1588 Human A-defensin-1 inhibits growth of human lung adenocarcinoma xenograft in nude mice Ning Xu,1 Yong-sheng Wang,1 Wu-bin Pan,1 treated mice through histologic analysis. These results Bo Xiao,1 Yan-jun Wen,1 Xian-cheng Chen,2 indicate that intracellularly expressed HNP1 induces tumor Li-juan Chen,1 Hong-xin Deng,1 Jia You,1 cell apoptosis, which inhibits tumor growth. The anti- Bing Kan,1 A-fu Fu,1 Dan Li,1 Xia Zhao,1 angiogenesis effect of HNP1 may contribute to its in vivo and Yu-quan Wei1 inhibitory activity , and HNP1 might involve the host immune response to tumor. These findings provide a 1State Key Laboratory of Biotherapy and Cancer Center, West rationale for developing HNP1-based gene therapy for China Hospital and 2Department of Gynecology and cancer. [Mol Cancer Ther 2008;7(6):1588–97] Obstetrics, Second West China Hospital, West China Medical School, Sichuan University, Chengdu, Sichuan, People’s Republic of China Introduction The human a-defensins, also known as the human neutrophil peptides (HNP1-3), are small cationic peptides Abstract found in azurophilic granules (1). In vitro, HNP show A Human -defensin-1 (HNP1), a small antimicrobial cytotoxicity to various types of eukaryotic cells and tumor in vitro peptide, shows cytotoxicity to tumor cells and cells (2–4). It was proposed that HNP could bind to and inhibitory activity for pathologic neovascularization damage cell membranes resulting in lethal damage (4, 5). in vivo . Here, we did a gene therapy with a plasmid that Due to the increased permeabilityof cell membranes, HNP expresses a secretable form of HNP1 for assaying its can also penetrate cells and cause a secondaryinjurythat is antitumor activity. The expression and secretion of HNP1 likelyrequired for tumor cell lysis(2). The mechanism of were determined by reverse transcription-PCR and ELISA HNP-induced apoptosis involves release of cytochrome in vitro . We found that expression of HNP1 in A549 tumor c from mitochondria, which is the keyevent of mitochon- cells caused significant growth inhibition. This effect is dria-mediated apoptosis (6). Moreover, recent evidence most likely cell autonomous, as a significant amount of shows that HNP are expressed in renal cell carcinomas and recombinant HNP1 protein was found to be accumulated influence the proliferation of renal malignant cells and in the cytoplasm by immunohistochemical staining using immune recognition (7) and also involved in host immune an anti-HNP1 antibody and the supernatant containing response to cervical human papillomavirus-associated secreted HNP1 failed to produce any noticeable antitumor neoplastic lesions (8). Based on these findings, we activity. Flow cytometry and Hoechst 33258 staining hypothesize that HNP may prove useful for cancer gene showed that the number of apoptotic cells among the therapy. A549 cells expressing recombinant HNP1 proteins was In addition, HNP can regulate angiogenesis byaffecting significantly greater than that of the nontransfected endothelial cell adhesion and migration in a fibronectin- control cultures, suggesting that this growth-inhibitory dependent manner as well as endothelial cell proliferation activity was due to an apoptotic mechanism triggered (9, 10). Inhibition of angiogenesis through HNP1 would by the intracellular HNP1. The antitumor activity of increase its antitumor effect, because antiangiogenesis in vivo intracellularly expressed HNP1 was also shown . has proven an effective strategyfor treatment of cancer Decreased microvessel density and increased lymphocyte patients (11). infiltration were observed in tumor tissue from HNP1- In this study, we construct a eukaryotic expression plasmid pSec-HNP1 to evaluate its antitumor effect in vitro and in vivo. Our data indicate that recombinant HNP1, expressed intracellularlybythe pSec-HNP1, exhib- Received 1/14/08; revised 3/17/08; accepted 3/23/08. its antitumor activities through induction of apoptosis and Grant support: National 973 Basic Research Program ofChina grants likelyinhibition of angiogenesis. 2006CB504303, 2004CB518800, and NSFC. Note: N. Xu and Y-s. Wang contributed equally to this work. The costs ofpublication ofthis article were defrayed in part by the Materials and Methods payment ofpage charges. This article must thereforebe hereby marked Tumor Cell Lines and Culture advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. The human lung adenocarcinoma cell line A549 Requests for reprints: Yu-quan Wei, Xia Zhao, State Key Laboratory of and monkeykidneycell COS-7 (purchased from the Biotherapy and Cancer Center, West China Hospital, West China Medical American Type Culture Collection) were maintained in School, Sichuan University, Keyuan Road 4, Chengdu, Sichuan, People’s Republic ofChina. Phone: 86-28-85164059; Fax: 86-28-85164060. DMEM (Life Technologies) supplemented with 10% E-mail: [email protected]; [email protected] heat-inactivated fetal bovine serum (Life Technologies) Copyright C 2008 American Association for Cancer Research. and antibiotics. The culture was maintained in 95% air- j doi:10.1158/1535-7163.MCT-08-0010 humidified atmosphere containing 5% CO2 at 37 C. MolCancerTher2008;7(6).June2008 Downloaded from mct.aacrjournals.org on September 27, 2021. © 2008 American Association for Cancer Research. Molecular Cancer Therapeutics 1589 Construction of the pSec-HNP1 Vector culturing for 48 h, MTT assaywas done. Untreated cells The nucleotide sequence encoding the mature region of served as the indicator of 100% cell viability. HNP1 was amplified from total RNA of human peripheral Detection of Tumor Necrosis Factor-A blood lymphocytes by reverse transcription-PCR using the HNP1 can promote lymphocytes to release tumor following primers: forward primer 5¶-GCGGCCCAGC- necrosis factor-a (TNF-a) that maymediate apoptosis of CGGCCGCCTGCTATTGCAGAATA-3¶ and reverse primer tumor cells (12, 13). To exclude that HNP1 promote the 5¶-GAGATATCAGCAGCAGAATGCCCAGAGTC-3¶.The release of TNF-a from A549, the TNF-a in supernatant was amplified mature HNP1 fragment was cloned into expres- tested with a commercial detection kit. sion vector pSecTag2B (Invitrogen), which contains a Hoechst 33258 Staining cytomegalovirus promoter and Ign-chain leader sequence. A549 cells were transfected with pSec-HNP1, pSecTag, The resultant recombinant plasmid was named pSec-HNP1 LipofectAMINE 2000, or left untreated. As described and verified byDNA sequencing. The emptyvector previously(14), 48 h after transfection, cells were fixed (pSecTag2B) was used as a control and named pSecTag. for 20 min in 4% paraformaldehyde in PBS and then The pSec-HNP1 and pSecTag were prepared with Endo- washed in PBS twice. Cells were stained with Hoechst Free kits from Qiagen. 33258 for 5 min and washed with PBS. Finally, apoptosis Detection of the Expression of HNP1 by Reverse was visualized with fluorescence microscope. Transcription-PCR and ELISA Flow Cytometry Assay To test the expression of HNP1 and the cytotoxicity of the A549 cells including both attached and floating cells were products from transfected cells, COS-7 and A549 cells were harvested 48 h after transfection. Flow cytometric analysis transfected with pSec-HNP1 or pSecTag vector. Briefly, was done to identifysub-G 1 cells/apoptosis cells. Briefly, cells were plated on a six-well plate (f2 Â 105 per well). cells were suspended in 1 mL hypotonic fluorochrome When cultivated to 70% confluence, cells were transfected solution containing 50 Ag/mL propidium iodide in 0.1% with 2 Ag pSec-HNP1 and pSecTag using LipofectAMINE sodium citrate plus 0.1% Triton X-100, and cells were 2000 reagent (Invitrogen) according to the manufacturer’s analyzed by a flow cytometer. Apoptosis cells appeared in instruction. Forty-eight hours after transfection, the total the cell cycle distribution as cells with DNA content less RNA was then extracted with Trizol reagent (Invitrogen) than that of G1 cells. and reverse transcription-PCR was done using One-Step Evaluation of Antitumor Effect Reverse Transcription-PCR Kit (Takara). The level of Human A549 lung cancer cells (5 Â 106) were implanted recombinant HNP1 in supernatant was determined by s.c. into the right flanks of 6- to 8-week-old female nude ELISA assayusing HNP1-3 ELISA Test Kit (Hbt, HK317). mice. When tumor diameter reached f5 mm (23 days after Detection of the Intracellular Expression of HNP1 in inoculation), animals were randomlydivided into three A549 Cells groups with five mice per group and were injected After 24 and 48 h of pSec-HNP1 and pSecTag (2 Ag) intratumorallyand around tumor with pSec-HNP1 treatment, respectively, cells were fixed with cold acetone (100 Ag), pSecTag vector (100 Ag), or PBS (100 AL). The and then treated with 0.1% Triton X-100 to increase the DNA was encapsulated in cationic liposome with a ratio of membrane permeability. The cells were then incubated 1:3. Because we showed previouslythat liposome has no with mouse anti-HNP1 monoclonal antibody(1:1,000; effect on tumor growth in vivo (15), we did not set the Serotec MCA1465) to determine the intracellular expression liposome group as a control. The DNA was administered of HNP1 in A549 tumor cells. once every3 daysin a volume of 100 AL for a total of five Tr y p a n B l u e S t a i n in g times, and the control injection in a volume of 100 AL PBS As described previously(2), briefly,A549 cells were solution was also done at the same time point. Tumor seeded in a six-well plate (f2 Â 105 per well). When volume was observed and tumor size was determined by cultured to 70% confluence, cells were transfected with caliper measurement of the largest and the smallest pSec-HNP1 (2 Ag), pSecTag (2 Ag), LipofectAMINE 2000, diameters once every3 days.Tumor volume ( V) was and left untreated, respectively. After 24 h, both attached calculated using the formula: V =1/2Â A Â B2, where and floating cells were harvested; 0.4% trypan blue (20 AL) A is the largest superficial diameter and B is the smallest was added to 20 AL cells and incubated for 5 min at room superficial diameter.