. 86, respectively, which were significantly lower than that of the untransfected group(15.43)and scrambled oligonucleotides (SODN)transfection group(14. 92, P<0.05). Trypan blue dye exclusion assay showed that miR-15a/miR-16-1 transfection group had obviously decreased the cell growth compared to miR-15a, miR-16-1 group, untransfected group and SODN transfected group; Hoechst dyeing demonstrated plenty of apoptotic cells. AnnexinV/PI double dyeing assays by FCM indicated that the cell apoptotic rates in earlier period and late period were 20. 93% and 25. 27% in the miR-15a+Ara-C group, and 20. 69% and 23. 13% in the miR-16-1 + Ara-C group, which were obviously higher than those in miR-15a group (6. 99%, 10. 08%), miR-16-1 group(4. 73%, 10. 64%), Ara-C group (10. 88%, 11. 83%) and control group (14. 39%, 11. 93%). Conclusion MiR-15a and miR-16-1 oligonucleotides can enhance the sensitivity of Raji cells to Ara-C.

Gambogic acid induces Raji cell apoptosis in vitro and its mechanism / 白血病·淋巴瘤

Objective To study the inducing apoptosis effect of a traditional Chinese medicine gambogic acid (GA) on Raji cell line and its mechanism. Methods The effect of GA on the proliferation of human peripheral blood mononuclear cells induced by lipopolysaccharide (LPS) was analyzed. Raji cells were treated with GA at different concentrations and times, and the inhibitory effect was detected by MTT assay.Apoptosis induced by GA was observed by Annexin V/PI doubling staining and flow cytometry assay.Mitochondrial membrane potential was measured by JC-1 assay. Activated Caspase-3, Caspase-8 and Caspase-9 in living Raji cells were measured by caspGLOWTM fluorescein staining kit and quantificated by flow cytometry. Results After incubation with GA, the proliferation rates of both normal blood mononuclear cells and Raji cells were dramatically inhibited in a concentration dependent manner. GA induced Raji cells to undergo apoptosis. GA decreased the mitochondrial membrane potential of Raji cells. GA increased the level of activated caspase 3, caspase 8, caspase 9 for 0.37 %, 33.57 %, 18.27 % in 24 h and 28.2 %, 69.2 %,76.7 % in 48 h respectively. Conclusion GA have an inhibitory effect on Raji cells, and can trigger apoptosis of Raji cells through both intrinsic and extrinsic pathways.

Influence of infrasound therapy on Raji cells / 中华物理医学与康复杂志

Objective To investigate the influence of infasound therapy on Raji cells. Methods The Raji cell line was cultivated routinely and grouped as an infrasound and a control groups.Infrasound 8TM was used as a therapeutic infrasound generator which worked in 3 modes(frequency range 4～20 Hz,infrasound energy less than 90 dB).The applicator of the infasound generator was put on 1.5～2.0 cm fom the surface of liquid in the dish containing Raji ceil.Raji cells would be treated for 15,30,60,and 90 minutes,then tested with trypan blue assay,MTT assay,flow cytometry anatysis,and scanning electron microscope(SEM)after 0,24,and 48 h cultivations,respectively. Results Trypan blue assay showed that there were no significant differences between the 2 groups(P>0.05).MTT assay showed that although optical density value of the infrasound groups seemed to be lower than that of the control group.the differences were not significant(P>0.05).Flow cytometry analysis showed that the rate of necrotic cells and apoptosis cells in all groups was less than 10%;and that the differences between all groups were not significant(P>0.05).The scanning electron microscopy showed that the cells treated by infasound exposure for 120 minutes and then cuhivated for 24 h showed that the prominent or micro-floss of the membrane become shorten and decreased.The surface of the membrane became smooth. Conclusion Infrasound(less than 90 dB)treatment in the experiment had no obvious influence on multiplication and apoptosis of Raji cells.But the membrane of Raji cell Would be affected directly by the infrasound,and the penetration of the membrane could be changed.

Anti-proliferation effect of peroxisome proliferator-activated receptor γ agonist on Raji cells and its mechanism / 白血病·淋巴瘤

Objective To investigate the anti-proliferation effect of peroxisome proliferator-activated receptor γ(PPARγ)agonist troglitazone(TGZ)on leukemic Raji cells and its mechanisms.Methods Raji cells in culture medium in vitro were given different concentrations of TGZ(0～60 μmol/L)for 24,48 and 72 h.The inhibitory rates of the cells were measured by MTT assay,cell apoptotic rate was detected by flow cytometry(FCM),agarose gel electrophoresis was used to observe the DNA ladder,and western blotting was used to analyzed the variation of apoptosis related proteins bcl-2,Bax and Survivin.Results TGZ(over 20 μmol/L)could inhibit the growth of Raji cells and cause apoptosis remarkably,the suppression was both in time-and dose-dependent manner.DNA ladder was observed after the cells treated by TGZ for 72 h,and western blotting analysis revealed that anti-apoptotie proteins Survivin and bcl-2 were decreased remarkably while pro-apoptotic protein Bax increased significantly after the cells were treated by TGZ for 48 h.Conclusion PPARγ agonist TGZ can inhibit the growth and induce apoptosis on Raji cells significantly,downregnlating the expression of Survivin and bcl-2 as well as upregulating of Bax expression of Raji cells may be one of its most important mechanisms.

Production of the Recombinant Single Chain Anti-B Cell Lymphoma Antibody and Evaluation of Immunoreactivity / 핵의학분자영상

PURPOSE: Recombinant ScFv lym-1 was produced, using pET vector system for large scale production. METHODS: ScFv lym-1 gene inserted pET-22b (+) vector, was expressed in E.coli BL-21 strain. ScFv lym-1 antibody extracted from periplasm, was purified with His-Taq column. To evaluated immunoreactivity with Raji cell, ScFv lym-1 was labeled with I-125 and I-125 ScFv lym-1 was purified with desalting column. Raji cell was injected into the C57BR/cdJ SCID mice. Gamma camera imaging were taken time point at 1, 8, 24, and 48 hr with 8 mm pinhole collimator. RESULTS: An active scFv lym-1 could be produced in E.coli with soluble from using pET vector system. Immunoreactivity and affinity constant of IgG lym-1 were 54% and 1.83 x 10(9) M(-1), respectively, and those of scFv lym-1 were 53.7% and 1.46 x 10(9) M(-1), respectively. Biodistribution of I-125 scFv lym-1 antibody showed faster clearance in blood, spleen, kidney and than I-125 IgG lym-1 antibody. Gamma camera image of I-125 scFv lym-1 antibody showed faster clearance and tumor targeting liver than I-125 IgG lym-1 antibody. CONCLUSIONS: In vitro properties of scFv lym-1 were similar to those of IgG lym-1. ScFv lym-1 showed faster blood clearance than IgG lym-1. These results suggest that scFv lym-1 antibody can be useful for tumor imaging agent.

Effect of Annonaceous Acetogenin on Proliferation and Apoptosis of Raji Cell / 中国中医药信息杂志

Objective To investigate the effects of annonaceous acetogenin on proliferation and apoptosis in Raji cells and its mechanism.Methods Raji cells cultured in vitro were divided into control group,annonaceous acetogenin group and adriamycin group.Raji cells were effected by 6.25,12.5,25,50 ?g/mL annonaceous acetogenin.Proliferation of Raji cells were evaluated by MTT assays,apoptosis percentage was assessed by flow cytometry.Caspase-9 protein was detected by immunohistochemistry.Results The Raji cell proliferation rate of annonaceous acetogenin decreased compared with the control,that of 25,50 ?g/mL group were lower than adriamycin group,and it was related to the concentration,relying on the incubating time.The apoptosis rate was higher than control,that of 25,50 ?g/mL group were higher than adriamycin group,and it was related to the concentration and the incubating time.The expression of caspase-9 protein of annonaceous acetogenin group was higher than control,and it had a positively relationship with the concentration and incubating time.Conclusions Annonaceous acetogenin could inhibit cell proliferation in a dose-dependent and time-dependent manner in Raji cells,and it may induce Raji cells apoptosis by up-regulating caspase-9 expression.

Anticancer Activities of Trichostatin A on Maligant Lymphoid Cells / 华中科技大学学报（医学）（英德文版）

The anticancer activity of trichostain A (TSA) on human B cell non-Hodgkin's lymphoma and its mechanism were explored. The effect of TSA on the growth of Raji cells and normal peripheral blood mononuclear cells (NPBMNC) was studied by MTT assay. The effect of TSA on the apoptosis of Raji cells and NPBMNC was studied by flow cytometry and TDT-mediated dUTP nick end labeling (TUNEL). The effect of TSA on the cell cycle of Raji cells was studied by propidium iodide method. The results showed that TSA potently inhibited proliferation of Raji cells at microgram concentrations and induced apoptosis of Raji cells in a time- and concentration-dependent manner.Treatment with TSA induced accumulation of cells in G0/G1 or G2/M and a concomitant decrease of cell population in S phase. However, NPBMNC was less sensitive to the cytotoxic effect of TSA than Raji cells. It was concluded that TSA may inhibit the proliferation of Raji cells by regulating the cell cycle and inducing the cell apoptosis. Moreover, TSA demonstrates low toxicity in NPBMNC but selectively induces apoptosis of Raji cells.

Anticancer Effect of Curcumin on B Cell non- Hodgkin's Lymphoma / 华中科技大学学报（医学）（英德文版）

To explore the anticancer effect of curcumin on human B cell non-Hodgkin's lymphoma and compare its effects on human B cell non-Hodgkin's lymphoma cells and normal peripheral blood mononuclear cells (NPBMNCs). MTT assay was used to study the effect of curcumin on the growth of Raji cells and NPBMNCs. The effect of curcumin on the apoptosis of Raji cells and NPBMNC were studied by flow cytometry and TDT-mediated dUTP nick and labeling (TUNEL). The effect of curcumin on the cell cycle of Raji cells were examined by propidium iodide staining flow cytometry. The results showed that curcumin strongly inhibited ±1.82 μmol/L and curcumin induced Raji cell apoptosis in a time- and dose-dependent manner. Raji cells treated with curcumin showed curcumin did not demonstrate apparent proliferation inhibition and apoptosis induction in NPBMNCs. It was concluded that curcumin is able to inhibit the proliferation of Raji cells by regulating the cell cycle and inducing the cell apoptosis. Morever, curcumin has low toxicity on NPBMNCs but can selectively induce apoptosis in Raji cells.

Inhibitory efectory of curcumin on transcriptional co-activator P300 expression of B-NHL cell line Raji cell / 中国药理学通报

0.05),but the expression of P300 decreased at intermedieate and high concentration(12.5,25,50 ?mol?L~(-1),P

Impact of dehydroepiandrosterone on antioxidant capacity of Raji cell cultured in vitro / 医学研究生学报

Objective: To investigate the influence of dehydroepiandrosterone(DHEA) on antioxidant capacity of Raji cell cultured in vitro. Methods: Raji cell line was cultured in vitro in the presence of DHEA and/or phenazine methosulfate(PMS), some antioxidant enzymes including glucose-6-phosphate dehydrogenase activity(G-6-PD),glutathione peroxidase activity(GPX), glutathione reductase activity(GR) and intracellular glutathione(GSH) concentration of the DHEA pretreatment cells were evaluated. meanwhile, these results were compared with that from the respective control cells. Results:(G-6-PD) activities were suppressed significantly and no changes in activities of GPX,GR and intracellular GSH concentration were occurred while Raji cells were cultured in the presence of DHEA at concentration more than 5.0?mol/L . On the other hand, the DHEA pretreatment cells show no changes in (G-6-PD,)GPX,GR activities and significantly decreased GSH concentration(P