ABSTRACT
Objective: To investigate the effect of bisindolylmaleimide derivative L6 on inducing apoptosis of leukemia cells and its molecular mechanism. Methods: MTT assay was used to determinate the killing effect of L6 on HELL, K562, and KG1a cells. Flow cytometry was used to detect the effects of L6 on apoptosis, cell cycle, and differentiation of HEL cells. Western blotting was used to detect the expression of apoptosis-related protein. Finally, the effect of L6 on leukemia mouse was studied in vivo. Results: MTT assay showed that L6 had a stronger inhibitory activity against HEL, K562, and KG1a cell lines than the positive control PKC412 compound, with IC50 of (0.05 ± 0.03), (0.32 ± 0.01), and (0.19 ± 0.10) μmol/L, respectively. L6 could induce the apoptosis, G2/M arrest, megakaryocyte differentiation of HEL cells with a dose effect. Western blotting revealed that L6 mainly performed apoptosis by activating Caspase-3, which is an apoptotic executive protein. Hematoxylin-eosin (HE) staining of liver tissue of mice showed a reduction in HEL cell infiltration, but the more significant reduction in group L6 was observed, indicating that L6 could delay the metastasis of leukemia, and its effect was better than that of PKC412. Conclusion: Bisindolylmaleimide derivative L6 has a strong anti-leukemia activity, providing new hope for the development of new leukemia drugs.
ABSTRACT
Objective • To investigate changes of immune thrombocytopenia (ITP) patients-derived bone marrow mesenchymal cells (BMCs) in cells survival, cytokines expression as well as the effects of BMCs on the biological behaviors of megakaryocytes. Methods • BMCs were collected from 7 ITP patients and 5 normal controls (NC), and cultivated by the whole marrow adherent method. Surface markers and basal apoptosis rate of BMCs were analyzed by flow cytometry (FCM). Proliferation of BMCs was assessed by CCK-8 method. Phorbol 12-myristate 13-acetate (PMA) was used to stimulate differentiation of HEL cells. The induced HEL cells (inHEL) were divided into 3 groups: inHEL cultured alone (group a), inHEL co-cultured with BMCs derived from ITP patients (group b), inHEL co-cultured with BMCs derived from NC (group c). After 72 h incubation, the expression of cell surface proteins (CD41a, CD42b) and cell apoptosis rate were analyzed by FCM. The mRNA and proteins expression levels of cytokines IL6, IL11, TPO, SCF were detected by real-time fluorescent quantitative PCR (RT-qPCR) and enzyme linked immunosorbent assay (ELISA), respectively. Results • Compared with NC, BMCs from ITP patients grew progressively slowly (Day 4, P=0.039; Day 6, 10, P=0.009; Day 8, P=0.007), cell basal apoptosis rates were increased [AV+PI- (early apoptosis rate), P=0.036; AV+PI+ (late apoptosis rate), P=0.003; AV+PI-/+ (total apoptosis rate), P=0.004]. Compared with group a, the expression of CD41a in group c was much higher (P=0.000). The expression of CD41a in group b was higher than that in group a (P=0.015), but still much less than that in group c (P=0.000). Compared with group a, the early and total apoptosis rate in group b, c and the late apoptosis rate in group c were decreased obviously (all P=0.000), whereas there was no obvious change of the late apoptosis rate in group b. However, compared with group c, the late and total apoptosis rate in group b were significantly increased (both P=0.000). The expression levels of IL6, SCF mRNA and IL6 protein were significantly decreased in ITP BMCs (all P=0.000), but there was no obvious difference in the expression levels of IL11 and TPO between ITP BMCs and NC BMCs. Conclusion • BMCs from ITP patients show some defects in supporting megakaryocytic differentiation and survival under co-culture conditions, which mechanisms are related to the reduction of IL6 and SCF expression.
ABSTRACT
Objective·To investigate changes of immune thrombocytopenia (ITP) patients-derived bone marrow mesenchymal cells (BMCs) in cells survival, cytokines expression as well as the effects of BMCs on the biological behaviors of megakaryocytes. Methods?·?BMCs were collected from 7 ITP patients and 5 normal controls (NC), and cultivated by the whole marrow adherent method. Surface markers and basal apoptosis rate of BMCs were analyzed by flow cytometry (FCM). Proliferation of BMCs was assessed by CCK-8 method. Phorbol 12-myristate 13-acetate (PMA) was used to stimulate differentiation of HEL cells. The induced HEL cells (inHEL) were divided into 3 groups: inHEL cultured alone (group a), inHEL co-cultured with BMCs derived from ITP patients (group b), inHEL co-cultured with BMCs derived from NC (group c). After 72 h incubation, the expression of cell surface proteins (CD41a, CD42b) and cell apoptosis rate were analyzed by FCM. The mRNA and proteins expression levels of cytokines IL6, IL11, TPO, SCF were detected by real-time fluorescent quantitative PCR (RT-qPCR) and enzyme linked immunosorbent assay (ELISA), respectively. Results?·?Compared with NC, BMCs from ITP patients grew progressively slowly (Day 4, P=0.039; Day 6, 10, P=0.009; Day 8, P=0.007), cell basal apoptosis rates were increased [AV+PI- (early apoptosis rate), P=0.036; AV+PI+(late apoptosis rate), P=0.003; AV+PI-/+(total apoptosis rate), P=0.004]. Compared with group a, the expression of CD41a in group c was much higher (P=0.000). The expression of CD41a in group b was higher than that in group a (P=0.015), but still much less than that in group c (P=0.000). Compared with group a, the early and total apoptosis rate in group b, c and the late apoptosis rate in group c were decreased obviously (all P=0.000), whereas there was no obvious change of the late apoptosis rate in group b. However, compared with group c, the late and total apoptosis rate in group b were significantly increased (both P=0.000). The expression levels of IL6, SCF mRNA and IL6 protein were significantly decreased in ITP BMCs (all P=0.000), but there was no obvious difference in the expression levels of IL11 and TPO between ITP BMCs and NC BMCs. Conclusion?·?BMCs from ITP patients show some defects in supporting megakaryocytic differentiation and survival under co-culture conditions, which mechanisms are related to the reduction of IL6 and SCF expression.