J Cancer Res Clin Oncol (2011) 137:19–28 DOI 10.1007/s00432-010-0855-2

ORIGINAL PAPER

Therapeutic eVects of survivin dominant negative mutant in a mouse model of prostate cancer Li Pan · Xing-Chen Peng · Fei Leng · Qing-Zhong Yuan · Yan Shan · Dan-Dan Yu · Zhi-Yong Li · Xiang Chen · Wen-Jing Xiao · Yuan Wen · Tian-Tai Ma · Li Yang · Yong-Qiu Mao · Han-Shuo Yang · Yu-Quan Wei · Chun-Ting Wang

Received: 30 October 2009 / Accepted: 19 February 2010 / Published online: 9 March 2010 © Springer-Verlag 2010

Abstract Purpose Patients with localized prostate cancer can usually achieve initial response to conventional treatment. However, most of them will inevitably progress to advanced disease stage. There is a clear need to develop innovative and eVective therapeutics for prostate cancer. Mouse survivin T34A (mS-T34A) is a phosphorylationdefective Thr34 ! Ala dominant negative mutant, which represents a potential promising target for cancer gene therapy. This study was designed to determine whether mS-T34A plasmid encapsuled by DOTAP-chol liposome (Lip-mS) has the anti-tumor activity against prostate cancer, if so, to further investigate the possible mechanisms. Methods In vitro, TRAMP-C1 cells were transfected with Lip-mS and examined for apoptosis by PI staining and Xow cytometric analysis. In vivo, subcutaneous prostate cancer models were established in C57BL/6 mice, which were randomly assigned into three groups to receive i.v. administrations of Lip-mS, pVITRO2-null plasmid complexed with DOTAP-chol liposome (Lip-null) or normal saline every 2 days for eight doses. Tumor volume was measured. Tumor

L. Pan, X.-C. Peng and F. Leng have equally contributed to the present study. L. Pan · X.-C. Peng · F. Leng · Q.-Z. Yuan · Y. Shan · D.-D. Yu · L. Yang · Y.-Q. Mao · H.-S. Yang · Y.-Q. Wei · C.-T. Wang (&) State Key Laboratory of Biotherapy and Cancer Center, West China Hospital, West China Medical School, Sichuan University, Keyuan Road 4, Chengdu, Sichuan, People’s Republic of China e-mail: [email protected] Z.-Y. Li · X. Chen · W.-J. Xiao · Y. Wen · T.-T. Ma State Key Laboratory of Biotherapy, West China Hospital, School of Life Science, Sichuan University, Chengdu, Sichuan, People’s Republic of China

tissues were inspected for apoptosis by TUNEL assay. Microvessel density (MVD) was determined by CD31 immunohistochemistry. Alginate-encapsulated tumor cell test was conducted to evaluate the treatment eVect on angiogenesis. Results Administration of Lip-mS resulted in signiWcant inhibition in the growth of mouse TRAMP-C1 tumors. The anti-tumor response was associated with increased tumor cell apoptosis and decreased microvessel density. Conclusions The present study may be of importance in the exploration of the potential application of Lip-mS in the treatment of a broad spectrum of tumors. Keywords Survivin · Apoptosis · Angiogenesis · Prostate cancer · Gene therapy

Introduction Prostate cancer is the most commonly diagnosed neoplasm and the second leading cause of cancer death in men in the United States. The epidemiologic studies predicated that 19,280 new cases of prostate cancer were to be diagnosed and 27,360 cases will die of this disease in American in 2009 (Jemal et al. 2009). Current therapies for localized prostate cancer include radical prostatectomy, cryoablation therapy, external beam radiation therapy (Koukourakis and Touloupidis 2006; Acher et al. 2006). Although recent advances in both early detection and therapeutic improvement have oVered potential chances of long-term cure, the therapeutic eVect is still unsatisfactory, and most of patients will experience local recurrence and unavoidably progress to advanced disease stage. In addition, local therapies are often associated with side eVects attributable to injury of adjacent tissues, and systemic treatments may have a negative impact on quality of life. To this end, development of

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more eVective therapeutics with fewer side eVects is pressing. One of such potential novel approach toward prostate cancer is gene therapy, which not only enables malignant cells to restore apoptosis but also increases sensitivity to existing treatments as well (Favrot et al. 1998). There has been accumulated evidence indicating that apoptosis and angiogenesis play crucial roles in the occurrence and the progression solid tumors. Thus, gene therapy, which targets anti-apoptotic and/or angiogenesis-related genes, holds great promise for prostate cancer treatment. Survivin, with the molecular weight of 16.5 kDa, is the smallest mammalian member of the inhibitory apoptotic protein (IAP) family (Salvesen and Duckett 2002). It has attracted great attention for its dual role in the control of apoptosis and the regulation of cell division. As an antiapoptotic protein, it is notably expressed in the majority of human neoplasms, whereas undetectable in terminally diVerentiated normal tissues (Ambrosini et al. 1997; Adida et al. 1998; Tanaka et al. 2000; Miller et al. 2001) with the exception of thymus, basal colonic epithelium (Altieri 2001) and endothelial cells during angiogenesis (O’Connor et al. 2000). The over expression of survivin has been related to reduced apoptosis, tumor aggression, tumor recurrence, poor survival (Moriai et al. 2001; Sarela et al. 2001) and drug resistance (Kato et al. 2001). Being a tumor-associated antigen, the integrity of survivin signal transduction pathway is required for cancer cell viability. Therefore, several studies have adopted it as a target for tumor therapy (Pennati et al. 2007) and developed molecular antagonists by variable strategies, such as antisense oligonucleotide, dominant negative mutants (Kanwar et al. 2001), interference RNA (Paduano et al. 2006; Jiang et al. 2006), cancer vaccines (Pisarev et al. 2003), to counteract endogenous survivin. A large number of clinical researches have conWrmed that down-regulation of survivin expression could eVectively prevent tumor growth and increase tumor cell sensitivity to anti-cancer agents. Although Survivin is expressed by some normal tissues such as thymus, basal colonic epithelium, several studies, which targeted survivin for cancer treatment showed little toxicity to these tissues (Li and Brattain 2006). It might be due to several reasons. First, survivin plays a more essential and important role in the regulation of apoptosis and control of cell division in cancer cells than in normal cells. Many signal interactions associated with survivin regulation and function are lacking or signiWcantly weaker in normal cells compared with those in cancer cells (Li and Ling 2006). Second, survivin is produced in small amount and transiently during mitosis in normal tissues, whereas it is constantly up-regulated in tumors to inhibit apoptosis and promote cell division. The diVerent expression and function proWle of survivin in tumor and normal tissue makes it a potentially selective target for cancer treatment.

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It has been reported that the survivin Thr34 ! Ala mutant can abolish a phosphorylation site of the cyclindependent kinase p34cdc2, which is essential for its antiapoptotic activity. Consequently, it induced spontaneous apoptosis in three diVerent melanoma cell systems (Grossman et al. 2001). There is no report on whether mS-T34A mutant can eVectively control prostate cancer. To test this concept, we made mS-T34A mutant compound to DOTAPchol liposome (Lip-mS) and carried out gene therapy by administering Lip-mS to the prostate tumor bearing mice. The anti-tumor eVect was evaluated on tumor growth, apoptosis and angiogenesis. At the same time, the empty vector (Lip-null) and normal saline (NS) were used as controls. Our Wndings showed that Lip-mS can be successfully used against prostate cancer without obvious side eVects and perhaps other solid tumors as well.

Materials and methods Experimental procedures were approved by the Animal Care Committee of Sichuan University. Cell and cell culture The TRAMP-C1 murine prostate cancer cell line derived from C57BL/6 TRAMP (transgenic adenocarcinoma mouse prostate) mice was purchased from the American Type Culture Collection (ATCC, Rockville, MD), cultured in Dulbecco’s modiWed Eagle medium (DMEM) (Gibco BRL, Grand Island, NY) and supplemented with 5% heatinactivated fetal bovine serum (FBS), 5% Nu-serum IV, 0.005 mg/ml bovine insulin, 100 g/ml amikaci, 0.01 nM dehydroisoandrosterone, 4 mM L-glutamine and maintained in a humidiWed incubator at 37°C in 5% CO2 atmosphere. Construction of recombinant plasmid The plasmid pVITRO2 (Invitrogen, San Diego, CA) expressing mS-T34A protein was constructed in our laboratory. BrieXy, cDNA clone encoding mS-T34A was PCR ampliWed with the following primers: forward, 5⬘-GATCA CGCGTCACCATGGGAGC-3⬘; reverse, 5⬘-GGCGGTCG ACAGCATTAGGCAG-3⬘. Then, it was digested with SalI/MluI and inserted into pVITRO2 plasmid digested with SalI/MluI to generate mS-T34A protein. The pVITRO2 plasmid without mS-T34A (pVITRO2-null) was used as a control. Colonies of Escherichia coli containing mS-T34A or null were cultured in Luria–Bertani broth containing 100 g of ampicillin/ml. Large-scale plasmid DNA was puriWed by using a Qiagen Endofree plasmid giga kit (Qiagen, Chatsworth, CA) according to the manufacture’s protocol. The OD260/280 ratios of the plasmid DNA prepared

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were among 1.8–2.0, and the concentration of plasmid was determined by ultraviolet spectrophotometer. Plasmids were stored at ¡20°C before use. Liposome preparation Liposome was prepared according to the procedure described (Lin et al. 2007). The lipids DOTAP and cholesterol (Avanti Polar Lipids Inc., AL, USA) (1:1 M ratio) were dissolved in chloroform in a 100 ml-round-bottomed Xask and rotated to make a thin Wlm. After that, the mixture was dried and put under vacuum for 2 h in order to remove the organic solvent. Then, the lipid Wlm was rehydrated in 5% dextrose (D5W) to give a Wnal concentration of 5 mg/ml and eddied for 30 min, at 60°C. Finally, the Wlm was extruded through a 100 nm polycarbonate Wlter by using an Avanti Polar Lipids Mini-Extruder. Liposomes were stored at 4°C. Western blot analysis The expression of wild-type survivin in TRAMP-C1 cells was analyzed by Western blot. BrieXy, the 293 cells and TRAMP-C1 cells were harvested, lysed and centrifuged. The supernatant was mixed with an equal volume of sodium dodecyl sulfate (SDS) sample buVer. The proteins were run on 12% SDS–polyacrylamide gel electrophoresis (PAGE). Gels were electroblotted onto a polyvinylidene diXuoride membrane (PVDF, Bio-Rad, Richmond, CA, USA). The membrane blots were blocked in 5% non-fat dry milk, washed and probed with rabbit-anti-murine survivin polyclonal antibodies, which is reactive against both native and mutant (Thr34 ! Ala) survivin (Pepro Tech House, USA) at 4°C overnight. Blots were washed and incubated with biotinylated secondary antibodies (biotinylated goatanti-rabbit IgG). The speciWc protein bands were detected using an enhanced chemiluminescence (ECL) detection system (Pierce, Rockford, IL, USA). The mS-T34A protein produced by pVITRO2-mS-T34A transfected human kidney cell 293 was also analyzed by Western blot. BrieXy, 293 cells were plated in six-well plates at 2 £ 105 cells per well and transfected with two-dose group: 2 g of plasmid and 6 g of DOTAP-chol liposome, 5 g of plasmid and 15 g of DOTAP-chol liposome, following the manufacture’s instructions. Cells were incubated for 48 h. Western blotting was performed as described earlier. Apoptosis assay in vitro

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complexes were added in serum-free DMEM medium without antibiotics, which contained 2 g DNA and 6 g DOTAP-chol liposome, then incubated at room temperature for 30 min. Meanwhile, NS was used as a control. After 6 h, the cells were rinsed with PBS, added 2 ml DMEM medium with FCS and antibiotics to each well and placed in a humidiWed incubator for 48 h. Cells were collected and analyzed. Morphological analysis BrieXy, cells were suspended in hypotonic propidium iodide solution containing 50 microgram PI per milliliter in 0.1% sodium citrate plus 0.1% Triton X-100, incubated in dark for 10 min and observed by Xuorescence microscopy. Flow cytometric analysis The TRAMP-C1 cells were collected, pelleted by centrifugation, resuspended in propidium iodide/RNase A solution (0.5 ml) and with a continued incubation for an additional 30 min. Samples were taken to analyze for DNA content and to estimate the percentage of sub-G1 cells by the use of a Xow cytometer (ESP Elite, Beckman Coulter, Fullerton, CA). Tumor model and therapy studies Healthy male C57BL/6 mice of 6–8 weeks old were obtained from the West China Experimental Animal Center and injected subcutaneously with 100 l freshly prepared TRAMP-C1 cell suspensions (3 £ 107 cells/ml) into the right Xank. When the tumor diameters reached about 0.5 cm, mice were assigned randomly to 3 groups, and each group contained 7 mice. The Wrst group (Lip-mS) was treated with 100 l mS-T34A complexed with DOTAPchol liposome (1:1 volume ratio, DNA at 50 g/ml) by intravenous administration via the tail vein. The second (Lip-null control) and the third (normal saline control) groups were administrated as described earlier. All groups were treated every 2 days for a total of 21 days (8 times). Tumor dimensions were measured with calipers every 3 days, and tumor volumes were calculated according to the formula: 0.52 £ length (largest dimension) £ width2 (perpendicular smallest diameter). Mice were killed on day 24 after the initiation of Lip-mS administration, tumors were excised, and the samples were Wxed in 4% neutral buVered formalin solution for histological analysis.

Cellular transfection Histology and apoptosis analysis TRAMP-C1 cells were seeded at aliquots of 2 £ 105 cells/ well in 6-well plates and incubated for 24 h to 70% conXuence. DNA (mS-T34A or null)/DOTAP-chol liposome

Tumors were Wxed in 4% paraformaldehyde in PBS for 24 h, 70% ethanol overnight and embedded in paraYn.

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Sections 3–5 m thick were cut, mounted and stained with hematoxylin and eosin (H&E). Apoptosis was evaluated by terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) assay using the Deadend Fluorometric TUNEL system (Promega, Madison, WI) according to the manufacture’s directions. Sections were examined with a Xuorescence microscope. Each sample was observed at a magniWcation of £400 and calculated the ratio of apoptotic cell number to the total cell number in 5 random Welds. Immunohistochemistry and alginate-encapsulated tumor cell test In order to verify whether the Lip-mS anti-tumor therapy involved the suppression of tumor angiogenesis, examination of angiogenesis in vivo and microvessel density (MVD) in tumor masses was performed. CD31 immunohistochemical assay BrieXy, the tumor sections (5 m) were dewaxed, rehydrated through grade ethanol immersion,antigen retrieval, incubated with 3% hydrogen peroxide for 15 min. After the earlier treatments, sections were incubated with polyclonal goat-anti-mouse epithelial cell marker CD31 antibodies (dilution 1:100; Santa Cruz Biotechnology) overnight at 4°C, then the biotinylated polyclonal rabbit-anti-goat antibodies (dilution 1:100; Santa Cruz Biotechnology) were added and left in a humidiWed chamber at 37°C for 1 h. After that the sections were treated with streptavidin– biotin–horseradish peroxidase agents at 37°C for another 1 h. Through the use of 3, 3-diamino-benzidine as chromogen (DAB visualization system; ZSJQ Biotechnology, Beijing, China), the positive reaction was visualized. Tumor sections were counterstained with ameliorative Gill’s hematoxylin and examined with microscope to count the number of microvessels at high magniWcation (£400) Weld.

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was centrifuged at 1,000 g £ 3 min, the supernatants, which representing the uptake of FITC-dextran into implanted alginate beads, were collected and measured with a Xuorescence spectrophotometer to qualify blood vessel formation as described (HoVmann et al. 1997). Statistical analyses ANOVA and an unpaired Student’s t test were performed to assay data and determine the statistical signiWcance of results in all of the experiments. Statistical signiWcance was deWned as P < 0.05.

Results Expression of survivin and mS-T34A in vitro Expression of survivin in TRAMP-C1 cells was conWrmed by Western blot assay. A distinct band of »16.5 kDa, corresponding to the size of survivin, was visualized in the TRAMP-C1 cells, but not in the 293 cells (Fig. 1a). Survivin expression by the plasmid was further tested by in vitro transfection of 293 cells with Lip-mS (2 or 5 g), Lip-null or NS for 48 h. Western blot analysis detected survivinspeciWc band in the Lip-mS-transfected cells, with denser band detected from 5 g group than that of 2 g group. There was no appearance of band in Lip-null-transfected or non-transfected cells (Fig. 1B). Anti-tumor eVect of Lip-mS-T34A in vitro Apoptotic morphological observation TRAMP-C1 cells treated with Lip-mS and other agents were Wxed, stained with PI and analyzed for the typical morphological changes of apoptosis (chromatin condensation

Alginate-encapsulated tumor cell assay Alginate beads containing 1 £ 106 TRAMP-C1 cells/bead were implanted subcutaneously into both dorsal sides of the C57B/6 male mice in the sterile environment. One the next day, all mice were assigned randomly into 3 groups (2 per group) and treated with Lip-mS (5 g plasmid, 100 l), Lip-null and NS, respectively. The treatments were performed every 2 days and continued for 2 weeks. After 14 days, mice were injected intravenously with 100 l of 1% Xuorescein isothiocyanate (FITC)-dextran (100 mg/kg, Sigma) solution via tail vein. Alginate beads were photographed with a SPOT FIEX camera after exposing surgically for 20 min and quickly removed then transferred to tubes containing 2 ml NS and ground down. The mixture

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Fig. 1 a Western blot analysis of secreted survivin by TRAMP-C1 cells. The survivin protein was expressed as a single band of 16.5 kDa in TRAMP-C1 cells (lane 1). b Expression of mS-T34A protein determined by Western blot. Conditioned media was obtained from 293 cells transfected with Lip-mS, Lip-null or from non-transfected cells. The mS-T34A protein was expressed as a band of »16.5 kDa in LipmS transfected 293 cells (lane 1 5 g, lane 2 2 g). While no band was detected in null (lane 3) transfected or untreated (lane 4) 293 cells

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Fig. 2 Induction of apoptosis in TRAMP-C1 cells by re-introduction of mS-T34A. a Nuclear morphology. TRAMP-C1 cells were treated with the indicated agents for 48 h and analyzed for nuclear apoptosis (DNA fragmentation, nuclear condensation) by PI stain. Large numbers of condensed and fragmented nuclei were detected in Lip-mStransfected cells but not the Lipnull-transfected and NS-treated cells. b Cellular apoptosis was further veriWed by Xow cytometric analysis. The percentage of apoptotic cells with hypodiploid DNA content was 2% (NS), 9.3% (Lip-null) and 42% (Lip-mS), respectively

and DNA fragmentation) by using a Xuorescence microscopy. Compared with the control groups (Lip-null and NS alone), there was more apoptosis induced in Lip-mS-treated cells (Fig. 2A). Flow cytometry analysis In order to quantitatively evaluate cellular apoptosis, we also used Xow cytometry to examine the apoptosispromoting eVect of Lip-mS in TRAMP-C1 cells (Fig. 2B). Lip-mS signiWcantly increased the proportion of apoptotic cells (42%) compared with other agents (Lip-null, 9.3%; NS, 2%). Anti-tumor eVect of Lip-mS-T34A in vivo We established a mouse subcutaneous prostate cancer model to study the eVect of Lip-mS on tumor growth inhibition. Tumor bearing mice were assigned randomly to 3 groups and treated with Lip-mS, Lip-null, NS, respectively. Tumor volume was recorded every 3 days (Fig. 3). Two days after the last administration, mice were killed, and tumors were excised and weighted. As shown here, the treatment with Lip-mS resulted in primary tumor growth regression of 70.8 and 64.5%, respectively, compared with NS and Lip-null groups (P < 0.05). Inhibition of tumor-induced angiogenesis and increased tumor apoptosis in mice The inhibition of tumor growth was closely related to the increased tumor cell apoptosis and decreased angiogenesis. Tumor sections of each group were stained with CD31 for

Fig. 3 Suppression of tumor growth in TRAMP-C1 subcutaneous tumors by Lip-ms, Lip-null and NS. Mice bearing prostate tumors were treated with NS (Wlled triangle), Lip-null (opened diamond) and Lip-mS (Wlled square) every 2 days for 8 times. There was a signiWcant diVerence in tumor volume (P < 0.05) between Lip-mS-treated mice and other control groups over 21 days of treatment. Data were presented as means § SE

microvessel density (MVD) evaluation and TUNEL for apoptosis evaluation. Lip-mS resulted in dramatic inhibition of angiogenesis in tumor (Fig. 4A, c), compared to the control groups (Fig. 4A, a, b). Lip-mS-treated tumor MVD showed signiWcant diVerence (Fig. 4B, P < 0.05) compared with the controls. The inhibition of angiogenesis was also conWrmed in the alginate-encapsulated tumor cell assay (Fig. 5A, a–c). Alginate beads in Lip-mS-treated mice had sparse new blood vessels compared to the control groups. In addition, the uptake of FITC-dextran from beads of Lip-mS-treated mice was signiWcantly decreased compared with control groups (Fig. 5B).

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Fig. 4 Inhibition of angiogenesis within tumors. a Mice bearing prostate tumors were treated with NS (a), Lip-null (b) and Lip-mS (c), and tumor sections from each group were tested by CD31 immunohistochemical assay. b Microvessel density of tumor tissues from mice treated with Lip-mS indicated a signiWcant decrease compared with control groups (P < 0.05)

Fig. 5 Inhibition of angiogenesis assay by alginate beads in vivo. a Alginate beads containing 1 £ 106 TRAMP-C1 cells/ bead were implanted s.c. into the both dorsal sides of the mice and treated with NS (a), Lip-null (b) and Lip-mS (c). b Beads were surgically removed 14 days later and FITC-dextran was quantiWed. The uptake of FITCdextran from beads of LipmS-treated mice showed a signiWcant decrease compared with control groups (P < 0.05)

We then examined the microscopic morphology of tumor from each group. There were many apoptotic cells in Lip-mS group, while in control groups, there were more tumor cells with apparently enlarged nuclei (Fig. 6A, a–c). To further conWrm the earlier phenomenon, we conducted TUNEL assay and as expected, a large number of apoptotic cells were detected in Lip-mS group while there were rare apoptotic cells in Lip-null or NS group (Fig. 6B, a–c). The earlier results suggested that Lip-mS could eVectively inhibit tumor growth, which might be due to the inhibition of tumor angiogenesis and induction of tumor cell apoptosis.

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Side eVect analysis We investigated the side eVect of the treatment by testing serum ALT and AST levels in mice, measuring the body weights of mice every 4 days and observing behavior. The body weight of mice in each group had no signiWcant diVerence, and the serum ALT and AST levels were among normal range. Besides, sections of heart, liver, spleen, lung, kidney, thymus and colon were stained with H&E and examined under microscope. No apparent organ damage was found (data not show).

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Fig. 6 a H&E staining of tumor tissue. Increased necrotic and apoptotic tumor cells were observed in tumor from LipmS-treated mice (c), while tumor grew well and no obvious necrosis and apoptosis were observed in control groups (NS a; Lip-null b). b Detection of apoptotic tumor cells by TUNEL assay. Tumor sections from mice treated with NS (a), Lip-null (b) and Lip-mS (c) were stained with FITC-DUTP. Cell nuclei with dark green Xuorescent staining were deWned as TUNEL-positive nuclei. c Percent of apoptosis in the Lip-mS groups markedly increased in comparison with controls (P < 0.05)

Discussion Previous researches indicated that tumor initiation and progression were correlated with two main factors: apoptosis and angiogenesis. Apoptosis, also known as programmed cell death, plays a central role in many processes, ranging from embryonic development to preservation of tissue homeostasis by eliminating injured or unwanted cells (Ellis et al. 1991). It is conWrmed that aberration of apoptosis can lead to the occurrence of several diseases including cancer (Reed 1999) and appears to be involved in tumor cell resistance to some anti-cancer agents (Borst et al. 2001; Brown and Wouters 2001; Roninson et al. 2001; Schmitt and Lowe 2001). In addition, it has been reported that the generation of new blood vessels was closely associated with a large variety of biological and pathological processes (Folkman 2006). The growth and progression of most solid cancers are angiogenesis dependent. Tumor cells release angiogenic factors, which can promote formation of new vascular network by basing on the pre-existing blood vessels. In return, these vascular-rich networks can feed the cancerous mass by providing tumor cells with nourishments and oxygen. So resistance to apoptosis and promotion of angiogenesis in tumor tissues are now increasingly recognized as crucial

mechanisms whereby tumors escape therapeutic intervention, often after a standard initial positive response. Most conventional anti-cancer agents only have a single antitumor mechanism, which results in an unsatisfactory curative eVect. Therefore, we consider that the ideal anti-cancer drugs should be enabled not only to promote apoptosis in tumor cells but also to inhibit tumor angiogenesis. Prostate cancer, like most other solid tumors, represents a very heterogeneous entity. At the time of clinical diagnosis, a majority of prostate cancers present themselves as mixtures of androgen-dependent and androgen-independent cells. Through the studies of recently accumulated experimental and clinical data, most of patients with primary prostate cancer would inevitably progress to androgen-independent status associated with a high mortality rate with ensuing 12 months even after conventional standard treatments (Denis 1993; Oh and KantoV 1998). Since androgenindependent prostate cancer cells do not undergo apoptosis upon androgen blocking, it presents a therapeutic challenge. However, these speciWc prostate cancer cells still do maintain the appropriate molecular machinery of apoptosis. On the other hand, although the speciWc mechanisms for the occurrence of androgen-independent prostate cancer still

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remained mysterious, some researches showed that the antiapoptotic mechanisms were participated in such process and became more compelling (Tang and Porter 1997). So it is conceivable that the utility of a proapoptotic drug for curing prostate cancer will be an excellent choice. Apoptosis is a complicated process involving diVerent gene families of inhibitors and stimulators of cell death. It is well known that survivin is one of the anti-apoptotic genes. Over the past few years, mutational analysis of survivin has helped to provide some insightful clues in characterizing the function of survivin and dissecting its downstream signaling events (Mesri et al. 2001). It has been conWrmed that survivin can preferentially block mitochondrial-dependent apoptosis by targeting caspase-9 and SMAC (second mitochondria-derived activator of caspase)/ DIABLO (direct inhibitory apoptotic protein-biding protein with a low isoelectric point), so that cells can keep on surviving. Previous studies indicated that survivin was up-regulated in virtually every human cancer and activated endothelial cells, but undetected in normal and terminally diVerentiated adult tissue. Therefore, altering the survivin pathway has been identiWed as a good choice for targeting a wide variety of tumors. Based on earlier point of view, in the present study, we established a mouse subcutaneous prostate cancer model and evaluated the anti-tumor eYcacy of a novel proapoptosis and anti-angiogenesis therapy by using the plasmid encoding the phosphorylation-defective mouse survivin threonine 34 ! alanine mutant (mS-T34A) encapsulated by DOTAP-chol liposome (Lip-mS). On day 21 after the initiation of administration, compared with the tumor size observed in control groups (Lip-null and NS), the volumetric reduction in tumor size of Lip-mS group was 64.5 and 70.8% (P < 0.05). It was quite obvious that tail vein injection of Lip-mS caused a signiWcant regression of tumor volume compared with injection of Lip-null or NS. The results of our experiments suggested that the Lip-ms indeed have a profound anti-prostate cancer eVect. In order to test the possible mechanisms underlying the anti-tumor property of Lip-mS, we made several studies to investigate the eVect of Lip-mS on TRAMP-C1 cells in vitro. PI staining Xuorescence microscopy and Xow cytometric analysis of Lip-mS-treated tumor cells were conducted. We found that tumor cells, which received Lip-mS displayed the highest apoptotic indices compared with control groups. These Wndings supported the apoptosis-inducing ability of mS-T34A reported earlier (Grossman et al. 2001). Meanwhile, tumor sections of each group were stained with TUNEL reagent. An apparent increase in the number of apoptotic cells was observed within the tumors treated with Lip-mS compared with the other treatments. These phenomena showed that the anti-tumor eVect of Lip-mS resulted from direct induction of apoptosis of tumor cells.

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We next examined whether Lip-mS could inhibit tumorassociated angiogenesis. It is reported that the tumor cells supported by the vessel would undergo apoptosis when endothelial cell apoptosis is induced (Dixelius et al. 2000). In this study, we investigated the eVect of Lip-mS on angiogenesis in tumor tissue and we found that CD31 staining of Lip-mS-treated group showed a decrease in the number of intratumoral microvessel in vivo and alginate-encapsulated tumor cell test also conWrmed that Lip-mS could indeed interfere with angiogenesis. These Wndings revealed that Lip-mS could result in tumor growth stasis by preventing new vessels from forming around a tumor and breaking up the existing network of abnormal capillaries that feed the cancerous mass. This seemed to be another potential mechanism of the anti-tumor eVect of Lip-mS. In fact, many of the earlier studies also demonstrated that the dominant negative mutant mS-T34A could signiWcantly retard the growth of tumor nodules in mice by causing caspase-dependent cell death and inhibiting angiogenesis (Xiang et al. 2005; Peng et al. 2008). In addition, unlike other members of the IAP family, survivin has a remarkable expression proWle. This feature is important to make it an ideal target in cancer therapy, because counteracting endogenous survivin will do no harm to normal cells. These conclusions were well consistent with our Wndings both in vivo and in vitro. Our present study has three characteristics compared with other previous researches. First, our study demonstrated the utility of TRAMP-C1 s.c. tumor model system for studying the anti-prostate cancer eVect of the dominant negative mutant mS-T34A. These TRAMP-C1 tumors are Wrm because a dense extracellular matrix formed by the tumor cells, and they exhibit a relatively slow doubling time. Both the traits resemble human prostate cancer and make these tumors more amenable to gene therapy strategies. Our study has, to our knowledge, Wrst demonstrated that Lip-mS could eVectively inhibit the development of prostate cancer. Secondly, our drugs were administered by tail vein injection. The approach to administration we used in present study undoubtedly has a better eVect on controlling systemic symptoms compared with intratumoral administration. Thirdly, we chose non-viral gene transfection as the delivery system, which provided a safer alternative to viral system in gene therapy. It is known that non-viral vectors have advantages in terms of simplicity of use, ease of large-scale production. Despite gene therapy has been used in the treatment of cancer for a long time, there are still a series of limitations led to the suboptimal eYcacy of existing gene therapies. Above all, low gene transfer eYciency by therapeutic vectors was the biggest disadvantage. In order to overcome this problem, we used the cationic liposome delivery system in this study, which represented the most common tool in gene therapy

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experiments (Hirko et al. 2003). It is reported that the cationic liposome has been used as carriers for delivery of a variety of drugs, including antibiotic, antifungal and cytotoxic agents (Allen and Cullis 2004; Batist et al. 2001). Furthermore, among various formulations of cationic liposome, it has been reported that DOTAP:Chol can achieve higher levels of expression in all kinds of organs via tail vein injection in mice, compared with other formulations such as DDAB:Chol, DDAB:DOPE. Besides, DOTAP:Chol can provide more eVective protection and delivery of DNA in circulation compared with other formulations, partly because of its more cohesive bilayer and its ability to eVectively internalize DNA (Templeton et al. 1997). As we know, survivin has emerged as a hot target for cancer therapy. So there are variable of strategies already articulated for nullifying survivin in tumors, including siRNA, peptide vaccine, dominant negative mutants of survivin. All of these methodologies have their own merits and limitations. The siRNA-survivin can reduce survivin expression at mRNA level (Hou et al. 2006). The antisense oligonucleotides can eVectively silence survivin expression (Rödel et al. 2008). Both of them can lead to tumor cells apoptosis. The survivin peptide vaccine can inhibit tumor growth by triggering eVective T cell-mediated immune response, but it cannot induce the apoptosis of tumor cells directly (Pisarev et al. 2003). The plasmid encoding dominant negative mutant compound with liposome can induce apoptosis of cancer cells by blocking interactions of wildtype survivin with critical partner proteins. Besides, it may evoke a cellular immune response (Decker et al. 2006). Furthermore, plasmid vector has advantages in terms of simplicity of use, ease of large-scale production. Therefore, we compounded mS-T34A with DOTAP-chol liposome to form modiWed Lip-mS nanoparticles and treated mice via tail vein injection of our drugs and ultimately received an exciting eVect. However, the majority of plasmid transfections are still less eYcient than viral vectors. Thus, innovation in applying the principles of physics, chemistry and biology to the development of a safe and eVective method for gene delivery is the key to make the needed breakthroughs in non-viral gene therapy. Taken together, our study demonstrates that the mouse survivin T34A plasmid complexed with cationic liposome can eYciently inhibit the growth of murine prostate cancer in vitro and in vivo. The mechanism probably involves two aspects: inducing the apoptosis of tumor cells and inhibiting tumor-associated angiogenesis. Despite that various survivin-targeting techniques have been reported, as a supplementary investigation, our successful targeting strategy using novel survivin dominant negative mutant mediated by DOTAP-chol liposome added an alternative approach to a growing list of survivin gene therapeutic strategies.

27 Acknowledgments The authors thank Han-shuo Yang, Xian-cheng Chen, zhi-xing Cao for technical help, and Dr. Chun-ting Wang for helpful discussions. This work is in part supported by the National 863 Project of China (2007AA021201), and partly by the Postdoctoral Science Fund of China (20070410390). ConXict of interest statement There is no duality of interest that could be perceived to bias our work, acknowledging all Wnancial support and any other personal connections.

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