J Huazhong Univ Sci Technol [Med Sci]
34(3):529-534,2014
10.1007/s11596-014-1310-4 J DOI Huazhong Univ Sci Technol［Med Sci］ 34(4):2014
529
Antitumor Activity of Recombinant Antimicrobial Peptide Penaeidin-2 against Kidney Cancer Cells Ming-xiang MENG (孟明翔)1, Jian-fang NING (宁建芳)2, Jing-you YU (于京佑)2, Dan-dan CHEN (陈丹丹)2, Xiao-lin MENG (孟小林)2, Jin-ping XU (徐进平)2#, Jie ZHANG (张 杰)1# 1 Department of Urology, Renmin Hospital of Wuhan University, Wuhan 430060, China 2 State Key Laboratory of Virology, College of Life Sciences, Wuhan University, Wuhan 430072, China © Huazhong University of Science and Technology and Springer-Verlag Berlin Heidelberg 2014
Summary: Penaeidin-2 (Pen-2) is an important antimicrobial peptide derived from the Pacific white shrimp, Penaeus vannamei, and possesses both antibacterial and antifungal activities. Recent studies suggest that recombinant penaeidins show similar activities to the native Pen-2 protein. Previous researches have shown that some antimicrobial peptides (AMPs) exhibit cytotoxic activity against cancer cells. To date, there have been no studies on the antitumor effects of Pen-2. This study evaluated the potential of recombinant pen-2 (rPen-2) in the selective killing of kidney cancer cell lines ACHN and A498, and its action mechanism. MTT assays found the maximal growth inhibition of HK-2, ACHN and A498 cells treated with 100 μg/mL rPen-2 at 48 h was 13.2%, 62.4%, and 70.4%, respectively. DNA-specific fluorescent dye staining showed a high percentage of apoptosis on cancer cells. Flow cytometry revealed that the apoptosis rate of HK-2, ACHN and A498 cells was 15.2%, 55.2%, and 61.5% at 48 h respectively, suggesting that rPen-2 induced higher apoptosis rate in cancer cells than in HK-2 cells. Laser confocal scanning microscopy demonstrated that the plasma membrane was the key site where rPen-2 interacted with and destroyed tumor cells. Scanning electron microscopy showed the morphologic changes of the cell membranes of kidney cancer cells treated with rPen-2. These results suggest that rPen-2 is a novel potential therapeutic agent that may be useful in treating kidney cancers. Key words: recombinant Penaeidin-2; kidney cancer cell; plasma membrane; apoptosis; lysis
Antimicrobial peptides (AMPs) are evolutionarily conserved components of the innate immune response that are found in almost all life forms. Penaeidin-2 belongs to Penaeidin family of AMPs, which was initially isolated from the hemolymph of the Pacific white shrimp, P. vannamei[1]. Penaeidin is the biggest AMP family in the shrimp, which can be classified into Pen-2, -3, and -4, based on the amino acid sequence and the position of special amino acids[2]. Like many other known crustacean AMPs[3], they are composed of an NH2-terminal proline-rich region with an isoelectric point ranging from 9.34 to 9.84. Antimicrobial assays using native penaeidins that were directly purified from shrimp hemolymph indicated that penaeidins have both antibacterial and antifungal activities[4]. The recombinant penaeidins also show almost indistinguishable antimicrobial activity as compared with native penaeidins[5, 6]. We have expressed recombinant Pen-2 (rPen-2) in E. coli Origami B (DE3) pLysS. Previous work from our laboratory demonstrated that rPen-2 has a broad-spectrum of antibacterial activity against both gram positive bacteria and gram negative bacteria[7]. Current researches on AMPs have shown that some of cationic AMPs have antitumor activity[8, 9]. Yet to date, there have been no studies on the potential antitumor effects of rPen-2. Ming-xiang MENG, E-mail:
[email protected] # Corresponding authors, Jie ZHANG, E-mail:
[email protected]; Jin-ping XU, E-mail:
[email protected]
Renal cell carcinoma (RCC) is the most common malignant tumor of the adult kidney, accounting for approximately 3% of all adult cancer cases[10]. More than 64 770 persons will be diagnosed with RCC, and 13 570 patients will die from the disease in USA in 2012[11]. Because of the resistance of RCC to radiotherapy and chemotherapy, the identification and development of novel therapeutic strategies and drugs are required. In this study, we evaluated the potency of rPen-2 as an anticancer agent against RCC. We treated two kidney cancer cell lines (ACHN and A498) and one normal kidney cell line (HK-2) with rPen-2, and conducted 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assays to determine cytotoxicity and the effects of rPen-2 on cell proliferation. Nuclear changes of cells were visualized by nuclear staining. Using confocal laser scanning, we observed the interaction between rPen-2 and cells. Scanning electron microscopy (SEM) found morphologic changes in the cell membranes of kidney cancer cells treated with rPen-2. 1 MATERIALS AND METHODS 1.1 Generation of Fusion Protein rPen-2 cDNA of Pen-2 was subcloned into the expression vector pET-32a (+) (Novagen, Germany). rPen-2 was produced as a result of recombinant protein expression of peptide Pen-2 with TrxA at the N-terminus in E. coli Origami B (DE3) pLysS. The rPen-2 was then purified by His-Band resin (Qiagen, Germany) chelating chroma-
530 tography. High-performance liquid chromatography (HPLC) showed that the purity of target protein reached more than 95% after purification. The protein was then dissolved in phosphate buffer solution (PBS) prior to use. 1.2 Cell Lines The established human kidney cancer cell line ACHN and the normal human kidney cell line HK-2 were purchased from China Center for Type Culture Collection (CCTCC, China). The established human kidney cancer cell line A498 was obtained from American Type Culture Collection (ATCC, USA). The two cancer cell lines were maintained in minimum essential medium (MEM) (Hyclone, USA), and HK-2 in Dulbecco′s minimal essential medium/Ham′s F12 (DMEM/F12) (Hyclone, USA). All media were supplemented with 10% fetal bovine serum (FBS) (Hyclone, USA). Cell lines were incubated in a humidified atmosphere of 5% CO2 and 95% air at 37°C. 1.3 Cell Proliferation Assay Cell proliferation was determined using MTT assays. Cells were seeded at 5×103 cells/well in a 96-well plate for 24 h followed by treatment with various concentrations of rPen-2. PBS served as a control. Following the 24 h incubation with rPen-2, 20 μL of MTT solution (0.5 mg/mL) was added to each well and incubated for 4 h at 37°C. Subsequently, the MTT was removed and 100 μL DMSO was added to each well to dissolve formazan crystals. The absorbance (A) at a wavelength of 570 nm was determined using an automated microplate reader. The A of cells incubated in the absence of rPen-2 was set as 100% proliferation. All results were expressed as inhibition rate of proliferation. 1.4 Hoechst 33258 Nuclear Staining Apoptotic cells were detected by staining with Hoechst 33258, a DNA specific fluorescent dye that stains the condensed chromatin of apoptotic cells more brightly than the chromatin of normal cells. The three cell types were incubated with rPen-2 (100 μg/mL) in 96-well plates for 24 h with PBS treatment as a control. The change in the apoptotic cells was visualized under a fluorescent microscope at the wavelength of 340 nm. 1.5 Acridine Orange/Ethidium Bromide Staining Acridine orange/ethidium bromide (AO/EB) staining was used to visualize nuclear changes and apoptotic body formation, both of which are crucial characteristics of apoptosis. Live cells with intact membranes were stained uniformly green, early apoptotic cells stained green while the pyknotic chromatin presented bright green dots in the nucleus. Late apoptotic cells showed extremely bright orange areas of condensed chromatin in the nucleus when compared with necrotic cells which had a uniform orange color. The three cell lines were seeded at 5×103 cells/well in a 96-well plate and incubated in the appropriate medium for 24 h. Cells were treated with rPen-2 (100 μg/mL) for 24 h, or PBS as a control. Cells were washed with PBS and stained with a solution of 100 μg/mL AO and 100 μg/mL EB in PBS in a 1:1 ratio. 1.6 Flow Cytometric Analysis of Cell Death Cell apoptosis was determined using an annexin-V-fluorescein isothiocyanate (FITC)/propidium iodide (PI) apoptosis detection kit (Invitrogen, USA). Each cell type was seeded at a density of 5×105 cells/well in 24-well plates and treated with rPen-2 (100
J Huazhong Univ Sci Technol［Med Sci］ 34(4):2014
μg/mL) for 24 and 48 h. After treatment, cells were harvested following the manufacturer’s protocol, washed with PBS, and incubated in 0.5 mL binding buffer containing 5 μL annexin-V-FITC and 10 μL PI for 5 min in the dark. Cells were then analyzed by flow cytometry (Epics XL, Beckman Coulter, USA); 10 000 events were recorded and represented as dot plots. 1.7 Laser Confocal Scanning Microscopy Cells were seeded in 35-mm Petri dishes and allowed to adhere. Subsequently, cells were treated with 100 μg/mL rPen-2 for 3, 6, and 12 h. After removing the medium, cells were fixed with 1% paraformaldehyde, rinsed with 0.1% Triton X-100, and incubated overnight at 4°C in 5% BSA. After serum blocking, cells were incubated with rPen-2 antibody (diluted 1:100 in PBS) for 30 min at 37C. Cells were then washed with PBS and incubated with secondary antibodies (FITC-labeled goat polyclonal anti-rabbit IgG diluted 1:100 in PBS) for 30 min at 37C. The cells were washed again with PBS and stained with PI for 10 min at 37C. Petri dishes were analyzed using laser confocal scanning microscope (FV1000, Olympus, Japan). 1.8 Scanning Electron Microscopy We placed sterilized cover slips at the bottom of wells in a 6-well plate, and ACHN, A498 and HK-2 cells were seeded at 5×105/well. Cells were then treated with rPen-2 (100 μg/mL) for 24 h. The medium containing rPen-2 was removed and a 4% glutaraldehyde solution (Sigma, USA) was added to each well. Dehydration was performed using an ethanol gradient (30%–100%), followed by counter fixation in 2% osmium tetroxide (Sigma, USA) for 2 h and drying in a freeze-drying device. Cells on cover slips were coated with gold and analyzed using a SEM (QUANTA 200, FEI, Holland). 1.9 Statistical Analysis Results were expressed as ±s . The differences between controls (untreated cells) and rPen-2 treated cells were analyzed by using one-way analysis of variance (ANOVA). Statistically significant differences were defined at P
