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OBJECTIVE To stud y the regulation mechanism of miRNA- 18a and miRNA-4802 on drug resistance of lymphoma cells via autophagy. METHODS Using human burkitt ’s lymphoma cell Daudi and human mantle cell lymphoma cell JeKo- 1 as the research objects ，adriamycin（ADR）and vincristine （VCR）as experimental drugs. After treatment of ADR and VCR ，relative cell viability was detected with CCK- 8 kit；the expression of apoptosis marker protein activated cleaved caspase- 9 and cleaved caspase-6 were detected by Western blot assay. The drug resistances of the two cells to ADR and VCR were investigated. The difference of autophagy activity between the two kinds of cells by expression detection of autophagy related proteins LC 3-Ⅱ and p62，autophagy flow experiment and transmission electron microscope observation. Fluorescence quantitative polymerase chain reaction was used to investigate the expression differences of miRNA- 18a and miRNA- 4802，ULK1 and ATG 7 mRNA in the two cells，and to detect the expression differences of ULK 1 and ATG 7 proteins. Taking JeKo- 1 cells as the research object ，the changes of autophagy activity and drug resistance were investigated after treatment with endogenous miRNAs （miRNA-18a mimics ， miRNA-4802 mimics）of two simulated organisms. RESULTS After ADR and VCR treatment ，compared with Daudi cells ，JeKo-1 cells had stronger drug resistance and autophagy activity. The expression of miRNA- 18a and miRNA- 4802 in JeKo- 1 cells were significantly lower than Daudi cells ，mRNA and protein expression of ULK 1 and ATG 7 were significantly higher than those of Daudi cells （P＜0.001）. After treatment of miRNA- 18a mimics and miRNA- 4802 mimics，the autophagy activities and drug resistances of JeKo- 1 cells were decreased significantly. CONCLUSIONS miRNA-18a and miRNA- 4802 can decrease drug resistances of lymphoma cells to ADR and VCR by reducing 2 the expression of autophagy-initiating genes ULK1 and ATG7， and inhibiting the autophagy activity of lymphoma cells.
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AIM: To observe the mechanism of celecoxib reversal adriamycin resistance in NK/T cell lymphoma cells. METHODS: SNK6 and SNK6/ADR cells were treated with celecoxib of different concentrations (10, 20, 40, 60, 80, 100 μmol/L), the growth inhibition rate of SNK6 and SNK6/ADR were measured by MTT method. The IC
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Objective To investigate the effect of TGF-β1 on the proliferation of lymphoma cells induced by M2 macrophages. Methods TGF-β1 was used to treat Jiyoye lymphoma cells, and MTT was used to determine the cell proliferation. Lymphoma cells were treated with M2 macrophage culture liquid supernatant and TGF-β1. MTT assay was used to assess the lymphoma cell proliferation, Transwell assay was used to assess the cell migration and invasion, and Western blot was perfomed to determine the levels of MMP-2, MMP-9, PCNA and Ki-67 proteins. Results The lymphoma cells after TGF-α treatment showed that the proliferation ability, the number of invaded cells and the number of migrated cells, and the levels of MMP-2, MMP-9, PCNA and Ki-67 were significantly decreased, compared with the untreated lymphoma cells (P＜ 0. 05). After treated with the supernatant of M2 type macrophage culture supernatant, the proliferation ability of lymphoma cells, the number of invaded cells and migrated cells, the levels of MMP-2, MMP-9, PCNA and Ki-67 proteins were significantly increased, compared with the untreated lymphoma cells (P ＜ 0. 05 ). Compared with the lymphoma cells treated with culture medium supernatant of M2 macrophages alone, the lymphoma cells treated with TGF-β1 and M2 macrophage culture medium supernatant showed that the proliferation ability, number of invaded and migrated cells, and the protein levels of MMP-2, MMP-9, PCNA and Ki-67 were significantly increased (P＜0. 05). Conclusions TGF-β1 can reduce the proliferation, invasion and migration of lymphoma cells induced by macrophages. The mechanism of action is related to the expression of MMP-2, MMP-9, PCNA and Ki-67 proteins.
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AIM:To observe the influences of IL-6 and AG490 on the growth of Raji cell line ( Burkitt lym-phoma cell, BL).METHODS:Raji cells were cultured.IL-6, an activator of signal transducer and activator of transcrip-tion 3 (STAT3), and AG490, a specific inhibitor of STAT3 were added into the medium respectively .The expression of STAT3 and survivin at mRNA and protein levels was detected by real-time PCR and Western blot .The cell viability was measured by MTT assay .Apoptosis and cell cycle were examined by flow cytometry .RESULTS:IL-6 or AG490 affected the growth of Raji cells significantly in a dose-dependent manner (P<0.05).The mRNA expression of STAT3 and sur-vivin in Raji cells was higher in IL-6 group, and lower in the AG490 group than that in the corresponding control group . The statistical differences were found in the mRNA expression of STAT 3 and survivin among different IL-6 or AG490 groups (P<0.05).The concentration dependent relationship was also presented in IL-6 and AG490 groups by the regression a-nalysis.The results of Western blot showed that the protein levels of phosphorylated STAT 3 (p-STAT3), STAT3 and sur-vivin were increased in IL-6 group, and decreased in AG490 group.The apoptotic rate of Raji cells was gradually reduced with the increasing concentration of IL-6.The opposite results were detected in the Raji cells treated with AG 490.There was significant difference in constitute of the cell cycle between the groups treated with IL -6 or AG490 and corresponding control group.The cells at G1-phase and G1/S were significantly increased , while those at S-phase had no obvious change under treating with AG490.The cells at S-phase decreased obviously in the Raji cells treated with IL-6.CONCLUSION:IL-6 and AG490 distinctly affect the growth of Raji cells .The mechanism may be associated with the activation of STAT 3 and survivin.
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Objective To find lymphoma cell markers characteristic of gas for breath monitoring application and provide experimental evidence for the early diagnosis of lymphoma patients,assessing solid phase micro extraction-gas chromatography / mass spectrometry( SPME-GC / MS)is applied to the lymphatic the feasibility of detecting tumor cells in the headspace. To investigate the value of VOCs in the diagnosis of hematologic malignancies. Meth-ods The air samples from the headspace of non-Hodgkin lymphoma cell JEOK,the human lymphoidcell lines and culture medium were collected by syringes,and then determined by means of SPME-GC / MS in order to have a bet-ter understanding of the concentration distributions and changes of VOCs in JEOK cells headspace. Using Mann-Whitney U test,we found the characteristic volatile markers of non-Hodgkin′s lymphoma cells. Results SPME-GC /MS for JEOK cell lines,human lymphocytecell line and a blank incubation were detected headspace analysis,found JEOK cell culture headspace can be detected dimethyl,sulfide,toluene,o-xylene,1,3-Di-tert-butylbenzene,aceto-pheno-ne,dodecane,representing human lymphocytes increased;and alcohol,benzaldehyde,hexanal is signifi-cantly reduced. Conclusion Non-Hodgkin′s lymphoma cell lines can cause flasks JEOK headspace change in the composition of VOCs,increased alkanes and aromatic compounds,aldehydes and reducing alcohol content,these substances may be used as acharacteristic of lymphoma cells markers;SPME-GC / MS as a trace substance detection method can be used to JEOK cell lines VOCs bottle headspace detection training.
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Objective:To investigate the in vitro effect of arsenic trioxide (As2O3) alone and in combination with dexamethasone (DXM), etoposide (VP-16), methotrexate (MTX), bortezomib (BTZ), and suberoylanilide hydroxamic acid (SAHA) on the growth of human cutaneous T cell lymphoma (CTCL) cells Hut-78 and Hut-102. Methods:Hut-78 and Hut-102 cells were cultured with different concentrations of As2O3, DXM, VP-16, MTX, BTZ, and SAHA alone and As2O3 in combination with DXM, VP-16, MTX, BTZ, or SAHA for 48 h. The effects of the different samples on Hut-78 and Hut-102 cell proliferation were determined by MTT assay. Analyses using CalcuSyn software were performed to determine whether the combination of As2O3 with DXM, VP-16, MTX, BTZ, or SAHA in-duced synergistic cytoxicity. Results:As2O3, DXM, VP-16, MTX, BTZ, and SAHA alone significantly inhibited the growth of Hut-78 and Hut-102 cells in a dose-dependent manner, with a 50%inhibiting concentration of 5μmol/L, 500μg/mL, 2.5μg/mL, 1μg/mL, 10μmol/L, and 2.5μmol/L individually after 48 h of culture. As2O3 with DXM, VP-16, MTX, BTZ, or SAHA showed remarkable antitu-mor efficacy compared with that of individual applications. Conclusion:As2O3 alone or combined with DXM, VP-16, MTX, BTZ, or SAHA significantly inhibited Hut-78 and Hut-102 cell growth in vitro. This study demonstrated that As2O3 with DXM, VP-16, MTX, BTZ, or SAHA presents synergistic antitumor effects on CTCL cells and may be an optimal regimen in clinical trials of CTCL.
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Objective To compare the incorporation method of 3H-TdR and 125Ⅰ-UdR on determining the proliferation effect of lymphocytes. Methods The proliferation effects of lymphocyte and Daudi lymphoma cells were estimated by 3H-TdR and 125Ⅰ-UdR incorporation. Results The incorporating fraction of 3H-TdR and 125Ⅰ-UdR into lymphocyte was 20.95% ± 1.06% and 1.00% ±0.04%,respectively, and the incorporating fraction for the lymphoma cells was 29. 94% ± 4. 10% and 6. 02% ±0. 73% ,respectively. The incorporation fractions of 3H-TdR into lymphocyte and lymphoma cells were much higher than those of 125Ⅰ-UdR, but the incorporating fractions of 3H-TdR or 125Ⅰ-UdR into the lymphoma cells were much higher than those of lymphocytes. Conclusions For lymphocytes, 125Ⅰ-UdR cannot substitute 3H-TdR as a tracer agent. But for lymphoma cells, whether 125Ⅰ-UdR could be replace 3H-TdR or not needs further research.
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Objective To evaluate the killing effect and the uptake of 125I-UdR on human lymphoma Raji and Daudi cell lines. Methods The amount of 125I-UdR in the cells and cell nuclei were determined after incubation of different time in RPMI 1640 culturing medium containing different concentrations of 125I-UdR. The killing effects of 125I-UdR on Raji and Daudi cell lines were estimated through MTT assay and cell cycle was analyzed by propidium iodide (PI) staining. Results The amounts of 125I-UdR in Raji and Dandi cells and cell nuclei were much higher than that of Na125I(P < 0.05). The amounts of 125l-UdR in Raji and Daudi cells were 14414±95 and (6916± 53.69) Bq/106 cell when the concentration was 100 kBq/ml. The amounts of Na125I were 68± 3.8 and (324±32.8) Bq/106 cell. The uptake of 125I-UdR in Raji and Daudi cells and cell nuclei increased with the 125I-UdR concentration and incubated time. The cell surviving fractions of 125I-UdR groups was much lower than that of Na125I groups (P < 0.05). When the concentration was 500 kBq/ml and incubated time was 48 hours, the Raji and Dandi cell surviving fractions of125I-UdR groups were (19.78 ± 1.39)% and (43.17 ± 2.69) % ;those of Na125I groups were (79.10 ± 1.79) % and (80.36 ± 6.12) %. The surviving fractions of 125I-UdR groups reduced with the 125I-UdR concentration. Conclusions 125I-UdR can be specially ingested by Raji and Daudi cells and incorporated into DNA, then the cells will be killed. The uptake of 125I-UdR is dose and time dependent.
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Objective To explore the effect and molecular mechanism of proteasome inhibitor in TNF-related apoptosis-inducing ligand (TRAIL)-induced apoptosis resistance on malignant lymphoma cells.Methods Raji cells were treated with TRAIL and proteasome inhibitor (PS-341) in vitro and the cell growth index was evaluated by MTT assay; cell cycle was analysed by flow cytometry; the protein and mRNA level of Bax were measured by Western blotting and real time RT-PCR. Results TRAIL inhibited proliferation of Raji cells at the concentration of 500 μg/L, but the inhibition rate was lower than that of the control cell:Hmy2.ciR.TRAIL arrested cell in G0/G1 phase. The Bax protein in Raji is degraded, but the Bax mRNA expression level does not change significantly .The effects of TRAIL was enhanced significantly 10 nmol/L PS-341 was added. Conclusion Raji cells are resistant in TRAIL-induced apoptosis. This effect may be related to the decrease of Bax protein. The Ubiquitin-proteasome pathway is involved in the degradation of Bax in TRAIL-treated Raji cells.
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Objective To explore the inhibition and apoptosis of the human cutaneous T-cell lymphoma cell lines Hut-78 by traditional Chinese medicine SVCⅢ. Methods After Hut-78 cells were treated with SVCⅢ of different concentration, the inhibition and apoptosis of Hut-78 cells was determined by MTT, agarose gel electrophoresis of DNA fragment and FACS. Results SVCⅢ could inhibit remarkably Hut-78 cells growth and DNA ladder was seen by agarose gel electrophoresis. The proliferation of Hut-78 cells were inhibited in G_1 stage by FACS. Conclusion SVCⅢ can promote growth retardation and apoptosis of human cutaneous T-cell lymphoma cell lines Hut-78, which suggests SVCⅢ has antineoplastic function.