Hemocyanin from Shrimp Litopenaeus vannamei Has Antiproliferative Effect against HeLa Cell In Vitro

Hemocyanin (HMC) has been shown to participate in multiple roles of immune defence. In this study, we investigated the antiproliferative effect and underpinning mechanism of HMC from Litopenaeus vannamei in vitro. Sulforhodamine B (SRB) assay indicated that HMC could dramatically inhibit the growth of HeLa cells, but not 293T cells under the same conditions. Moreover, typical morphological features of apoptosis in HeLa cells including the formation of apoptotic body-like vesicles, chromatin condensation and margination were observed by using 4, 6-diamidino-2- phenylindole dihydrochloride (DAPI) staining and fluorescence analysis. An apoptotic DNA ladder from 180 to 300 bp was also detected. Furthermore, 10 variation proteins associated with apoptosis pathway, viz. G3PDH isoforms 1/2 (G3PDH1/2), aldosereductase, ectodemal dysplasia receptor associated death receptor domain isoform CRA_a (EDARADD), heat shock 60kD protein 1 variant 1 (HSP60), heat shock 70kDa protein 5 precursor (HSP70), heat shock protein 90kDa beta member 1 precursor (HSP90), 14-3-3 protein ζ/δ, Ran and ubiquitin activating enzyme E1(UBE1), were identified from HMC-treated HeLa cells by the proteomic and quantitative real-time RT-PCR strategies. Importantly, the reactive oxygen species (ROS), mitochondrial membrane potential (Δψm) and caspase-9/3 activities were changed significantly in HMC-treated HeLa cells. Together, the data suggests that L. vannamei HMC mediates antiproliferative properties through the apoptosis mechanism involving the mitochondria triggered pathway.


Introduction
Hemocyanin (HMC) is an extracellular giant copper-containing glycoprotein found in the hemolymph of both mollusk and arthropod. As the main protein component of hemolymph, it typically represents up to 95% of the total amount of protein. It is colorless in the deoxy form and blue in the oxygenous form, whose traditional function is responsible for oxygen transport [1]. Subsequently, it is demonstrated that HMC is also involved in several physiological processes, such as energy storage, osmoregulation, molt cycle and exoskeleton formation [2][3][4]. Later studies reveal that HMC can act as a multifunctional protein associated with the immune defense in invertebrates [5][6]. HMC from about 45 species, such as Tachypleus tridentatus, Porcellio scaber, Cherax quadricarinatus, and so on, could be functionally converted into a phenoloxidase-like enzyme [7][8][9]. Moreover, Zhang et al. reported that Penaeus monodon HMC could act as an antiviral agent against a variety of viruses including DNA and RNA viruses [10]. Zanjani et al. found Haliotis rubra HMC served as a new antiviral candidate for herpes simplex virus (HSV) infections [11]. Jiang et al. indicated that HMC from horseshoe crab Carcinoscorpius rotundicauda could possess a strong antimicrobial defense by the production of reactive oxygen species (ROS) activated with microbial proteases [12]. Destoumieux-Garzón reported that C-terminal fragments from HMCs in penaeid shrimps, Penaeus vannamei and Penaeus stylirostris, had broad antifungal activities [13]. Additionally, Oncomelania hupensis HMC could function as a vaccine in combination with Freund's adjuvant to evaluate the induction of immune responses and protection against Schistosoma japonicum infection in mice [14]. HMCs isolated from marine gastropods Rapana thomasiana and Megathura crenulata were doucumented to be acted as a potential bio-adjuvant for subunit vaccines [15]. Further, our previous evidence indicated that HMC from Litopenaeus vannamei could react with human IgG or IgA as an antigen [16,17], bind to eight bacteria as an agglutinin [17], interact with inhomogeneous erythrocytes as a hemolysin [18], and enhance shrimp's immune response to immunostimulants as a related immune-enhancing protein [19].
Notably, in contrast to the similarities in binding oxygen mollusk and arthropod HMCs are profoundly different in their molecular structure, size, and subunit organization. Generally, mollusk HMCs exist as decamers of several subunits with approximate masses of 350-450 kDa, each consists of 7 or 8 globular "functional units" connected by linker peptide strands, forming hollow cylindrical arrays with 5-or 10-fold axial symmetry. While arthropod HMCs are built on an entirely different plan, it consists of multiples of hexamers, each hexamer made of monomers of about 75 kDa. Because of these differences, it has now become customary to consider the mollusk and arthropod HMCs as different proteins [29][30][31]. However, so far little is known about the antitumor effects of HMC in arthropod.
In this study, the antiproliferative properties of HMC from shrimp L. vannamei against HeLa cells in vitro were investigated. Furthermore, the underlying mechanism was investigated via cellular, proteomics and molecular biology strategies. Our data will assist in the investigation of multifunctionality of HMC and help to establish a potential strategy for cancer control.
Penaeid shrimps (L.vannamei), weighing about 18g, were obtained from Shantou Huaxun Aquatic Product Corporation (Shantou, Guangdong, China). They were cultured in tanks filled with 25 L of open-circuit filtered seawater at room temperature with free access to regular food and enough oxygen. Shrimps were acclimatized to laboratory conditions for 2 d before the experiments. Hemolymph was drawn directly from L.vannamei pericardial sinus and sera were separated as our previous descriptions [17]. The study protocol was approved by the Institutional Animal Care and Use Committee of Shantou University.
Purification and identification of HMC HMC purification was performed by affinity chromatography as described previously with modifications [18]. Briefly, a affinity chromatography column with a ligand of rabbit antishrimp HMC antibodies was installed according to the conventional method. After loading with L. vannamei sera (200 μL), the column was washed with PBS (0.01 M, pH 7.4) until the absorbance at 280 nm reached baseline. Bound HMC was eluted with glycine-HCl buffer (0.1 M, pH 2.4), and neutralized immediately with Tris-HCl buffer (1M, pH 8.0). After being concentrated, the total protein concentration was determined by the Bicinchoninic Acid assay (Genstar, China), and then stored in 0.01M pH7.4 PBS at -20°C until further identification and analysis.
Purified HMC was identified by gel electrophoresis and immunoblotting assays. SDS polyacrylamide gel electrophoresis (SDS-PAGE) was carried out under reducing conditions on a 10% polyacrylamide separating gel with a 5% stacking gel. The gel was stained with Coomassie Brilliant Blue R-250. For Western blotting, the proteins were transferred to a polyvinylidene fluoride (PVDF) membrane after SDS-PAGE with a semi-dry transfer apparatus according to the manufacturer's instructions. The membrane was blocked for 1 h with 5% skim milk in TBS (20 mM Tris, 0.15 M NaCl, pH 7.4) at room temperature, then incubated with rabbit antishrimp HMC antisera (1:1500 dilution) and goat anti-rabbit IgG-HRP (1:3000 dilution) antibodies at room temperature for 1 h and 40 min, respectively. Finally, the membrane was washed and developed with substrate (3'3-diminobenzidine, DAB) until optimum color was observed. In addition, bovine serum albumin (BSA) was used as a negative control to exclude potential non-specific reaction errors.

Cell culture
Cervical carcinoma line HeLa cells were provided by Research Institute for Biomedical and Advanced Materials, Shantou University, Shantou 515063, China. Human embryonic kidney 293T cells, as a negative control, were given by Medical College, Shantou University, Shantou 515063, China. The cells were maintained in Dulbecco's Modification of Eagle's Medium (DMEM, Thermo, USA) supplemented with 10% fetal bovine serum (Gibco, USA), 100 IU/mL penicillin and 100 μg/mL streptomycin. Cells were cultivated in a humidified incubator containing 5% CO 2 at 37°C.

Sulforhodamine B (SRB) assay
The SRB assay was performed according to the report by Sagias et al [32]. In brief, HeLa or 293T cells were planted into a 96-well plate (5×10 3 /well) overnight. The cells were then treated with 10, 20, 30, 40 or 50μg/mL of HMC. PBS (0.01M pH 7.4) was used as a negative control, while 5-fluorouracil (5-FC, 20 μg/mL) was used as a positive control. After 24h or 48 h treatment, the supernatant was discarded, each well was then fixed with 50 μL of cold trichloroacetic acid (500 mg/mL) at 4°C for 1 h. After washing four times with deionized water, cells were incubated with 100 μL /well of SRB (4 mg/mL) for 30 min. Finally, the cells were washed with 1% acetic acid to remove excess dye, and then processed to solubilize the bound protein dye by adding 200 μL/ well of 10 mM unbuffered Tris base. The absorbance at 515 nm was measured with a microplate reader (BioTek, USA). The inhibition rate (%) = (OD 0h /OD 24h or 48h -1) × 100%. All samples were prepared in triplicate and data are expressed as means ± standard error (SD). The p values were determined using Student's t-test.
At the same time, the nuclear staining of HeLa cells by 4,6-diamidino-2-phenylindole dihydrochloride (DAPI) was carried out as previously described [33]. Briefly, HeLa cells (5×10 3 /well) were treated with 10-50 μg/mL of HMC for 48 h. The cells were washed with PBS (0.01M, pH 7.4) twice and fixed with 2.5% glutaraldehyde for 20 min, and then washed two times with PBS (0.01M, pH 7.4). The fixed cells were stained with 1 μg/mL of DAPI for 10 min at room temperature. Cells were examined under a fluorescence microscope (Olympus, Tokyo, Japan) equipped with a CCD camera.

Cell immunofluorescence assay
HeLa cells in log phase were harvested and incubated with 30 μL 3.5mg/mL HMC into immunofluorescence chamber at 37°C for 48 h. After washing with 0.01M pH7.4 PBS for twice, followed by 1% BSA for 30 min, then the cells were probed with rabbit anti-shrimp HMC (dilution 1:500) overnight at 4°C. After washing with PBS for three times, added cy3-conjugated anti-rabbit IgG (dilution 1:150) for 1 h. Then the nucleus is properly stained with 100 μL DAPI for 1-2 min, the antibody staining was visualized and images were obtained using an upright fluorescence microscope (Eclipse 90i, Nikon) under ×60 magnification.

DNA ladder assay
DNA ladder assay was carried out as previously described [34]. HeLa cells (1 mL), treated with PBS (0.01 M, pH 7.4), HMC (50 μg/ml) and 5-FC (20 μg/ml) for 48 h, were fixed with 10 mL of 70% ethanol diluted in hanks´buffered salt solution (HBSS) for 24-72 h at room temperature. Cells were collected by centrifugation at 800 g for 5 min, and resuspended in 200 μL of 0.2 M phosphate-citrate (PC) buffer at room temperature for more than 30 min. After centrifugation at 1,000 g for 5 min, the supernatant was dried in a vacuum oven for 15 min. The sample was then incubated with 3 μL of 0.25% Nonidet NP-40 (FLUKA, U.S.A) and 3 μL of 1 mg/mL RNase A (Sigma, U.S.A) for 30 min, and 3 μL of 1 mg/mL proteinase K (Sigma, U.S.A) for an additional 30 min at 37°C. The DNA was eluted by addition of 70μL elution buffer (10 mM Tris-HCl, 0.1 mM EDTA; pH 8.8) followed by centrifugation at 13,000g for 1 min. After extraction and precipitatation, DNA was analyzed by electrophoresis in a 5% polyacrylamide gel at 140 V for 1-2 h. After rinsing with distilled water twice, the gel was stained with 0.2% aqueous ethanolic silver nitrate for 10 min. Finally, the gel was destained in distilled water until optimum DNA bands were observed.

Imaging and mass spectrometry analysis
Following 2-DE analysis, the gel images and differentially expressed proteins between the experimental group and control group were further analyzed with PDQuest software version 8.0 (Bio-Rad, Hercules, CA) and MALDI-TOF-TOF/MS, respectively. The detailed procedures were same as our previous descriptions [35].

Quantitative real-time RT-PCR analysis
For further verification of the above results from mass spectrometry analysis, ten identified genes, viz. G3PDH1, G3PDH2, aldosereductase, EDARADD, HSP60, HSP70, HSP90, 14-3-3 protein z/δ, Ran and UBE1, were amplified by using quantitative real-time RT-PCR assay. Total RNA was extracted from HeLa cells by using Trizol Reagent (Invitrogen, U.S.A). 5 μg of total RNA was reverse transcribed to cDNA using the PrimeScrip RT reagent Kit (TaKaRa, Dalian, China) following the manufacturer's instructions. The specific primers of target genes are listed in Table 1. The PCR procedures include 94°C for 2 min and 30 cycles of 94°C for 30 s and 58°C for 30 s and 72°C for 1 min, then a final elongation at 72°C for 10 min. As an internal loading control, beta-actin transcripts were amplified using the same amplification conditions. Data from the quantitative real-time RT-PCR analysis were subjected to the one-way analysis (oneway ANOVA) followed by an unpaired, two-tailed t-test. The fold change was calculated by the formula of 2 -ΔΔCt , the mean of ΔΔCt was [(Ct. target gene -Ct. beta-actin) treated -(Ct. target gene -Ct. beta-actin) control [35,36]. All samples were prepared in triplicate, each including three technical replicates, and data are expressed as means±standard error (SD). Significant p-values were <0.05 or 0.01 on 2-tailed analysis.

Reactive oxygen species (ROS) measurement
ROS analysis of HeLa cells treated with HMC was carried out as previously described [37]. In brief, HeLa cells were planted at a density of 5×10 3 /well in a 96-well plate. One day after seeding, the culture dishes were treated with HMC (20-

Mitochondrial membrane potential assay
Mitochondrial membrane potential (Δψm) in HeLa cells was measured using the Tetrechlorotetraethylbenzimidazol carbocyanine iodide (JC-1) mitochondrial membrane potential assay kit

HMC possessed antiproliferative effect aganist HeLa cells in vitro
HMC from the haemolymph of L. vannamei was purified by affinity chromatography as shown in Fig 1A. In consistent with our previous findings [18], two bands at molecular weights around  75 and 77 kDa were observed by SDS-PAGE, which could react with anti-shrimp HMC antibody specifically. Then, SRB assay was used to characterize the antiproliferative effect of HMC on HeLa cells viability. As shown in Fig 1B, an obvious inhibitory effect (57-71%) was observed in HeLa cells treated with 10-50 μg/mL of HMC for 48 h as compared to the untreated groups (p<0.01). In contrast, there was no significant difference in growth of 293T cells between the experimental group and negative group under the same conditions. In addition, a similar result was also found by using MTT analysis (data not shown). Thus, these data suggested that shrimp HMC exhibited antiproliferative effect on HeLa cells in vitro as an antitumor agent.

Apoptosis was induced in HMC-treated HeLa cells
To investigate the underlying mechanism of the antiproliferative effect of HMC at the cellular level, cell morphology, chromatin formation and immunofluorescence assay were determined. As shown in Fig 2A, HeLa cells were found to shrink and round up after 48 h exposure to HMC, accompanying with the appearance of membrane blebbing. Similar observations were found in 5-FC treated control, but not in PBS treated HeLa cells. Moreover, chromatin condensation, DNA fragmentation, chromatin margination and apoptotic body-like vesicles were revealed by DAPI staining in HMC-treated and 5-FC-treated HeLa cells but without the same phenomenon in PBS-treated group (Fig 2A). Notably, HMC (red) that distributed on the surface of HeLa cells (blue) by immunofluorescence assay, indicating that the interaction between HMC and HeLa cells (Fig 2B). In addition, a representative apoptotic DNA ladder around 180 to 300 bp was also observed in HeLa cells treated with HMC (50 μg/mL) for 48 h (Fig 2C). Thus, these results indicated that apoptosis was induced in HeLa cells treated with HMC.

Apoptosis was mediated by a mitochondria-initiated intrinsic pathway
To test if the mitochondria-initiated pathway was involved in the HMC-induced apoptosis, the content of ROS in HeLa cells was measured. Incubation of HeLa cells with HMC at the concentration of 20-100 μg/mL for 48 h significantly (p<0.05 or 0.01) increased the intracellular level of ROS as compared to those in the control group of PBS (Fig 4A). Given that a high level of ROS could change mitochondrial permeability, we measured the membrane potential (Δψm) in mitochondria. As shown in Fig 4B, the ratio of green and red fluorescence increased significantly (p<0.05 or 0.01) in HeLa cells treated with HMC (20-100 μg/mL) for 48 h when compared to that of negative control group, suggesting the decrease of Δψm in cancer cells. Furthermore, we analyzed the activity of caspase-9/3, showing that the value in HeLa cells exposed to the different concentration of HMC (20-60 μg/mL) for 48 h were significantly (p<0.05 or 0.01) increased compared with those of negative control groups (Fig 4C). Therefore, these data suggested that HMC induced HeLa cells apoptosis might be involved in a mitochondria-initiated intrinsic pathway (Fig 4E). In Vitro Antiproliferative Effect of Hemocyanin in Shrimp

Discussion
It has been documented that cancers of the breast and cervix are the most common causes of cancer death worldwide for women, with cervical cancer the most common cause in 55 countries [38]. HeLa cell is the most widely used model cell line for studying human cellular and molecular biology [39,40]. Evidence suggests that Rapana thomasiana HMC showed antiproliferative activity against human breast cancer MCF-7 and cervical cancer HeLa cells [41]. However, the antiproliferative effect of arthropod HMCs have not been reported. Here, we found for the first time HMC from L. vannamei could inhibit HeLa cells proliferation obviously ( Fig  1). Further, morphological features, bound characterization between HMC and cells, and DNA fragmentation pattern (Fig 2) indicated that HeLa cells bound with HMC died of apoptosis. Moreover, a total of 17 altered proteins were found from HeLa cells treated with HMC by comparative proteomics analysis (Fig 3A). Of these, 10 protein spots were successfully identified as G3PDH1, G3PDH2, aldosereductase, EDARADD, HSP60, HSP70, 14-3-3 protein z/δ, HSP90, Ran and UBE1 (Fig 3A and Table 2). In addition, the transcriptions of HSP60, HSP70, 14-3-3 protein z/δ, HSP90, EDARADD and UBE1 increased, while that of Ran, G3PDH1, G3PDH2 and aldosereductase decreased significantly 48 h post HMC treatment (Fig 3B).
Evidence suggests that these differentially expressed proteins play a significant role in the induction of apoptosis in tumor cells [42][43][44][45][46][47]. For examples, silencing of Ran in various tumor cell types could cause aberrant mitotic spindle formation, mitochondrial dysfunction and apoptosis [43]. EDAR in MCF7 cells has been shown to be capable of activating the nuclear factor-κB, JNK, and caspase-independent cell death pathway [44]. Study by Chandra et al. [45] indicates that HSP60 has a pro-death function probably related to mitochondria-initiated caspasedependent apoptosis. Liossis et al. [46] found that the over expression of HSP70 could enhance the TCR/CD3-and Fas/Apo-1/CD95-mediated apoptotic cell death in Jurkat T cells.
It has been documented that HMC is associated with ROS production in invertebrate [48,12]. Coates et al found. [48] that HMC-derived phenoloxidase (PO) in amebocytes that is under pathologic condition to help them undergoing apoptosis induced by phagocytosis. Jiang et al. [12] reported that HMC from horseshoe crab was cleaved into PO, resulting in the production of ROS intermediate. Previous reports have indicated that ROS plays an important role in regulating and trigging mitochondria-initiated apoptosis [49][50][51]. Excessive amounts of ROS accumulated in mitochondrial could cause mitochondrial swelling and damage to the inner membrane [49,50], subsequently, intermembrane proteins, such as apoptosis inducing factors and cytochrome (Cytc), would be released, and ultimately triggering the caspase cascade [51]. In this study, we showed clearly a significant increase of ROS concentration (Fig 4A), dissipation of Δψm ( Fig 4B) and upregulation of caspase-9/3 activity (Fig 4C) in HeLa cells after 48 h treatment with HMC. These data indicated that mitochondria pathway was involved in the process of HMC-mediated HeLa cell apoptosis.
In summary, the present study showed that HMC from L. vannamei would be effective against HeLa cell growth in vitro. The likely molecular mechanisms underpinning the antiproliferative effect of HMC could be mitochondria mediated apoptosis pathway (Fig 4D). Further investigation will be required to explore the antitumor properties of L.vannamei HMC in vivo by use of the nude mice model. Measurement of caspase-9 (a) and caspase-3 (b) activities in HeLa cells treatment with HMC, and PBS for 48 h. Caspase-9/3 activities were represented with the concentration of pNA. The significance difference between the HMC-(or Rosup-, or CCCP-) and PBS-treated cells was indicated with one (p < 0.05) or two asterisks (p < 0.01). (D) Schematic diagram of the likely molecular mechanism of HMC against HeLa cells in vitro. doi:10.1371/journal.pone.0151801.g004