Silvestrol exhibits significant in vivo and in vitro antileukemic activities and inhibits FLT3 and miR-155 expressions in acute myeloid leukemia

Abstract

Background: Activating mutations [internal tandem duplication (ITD)] or overexpression of the FMS-like tyrosine kinase receptor-3 (FLT3) gene are associated with poor outcome in acute myeloid leukemia (AML) patients, underscoring the need for novel therapeutic approaches. The natural product silvestrol has potent antitumor activity in several malignancies, but its therapeutic impact on distinct molecular high-risk AML subsets remains to be fully investigated. We examined here the preclinical activity of silvestrol in FLT3-ITD and FLT3 wild-type (wt) AML.

Methods: Silvestrol in vitro anti-leukemic activity was examined by colorimetric cell viability assay, colony-forming and flow cytometry assays assessing growth inhibition and apoptosis, respectively. Pharmacological activity of silvestrol on FLT3 mRNA translation, mRNA and protein expression was determined by RNA-immunoprecipitation, qRT-PCR and immunoblot analyses, respectively. Silvestrol in vivo efficacy was investigated using MV4-11 leukemia-engrafted mice.

Results: Silvestrol shows antileukemia activity at nanomolar concentrations both in FLT3-wt overexpressing (THP-1) and FLT3-ITD (MV4-11) expressing AML cell lines (IC50 = 3.8 and 2.7 nM, respectively) and patients' primary blasts [IC50 = ~12 nM (FLT3-wt) and ~5 nM (FLT3-ITD)]. Silvestrol increased apoptosis (~4fold, P = 0.0001), and inhibited colony-formation (100%, P < 0.0001) in primary blasts. Silvestrol efficiently inhibited FLT3 translation reducing FLT3 protein expression by 80-90% and decreased miR-155 levels (~60%), a frequently co-regulated onco-miR in FLT3-ITD-positive AML. The median survival of silvestrol-treated vs vehicle-treated mice was 63 vs 29 days post-engraftment, respectively (P < 0.0001).

Conclusions: Silvestrol exhibits significant in vivo and in vitro antileukemic activities in AML through a novel mechanism resulting in inhibition of FLT3 and miR-155 expression. These encouraging results warrant a rapid translation of silvestrol for clinical testing in AML.

Figure 1

Antileukemic activity of silvestrol in vitro: (a) MV4-11 cells were incubated with 5 to 160 nM silvestrol. Cell viability was evaluated by a MTS assay at 24, 48 and 72 hours. (b) AML cell lines were incubated with 5 to 160 nM silvestrol, and viability was evaluated by a MTS assay 48 hours later. (c) AML primary cells unmutated FLT3 or with FLT3-ITD were incubated with 5 to 320 nM silvestrol. Cell viability was evaluated by MTS assay 48 hours following treatment. (d) Colony forming ability in primary blasts from FLT3-ITD positive and negative AML patients treated with 25 nM of silvestrol and scored 14 days later. (e) Primary blasts from FLT3-ITD positive and negative AML patients treated with 10, 30 and 50 nM of silvestrol for 48 hours and analyzed by annexin/PI flow cytometry. Values are presented as mean ± SEM.

Figure 2

Silvestrol down-regulates FLT3: (a) FLT3 mRNA expression in eIF4E immunoprecipitates from 50 nM silvestrol treated MV4-11 cells compared with that from untreated cells. Values are presented as mean ± SEM. (b) Expression of FLT3 examined by flow cytometry in MV4-11 and THP-1 cells after 24 hours exposure to 50 nM silvestrol. Blue = silvestrol-treated samples; Red = vehicle-treated control. (c) AML cells treated with 50 nM silvestrol and examined for FLT3 protein expression by immunoblotting. (d) MV4-11 cells treated with silvestrol or PKC412 (50 nM each) for 6 hours, and assessed for STAT5 phosphorylation by immunoblotting. (e) Primary AML cells incubated with increasing concentrations of silvestrol for 24 hours, and assessed for FLT3 protein expression by immunoblotting.

Figure 3

Silvestrol down-regulates miR-155 and upregulates miR-155 target PU.1: (a) MV4-11 cells were treated with 50 nM of silvestrol and miR-155 expression was measured and normalized to U44 expression at 6, 12 and 24 hours following silvestrol treatment. (b) MV4-11 cells were treated with 50 nM of silvestrol for 24 hours, and miR-34a expression was measured thereafter. (c,d) MV4-11 cells were treated with 50 nM of silvestrol for 24 hours, and miR-155 (c) and PU.1 expression (d) levels were measured thereafter. (e,f) FLT3-ITD positive primary cells were treated with 50 nM of silvestrol for 24 hours, and miR-155 expression (e) and PU.1 expression (f) levels were measured thereafter. (g) MV4-11 and THP-1 cells were treated with 50 nM of silvestrol for 24 hours and assessed for P65 protein expression by immunoblotting. Values are presented as mean ± SEM.

Figure 4

In vivo antileukemic activity of silvestrol in FLT3-ITD positive MV4-11 xenograft leukemia mouse model: (a) FLT3 expression by flow cytometry evaluated 48 hours after 1^st and 2^nd doses. (b) Spleen size after 3 silvestrol doses (day 6 of treatment) (c) Immunoblot of FLT3 protein expression in spleens from silvestrol-treated mice and vehicle-treated controls. (d) Cytospins of bone marrow cells and (e) histopathology of bone marrow (sternum), spleen, and liver from the silvestrol-treated and vehicle-treated leukemic mice and age-matched (non-leukemic) control mice showing the infiltration of leukemic blasts in the vehicle treated and signs of differentiation in the silvestrol treated mice (see red pointing arrows). Stain: H&E; magnifications, 400×. (f) Survival analysis of silvestrol-treated leukemic mice (N = 10) compared with the vehicle-treated controls (N = 6). Values are presented as mean ± SEM.