Role of Ca-NFAT Signaling Pathway in Ph ALL Drug-resistance Mediated by Bone Marrow Stromal Cells / 中国实验血液学杂志

OBJECTIVE@#To explore the role of Ca-NFAT signaling pathway in Ph-ALL drug resistance mediated by bone marrow stromal cells.@*METHODS@#The transcription level of NFAT mRNA in Sup-B15 cells and Ph ALL primary cells was detected by polymerase chain reaction. The expression of P-glycoprotein in Sup-B15 cells was detected by flow cytometry. The change of NFAT protein in Sup-B15 cells was detected by Western blot. AnnexinV/7-AAD was used to label cells. Flow cytometry was used to detect cell apoptosis; Fluo 3-AM dye was used to label cells, and flow cytometry used to detect changes of Ca concentration in leukemia cells.@*RESULTS@#NFAT expression could be detected in both Sup-B15 and Ph ALL primary cells; P-glycoprotein could not be detected by flow cytometry; CAS could significantly inhibit NFAT protein expression in clinically applied drug concentrations (2.5, 5 μmol/L); Clinically applied concentration of CAS (2.5, 5 μmol / L) has no significant effect on the apoptosis of Sup-B15 cells, while higher concentration of CAS (10 μmol / L) could induce apoptosis of Sup-B15 cells. Bone marrow stromal cells OP9 could, decrease the sensitivity of Sup-B15 cells and Ph ALL primary cells to imatinib (IM); After co-culture with bone were marrow stromal cells, the Ca concentration in Sup-B15 cells was enhanced, the levels of NFAT protein and nullear protein in sup-B15 cells also were enhanced. The addition of CAS in co-culture system could inlibit the Ca-NFAT signaling pathway, reduce the protective effect of OP9 on Sup-B15 cells.Conclution:The Ca-NFAT sigualing pathway, contributes to the survival of Ph ALL cells. Bone marrow stromal cells can mediate the resistance of Ph ALL cells to IM by activating Ca-NFAT signaling pathway.

Statins Regulate the Proliferation and Apoptosis of T-ALL Cells through the Inhibition of Akt Pathway / 中国实验血液学杂志

<p><b>OBJECTIVE</b>To investigate the effect of Statins on proliferation and apoptosis in human acute T lymphocytic leukemia (T-ALL) cells and its possible mechanism.</p><p><b>METHODS</b>Jurkat and CCRF-CEM cells were cultured in different concentrations of Fluvastatin and Simvastatin for 24 h respectively. Then, the cell growth inhibition level was defected by CCK-8; the DNA replication was analyzed by EdU; the cell apoptosis was analyzed by Annexin V/7-AAD double labeling; the cell cycle changes were analyzed by flow cytometry; the expressions of Cyclin D1, p21, p27, BAX, BCL-2 and p-Akt were determined by Western blot.</p><p><b>RESULTS</b>Fluvastatin and Simvastatin both significantly inhibited the growth of Jurkat and CCRF-CEM cells in a dose-dependent manner. The inhibitory rate of Jurkat and CCRF-CEM cells at 0.2 mmol/L Fluvastatin was 41.14% and 57.08% respectively, while the 0.2 mmol/L Simvastatin could supress 68.42% of Jurkat and 77.10% of CCRF-CEM cells. Half or more than half of cell inhibition were observed in Statins-treated groups with significantly statistical differences, compared with the control groups (P<0.05). After the Jurkat and CCRF-CEM cells were treated with Fluvastation and Simvastation of different concentrations for 24 hours, the proportion of early and later apoptotic cells both increased; moreover, the total apoptotic rate increased significantly(P<0.05) at 0.2 mmol/L and 0.3 mmol/L concentration of Fluvastatin and Simvastatin. The detection of cell cycle showed that both of Jurkat and CCRF-CEM cells were arrested in G phase. Western blot revealed that, in comparison with the control group, the expressions of BAX, p21 and p27 in cells treated with Statins were up-regulated, while Cyclin D1, BCL-2 and p-Akt expressions were down-regulated.</p><p><b>CONCLUSION</b>Statins can suppress T-ALL cell proliferation and induce cell apoptosis through the inhibition of Akt pathway.</p>

Effect of Alantolactone on Proliferation of RPMI-8226 Cells and Its Possible Mechnism / 中国实验血液学杂志

<p><b>OBJECTIVE</b>To investigate the effect of alantolactone on perliferation and apoptosis of multiple myeloma (MM) RPMI-8226 cells, and to explore its possible mechism in vitro and in vivo.</p><p><b>METHODS</b>The RPMI-8226 cells were treated with alantolactone (1, 2.5, 5, 7.5 and 10 µmol/L) for 48 h, cell viability was detected by CCK-8 assay and the value of IC50 was calculated; The RPMI-8226 cells were treated with alantolactone (2.5, 5 and 7.5 µmol/L) for 48 h, the apoptotic rate was detected by flow cytmetry with Annexin V/PI staining; the expression level of cleaved caspase-3 and phosphorylation of ERK were measured by Western blot; the nude mice was used to further confirm the proapoptotic effect of alantolactone on MM cells in vivo.</p><p><b>RESULTS</b>The alantolactone inhibited RPMI-8226 cell viability remarkably with a dose-dependent manner; the IC50 value of RPMI-8226 cells at 48 h was 4.32 ± 0.15 µmol/L; the apoptotic rate increased observably with a dose-dependent manner; the levels of cleaved-caspase-3 increased and the phosphorylation of ERK decreased significantly; as compared to control, the volum of tumor was much smaller, the expression levels of Ki67 and p-ERK decreased.</p><p><b>CONCLUSION</b>The alantolactone can efficiently inhibit the proliferation and induce the apoptosis of multiple myeloma RPMI-8226 cells in vitro and in vivo through inhibiting the activation of ERK signal pathway.</p>

Effect of AZD8330 on proliferation and apoptosis of multiple myeloma cells / 中国实验血液学杂志

This study was aimed to investigate the effect of MEK inhibitor AZD8330 on proliferation and apoptosis of multiple myeloma IM9 and NCI-H929 cell lines and its possible mechanism. These two cell line cells were exposed to different concentrations of AZD8330 for 48 h. The CCK-8 assay was used to detect cell viability and the IC50 value at 48 h. These above-mentioned IM9 and NCI-H929 cells were treated with 5,10 and 100 nmol/L of AZD8330, then the change of cell cycle was analysed by flow cytometry with PI staining. The Wester blot was used to detect the expression levels of cyclin D and cyclin E, and multiple myeloma cells were treated with 10, 100, 1000 and 2000 nmol/L of AZD8330, the AnnexinV/7-AAD double staining was used to analyse cell apoptosis and the Western blot was used to detect the expression level of caspase-3. The results showed that AZD8330 could significantly inhibit the cell viability of IM9 and NCI-H929 cell lines in a time-and dose-dependent manner, the IC50 value (48 h) of IM9 and NCI-H929 were 19.88 ± 2.7 nmol/L and 29.3 ± 2.03 nmol/L respectively, these two cell lines were arrested on G1 phase of cell cycle, the apoptosis cells increased along with enhancement of AZD8330 concentration, and the expression level of cleaved caspase-3 protein was up-regulated. It is concluded that AZD8330 can efficiently inhibit the proliferation of NCI-H929 and IM9 cell lines, and induce apoptosis, suggesting that the AZD8330 may be a potential chemotherapeutic candidate for multiple myeloma therapy.