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Water Stress Proteins from Nostoc commune Vauch. Exhibit AntiColon Cancer Activities in Vitro and in Vivo Songjia Guo,†,§ Shuhua Shan,†,∥ Xiaoting Jin,† Zongwei Li,† Zhuoyu Li,*,†,‡ Liangqi Zhao,† Quan An,⊥ and Wei Zhang⊥ †

Institute of Biotechnology, Key Laboratory of Chemical Biology and Molecular Engineering of National Ministry of Education, Institute of Biotechnology, Shanxi University, Taiyuan 030006, China ‡ College of Life Science, Zhejiang Chinese Medical University, Hangzhou 310053, China § Department of Medical Laboratory, Fenyang College of Shanxi Medical University, Fenyang 032200, China ∥ Department of Biology, Taiyuan Normal University, Taiyuan 030031, China ⊥ China Institute for Radiation Protection, Taiyuan 030031, China S Supporting Information *

ABSTRACT: Nostoc commune has been traditionally used in China as a health food and medicine. The water stress proteins (WSP) of Nostoc commune are the major component of the extracellular matrix. This study purified and identified the water stress proteins (WSP1) from Nostoc commune Vauch., which could inhibit the proliferation of human colon cancer cell lines. The IC50 values of WSP1 against DLD1, HCT116, HT29, and SW480 cells were 0.19 ± 0.02, 0.21 ± 0.03, 0.39 ± 0.05, and 0.41 ± 0.01 μg/μL, respectively. Notably, it displayed very little effect on the normal human intestinal epithelial FHC cell line. The IC50 value of WSP1 against FHC cells was 0.67 ± 0.05 μg/μL. Moreover, the growth of DLD1 xenografted tumors in nude mice were significantly suppressed in the WSP1 treated group. Mechanistically, the cell-cycle analysis revealed that WSP1 induced growth inhibition by G1/S arrest. Meanwhile, Western blotting and immunohistochemistry assays showed WSP1 could activate caspase8, -9, and -3, along with subsequent PARP cleavage. Furthermore, the pan-caspase inhibitor, z-VAD-FMK, partly reversed the effect caused by WSP1, confirming that WSP1 induced cell apoptosis through caspase-dependent pathway. Collectively, WSP1 has targeted inhibition for colon cancer proliferation both in vitro and in vivo and it is valuable for future exploitation and utilization as an antitumor agent. KEYWORDS: Nostoc commune Vauch, water stress protein 1 (WSP1), colorectal cancer (CRC), apoptosis, nude mice

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INTRODUCTION Colorectal cancer (CRC) is a worldwide disease with an annual incidence of 1 million cases. It is the second most common cause of cancer mortality and comprises 9% of the cancer burden globally.1 In China, CRC ranks fourth among the most common cancers and the mortality rate is 57.5%.2 Chemotherapy is used in the treatment of cancer with one or more antineoplastic drugs. Patients often receive the combination of chemotherapy with various chemical agents.3 However, the toxicity and side effects greatly restrict the application of chemotherapeutic agents.4 Efforts have focused on developing effective therapeutic regimens to combat colon cancer with low toxicity and side effects. A large number of previous studies have shown that many extracts from plants or microorganisms have exhibited strong inhibitory properties on several types of cancers, especially on colon epithelial tumors.5 For example, compounds from colorfleshed potatoes have shown pro-apoptotic properties in human colon cancer cells,6 major metabolites of 6-gingerol had cytotoxic effects on human cancer cells,7 and dietary black and brown rice bran displayed antitumor effects in tumorbearing mice.8 Nostoc commune Vauch. (NCV) is an edible terrestrial cyanobacterium and is a photosynthetic prokaryote. In China, NCV is a popular food and is rich in protein, calcium, iron, vitamin C, and other nutrients.9,10 Accumulated evidence © 2014 American Chemical Society

indicates that substances from NCV could prevent fatty liver, kidney stones, and heart diseases.11,12 The bioactive fractions from NCV showed multiantioxidant and antitumor activities;13,14 however, the molecular mechanisms remain poorly understood. In the present study, we purified active proteins from NCV, which had remarkable inhibitory effects on cell proliferation of colon cancer. Mass spectrometry analysis indicated that the protein belonged to the water stress proteins (WSP). Nostoc commune typically synthesizes water stress proteins when they are subjected to acute water stress in nature. WSP is a group of acidic polypeptides with cryptic functions and PI values between 4.3 and 4.8. WSP is a major extracellular matrix protein, which maintains the structure and function of the extracellular matrix.15 The WspA from Nostoc commune possesses β-D-galactosidase activity16 and is associated with 1,4-β-D-xylanxylanohydrolase activity.17 Previous reports have shown that wspA is a single copy gene,18 while WspA has microheterogeneity characterized by two or more proteins of 30−40 kDa but with identical N-terminal sequence.19 These Received: Revised: Accepted: Published: 150

July 11, 2014 December 18, 2014 December 19, 2014 December 19, 2014 DOI: 10.1021/jf503208p J. Agric. Food Chem. 2015, 63, 150−159

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Journal of Agricultural and Food Chemistry

systems USA), a peptide mass fingerprint (PMF), and the NCBI database. Cloning of the WSP1 Gene. Specific primers for PCR amplification of the full-length WSP1 gene in NCV were designed according to the wspA gene sequence of Nostoc commune (accession no. AB518000). The primer sequences were as follows: Forward: 5′-CGCGGATCCATGGCTCTTTACGGCT-3′. Reverse: 5′-CCGCTCGAG TTATTCATTAACAATCGT-3′. PCR was performed under the following conditions: initial denaturation at 95 °C for 3 min, followed by 30 cycles of 95 °C for 1 min, 60 °C for 1 min, 72 °C for 1 min, and a final extension step at 72 °C for 10 min. Cell Culture. Human colon cancer cell lines (DLD1, SW480, HT29, and HCT116) and a normal colonic epithelial cell line (FHC) were obtained from the Chinese Type Culture Collection. Human colon cancer cell lines were cultured in RPMI 1640 medium supplemented with 10% (v/v) heat-inactivated FBS. The FHC was cultured in F12 medium with 10% FBS. All media were supplemented with 100 U/mL penicillin (Sigma, Germany) and 100 mg/mL streptomycin (Sigma, Germany). All cultures were incubated at 37 °C with 5% fully humidified CO2. MTT Assay. Growth inhibitory effects of WSP1 on DLD1, HCT116, SW480, HT-29, and FHC were determined using a MTT assay. Cells (1 × 104) were seeded into 96-well plates and allowed to grow for 24 h for acclimatization. The cells were then incubated with treatments of 0, 0.05, 0.15, and 0.25 μg/μL WSP1 for 48 h. Then 20 μL of MTT (5 mg/mL) was added to each well and further incubated for 4 h. The blue formazan produced was dissolved by DMSO, and the absorbance was measured at 570 nm with an ELISA plate reader. The percentage of cell growth inhibition was calculated with the formula: (A570 Control − A570 Experiment/A570 Control) × 100%. After the preliminary screening, in order to determine the time-dependent manner of WSP1 induced cell growth inhibition, DLD1 was treated with 0.05, 0.15, and 0.25 μg/μL WSP1 for 24 and 48 h. Assays were performed in triplicate independent experiments. The 50% inhibitory concentration (IC50) of 48 h was determined as the anticancer drug concentration, causing a 50% reduction in cell viability and calculated by regression analysis. Clonogenic Survival Assay. DLD1 cells were counted and plated onto 6-well tissue culture plates (1000 cells/well) in 1640 medium supplemented with 10% FBS. After attachment, the cells were treated with 0.15 μg/μL WSP1 for 10 days. The cells were then fixed with immune dye fixative and stained with 0.1% crystal violet. The results were observed under a stereomicroscope (OLYMPUS SZX16, Japan). The number of clones (more than 50 cells each) counted in 10 different microscopic fields were used to calculate the colon formation rate: Colon Formation Rate = (Clones/Seeded Cells) × 100%. DAPI Staining and Quantification of Apoptotic Cells. To examine the effects of WPS1 on cancer cell apoptosis, a DAPI nuclear staining assay was performed. DLD1, HCT116, and FHC cells were plated in 6-well plates with glass slides (1 × 106 cells/well). After treatment with 0.15 μg/μL WSP1 for 48 h, the cells were fixed with immunostaining fixative for 60 min at 4 °C. Fixed cells were washed twice with PBS and incubated with DAPI for 1 h at 4 °C in the dark. Then the slides were washed with PBS to remove the excess DAPI and the cell nuclei were observed under a laser confocal scanning microscope (LCSM; OLYMPUS FV1000, Japan). The apoptosis rate of colon cancer cells treated with WSP1 was determined by scoring the number of cells with nucleus phenotypic changes in 20 different microscopic fields under LCSM. Analysis of Cell Apoptosis and Cell Cycle by Flow Cytometry. WSP1 induced apoptosis in DLD1 was analyzed by flow cytometry using the Annexin V-FITC and propidium iodide (PI) double staining method. Briefly, after incubation with 0.15 μg/μL WSP1 for 48 h, cells were trypsinized and washed with PBS. Cells were then stained with 200 μL of Annexin V solution (10 μL of Annexin V + 200 μL of binding buffer) and 300 μL of PI (5 μL PI + 300 μL binding buffer) in the dark (RT) for 30 min. The population of Annexin V positive cells was examined using a FACSort flow cytometer. The cell cycle of DLD1 cells was assessed with a cell

isoforms may arise from deamidation, covalent modification, protease degradation, etc. In this study, we purified three predominate proteins (39, 37, and 35 kDa). They were all identified as WSP by MALDI-TOF analysis and were named WSP1. On the basis of the above data, the single copy gene (accession KF003026) that encoded the water stress protein (WSP1) was cloned into pET28a, and the recombinant protein of WSP1 (GI: 530891432) was obtained by genetic engineering technology.20 The molecular weight of the recombinant WSP1 was found to be 39 kDa and could significantly inhibit the proliferation of human colon cancer cells.20 We report that WSP1 possessed potent antitumor effects on human colon cancer cell lines (DLD1, HCT-116, HT-29, and SW480). More importantly, WSP1 could suppress DLD1 xenografted tumors in nude mice. The results further indicated that the WSP1 treatment induced colon cancer cell apoptosis by triggering the caspase-dependent pathway and suppressed cell growth by G1/S arrest. Interestingly, WSP1 specifically suppressed proliferation and induced apoptosis in colon cancer cells in vitro and in vivo but not in normal human colon epithelial cells. The data presented in this study provides evidence for the potential of WSP1 as an antitumor agent against colon cancer.

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MATERIALS AND METHODS

Chemicals. RPMI-1640 medium and fetal bovine serum (FBS) were obtained from GIBCO (Grand Island, NY). 3-4,5-Dimethylthiazol-2-yl-2,5-diphenyltetrazolium bromide (MTT) and DAPI (4′,6′diamidino-2-phenylindole) were obtained from Sigma (St. Louis, MO). 5,5′,6,6′-Tetrachloro-1,1′,3,3′-tetraethyl-imidacarbocyanine iodide (JC-1) and the pan-caspase inhibitor (z-VAD-FMK) were provided by Beyotime Institute of Biotechnology (Haimen, China). The annexin V-FITC apoptosis detection kit was purchased from Oncogene (San Diego, CA). The antibodies used in this study were as follows: GAPDH and β-actin antibodies were purchased from Abmart (Arlington, MA); pRb was obtained from Cell Signaling Technology (Danvers, MA); c-Myc, cyclineD1, and cyclineE1 antibodies were obtained from Bioworld Technology (Minneapolis, MN); AIF, Bax, Bcl-2, caspase-3, -8, and -9, and polymerase (PARP) antibodies were obtained from the Beyotime Institute of Biotechnology (Haimen, China). Isolation and Purification of WSP1 Polypeptides from Nostoc commune Vauch. Desiccated colonies of NCV were collected from Shanxi, China, in 2012. Then 20 g of desiccated colonies (unground) were cleaned with double distilled water, then suspended in 1000 mL of PBS (phosphate-buffered saline, pH 7.4, containing 1 mM PMSF) for 48 h, followed by centrifugation at 12000 rpm for 25 min at 4 °C. Then, the supernatant was precipitated with acetone (V/V = 1:2) at −20 °C for 2 h. The obtained precipitation was dissolved with appropriate volumes of PBS and subjected to ammonium sulfate fractionation. The precipitation from 30−60% ammonium sulfate was resuspended in PBS, dialyzed, and concentrated by ultrafiltration through a 10 kDa MWCO hollow fiber membrane (Millipore, USA). Proteins were analyzed with a 10% SDSPAGE and a 10% nondenature PAGE. The collected active fractions showed three bands on both the SDS-PAGE and nondenature PAGE gels. Gels were stained using CBB R-250. The relative gray value of WSP1 was calculated using Image-J software. Mass Spectrometry Analysis and Protein Identification. The purified target proteins were resolved using a 10% SDS-polyacrylamide gel. The SDS-PAGE was performed using standard methods on a Mini-Protean II system 3 (Bio-Rad, USA). The target protein band was cut from the SDS-PAGE gel and sent to Shanghai Applied Protein Technology Co. Ltd., China, for protein identification. Protein identification was performed using a matrix-assisted laser desorption/ionization-time-of-flight method (MALDI-TOF, Applied Bio151

DOI: 10.1021/jf503208p J. Agric. Food Chem. 2015, 63, 150−159

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Figure 1. Purification of WSP1 from Nostoc commune Vauch. (A) Total proteins precipitated from 100% ammonium sulfate analyzed by 10% SDSPAGE. (B) Precipitation from 30−60% ammonium sulfate analyzed by 10% SDS-PAGE. (C) Precipitation from 30−60% ammonium sulfate analyzed by 10% nondenaturing PAGE. (D) Relative gray values of three bands of WSP1 based in (B). (E) Amino acid sequence of WSP1 protein of 39 kDa. cycle detection kit by FACScan. DLD1 cells were plated into 60 mm dishes (1 × 106 cells/dish) and incubated overnight. Cells were treated with 0.15 μg/μL WSP1 for 48 h and then trypsinized. These cells were washed with PBS and fixed in 70% ethanol at −20 °C for 12 h. The ethanol was removed after centrifugation, and the cells were resuspended in 1 mL of PBS, 100 μL of ribonuclease A (100 μg/ mL PBS), and 400 μL of propidium iodide (400 μg/mL PBS) in the dark (RT) for 30 min. The percentages of cells in different cell cycle phases were analyzed using FACScan, and experiments were performed in triplicate. Measurement of Changes of Mitochondrial (Δψm). Measurement of mitochondrial transmembrane potential was performed with DLD1 cells. First, 5 × 105 cells/well were seeded into 6-well plates. After treatment with WSP1 (0.15 μg/μL) for 48 h, the cells were then harvested and stained with 10 mg/mL JC-1 at 37 °C for 10 min. Then, the stained cells were washed, resuspended with PBS, and analyzed for their red and green fluorescence from JC-1 using a flow cytometer. Data are reported as the percentage of cells with depolarized mitochondrial membranes, and experiments were performed in triplicate. Western Blotting Analysis. The human colon cancer cell line DLD1 (1 × 106) was treated with 0.15 μg/μL WSP1 for 24 and 48 h. After the treatment, cells were incubated with lysis buffer for 30 min on ice and then centrifuged for 10 min at 13000 rpm. Protein concentrations were measured with Bradford reagent (Bio-Rad, USA), and an equal amount of protein (60 μg) from each sample was separated by 10% SDS-PAGE and then transferred to PVDF membranes. The membranes were blocked using Tris-buffered saline with 0.1% Tween-20 and 5% skimmed milk for 1.5 h at room temperature. The membranes were probed with primary antibodies at 4 °C overnight. The membranes were then washed three times with Tris-buffered saline containing 0.1% Tween-20 and probed for 2 h at room temperature with HRP conjugated secondary antibodies. Protein bands were visualized using enhanced chemiluminescence, and experiments were performed in triplicate. In Vivo Studies in Nude Mice. All animal experiments were carried out following procedures approved by the Institutional Animal Care and Use Committee of China Institute for Radiation Protection. The review board and ethics committee of the institution specifically approved this study. Sixteen BALB/c nude mice (five-week old, female, weighing between 18 and 21 g) were purchased from the Institute of Zoology, Chinese Academy of Sciences. The nude mice were maintained under

specified-pathogen free (SPF) conditions in the laboratory animal service center of the China Institute for Radiation Protection. DLD1 cells in 0.2 mL of PBS (5 × 106) were injected subcutaneously into the right oxter of each nude mouse. Fourteen days later, solid tumors were apparent in all injected nude mice and the 16 nude mice were randomly divided into two groups (each group with 8 nude mice) with no significant difference of tumor volumes between the groups. Mice in the WSP1 group received an intraperitoneal injection of 60 μg WSP1/g body weight every 3 days (total six injections), and the control mice were treated with PBS. The body weight and tumor diameters of nude mice were measured twice a week. Tumor volumes were calculated using the formula: tumor volume (cm3) = 0.5 tumor length (cm) × tumor width2 (cm2). Then 21 days later, all mice were sacrificed according to ethical demands and tumors were excised, weighed, and frozen at −80 °C for further Western blotting and immunohistochemical analysis. Immunohistochemical Analysis. Tumors were fixed in 10% formalin, embedded in paraffin wax, and used for immunohistochemical analysis. The paraffin sections were dewaxed in xylene and dehydrated in serially diluted ethanol. Antigen retrieval was carried out using citrate buffer (pH 6.0). All sections were stained with antihuman Ki-67, activated-caspase-8, activated-caspase-9, and PARP. Staining was performed using a universally labeled streptavidin−biotin kit according to the standard protocol. The percentage of cells was calculated by Imagepro-Plus 6.0 software. Statistical Analysis. Data analysis was carried out using SPSS 17.0 software. Results were expressed as mean ± SD. IC50 values were calculated by regression analysis. Differences in means between the control and treatment groups were determined using a two-tailed unpaired Student’s t test or a One-Way ANOVA followed by Tukey’s multiple comparison tests. Values of less than 0.05 (p < 0.05) and 0.01 (p < 0.01) were considered statistically significant and highly significant, respectively.

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RESULTS Purification and Identification of WSP1. Total extracellular matrix proteins of NCV were prepared by 100% (m/v) ammonium sulfate precipitation (Figure 1A). After ammonium sulfate grading precipitation, three proteins (39, 37, and 35 kDa) dominated in both the SDS-PAGE (Figure 1B) and the nondenaturing PAGE gels (Figure 1C). The three proteins were all identified as WSP1 by MALDI-TOF analysis

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Figure 2. WSP1 antiproliferative activities. (A) Dose-dependent cell proliferation assays. FHC, HT29, SW480, HCT116, and DLD1 cells were treated with different doses (0, 0.05, 0.15, and 0.25 μg/μL) of WSP1 for 48 h, and cell proliferation was measured by MTT assay. (B) Timedependent cell proliferation assays. DLD1 cells were seeded in the presence of WSP1 for 24 and 48 h. (C) Clonogenic survival assays. DLD1 cells (1000/well) were seeded in 6-well plates in the absence or presence of WSP1 for 10 d, then the clonogenic assay was carried out. (D) Colon Formation Rate = (Clones/Seeded Cells) × 100%. (E,F) Effects of WSP1 on cell cycle distribution in DLD1 cells. Cells were treated with WSP1 (0.15 μg/μL) for 48 h. DNA content was analyzed by cytometry. Accumulated cells in the G1 phase were compared with the control. (G) The percent of cell population in G0/G1, G1, S, and G2/M phases of the cell cycle. (H) Effects of WSP1 treatment on cell cycle proteins as shown by Western blotting. Antibodies were used against cyclin D1, cyclin E1, c-Myc, and pRb. GAPDH antibody was used as an internal control. The results represent mean ± SD of three independent experiments. *p < 0.05, **p < 0.01 compared with untreated cells.

sequence identity to WspA of N. commune KU002 (accession no. AB518000). WSP1 Suppresses Cell Growth of Human Colon Cancer. This study investigated the sensitivity of colon cancer cells (HT29, SW480, DLD1, and HCT116) and normal colon epithelial cells (FHC) to WSP1. The proliferation of those cells treated with various concentrations of WSP1 for 24 or 48 h was monitored using a MTT assay. As shown in Figure 2A,B, WSP1 markedly suppressed colon cancer cells growth in a time- or dose-dependent manner; however, it displayed less effect on FHC cell proliferation. The IC50 values of WSP1 for DLD1, HCT116, HT29, and SW480 cells were 0.19 ± 0.02, 0.21 ± 0.03, 0.39 ± 0.05, and 0.41 ± 0.01 μg/μL, respectively, and the IC50 against FHC was 0.67 ± 0.05 μg/μL (Table 1). The ability of individual cells to aggregate into viable colony clusters can be measured by a clonogenic survival assay; therefore, this assay was performed with DLD1 cells to evaluate the long-term

(Supporting Information, Figure S1), and the three peptide fragments with identical amino acid sequences were detected at 55.8% (39 kDa), 45.3% (37 kDa), and 42.8% (35 kDa) coverage to WSP1 (GI: 530891431) of NCV (Supporting Information, Figure S1). The microheterogeneity of WSP1 was consistent with previous reports.18,19 On the basis of the band intensity analysis, the three bands of WSP1 in Figure 1B were estimated to account for 31.5% (39 kDa), 23.6% (37 kDa), and 28.6% (35 kDa) of the total purified protein (Figure 1D). Additionally, the amino acid sequence of WSP1 protein of 39 kDa is shown in Figure 1E. Cloning of WSP1 Gene. The full-length coding sequence of WSP1 gene was amplified by PCR from the total cDNA of NCV. The identified WSP1 gene sequence has been submitted to GenBank under accession number KF003026. It encoded a polypeptide of 337 amino acids (GI: 530891431) with an 83% 153


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time-dependent cell percentage reduction in the S-phase and accumulation in the G1-phase. The percentage of cells in different phases is shown in Figure 2G. The expression levels of cell cycle associated proteins were analyzed by Western blotting. Compared with the control, the levels of c-Myc, cyclin D1, cyclin E1, and p-Rb decreased in DLD1 cells with the WSP1 treatment (Figure 2H), suggesting that the induced G1 phase arrest was attributed to the inhibition of c-Myc expression, followed by decreased cyclin D1, cyclin E1, and Rb phosphorylation expression. Treatment of WSP1 Induces the Apoptosis of Human Colon Cancer Cells. Because many chemotherapeutic drugs have been used to treat tumors by inducing apoptosis,21,22 we questioned whether WSP1 inhibited the colon cancer cell proliferation by inducing apoptosis. DLD1, HCT116, and FHC cells were treated with 0.15 μg/μL WSP1 for 48 h, and apoptotic bodies were detected by DAPI staining. The results showed WSP1-treated cells exhibited the apoptotic characteristics of chromatin condensation with typical apoptotic bodies. The number of apoptotic nuclei significantly increased in the group of colon cancer cells with the WSP1 treatment. In contrast, the WSP1-treated FHC cells did not display any obvious apoptotic bodies (Figure 3A,B).

Table 1. Cytotoxic Activity of WSP1 against Various Human Cell Lines cell lines DLD1 (colon cancer cell) HCT116 (colon cancer cell) HT29 (colon cancer cell) SW480 (colon cancer cell) FHC (colon epithelial cell)

IC50 (μg/μL) ± SD 0.19 0.21 0.39 0.41 0.67

± ± ± ± ±

0.02 0.03 0.05 0.01 0.05

effects of WSP1 on cell survival. The results showed that the clone formation rate of each group was 62.2 ± 5.1% in control group of 10 days, 2.8 ± 0.1% in WSP1-treated group of 10 days (Figure 2D). The data showed that the cell clone formation inhibiting effect of all the WSP1-treated groups were extremely significantly different from the control groups (p < 0.01). The result showed that clonogenicity was significantly inhibited in colon cancer cells cultured in the presence of WSP1 (Figure 2C). WSP1 Inhibits Colon Cancer Cell Growth by G1-Phase Arrest. To determine the mechanisms by which WSP1 inhibits the proliferation of colon cancer cells, analysis of cell cycle by flow cytometry was applied with WSP1 treated DLD1 cells. As shown in Figure 2E,F, DLD1 exposed to WSP1 resulted in a

Figure 3. Effects of WSP1 on apoptosis. (A) DLD1 and HCT116 were treated with WSP1 (0.15 μg/μL) for 24 or 48 h, and monolayer cultures were stained with DAPI to reveal apoptotic changes in the cell nuclei. Apoptotic bodies were pointed out by arrows. (B) Apoptotic cells of DLD1 and HCT116 were calculated. (C,D) DLD1 cells were either untreated or treated with WSP1 (0.15 μg/μL) for 48 h. Effects of WSP1 on cell apoptosis induction assessed by Annexin V/PI method using flow cytometry. The bottom right quadrant represented cells stained mainly by Annexin V (early apoptotic cells). (E) The early apoptosis ratio of DLD1 was shown in the bar graph. All experiments were performed at least three times in independent cell culture. *p < 0.05, **p < 0.01 compared cells untreated with WSP1. 154


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suggested that WSP1 caused mitochondrial dysfunction and the alteration of the Bax/Bcl-2 ratio that accounted for the WSP1induced decline of Δψm. WSP1 Treatment Induces the Caspase-Dependent Apoptosis in DLD1 Cells. To further explore the molecular mechanisms of WSP1 to induce cell apoptosis, the detection of PARP cleavage25 was performed using Western blotting. The result showed WSP1 induced proteolytic inactivation of PARP and the extent of cleavage was elevated time-dependently (Figure 5A). Because PARP is a substrate of executioner caspases, its cleavage indicates that caspases might be activated during the cell death process. It is been known that caspase activation is involved in the cleavage of its pro-caspase.26 After the WSP1 treatment, there were more pro-caspases-8 and -9 cleaved into caspases-8 and -9 (Figure 5A). In addition, the caspase-3 was further activated and led to apoptosis in DLD1 cells with the WSP1 treatment (Figure 5A). Moreover, the pancaspase inhibitor, z-VAD-FMK, could partially reverse WSP1inhibited cell proliferation in a dose dependent manner (Figure 5B); 20 μM z-VAD-FMK had inhibitory effects on the activation of caspase-9 (Figure 5C). These results confirmed that WSP1 induced apoptosis can be at least partially attributed to the activation of the caspase signaling pathway. WSP1 Inhibits DLD1 Xenografted Tumor Growth in Nude Mice. To determine the anticolon cancer effects of WSP1 in vivo, we performed xenograft studies with DLD1bearing nude mice. The nude mice received intraperitoneal injections of 60 μg WSP1/g body weight every 3 days. As shown in Figure 6, the WSP1-treatment for five injections resulted in a significantly reduced tumor volume and mass compared with the untreated group (p < 0.01). This phenomenon achieved a higher significance (p < 0.01) after six times injections. Furthermore, histology analyses revealed that WSP1-treated tumors had massive growth inhibition and apoptosis, as indicated by the significant reduction of Ki-67 and increase of actived-caspase-8, -9, and PARP (Figure 7). In addition, no mice died during the experiment. The body weights of the control group and the experimental group had no obvious difference (Figure 6A).

The degree of apoptosis was further determined by monitoring Annexin V-stained cells. DLD1 cells were treated with WSP1 for 48 h and then subjected to a flow cytometry analysis using Annexin V/propidium iodide double-staining. Compared to the control group, the ratio of Annexin V-stained cells was significantly increased in the group in the WSP1 treatment (Figure 3C,D). The early apoptotic cell percentage increased from 1.99 ± 0.38% to 14.78 ± 1.37% for DLD1 cells in the WSP1 treatment (Figure 3E). WSP1 Alters Mitochondrial Membrane Potential and Induces Cell Apoptosis. Mitochondrial membrane depolarization23 usually occurs in the stage of cell apoptosis. The collapse of mitochondrial membrane potential (Δψm), which reflects changes in mitochondrial membrane permeability, plays a vital role in the apoptotic process. Next, the changes of mitochondrial membrane potential were measured using JC-1 by flow cytometry. Under normal circumstances, JC-1 accumulates in active mitochondria in which it fluoresces red. A reduction in mitochondrial membrane potential results in the replacement of red fluorescence by green JC-1 monomers. The result indicated that there was an elevated percentage of DLD1 cells with depolarized mitochondrial membranes (time-dependently) after treatment with WSP1 for 48 h (Figure 4A). The fluorescent intensity ratio of red and green is shown in Figure 4B.

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DISCUSSION Studies have revealed that microorganisms serve as rich sources of anticancer therapeutics.27−30 Previous studies have shown that Nostoc commune Vauch. contains various nutritional ingredients and has been used as food and medicine for centuries in China.12 In this study, we found that the WSP1 from Nostoc commune Vauch. has anticolon cancer activity both in vitro and in vivo. To prepare WSP1, Nostoc commune Vauch. does not need to be ground because water stress protein is the major component of the extracellular matrix.16,18 The proteins obtained in this study had a relatively high purity. As shown in Figure 1D, WSP1 accounted for 83.6% of the total extracellular matrix protein. The three proteins of different molecular weights in the SDS-PAGE were all identified as WSP1 by mass spectrum. These isoforms may arise from deamidation, covalent modification, protease degradation, etc.19 On the basis of our previous studies,20 the WSP1 of 39 kDa was the main anticolon cancer protein and its amino acid sequence is shown in Figure 1E. Malignant tumor growth is due to the abnormality of cell proliferation or regulation defects of cell death. Cell cycle arrest and apoptosis induction are concurrent.31 The occurrence of

Figure 4. WSP1 induced mitochondrial membrane depolarization and releases of apoptotic factors in DLD1 cells. (A,B) Cells treated with WSP1 (0.15 μg/μL) for 48 h were harvested and subjected to flow cytometric analysis of mitochondrial membrane potential by JC-1 staining. Results were shown as mean values from three independent experiments. Treatment caused significant increases in the percentage of cells with depolarized mitochondrial membrane compared with the control (*p < 0.05, **p < 0.01). (C) Red and green fluorescence ratio of DLD1 cells treated and untreated by flow cytometric analysis. (D) Expressions of Bax and Bcl-2 were analyzed by Western blotting. GAPDH was used as the loading control. Representative Western blotting from two independent experiments was shown.

It is recognized that Bcl-2 family proteins regulate the mitochondrial membrane potential and the Bax/Bcl-2 ratio determines the cellular survival or apoptotic cell death.24 The results showed that WSP1 could inhibit the antiapoptotic Bcl-2 protein and elevate the pro-apoptotic Bax protein (Figure 4D). With this, the Bax/Bcl-2 ratio was significantly increased (Figure 4D) and there was a subsequent reduction of mitochondrial membrane potential (Figure 4B,C). These data 155


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Figure 5. WSP1 induced caspase-dependent apoptosis in DLD1 cells. (A) Caspases and PARP were determined by Western blotting after treatment with WSP1 (0.15 μg/μL). GAPDH was used as the loading control. (B,C) The pan-caspase inhibitor reversed WSP1-suppressed cell proliferation. DLD1 cells were preincubated for 1 h in the absence or presence of 20 μM pan-caspase inhibitor, z-VAD-fmk, and were treated with WSP1 (0.15 μg/μL) for 48 h. Proliferation inhibitions of DLD1 cells were calculated by MTT, and the expression of caspase-9 was analyzed by Western blotting. **p < 0.01 vs cells untreated with WSP1.

Figure 6. Effects of WSP1 on the growth of xenografted tumor in nude mice. DLD1 cells (5 × 106) were injected subcutaneously into the right oxter of the nude mice. After tumor formation, the mice were divided into two groups and treated with WSP1 (60 μg/g body weight) every 3 days or with PBS as control. (A) Curve of average body weight in two groups within 21 days. (B) Tumor volumes of both groups were calculated. (C) After six treatments with WSP1, mice were sacrificed and five representative tumors of each group were shown. (D) Mass of dissected tumors was excised. Results represent mean ± SD (n = 8). **p < 0.01 vs control group.

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Figure 7. Immunohistochemical analysis of the expression of Ki-67, activated-caspase-8, activated-caspase-9, and PARP in the dissected tumor tissues by WSP1 treatment or control. There were significant differences between control and WSP1-treated group (**p < 0.01).

cell death is not caused by biochemical abnormalities but rather to completely split cell cycle events. Morphological change is considered the most reliable standard of apoptosis. The results showed that WSP1 dramatically inhibited proliferation of CRC cells in vitro, and the typical morphological changes of apoptosis were observed with WSP1 treatment (Figure 3A). Especially, WSP1 could induce the cell cycle arrest and apoptosis significantly. Moreover, it exerted obvious negative effects on normal colonic epithelial cell FHC, suggesting WSP1 has targeted antitumor effects on colon cancer cells. It is known that c-Myc plays an important role in the cell cycle by activating cyclin D1 and cyclin E1, which in turn lead to phosphorylation of the retinoblastoma protein (Rb) and facilitate the cells entering the G1 phase.32 Here, WSP1 induced cell cycle arrest at the G1/S phase in DLD1 cells. The corresponding Western blotting demonstrated that c-Myc expression was reduced, followed by the down regulations of cyclin D1, cyclin E1, and pRb expression. Thus, WSP1 could induce cell growth inhibition through the induction of G1arrest in colon cancer cells (Figure 8). Tumor formation often results from an increase in cell proliferation and a decrease in cell apoptosis.33 The ability to induce apoptosis in tumor cells is as important as antitumor drugs.34 Apoptosis usually initiated by caspase activation, even though not all cell apoptosis is linked to caspase activation.35 Furthermore, PARP, a nuclear enzyme involved in DNA repair, is a well-known substrate for caspase-3 cleavage during apoptosis. Studies showed that PARP cleavage reduced DNA repair function and is considered to be a hallmark of apoptosis.36 In the process of identifying the pathways by

Figure 8. Proposed the possible signal pathway for growth inhibition and apoptosis induced by WSP1 in DLD1 cells.

which WSP1 induced colon cancer cell apoptosis, PARP was found to be cleaved and mediated by a cascade of caspases, including caspase-8 of the extrinsic apoptosis pathway and caspase-9 of the intrinsic pathway. These initiator caspases then lead to the activation of capsase-3. The present study showed that caspase-8, -9, and -3 in DLD1 cells were all activated with the WSP1 treatment. Consistently, WSP1-induced DLD1 cell apoptosis was partially suppressed and the cleavage of procaspase-9 was reversed by the addition of the pan-caspase inhibitor. The immunohistochemistry of DLD1 xenografted nude mice further confirmed that actived-caspase-8, -9, and PARP had strong expression in the cells treated with WSP1. These data confirmed that the caspase-dependent pathway was 157


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Journal of Agricultural and Food Chemistry Funding

responsible for the apoptotic process caused by WSP1 (Figure 8). Cell survival depends on the maintenance of mitochondrial membrane potential.37 The Bcl-2 family members consist of pro- and antiapoptotic proteins, which work together with other proteins to maintain a dynamic balance between the survival and death of cells by regulating the function of mitochondria. In human tumors, the expressions of Bcl-2, BclxL, or other antiapoptotic homologues are up-regulated and the expression of pro-apoptotic protein Bax or Bak is downregulated.38 This study showed that WSP1 induced precocious apoptosis in DLD1 cells, as evidenced by a decrease in the expression of Bcl-2 and an increase in Bax, accompanied by a decrease in Δψm (Figure 8). The water stress protein, WSP1 from Nostoc commune Vauch. was prepared and studied in the present investigation. It indicated that WSP1 could suppress the growth of colon cancer cells by inducing cell cycle G1-phase arrest. Its apoptosispromoting activity is attributed to the activation of caspasedependent pathway. In agreement with the in vitro data, WSP1 could also suppress the growth of xenografted DLD1 tumors in vivo, indicating that it may be developed as an effective therapeutic agent for patients with colon cancers. Intraperitoneal injection was applied in vivo in our study. It is an effective administration method for protein drugs in mice models. However, although it has good clinical characteristics such as a good curative effect and low side effects, the bioavailability of protein drugs is only a small percentage of their short half-life, they have large molecular weights and poor trans-membrane ability using methods other than injection. In contrast, using intravenous injection, protein drugs easily cause an immune response. Therefore, it has been an urgent task for scientists to research and develop protein drugs that are stable, have a long half-life, and have good biocompatibility and easy absorption. Studies 39−41 have shown that a chemical modification or a prodrug, such as an enzyme inhibitor, working as an absorption accelerator, could improve the absorption and reduce the immune response of protein drugs delivered via intravenous injection. For example, PEG (polyethylene glycol) is often used to carry modified protein structures and it could prolong the half-life of protein drugs and control their velocity into the blood. In addition, the injectable microspheres can achieve slow-release effects. Other noninjection methods such as nasal and pulmonary routes of administration can also improve the absorption and utilization of protein drugs. To improve the bioavailability of protein drugs, new biotechnology such as nanoliposomes are being used as oral and transdermal protein drug delivery carriers. Collectively, WSP1 has specific antitumor activities on colon cancer and may have great potential for medicinal applications.

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This study was supported by the National Natural Science Foundation of China (nos. 31271516 and 31201072), the Shanxi Province Science Foundation for Youths (20120210284), the Oversea Scientists’ Funds (20111009/10), and the Research Fund for the Doctoral Program of Higher Education of China (20111401110011). Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We thank Dr. Enmin Zou of the Department of Biological Sciences of Nicholls State University, USA, for improving the readability of the paper.

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ASSOCIATED CONTENT

S Supporting Information *

Summary of WSP1 identification by MALDI-TOF analysis. This material is available free of charge via the Internet at http://pubs.acs.org.

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REFERENCES

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*For Zhuoyu Li: phone, 86-351-7018268; fax, 86-351-7018268; E-mail, [email protected]. 158


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