1-Cinnamoyltrichilinin from Melia azedarach Causes Apoptosis through the p38 MAPK Pathway in HL-60 Human Leukemia Cells

Acute myeloid leukemia (AML) is an aggressive type of human leukemia with a low survival rate, and its complete remission remains challenging. Although chemotherapy is the first-line treatment of AML, it exerts toxicity in noncancerous cells when used in high doses, thus necessitating the development of novel compounds with a high therapeutic window. This study aimed to investigate the anticancer effects of several compounds derived from the fruits of Melia azedarach (a tree with medicinal properties). Among them, 1-cinnamoyltrichilinin (CT) was found to strongly suppress the viability of HL-60 human leukemia cells. CT treatment induced apoptosis and increased nuclear fragmentation and fractional DNA content in HL-60 cells in a dose-dependent manner. CT induced phosphorylation of p38 mitogen-activated protein kinases (p38), though not of c-Jun N-terminal kinase (JNK) and extracellular signal-regulated kinase (ERK), and activated Bcl-2 family proteins towards the proapoptosis and cleavage of caspase-3 and poly (ADP-ribose) polymerase. Both CT-mediated apoptosis and apoptotic protein expression were reversed by treatment with the p38 inhibitor, thereby indicating the p38 pathway to be critical in CT-stimulated apoptosis. The results collectively indicated CT to suppress HL-60 survival by activating the p38 pathway and inducing apoptosis, hence being a novel potential therapeutic agent for AML.


Introduction
Acute myeloid leukemia (AML) is an aggressive cancer characterized by abnormal proliferation of myeloid cells in the bone marrow or blood [1]. Myeloid progenitor cell differentiation is impaired in AML, thereby disrupting the balance of blood cell populations, resulting in immune dysfunction. As one of the most common leukemias among adults, AML is primarily caused by the accumulation of genetic mutations in hematopoietic stem cells, a progenitor cell type for all blood cells, displaying pervasive aggressiveness in patients with AML, whose five-year overall survival rate is <30% [2]. First-line treatment of AML includes chemotherapy, targeting cell division, which successfully induces recovery [3], since noncancerous cells continue to multiply through cell division; however, the applications are limited in some patients with cancer, especially the older individuals with health risk

CT Induced Apoptosis in HL-60 Cells
To determine whether CT promotes apoptosis in HL-60 cells and decreases their viability, we performed Hoechst 33342 staining to detect nuclear fragments, which is an indicator of apoptotic progression in cancer cells [11]. As shown in Figure 3A, nuclear fragmentation, including the appearance of apoptotic bodies, was observed in HL-60 cells upon treatment with CT in a dose-dependent manner. Similar results were obtained by assessing the sub-G 1 population by flow cytometry in response to CT treatment [12]. CT treatment increased the amount of sub-G 1 DNA content from 1.5% to 61.2% in a dose-dependent manner ( Figure 3B), hence suggesting apoptosis in HL-60 cells to be stimulated by CT, thereby decreasing cell viability.

CT Induced Apoptosis in HL-60 Cells
To determine whether CT promotes apoptosis in HL-60 cells and decreases their viability, we performed Hoechst 33342 staining to detect nuclear fragments, which is an indicator of apoptotic progression in cancer cells [11]. As shown in Figure 3A, nuclear fragmentation, including the appearance of apoptotic bodies, was observed in HL-60 cells upon treatment with CT in a dosedependent manner. Similar results were obtained by assessing the sub-G1 population by flow cytometry in response to CT treatment [12]. CT treatment increased the amount of sub-G1 DNA content from 1.5% to 61.2% in a dose-dependent manner ( Figure 3B), hence suggesting apoptosis in HL-60 cells to be stimulated by CT, thereby decreasing cell viability.

CT Activated p38 Mitogen-Activated Protein Kinase in HL-60 Cells
Apoptosis is mediated by the activation of mitogen-activated protein kinases (MAPKs), including p38 MAPK (p38), c-Jun N-terminal kinase (JNK), and extracellular signal-regulated kinase (ERK) [13]. To determine the type of MAPK signaling pathway involved in the apoptosis of HL-60 cells exposed to CT, we performed Western blotting for phosphorylated p38, JNK, and ERK in HL-60 cells. Based on the finding of dose-dependent toxicity in HL-60 cells ( Figure 2B), we determined 10 μM as the concentration suppressing approximately half the growth of HL-60 cells and used it for monitoring cell growth improvement in subsequent experiments. The phosphorylation level of only p38 was significantly increased upon CT treatment, whereas those of JNK and ERK were not affected significantly ( Figure 4); this indicated the selective induction of p38 phosphorylation, though not of JNK or ERK, by CT.
To determine the type of MAPK signaling pathway involved in the apoptosis of HL-60 cells exposed to CT, we performed Western blotting for phosphorylated p38, JNK, and ERK in HL-60 cells. Based on the finding of dose-dependent toxicity in HL-60 cells ( Figure 2B), we determined 10 µM as the concentration suppressing approximately half the growth of HL-60 cells and used it for monitoring cell growth improvement in subsequent experiments. The phosphorylation level of only p38 was significantly increased upon CT treatment, whereas those of JNK and ERK were not affected significantly ( Figure 4); this indicated the selective induction of p38 phosphorylation, though not of JNK or ERK, by CT.

CT-Induced Apoptosis Was Prevented by p38 Inhibition
To evaluate the role of MAPKs in the growth inhibitory effect of CT, HL-60 cells were treated with MAPK inhibitors after CT treatment. As shown in Figure 5A, SP600125 and PD98059, the specific inhibitors of JNK and ERK, respectively, did not influence the reduced viability due to CT treatment; however, SB203580, a specific p38 inhibitor, significantly improved cell viability after CT treatment, thus indicating p38 to be critical for cell death upon treatment with CT. To investigate whether CTinduced apoptosis is hindered by the p38 inhibitor, we performed Hoechst 33342 nuclear staining and flow cytometry. Cotreatment with SB203580 and CT in HL-60 recovered apoptotic bodies ( Figure  5B) and remarkably decreased the population of sub-G1 cells from 34.1% to 9.8% compared to the CT condition alone ( Figure 5C). The results suggested p38 to be critical for the apoptosis of HL-60 cells upon treatment with CT.

CT-Induced Apoptosis Was Prevented by p38 Inhibition
To evaluate the role of MAPKs in the growth inhibitory effect of CT, HL-60 cells were treated with MAPK inhibitors after CT treatment. As shown in Figure 5A, SP600125 and PD98059, the specific inhibitors of JNK and ERK, respectively, did not influence the reduced viability due to CT treatment; however, SB203580, a specific p38 inhibitor, significantly improved cell viability after CT treatment, thus indicating p38 to be critical for cell death upon treatment with CT. To investigate whether CT-induced apoptosis is hindered by the p38 inhibitor, we performed Hoechst 33342 nuclear staining and flow cytometry. Cotreatment with SB203580 and CT in HL-60 recovered apoptotic bodies ( Figure 5B) and remarkably decreased the population of sub-G 1 cells from 34.1% to 9.8% compared to the CT condition alone ( Figure 5C). The results suggested p38 to be critical for the apoptosis of HL-60 cells upon treatment with CT.

Inhibition of p38 Improved HL-60 Viability by Regulating Apoptotic Factors
Apoptosis progresses through the activation of Bcl-2 family protein and cleavage of caspase-3 and poly (ADP-ribose) polymerase (PARP) [14]. To determine the effect of CT on apoptosis, we quantified the expression levels of apoptosis-related proteins, including Bcl-xL (antiapoptotic), Bax (proapoptotic), cleaved caspase-3, and PARP, using Western blotting. First, p38 MAPK phosphorylation, enhanced by CT, was recovered after cotreatment with SB203580 ( Figure 6). Next, CT decreased Bcl-xL and increased Bax, cleaved caspase-3, and cleaved PARP, thus activating proapoptotic factors; however, SB203580 partially or completely reversed the expression of all proteins to the baseline levels ( Figure 6). These results suggested the activation of apoptotic signaling cascades in HL-60 cells by CT via p38 phosphorylation.

Inhibition of p38 Improved HL-60 Viability by Regulating Apoptotic Factors
Apoptosis progresses through the activation of Bcl-2 family protein and cleavage of caspase-3 and poly (ADP-ribose) polymerase (PARP) [14]. To determine the effect of CT on apoptosis, we quantified the expression levels of apoptosis-related proteins, including Bcl-xL (antiapoptotic), Bax (proapoptotic), cleaved caspase-3, and PARP, using Western blotting. First, p38 MAPK phosphorylation, enhanced by CT, was recovered after cotreatment with SB203580 ( Figure 6). Next, CT decreased Bcl-xL and increased Bax, cleaved caspase-3, and cleaved PARP, thus activating proapoptotic factors; however, SB203580 partially or completely reversed the expression of all proteins to the baseline levels ( Figure 6). These results suggested the activation of apoptotic signaling cascades in HL-60 cells by CT via p38 phosphorylation. Figure 6. Effect of CT and a p38 inhibitor on apoptotic factors. Western blot using HL-60 cells treated with CT (10 μM) and/or SB203580 (SB, 15 μM) for 6 h to assess phospho-p38 (p-p38) and apoptotic proteins, including Bcl-xL, Bax, cleaved caspase-3, and poly (ADP-ribose) polymerase (PARP). β-Actin was used as the loading control. The relative band intensity of proteins to that of β-actin is expressed as the fold change compared to the control (no treatment) and is indicated below each band.

Discussion
This study provided clear evidence of CT inducing apoptosis in HL-60 cells via p38 phosphorylation. CT is an oxygenated triterpenoid derivative containing a furan ring [15]. Limonoids, identified as the core component causing bitterness in citrus fruits, exhibit anticancer activity both in vitro and in vivo [16,17]. In neuroblastoma cells, bioactive limonoid compounds induce apoptosis by increasing caspase-3/7 activity [16]. Limonoids from citrus fruits have been reported to suppress chemically induced hepatocarcinogenesis in rats by decreasing lipid peroxidation and oxidative stress [17]. Some limonoid compounds isolated from M. azedarach have been shown to induce cell cycle arrest and apoptosis in human leukemia cells via ERK1/2 activation [18]. The present results suggested a trichilin-type limonoid to trigger apoptosis and hinder cell growth in cancer cells.
Apoptosis is a type of programmed cell death that maintains tissue homeostasis during aging and development [11]. During apoptosis, cells shrink and display nuclear and organellar lysis, contrary to necrosis, in which they undergo energy-independent cell death, accompanied by swelling. Necrosis is characterized as an accidental cell death, with an uncontrolled release of intracellular contents, thus initiating inflammatory responses in surrounding tissues [11]. Thus, examining therapeutic compounds inducing apoptosis rather than necrosis is considered an appropriate strategy in cancer research. Here, we treated HL-60 cells with CT and examined whether it triggers apoptosis in these cells. The breakdown of cellular components yields small membranebound compartments containing DNA, called apoptotic bodies, which are detectable through nuclear staining [11]. DNA multimers of <200 bp are formed, owing to the extracellular leakage of apoptotic nuclear fragments, thus reducing the overall DNA content; such cells are referred to as sub-G1 cells, and confirmed by flow cytometry [12]. Cancer cells evade apoptosis via defective apoptotic signaling due to accumulated mutations [19]. Half of all tumor types present overexpressed antiapoptotic Bcl-2 proteins and the loss of proapoptotic factors, resulting in resistance to apoptotic stimuli, including Figure 6. Effect of CT and a p38 inhibitor on apoptotic factors. Western blot using HL-60 cells treated with CT (10 µM) and/or SB203580 (SB, 15 µM) for 6 h to assess phospho-p38 (p-p38) and apoptotic proteins, including Bcl-xL, Bax, cleaved caspase-3, and poly (ADP-ribose) polymerase (PARP). β-Actin was used as the loading control. The relative band intensity of proteins to that of β-actin is expressed as the fold change compared to the control (no treatment) and is indicated below each band.

Discussion
This study provided clear evidence of CT inducing apoptosis in HL-60 cells via p38 phosphorylation. CT is an oxygenated triterpenoid derivative containing a furan ring [15]. Limonoids, identified as the core component causing bitterness in citrus fruits, exhibit anticancer activity both in vitro and in vivo [16,17]. In neuroblastoma cells, bioactive limonoid compounds induce apoptosis by increasing caspase-3/7 activity [16]. Limonoids from citrus fruits have been reported to suppress chemically induced hepatocarcinogenesis in rats by decreasing lipid peroxidation and oxidative stress [17]. Some limonoid compounds isolated from M. azedarach have been shown to induce cell cycle arrest and apoptosis in human leukemia cells via ERK1/2 activation [18]. The present results suggested a trichilin-type limonoid to trigger apoptosis and hinder cell growth in cancer cells.
Apoptosis is a type of programmed cell death that maintains tissue homeostasis during aging and development [11]. During apoptosis, cells shrink and display nuclear and organellar lysis, contrary to necrosis, in which they undergo energy-independent cell death, accompanied by swelling. Necrosis is characterized as an accidental cell death, with an uncontrolled release of intracellular contents, thus initiating inflammatory responses in surrounding tissues [11]. Thus, examining therapeutic compounds inducing apoptosis rather than necrosis is considered an appropriate strategy in cancer research. Here, we treated HL-60 cells with CT and examined whether it triggers apoptosis in these cells. The breakdown of cellular components yields small membrane-bound compartments containing DNA, called apoptotic bodies, which are detectable through nuclear staining [11]. DNA multimers of <200 bp are formed, owing to the extracellular leakage of apoptotic nuclear fragments, thus reducing the overall DNA content; such cells are referred to as sub-G 1 cells, and confirmed by flow cytometry [12]. Cancer cells evade apoptosis via defective apoptotic signaling due to accumulated mutations [19]. Half of all tumor types present overexpressed antiapoptotic Bcl-2 proteins and the loss of proapoptotic factors, resulting in resistance to apoptotic stimuli, including anticancer therapeutic drugs [20]. One method of treating tumors is to terminate their uncontrollable growth and activate apoptosis. Targeting apoptotic factors is an attractive method for inducing cell death in tumors. In this study, CT significantly increased nuclear fragmentation and sub-G 1 DNA content and triggered apoptosis in HL-60 cells.
MAPK proteins, key regulators of cellular proliferation, differentiation, and apoptosis, are activated by cytokines or extracellular stresses [13]. Upon stimulation, serine/threonine kinases are phosphorylated and regulate numerous substrates, leading to differential signal transduction, depending on the type of stimulus. The three primary components of MAPKs are p38, JNK, and ERK; ERK is primarily involved in survival, whereas p38 and JNK are responsive to stress conditions, including the apoptotic phenotype [21]. In cancer cells, however, MAPK signaling cascades are dysregulated, owing to genetic mutations or environmental stimuli; hence, numerous studies have focused on developing therapeutic agents to restore the balance of MAPK function [22]. In this study, p38, though not JNK and ERK, was phosphorylated during CT-mediated HL-60 apoptosis. To evaluate the direct association of the MAPK pathway in cancer apoptosis, many researchers have determined whether MAPK inhibitors can reverse compound-mediated apoptosis. We observed that the p38 inhibitor restored the elevated levels of apoptotic bodies and sub-G 1 DNA content to the baseline levels, whereas JNK and ERK inhibitors had no such effect, suggesting p38 to play an important role in the apoptosis of HL-60 cells upon CT treatment.
The apoptotic pathway is triggered by mitochondrial cytochrome c release [23]. Once released, cytochrome c binds to apoptotic protease activating factor-1, generating an apoptosome, which, in turn, initiates the caspase cascade, cleaving pro-caspase-9 and pro-caspase-3 to their active forms, thereby leading to PARP cleavage [14]. Mitochondrial cytochrome c release is mediated by Bcl-2 family proteins. Under physiological conditions, proapoptotic proteins, including Bax, Bak, and Bad, are downregulated. In the presence of apoptotic signals, however, the ratio of proapoptotic to prosurvival proteins, including Bcl-xL and Bcl-2, is increased, resulting in the permeabilization of the mitochondrial outer membrane and the release of cytochrome c [23]. The present results showed CT to upregulate Bax, cleaved caspase-3, and PARP and downregulate Bcl-xL, implying the apoptotic caspase cascade to be triggered by CT. This activation could be reversed by the p38 inhibitor, hence validating the essential role of p38 activation in CT-induced apoptosis in HL-60 cells.
In summary, this study demonstrated the anticancer activity of CT in HL-60 human leukemia. CT induced apoptosis in HL-60 cells by upregulating apoptotic proteins, including Bax, caspase-3, and PARP. Mechanistically, p38 activation was demonstrated to be important for CT-mediated apoptosis, which could be prevented by the p38 inhibitor. The results together suggested CT to be a potent anticancer therapeutic agent, owing to its ability to induce p38 phosphorylation and apoptosis via Bcl-xL, Bax, and caspase-3. To investigate how CT activates p38 and specifically affects human leukemia cells, we plan to perform further mechanistic studies. We believe that our findings would be valuable as fundamental data for developing CT as a novel therapeutic agent to treat AML.