Effect of Pulsatilla Saponin A on Proliferation and Apoptosis of Burkitt Lymphoma Cells Based on JAK2/STAT3 Signaling Pathway / 中国实验方剂学杂志

ObjectiveTo investigate the effect of pulsatilla saponin A （PSA） on proliferation and apoptosis of human Burkitt lymphoma （BL） cell line Raji cells and expression of related pathway proteins. MethodWith Raji cells as the research object， the cell proliferation was detected by cell counting kit-8 （CCK-8） method， and the half-maximal inhibitory concentration （IC50） values of 24 h， 48 h and 72 h were calculated to be 19.77， 18.31， 16.70 μmol·L-1， respectively. In subsequent related experiments， 0， 8， 16， 32 μmol·L-1 PSA were selected according to the IC50 value of Raji cells treated with PAS for 72 h. After 0， 8， 16， 32 μmol·L-1 PSA acted on Raji cells for 24， 48， 72 h， the optical density values of cell growth curve were detected by CCK-8 method. The zymogen activities of cysteine aspartate-specific protease （Caspase）-3， Caspase-8 and Caspase-9 in Raji cells treated with 0， 8， 16 and 32 μmol·L-1 PSA for 24 h were measured by Caspase-3， Caspase-8 and Caspase-9 colorimetric assay kit. The apoptosis rate and cell cycle of Raji cells treated with different concentrations of PSA after 24 h were detected by flow cytometry. The expression of B-cell lymphoma 2 （Bcl-2）， Bcl-2-associated X protein （Bax）， cleaved poly（ADP-ribose） polymerase （cleaved PARP）， cleaved cysteinyl aspartate-specific protease-3 （cleaved Caspase-3） apoptosis related protein and Janus kinase 2 （JAK2）， signal transducer and activator of transcription 3 （STAT3）， phosphorylated-JAK2 （p-JAK2）， and phosphorylated- STAT3 （p-STAT3） pathway proteins in Raji cells after 24 h of treatment with 0， 8， 16 and 32 μmol·L-1 PSA were tested by Western blot. ResultCompared with control group， decreased cell survival rate， inhibited cell proliferation， activated zymogens of Caspase-3， Caspase-8 and Caspase-9 （P<0.01）， increased apoptosis （P<0.05， P<0.01）， and enhanced cell cycle arrest in Gap phase 2 （G2） were observed in 8， 16 and 32 μmol·L-1 PSA groups（P<0.05， P<0.01）. Compared with control group， cells treated with 8， 16 and 32 μmol·L-1 PSA had lower expression of Bcl-2， p-JAK2， p-STAT3 proteins （P<0.05， P<0.01）， and higher expression of Bax， cleaved PARP and cleaved Caspase-3 protein （P<0.01）， while no significant change was found in the expression of JAK2 and STAT3 proteins. ConclusionPSA could inhibit proliferation and induce apoptosis of Raji cells， and its potential mechanism might be related to the regulation of JAK2/STAT3 signaling pathway.

Effects of PTEN Combined with Adriamycin on the Proliferation, Migration and Invasion of Adriamycin in Non Hodgkin lymphoma Raji Cells / 中国药学杂志

OBJECTIVE: To investigate the inhibitiom effect of PTEN(gene of phosphate and tension homology deleted on chromsome ten) combined with adriamycin on proliferation, migration and invasion of non-Hodgkin lymphoma cell line Raji in vitro. METHODS: Cell proliferation was determined by MTT in Raji cells response to adriamycin with different concentrations. Transwell assay was performed to evaluate the effects of adriamycin and PTEN on migration and invasion of Raji cells. RT-qPCR was conducted to measure the expression of PTEN in Raji cells after adriamycin treatment. RESULTS: Adriamycin significantly inhibited the proliferation of Raji cells in a concentration dependent manner (r=-0.925, P<0.001). Adriamycin inhibited invasion and migration in Raji cells. Moreover, adriamycin promoted the expression of PTEN. Overexpression of PTEN markedly suppressed invasion and migration in Raji cells. The combination of adriamycin and PTEN strikingly decreased the proliferation, invasion and migration of Raji cells. CONCLUSION: Adriamycin and PTEN would inhibite the proliferation, invasion and migration of Raji cells. PTEN drastically enhances the inhibition of adriamycin on the proliferation, migration and invasion of Raji cells.

The sequential switch from macroautophagy to chaperone-mediated autophagy in Raji cells induced by starvation / 中国药理学通报

Aim To research the cross-talk and conversion between macroautophagy and chaperone-media-ted autophagy ( CMA) in cultured Burkitt lymphoma Raji cells induced by starvation. Methods The autophagic vacuoles were observed by fluorescence microscopy and confocal laser-scanning microscopy with monodansylcadaverine staining. The expression of autophagy associated-proteins were determined by West-em blot. Results Both macroautophagy and CMA were activated sequentially instead of simultaneously in starvation-induced Raji cells, and macroautophagy was quickly activated and peaked during the first hours of near baseline. With starvation persisted, CM A progressively increased along with the decline of macroautoph- A gy. Conclusions Macroautophagy and CMA are maximally activated during different stages of starvation. Activation of these two pathways is often sequential. The sequential switch from macroautophagy to CMA might be conducive to the adaption of cancer cells to miscellaneous intracellular or extracellular changes, maintaining their own growth and proliferation.

Role and mechanism of autophagy in the arsenic trioxide-induced death of Burkitt lymphoma Raji cells / 中国药理学通报

Aim To investigate the role of autophagy and its mechanism in Raji cell death induced by arse-nic trioxide. Methods Transmission electron micros-copy ( SEM) and MDC fluorescence staining were used to observe autophagy. MTT colorimetry was employed to assay the cellular proliferating activity. Cell apopto-sis and cell cycle analysis were performed using FITC-Annexin-V/PI double staining and flow cytometry ( FCM) . The expressions of LC3 and the conversion of LC3-I to LC3-II were measured by western bloting. The expression of bcl-2 mRNA and p53 mRNA were detected by reverse transcription-polymerase chain re-action ( RT-PCR ) . Results Arsenic trioxide could obviously inhibit the proliferation of Raji cells, arrest the cells at G2/M phase and induce apoptosis. Mean-while, arsenic trioxide markedly inhibited the expres-sion of bcl-2 mRNA and enhanced the expression of p53 mRNA in Raji cells. Arsenic trioxide also induced autophagy synchronously which paralleled with the in-duction of apoptosis in Raji cells, and 3-MA, an auto-phagy inhibitor, was able to reverse the arsenic triox-ide-activated autophagic activity, up-regulate bcl-2, down-regulated p53 expression and suppress the lethal effect of arsenic trioxide on Raji cells to reduce their sensitivity to arsenic trioxide. In contrast, the Rapamy-cin, an autophagy inducer, possessed the completely opposite effects on Raji cells compared with 3-MA. Conclusions The apoptosis and autophagic cell death are coexistent in arsenic trioxide-triggered death of Raji lymphoma cells, and Bcl-2 and p53 may play a key regulating role in this process.

Effect of combination with magnetic nanoparticle of Fe3O4 and adriamycin on Raji cell line / 白血病·淋巴瘤

Objective To investigate the effect of combination with magnetic nanoparticle of Fe3O4and adriamycin (ADM) on Raji cell line.Methods Raji cells were cultured with Fe3O4-magnetic nanoparticle and ADM after using mechanical absortion polymerization,and the cell viability was detected by MTT and trypan-blue exclusion.The apoptosis was detected by flow cytometry,and the expression of p53 and NF-κB were measured by Western blot. Results Raji cell proliferation ratio was significantly inhibited in concentration-and time-dependent manners in both ADM and Fe3O4-MNP-ADM groups (r =0.412,P =0.027;r =0.523,P =0.014).The percentages of apoptotic cells induced by ADM and Fe3O4-MNP-ADM were 8.76 ％ vs 14.85 ％,35.08 ％ vs 44.50 ％,44.00 ％ vs 69.40 ％ in 12,24 and 48 hours,respectively (t =-9.137,-4.808,-6.337; P =0.012,0.041,0.024).The grey straps of NF-κB measured by Western blot were 4.22±0.32,3.31±0.28 in ADM and Fe3O4-MNP-ADM group (t =-54.416,P =0.035),whereas the grey straps of p53 were 1.042±0.114,1.270±0.091,respectively (t =33.963,P =0.047).Conclusion Combination therapy of Fe3O4-MNP and ADM could inhibit proliferation and induce apoptosis of Raji cells line,which may due to upregulation of p53 and down-regulation of NF-κB.

Small interfering RNA targeting Bcl-2 enhances the inhibitory effects of MTX on transplanted human lymphoma in nude mice / 第二军医大学学报

Objective: To explore whether short hairpin RNA (shRNA) targeting Bcl-2 can enhance the inhibitory effect of methotrexate (MTX) on growth of subcutaneously-transplanted human lymphoma in nude mice. Methods: Recombinant shRNA expression vector targeting the coding region of Bcl-2 mRNA was constructed and preserved in our lab. Human lymphoma Raji cells were injected subcutaneously into 45 nude mice to establish lymphoma models. The polyethylenimine (PEI)/shRNA complex and (or) MTX were injected into tumors. The influence of Bcl-2 shRNA and (or) MTX on tumor growth was observed. The animals were sacrificed 21 days after administration of drugs and the tumors were removed and weighed; the tumor inhibitory rate was calculated. H-E staining was used to observe the pathological morphology of the tumor. The expression of Bcl-2 mRNA in the tumor tissues was examined by RT-PCR. Results: The tumor growth was significantly slower in Bcl-2 shRNA/MTX group than in Bcl-2 shRNA or MTX alone groups (P<0.05). The tumor weight of mice in Bcl-2 shRNA plus MTX group was significantly lower than those in negative shRNA and blank plasmid group (P<0.05). The inhibition rate of tumor growth in Bcl-2 shRNA/MTX was significantly higher than those in the Bcl-2 shRNA or MTX alone groups (P< 0.05). H-E staining showed obvious apoptosis and necrosis in Bcl-2 shRNA group and MTX group. RT-PCR result showed that the expression of Bcl-2 mRNA in tumor cell suspension was significantly decreased in Bcl-2 shRNA group (P<0.05), and kept unchanged in the control group. Conclusion: The shRNA targeting Bcl-2 can enhance the inhibitory effect of MTX on the growth of subcutaneously transplanted human lymphoma in nude mice.

The study on effects of Bcl-2 shRNA in enhancing methotrexatE-induced apoptosis and inhibiting cell proliferation in Raji cells / 肿瘤

Objective:To study the effect of Bcl-2 short hairpin RNA (shRNA) in enhancing methotrexate (MTX)-induced apoptosis of Raji cells. Methods:Expression plasmid containing Bcl-2 shRNA was transfected into Raji cells by lipofectmine 2000 and then the transfected cells were treated with MTX. The expression levels of Bcl-2 mRNA and protein were evaluated by RT-PCR and immunofluorescence method at 48 h of transfection. MTT assay was used to analyze cell proliferation at 24, 48 and 72 h. Apoptosis was detected by Giemsa staining and flow cytomertric cell cycle analysis. Results:After transfection with Bcl-2 shRNA, the expression levels of Bcl-2 mRNA and protein in Raji cells were significantly decreased (P<0.05). Bcl-2 shRNA transfection plus MTX treatment induced marked apoptosis, decreased in cell proliferation activity, and increased in apoptotic rate. The difference was significant compared with MTX group, negative shRNA plus MTX group, Bcl-2 shRNA group, and empty plasmid plus MTX group (P<0.05). Conclusion:Bcl-2 shRNA could enhance MTX-induced apoptosis and inhibition of cell proliferation in Raji cells.

Construction of eukaryotic expression vector of pre-miR-15a and its inhibitory effect on Raji cells proliferation / 第三军医大学学报

Objective To construct an eukaryotic expression vector of pre-miR-15a,and to investigate the inhibitory effect of pre-miR-15a to Raji cells. Methods The pGCSIL-GFP vector encoding pre-miR-15a nucleotides was transfected into the bacterial competent cells,and then confirmed by PCR and sequencing analysis. The identified vector was transfected into Raji cells with oligofectamine 2000. The cells were divided into 3 groups,blank,negative control and pre-miR-15a group. Semi-quantitative RT-PCR was used to detect the expression of Bcl-2 mRNA,and immunofluorescence indirect for Bcl-2 protein expression. The growth of Raji cells was assayed by trypan blue dye exclusion method. Results PCR and sequences analysis indicated that the recombinant clones was identical with target sequences. Many green fluorescent cells were observed under fluorescent microscopy. The levels of Bcl-2 mRNA at every group had no obviously difference. Bcl-2 protein expression was obviously decreased at pre-miR-15a group compared with the other groups. Trypan blue dye exclusion method showed the cell growth was inhibited at 48 h and 72 h post-transfection. Conclusion We successfully construct the eukaryotic expression vector of pre-miR-15a,and it can inhibit the growth of Raji cells.

Effect of betulinic acid on proliferation,apoptosis,and cell cycle of human lymphoma cell line Raji / 中草药

Objective To investigate the anticancer effects and molecular mechanism of betulinic acid (BA)on Raji cells in vitro.Methods The effects of BA on the growth of Raji cells were studied by MTT assay.Apoptosis was assessed by Annexin-V/PI double-labeled cytometry.The influence on cell cycle was studied by flow cytometer.The cyclin D3 mRNA expression was checked by Western blotting and RT-PCR techniques.Results BA showed obvious inhibition on proliferation,as well as induction potency of apoptosis on Raji cells in vitro in a time-and dose-dependent manner by Annexin-V/PI double-labeled method.With the IC_(50)value for 24 h being(39.44?0.65)?g/mL,Raji cells treated with BA showed ac- cumulation in G_0/G_1 phase and reduction in the percentage of cells in S phase.The cyclin D3 mRNA ex- pression and protein were sharply decreased in Raji cells treated with BA.Conclusion BA could inhibit the proliferation of Raji cells by regulating the cell cycle that arrests cells at G_0/G_1 phase and induces apop- tosis of Raji cells.The antitumor effects of BA may be related to down-regulation of the expression of cy- clin D3.

Effects of hTERT antisense oligodeoxynucleotides on telomerase activity in Raji cells / 中国病理生理杂志

AIM: To explore the effect of hTERT gene antisense phosphorothioate oligodeoxynucleotide (ASODN) on telomerase activity in Raji cells. METHODS: Polymerase chain reaction enzyme-linked immuonassay (PCR-ELISA) was used to determine telomerase activity. The expression levels of hTERT mRNA and protein were assayed by reverse transcriptase polymerase chain reaction (RT-PCR) and immunofluorescence using fluoresce isothiocyanate (FITC) label,respectively. RESULTS: RT-PCR and immunofluorescence assay showed that the expression levels of hTERT mRNA and protein from Raji cells decreased with time after hTERT ASODN treatment. There was no difference in hTERT mRNA and protein levels between control and SODN-treated cells. Telomerase activity decreased when Raji cells were treated with ASODN for 48 h. Telomerase activity of Raji cells was significantly inhibited when treated with ASODN for 72 h. There was no difference in telomerase activity levels between control and hTERT SODN-treated cells. CONCLUSION: hTERT ASODN could inhibit telomerase activity in Raji cells.

Cloning and sequence analysis of death associated protein kinase gene ORF and DAPK1 inducing Raji cell apoptosis / 中国病理生理杂志

AIM: Open reading frame(ORF) of death associa ted protein kinase1(DAPK1) gene was cloned for studying on tumor forming and met astasis.METHODS: Based on nucleotide sequence of DAPK1 gene f rom GenBank, a pair of primers was designed. DAPK1 gene ORF was transfected into Raji cells in expression vector pcDNA3.1(+) with lipofectamine reagent. Morphol ogic assessment of apoptosis was performed with fluorescence microscope cytotoxi city and cell viability was assayed by MTT. RESULTS: DAPK1 gene ORF was amprified from K562 cells by RT-PCR. It was cloned into plasmid pMD18-T and sequenced. There were seven mutation in 4 300 bp nucleotide sequence rel ativel y to DAPK1 nucleotide sequence from GenBank, but six was synonymous mutation and one was single nucleotide polymorphism. 4 300 bp nucleotide of DAPK1 gene O RF was transfected into Raji cells. DAPK1 gene expression was detected in 48 h a fter it was transfected into Raji cells. Then Raji cells showed apoptosis.CONCLUS ION: Large fragment gene was cloned by RT-PCR and transfected into Raji cells successfully. Over-expression of DAPK1 gene induced Raji cells apoptosis.

Inhibitory effects of triptolide on cell proliferation and metastasis in Raji cells in vitro / 中国病理生理杂志

AIM:To investigate the inhibitory effects of triptolide on cell proliferation and metastasis in Burkitt's lymphoma cell line Raji cells.METHODS: The effects of triptolide on the growth of Raji cells were studied by 3-(4,5-Dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium(MTT) assay.The effects of triptolide on the cell apoptosis of Raji cells were detected by using Annexin Ⅴ/PI double-labled cytometry.The effects of triptolide on CXCR4 expression on Raji cells were studied by flow cytometric analysis.Chemotaxis assays were performed to observe the effects of triptolide on migration of Raji cells towards recombinant human SDF-1?(rhSDF-1?)in vitro.RESULTS: Triptolide inhibited the proliferation of Raji cells in a dose-and time-dependent way with a 24 h IC50 value of 43.06 nmol/L and a 36 h IC50 value of 25.08 nmol/L.Following the treatment of triptolide,the cell apoptosis rate was increased as the treatment concentration increased and the culture time extended.The effects were dose-and time-dependent.Triptolide could downregulate the expression of CXCR4 on Raji cells in a dose-dependent manner.Moreover,chemotaxis assay suggested that triptolide could block the migration of Raji cells to rhSDF-1? in vitro,and the inhibition was dose-dependent.CONCLUSION: Triptolide could inhibit the cell proliferation and induce the cell apoptosis of Raji cells.Furthermore,it could block the cell metastasis of Raji cells in vitro and the underlying mechanism might be related to the inhibition of the SDF-1/CXCR4 axis.