Kuguaglycoside C is a triterpene glycoside isolated from the leaves of Momordica charantia, and the biological effects of this compound remain almost unknown. We investigated the anti;?>cancer effect of kuguaglycoside C against human neuroblastoma IMR-32 cells. In the MTT assay, kuguaglycoside C induced significant cytotoxicity against the IMR-32 cells (IC50: 12.6 μM) after 48 h treatment. Although examination by Hoechst 33342 staining revealed that kuguaglycoside C induced nuclear shrinkage at a high concentration (100 μM), no apoptotic bodies were observed on flow cytometry. No activation of caspase-3 or caspase-9 was observed at the effective concentration (30 μM) of kuguaglycoside C. On the other hand, the substance significantly decreased the expression of survivin and cleaved poly (ADP-ribose) polymerase (PARP). Kuguaglycoside C also significantly increased the expression and cleavage of apoptosis-inducing factor (AIF). Moreover, kuguaglycoside C was found to induce caspase-independent DNA cleavage in the dual-fluorescence apoptosis detection assay. These results suggest that kuguaglycoside C induces caspase-independent cell death, and is involved, at least in part, in the mechanism underlying cell necroptosis.
Neuroblastoma is one of the most common solid tumors of childhood, accounting for 10% of all tumors in the pediatric age group. According to an analytic cohort study of the International Neuroblastoma Risk Group, 40% of patients with neuroblastoma already have metastasis by the time of diagnosis, and the 5-year event-free survival rate is only 35%. These high-risk patients are treated by a multidisciplinary approach, including intensive chemotherapy, irradiation, surgery and transplantation; however, the cure rates remain very low. Moreover, survivors after intensive therapy may suffer from sequelae, such as growth hormone deficiency, sensorineural hypacusis, loss of kidney function, and second malignancies. Thus, the development of novel effective and safe therapeutic agents for the treatment of malignant neuroblastoma is urgently needed.
The genetic and molecular biological features of neuroblastomas and their relationship to the prognosis have been reported previously. Gene amplification of MYCN (v-myc myelocytomatosis viral related oncogene, neuroblastoma derived [avian]) is known as the worst prognostic indicator in neuroblastomas, while Trk A expression has been shown to be associated with a favorable prognosis. Although most human cancers are known to show mutation of p53, absence of p53 gene mutation is characteristic in primary neuroblastomas. Moreover, aggressive neuroblastomas do not express caspase-8, a key molecule in the extrinsic pathway of apoptosis.
Classically, cell death has been divided into apoptosis and necrosis. A recent study also revealed a novel type of cell death called programmed necrotic cell death, referred to as necroptosis. Signal transduction for necroptosis is known to be caspase-independent. When a ligand binds to the death receptor in the cell under the condition of apoptosis suppression, activated Rip 1 forms a complex with Rip 3 and Fas-associated death domain (FADD), triggering necroptotic signaling. Mitochondrial protein apoptosis-inducing factor (AIF) is also shown as an important executor of necroptosis.
Momordica charantia (bitter melon) is a Cucurbitaceae plant cultivated in Asia, Africa and South America, that has been used as a traditional medicine and vegetable source. Antidiabetic, antibiotic, antiviral and anticancer effects of M. charantia have been reported previously.[9-11] Extracts of the fruits and the leaves of the M. charantia plant have been shown to suppress proliferation of breast, prostate and adrenocortical cancers.[12-14] Moreover, kuguacin J, a triterpenoid derived from the leaves of M. charantia, has been shown to induce G1 phase cell cycle arrest and apoptosis in a prostate cancer cell line.
In our attempts to identify a novel effective chemotherapeutic agent against aggressive neuroblastoma, we investigated the anticancer activity of kuguaglycoside C, a triterpene glycoside derived from the leaves of M. charantia.
Materials and Methods
Kuguaglycoside C (Fig. 1A) was purified and isolated from the leaves of M. charantia, and its characteristics were compared with its previously described chemical profile.
Human neuroblastoma IMR-32 cells (Riken Cell Bank, Ibaraki, Japan) were cultured in RPMI 1640 medium (Life Technologies Invitrogen, Carlsbad, CA, USA) supplemented with 100 U/mL penicillin, 100 μg/mL streptomycin, and 10% heat-inactivated FBS (Thermo Scientific HyClone, Logan, UT, USA) at 37°C in a humidified incubator in a 95% air/5% CO2 atmosphere. Normal human dermal fibroblasts (NHDF) and HUVEC (Lonza Japan, Tokyo, Japan) were cultured in FGM-2 (Lonza Japan) and EGM-2 (Lonza Japan), respectively, in the environment described above in a 95% air/5% CO2 atmosphere.
IMR-32 cells (1 × 104 cells/100 μL per well) were plated onto the wells of a 96-well culture plate containing phenol red-free RPMI 1640 medium supplemented with 10% FBS, and incubated for 24 h. Then, the cells were treated with kuguaglycoside C (final concentration 0.3–100 μM) or vehicle for 48 h. After the addition of 0.5% MTT solution at 10% of the total well volume, the incubation was continued for an additional 3 h at 37°C/5% CO2. A stop solution (0.04 M HCl in isopropanol) was added at an equal volume to the culture medium in each well, and the absorbances were measured at 570 nm (peak) and 655 nm (trough), after thorough pipetting to disperse the generated blue formazan. The survival rate was calculated as the percentage of that in the vehicle control. In the normal cell study, NHDF or HUVEC cells (2 × 104 cells/100 μL per well) were plated into the respective culture media, and the assay was performed as described above.
Cell cycle analysis
IMR-32 cells (1 × 106 cells/2 mL per well) were plated onto the wells of a six-well culture plate containing RPMI 1640 medium supplemented with 10% FBS, and incubated at 37°C/5% CO2 for 24 h. Kuguaglycoside C (final concentration 1–100 μM) or vehicle was added and the cells were incubated for a further 48 h. The cells were collected and washed twice with ice-cold PBS, then fixed with ice-cold 70% ethanol for 2 h at 4°C. After being washed twice with PBS, the cells were treated with 0.25 mg/mL RNase solution for 30 min at 37°C. Then, propidium iodide (PI) solution was added (final concentration 50 μg/mL) and the cells were incubated for 30 min at 20°C in the dark. The cell samples were filtered through a 40-μm nylon mesh and analyzed with a FC500 flow cytometer (Beckman Coulter, Brea, CA, USA), using the FL3 range.
Hoechst 33342 staining
Staining with Hoechst 33 342 is used to detect apoptotic nuclear morphology. IMR-32 cells (2 × 105 cells/2 mL per well) were plated onto the wells of a six-well culture plate containing RPMI 1640 medium supplemented with 10% FBS, and incubated at 37°C under 5% CO2 for 24 h. Then, kuguaglycoside C (final concentration 10–100 μM) or cisplatin (final concentration 30 μM) was added and the incubation continued for a further 48 h. Hoechst 33 342 solution (final concentration, 0.001% of the medium) was added to the wells, and the wells were allowed to stand for 15 min. The cells were then observed under a fluorescence microscope (IX71; Olympus, Tokyo, Japan).
IMR-32 cells (2 × 106 cells/5 mL per dish) were plated onto 60-mm dishes containing RPMI 1640 medium supplemented with 10% FBS, and incubated at 37°C under 5% CO2 for 24 h. The cells were then treated with kuguaglycoside C (30 μM) for 0–48 h (0 h refers to untreated dishes), collected, washed with TBS, and lysed in an extraction buffer containing 20 mM Tris–HCl (pH 8.0), 137 mM NaCl, 1% NP-40, 10% glycerol, protease inhibitor cocktail I (1:200; Sigma-Aldrich, St. Louis, MO, USA), phosphatase inhibitor cocktail II (1:100; Sigma-Aldrich), 1 mM PMSF, and 1 mM DTT. The cells were disrupted by sonication for 30 s twice, and the supernatants were obtained by centrifugation at 20 630g for 10 min at 0°C. The supernatants were mixed with 3 × sample buffer (0.24 M Tris–HCl (pH 6.8), 9% SDS, 30% glycerol, 15% 2-mercaptoethanol and traces of bromophenol blue) at the mixing ratio of 2:1, and boiled for 3 min at 100°C. The supernatants, as loading samples, were obtained by centrifugation at 20 630g for 1 min at 0°C. Equal amounts (20 μg) of proteins were separated by SDS-PAGE and transferred on to PVDF membranes (GE Healthcare, Tokyo, Japan). After being blocked with 5% skim milk, the membranes were probed with the primary antibodies overnight at 4°C. After another wash, the membranes were incubated with HRP-conjugated secondary antibody for 1 h at room temperature. The blots were detected with an ECL system (GE Healthcare).
The primary antibodies used were as follows: anti-apoptosis-inducing factor (AIF) (4642), anti-caspase-3 (9665), anti-cleaved caspase-3 (9664), anti-caspase-7 (9494), anti-caspase-9 (9502), anti-cytochrome c (4272), anti-NF-κB p65 (4764), anti-survivin (2808X), anti-X-linked inhibitor of apoptosis protein (XIAP) (2042), anti-phospho-p38 MAPK (4092), anti-phospho-JNK (9255S) (Cell Signaling Technology, Danvers, MA, USA), anti-DNA fragmentation factor (DFF) 45 (611036), anti-Fas (610197), anti-Fas-associated death domain protein (FADD) (610399), anti-poly(ADP-ribose) polymerase (PARP) (611038), anti-receptor-interacting protein (RIP) 1 (610458), anti-Smac/DIABLO (612246), anti-tumor necrosis factor receptor-associated death domain protein (TRADD) (610572) (BD Biosciences, San Jose, CA, USA), anti-Bax (B8554), anti-Bcl-2 (B3170), and anti-β-tubulin (T4026) (Sigma-Aldrich).
Caspase activity assay
IMR-32 cells (1 × 106 cells/2 mL per well) were plated onto the wells of a six-well cell culture plate containing RPMI 1640 medium, and incubated at 37°C under 5% CO2 for 24 h. Then, the cells were treated with kuguaglycoside C (30 μM), cisplatin (positive control; 100 μM) or vehicle for 48 h. The experiment was performed according to the manufacturer's protocol prescribed in the caspase-3 and caspase-9 colorimetric assay kits (Biovision, Milpitas, CA, USA). Briefly, the cells were collected and lysed in cell lysis buffer with pipetting and incubated for 10 min on ice, and the supernatants were obtained by centrifugation at 10 000g for 1 min at 4°C. The protein samples were diluted to obtain equal amounts of proteins (150 μg/75 μL), and the same amount of reaction buffer and substrate were added, followed by incubation for 1 h at 37°C. The reacted samples were plated onto the wells of a 96-well plate and the absorbance at 405 nm was measured.
Annexin V and PI staining analysis
Early apoptosis was detected using the Alexa Fluor 488 Annexin V/Dead Cell Apoptosis Kit (Life Technologies Invitrogen). IMR-32 cells (1 × 106/2 mL per well) were plated onto the wells of a six-well cell culture plate. After 24-h incubation, the cells were treated with kuguaglycoside C (30 μM), cisplatin (30 μM), or vehicle for 48 h. To prepare the cell samples for flow cytometry, the cells were washed with annexin-binding buffer and stained with annexin V-Alexa Fluor 488 and PI for 15 min. The cell samples were analyzed using a FC500 flow cytometer (Beckman Coulter) using the FL1 and FL4 range for annexin V-Alexa Fluor 488 and PI, respectively.
Dual-fluorescence apoptosis detection assay
IMR-32 cells (2 × 106 cells/5 mL per dish) were plated onto a 60-mm dish (two dishes per sample) containing RPMI 1640 medium and incubated at 37°C under 5% CO2 for 24 h. Then, the cells were treated with kuguaglycoside C (30–100 μM), cisplatin (positive control; 100 μM) or vehicle for 48 h. The experiment was performed according to the manufacturer's protocol prescribed in the ApopTag ISOL dual-fluorescence apoptosis detection kit (DNase Types I & II) (Merck Millipore, Billerica, MA, USA). Briefly, the cells were collected and fixed with 1% paraformaldehyde for 10 min at room temperature. After being washed twice with PBS, the cells were treated with the reaction mixture and incubated for 15 h at 20°C in the dark. After a further wash with PBS and three washes with purified water, DAPI solution was added. The samples were dropped onto glass slides and the fluorescence images were photographed with a camera fitted to the fluorescence microscope (FSX100; Olympus).
Data are expressed as mean ± SEM (n = 3). Significance testing was performed using one-way anova followed by Bonferroni's test.
To assess the anticancer activity of kuguaglycoside C against neuroblastoma cells, we used human neuroblastoma IMR-32, a cell line carrying no mutation of p53, an amplified MYCN gene, and silenced caspase-8. Kuguaglycoside C induced significant cytotoxicity against IMR-32 cells, and the IC50 value was 12.6 μM (Fig. 1). On the other hand, the cytotoxicity (IC50) values against NHDF and HUVEC were 16.4 μM and 27.0 μM, respectively (data not shown).
Kuguacin J, an aglycon of kuguaglycoside C, has been reported to induce G1 arrest in prostate cancer cells. Here, we examined the effect of kuguaglycoside C on the cell cycle in a neuroblastoma cell line. In contrast to kuguacin J, treatment of the cells with kuguaglycoside C for 48 h had no effect on the cells of any phase of the cell cycle in the neuroblastoma cell line (Fig. 2).
In the cell morphological observation, nuclear shrinkage, a feature of apoptosis, was observed following treatment with kuguaglycoside C, in a concentration-dependent manner (Fig. 3). Apoptosis is typically known to be controlled by the caspases; however, treatment with kuguaglycoside C at 30 μM, a concentration found to be cytotoxic in the MTT assay, did not increase the activity of either caspase-3 or caspase-9 in the colorimetric assay (Fig. 4). To clarify whether or not kuguaglycoside C-induced cell death involved apoptosis, early apoptosis was examined by flow cytometry after annexin V and PI double staining (Fig. 4). Kuguaglycoside C (30 μM) did not show an early apoptotic cell population, in contrast to the cisplatin (30 μM)-treated cells.
Protein levels of cleaved caspase-3, -7 and -9 were not increased by treatment with kuguaglycoside C (30 μM), consistent with the results of the colorimetric assay (Fig. 5). Moreover, the expression levels of p-p38 MAPK, p-JNK, Bax, Bcl-2 and cytochrome c remained unchanged following treatment with kuguaglycoside C (Fig. 5), although significant PARP cleavage was observed (Fig. 5). Increase in the levels of TRADD, AIF and DFF45, and decrease in the levels of RIP1 and survivin were observed (Fig. 5). Moreover, truncated AIF, an activated form of the protein, was observed after treatment of the cells with kuguaglycoside C for 48 h (Fig. 5). To clarify the contribution of type II DNase activation in the cell death induced by kuguaglycoside C, we conducted the dual-fluorescence apoptosis detection assay. Whereas cisplatin activated only type I (caspase-dependent) DNase, kuguaglycoside C activated both type I (caspase-dependent) and type II (caspase-independent) DNases (Fig. 6).
We investigated the anticancer activity of kuguaglycoside C, a triterpene glycoside derived from M. charantia, against neuroblastoma cells. Biological activities, including anticancer effect, of kuguaglycoside C have not been reported previously. Here, we showed that kuguaglycoside C induces caspase-independent cell death of neuroblastoma IMR-32 cells.
Anticancer effects of kuguacin J, an aglycon of kuguaglycoside C, have been reported. Kuguacin J induces significant G1 phase cell cycle arrest, with downregulation of cyclin D1, cyclin E, cyclin-dependent kinase (CDK) 2, and CDK4 in both LNCaP and PC3 prostate cancer cells.[15, 20] Kuguacin J has been shown to induce apoptosis with caspase-3 activation and PARP cleavage in LNCaP prostate cancer cells. On the other hand, caspase-3 activation and PARP cleavage were not found to be involved in the cell death induced by kuguacin J in the PC3 prostate cancer cells. These findings suggest that caspase dependency of the cytotoxicity of kuguacin J may depend on the cancer cell type. In this study, kuguaglycoside C induced significant cell death at 30 μM, in the absence of activation of caspases or cell cycle arrest in the IMR-32 cells, indicating that kuguaglycoside C induces caspase-independent cell death by a mechanism(s) different from that underlying the cytotoxicity of kuguacin J. It is likely that the glycoside constitution in kuguaglycoside C contributes to the lack of its cell cycle arrest activity.
Aberrant survivin expression has been reported to be observed and involved in the malignant behavior of common human cancers, including neuroblastomas. Therefore, survivin is regarded as a target to regulate the malignant potential of cancers. Survivin is one of the inhibitors of apoptosis protein (IAP) family proteins. It exerts multimodal molecular biological functions, such as inhibition of apoptosis via blockade of Smac/DIABLO and stabilization of XIAP, and inhibition of caspase-independent necroptosis via suppression of AIF. In this study, we showed that kuguaglycoside C significantly suppressed the expression of survivin, although the expression levels of Smac/DIABLO and XIAP did not change after treatment of the cells with kuguaglycoside C. Taken together with our findings, that neither caspase-3, -7, -9 cleavage nor enzymatic activation of caspase-3, -9 were induced by kuguaglycoside C, it would seem that suppression of survivin expression activates only the caspase-independent pathway in this model. Previously, it was reported that silencing of survivin gene expression by siRNA resulted in the induction of caspase-independent cell death, involving AIF activation, in human breast cancer cells. The downstream mechanism of cell death following survivin suppression induced by kuguaglycoside C in neuroblastoma cells seems to be related to the evidence obtained from the survivin-silencing study.
Findings in respect of the molecular bases of caspase-independent cell death have been accumulating during the past decade. In recent years, the concept of necroptosis, a mode of programmed cell death, has come to be widely accepted. AIF is known as a key molecule involved in caspase-independent necroptosis. AIF is embedded in the inner mitochondrial membrane, and its activation requires a caspase-independent proteolytic cleavage that would allow translocation from the mitochondrium to the nucleus. In our study, not only was AIF expression increased, but it was also cleaved after 48 h treatment of the cells with kuguaglycoside C.
Complex formation, including that of TRADD and RIP1, is the key initial mechanism of TNF-induced necroptosis. In this study, while kuguaglycoside C increased TRADD formation, it decreased the formation of RIP1. Although RIP1 is known as a key regulator of necroptosis, it has been shown to be not involved in the necroptosis induced by TNF under particular conditions. Despite continuing debate, cell death induced by kuguaglycoside C seems to be involved in the necroptotic mechanism, at least in the downstream part of the cascade.
In the final phase of the caspase-dependent apoptosis pathway, DFF45 is degraded by caspase-3 and -7 to activate DFF40, a type I DNase, which hydrolyses DNA into oligo;?>nucleotides bearing a 3′-OH end. On the other hand, oligonucleotides generated by caspase-independent type II DNases possess a 3′-phosphate end. The dual-fluorescence apoptosis detection assay allows detection of the difference in the cleavage site. Kuguaglycoside C activated type II as well as type I DNase, in contrast to cisplatin, which activated only type I DNase. Type I DNase is activated via the caspase-dependent pathway of apoptosis. On the other hand, Type II DNase is activated in the absence of cations under acidic pH, in a caspase-independent manner. Because cell death induced by kuguaglycoside C is mainly caspase-independent, and may occur under a condition conducive to easy activation of type II DNase, both DNases are thought to be activated. It may be advantageous to induce robust cell death even if the cancer cells have acquired resistance to apoptosis induced by the commonly-used anticancer drugs, such as cisplatin. Moreover, kuguaglycoside C induced DFF45 expression, suggesting that DFF40 was suppressed. Poly (ADP-ribose) polymerase plays a key role in base excision repair of DNA. Although caspase-induced PARP cleavage is considered as a prominent marker of apoptosis, caspase-independent PARP cleavage has also been reported in some experiments.[28, 29] Kuguaglycoside C induced caspase-independent PARP cleavage in this study. Taken together, kuguaglycoside C exerts a facilitatory influence on DNA cleavage in the absence of caspase activation.
Caspase-8 is silenced in malignant neuroblastomas, and IMR-32 is reported as a caspase-8 silenced cell line. Recently, it was reported that necroptosis is inhibited by caspase-8.[30, 31] Thus, necroptosis induction is beneficial in malignant neuroblastoma therapeutics, and kuguaglycoside C appears to be a promising necroptosis-inducing-type anticancer agent.