Hemocyanin (HMC) is a respiratory protein, which is also reported to be multi-functional, and thus plays essential roles in mollusk and arthropods [1
]. A growing number of immune-related functions have been ascribed to hemocyanin [3
], including phenoloxidase [5
], antiviral [6
], agglutinative [7
], anticancer [9
], reaction with anti-human Ig (immunoglobulin) as an antigen [10
], hemolysin activity [11
], and as an immune-enhancement protein in shrimp [12
]. Moreover, in different crustaceans, hemocyanin is reported to generate antimicrobial peptides (AMPs) in response to microbial challenge [13
]. Destoumieux-Garzón et al. reported that the C-terminal fragment of hemocyanin from penaeid shrimps Penaeus vannamei
and Penaeus stylirostris
had broad antifungal activities [14
]. An antibacterial peptide with 16 amino acid residues was also found in the plasma of freshwater crayfish Pacifastacus leniusculus
]. Similarly, we previously found an 18.4-kDa fragment of hemocyanin with antimicrobial activity in Litopeneus vannamei
infected with Vibrio parahaemolyticus
]. Generally, AMPs are small cationic peptides characterized by positive charges and hydrophobic amino acids, as well as amphipathic features [17
]. Since AMPs are positively charged, they are able to bind to negatively charged bacteria cell membranes, resulting in the disruption of the membrane and bacteria death [18
]. These features and properties of AMPs makes them important components of the innate immune system in a variety of organisms, including plants and animals [19
Several recent studies have shown that AMPs also have anticancer activity [21
]. For instance, Rodrigues et al. reported that a cream that is mixed with the AMP gomesin, and used as a topical drug to smear over the external surface of tumors, successfully treated intradermal and intraepithelial cancers [22
]. A synthetic 21-mer AMP (Epinecidin-1) from grouper (Epinephelus coioides
) has been reported to have in vitro antitumor activity against human fibrosarcoma cells (HT1080) [23
]. Similarly, the pleurocidin family of AMPs (NRC-03 and NRC-07) derived from Atlantic flounder
were found to kill breast carcinoma cells, including drug-resistant and slow-growing breast cancer cells [24
]. Interestingly, our recent studies involving the screening of L. vannamei
hemocyanin identified 20 potential AMPs ranging from 1.5 to 1.9 kDa [25
]. While the antibacterial activities of these hemocyanin-derived peptides have been ascertained, whether or not these peptides also have anticancer effects is not known.
In the current study, we report on the antiproliferative and potential anticancer activity of one of these L. vannamei hemocyanin-derived AMPs (designated B11). Peptide B11 could inhibit the proliferation of three cancer cell lines by permeabilizing, entering, and inducing apoptotic cell death. Given the properties exhibited by peptide B1, it could be used for anticancer agents, while the knowledge gained from this study could provide the basis for developing therapeutic peptides from marine resources into anticancer therapeutic agents.
Several recent studies have reported on the development and sophisticated resistance being mounted by cancer cells and bacteria against most of the traditional drugs [33
]. Therefore, researchers are exploring alternative more efficacious drugs, with more attention being drawn toward the use of naturally occurring antimicrobial peptides (AMPs). Several AMPs have so far been evaluated in both preclinical and clinical studies [36
], with data suggesting that AMPs could substitute traditional antibiotics in combatting microbial infections, especially multidrug-resistant microbes [17
]. Most importantly, some AMPs have been shown to have antitumor activity [39
]. In the current study, an in silico predicted AMP (designated peptide B11) derived from the hemocyanin protein of the shrimp L. vannamei
, was synthesized manually via solid phase peptide synthesis, and shown to significantly inhibit the proliferation of cancer cells. The antiproliferative effect of peptide B11 is because it causes mitochondrial dysfunction and induces apoptosis, therefore suggesting its anticancer potential. The research approach that was used in this study implies that bioinformatics prediction tools could be leveraged to explore anticancer therapeutic agents followed by cell-based validation.
While a number of studies have explored the discovery of AMPs, and studied their structures and mechanisms of action [41
], recent reviews have reported on the anticancer activities and efficacy of AMPs from terrestrial animals and plants [21
]. The shrimp L. vannamei
is reported to generate different AMPs from hemocyanin, both naturally as part of the innate immune system and in response to specific pathogen challenge [13
]. The antimicrobial activities of these shrimp hemocyanin-derived AMPs have been demonstrated [25
]; however, their effect on cell proliferation and potential use as antiproliferative or antitumor agents has not been explored.
In this study, the antitumor potential of a 15 amino acids hydrophobic cationic antimicrobial peptide, peptide B11, which is derived from the large molecular weight hemocyanin protein of L. vannamei
, and has α-helical and β-sheet structure, was explored. The structure and physicochemical properties of AMPs are reported to play a significant role in their ability to induce apoptosis in cancer cells [21
]. Cationic AMPs are able to physically associate with the negatively charged membranes of cancer cells, destabilizing the lipid membrane and subsequently binding to intracellular targets, resulting in cell death [24
]. On the other hand, AMPs rich in hydrophobic amino acids are able to insert into cell membranes to form a stable structure, thereby disrupting the cell membrane and forming pores, leading to changes in cell membrane charge and therefore interfering with cell death pathways [21
]. Given that the features of peptide B11 (Table 1
and Figure 4
A) are synonymous with cationic anticancer peptides, this suggests that peptide B11 might share similar properties and/or functions. Since the outer membranes of cancer cells are negatively-charged due to an abundance of anionic molecules, while the membranes of noncancerous or normal cells are neutral [44
], cationic peptides such as B11 could exert antiproliferative effects targeted at only cancer cells, due to this membrane charge difference. In the current study, rhodamine-labeled B11 was able to permeate HeLa cells (Figure 4
D), just as other anticancer peptides [40
], and localized to the mitochondria (Figure 5
A), where it induced a loss of mitochondrial membrane potential (Figure 5
B), and consequently mitochondrial dysfunction. Dysfunctional mitochondria might eventually lead to the induction of apoptosis via the mitochondrial-dependent pathway [49
In the apoptosis pathway, caspases (cysteine proteases) act in concert in a cascade to trigger apoptosis [50
]. The apoptosis-related caspases are generally divided into initiator caspases (including caspase-2, caspase-8, caspase-9, and caspase-10), and effector or executioner caspases (including caspase-3, caspase-6, and caspase-7) [51
]. As the initiator caspase in the mitochondrial-dependent apoptotic pathway [54
], activated caspase-9 in turn activates caspase-3/7 potentiating the apoptosis cascade, and thereby catalyzing the cleavage of many key cellular proteins that commit cells to apoptosis. To ascertain that there was an induction of apoptosis due to the mitochondrial dysfunction, an increase in the protein expression levels of caspase-9 and caspase-3 (determined by Western blot) was observed following the treatment of HeLa cells with peptide B11 (Figure 5
C(i)). Since the Bcl-2 family proteins, which govern mitochondrial outer membrane permeability, are either proapoptotic (Bax, BAD, Bak, Bok, etc.) or antiapoptotic (Bcl-2, Bcl-xL, Bcl-w, etc.) [55
], we went on to determine changes in the protein expression of Bcl-2 and Bax, given their important role in mitochondrial-dependent apoptosis [31
]. Interestingly, peptide B11 treatment caused a decrease in antiapoptotic Bcl-2 protein, but an increase in proapototic Bax in HeLa cells (Figure 5
C(ii),(iii)). This observation is similar to that previously reported by Wang et al., where an oligopeptide from Sepia
ink induced the apoptosis of lung cancer cells via the mitochondrial pathway [57
]. The antiapoptotic Bcl-2 family proteins prevent cell death induced by various apoptotic stimuli by inhibiting the release of mitochondrial cytochrome C, and inhibiting the activation of caspase-9 and caspase-3 [58
]. Thus, a decrease in the expression of Bcl-2 protein coupled with an increase in the expression of Bax often facilitates apoptosis [59
]. The studies presented thus far provide evidence that suggest peptide B11 exerts an antiproliferative effect in cancer cells by targeting the mitochondria, causing mitochondrial dysfunction through a loss in membrane potential, and consequently inducing mitochondrial-dependent apoptosis.
4. Materials and Methods
4.1. Peptide Synthesis
Peptide B11 (RIRDAIAHGYIVDKV) and rhodamine-labeled B11 were synthesized by a commercial company (Scilight Biotechnology, Beijing, China) using the solid-phase procedure as reported by Huertas et al. [61
]. Briefly, 9-fluorenylmethoxycarbonyl amino acid (Fmoc amino acid) and 2,6-dichlorobenzenoylchloride (DCB) were added to the resin to attach the first amino acid. The deprotection was conducted by adding piperidine to remove the protection group, and then the activated amino acid was attached to the peptidyl resin with agitation to couple the next residue. The cycle of deprotection and coupling was repeated until the target peptide was completely synthesized. Rhodamine-labeling was carried out after the last deprotection and coupling step. The peptide was then cleaved from the resin with trifluoroacetic acid (TFA). The disulfide bridge in the peptide was formed by DMF, and C-terminal amidation was conducted by the amidating enzyme. The synthetic peptides were purified by reversed-phase high performance liquid chromatography (RP-HPLC, Omaha, NE, USA) with an Agela C18 column (Agilent, CA, USA). The purity and molecular masses of the purified synthetic peptides were determined using RP-HPLC and MALDI-TOF mass spectrometry (Bruker Daltonics, Bremen, Germany).
4.2. Cell Culture
Human cervical cancer cells (HeLa cells), human hepatocellular carcinoma cells (HepG2 cells), human esophageal cancer cell (EC109 cells), and normal liver cell lines (THLE-3 cells) were kind gifts from Dr. En Min Li of Shantou University Medical College, Shantou University, China. All of the cell lines were cultured in Dulbecco’s Modified Eagle Medium (DMEM, Thermo Fisher Scientific Inc., Waltham, MA, USA) supplemented with 10% fetal bovine serum (FBS, Gibco, Carlsbad, CA, USA) and 1% penicillin/streptomycin (Gibco), and then maintained in a 5% CO2 incubator at 37 °C.
4.3. Cell Proliferation Assay
Cell proliferation was determined using the MTS assay as reported by Kong et al. [62
]. Briefly, 5 × 103
cells were seeded onto 96-well plates overnight. Next, cells were treated with 50 μg/mL of peptide B11 or 50 μg/mL 5-fluorouracil (5-FU) as a positive control, with PBS (0.01 M, pH 7.4) used as the negative control. After 24 h, 20 µL/well of CellTiter 96 AQueous
One Solution (MTS) solution (Promega, Madison, WI, USA) was added and incubated at 37 °C for 3 h. Optical density (OD) was measured using a microplate reader (BioTek, Winooski, VT, USA) at 490 nm. The viability rate was calculated as, cell viability% = ODB11
× 100%. Triplicate samples were analyzed, and data were represented as means ± standard error (SD). Experiments were repeated at least three times and the p
-values were determined using Student’s t
4.4. Cytological Effect of Peptide B11 on HeLa Cells
To examine the cytological effects of peptide B11 on cells, HeLa cells were plated onto 96-well plates (5 × 103
/well) overnight, followed by treatment with peptide B11 (50 μg/mL), 5-FU (50 μg/mL), or PBS (0.01 M, pH 7.4) for 24 h. Next, media was removed, and cells were washed two times with PBS (0.01 M, pH 7.4). Following this, cells were fixed for 20 min with 2.5% glutaraldehyde, and then washed twice with PBS (0.01 M, pH 7.4). The fixed cells were stained for 8 min with 1 μg/mL of 4,6-diamidino-2-phenylindole dihydrochloride (DAPI) at 37 °C. Cells were washed four times with PBS (0.01 M, pH 7.4), and finally examined under a fluorescence microscope (Olympus, Tokyo, Japan) as previously described [63
4.5. Annexin V-FITC/PI Apoptosis Detection Assay
The ability of peptide B11 to induce cell death in terms of apoptosis was determined using flow cytometry with Annexin V-FITC apoptosis detection kit (Beijing Beyotime Corp, Beijing, China) following the manufacturers’ instructions. Briefly, HeLa cells were plated onto 24-well plates (1 × 105/well) overnight. Next, cells were treated with peptide B11 (50 μg/mL), PBS (0.01 M, pH 7.4), and 5-FU (100 μg/mL) for eight to 48 h. At each time point, cells were washed with PBS (0.01 M, pH 7.4) and re-suspended in 195 μL of binding buffer (10 mM of Hepes/NaOH, pH 7.4, 140 mM of NaCl, 2.5 mM of CaCl2); then, five μL of Annexin V-FITC and 10 μL of propidium iodide (PI) were added to stain for 15 min at room temperature in the dark. Cells were analyzed on an Accuri C6 flow cytometer (BD bioscience, SanDiego, CA, USA).
4.6. Prediction of the Structural Characteristics and Features of Peptide B11
The physicochemical properties of peptide B11 were analyzed using the antimicrobial peptide database web server (APD3, Omaha, NE, USA), while the helical properties of peptide B11 were determined by Schiffer Edmundson wheel modeling using the DNAstar Lasergene 7.1 programme. The 3D structure of peptide B11 was predicted using the I-TASSER program (Version 5.1, zhanglab, Ann Arbor, MI, USA) (http://zhanglab.ccmb.med.umich. edu/I-TASSER
4.7. Cell Uptake Studies
The uptake of peptide B11 by cells was performed according to the method described by Wang et al. [48
]. Briefly, HeLa cells (1 × 105
) were plated onto a 35-mm glass-bottom dish (In Vitro Scientific, Sunnyvale, CA, USA) overnight, followed by treatment with 50 μg/mL of rhodamine-labeled B11 for eight to 24 h, and placed in a CO2
incubator at 37 °C. The media was removed, and the cells were washed three times with PBS (0.01 M, pH 7.4). Following this, cells were stained with one μg/mL of Hoechst 33342 (Beijing Beyotime Corp) for 10 min at 37 °C, before being washed three times with PBS (0.01 M, pH 7.4). Cells were imaged using a confocal microscope (LSM 800, Carl Zeiss, Jena, Germany).
4.8. Analysis of Subcellular Localization
HeLa cells (1 × 105) were plated onto a 35-mm glass-bottom dish (In Vitro Scientific) overnight, and then treated with 50 μg/mL of rhodamine-labeled B11 for eight h, and placed in a CO2 incubator at 37 °C. The media was removed, and the cells were washed three times with PBS (0.01 M, pH 7.4). Following this, cells were incubated for 30 min with 200 nM of MitoTracker Green (Beijing Beyotime Corp), and then for 10 min with one μg/mL Hoechst 33342 at 37 °C to visualize the mitochondria and nuclei, respectively. Next, the reagents were removed, and the monolayer of cells washed three times with ice-cold PBS before being examined by a confocal laser-scanning microscope (LSM 800, Carl Zeiss, Germany).
4.9. JC-1 Dye Staining for Mitochondrial Membrane Potential Analysis
The loss of mitochondrial membrane potential (∆Ψm) was examined by confocal laser-scanning microscopy (LSM 800, Carl Zeiss, Germany) using 5,5′,6,6′-tetrachloro-1,1′, 3,3′-tetraethylbenzimidazole-carbocyanide iodine (JC-1; Beijing Beyotime Corp) staining. Briefly, HeLa cells were seeded at a density of 1 × 105/well onto a 35-mm glass-bottom dish (In Vitro Scientific). After overnight incubation, cells were treated for 24 h with peptide B11 (50 μg/mL) and PBS (0.01 M, pH 7.4); then, they were placed in a carbon dioxide (CO2) incubator at 37 °C. The spent media was removed, and cells washed once with PBS. Next, one mL of complete culture media and one mL of JC-1 working solution were added to each well, and then they were incubated for 20 min at 37 °C protected from light according to the manufacturer’s instructions. After being washed twice with buffer solution, cells were examined using a laser-scanning confocal microscope (LSM 800, Carl Zeiss, Germany) at room temperature. Cells were treated with 10 μM of carbonyl cyanide m-chlorophenylhydrazone (CCCP), which is a protonophore that can cause the dissipation of ∆Ψm, and was used as positive control.
4.10. Western Blot Analysis
Western blot analysis was performed as described previously [64
]. Briefly, HeLa cells were plated onto six-well plates (1 × 106
/well) overnight, and then treated with peptide B11 (50 μg/mL) and PBS (0.01 M, pH 7.4) for 24 h and collected, washed, and pelleted by centrifugation. Cell pellets were lysed with Radioimmunoprecipitation assay (RIPA) buffer (150 mM of sodium chloride, 1% Triton X-100, 1% sodium deoxycholate, 0.1% SDS, 50 mM of Tris–HCl, pH 7.5, and 2 mM of ethylenediaminetetraacetic acid (EDTA)) with protease inhibitor (Roche, Indianapolis, IN, USA). Total protein extracts were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) using a 12% SDS-PAGE gel, and then transferred onto a polyvinylidene difluoride (PVDF) membrane with a wet transfer apparatus according to the manufacturer’s instructions (Bio-Rad, Richmond, CA, USA). The membrane was blocked for 1 h with 5% skimmed milk in Tris Buffered Saline with Tween (TBST) (0.1% Tween-20, 20 mM Tris, 0.15 M NaCl, pH 7.4) at room temperature. Next membranes were probed with primary antibodies: anti-caspase-9 antibody (Rabbit monoclonal, Abcam; 1:1000), anti-caspase-3 antibody (Rabbit monoclonal, Abcam; 1:1000), anti-Bax (Rabbit monoclonal, Abcam; 1:1000), anti-β-actin antibody (Rabbit polyclonal, Abbkine; 1:1000) or anti-Bcl-2 antibody (Mouse monoclonal, Abbkine; 1:1000). After being washed three times with TBST, membranes were then incubated with horseradish peroxidase (HRP) linked goat anti-mouse or goat anti-rabbit secondary antibodies (Sigma-Aldrich, St Louis, MO, USA 1:5000) for 1 h at room temperature. Signals were detected by chemiluminescence using an enhanced chemiluminescence (ECL)-detecting reagent, and images were captured using the GE Amersham Imager 600 imaging system (GE, Boston, MA, USA).