# Antiproliferative and Proapoptotic Effects of a Protein Component Purified fromAspongopus chinensis Dallas on Cancer CellsIn Vitro andIn Vivo

Authors: Jun Tan; Ying Tian; Renlian Cai; Tianci Yi; Daochao Jin; Jianjun Guo
Journal: Evidence-Based Complementary and Alternative Medicine
 (2019)

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## Abstract

Aspongopus chinensisDallas is used as a traditional Chinese medicine. In China, clinical evidence suggests that it has anticancer activity. However, the anticancer active components are not fully elucidated. In the present study, we purified an anticancer active component (named CHP) fromA. chinensis. To gain a comprehensive insight into the protein components, shotgun proteomic analysis was conducted. The anticancer active protein band was cut from the sodium dodecyl sulphate-polyacrylamide gel electrophoresis gel and digested with trypsin to generate peptide mixture. The peptide fragments were then analysed by liquid chromatography tandem mass spectrometry; 18 proteins were identified. In addition, we evaluated the effects of CHP on the proliferation and apoptosis of two human gastric cancer cell lines (SGC-7901 and BGC-823). The cultured cells were treated with CHP at concentrations of 20, 30, and 40 μg/mL. Inhibition of cell growth was determined by the MTT assay. Hoechst 33258 staining was adopted to detect apoptosis morphologically. Apoptotic cells were quantified by Annexin V-FITC/propidium iodide staining and flow cytometry. Tumour growth was assessed by subcutaneous inoculation of 4T1 cells into BALB/c mice. There was a concentration- and time-dependent decrease in the proliferation of both cell lines at CHP concentrations of 20–40 μg/mL. Apoptotic characteristics, such as karyopyknotic pyknic hyperfluorescence bolus and nuclear fragmentation, were observed in both the cell lines by Hoechst 33258 staining. Flow cytometry showed that CHP induced significant (P< 0.01) concentration-dependent apoptosis of SGC-7901 cells.In vivo assay showed that CHP can partially inhibit tumour growth derived from 4T1 cellsin vivo. The present study is the first to report that CHP inA. chinensis inhibits the proliferation of cancer cell lines via the suppression of cancer cell proliferation and acceleration of apoptosis.


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## Body

## 1. Introduction

Based on GLOBOCAN estimates, approximately 14.1 million new cancer cases and 8.2 million deaths occurred in 2012 worldwide [1]. Gastric cancer is the fourth major malignancy in the world and the second most common cancer in China, with approximately 679,100 new cases and 498,000 related deaths in 2015 [2], and is the third leading cause of cancer-related deaths in China [3]. Approximately 70% of patients with gastric cancer are diagnosed at an advanced stage [4]. Breast cancer is the most common malignancy in women, comprising approximately 18% of all female cancers [5]. In the past decade, the incidence of breast cancer in China increased by 27%, among which, 40% of the patients diagnosed died within five years [6].Several chemotherapy agents offer significant advantages in the treatment of cancer. However, some of these drugs are often considered ineffective or excessively toxic. Therefore, highly effective and less toxic drugs are urgently required. During recent years, some investigators have focused on crude drugs and health products from insects, which are the most diverse and abundant of all terrestrial animals [7].Aspongopus chinensis Dallas is an insect belonging to Pentatomidae and is widely distributed in China, including Guizhou, Sichuan, and Yunnan Provinces [8]. DriedA.chinensis is a common traditional Chinese medicine used to relieve pain, warm the stomach, and treat nephropathy. It is also a popular and unique food in some regions of China [9]. Extracts ofA.chinensis possess strong anticancer and antibacterial activities that have been demonstrated by pharmacological studies [10, 11]. It has also been reported thatA. chinensis has anticancer effects. Yang et al. (1999) used Xianglong powder (composed ofPinellia ternata,Scolopendra subspinipes,A.chinensis, and RhizomaAtractylodis Macrocephalae) and observed its ability to induce apoptosis in human gastric cancer cells [12]. Fan et al. (2011) investigated the effect of serum containingA. chinensis on the expression of the apoptosis-related factors, Fas-associated death domain and p53, in human SW480 colon cancer cell line. They found that it had a significant inhibitory effect on the proliferation of SW480 and that the mechanism of inducing SW480 cell apoptosis might be associated with the stimulation of FADD and suppression of p53 [11]. These previous studies used driedA. chinensis, and the anticancer active components have not been fully elucidated.In the past, studies focused on the isolation and identification of small molecule natural products from driedA.chinensis[13, 14]. Four new norepinephrine derivatives, three new sesquiterpenoids, one new lactam, and twenty-three known compounds have been identified, most of which were isolated fromA.chinensis for the first time. Selected members of insect-derived substances were evaluated for their biological activities against renal protection in high-glucose-induced mesangial cells and COX-2 inhibition [15]. In addition, four pairs of enantiomeric hypoxanthine analogues, two pairs of enantiomeric adenine analogues, two pyrazines, aspongpyrazines A and B, and a pair of glycerides, along with 28 known substances, were isolated fromA. chinensis. Several newA. chinensis-derived natural products have been reported to promote the proliferation of neural stem cells (NSCs) [16]. However, information on the effects of peptides and proteins present in liveA.chinensisis limited. Furthermore, the anticancer active components are not fully elucidated.Our previous study results demonstrated that the anticancer active components were mainly distributed in the haemolymph and abdomen [17], and the haemolymph ofA. chinensis can inhibit the proliferation of human gastric cancer SGC-7901 cells and human breast cancer MCF-7 cells in a dose- and time-dependent manner [18–20]. In the present study, an anticancer active component (named CHP) was isolated from the haemolymph ofA. chinensis. The results of this study will provide a theoretical basis for investigating the anticancer effects ofA. chinensis and support further use of this medicinal insect resource.


## 2. Materials and Methods

### 2.1. Chemicals and Reagents

The Annexin V-FITC apoptosis detection kit was purchased from Nanjing KeyGen Biotech (Nanjing, China). 3-(4,5-Dimethyl-2-thiazolyl)-2, 5-diphenyl-2H-tetrazolium bromide (MTT) was obtained from Beyotime (Beyotime Institute of Biotechnology, Shanghai, China). Dulbecco’s modified Eagle’s medium was purchased from Hyclone (GE Healthcare, Logan, UK).


### 2.2. Collection of Haemolymph from A. chinensis

Specimens ofA. chinensis were caught in Zunyi, Guizhou Province, China. The specimens were authenticated by Prof. Jian-jun Guo (Institute of Entomology, Guizhou University, Guiyang, China) and washed thrice with distilled water. The samples were then dried with filter paper and the haemolymph was extracted by squeezing the insects through cheese cloth. The fluid was centrifuged at 28,000 × g for 10 min at 4°C and then transferred into another centrifuge tube containing the protease inhibitor phenylmethylsulfonyl fluoride (0.5 mM), lyophilised, and stored at -80°C for later use.


### 2.3. Haemolymph Purification

The haemolymph ofA. chinensis was purified by heat treatment, ammonium sulphate precipitation, ultrafiltration, and reversed-phase high-performance liquid chromatography (RP-HPLC). Freshly prepared haemolymph was incubated at 20°C, 40°C, 60°C, 80°C, and 100°C for 30 min and then centrifuged at 28,000 × g for 10 min. The active anticancer components were collected, mixed with 10 mL of 10 mM Tris HCl buffer (pH 7.4), and then subjected to ammonium sulphate precipitation (0%–20%, 20%–40%, 40%–60%, 60%–80%, and 80%–100%). The pellet resulting from 40%–60% precipitation was dialysed and then concentrated and lyophilised. Three peptide fractions (> 50, 5–50, and < 5 kDa) were obtained by ultrafiltration using 5- and 50-kDa cut-off hydrophilic membranes (EMD Millipore, Billerica, MA, USA). Subsequently, the 5–50-kDa fraction was further purified by semi-preparative RP-HPLC on a C18 column (4.6 mm × 250 mm). The 5–50-kDa fraction was dissolved in solvent A (water: trifluoroacetic acid (TFA), 1000:1, v/v) and subjected to HPLC (Waters Corp., Mildford, MA, USA). The fraction (400 μL) was injected and eluted with a linear gradient of 0%–30% acetonitrile at a flow rate of 0.5 mL·min−1. The data-processing software was Empower 2 (Waters Corp.). To isolate the active and stable components, each fraction was collected, dialysed, and lyophilised. The anticancer active protein levels were determined by Bradford method using bovine serum albumin as the standard [21].


### 2.4. Sodium Dodecyl Sulphate Polyacrylamide Gel Electrophoresis (SDS-PAGE) for Separation of Anticancer Active Components

Molecular mass of the anticancer active components was determined by SDS-PAGE as described by Laemmli [22]. SDS-PAGE was executed using an electrophoresis unit with 5% stacking and 15% separating polyacrylamide gels in 25 mM Tris HCl of pH 8.3, containing 0.18 M glycine and 0.1% SDS. The protein bands were identified by staining the gel with Coomassie Brilliant Blue R-250.


### 2.5. In-Gel Digestion

The protein band in SDS-PAGE gel was cut into a cube (approximately 1 mm × 1 mm) and washed twice with 400μL of 50 mM of NH4HCO3/ACN (50% v/v). The Cys residues were reduced by 100 μL of 50 mM DTT at 56°C for 30 min and then alkylated by 100 μL of 100 mM of iodoacetamide at 25°C for 30 min. After dehydration of the gel band with acetonitrile (ACN), the proteins were digested in gel overnight with 10 μL of 0.05 μg/μL of trypsin (Promega, Madison, US) in 50 mM of NH4HCO3 at 25°C. The generated peptides were extracted with 60% ACN (v/v) in 0.1% formic acid (v/v). The peptide mixture samples were then completely dried in a vacuum centrifuge. For LC-MS/MS, the samples were reconstituted in 0.1% formic acid.


### 2.6. Protein Analysis by LC–MS/MS

We diluted each filtrate sample to 1μg/μL peptide, and 5 μL of each diluted sample was analysed via LC-MS/MS using the TripleTOF® 5600 mass spectrometer (AB SCIEX, Framingham, US). The mass spectrometer was coupled to an Eksigent UPLC425 system (AB SCIEX, Framingham, US). HPLC was performed with a trap and elution configuration using a C18 column (150 mm × 75 μm, 3μm). The LC solvents included solvent B (95% CAN + 0.1% formic acid). The sample was loaded in the trap column at a flow rate of 300 nL/min for 10 min using 100% solvent A and eluted at a flow rate of 300 μL/min using a stepwise gradient (0–1 min, 5%–7% B; 1–45 min, 7%–40% B). The full-scan mass spectra obtained by the data-dependent acquisition mode were used for protein identification, and parallel reaction monitoring technique was used for quantitative analysis of the target signature peptide. The RAW data files were analysed using Protein Pilot 4.5 (AB SCIEX, Framingham, US), and high-confidence peptides were used for protein identification by setting a target false discovery rate threshold of 1% at the peptide level. Only unique peptides with high confidence were used for protein identification.


### 2.7. Cell Culture

Human gastric cancer cell lines SGC-7901 and BGC-823 and murine mammary adenocarcinoma 4T1 cells were provided by the Stem Cell Bank, Chinese Academy of Sciences. The cells were maintained as adherent monolayer cultures in DMEM medium (SGC-7901 and BGC-823 cells) and RPMI-1640 medium (4T1 cells), containing 100 U/mL penicillin, 100μg/mL streptomycin (Life Technology, Shanghai, China), and 10% foetal bovine serum (GE Healthcare, Logan, UK). The cells were cultured in an incubator with 5% CO2 at 37°C.


### 2.8. Cell Viability Assay

SGC-7901 and BGC-823 cells (5 × 103/well) were seeded in 96-well plates and incubated for 24 h. After treatment with CHP (0, 20, 30, or 40 μg/mL) for 24, 48, and 72 h, viability was determined by the MTT assay. Twenty microliters of MTT solution (5 mg/mL in phosphate-buffered saline) was added to each well, and the mixtures were incubated for 4 h at 37°C. The MTT solution was then removed and 150 μL of dimethyl sulfoxide was added to the wells. The absorbance of the solution at 570 nm was measured using an automated microplate reader (Bio-Rad, Hercules, CA, US). The control values for dimethyl sulfoxide alone were subtracted from all readings and the mean absorbance was calculated. The 50% inhibition of growth (IC50) value for CHP was determined using the relative viability from the CHP concentration curve. The experiment was repeated five times. The proliferation index was calculated as the percent of viable cells in the drug-treated group versus untreated control cells using the following equation: proliferation index (%) = absorbance (treated) / absorbance (control) × 100.


### 2.9. Morphological Study

SGC-7901 and BGC-823 cells were grown on coverslips (1 × 105 cells/coverslip), incubated with CHP (0, 20, 30, or 40 μg/mL), and then fixed in ethanol:acetic acid solution (3:1, v/v). The coverslips were mounted on glass slides for the morphometric analysis. Three monolayers per experimental group were photographed. The morphological changes of SGC-7901 and BGC-823 cells were determined using a Nikon bright-field inverted light microscope (Tokyo, Japan) at 400× magnification.


### 2.10. Hoechst 33258 Staining

Apoptosis was analysed using the Hoechst Staining Kit (Beyotime, Shanghai, China) according to the manufacturer’s protocol. The cells were grown on coverslips (1 × 105 cells/coverslip) and then treated with CHP (0, 20, 30, or 40 μg/mL) for 36 h. The harvested cells were washed with PBS, treated with fixing solution for 10 min, and then stained with Hoechst 33258 fluorescent dye for 5 min at 25°C. The morphological nuclear changes were observed and captured using a fluorescence microscope.


### 2.11. Cell Apoptosis by Flow Cytometry

The extent of apoptosis was analysed using the Annexin V-FITC Apoptosis Detection Kit according to the manufacturer’s instructions (Nanjing KeyGen Biotech, Nanjing, China). The cells were seeded in six-well plates and then treated with CHP (10, 15, and 20μg/mL) or PBS for 24 h. The harvested cells were washed twice with ice-cold PBS and then resuspended in 500 μL of binding buffer containing 5 μL of Annexin V-FITC staining solution. The cells were incubated in dark for 20 min. Then, 5 μL of propidium iodide staining solution was added to the cell suspension. The cells were mixed gently and incubated for 15 min in dark at 25°C. The stained cells were analysed immediately. The number of apoptotic cells was quantified using a flow cytometer (Accuri C6; BD Biosciences, San Jose, CA, US) that collected 10,000 events for analysis. The data were analysed with CFlow Plus software (BD Biosciences, San Jose, CA, US). Each assay was performed in triplicate.


### 2.12. In Vivo Assays of Antitumour Activity

Six-week-old female BALB/c mice of body weight approximately 18 g (Byrness Weil Biotech Ltd., Chongqing, China) were housed in individually ventilated cages. After approximately one week of acclimatization, the cells (1.0 × 106) were resuspended in PBS (0.1 mL) and inoculated subcutaneously into the second breast of each mice. When the tumour volume reached approximately 60 mm3, the animals were randomised to the following treatment groups—NS (normal PBS, 0.02 mL/kg/2 d, in situ injection), CHP (40 mg/kg/2 d, in situ injection); each group contained 8 mice. Tumour length and width and animal body weight were measured every two days. The tumour volume was calculated using the following formula: (a × b2) / 2; where “a” is the smallest diameter and “b” is the widest one. The mice were sacrificed at the end of drug treatment and the tumours were harvested and then weighed and photographed. Thein vivo assays were carried out in accordance with the institutional guidelines that comply with national and international laws and policies. All animal experiments were approved by the Ethics Committee of Guizhou University.


### 2.13. Statistical Analysis

All data are presented as mean ± SD. The significance of differences was evaluated by the one-way analysis of variance, followed by Fisher’s least significant difference (LSD) comparison using SPSS 18.0 software (IBM, Chicago, IL, USA), The P value of <0.01 was considered statistically significant.


## 2.1. Chemicals and Reagents

The Annexin V-FITC apoptosis detection kit was purchased from Nanjing KeyGen Biotech (Nanjing, China). 3-(4,5-Dimethyl-2-thiazolyl)-2, 5-diphenyl-2H-tetrazolium bromide (MTT) was obtained from Beyotime (Beyotime Institute of Biotechnology, Shanghai, China). Dulbecco’s modified Eagle’s medium was purchased from Hyclone (GE Healthcare, Logan, UK).


## 2.2. Collection of Haemolymph from A. chinensis

Specimens ofA. chinensis were caught in Zunyi, Guizhou Province, China. The specimens were authenticated by Prof. Jian-jun Guo (Institute of Entomology, Guizhou University, Guiyang, China) and washed thrice with distilled water. The samples were then dried with filter paper and the haemolymph was extracted by squeezing the insects through cheese cloth. The fluid was centrifuged at 28,000 × g for 10 min at 4°C and then transferred into another centrifuge tube containing the protease inhibitor phenylmethylsulfonyl fluoride (0.5 mM), lyophilised, and stored at -80°C for later use.


## 2.3. Haemolymph Purification

The haemolymph ofA. chinensis was purified by heat treatment, ammonium sulphate precipitation, ultrafiltration, and reversed-phase high-performance liquid chromatography (RP-HPLC). Freshly prepared haemolymph was incubated at 20°C, 40°C, 60°C, 80°C, and 100°C for 30 min and then centrifuged at 28,000 × g for 10 min. The active anticancer components were collected, mixed with 10 mL of 10 mM Tris HCl buffer (pH 7.4), and then subjected to ammonium sulphate precipitation (0%–20%, 20%–40%, 40%–60%, 60%–80%, and 80%–100%). The pellet resulting from 40%–60% precipitation was dialysed and then concentrated and lyophilised. Three peptide fractions (> 50, 5–50, and < 5 kDa) were obtained by ultrafiltration using 5- and 50-kDa cut-off hydrophilic membranes (EMD Millipore, Billerica, MA, USA). Subsequently, the 5–50-kDa fraction was further purified by semi-preparative RP-HPLC on a C18 column (4.6 mm × 250 mm). The 5–50-kDa fraction was dissolved in solvent A (water: trifluoroacetic acid (TFA), 1000:1, v/v) and subjected to HPLC (Waters Corp., Mildford, MA, USA). The fraction (400 μL) was injected and eluted with a linear gradient of 0%–30% acetonitrile at a flow rate of 0.5 mL·min−1. The data-processing software was Empower 2 (Waters Corp.). To isolate the active and stable components, each fraction was collected, dialysed, and lyophilised. The anticancer active protein levels were determined by Bradford method using bovine serum albumin as the standard [21].


## 2.4. Sodium Dodecyl Sulphate Polyacrylamide Gel Electrophoresis (SDS-PAGE) for Separation of Anticancer Active Components

Molecular mass of the anticancer active components was determined by SDS-PAGE as described by Laemmli [22]. SDS-PAGE was executed using an electrophoresis unit with 5% stacking and 15% separating polyacrylamide gels in 25 mM Tris HCl of pH 8.3, containing 0.18 M glycine and 0.1% SDS. The protein bands were identified by staining the gel with Coomassie Brilliant Blue R-250.


## 2.5. In-Gel Digestion

The protein band in SDS-PAGE gel was cut into a cube (approximately 1 mm × 1 mm) and washed twice with 400μL of 50 mM of NH4HCO3/ACN (50% v/v). The Cys residues were reduced by 100 μL of 50 mM DTT at 56°C for 30 min and then alkylated by 100 μL of 100 mM of iodoacetamide at 25°C for 30 min. After dehydration of the gel band with acetonitrile (ACN), the proteins were digested in gel overnight with 10 μL of 0.05 μg/μL of trypsin (Promega, Madison, US) in 50 mM of NH4HCO3 at 25°C. The generated peptides were extracted with 60% ACN (v/v) in 0.1% formic acid (v/v). The peptide mixture samples were then completely dried in a vacuum centrifuge. For LC-MS/MS, the samples were reconstituted in 0.1% formic acid.


## 2.6. Protein Analysis by LC–MS/MS

We diluted each filtrate sample to 1μg/μL peptide, and 5 μL of each diluted sample was analysed via LC-MS/MS using the TripleTOF® 5600 mass spectrometer (AB SCIEX, Framingham, US). The mass spectrometer was coupled to an Eksigent UPLC425 system (AB SCIEX, Framingham, US). HPLC was performed with a trap and elution configuration using a C18 column (150 mm × 75 μm, 3μm). The LC solvents included solvent B (95% CAN + 0.1% formic acid). The sample was loaded in the trap column at a flow rate of 300 nL/min for 10 min using 100% solvent A and eluted at a flow rate of 300 μL/min using a stepwise gradient (0–1 min, 5%–7% B; 1–45 min, 7%–40% B). The full-scan mass spectra obtained by the data-dependent acquisition mode were used for protein identification, and parallel reaction monitoring technique was used for quantitative analysis of the target signature peptide. The RAW data files were analysed using Protein Pilot 4.5 (AB SCIEX, Framingham, US), and high-confidence peptides were used for protein identification by setting a target false discovery rate threshold of 1% at the peptide level. Only unique peptides with high confidence were used for protein identification.


## 2.7. Cell Culture

Human gastric cancer cell lines SGC-7901 and BGC-823 and murine mammary adenocarcinoma 4T1 cells were provided by the Stem Cell Bank, Chinese Academy of Sciences. The cells were maintained as adherent monolayer cultures in DMEM medium (SGC-7901 and BGC-823 cells) and RPMI-1640 medium (4T1 cells), containing 100 U/mL penicillin, 100μg/mL streptomycin (Life Technology, Shanghai, China), and 10% foetal bovine serum (GE Healthcare, Logan, UK). The cells were cultured in an incubator with 5% CO2 at 37°C.


## 2.8. Cell Viability Assay

SGC-7901 and BGC-823 cells (5 × 103/well) were seeded in 96-well plates and incubated for 24 h. After treatment with CHP (0, 20, 30, or 40 μg/mL) for 24, 48, and 72 h, viability was determined by the MTT assay. Twenty microliters of MTT solution (5 mg/mL in phosphate-buffered saline) was added to each well, and the mixtures were incubated for 4 h at 37°C. The MTT solution was then removed and 150 μL of dimethyl sulfoxide was added to the wells. The absorbance of the solution at 570 nm was measured using an automated microplate reader (Bio-Rad, Hercules, CA, US). The control values for dimethyl sulfoxide alone were subtracted from all readings and the mean absorbance was calculated. The 50% inhibition of growth (IC50) value for CHP was determined using the relative viability from the CHP concentration curve. The experiment was repeated five times. The proliferation index was calculated as the percent of viable cells in the drug-treated group versus untreated control cells using the following equation: proliferation index (%) = absorbance (treated) / absorbance (control) × 100.


## 2.9. Morphological Study

SGC-7901 and BGC-823 cells were grown on coverslips (1 × 105 cells/coverslip), incubated with CHP (0, 20, 30, or 40 μg/mL), and then fixed in ethanol:acetic acid solution (3:1, v/v). The coverslips were mounted on glass slides for the morphometric analysis. Three monolayers per experimental group were photographed. The morphological changes of SGC-7901 and BGC-823 cells were determined using a Nikon bright-field inverted light microscope (Tokyo, Japan) at 400× magnification.


## 2.10. Hoechst 33258 Staining

Apoptosis was analysed using the Hoechst Staining Kit (Beyotime, Shanghai, China) according to the manufacturer’s protocol. The cells were grown on coverslips (1 × 105 cells/coverslip) and then treated with CHP (0, 20, 30, or 40 μg/mL) for 36 h. The harvested cells were washed with PBS, treated with fixing solution for 10 min, and then stained with Hoechst 33258 fluorescent dye for 5 min at 25°C. The morphological nuclear changes were observed and captured using a fluorescence microscope.


## 2.11. Cell Apoptosis by Flow Cytometry

The extent of apoptosis was analysed using the Annexin V-FITC Apoptosis Detection Kit according to the manufacturer’s instructions (Nanjing KeyGen Biotech, Nanjing, China). The cells were seeded in six-well plates and then treated with CHP (10, 15, and 20μg/mL) or PBS for 24 h. The harvested cells were washed twice with ice-cold PBS and then resuspended in 500 μL of binding buffer containing 5 μL of Annexin V-FITC staining solution. The cells were incubated in dark for 20 min. Then, 5 μL of propidium iodide staining solution was added to the cell suspension. The cells were mixed gently and incubated for 15 min in dark at 25°C. The stained cells were analysed immediately. The number of apoptotic cells was quantified using a flow cytometer (Accuri C6; BD Biosciences, San Jose, CA, US) that collected 10,000 events for analysis. The data were analysed with CFlow Plus software (BD Biosciences, San Jose, CA, US). Each assay was performed in triplicate.


## 2.12. In Vivo Assays of Antitumour Activity

Six-week-old female BALB/c mice of body weight approximately 18 g (Byrness Weil Biotech Ltd., Chongqing, China) were housed in individually ventilated cages. After approximately one week of acclimatization, the cells (1.0 × 106) were resuspended in PBS (0.1 mL) and inoculated subcutaneously into the second breast of each mice. When the tumour volume reached approximately 60 mm3, the animals were randomised to the following treatment groups—NS (normal PBS, 0.02 mL/kg/2 d, in situ injection), CHP (40 mg/kg/2 d, in situ injection); each group contained 8 mice. Tumour length and width and animal body weight were measured every two days. The tumour volume was calculated using the following formula: (a × b2) / 2; where “a” is the smallest diameter and “b” is the widest one. The mice were sacrificed at the end of drug treatment and the tumours were harvested and then weighed and photographed. Thein vivo assays were carried out in accordance with the institutional guidelines that comply with national and international laws and policies. All animal experiments were approved by the Ethics Committee of Guizhou University.


## 2.13. Statistical Analysis

All data are presented as mean ± SD. The significance of differences was evaluated by the one-way analysis of variance, followed by Fisher’s least significant difference (LSD) comparison using SPSS 18.0 software (IBM, Chicago, IL, USA), The P value of <0.01 was considered statistically significant.


## 3. Results

### 3.1. Purification of Anticancer Active Component from A. chinensis

The haemolymph extracted fromA. chinensis inhibited the proliferation of SGC-7901 cells in a dose-dependent manner (IC50 = 42.912 mg/mL) (Figure 1(a)). We found that when the haemolymph (2086.368 mg) was treated at high temperature, a large amount of proteins was denatured. The haemolymph treated at 60°C still exhibited anticancer activity, which was detected by the MTT assay (IC50 = 3.145 mg/mL) (Table 1). Therefore, the components of haemolymph treated at 60°C (named Component I) were selected for further purification.Table 1
Summary of purification of CHP fromA. chinensis Dallas.
No. Purification Step Total protein (mg) Yield (%) IC50 (mg/mL) 1 Haemolymph 2086.368 100 42.912  2 Heat treatment (60°C) 266.251 12.761 3.145  3 Ammonium sulphate (40-60%) 67.492 3.235 1.435  4 Ultrafiltration (5-50 kDa) 17.158 0.822 0.337  5 Semi-preparative RP-HPLC 1.162 0.056 0.026Figure 1
Effect of haemolymph (a) and ammonium sulphate-precipitated components (b) on the proliferation of SGC-7901 cells. The values are presented as mean ± SD of five independent experiments. Different upper case letters indicate significant difference between groups (Fisher’s LSD,P< 0.01).
(a) (b)Component I was precipitated by ammonium sulphate salting-out method, with 0%–20%, 20%–40%, 40%–60%, 60%–80%, and 80%–100% saturation gradient. The five groups of proteins were precipitated and dissolved with PBS, dialysed, and freeze-dried. The concentration of protein was determined by Bradford method and the inhibition of cell proliferation was detected by the MTT assay. The results showed that 40%–60% ammonium sulphate saturation precipitated the components that could significantly inhibit the proliferation of SGC-7901 cells (IC50 = 1.435 mg/mL) (Figure 1(b)). Therefore, the 40%–60% ammonium sulphate-precipitated fraction (hereafter referred to as Component II) was selected for the downstream separation and purification experiment.To determine the effect of molecular weight on the antiproliferative activity of proteins, Component II was fractionated to obtain three molecular weight fractions (< 5, 5–50, and > 50 kDa), which were screened for the antiproliferative activity against SGC-7901 cells. Among these fractions, the 5–50-kDa proteins showed higher potency (IC50 = 0.337 mg/mL) to inhibit the proliferation of SGC-7901 cells than that of the > 50-kDa (IC50 = 0.459 mg/mL) and < 5-kDa (IC50 = 4.425 mg/mL) proteins (Figure 2(a)). This indicates that haemolymph proteins of intermediate molecular weight (5–50-kDa fractions) are more effective in inhibiting the proliferation of SGC-7901 cells than smaller molecular weight proteins (< 5 kDa) and high molecular weight proteins (> 50 kDa). The 5–50-kDa fractions were fractionated on a C18 column and the protein peaks were resolved (Figure 2(b)). Finally, the anticancer active component was subjected to SDS-PAGE. A band (15 kDa) was observed in the gel (Figure 2(c)).Figure 2
Purification of the anticancer active component. (a) Effect of three molecular weight fractions (< 5, 5–50, and > 50 kDa) on the proliferation of SGC-7901 cells. (b) Semi-preparative reversed-phase high-performance liquid chromatograms of CHP (indicated by an arrow) purified fromA. chinensis. (c) SDS-PAGE reveals the anticancer active component. Lane A: purified proteins (CHP) (indicated by an arrow); Lane B: 5–50-kDa ultrafiltration fractions; Lane M: protein marker.


### 3.2. LC–MS/MS Analysis

The most active subfraction (CHP) was selected for peptide identification by LC–MS/MS. A total of 59 peptides of different lengths, ranging from 6 to 19 amino acids, were identified (Figure3). We identified 18 proteins by LC-MS/MS analysis. Six of these had at least six unique peptides and were thus recognised with high confidence (Table 2), namely, cytochrome c (Cyt c), ferritin, superoxide dismutase, ATP synthase subunit a, NADH-ubiquinone oxidoreductase chain 1, and cytochrome b. Among these proteins,Cyt c might be a noteworthy anticancer protein, which might play the main role in anticancer effect. The release of cytochrome c from mitochondria was necessary for the formation of the Apaf-1 apoptosome and subsequent activation of caspase-9 in mammalian cells, and apoptosis was initiated [23]. This was consistent with the findings of our previous study, and the expression of caspase 3, caspase 9, and Bax proteins was significantly upregulated when SGC-7901 cells were treated with haemolymph, which indicated that apoptosis of SGC-7901 cells induced by the haemolymph might be via the mitochondrial signal pathway [24].Table 2
Proteins identified with high confidence from the CHP ofA. chinensis Dallas.
Peptide sequence Prec MW Prec m/z Theor MW Theor m/z Protein DTLFEYLENPKK 1552.774414 777.3945 1552.777344 777.3959351 Cytochrome c MVFAGIK 807.4334717 404.724 807.4312744 404.7229309 LENPKK 727.4298706 364.7222 727.4228516 364.718689 ADLIAYLEQATK 1391.730103 696.8723 1391.729614 696.8720703 DTLFEYLENPKK 1495.751831 748.8832 1495.755859 748.8851929 KPQERADLIAYLEQATK 2032.07959 678.3671 2032.05896 678.3602295 VGPNLSGLIGR 1139.647217 570.8309 1139.629883 570.8222046  EGLGIFVLDK 1090.612305 546.3134 1089.607056 545.810791 Ferritin QNFHADSEEAINK 1501.667236 751.8409 1501.679688 751.847168 SLGDLLTNVR 1086.604492 544.3095 1086.603271 544.30896 VKEGLGIFVLDK 1316.761719 659.3881 1316.770386 659.3924561 AVEAALQLEK 1070.595215 536.3049 1070.597168 536.3058472 LAFVEIDR 977.5206909 489.7676 977.5181885 489.7663879 TLALLHR 822.5070801 412.2608 822.5075684 412.2610474 LFNLFYFAK 1162.605713 582.3101 1162.606323 582.3104248  MISLCGPLSIIGR 1431.728638 716.8716 1431.757813 716.8861694 Superoxide dismutase TIVVHADPDDLGK 1379.704834 690.8597 1378.709229 690.3618774 VACGVIGITK 1073.583618 537.7991 1073.590332 537.8024292 HVGDLGNICAEANGVAK 1752.820435 877.4175 1752.846436 877.430542 YDTPELVEK 1149.551025 575.7828 1149.55542 575.7849731 AGGQVAWTK 916.4700928 459.2423 916.4766846 459.2456055 EAFSLFDKDGDGTITTK 1843.888916 615.6369 1843.884033 615.6352539  LFDLFFFAK 1203.633667 602.8241 1203.632813 602.8236694 Secreted Hemocyanin-like protein  AVGVDLNLK 927.5370483 464.7758 927.5389404 464.7767334 Glutathione s-transferase  KASGQKIGQK 1044.60791 523.3112 1044.592773 523.3036499 Tyrosine-protein phosphatase corkscrew isoform x2 LVSLSEQQLIDCSR 1704.82605 853.4203 1704.835205 853.4249268  LVSLSEQQLIDCSR 1704.82605 853.4203 1704.835205 853.4249268 Cysteine protease  INNPLK 698.3964844 350.2055 698.3963013 350.2054138 Death-associated protein 1  KLNTNSLQK 1044.60791 523.3112 1044.592773 523.3036499 Telomerase reverse transcriptase  VLHGTVVAVGPGAR 1331.76123 444.9277 1331.767334 444.929718 Heat shock protein, putative  LDEALEAAR 986.5010986 494.2578 986.5032959 494.2589111 TATA element modulatory factor  RAKEASAAEGGIK 1286.706421 644.3605 1286.694214 644.3544312 6S proteasome non-ATPase regulatory subunit 4  SILSTLYASEVMYENT 1819.820435 607.6141 1819.85498 607.6256104 ATP synthase subunit a SLYNEFKLLTGPNNK 1736.867798 435.2242 1736.90979 435.2347107 LLTGPNNK 856.4470825 429.2308 856.4654541 429.2399902 INIIMNNMLNSLYNEFK 2070.905273 1036.46 2071.011719 1036.513184 TNLFSTFDPATSIK 1541.681274 771.8479 1541.761353 771.8879395 PFMVLIETISNLIR 1644.788452 549.2701 1644.927246 549.3164063 STLYASEVMYE 1291.598267 646.8064 1291.564209 646.7893677 INIIMN 716.3768921 359.1957 716.3890991 359.2018127 MTNLFSTFDPAT 1343.644287 672.8294 1343.606812 672.8106689  MVFIMFWFIWVR 1673.763306 837.8889 1673.86145 837.9379883 LMYLTWK 953.5025024 477.7585 953.5044556 477.7595215 TGLMGLLQPFADGLK 1559.709106 780.8618 1559.838135 780.9263306 SVLGYIQLRK 1176.579468 589.297 1176.686646 589.3505859 NADH-ubiquinone oxidoreductase chain 1 IFMSFPLFF 1147.543457 574.779 1147.577637 574.7960815 MSFPLFFCWLSSCLAETNR 2364.968506 592.2494 2365.069336 592.2745972 SEYMNIIFM 1146.536621 574.2756 1146.508911 574.2617798  FQGNKFYPLNK 1355.621094 678.8178 1355.687378 678.8509521 Cytochrome b DVNNGWLMR 1104.517456 553.266 1104.502197 553.2583618 AGRGLYYGSYK 1233.599121 617.8068 1233.614258 617.8143921 WLWGGFSVDNATLTR 1722.773315 862.3939 1722.836548 862.4255371 SNYDKTPFHPYF 1514.661133 758.3378 1514.682983 758.3488159 MLGDPENFIP 1147.543457 574.779 1147.521973 574.7682495  ISFINQNIK 1075.515259 538.7649 1075.602539 538.8085938 NADH dehydrogenase subunit 6  NDIICSQMNWK 1407.588623 704.8016 1407.627563 704.8210449 ATP synthase protein 8  TASYDREK 968.3950806 485.2048 968.4562988 485.2354431 NADH-ubiquinone oxidoreductase chain 3 TICSIISK 920.4076538 461.2111 920.5001221 461.2573242 MMMYVMMTTI 1266.534912 634.2747 1266.519043 634.2667847Figure 3
LC-MS/MS spectrogram reveals the anticancer active component (CHP).


### 3.3. Cell Viability

Morphological characterization of SGC-7901 and BGC-823 cells was performed after treatment with CHP. A light microscope was used to evaluate the extent of cell shrinkage at 48 h after treatment with different concentrations of the CHP protein. The results were compared with those of untreated gastric cancer cells. The most recognizable morphological changes of CHP-treated cells were cytoplasmic condensation and cell shrinkage (Figures4(a) and 5(a)).Figure 4
Effect of CHP on the proliferation of gastric cancer SGC-7901 cells. (a) Morphologic characterization of SGC-7901 cells treated with different concentrations of CHP (0, 20, 30, and 40μg/mL; A-D, respectively). (b) The viability of SGC-7901 cells treated with 0 (control), 20, 30, and 40 μg/mL CHP. The viability of cells was determined by the MTT assay. The values are presented as mean ± SD of five independent experiments. Different upper case letters indicate significant difference between the groups (Fisher’s LSD,P < 0.01).
(a) (b)Figure 5
Effect of CHP on the proliferation of gastric cancer BGC-823 cells. (a) Morphologic characterization of BGC-823 cells treated with different concentrations of CHP (0, 20, 30, and 40μg/mL; A–D, respectively). (b) The viability of BGC-823 cells treated with 0 (control), 20, 30, and 40 μg/mL CHP. The viability of cells was determined by the MTT assay. The values are presented as mean ± SD of five independent experiments. Different upper case letters indicate significant difference between the groups (Fisher’s LSD,P < 0.01).
(a) (b)To determine the antiproliferative effects of CHP on gastric cancer cells, their viability was determined by the MTT assay. The MTT assay was used as a sensitive and reliable colorimetric assay to measure the viability and proliferation of cells. The cleavage of MTT has several desirable properties for assaying cell survival and proliferation. MTT is cleaved by all living, metabolically active cells, but not by dead cells or erythrocytes. The amount of formazan generated is directly proportional to the cell number over a wide range, using a homogeneous cell population. Activated cells produce more formazan than the resting cells, which enable the measurement of activation even in the absence of proliferation. These properties are all consistent with the cleavage of MTT only by active mitochondria [25]. The MTT assay was performed to determine the IC50 of CHP in SGC-7901 and BGC-823 cell lines. The viability of gastric cancer SGC-7901 and BGC-823 cells treated with different concentrations of CHP (0, 20, 30, and 40 μg/mL) for 24, 48, and 72 h is shown in Figures 4(b) and 5(b). The treated cells showed a reduced proliferation capacity in a concentration-dependent manner (P< 0.01), although SGC-7901 (IC50 = 25.462 μg/mL) was more sensitive than BGC-823 (IC50 = 29.003 μg/mL) cells.


### 3.4. Hoechst 33258 Staining

The nuclei were stained with Hoechst 33258 to determine the incidence of apoptosis morphologically. Representative photomicrographs showed the morphological characteristics of apoptosis, such as chromatin condensation and nuclear fragmentation (indicated by arrows), in SGC-7901 and BGC-823 cells treated with different concentrations of CHP. All data were obtained within 36 h of treatment. The CHP 20, 30, and 40μg/mL groups showed significant increases in apoptosis compared with that of the control group (Figure 6), indicating that CHP increases the extent of apoptosis.Figure 6
Apoptosis induced by treatment of BGC-823 (A1-A4) and SGC-7901 (B1-B4) cells with 0 (control), 20, 30, or 40 μg/mL CHP for 36 h. Panels 1-4 represent treatment with 0, 20, 30, or 40 μg/mL CHP, respectively.


### 3.5. Flow Cytometry

Apoptosis is frequently dysregulated during the progression of cancer [23]. After treatment of SGC-7901 cells with CHP for 24 h, flow cytometry showed that the percent of apoptotic cells was 2.67 ± 0.09, 7.7 ± 0.23, 14.07 ± 0.37, and 26.3 ± 0.95 %, for 0, 10, 15, and 20 μg/mL CHP, respectively (Figure 7). Quantification of early and late apoptosis after treatment with various concentrations of CHP showed significantly increased apoptosis in the groups treated with 10, 15, and 20 μg/mL CHP compared with that in the control group (P < 0.01).Figure 7
Flow cytometry analysis of SGC-7901 cell apoptosis by double staining with Annexin V-FITC and propidium iodide (PI). Values are presented as the mean ± SD of three independent experiments. Different upper case letters indicate significant difference between groups (Fisher’s LSD,P < 0.01).
(a) (b)


### 3.6. CHP Inhibited In Vivo Tumour Growth in BALB/c Mice

To elucidate whether CHP suppressesin vivo tumour growth, we established the live tumour model by subcutaneously transplanting 4T1 cells into BALB/c mice, which werein situ injection of CHP (20 mg/kg/2 d), PDD (1 mg/kg/2 d), or PBS (0.02 mL/kg/2 d). Two weeks later, the CHP-treated BALB/c mice developed smaller tumours in terms of size and weight compared with those of PBS group (Figures 8(a) and 8(b)). These results implicated that CHP can partially inhibit tumour growth derived from 4T1 cellsin vivo.Figure 8
The antitumour effects of CHPin vivo. (a) 4T1 cells were subcutaneously injected into the BALB/c mice. Tumour volume measurements for the BALB/c mice during treatment. (b) Tumour weight at the end of treatment (Day 14). Values are presented as the mean ± SD of three independent experiments. Different upper case letters indicate significant difference between groups (Fisher’s LSD,P < 0.01).
(a) (b)


## 3.1. Purification of Anticancer Active Component from A. chinensis

The haemolymph extracted fromA. chinensis inhibited the proliferation of SGC-7901 cells in a dose-dependent manner (IC50 = 42.912 mg/mL) (Figure 1(a)). We found that when the haemolymph (2086.368 mg) was treated at high temperature, a large amount of proteins was denatured. The haemolymph treated at 60°C still exhibited anticancer activity, which was detected by the MTT assay (IC50 = 3.145 mg/mL) (Table 1). Therefore, the components of haemolymph treated at 60°C (named Component I) were selected for further purification.Table 1
Summary of purification of CHP fromA. chinensis Dallas.
No. Purification Step Total protein (mg) Yield (%) IC50 (mg/mL) 1 Haemolymph 2086.368 100 42.912  2 Heat treatment (60°C) 266.251 12.761 3.145  3 Ammonium sulphate (40-60%) 67.492 3.235 1.435  4 Ultrafiltration (5-50 kDa) 17.158 0.822 0.337  5 Semi-preparative RP-HPLC 1.162 0.056 0.026Figure 1
Effect of haemolymph (a) and ammonium sulphate-precipitated components (b) on the proliferation of SGC-7901 cells. The values are presented as mean ± SD of five independent experiments. Different upper case letters indicate significant difference between groups (Fisher’s LSD,P< 0.01).
(a) (b)Component I was precipitated by ammonium sulphate salting-out method, with 0%–20%, 20%–40%, 40%–60%, 60%–80%, and 80%–100% saturation gradient. The five groups of proteins were precipitated and dissolved with PBS, dialysed, and freeze-dried. The concentration of protein was determined by Bradford method and the inhibition of cell proliferation was detected by the MTT assay. The results showed that 40%–60% ammonium sulphate saturation precipitated the components that could significantly inhibit the proliferation of SGC-7901 cells (IC50 = 1.435 mg/mL) (Figure 1(b)). Therefore, the 40%–60% ammonium sulphate-precipitated fraction (hereafter referred to as Component II) was selected for the downstream separation and purification experiment.To determine the effect of molecular weight on the antiproliferative activity of proteins, Component II was fractionated to obtain three molecular weight fractions (< 5, 5–50, and > 50 kDa), which were screened for the antiproliferative activity against SGC-7901 cells. Among these fractions, the 5–50-kDa proteins showed higher potency (IC50 = 0.337 mg/mL) to inhibit the proliferation of SGC-7901 cells than that of the > 50-kDa (IC50 = 0.459 mg/mL) and < 5-kDa (IC50 = 4.425 mg/mL) proteins (Figure 2(a)). This indicates that haemolymph proteins of intermediate molecular weight (5–50-kDa fractions) are more effective in inhibiting the proliferation of SGC-7901 cells than smaller molecular weight proteins (< 5 kDa) and high molecular weight proteins (> 50 kDa). The 5–50-kDa fractions were fractionated on a C18 column and the protein peaks were resolved (Figure 2(b)). Finally, the anticancer active component was subjected to SDS-PAGE. A band (15 kDa) was observed in the gel (Figure 2(c)).Figure 2
Purification of the anticancer active component. (a) Effect of three molecular weight fractions (< 5, 5–50, and > 50 kDa) on the proliferation of SGC-7901 cells. (b) Semi-preparative reversed-phase high-performance liquid chromatograms of CHP (indicated by an arrow) purified fromA. chinensis. (c) SDS-PAGE reveals the anticancer active component. Lane A: purified proteins (CHP) (indicated by an arrow); Lane B: 5–50-kDa ultrafiltration fractions; Lane M: protein marker.


## 3.2. LC–MS/MS Analysis

The most active subfraction (CHP) was selected for peptide identification by LC–MS/MS. A total of 59 peptides of different lengths, ranging from 6 to 19 amino acids, were identified (Figure3). We identified 18 proteins by LC-MS/MS analysis. Six of these had at least six unique peptides and were thus recognised with high confidence (Table 2), namely, cytochrome c (Cyt c), ferritin, superoxide dismutase, ATP synthase subunit a, NADH-ubiquinone oxidoreductase chain 1, and cytochrome b. Among these proteins,Cyt c might be a noteworthy anticancer protein, which might play the main role in anticancer effect. The release of cytochrome c from mitochondria was necessary for the formation of the Apaf-1 apoptosome and subsequent activation of caspase-9 in mammalian cells, and apoptosis was initiated [23]. This was consistent with the findings of our previous study, and the expression of caspase 3, caspase 9, and Bax proteins was significantly upregulated when SGC-7901 cells were treated with haemolymph, which indicated that apoptosis of SGC-7901 cells induced by the haemolymph might be via the mitochondrial signal pathway [24].Table 2
Proteins identified with high confidence from the CHP ofA. chinensis Dallas.
Peptide sequence Prec MW Prec m/z Theor MW Theor m/z Protein DTLFEYLENPKK 1552.774414 777.3945 1552.777344 777.3959351 Cytochrome c MVFAGIK 807.4334717 404.724 807.4312744 404.7229309 LENPKK 727.4298706 364.7222 727.4228516 364.718689 ADLIAYLEQATK 1391.730103 696.8723 1391.729614 696.8720703 DTLFEYLENPKK 1495.751831 748.8832 1495.755859 748.8851929 KPQERADLIAYLEQATK 2032.07959 678.3671 2032.05896 678.3602295 VGPNLSGLIGR 1139.647217 570.8309 1139.629883 570.8222046  EGLGIFVLDK 1090.612305 546.3134 1089.607056 545.810791 Ferritin QNFHADSEEAINK 1501.667236 751.8409 1501.679688 751.847168 SLGDLLTNVR 1086.604492 544.3095 1086.603271 544.30896 VKEGLGIFVLDK 1316.761719 659.3881 1316.770386 659.3924561 AVEAALQLEK 1070.595215 536.3049 1070.597168 536.3058472 LAFVEIDR 977.5206909 489.7676 977.5181885 489.7663879 TLALLHR 822.5070801 412.2608 822.5075684 412.2610474 LFNLFYFAK 1162.605713 582.3101 1162.606323 582.3104248  MISLCGPLSIIGR 1431.728638 716.8716 1431.757813 716.8861694 Superoxide dismutase TIVVHADPDDLGK 1379.704834 690.8597 1378.709229 690.3618774 VACGVIGITK 1073.583618 537.7991 1073.590332 537.8024292 HVGDLGNICAEANGVAK 1752.820435 877.4175 1752.846436 877.430542 YDTPELVEK 1149.551025 575.7828 1149.55542 575.7849731 AGGQVAWTK 916.4700928 459.2423 916.4766846 459.2456055 EAFSLFDKDGDGTITTK 1843.888916 615.6369 1843.884033 615.6352539  LFDLFFFAK 1203.633667 602.8241 1203.632813 602.8236694 Secreted Hemocyanin-like protein  AVGVDLNLK 927.5370483 464.7758 927.5389404 464.7767334 Glutathione s-transferase  KASGQKIGQK 1044.60791 523.3112 1044.592773 523.3036499 Tyrosine-protein phosphatase corkscrew isoform x2 LVSLSEQQLIDCSR 1704.82605 853.4203 1704.835205 853.4249268  LVSLSEQQLIDCSR 1704.82605 853.4203 1704.835205 853.4249268 Cysteine protease  INNPLK 698.3964844 350.2055 698.3963013 350.2054138 Death-associated protein 1  KLNTNSLQK 1044.60791 523.3112 1044.592773 523.3036499 Telomerase reverse transcriptase  VLHGTVVAVGPGAR 1331.76123 444.9277 1331.767334 444.929718 Heat shock protein, putative  LDEALEAAR 986.5010986 494.2578 986.5032959 494.2589111 TATA element modulatory factor  RAKEASAAEGGIK 1286.706421 644.3605 1286.694214 644.3544312 6S proteasome non-ATPase regulatory subunit 4  SILSTLYASEVMYENT 1819.820435 607.6141 1819.85498 607.6256104 ATP synthase subunit a SLYNEFKLLTGPNNK 1736.867798 435.2242 1736.90979 435.2347107 LLTGPNNK 856.4470825 429.2308 856.4654541 429.2399902 INIIMNNMLNSLYNEFK 2070.905273 1036.46 2071.011719 1036.513184 TNLFSTFDPATSIK 1541.681274 771.8479 1541.761353 771.8879395 PFMVLIETISNLIR 1644.788452 549.2701 1644.927246 549.3164063 STLYASEVMYE 1291.598267 646.8064 1291.564209 646.7893677 INIIMN 716.3768921 359.1957 716.3890991 359.2018127 MTNLFSTFDPAT 1343.644287 672.8294 1343.606812 672.8106689  MVFIMFWFIWVR 1673.763306 837.8889 1673.86145 837.9379883 LMYLTWK 953.5025024 477.7585 953.5044556 477.7595215 TGLMGLLQPFADGLK 1559.709106 780.8618 1559.838135 780.9263306 SVLGYIQLRK 1176.579468 589.297 1176.686646 589.3505859 NADH-ubiquinone oxidoreductase chain 1 IFMSFPLFF 1147.543457 574.779 1147.577637 574.7960815 MSFPLFFCWLSSCLAETNR 2364.968506 592.2494 2365.069336 592.2745972 SEYMNIIFM 1146.536621 574.2756 1146.508911 574.2617798  FQGNKFYPLNK 1355.621094 678.8178 1355.687378 678.8509521 Cytochrome b DVNNGWLMR 1104.517456 553.266 1104.502197 553.2583618 AGRGLYYGSYK 1233.599121 617.8068 1233.614258 617.8143921 WLWGGFSVDNATLTR 1722.773315 862.3939 1722.836548 862.4255371 SNYDKTPFHPYF 1514.661133 758.3378 1514.682983 758.3488159 MLGDPENFIP 1147.543457 574.779 1147.521973 574.7682495  ISFINQNIK 1075.515259 538.7649 1075.602539 538.8085938 NADH dehydrogenase subunit 6  NDIICSQMNWK 1407.588623 704.8016 1407.627563 704.8210449 ATP synthase protein 8  TASYDREK 968.3950806 485.2048 968.4562988 485.2354431 NADH-ubiquinone oxidoreductase chain 3 TICSIISK 920.4076538 461.2111 920.5001221 461.2573242 MMMYVMMTTI 1266.534912 634.2747 1266.519043 634.2667847Figure 3
LC-MS/MS spectrogram reveals the anticancer active component (CHP).


## 3.3. Cell Viability

Morphological characterization of SGC-7901 and BGC-823 cells was performed after treatment with CHP. A light microscope was used to evaluate the extent of cell shrinkage at 48 h after treatment with different concentrations of the CHP protein. The results were compared with those of untreated gastric cancer cells. The most recognizable morphological changes of CHP-treated cells were cytoplasmic condensation and cell shrinkage (Figures4(a) and 5(a)).Figure 4
Effect of CHP on the proliferation of gastric cancer SGC-7901 cells. (a) Morphologic characterization of SGC-7901 cells treated with different concentrations of CHP (0, 20, 30, and 40μg/mL; A-D, respectively). (b) The viability of SGC-7901 cells treated with 0 (control), 20, 30, and 40 μg/mL CHP. The viability of cells was determined by the MTT assay. The values are presented as mean ± SD of five independent experiments. Different upper case letters indicate significant difference between the groups (Fisher’s LSD,P < 0.01).
(a) (b)Figure 5
Effect of CHP on the proliferation of gastric cancer BGC-823 cells. (a) Morphologic characterization of BGC-823 cells treated with different concentrations of CHP (0, 20, 30, and 40μg/mL; A–D, respectively). (b) The viability of BGC-823 cells treated with 0 (control), 20, 30, and 40 μg/mL CHP. The viability of cells was determined by the MTT assay. The values are presented as mean ± SD of five independent experiments. Different upper case letters indicate significant difference between the groups (Fisher’s LSD,P < 0.01).
(a) (b)To determine the antiproliferative effects of CHP on gastric cancer cells, their viability was determined by the MTT assay. The MTT assay was used as a sensitive and reliable colorimetric assay to measure the viability and proliferation of cells. The cleavage of MTT has several desirable properties for assaying cell survival and proliferation. MTT is cleaved by all living, metabolically active cells, but not by dead cells or erythrocytes. The amount of formazan generated is directly proportional to the cell number over a wide range, using a homogeneous cell population. Activated cells produce more formazan than the resting cells, which enable the measurement of activation even in the absence of proliferation. These properties are all consistent with the cleavage of MTT only by active mitochondria [25]. The MTT assay was performed to determine the IC50 of CHP in SGC-7901 and BGC-823 cell lines. The viability of gastric cancer SGC-7901 and BGC-823 cells treated with different concentrations of CHP (0, 20, 30, and 40 μg/mL) for 24, 48, and 72 h is shown in Figures 4(b) and 5(b). The treated cells showed a reduced proliferation capacity in a concentration-dependent manner (P< 0.01), although SGC-7901 (IC50 = 25.462 μg/mL) was more sensitive than BGC-823 (IC50 = 29.003 μg/mL) cells.


## 3.4. Hoechst 33258 Staining

The nuclei were stained with Hoechst 33258 to determine the incidence of apoptosis morphologically. Representative photomicrographs showed the morphological characteristics of apoptosis, such as chromatin condensation and nuclear fragmentation (indicated by arrows), in SGC-7901 and BGC-823 cells treated with different concentrations of CHP. All data were obtained within 36 h of treatment. The CHP 20, 30, and 40μg/mL groups showed significant increases in apoptosis compared with that of the control group (Figure 6), indicating that CHP increases the extent of apoptosis.Figure 6
Apoptosis induced by treatment of BGC-823 (A1-A4) and SGC-7901 (B1-B4) cells with 0 (control), 20, 30, or 40 μg/mL CHP for 36 h. Panels 1-4 represent treatment with 0, 20, 30, or 40 μg/mL CHP, respectively.


## 3.5. Flow Cytometry

Apoptosis is frequently dysregulated during the progression of cancer [23]. After treatment of SGC-7901 cells with CHP for 24 h, flow cytometry showed that the percent of apoptotic cells was 2.67 ± 0.09, 7.7 ± 0.23, 14.07 ± 0.37, and 26.3 ± 0.95 %, for 0, 10, 15, and 20 μg/mL CHP, respectively (Figure 7). Quantification of early and late apoptosis after treatment with various concentrations of CHP showed significantly increased apoptosis in the groups treated with 10, 15, and 20 μg/mL CHP compared with that in the control group (P < 0.01).Figure 7
Flow cytometry analysis of SGC-7901 cell apoptosis by double staining with Annexin V-FITC and propidium iodide (PI). Values are presented as the mean ± SD of three independent experiments. Different upper case letters indicate significant difference between groups (Fisher’s LSD,P < 0.01).
(a) (b)


## 3.6. CHP Inhibited In Vivo Tumour Growth in BALB/c Mice

To elucidate whether CHP suppressesin vivo tumour growth, we established the live tumour model by subcutaneously transplanting 4T1 cells into BALB/c mice, which werein situ injection of CHP (20 mg/kg/2 d), PDD (1 mg/kg/2 d), or PBS (0.02 mL/kg/2 d). Two weeks later, the CHP-treated BALB/c mice developed smaller tumours in terms of size and weight compared with those of PBS group (Figures 8(a) and 8(b)). These results implicated that CHP can partially inhibit tumour growth derived from 4T1 cellsin vivo.Figure 8
The antitumour effects of CHPin vivo. (a) 4T1 cells were subcutaneously injected into the BALB/c mice. Tumour volume measurements for the BALB/c mice during treatment. (b) Tumour weight at the end of treatment (Day 14). Values are presented as the mean ± SD of three independent experiments. Different upper case letters indicate significant difference between groups (Fisher’s LSD,P < 0.01).
(a) (b)


## 4. Discussion

Malignant tumours are a major public health problem worldwide. These tumours exhibit uncontrolled proliferation, leading to death. Disorders in the control of cancer cell apoptosis play an important role in the development of cancer. The key approaches to prevent cancer are the suppression of cancer cell proliferation and acceleration of apoptosis [23]. A number of chemotherapy agents have offered significant advantages in the treatment of cancer. Owing to the advantages of high bioactivities and low toxicity, the use of natural products as a new source of anticancer agents is becoming popular [7];A. chinensis is such a natural source. DriedA. chinensis is a traditional Chinese medicine utilised to treat pain, poor digestion, and kidney diseases. Previous investigations have shown thatA. chinensis possesses pronounced antibacterial, anticancer, analgesic, antifibrotic, and angiogenesis-promoting activities [8–10, 12]. Our previous study revealed that the anticancer active components were mainly distributed in the haemolymph and abdomen [17] and that the haemolymph fromA. chinensis can inhibit the proliferation of human gastric cancer cell lines SGC-7901 and BGC-823, and human breast cancer cell line MCF-7 in a dose- and time-dependent manner [18–20]. Our further studies showed that the expression of caspase 3, caspase 9, and Bax proteins was significantly upregulated, and there was no significant difference in the expression of caspase 8 when SGC-7901 cells were treated with haemolymph. This indicated that apoptosis of SGC-7901 cells induced by the haemolymph might be via the mitochondrial signal pathway [24]. Although increasing evidence supports the promising anticancer effect ofA. chinensis,several questions regarding the anticancer active components remain unanswered.In the present study, our results demonstrated that the haemolymph extracted fromA. chinensis inhibited the proliferation of SGC-7901 cells in a dose-dependent manner (Figure 1(a)), which was consistent with the findings of a previous study [20]. In the present study, we found that when the haemolymph was treated at 60°C, a large amount of proteins were denatured. The results showed that haemolymph proteins treated at 60°C are more effective (IC50 = 3.145 mg/mL) at inhibiting the proliferation of SGC-7901 cells than the untreated haemolymph (IC50 = 42.912 mg/mL) (Table 1), indicating the anticancer active component may be thermostable. Interestingly, the proteins identified with high confidence from the CHP ofA. chinensis were thermostable, such as cytochrome c, ferritin, and superoxide dismutase (Table 2) [26].In the present study, ammonium sulphate was added to the extract by gradually increasing the ratio. In the early stage of purification experiment, neutral salt can affect the nonpolar, hydrophobic, and electrostatic interactions among proteins that need to be concentrated [27]. The electrostatic charge induction on the surface of protein induces conformational change and therefore the protein cannot fully solvate to prevent aggregation. Consequently, the protein can dissolve in a low concentration of salt solution, but rarely soluble or insoluble in water [28]. The electric charge of the protein can be increased by the addition of a small amount of salts to neutralise the surface electrostatic force. The increase in salt concentration can not only neutralise the hydrostatic power on the surface of the protein, but also remove the water molecules from the protein. These water molecules are necessary for the dissolution of proteins, and the salt competes with water molecule [29]. To ensure repeatability of the results of different batches of experimental materials, a relatively wide range of 40%–60% was selected according to the results of the MTT assay (Figure 1(b)), but some target components might be inevitably lost and experimental deviation might be caused.In the present study, two kinds of ultrafiltration membranes, 5 and 50 kDa, were used to divide the protein solution into three parts, removing most of the impurities and preparing the sample for the subsequent column chromatography. Ultrafiltration (UF) is a membrane separation process that uses a pressurised active membrane to separate particles according to their molecular weight. Under external pressure, dissolved substances and substances with pore size smaller than the membrane will be used as permeable liquid through the membrane to achieve the purpose of solution purification, separation, and concentration [30]. To improve the permeation flux of the membrane and ensure the normal and stable operation of the ultrafiltration membrane, ultrafiltration was performed after heat treatment, salting out, and preferably centrifugation at high speed to remove denatured protein from the intermediate process. The results suggest that the haemolymph proteins with intermediate molecular weight (5–50 kDa fractions) are more effective in inhibiting the proliferation of SGC-7901 cells than the low molecular weight proteins (< 5 kDa) and high weight proteins (> 50 kDa). Therefore, the 5–50 kDa fraction was selected for further purification (Figures 2(a) and 2(b)). The results of SDS-PAGE revealed that the molecular weight of the anticancer active component proteins is approximately 15 kDa (Figure 2(c)). Furthermore, most predicted molecular weights of peptides identified with high confidence were distributed from 6 to 24 kDa (Table 2), which was consistent with the results of ultrafiltration fractions (5–50 kDa).Reversed phase chromatography is based on the hydrophobic effect between the solute, polar flow phase, and nonpolar stationary phase surface [31]. Any organic molecule has a nonpolar hydrophobic part in the structure, which is the most widely used separation mode in HPLC. The mobile phase uses acidic and low ionic strength aqueous solutions and a certain proportion of the organic modifiers such as isopropanol, acetonitrile, and methanol, which can be dissolved with water [32]. In the present study, the fraction was eluted with acetonitrile. The length of alkyl chain has no significant effect on the reverse phase retention of proteins, but the reaction of short-chain alkyl groups (such as C4 and C8) and long-chain alkyl groups (such as C18 and C22) is different in the recovery of protein activity [33]. The longer the alkyl chain is, the stronger the hydrophobicity of the stationary phase is. Therefore, it is necessary to increase the organic components of the mobile phase to rapidly elute the protein. In general, reversed phase chromatography on the alkyl-bonded silica gel is one of the methods for the separation, analysis, and purification of proteins because of its high column efficiency, good separation, and clear retention mechanism [34]. In this study, C18 column was used, and the results of MTT detection showed that the activity of antigastric cancer SGC-7901 cells was obviously enhanced after the separation and purification (Table 1).Among the proteins identified with high confidence from the CHP ofA. chinensis(Figure 3),Cyt c might be a noteworthy antitumour protein (Table 2), which has an intermediate role in apoptosis [23]. Previous investigations have shown that the apoptotic program can be initiated by the addition of dATP and Cyt c in cell-free extracts [35]. Once released into the cytosol, Cyt c binds to apoptotic protease activating factor-1 (Apaf-1) and procaspase-9, apoptosome was formed, and apoptosis was initiated [36]. The results of the present study demonstrated that CHP can inhibit the proliferation and increase the rate of apoptosis in gastric cancer cells (Figures 4, 5, 6, and 7). Representative microphotographs showed that morphological characteristics of apoptosis, such as chromatin condensation and nuclear fragmentation, were evident in SGC-7901 cells treated with CHP (Figure 6), and Annexin V-FITC/PI flow cytometric analysis further validated the apoptosis-inducing effect of CHP in a concentration-dependent manner (Figure 7). On account of stage IV breast cancer leads to death in most patients with breast cancer [37], and the growth and distant metastasis of 4T1-transplanted tumour model cells of breast cancer are highly similar to those of stage IV human breast cancer [38]. This animal model has been successfully applied to study drugs for the treatment of malignant tumours [39]. Moreover, the second breast in mice with the same anatomical site can better imitate the development of human breast cancer [40]. Therefore, in the present study, the 4T1 cells (1.0 × 106) were inoculated subcutaneously into the second breast of each mice, and we demonstrated that CHP can suppress the growth of breast cancer 4T1 cell xenograft tumoursin vivo (Figure 8).Overall, to the best of our knowledge, the present study is the first to report the antiproliferative and proapoptotic effects of newly isolated and identified bioactive protein component fromA. chinensis in cancer cellsin vitro andin vivo. However, changes in apoptosis-related genes and the generation of apoptosis signals during the induction of cancer cell apoptosis require further investigation. In addition, thein vivo safety of these active peptides should be verified.



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