Simalikalactone E (SkE), a new weapon in the armamentarium of drugs targeting cancers that exhibit constitutive activation of the ERK pathway

Abstract

Simalikalactone E (SkE) is a quassinoid extracted from a widely used Amazonian antimalarial remedy. Although SkE has previously been shown to have cytostatic and/or cytotoxic activities in some tumor cell lines, its mechanism of action has not yet been characterized. We show here that SkE in the high nanomolar range inhibited the growth of various leukemic and solid tumor cell lines. Importantly, SkE was highly efficient at inhibiting chronic myelogenous leukemia (CML) cells that exhibit constitutive activation of the MAPK pathway and, accordingly, it impaired the phosphorylation of ERK1/2. SkE also abrogated MEK1/2 and B-Raf phosphorylation but had no effect on Ras activity. Moreover, SkE was particularly effective against melanoma cell lines carrying the B-Raf-V600E mutation. Importantly, SkE resensitized the PLX-4032-resistant 451Lu melanoma cell line (451Lu-R) and was more efficient than U0126, a MEK inhibitor, and PLX-4032 (PLX) at inducing the apoptosis of two hairy cell leukemia (HCL) patient samples carrying the B-Raf-V600E mutation. Finally, SkE was as efficient as imatinib at inhibiting tumor formation in a xenograft model of CML cells in athymic mice. In conclusion, we show that SkE, a very potent inhibitor of B-Raf-V600E, is highly effective against cancer cell lines that exhibit constitutive activation of the ERK1/2 pathway.

Figure 1. SkE treatment induces cell death of CML cell lines and primary CML CD34+ cells

(A) SkE in the 100-500 nM range was added to K562 CML cell lines growing in semi-solid methyl cellulose medium (0.5'10³ cells/ml). Imatinib (1 μM) was used as an internal control. Colonies were detected after 10 days of culture by adding 1 mg/ml of MTT reagent and were scored by Image J quantification software. Results are expressed as the number of colony forming cells per well after drug treatment. Results are given as the mean ± SD of 3 different determinations made in triplicate. Error bars = 95% confidence intervals. (B) Primary CML CD34+ cells were incubated for 48 h at 37°C with increasing concentrations of SkE. The cell metabolism was measured by the XTT assay, as described in the Materials and Methods section. Results are given as the mean ± SD of 3 different determinations made in triplicate. Error bars = 95% confidence intervals.

Figure 2. SkE treatment impairs ERK1/2 phosphorylation

(A) K562 cells were treated with 250 nM of SkE for 2 h; then, cells were lysed, and cell lysates were loaded on a Pathscan multikinase® membrane. (B) Histograms represent the relative intensity quantification of the most regulated dot with Image J software. Results are expressed as the percentage of kinase phosphorylation in SkE-treated cells versus control cells. (C) K562 cells were incubated at 37°C with 250 nM SkE for the indicated times. Whole-cell lysates were prepared, and the expression of Phospho-C-Abl, C-Abl, Phospho-STAT5, Phospho-CRKL, PARP, Phospho-S6RP, S6RP, Phospho-ERK1/2, ERK1/2 and LC3b was visualized on a Western blot.

Figure 3. SkE can inhibit the RAF/MEK/ERK signaling pathway

HEK Raf-ER cells were pre-treated with increasing doses of SkE (A) or U0126 (B) for 1 h. Cells were then treated with tamoxifen (1 μM) for one additional hour. Protein samples were separated by electrophoresis, and the expression of Phospho-ERK and ERK was visualized on a Western blot. (C) K562 leukemic cells were treated for different times with 250 nM SkE. The status of phosphorylation of BRAF, MEK and ERK was visualized by Western blot. (D) K562 cells were incubated at 37°C with 250 nM SkE for the indicated times. Ras activity was determined after GST-pull-down. Ras-GTP levels were determined using GST-c-Raf RBD to pull down active GTP-bound Ras from cell extracts by glutathione beads. The beads were washed 4 times and subjected to SDS/PAGE (12% polyacrylamide). Ras and Phospho-ERK1/2 proteins were detected by Western blot analysis. (E) K562 cells were treated with 250 nM SkE for 3 h. Cells were then washed and placed in fresh medium for 1 h, 3 h or 24 h. The BRAF, MEK and ERK1/2 protein levels and their phosphorylation status were analyzed by Western blot. HSP60 and HSP90 were used as the loading controls.

Figure 4. SkE induces cell death of tumors exhibiting the BRAF V600E mutation

(A) PLX-sensitive and PLX-resistant 451Lu melanoma cell lines were treated with either 2 μM PLX or increasing doses of SkE. Forty-eight hours later, cell metabolism was measured by the XTT assay as described in the Materials and Methods section. (B) Primary blood cells from patients suffering from hairy cell leukemia were treated with 2 μM PLX, 10 μM U0126 or increasing doses of SkE. Twenty-four hours later, cells were stained using propidium iodide, and cell death was analyzed with a cytometer.

Figure 5. Chronic treatment with SkE is as effective as imatinib in inhibiting the growth of tumors derived from CML cells in athymic mice

A total of 5'10⁶ K562 leukemic cells were implanted in both flanks in athymic mice. After tumor establishment, when the tumors reached 100 mm3, animals received a daily intraperitoneal injection of vehicle, imatinib (60 mg/kg body weight) or SkE (1 mg/kg body weight). (A) This picture shows the size of tumors at day 16 post injection with vehicle (left panel), Imatinib (middle panel) and SkE (right panel). (B) This panel shows the evolution of tumor growth, expressed in cpm at different times post-injection of the same treatments. (C) Tumors were removed, frozen and cut to perform immune staining. Slides containing a representative section of each tumor were incubated with either an anti-phospho-ERK1/2 or anti-ERK1/2 antibody. Slides were finally mounted and analyzed under a fluorescence microscope.