A Litopenaeus vannamei Hemocyanin-Derived Antimicrobial Peptide (Peptide B11) Attenuates Cancer Cells’ Proliferation

Antimicrobial peptides play important roles in the immune response to pathogens and tumor cells; for this reason, they are being exploited for therapeutic use. In this study, we describe a Litopenaeus vannamei hemocyanin-derived peptide, denoted B11, which shares similar features with other anticancer peptides and attenuates the proliferation of cancer cells. Cell viability assay revealed that B11 significantly inhibited the proliferation of human cervical (HeLa), human hepatocellular carcinoma (HepG2), and human esophageal cancer (EC109) cancer cell lines, but not normal liver cell lines (T-antigen-immortalized human liver epithelial (THLE) cells or THLE-3), by inducing morphological changes, nuclear condensation, and margination, features which are indicative of apoptosis. Besides, peptide B11-induced apoptosis was confirmed by isothiocyanate-labeled Annexin V/propidium iodide (Annexin V-FITC/PI) double staining of HeLa cells. Moreover, cell uptake studies, confocal microscopy, and Western blot analysis revealed that rhodamine-labeled B11 permeated HeLa cells and localized to the mitochondria, causing mitochondria dysfunction through lost mitochondrial membrane potential, which consequently triggered the induction of apoptosis. Increased expression levels of caspase-9, caspase-3, and Bax (Bcl-2-associated X) proteins, coupled with a decrease in Bcl-2 (B-cell lymphoma 2) protein, confirmed that peptide B11 induced apoptosis via the mitochondrial pathway. Thus, the hemocyanin-derived peptide, B11, inhibits the proliferation of cancer cells by causing mitochondrial dysfunction and inducing apoptotic cell death, for which reason it could be explored as an anticancer peptide.


Effect of Peptide B11 on Cancer Cells' Proliferation
The antiproliferative activity of peptide B11 against some cancer cell lines, including HeLa cells (human cervical cancer cells), HepG2 cells (human hepatocellular carcinoma cells), and EC109 cells (human esophageal cancer cells) was examined. When the cell proliferation or viability following treatment with peptide B11, 5-fluorouracil (5-FU), or PBS (Phosphate-buffered saline) was determined using the MTS assay (Figure 2), it was observed that the proliferation of all three cancer cell types was significantly decreased 24 h post-treatment with peptide B11 or with the anticancer drug 5-FU compared with PBS. For instance, peptide B11 significantly (p < 0.05) decreased HeLa cells viability by 20.0% (Figure 2A), while that of HepG2 cells decreased by 23.0% ( Figure 2B) and EC109

Effect of Peptide B11 on Cancer Cells' Proliferation
The antiproliferative activity of peptide B11 against some cancer cell lines, including HeLa cells (human cervical cancer cells), HepG2 cells (human hepatocellular carcinoma cells), and EC109 cells (human esophageal cancer cells) was examined. When the cell proliferation or viability following treatment with peptide B11, 5-fluorouracil (5-FU), or PBS (Phosphate-buffered saline) was determined using the MTS assay (Figure 2), it was observed that the proliferation of all three cancer cell types was significantly decreased 24 h post-treatment with peptide B11 or with the anticancer drug 5-FU compared with PBS. For instance, peptide B11 significantly (p < 0.05) decreased HeLa cells viability by 20.0% (Figure 2A), while that of HepG2 cells decreased by 23.0% ( Figure 2B) and EC109 cells decreased by 13.0% ( Figure 2C) relative to PBS treatment. On the contrary, peptide B11 had no significant effect on the proliferation of normal liver cell lines (THLE-3) ( Figure 2D), as treatment with B11 or PBS for 24 h had almost the same cell viability (97.92% and 100%, respectively). Thus, these results suggest that peptide B11 selectively inhibits the in vitro proliferation of only cancer cell lines, and could potentially be used as an antitumor agent.
Molecules 2018, 23, x FOR PEER REVIEW 4 of 15 cells decreased by 13.0% ( Figure 2C) relative to PBS treatment. On the contrary, peptide B11 had no significant effect on the proliferation of normal liver cell lines (THLE-3) ( Figure 2D), as treatment with B11 or PBS for 24 h had almost the same cell viability (97.92% and 100%, respectively). Thus, these results suggest that peptide B11 selectively inhibits the in vitro proliferation of only cancer cell lines, and could potentially be used as an antitumor agent. Data represent means ± SD for three independent experiments (n = 3). Significant difference relative to control (PBS-treated cells) were determined by one-way ANOVA and indicated by asterisks (* p < 0.05, ** p < 0.01).

Peptide B11 Induces Apoptosis in HeLa Cells
The induction of apoptosis induction and changes to cell morphology were examined to ascertain if this could be responsible for the antiproliferative effect of peptide B11. As shown in Figure  3A, HeLa cells treated with peptide B11 (50 μg/mL) for 24 h were changed morphologically, including cell shrinkage and rounding up. A similar observation was found in 5-FU-treated cells, but not in the PBS-treated cells. In addition, the typical characteristics of apoptosis, including nuclei shrinkage, condensation, margination and the formation of apoptotic body-like vesicles were observed in cells 24 h post-treatment with peptide B11 and 5-FU, but not in PBS ( Figure 3B). Moreover, flow cytometry analysis with Annexin V/propidium iodide (Annexin V/PI) staining revealed that the ratio of viable cells to total cells gradually decreased in peptide B11-treated cells, while there was a time-dependent gradual increase in the percentage of late apoptotic cells ( Figure 3C). In fact, the average percentage of apoptotic cells in peptide B11-treated cells was 40.4% at 48 h post-treatment, which is similar to The cancer cell lines (A) human cervical cancer (HeLa) cells, (B) human hepatocellular carcinoma (HepG2) cells, (C) human esophageal cancer (EC109) cells, and immortalized normal human liver cells (D) THLE-3 cells (T-antigen-immortalized human liver epithelial (THLE) cells) were grown for 24 h in the presence of 50 µg/mL of peptide B11. Cell proliferation was analyzed using the MTS assay with PBS used as the negative control, while 5-fluorouracil (5-FU) was used as the positive control. Data represent means ± SD for three independent experiments (n = 3). Significant difference relative to control (PBS-treated cells) were determined by one-way ANOVA and indicated by asterisks (* p < 0.05, ** p < 0.01).

Peptide B11 Induces Apoptosis in HeLa Cells
The induction of apoptosis induction and changes to cell morphology were examined to ascertain if this could be responsible for the antiproliferative effect of peptide B11. As shown in Figure 3A, HeLa cells treated with peptide B11 (50 µg/mL) for 24 h were changed morphologically, including cell shrinkage and rounding up. A similar observation was found in 5-FU-treated cells, but not in the PBS-treated cells. In addition, the typical characteristics of apoptosis, including nuclei shrinkage, condensation, margination and the formation of apoptotic body-like vesicles were observed in cells 24 h post-treatment with peptide B11 and 5-FU, but not in PBS ( Figure 3B). Moreover, flow cytometry analysis with Annexin V/propidium iodide (Annexin V/PI) staining revealed that the ratio of viable cells to total cells gradually decreased in peptide B11-treated cells, while there was a time-dependent gradual increase in the percentage of late apoptotic cells ( Figure 3C). In fact, the average percentage of apoptotic cells in peptide B11-treated cells was 40.4% at 48 h post-treatment, which is similar to that observed in the 5-FU treated cells (41.1%) at the same time point. These results suggest that peptide B11 exerts a proapoptotic effect on cancer cells, which is synonymous with 5-FU, and that this might account for the antiproliferative effects of peptide B11 on HeLa cells.

Physicochemical Properties of B11 and Cell Permeabilization Analysis
Peptide B11 is located within the copper-containing domain of the large molecular weight L. vannamei hemocyanin protein (GeneBank accession No.: CAB85965.1), and is composed of 15 amino acids, half of which are hydrophobic ( Figure 4A). The physicochemical properties of peptide B11, including molecular weight, total hydrophobic ratio, hydrophobicity, hydrophobic moment, average hydropathy value, molar extinction coefficient, charge and isoelectric point are summarized in Table 1. Peptide B11 has a molecular weight of 1767 Da, with a net positive charge, and a total hydrophobic ratio of 46%. Modeling using the Schiffer-Edmundson helical wheel on the DNAstar (Lasergene 7.1, Madison, WI, USA) program revealed that peptide B11 has an amphipathic α-helical conformation ( Figure 4B), while three-dimensional (3D) model prediction using the I-TASSER program (http:// zhanglab.ccmb.med.umich.edu/I-TASSER) showed that it has α-helical and one β-sheet structure ( Figure 4C). Thus, given the size of peptide B11, its net positive charge, α-helix, and β-sheet structure, as well as other features that are typical of therapeutic peptides and/or AMPs [27,28], it should be able to permeable cells [29]. Peptide B11 is located within the copper-containing domain of the large molecular weight L. vannamei hemocyanin protein (GeneBank accession No.: CAB85965.1), and is composed of 15 amino acids, half of which are hydrophobic ( Figure 4A). The physicochemical properties of peptide B11, including molecular weight, total hydrophobic ratio, hydrophobicity, hydrophobic moment, average hydropathy value, molar extinction coefficient, charge and isoelectric point are summarized in Table  1. Peptide B11 has a molecular weight of 1767 Da, with a net positive charge, and a total hydrophobic ratio of 46%. Modeling using the Schiffer-Edmundson helical wheel on the DNAstar (Lasergene 7.1, Madison, WI, USA) program revealed that peptide B11 has an amphipathic α-helical conformation ( Figure 4B), while three-dimensional (3D) model prediction using the I-TASSER program (http://zhanglab.ccmb.med.umich.edu/I-TASSER) showed that it has α-helical and one β-sheet structure ( Figure 4C). Thus, given the size of peptide B11, its net positive charge, α-helix, and β-sheet structure, as well as other features that are typical of therapeutic peptides and/or AMPs [27,28], it should be able to permeable cells [29]. Cell uptake studies were carried out to determine whether peptide B11 could indeed penetrate cell membranes. Confocal microscopy analysis showed that rhodamine-labeled B11 was readily taken up by HeLa cells, with the number of fluorescent cells higher at 24 h post-treatment compared to 8 h, suggesting that peptide B11 penetrates cells time-dependently ( Figure 4D(i)). Moreover, analysis of the X-Z and Y-Z sections of the three-dimensional (3D) reconstructed confocal images confirmed that peptide B11 was found inside the cytoplasm of cells ( Figure 4D (ii)), further suggesting that as a cationic peptide, B11 could permeate cells to exert its effects.

Peptide B11 Induces Mitochondrial-Dependent Apoptotic Cell Death in HeLa Cells through Lost of Mitochondrial Membrane Potential
The integrity of mitochondrial function is crucial for the maintenance of cell viability [30]. Thus, given that peptide B11 has a net positive charge, induces apoptosis, and could permeate cells, we went about determining the effect of peptide B11 on the mitochondria. First, confocal microscopy revealed that rhodamine-labeled B11 colocalized with MitoTracker in the mitochondria of HeLa cells ( Figure 5A). Moreover, peptide B11 not only localized to the mitochondria, but was able to cause mitochondrial dysfunction in the form of a loss in mitochondrial membrane potential ( Figure 5B). In addition, Western blot analysis was used to further examine the expression of proteins involved in the mitochondrial-dependent apoptosis pathways following treatment with peptide B11. As shown in Figure 5C(i), at 24-h post-treatment of HeLa cells with B11 (50 μg/mL), there was an increase in the protein expression levels of caspase-3 (an executioner caspase in the apoptosis cascade) and caspase-9 (an initiator caspase in the mitochondrial-dependent or intrinsic apoptosis pathway) compared to PBS treatment. Besides, there was an increase in the protein expression level of proapototic Bax ( Figure 5C(ii)) and a decrease in the protein expression level of prosurvival Bcl-2 ( Figure 5C(iii)), two Bcl-2 family members that play a central role in regulating changes in mitochondrial outer membrane permeability [31,32]. All of these results indicate that peptide B11 was able to cause mitochondrial dysfunction and induced apoptosis via the mitochondrial-dependent pathway. Cell uptake studies were carried out to determine whether peptide B11 could indeed penetrate cell membranes. Confocal microscopy analysis showed that rhodamine-labeled B11 was readily taken up by HeLa cells, with the number of fluorescent cells higher at 24 h post-treatment compared to 8 h, suggesting that peptide B11 penetrates cells time-dependently ( Figure 4D(i)). Moreover, analysis of the X-Z and Y-Z sections of the three-dimensional (3D) reconstructed confocal images confirmed that peptide B11 was found inside the cytoplasm of cells ( Figure 4D(ii)), further suggesting that as a cationic peptide, B11 could permeate cells to exert its effects.

Peptide B11 Induces Mitochondrial-Dependent Apoptotic Cell Death in HeLa Cells through Lost of Mitochondrial Membrane Potential
The integrity of mitochondrial function is crucial for the maintenance of cell viability [30]. Thus, given that peptide B11 has a net positive charge, induces apoptosis, and could permeate cells, we went about determining the effect of peptide B11 on the mitochondria. First, confocal microscopy revealed that rhodamine-labeled B11 colocalized with MitoTracker in the mitochondria of HeLa cells ( Figure 5A). Moreover, peptide B11 not only localized to the mitochondria, but was able to cause mitochondrial dysfunction in the form of a loss in mitochondrial membrane potential ( Figure 5B). In addition, Western blot analysis was used to further examine the expression of proteins involved in the mitochondrial-dependent apoptosis pathways following treatment with peptide B11. As shown in Figure 5C(i), at 24-h post-treatment of HeLa cells with B11 (50 µg/mL), there was an increase in the protein expression levels of caspase-3 (an executioner caspase in the apoptosis cascade) and caspase-9 (an initiator caspase in the mitochondrial-dependent or intrinsic apoptosis pathway) compared to PBS treatment. Besides, there was an increase in the protein expression level of proapototic Bax ( Figure 5C(ii)) and a decrease in the protein expression level of prosurvival Bcl-2 ( Figure 5C(iii)), two Bcl-2 family members that play a central role in regulating changes in mitochondrial outer membrane permeability [31,32]. All of these results indicate that peptide B11 was able to cause mitochondrial dysfunction and induced apoptosis via the mitochondrial-dependent pathway.

Discussion
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][34][35]. 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 [36,37], with data suggesting that AMPs could substitute traditional antibiotics in combatting microbial infections, especially multidrug-resistant microbes [17,38]. Most importantly, some AMPs have been shown to have antitumor activity [39,40]. 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 [41,42], recent reviews have reported on the anticancer activities and efficacy of AMPs from terrestrial animals and plants [21,[43][44][45]. 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 [13,14,46]. 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 [21,43]. 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 4A) 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 [44,47], 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 4D), just as other anticancer peptides [40,48], and localized to the mitochondria ( Figure 5A), where it induced a loss of mitochondrial membrane potential ( Figure 5B), 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 [50,51]. 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][52][53]. 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 5C(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 [31,56]. Interestingly, peptide B11 treatment caused a decrease in antiapoptotic Bcl-2 protein, but an increase in proapototic Bax in HeLa cells ( Figure 5C(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 [59,60]. 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.

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).

Cell Proliferation Assay
Cell proliferation was determined using the MTS assay as reported by Kong et al. [62]. Briefly, 5 × 10 3 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 AQ ueous 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% = OD B11 /OD PBS × 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-test.

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 × 10 5 /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 CaCl 2 ); 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).

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).

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 × 10 5 ) 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 CO 2 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).

Analysis of Subcellular Localization
HeLa cells (1 × 10 5 ) 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 CO 2 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).

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 × 10 5 /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 (CO 2 ) 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.

Conclusions
Collectively, the data generated in the current study indicates that antimicrobial peptide B11, which is derived from the large molecular weight hemocyanin protein of L. vannamei, has antiproliferative effects on cancer cells, as it was able to cause mitochondrial dysfunction and induce apoptosis. In view of these interesting findings, future studies would explore the antitumor potential of peptide B11 using an animal model.

Patents
Zhang YL, Liu SJ, Zheng LY and Wang F have filed a patent that relates to the use of the peptide (B11) for tumor suppression.