Antineoplastic effects of CPPTL via the ROS/JNK pathway in acute myeloid leukemia

Abstract

Drug resistance and human leukocyte antigen (HLA) matching limit conventional treatment of acute myeloid leukemia (AML). Although several small molecule drugs are clinically used, single drug administration is not sufficient to cure AML, which has a high molecular diversity. Metabolic homeostasis plays a key role in determining cellular fate. Appropriate levels of reactive oxygen species (ROS) maintain the redox system balance, and excessive amounts of ROS cause oxidative damage, thus providing a strategy to eliminate cancer cells. CPPTL is a novel analogue of parthenolide that exhibited significant cytotoxicity to AML cells in vitro and induced apoptosis in a dose-dependent manner. Additionally, CPPTL's prodrug DMA-CPPTL decreased the burden of AML engraftment and prolonged survival in a mouse model administered human primary AML cells in vivo. CPPTL induced apoptosis of AML cells by stimulating ROS production, and accumulation of ROS then activated the JNK pathway, thereby promoting mitochondrial damage. These results demonstrated that CPPTL effectively eradicated AML cells in vitro and in vivo and suggested that CPPTL may be a novel candidate for auxiliary AML therapy.

Keywords: CPPTL; JNK pathway; ROS; acute myeloid leukemia.

Figure 1. CPPTL inhibited leukemic cell proliferation and induced apoptosis

(A) The chemical structure of CPPTL. (B) Cytotoxicity of cell lines exposed to CPPTL, as determined by MTT assays. We used MTT assays to determine the concentration that inhibited approximately 50% cell proliferation. All IC₅₀ values are the average of three independent experiments. (C) Percentage of viable KG1a cells and (D) percentage of viable primary AML cells, assessed after treatment with CPPTL for 24 h. CON represents the vehicle control. (E) Percentage of apoptosis was assessed in MNCs isolated from human umbilical cord blood and treated with CPPTL for 24 h. (F) Representative flow cytometry images of primary AML samples treated with CPPTL. All data represent the mean ± SD. *p < 0.05, **p < 0.01, ***p < 0.005, ****p < 0.001.

Figure 2. CPPTL eliminated AML cells in vivo and prolonged survival in a mouse model

(A) Schematic of the xenotransplantation experiment. NOD/SCID mice received 2.5 Gy irradiation before transplantation of 1 × 10⁷ primary human AML MNCs via the tail vein. AML mice were randomly distributed to groups receiving 100 mg/kg of DMA-CPPTL via i.g. administration or vehicle every 48 h for 7 treatments. The positive control group received 2 mg/kg of ADR, a traditional chemotherapy drug, via i.v. injection every 72 h for 4 treatments. After 8 weeks, we detected CD45⁺ cells in peripheral blood to assess the success of AML engraftment. The survival curve shows only mice transplanted with AML successfully. (B) Synthesis of DMA-CPPTL. (C) Survival curves of 2.5 Gy-irradiated NOD/SCID mice transplanted with primary AML MNCs receiving treatment with DMA-CPPTL (n = 7, shown in blue), ADR (n = 9, shown in red) and vehicle control (n = 9, shown in black). (D) FACS analysis of the percentage of CD45⁺ cell engraftment in bone marrow after natural death of AML mice. (E) Percentage of mice exhibiting varying degrees of leukemic cell engraftment.

Figure 3. CPPTL induced apoptosis by stimulating ROS generation

(A) Western blot analysis of KG1a cells treated with or without the ROS inhibitor NAC, or treated with NAC alone for 24 h. All lanes contained aliquots of protein extract samples. (B) ΔΨm levels were measured on the basis of TMRE fluorescence in KG1a cells treated with CPPTL for 24 h. (C) ROS levels were measured on the basis of DCF-DA fluorescence in KG1a cells treated with CPPTL for 1 h. (D) Representative flow cytometry images of ROS detection. (E) ROS levels in KG1a cells after co-treatment with CPPTL and NAC for 1 h. (F) Percentage of viable KG1a cells, assessed after co-treatment with CPPTL and NAC for 24 h. (G) ROS levels in KG1a cells treated with 5 μM CPPTL at different time points. All data represent the mean ± SD. *p < 0.05, **p < 0.01, ***p < 0.005, ****p < 0.001.

Figure 4. JNK activation contributed to CPPTL-induced apoptosis of AML cells

Western blot analysis of KG1a cells (A) treated with different concentrations of CPPTL for 24 h, (B) treated with or without the ROS inhibitor NAC, or treated with NAC alone for 24 h. All lanes contained aliquots of protein extract samples.

Figure 5. Hypothesized model of hierarchical signaling events leading to apoptosis of AML cells after treatment with CPPTL