The natural alkaloid Jerantinine B has activity in acute myeloid leukemia cells through a mechanism involving c-Jun

Abstract

Background: Acute myeloid leukemia (AML) is a heterogenous hematological malignancy with poor long-term survival. New drugs which improve the outcome of AML patients are urgently required. In this work, the activity and mechanism of action of the cytotoxic indole alkaloid Jerantinine B (JB), was examined in AML cells.

Methods: We used a combination of proliferation and apoptosis assays to assess the effect of JB on AML cell lines and patient samples, with BH3 profiling being performed to identify early effects of the drug (4 h). Phosphokinase arrays were adopted to identify potential driver proteins in the cellular response to JB, the results of which were confirmed and extended using western blotting and inhibitor assays and measuring levels of reactive oxygen species.

Results: AML cell growth was significantly impaired following JB exposure in a dose-dependent manner; potent colony inhibition of primary patient cells was also observed. An apoptotic mode of death was demonstrated using Annexin V and upregulation of apoptotic biomarkers (active caspase 3 and cleaved PARP). Using BH3 profiling, JB was shown to prime cells to apoptosis at an early time point (4 h) and phospho-kinase arrays demonstrated this to be associated with a strong upregulation and activation of both total and phosphorylated c-Jun (S63). The mechanism of c-Jun activation was probed and significant induction of reactive oxygen species (ROS) was demonstrated which resulted in an increase in the DNA damage response marker γH2AX. This was further verified by the loss of JB-induced C-Jun activation and maintenance of cell viability when using the ROS scavenger N-acetyl-L-cysteine (NAC).

Conclusions: This work provides the first evidence of cytotoxicity of JB against AML cells and identifies ROS-induced c-Jun activation as the major mechanism of action.

Keywords: Acute myeloid leukemia; Jerantinine; c-Jun; reactive oxygen species.

Fig. 1

Cytotoxicity of jerantinine B in AML cell lines.a. Structure of JB and JBa. b. Mean IC₅₀ values of JB at 24 h. Columns, mean of three independent experiments; bars, SD

Fig. 2

Induction of apoptosis in IC₅₀JB exposed AML cells.a. Flow cytometric analysis of Annexin V/propidium iodide staining following IC₅₀-JB treatment for 24 and 72 h. Representative flow cytometry plots and summary histograms are shown. A+/PI− indicates cells undergoing early stage apoptosis, while A+/PI+ defines late stage apoptotic populations. b. Summary bar chart of flow cytometric analysis of cleaved PARP and active caspase 3 apoptotic markers following 24 h IC₅₀ JB exposure. c. BH3 profiling following 4 h IC₅₀-JB treatment in MV4–11 cells. Cytochrome C release demonstrates PUMA- and BAD-BH3 peptides prime the cells to apoptosis. PUMA2A is a mutated peptide which acts as a negative control . Columns, mean of at least three independent experiments; bars, SD. * P < 0.05, ** P < 0.01, *** P < 0.001

Fig. 3

JB activates c-Jun through ROS induction.a. Western blot (cropped) demonstrating 4 h JB exposure results in a strong upregulation of total c-Jun and activation of c-Jun (S63 phosphorylation) in AML cell lines. Lamin is shown as the loading control and the figure is representative of three independent experiments. b. JB induced intracellular ROS in AML cell lines. The bar charts indicate the fold change in median fluorescence intensity compared to untreated controls upon addition of the oxidative stress indicator CM-H2DCFDA, with the elimination of ROS seen when the anti-oxidant NAC is included. Representative flow cytometry plots of ROS measurements are shown above the corresponding bar charts. c. Western blot results (cropped) showing elimination of JB-dependent c-Jun activation by either ROS scavenger or JNKI, representative of three independent experiments. d. cell counts after 24 h incubation showing a combination of ROS scavenger or JNKI with JB treatment reversed JB-induced cell death, displayed as % viability of untreated control. e. DNA damage, assessed by the response marker γH2AX, is increased in JB-treated cells. Bars represent the mean of the Median Fluorescence Intensity (MFI) in respect to the negative untreated control. Etoposide was used as a positive control. Columns, mean of at least three independent experiments; bars, SD. * P < 0.05 and ** P < 0.01. Full length blots for the westerns in this figure are shown in additional file 3

Fig. 4

Effect of JB on colony formation of AML primary cells.a. Survival fraction of four AML patient samples when treated with JB. Results are displayed as mean ± SD of survival fraction percent. b. Representative images of AML cells from a patient sample showing the effect of JBA on colony formation at a range of drug concentration

Fig. 5

Suggested mechanism of action of JB in AML cells. JB in AML exerts its effect through increasing ROS level that cause c-Jun/JNK activation as well as DNA damage. JB also targets PLK1 that contributes to G2/M arrest. Activated c-Jun/JNK may contribute to microtubule disruption and ultimately G2/M arrest. JB was reported to bind directly to the colchicine site on microtubule and inhibits microtubule polymerisation but this was not tested in this study