Artesunate suppresses tumor growth and induces apoptosis through the modulation of multiple oncogenic cascades in a chronic myeloid leukemia xenograft mouse model

Abstract

Artesunate (ART), a semi-synthetic derivative of artemisinin, is one of the most commonly used anti-malarial drugs. Also, ART possesses anticancer potential albeit through incompletely understood molecular mechanism(s). Here, the effect of ART on various protein kinases, associated gene products, cellular response, and apoptosis was investigated. The in vivo effect of ART on the growth of human CML xenograft tumors in athymic nu/nu mice was also examined. In our preliminary experiments, we first observed that phosphorylation of p38, ERK, CREB, Chk-2, STAT5, and RSK proteins were suppressed upon ART exposure. Interestingly, ART induced the expression of SOCS-1 protein and depletion of SOCS-1 using siRNA abrogated the STAT5 inhibitory effect of the drug. Also various dephosphorylations caused by ART led to the suppression of various survival gene products and induced apoptosis through caspase-3 activation. Moreover, ART also substantially potentiated the apoptosis induced by chemotherapeutic agents. Finally, when administered intraperitoneally, ART inhibited p38, ERK, STAT5, and CREB activation in tumor tissues and the growth of human CML xenograft tumors in mice without exhibiting any significant adverse effects. Overall, our results suggest that ART exerts its anti-proliferative and pro-apoptotic effects through suppression of multiple signaling cascades in CML both in vitro and in vivo.

Figure 1. The Human Phospho-Kinase Array detection kit reveals that ART suppresses phosphorylation of p38, ERK, STAT5, and CREB in KBM-5 cells

(A) The chemical structure of Artesunate (ART). (B) KBM-5 human myeloid leukemia cells (1 × 10⁷ cells/well) were either untreated or treated with 50 μM of ART for 4 h. Parts A and B of the array were each incubated with 300 μg of cell lysate. Arrays were done according to the manufacturer's protocols using Human Phospho-Kinase Array Kit (R&D Systems, Minneapolis, MN), and array images are shown. (C) Array profiles created by quantifying the mean spot pixel densities are shown. Graphs represent spot intensities of indicated proteins. (D) After KBM-5 cells (1 × 10⁶ cells/well) were seeded onto 6-well plates, they were treated with various indicated concentrations of ART for 4 h. Thereafter, equal amounts of lysates were analyzed by Western blot analysis using antibodies against p-p38, p38, p-ERK, ERK, p-STAT5 and STAT5. (E) ART suppresses STAT5 and CREB-DNA binding activity. KBM-5 cells (1 × 10⁶ cells/well) were treated with various indicated concentrations of ART for 4 h, analyzed for nuclear STAT5 and CREB levels by EMSA. (F) KBM-5 cells (1 × 10⁶ cells/well) were treated with various indicated concentrations of ART for 4 h. After that, nuclear proteins were extract, equal amounts of lysates were analyzed by Western blot analysis using antibodies against p-STAT5, STAT5, p-CREB, and CREB. (G) After KBM-5 cells (1 × 10⁶ cells/well) were seeded onto 6-well plates, they were treated with various indicated concentrations of ART for 4 h. Thereafter, equal amounts of lysates were analyzed by Western blot analysis using antibodies against p-JAK2, and JAK2. (H) KBM-5 cells (1 × 10⁶ cells/well) were treated with various indicated concentrations of ART for 4 h, after which whole-cell extracts were prepared and 10 μg portions of those extracts were resolved on 8% SDS-PAGE, electrotransferred onto nitrocellulose membranes, and probed for SOCS-1 antibody. The same blots were stripped and reprobed with β-actin antibody to verify equal protein loading (H, left panels). KBM-5 cells (1 × 10⁶ cells/well) were treated with various indicated concentrations of ART for 4 h, and total RNA was extracted and examined for expression of SOCS-1 by RT-PCR. GAPDH was used as an internal control to show equal RNA loading (H, right panels). (I) Effect of SOCS-1 knockdown on ART induced expression of SOCS-1. KBM-5 cells were transfected with either SOCS-1 siRNA or scrambled siRNA (50 nM). After 48 h, cells were treated with 100 μM ART for 4 h and whole-cell extracts were subjected to Western blot analysis. (J) ART causes the inhibition of translocation of STAT5 to the nucleus. After 4 h of ART treatment, the cells were fixed and permeabilized. STAT5 (green) was immunostained with mouse anti-STAT5 followed by FITC-conjugated secondary antibodies and the nuclei (blue) were stained with DAPI. The third panels show the merged images of the first and second panels. The results shown are representative of two independent experiments.

Figure 2. ART induces apoptosis by PARP cleavage through activation of caspase-3

(A) and (B) After KBM-5, U266, MM1.S, AsPC-1, H1299, DU145, and MCF-7 cells (1 × 10⁶ cells/well) were seeded onto 6-well plates, they were either untreated or treated with 50 μM of ART for 24 h. Then, the cells were fixed and analyzed using a flow cytometry. (C) and (D) Various tumor cells (1 × 10⁶ cells/well) were treated with indicated concentrations of ART for 24 h. Thereafter, equal amounts of lysates were analyzed by Western blot analysis using antibodies against caspase-3 and PARP. The same blots were stripped and reprobed with β-actin antibody to verify equal protein loading.

Figure 3. ART and chemotherapeutic agents induce apoptosis

(A) Inhibition of bcl-2, bcl-xL, and survivin mRNA expressions by ART in KBM-5 cells. Cells (1 × 10⁶ cells/well) were treated with indicated concentrations of ART for 24 h. Total RNA was isolated, bcl-2, bcl-xL, and survivin mRNA expressions were examined by RT-PCR analysis. GAPDH was performed to control for a similar initial cDNA content of the sample. The results shown are representative of the three independent experiments. (B) KBM-5 cells (1 × 10⁶ cells/well) were treated with indicated concentrations of ART for 24 h. Thereafter, equal amounts of lysates were analyzed by Western blot analysis using antibodies against bcl-2, bcl-xL, survivin, and IAP-1/2. The same blots were stripped and reprobed with β-actin antibody to verify equal protein loading. (C) Cells (1 × 10⁶ cells/well) were treated with indicated concentrations of ART for 24 h. Total RNA was isolated, bax and p21 mRNA expressions were examined by RT-PCR analysis. GAPDH was performed to control for a similar initial cDNA content of the sample. The results shown are representative of the three independent experiments. (D) Cells (1 × 10⁶ cells/well) were treated with indicated concentrations of ART for 24 h. Thereafter, equal amounts of lysates were analyzed by Western blot analysis using antibodies against bax and p21. The same blots were stripped and reprobed with β-actin antibody to verify equal protein loading. (E) KBM-5 cells were treated with ART at 50 μM and 100 μM concentrations for 24 h and the cells were incubated with an FITC-conjugated Annexin V antibody and then analyzed by a flow cytometry. (F) After treatment of ART for 24 h, the cells were fixed and incubated using TUNEL reaction solution and then analyzed by a flow cytometry. (G) KBM-5 cells (1 × 10⁴ cells/well) were incubated at 37°C with 30 μM doxorubicin, 1.5 nM paclitaxel, and 1.5 nM docetaxel in the presence and absence of 10 μM ART as indicated for 24 h, and the viable cells were assayed using the MTT reagent. (H) KBM-5 cells (1 × 10⁶ cells/well) were treated 30 μM doxorubicin, 1.5 nM paclitaxel, and 1.5 nM docetaxel in the presence and absence of 10 μM ART as indicated for 24 h. Thereafter, equal amounts of lysates were analyzed by Western blot analysis using antibodies against survivin and PARP. The same blots were stripped and reprobed with β-actin antibody to verify equal protein loading.

Figure 4. Effects of ART in human myeloid leukemia cells growth in nude mice induced by KBM-5

(A) Schematic representation of experimental protocol described in “Materials and Methods.” KBM-5 cells (4 × 10⁶ cells/mice) were injected subcutaneously into the right flank of the mice. The animals were randomized after 1 week of tumor cell injection into four groups based on tumor volume. Group I was given PBS (200 μL, i.p. thrice/week), group II was given ART (50 mg/kg body weight, i.p. thrice/week), group III was given ART (100 mg/kg body weight, i.p. thrice/week), and group IV was given ART (200 mg/kg body weight, i.p. thrice/week). (B) Necropsy photographs of mice bearing subcutaneously implanted myeloid leukemia tumors. (C) Tumor volumes in mice measured during the course of experiment and calculated using the formula V = 4 / 3 πr³, ** indicates p < 0.01. (D) Tumor volumes in mice measured on the last day of the experiment at autopsy using Vernier calipers and calculated using the formula V = 4 / 3 πr³ (n = 5). Columns, mean; bars, SE. ** indicates p < 0.01.

Figure 5. ART exerts the effect against tumor cell proliferation and angiogenesis in myeloid leukemia

(A) Immunohistochemical analysis of proliferation marker Ki-67⁺ cell indicates the inhibition of human myeloid leukemia cells proliferation by ART dose-dependent treated groups of animals. Samples from 3 animals in each treatment group were analyzed, and representative data are shown (A, left panel). Quantification of Ki-67 proliferation index as described in “Materials and Methods.” Values are represented as mean ± SE of triplicate (A, right panel). Columns, mean of triplicate; bars, SE. (B) Immunohistochemical analysis of VEGF and cleaved caspase-3 in myeloid leukemia tumors. Samples from 3 animals in each treatment group were analyzed. (C–E) Western blot analysis showed the inhibition of p-p38, p-ERK, p-CREB, p-STAT5, p-JAK2, and SOCS-1 by ART in whole cell extracts from animal tissue. The same blots were stripped and reprobed with p38, ERK, CREB, STAT5, JAK2, and β-actin antibody to verify equal protein loading. (F) and (G) Equal amounts of lysates were analyzed by Western blot analysis using antibodies against bcl-2, bcl-xL, survivin, IAP-1, IAP-2, bax, and p21. β-actin was used as a loading control. Western blotting samples from three mice in each group were analyzed and representative data are shown.