Jellyfish extract induces apoptotic cell death through the p38 pathway and cell cycle arrest in chronic myelogenous leukemia K562 cells

Extraction of Jellyfish hexane extract (Jellyfish-HE)

Stomolophus nomurai (Nomura’s jellyfish) were harvested from the shore near Busan, Korea. The voucher specimen has been deposited after classical identification in the invertebrate animals stocks of College of Fisheries Sciences, Pukyung National University, Busan, Korea (Prof NG Park). In order to dry the raw materials, the jellyfish has been harvested from coastal fishery and the water content was naturally removed using a house sieve. Then, the roughly dried jellyfish (100 g) was vacuum-dried using a freezing dryer (Ilshin Lab Co., LTD, Seoul, Korea). Dried jellyfish (36 g) fragmentized were extracted with 300 ml of 50% ethanol (EtOH) three times under reflux at 50 °C for 24 h, then filtered and concentrated to yield the EtOH extract (25 g). The EtOH extract was suspended in 100 ml H₂O and extracted successively with n-hexane (Hex), ethylacetate (EtOAc; EA), and n-butanol (n-BuOH) to yield an n-hexane fraction (34 mg), an EA fraction (42 mg), an n-BuOH fraction (1.9 g), and water residue (18.4 g). The concentrated extract (34 mg) was then lyophilized, resulting in 14.9 mg of powder. Dried HE was subsequently dissolved in dimethyl sulfoxide (DMSO) diluted with DMEM media. The final concentration of DMSO was adjusted to 0.1% (v/v) in the culture media.

Cell culture and reagents

The human CML K562 cell line, human colon cancer HCT116 cells and human liver cancer Huh-7 cells were purchased from ATCC (American Type Culture Collection; Rockville, MD, USA). The human CML K562 cell line was cultured in RPMI1640, HCT116 cells and Huh-7 cells were cultured in DMEM (WelGENE Co., Daegu, Korea) containing 10% fetal bovine serum (FBS), penicillin (100 U/mL), and streptomycin (100 mg/mL) at 5% CO2 in a humidified incubator at 37 °C. Z-VAD-FMK (a pan-caspase inhibitor) (catalog no. 219007) was purchased from Calbiochem (Darmstadt, Germany). 3-(4,5-dimethylth-iazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) (catalog no. M2128) was purchased from Sigma–Aldrich (St. Louis, MO, USA). 6-diamidino-2-phenylindole dihydrochloride (DAPI) (catalog no. D9542) was purchased from Sigma-Aldrich (St. Louis, MO, USA). SB203580 (catalog no. 559389) and SP600125 (catalog no. 420119) were purchased from Calbiochem (Darmstadt, Germany). U0126 (catalog no. V1121) was purchased from Promega (Madison, WI, USA). Antibodies against caspase-3 (catalog no. 9661), caspase-8 (catalog no. 9746), cleaved caspase-9 (catalog no. 9501), p-JNK (catalog no. 9251), JNK (catalog no. 9252), and p-p38 (catalog no. 9211) were purchased from Cell Signaling Technology (Dancers, MA, USA). Antibodies against β-actin (catalog no. sc-47778), PARP-1 (catalog no. sc-7150), Bcl-2 (catalog no. sc-492), BAX (catalog no. sc-493), p38 (catalog no. sc-535), CDK2 (catalog no. 163), CDK4 (catalog no. sc-264), cyclin A (catalog no. sc-596), and cyclin D1 (catalog no. sc- 450) were purchased from Santa Cruz Biotechnology (Paso Robles, CA, USA). The Bio-Rad protein assay kit (catalog no. 500-0114 and 500-0113) was purchased from Bio-Rad (Richmond, CA, USA). The Annexin V-FITC/PI apoptosis detection kit (catalog no. 556547) was purchased from BD Biosciences (San Jose, CA, USA).

MTT assay

Cell were plated in a 96-well culture plate (5 × 10⁴ cells/well) and treated with various concentrations (0, 10, 20, 30, 40, and 50 µg/ml) of Jellyfish-HE. After 24 h, the media was removed and MTT (0.5 mg/ml) was added to each well for 4 h. Formazan crystals from MTT reduction were dissolved in DMSO and the OD value was read at 590 nm with a Versamax microplate reader (Molecular Devices, Sunnyvale, CA, USA).

DAPI stain assay

After treatment with Jellyfish-HE, to confirm nuclear condensation, cells were stained with DAPI. Before treatment with Jellyfish-HE, cover slides were coated with lysine to encourage attachment of K562 cells. Cells were spread in 24-well culture plates (4 × 10⁵ cells/well) and treated with Jellyfish-HE (40 µg/mL) for 24 h. Then, cells were washed with 1 X PBS and fixed with 4% paraformaldehyde. After 20 mins at 4 °C, the cells were washed with 1 X PBS and stained with DAPI (1 mg/mL) for 10 mins at room temperature in the dark. Then, the cells were washed with 1 X PBS and mounted with mounting solution (Dako, Glostrup, Denmark). Nuclei were detected under a fluorescence microscope TMS (Nikon, Tokyo, Japan).

Annexin V and PI staining

After treatment with 40 µg/ml of jellyfish hexane extract for 8 h, K562 cells were harvested and cell were washed with PBS and suspended with binding buffer (1X). After that 4 µl Annexin V-FITC and 2 µl propidium iodide (PI) were added in the cells for 15 min at 37 °C in the dark. Then, cells were analyzed with a flow cytometer, FACS Canto II (BD Biosciences, San Jose, CA, USA). Using this data, Living cells (Annexin V−/PI−), early apoptotic cells (Annexin V+/PI−), late apoptotic cells (Annexin V+/ PI+), necrotic cells (Annexin V−/PI−) cells were measured.

Western blot analysis

K562 cells were lysed in a buffer containing 50 mmol/L Tris–HCl (pH 8.0), 150 mmol/L NaCl, 100 m mol/L NaF, 100 mg/mL phenylmethylsulfonyl fluoride (PMSF), 1 mg/mL aprotinin, and 1% Triton X-100. After 30 mins at 4 °C, cells were centrifuged at 13,000 rpm at 4 °C. Then, protein concentration in the supernatant was quantified using the Bio-Rad protein assay (Bio-Rad, Berkeley, CA, USA). Equal amounts of whole cell lysates were fractionated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). After electrophoresis, gels were transferred to polyvinylidene difluoride (PVDF) membranes using the Hoefer electrotransfer system (Amersham Biosciences, Buckinghamshire, UK). To visualize the target protein, the membranes were incubated at 4 °C overnight with each primary antibody. Then, the membranes were washed and incubated at room temperature for 1 h with the appropriate secondary antibody. Detection was carried out using a secondary horseradish peroxidase-linked antibody and the ECL chemiluminescence system (Amersham Biosciences, Buckinghamshire, UK). Films were scanned and densitometry analysis was performed using Image J software.

Cell cycle analysis

To analyze cell cycle, K562 cells were treated with 40 µg/ml of Jellyfish-HE for 12h and cells were harvested, fixed with 70 % ethanol at 4 °C for 30 min. Fixed cells were washed with PBS and then stained PI buffer containing propidium iodide, RNase, Nacitrate and NP-40 for 30 min in the dark. After that cells were analyzed by flow cytometer, FACS Canto II (BD Biosciences, San Jose, CA, USA).

Statistical analysis

All data are expressed as means ± SEM of three independent replicates for each group. Comparisons were made by Student’s t-test. *P < 0.05 was considered statistically significant. Statistical analysis was measured using GraphPad Prism software 5.0.

Jellyfish hexane extract specifically induces cell death in K562 cancer cells

In order to examine whether Jellyfish extracts can affect cell viability in the K562 cancer cell line, jellyfish were extracted with a variety of organic solvents (EtOH, BuOH, Ethyl Acetate, and hexane), as well as distilled water, as described in the Materials and Methods section. Then, human chronic myeloid leukemia K562 cells were treated for 3 days with 50 µg/ml EtOH, BuOH, ethyl acetate, or hexane extract. Only Jellyfish-HE obviously exhibited the reduced cell viability at the concentrations used. Cell viability was measured using the MTT assay. As shown in Fig. 1, cell viability of K562 cells was specifically decreased in Jellyfish-HE-treated cells. The results demonstrated that Jellyfish-HE specifically decreases cell viability in K562 cells, whereas the other extracts do not.

Furthermore, K562 cells have been treated with the H2O extracts, EtOH extracts, BuOH extracts, EA extracts and hexane extracts for 1 to 3 days to evaluate possible changes in acting compounds. The results have also shown that the H2O extracts, EtOH extracts, BuOH extracts and EA extracts do not affect cell viabilities, in contrast to hexane extracts in K562 cells (Fig. S1). These results indicated that the long term treatment such as 3 days does not seem to cause loss of activity of the active compounds.

Jellyfish hexane extract induces cell death in a dose-dependent manner in various cancer cell lines

Because Jellyfish-HE inhibits the growth of human chronic myeloid leukemia K562 cells, we investigated its effects in other cancer cell lines. Several cancer cell lines, including human colon cancer HCT116 cells and human liver cancer Huh-7 cells, were treated with Jellyfish-HE and assessed for cytotoxicity. Cells were treated with various concentrations (0, 10, 20, 30, 40, and 50 µg/ml) of Jellyfish-HE for 24 h, as shown in Fig. 2. As shown in Fig. 2, the growth rate of HCT116 human colon cancer cells (Fig. 2B) and Huh-7 human liver cancer cells (Fig. 2C) were examined using an MTT assay. When half-maximal inhibition concentrations (IC50) were measured on the cancer cell lines, each IC50 value was calculated to be 49.51 µg/ml, 62.85 µg/ml and 67.28 µg/ml for K562 cells, HCT116 cells and Huh-7 cells, respectively (Fig. 2). For the calculation of IC50, the computed maximum effects on cell viability were calculated to be 33.85 % (84.23 µg/ml) in K562 cells, 34.34% (101.0 µg/ml) in HCT116 cells and 39.66 % (104.9 µg/ml) in Huh-7 cells. The results of the MTT assays showed that Jellyfish-HE inhibits cell proliferation in a dose-dependent manner in all cancer cell lines. K562 cells were especially susceptible to Jellyfish-HE-induced growth suppression, although growth of all of the tested cancer cells was inhibited.

Jellyfish hexane extract induces apoptosis characterized by typical biochemical and morphological changes, such as cell shrinkage, DNA fragmentation, chromatin condensation, and formation of apoptotic bodies in K562 cells

Apoptosis is typically characterized by cell death-related biochemical and morphological changes such as cell shrinkage, DNA fragmentation, chromatin condensation, and formation of apoptotic bodies (Haefner, 2003). In this study, to determine whether Jellyfish-HE induces cell death by apoptosis, K562 cells were treated with various concentrations (0, 10, 20, 30, 40, and 50 µg/ml) of Jellyfish-HE for 24 h, and then morphological changes were observed using microscopy. Apoptotic cell body-like cell morphologies were easily observed in Jellyfish-HE-treated K562 cells in a dose-dependent manner (Fig. 3A). To further examine the nuclear morphological changes in K562 cells, DAPI staining was performed after treatment with 40 µg/ml Jellyfish-HE (Fig. 3B). As shown in Fig. 3B, apoptotic body-like shapes and chromatin condensation were observed in cells treated with 40 µg/ml of Jellyfish-HE compared with untreated control cells. Next, to confirm that Jellyfish-HE-induced apoptosis is directly under the control of Jellyfish-HE, cells were treated with 40 µg/ml Jellyfish-HE for 8 h and then assessed using Annexin V-FITC/PI staining and quantitative analysis (Fig. 3C). When the statistical significance has been analyzed using Student’s t-test, the Annexin V-positive, PI-positive and Annexin V/PI-double positive cells were significantly increased (Fig. 3D). The 3 positive levels are similar to that of the known apoptotic agents such as lactose-binding lectins (Kwak et al., 2015). The results clearly show that the levels of Annexin V and Annexin V-PI positive cells were increased by treatment with 40 µg/ml Jellyfish-HE. These results suggest that Jellyfish-HE induces apoptotic cell death in K562 cells.

Jellyfish hexane extract-mediated apoptosis acts through the intrinsic and extrinsic apoptotic pathways in K562 cells

Caspases are vital in apoptosis. Therefore, after treatment of K562 cells with 40 µg/ml Jellyfish-HE, the cleaved forms of PARP and procaspase-3 through activation of caspase-8 and caspase-9 were investigated by immunoblotting analysis and densitometry. Cleaved PARP and mature caspase-3 levels were normalized to those of β-actin as an internal control (Figs. 4A, 4B). The results demonstrated that Jellyfish-HE induces apoptosis in a dose-dependent manner through both the extrinsic and intrinsic apoptotic pathways in K562 cells. The BCL-2 family is a group of proteins that have both pro- (BAX, BAD, and others) and anti- (BCL-2, Bcl-xL, and others) apoptotic functions. Thus, the relative ratio of those proteins expressed during the apoptotic process helps to determine the type of cell death signaling (Gross, McDonnell & Korsmeyer, 1999). As shown in Figs. 4C and 4D, after treatment with 40 µg/ml Jellyfish-HE for 24 h, the levels of BCL-2 and BAX were analyzed by immunoblotting and densitometry. The level of BCL-2 was decreased, while that of BAX was slightly increased and the ratio of BAX/BCL-2 was significantly increased. To further confirm that Jellyfish-HE induces apoptosis that depends on the caspase cascade, K562 cells were treated with a pan-caspase inhibitor, Z-VAD, along with 40 µg/ml Jellyfish-HE for 12 h, and cell viability was measured with the MTT assay (Fig.  4E). In the same conditions as shown in Fig. 4E, the levels of PARP and caspase-3 were also analyzed by immunoblotting (Fig. 4F). Treatment with Z-VAD and Jellyfish-HE resulted in an increase in the viability of K562 cells compared to cells incubated in the absence of Z-VAD and the presence of Jellyfish-HE. Likewise, the levels of cleaved caspase-3 and PARP were increased in the presence of Z-VAD and Jellyfish-HE in K562 cells. Overall, these results suggest that Jellyfish-HE induces caspase-dependent apoptosis through both the extrinsic and intrinsic apoptotic pathways.

Jellyfish hexane extract induces apoptosis through the p38 pathway

In caspase-regulated apoptotic cell death, the MAPK signaling pathway, which involves ERK1/2, p38, and JNK, has been reported to play important roles, and it controls apoptosis in human cancers (Zhang et al., 2014). In the present study, in order to examine whether the MAPK signaling pathway activates Jellyfish-HE-induced apoptosis, we analyzed the phosphorylation level of MAPK by Western blot analysis after treatment of K562 cells with 40 µg/ml of Jellyfish-HE for various times up to 2 h and in different doses for 12 h. As shown in Figs. 5A and 5C, the levels of phosphorylated ERK, phosphorylated JNK, and phosphorylated p38 were significantly increased in dose- and time-dependent manners, while the total levels of such proteins remained unchanged, indicating increased phosphorylation of the signaling molecules. To compare the relative ratio between the phosphorylated forms and total protein forms, each was analyzed by densitometry using Image J software (Figs. 5B, 5D). Although the three different MAPKs, ERK, JNK, and p38, were all activated by Jellyfish-HE, a more precise role of each one may be important in the subsequent process of apoptosis. Therefore, to confirm which MAPK affects Jellyfish-HE-induced apoptosis, K562 cells were examined using specific inhibitors of MAPK signaling. Cells were pretreated for 1 h with 10 µM of specific inhibitors, U0126 (ERK inhibitor), SP600125 (JNK inhibitor), and SB203580 (p38 inhibitor), and then treated with 40 µg/ml of Jellyfish-HE for a further 12 h. The viability of the treated cells was then measured using the MTT assay. Treatment of the cells with Jellyfish-HE and SB203580 resulted in a slight increase in cell viability compared with treatment with Jellyfish-HE only (Fig. 5E). In contrast, treatment with U0126 or SP60015 failed to inhibit Jellyfish-HE-induced apoptosis (Fig. S2). To further investigate the mechanisms of apoptosis, we analyzed the expression levels of apoptotic proteins, which are specifically related to the biochemical and enzymatic processes, by Western blotting. As shown in Fig. 5F, the level of cleaved caspase-3 (detected at 17 kD and 19 kD), a well-known indicator of apoptosis, is decreased when 10 µM of SB203580 was applied for 1 h followed by 40 µg/ml of Jellyfish-HE treatment for 12 h. These results indicated that p38 MAPK signaling is directly related to Jellyfish-HE-induced apoptosis in K562 cells.

Jellyfish hexane extract induces cell cycle arrest

As shown in Fig. 1, Jellyfish-HE decreased the growth rate of K562 cells. Therefore, we investigated whether the Jellyfish-HE extract affects cell cycle. K562 cells were treated with 40 µg/ml of Jellyfish-HE or absence of Jellyfish-HE for 12h. Jellyfish-HE treated K562 cells were arrested in G0/G1phase (Figs. 6A and 6B). To further investigate, whether the cell cycle-related proteins CDK2, CDK4, cyclin A, and cyclin D1 are altered in Jellyfish-HE-treated cells. K562 cells were treated with various concentrations (0, 10, 20, 30, and 40 µg/ml) of Jellyfish-HE for 24 h and cell extracts were subjected to immunoblotting and densitometry analysis. Cyclin D1 and CDK4 are known to be expressed in the initial stages of the G1 and S1 phase transitions (Liu et al., 2014). Therefore, downregulation of Cyclin D1 and CDK4 is regarded as an indicator of G0/G1 cell cycle arrest. In this study, as shown in Fig. 6C, treatment of the cells with various concentrations of Jellyfish-HE significantly decreased the expression levels of the G0/G1 cell cycle-related proteins Cyclin D1 and CDK4 in a dose-dependent manner. Other proteins involved in the cell cycle, CDK2 and cyclin A, were also downregulated by Jellyfish-HE treatment (Fig. 6D). Although the expression levels of CDK2, CDK4, and Cyclin D1 proteins were up-regulated by HE extract treatment at low dose (10 µg/ml), the adaptation state of the cells is suggested. In general, most cells respond to environmental changes such as stress, which called “Adaptation” for cell survival, as previously reported (Brooks et al., 2011). However, high doses of Jellyfish-HE directly induce cell injury, cell death and cell cycle arrest. These results show that Jellyfish-HE induces G0/G1 cell cycle arrest that is regulated by cell cycle-related proteins in K562 cells.