The impact of meisoindigo on apoptosis and proliferation of SET2 cell line by JAK-STAT pathway / 中华血液学杂志

Objective: To observe the effect of meisoindigo on apoptosis and proliferation of JAK2/V617F heterozygous mutation cell line-SET2 cell line to further explore the role of JAK-STAT pathway in this effect. Methods: Cell apoptosis after treated with different concentration of meisoindigo (0, 5, and 10 μmol/L) was evaluated by flow cytometry at different time points (24, 48, 72 h). Cell proliferation with CCK8 test was evaluated at different time points (24, 48, 72, 96 h) after administered with different concentration of meisoindigo (0, 5, 10, and 20 μmol/L). After treatment with different concentration of meisoindigo (0, 5, 10, and 20 μmol/L), SET2 cells were collected after 12 h, and then cultured in incomplete methylcellulose-based medium for clone formation. JAK-STAT signaling pathway and apoptosis related protein by Western blot test were evaluated 12 h after administered with different concentration of meisoindigo (0, 5, 10, and 20 μmol/L). Results: At different time points after treated with meisoindigo, the apoptosis rate of SET2 cell lines increased (P<0.01) with the inhibited proliferation (P<0.01), and the decreased clone formation rate of SET2 cell lines [0 μmol/L meisoindigo: (4.48±1.19)%, 20 μmol/L meisoindigo: (2.55±0.36)%; Dunnett P=0.020] in the presence of augmented concentrations of meisoindigo. At 12 hours after administration with meisoindigo, the reduced expressions of JAK2, P-JAK2, P-STAT1, P-STAT3, P-STAT3, STAT5, the decreased anti-apoptosis proteins BCL-2, BCL-XL and the increased pro-apoptosis protein BID, BIM were observed in the presence of increased concentrations of meisoindigo. Conclusion: Meisoindigo played an important role during the apoptosis and the inhibition of proliferation in ph-negative myeloproliferative neoplasm cell-SET2 cell lines, which might be related to the inhibition of JAK-STAT signaling pathway with up-regulation of pro-apoptosis protein and down-regulation of anti-apoptosis protein.

The anti-proliferative and anti-inflammatory mechanisms of JAK1 inhibitor SHR0302 versus Ruxolitinib in SET2 cell line and primary cells / 中华血液学杂志

Objective: To explore the effects and molecular mechanism of the selective JAK1inhibitor SHR0302 and Ruxolitinib on myeloproliterative neoplasms (MPN) cell line SET2 and primary cells in vitro. Methods: Cell proliferation was detected by CCK8 kit. Colony forming experiment was conducted to evaluate erythroid burst colony formation unit (BFU-E) of primary cells from MPN patients. Multi-factor kits were used to detect six inflammatory cytokines. Phosphorylated proteins of Jak-Stat signaling pathway were tested by Western blot. Results: At different time points after treated with SHR0302 and Ruxolitinib, the inhibition of cell proliferation was dose dependent by both drugs (P<0.01) . The inhibitory rates of 2.5 μmol/L SHR0302 and 0.1 μmol/L Ruxolitinib on SET2 cells for 72 h were comparable, i.e. (59.94±0.60) % and (64.00±0.66) %, respectively, suggesting that the inhibitory effect of SHR0302 was weaker than that of Ruxolitinib. Similarly, both SHR0302 and Ruxolitinib inhibited BFU-E in primary marrow cells from MPN patients in a dose-dependent manner. SHR0302 1.0 μmol/L produced similar degree of inhibition compared to Ruxolitinib 0.2 μmol/L. Except IL-12, the expression of other 5 cytokines (IL-6, TNF-α, IL-1β, IL-2, IL-8) was significantly inhibited by 1.6 μmol/L SHR0302 in SET2 cells at 24 h (P<0.01) , while Ruxolitinib 1.0 μmol/L had the same effect. Several phosphorylated molecules of Jak-Stat signaling pathway were significantly inhibited by SHR0302 in SET2 cells only for 3 h. P-stat1 (Tyr701) , p-stat3 (Tyr705) were down-regulated when treated with SHR0302 1.0 μmol/L (P<0.05) , p-jak1 (tyr1022/1023) and p-stat5 (Tyr694) were inhibited at 5.0 μmol/L (P<0.05) . Ruxolitinib significantly inhibited the downstream STAT protein at 0.1 μmol/L. Again, the inhibitory effect of SHR0302 on protein expression was weaker than that of Ruxolitinib. Conclusion: SHR0302 can effectively inhibit the proliferation of MPN cell line and patients' primary cells, as well as the expression of inflammatory factors. The molecular mechanism is possibly related to the down-regulation of phosphorylated proteins of Jak-Stat signaling pathway. Overall, the anti-proliferative and anti-inflammatory effects of SHR0302 are weaker than those of Ruxolitinib.

Targeted sequencing analysis of hyper-eosinophilic syndrome and chronic eosinophilic leukemia / 中华血液学杂志

Objective: Analysis of the molecular characteristics of eosinophilia. Methods: Targeting sequence to 24 patients with chronic eosinophilic leukemia (CEL) with rearrangement of PDGFRA, PDGFRB, or FGFR1 and 62 patients with hyper-eosinophilic syndrome (HES). Mutation annotation and analysis of amino acid mutation using authoritative databases to speculate on possible pathogenic mutation. Results: Thirty-seven kinds of clonal variant were detected from 17 patients with CEL, no recurrent mutation site and hot spot region were found. No pathogenic mutation was detected in 19 patients with PDGFRA rearrangement, but pathogenic mutations of ASXL1, RUNX1 and NRAS were detected from 2 patients with FGFR1 rearrangement who progressed to acute myeloid leukemia and 1 patient with PDGFRB rearrangement who progressed to T lymphoblastic lymphoma, respectively. One hundred and two kinds of clonal abnormalities were detected in 49 patients with HES. The main hot spot mutation regions included: CEBPA Exon1, TET2 Exon3, ASXL1 Exon12, IDH1 Y208C, and FGFR3 L164V. CRRLF2 P224L and PDGFRB R370C point mutations were detected separately in 2 patients with HES who treated with imatinib monotherapy and achieved hematologic remission. Conclusion: The pathogenesis of CEL with PDGFRA, PDGFRB or FGFR1 rearrangement is usually single, and the progression of the disease may involve other driver mutation. A variety of genes with hot mutation regions may be involved in the pathogenesis of HES, and some mutation sites are sensitive to tyrosine kinase inhibitors.