Figure 2 | Blood Cancer Journal

Figure 2

From: Macrolide antibiotics exert antileukemic effects by modulating the autophagic flux through inhibition of hERG1 potassium channels

Figure 2

Effects of combination treatment with MAs and conventional antileukemic agents in ALL and AML cell lines in vitro and in vivo. A panel of leukemic cell lines were cultured with or without MSC (suspension) and exposed to LD50 of doxorubicin (Doxo; 0.1 μg/ml) or cytarabine (Cyt; 45 nm) with or without the corresponding LD50 dose of the MAs antibiotic Cla/Er for 48 h. (a) ALL cell lines (BCP-ALL: 697, REH) exposed to LD50 of Doxo in the presence of LD50 of Cla. (b) AML cell lines (FLG 29.1; HL60) exposed to LD50 of Cyt in the presence of LD50 of Cla. The percentage of Annexin V+/propidium iodide (PI)− cells was measured. Values are mean±s.e.m. of three indipendent experiments each performed in triplicate. (c) 697 cells were cultured with or without MSC and exposed to LD50 of prednisone (5 μm) with or without the LD50 dose of Er for 48 h. The percentage of Annexin V+/PI− cells was measured. Values are mean±s.e.m. of two indipendent experiments each performed in triplicate. (d) Three representative pediatric AML primary samples were cultured onto MSCs and treated with LD50 doses relative to FLG 29.1 of either Cyt (45 nm, see Supplementary Information), MA antibiotic Cla (56 μm) and E4031 (50 μm) for 48 h. The percentage of Annexin V+/PI− cells was measured. Values are mean±s.e.m. of one experiment performed in triplicate. Statistical analysis was carried out with the Student’s t-test (AML-1: Cyt+Cla vs Cyt, P<0.01; AML-2: Cyt+Cla vs Cyt, P<0.01; AML-3: Cyt+Cla vs Cyt, P<0.01). (e) SCID mice were injected with HL60-luc2 cell line (5 × 106 cells intraperitonially (i.p.)) and starting from day 5, animals were treated daily for 14 consecutive days with saline (control, n=4), Cyt (6.25 mg/kg, i.p., n=4); Cla (15 mg/kg, by oral gavage, n=4). Images were acquired with Photo Acquisition software (Biospace Laboratory, Paris, France) and processed with M3 Vision software (Biospace Laboratory). Median values of counts per minutes (c.p.m.) reported for each group of treatment at different time points are shown in the right panel. (f) NOD SCID mice were inoculated with REH cells on day 0 and after one week treated for 2 weeks with saline (Con, n=4) and Er (Er15, 15 mg/kg, n=4) and sacrificed 3 weeks after cell injection. Leukemia BM engraftment and PB burden were evaluated by FACS analysis estimating the hCD45+/mCD45+ ratio and were reported as percentage of the control for each treatment group. (g) SCID mice were injected with HL60-luc2 cell line (5 × 106 cells i.p.) and starting from day 5, animals were treated daily for 14 consecutive days with saline (control, n=4), Cyt (6.25 mg/kg, ip, n=4); Cla (15 mg/kg, by oral gavage, n=4) and Cyt (6.25 mg/kg, i.p.) plus Cla (15 mg/kg, by oral gavage, n=4). Survival curves of each experimental group, estimated by Kaplan and Meier analysis are reported (P=0.0024). (h) NOD SCID mice were inoculated with REH cells on day 0 and treated for 14 consecutive days with saline (Con, n=5), Dexa (15 mg/kg, n=5) and Dexa (15 mg/kg) plus Er (15 mg/kg, n=5). Survival curves of Control, Dexa and Dexa+Er experimental group, estimated by Kaplan and Meier analysis are reported (P=0.208): median survival is 22 days in control group, 48.5 days in Dexa group and 108 days in Dexa+Er group. Inset. An additional group of mice (n=3 per each group of treatment, treated as reported above) were analyzed 3 weeks after cell injection and BM and PB collected. Leukemia BM engraftment and PB burden were evaluated by FACS analysis estimating the hCD45+/mCD45+ ratio and were reported as percentage of the control for each treatment group.

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