Induction of Apoptosis, G0/G1 Phase Arrest and Microtubule Disassembly in K562 Leukemia Cells by Mere15, a Novel Polypeptide from Meretrix meretrix Linnaeus

Mere15 is a novel polypeptide from Meretrix meretrix Linnaeus with cytotoxicity in solid cancer cells. In this study, we investigated its activity on human K562 chronic myelogenous leukemia cells. Mere15 inhibited the growth of K562 cells with IC50 values of 38.2 μg/mL. Mere15 also caused concentration dependent induction of apoptosis, with overproduction of reactive oxygen species and loss of mitochondrial membrane potential. Moreover, Mere15 arrested cell cycle progression at G0/G1 phase of K562 cells in a concentration dependent manner. In addition, Mere15 caused the disassembly of the microtubule cytoskeleton in K562 cells and inhibited the polymerization of tubulin in a cell free system via interaction with tubulin. We concluded that Mere15 was cytotoxic to K562 leukemia cells and the cytotoxicity was related to the apoptosis induction, cell cycle arrest and microtubule disassembly. These results implied that Merer15 was a broad spectrum anticancer polypeptide, not only cytotoxic to various solid cancer cells but also to the chronic myelogenous leukemia cells. Mere15 may have therapeutic potential for the treatment of leukemia.


Introduction
Marine organisms are a rich resource for discovering novel anticancer drugs [1,2]. A lot of peptides from marine source display potent anticancer activity [3]. For example, Didemnin, a cyclic depsipeptides isolated from the Caribbean tunicate Trididemnun solidum, is the first marine peptide that entered in clinical trials in US for the treatment of cancer [4]. Kahalalide F, a depsipeptide isolated from the herbivorous marine mollusk Elysia rufescens, shows strong antitumor activity both in preclinical and clinical studies [5]. Other marine obtained anticancer peptides such as hemiasterlin, dolastatins, cemadotin, soblidotin and aplidine have also entered in clinical trials [4]. The discovery of Dolastatin 10, originally isolated from the Indian Ocean sea hare, Dolabella auricularia, has lead to the development of brentuximab vedotin, which has been recently approved by the US Food and Drug Administration for the treatment of Hodgkin's lymphoma and systemic anaplastic large cell lymphoma [6]. These researches suggest that it is a promising strategy to search anticancer peptides/proteins from the marine organisms.
The marine mollusk Meretrix meretrix Linnaeus has been used by traditional Chinese medicine for the treatment of cancer for a long time. Recent studies have shown that certain peptides and/or proteins derived from M. meretrix are contributed to their anticancer activity. For example, a peptide from M. meretrix has been reported to strongly inhibit the growth of human gastric gland carcinoma cells [7]. In addition, two M. meretrix derived glycoproteins, MGP0405 (MW: 9655 Da) [8] and MGP0501 (MW: 15,878 Da) [9], also possess anticancer activity in vitro. In our previous studies, we have isolated a novel 15 kDa polypeptide (Mere15) from M. meretrix. Mere15 exhibits significant cytotoxicity to several solid cancer cell lines via apoptotic pathway [10]. Moreover, Mere15 inhibits metastasis of human lung cancer A549 cell line via down-regulating matrix metalloproteinases [11]. However, the mode of action of Mere15 induced apoptotic pathways needs to be further elucidated. On the other hand, our previous research was only confined to the solid cancer cell lines; whether Mere15 could also be cytotoxic to hematological malignant tumors remains unknown.
Unlike solid tumors, hematological malignant tumors could not be cured by surgical treatment or radiation therapy and therefore largely depend on the chemotherapy. There have been considerable advances in the treatment of leukemia over the last decade; however, the major obstacle associated in chemotherapy is the resistance of leukemic cells to various chemotherapeutic agents and drug intolerance [12]. To reverse the resistance and deduce the side effects, new therapeutic agents are still needed. In the present study, we evaluated the cytotoxicity of Mere15 on human K562 chronic myelogenous leukemia cells. Moreover, we investigated the possible mechanisms underlying their cytotoxicity.

Mere15 Inhibits K562 Cell Proliferation
The cytotoxicity of Mere15 on K562 cell lines was evaluated by MTT assay. As shown in Figure 1A, Mere15 displayed obvious cytotoxicity on K562 cell lines in a concentration dependent manner with the IC 50 value of 38.2 μg/mL. Treatment with certain concentrations of Mere15 induced decreased population and obvious cell morphological shrinkage ( Figure 1B). These results implied that Merer15

Mere15
Since apo apoptosis-in showed that were stained more nuclei   ROS overproduction and ROS-mediated mitochondrial depolarization has been widely reported in other natural products induced apoptosis in cancer cells [16][17][18]. In our previous study, we have found that Mere15 could induce cell death via apoptotic pathway on human lung cancer A549 cells. The present study showed that Mere15 also induced K562 cell death in a similar manner, and ROS overproduction and MMP disruption were all involved in Mere15 induced cell apoptotic pathway.

Mere15 Induces a G 0 /G 1 Phase Arrest
To examine whether Mere15 induced effect of anti-proliferation was also associated with cell cycle arrest, cell cycle distribution was analyzed by flow cytometry. As shown in Figure 5, treatment of K562 cells with Mere15 induced accumulation of cells at G 0 /G 1 phase in a concentration dependent manner. The amount of cells in the G 0 /G 1 phase increased from 27% in untreated cells to 31%, 37% and 40% in K562 cells treated with 20, 40, and 80 μg/mL Mere15, respectively. These results suggested that Mere15 induced G 0 /G 1 phase arrest in K562 cells. It should be noted that cells arrested at G 0 /G 1 phase around Mere15 IC 50 concentration (80 μg/mL) differs only by 10% from the control.

Mere15
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Cell Lines and Cell Culture
K562 cells, purchased from ATTCC, were grown on RPMI 1640 (with 300 mg/L glutamine) supplemented with 10% fetal bovine serum and 1× antibiotics at 37 °C in a 5% CO 2 incubator.

Cell Viability Assessment
The cytotoxicity of Mere15 was determined by MTT assay [24]. Briefly, cancer cells were untreated or treated with certain concentrations of Mere15 (0-160 μg/mL) for 48 h. MTT (20 μL, 0.5 mg/mL) was added and incubated for another 4 h, and then the supernatant was removed and the dye crystals were dissolved in 200 μL DMSO. Absorbance was measured at 490 nm using a microplate reader (BioTek, USA). The cytotoxicity of Mere15 against K562 cells was expressed as an IC 50 , which was determined from the concentration-response curve.

Hoechst33342/Propidium Iodide (PI) Dual Staining Assays
The apoptotic cells were stained using Hoechst33342/PI double staining as we described previously [25]. The K562 cells were seeded in 6-well plates (2 × 10 5 cells/well) and treated with certain concentrations of Mere15 (0-80 µg/mL). After incubation for 24 h, the cells were collected and washed with PBS. Cells were stained with Hoechst33342 and PI using the dual staining kit (Beyond, China). Then the cells were spread on slides and observed under the fluorescence microscopy (Zeiss, Germany).

Measurement of Reactive Oxygen Species (ROS) Generation
Generation of ROS was measured by the oxidation-sensitive fluorescent probe DCFH-DA (Beyond, China) according to the manufacturer's instructions [26]. Briefly, K562 cells were seeded in 6-well plates and treated with certain concentrations of Mere15 (0-80 μg/mL) for 24 h. Then cells were incubated in serum-free culture medium with DCFH-DA (10 μM) for 30 min at 37 °C. Cells were washed with serum-free medium three times and analyzed by fluorescence microscope (Zeiss, Germany) and fluorescence spectrophotometer (F-4500, HITACHI).

Assessment of Mitochondrial Membrane Potential (MMP)
Mitochondrial membrane potential was determined using Mitochondrial Membrane Potential Detection Kit according to the manufacturer's instructions (Beyond, China) as described previously [27]. Briefly, K562 cells were seeded in 6-well plates and treated with certain concentrations of Mere15 (0-80 μg/mL) for 24 h. Then the cells were stained with JC-1 for 20 min at 37 °C. MMP were analyzed by fluorescence microscope (Zeiss, Germany) and fluorescence spectrophotometer (F-4500, HITACHI), respectively. The ratio of red to green fluorescence intensity was measured according to the integration of the peaks in the fluorescence spectra.

Cell Cycle Analysis
The K562 cells were treated with certain concentrations of Mere15 (0-80 µg/mL) for 24 h. The cells were harvested and fixed in ice-cold 70% (v/v) ethanol for 24 h at 4 °C. The cell pellet was collected by centrifugation at 5000× g, resuspended in PBS, and stained with a mixture of RNase (10 μg/mL) and PI (50 μg/mL) in sodium citrate containing 0.5% Triton X-100 for 20 min in the dark. Cell cycle analysis was performed using an Aria FACS flow cytometry system (Bection Dickinson, USA), and cell cycle distribution was analyzed with ModFit LT software (Verity Software House. Inc., USA).

Data Analysis
One-way ANOVA with Tukey's post hoc test was used for statistical analysis, and values were expressed as mean ± SD. Differences of P < 0.05 were considered statistically significant.

Conclusions
In conclusion, our present study provides solid evidence that Mere15 could inhibit the growth of K562 cells in a concentration dependent manner, resulted from apoptosis and cell cycle arrest. Mere15 induced overproduction of ROS and disputed the MMP, and therefore resulted in apoptosis in K562 cells. In addition, Mere15 interacted with tubulin, inhibited the polymerization of microtubules and arrested the K562 cells in G 0 /G 1 phase. In the past decades, thousands of anticancer agents from marine source have been identified. However, most of the agents belong to small chemical molecules. Our study also suggests that the macromolecule polypeptide or proteins, a rich source in marine organisms, could also be important therapeutic candidates for the treatment of cancer diseases.