Gene therapy for colon cancer by adeno-associated viral vector-mediated transfer of survivin Cys84Ala mutant
2005; Elsevier BV; Volume: 128; Issue: 2 Linguagem: Inglês
10.1053/j.gastro.2004.11.058
ISSN1528-0012
AutoresShui Ping Tu, Jian Cui, Peter Liston, Xiao Hua Jiang, Ruian Xu, Marie C.M. Lin, Yan Zhu, Bing Zou, Samuel S. Ng, Shi Hu Jiang, Harry Hua‐Xiang Xia, Wai Man Wong, Annie O.O. Chan, Man‐Fung Yuen, Shiu Kum Lam, Hsiang Fu Kung, Benjamin C.Y. Wong,
Tópico(s)RNA Interference and Gene Delivery
ResumoBackground & Aims: Reactivation of survivin expression is involved in carcinogenesis and angiogenesis in colon cancer. Previous in vitro studies showed that mutation of the cysteine residue at position 84 (Cys84Ala) of survivin generates a dominant-negative mutant that triggers mitotic catastrophe and apoptosis. We investigated the therapeutic effect of the adeno-associated virus (AAV)-mediated survivin mutant (Cys84Ala) on colon cancer. Methods: Survivin mutant (Cys84Ala) (Sur-Mut(Cys84Ala)) was cloned into the AAV expression vector pAM/CAG-WPRE.poly(A) to generate recombinant AAV-Sur-Mut(Cys84Ala) virus. Cell proliferation, apoptosis, mitotic catastrophe, and tumor growth were measured in vitro and in vivo. Results: Transduction of colon cancer cells with rAAV-Sur-Mut(Cys84Ala) inhibited cell proliferation and induced apoptosis and mitotic catastrophe in vitro. rAAV-Sur-Mut(Cys84Ala) sensitized colon cancer cells to chemotherapeutic drugs. Furthermore, expression of survivin mutant mediated by AAV inhibited tumorigenesis in colon cancer cells. Intratumoral injection of rAAV-Sur-Mut(Cys84Ala) significantly induced apoptosis and mitotic catastrophe and inhibited angiogenesis and tumor growth in a colon cancer xenograft model in vivo. No obvious cytotoxicity to other tissues was observed. More importantly, rAAV-Sur-Mut(Cys84Ala) expression strongly enhanced the antitumor activity of 5-Fluorouracil (5-FU), resulting in regression of established tumors. Conclusions: Our results showed that rAAV-Sur-Mut(Cys84Ala) induced apoptosis and mitotic catastrophe and inhibited tumor angiogenesis and tumor growth. Thus, use of AAV-mediated survivin mutant (Cys84Ala) is a promising strategy in colon cancer gene therapy. Background & Aims: Reactivation of survivin expression is involved in carcinogenesis and angiogenesis in colon cancer. Previous in vitro studies showed that mutation of the cysteine residue at position 84 (Cys84Ala) of survivin generates a dominant-negative mutant that triggers mitotic catastrophe and apoptosis. We investigated the therapeutic effect of the adeno-associated virus (AAV)-mediated survivin mutant (Cys84Ala) on colon cancer. Methods: Survivin mutant (Cys84Ala) (Sur-Mut(Cys84Ala)) was cloned into the AAV expression vector pAM/CAG-WPRE.poly(A) to generate recombinant AAV-Sur-Mut(Cys84Ala) virus. Cell proliferation, apoptosis, mitotic catastrophe, and tumor growth were measured in vitro and in vivo. Results: Transduction of colon cancer cells with rAAV-Sur-Mut(Cys84Ala) inhibited cell proliferation and induced apoptosis and mitotic catastrophe in vitro. rAAV-Sur-Mut(Cys84Ala) sensitized colon cancer cells to chemotherapeutic drugs. Furthermore, expression of survivin mutant mediated by AAV inhibited tumorigenesis in colon cancer cells. Intratumoral injection of rAAV-Sur-Mut(Cys84Ala) significantly induced apoptosis and mitotic catastrophe and inhibited angiogenesis and tumor growth in a colon cancer xenograft model in vivo. No obvious cytotoxicity to other tissues was observed. More importantly, rAAV-Sur-Mut(Cys84Ala) expression strongly enhanced the antitumor activity of 5-Fluorouracil (5-FU), resulting in regression of established tumors. Conclusions: Our results showed that rAAV-Sur-Mut(Cys84Ala) induced apoptosis and mitotic catastrophe and inhibited tumor angiogenesis and tumor growth. Thus, use of AAV-mediated survivin mutant (Cys84Ala) is a promising strategy in colon cancer gene therapy. Survivin is a member of the inhibitor of apoptosis (IAP) gene family. It is expressed in the G2-M phase, and its interaction with the mitotic spindle apparatus is essential for its antiapoptotic function.1Ambrosini G. Adida C. Altieri D.C. 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Adeno-associated virus for cancer gene therapy.Cancer Res. 2001; 61: 6313-6321PubMed Google Scholar, 27Davidoff A.M. Nathwani A.C. Spurbeck W.W. Ng C.Y. Zhou J. Vanin E.F. rAAV-mediated long-term liver-generated expression of an angiogenesis inhibitor can restrict renal tumor growth in mice.Cancer Res. 2002; 62: 3077-3083PubMed Google Scholar AAV vectors penetrate human solid tumor tissue in vivo more effectively than adenoviral vectors.28Enger P.Q. Thorsen F. Lønning P.E. Bjerkvig R. Hoover F. Adeno-associated viral vectors penetrate human solid tumor tissue in vivo more effectively than adenoviral vectors.Hum Gene Ther. 2002; 13: 1115-1125Crossref PubMed Scopus (55) Google Scholar As a result, AAV vectors may meditate gene transfer to malignant tumors with better safety, efficiency, and consistency than other available gene delivery system. Survivin is overexpressed in 53%–64% of colorectal cancers,29Kawasaki H. Altieri D.C. Lu C.D. Toyoda M. Tenjo T. Tanigawa N. 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In this study, we generated rAAV-survivin mutant (Cys84Ala) and evaluated the effect of the AAV-mediated survivin mutant (Cys84Ala) in the treatment of experimental colon cancer. Human colon cancer cell lines SW1116, Colo 205, HT-29, and CRL-238 and human embryonic kidney cells (HEK293) (American Type Culture Collection, Rockville, MD) were maintained in RPMI-1640 containing 10% fetal bovine serum (FBS), 100 U/mL penicillin, and 100 μg/mL streptomycin (Gibco BRL, Life Technologies, NY). Cisplatin (2 μg/mL) and 5-fluorouracil (10 μg/mL) (Pharmacia & Upjohn Limited Corp, Australia) were dissolved in DMSO. The 293 cell line was used for the construction and amplification of AAV vectors. We used reverse-transcriptase polymerase chain reaction (RT-PCR) and an overlap extension PCR to construct pcDNA3-survivin and pcDNA3-dominant-negative mutant (Cya84Ala) plasmids as previously described.20Tu S.P. Jiang X.H. Lin M.C. Cui J.T. Yang Y. Lum C.T. Zou B. Zhu Y.B. Jiang S.H. Wong W.M. Chan A.O. Yuen M.F. Lam S.K. Kung H.F. Wong B.C. Suppression of survivin expression inhibits in vivo tumorigenicity and angiogenesis in gastric cancer.Cancer Res. 2003; 63: 7724-7732PubMed Google Scholar All constructs were confirmed by sequencing. We constructed 2 recombinant AAV type 2 plasmids34Xu R. Sun X.Y. Tse L.Y. Li Hu Chan P.C. Xu S. Xiao W.D. Kung H.F. Krissansen G.W. Fan S.T. Long-term expression of angiostatin suppresses metastatic liver cancer in mice.Hepatology. 2003; 37: 1451-1460Crossref PubMed Scopus (56) Google Scholar encoding mutant survivin (Cys84Ala) and EGFP, respectively. Briefly, full-length Sur(wt) and Sur-Mut(Cys84Ala) cDNAs were cut with BamHI and XhoI from pcDNA3-Survivin and pcDNA3-Sur-Mut(Cys84Ala), respectively, and subcloned into the corresponding BamHI and XhoI sites of pAM/CAG-WPRE-BGH-polyA to generate pAM/CAG-Sur-Mut (Cys84Ala). The 785-bp EGFP cassette was removed from pAV/NSE-EGFP-WPRE.poly(A) with XhoI and EcoRI and subcloned into the XhoI and EcoRI sites of pAM/CAG to generate pAM/CAG-EGFP. Recombinant AAV virus stocks were generated and purified by HiTrap Heparin column chromatography (Sigma Chemical Co., St. Louis, MO).34Xu R. Sun X.Y. Tse L.Y. Li Hu Chan P.C. Xu S. Xiao W.D. Kung H.F. Krissansen G.W. Fan S.T. Long-term expression of angiostatin suppresses metastatic liver cancer in mice.Hepatology. 2003; 37: 1451-1460Crossref PubMed Scopus (56) Google Scholar The AAV viral genome titer was quantified by real-time PCR using TaqMan (Perkin-Elmer Biosystems, Foster City, CA).34Xu R. Sun X.Y. Tse L.Y. Li Hu Chan P.C. Xu S. Xiao W.D. Kung H.F. Krissansen G.W. Fan S.T. Long-term expression of angiostatin suppresses metastatic liver cancer in mice.Hepatology. 2003; 37: 1451-1460Crossref PubMed Scopus (56) Google Scholar The viral vector was stored at −80°C before experiments. Cells were pretreated with 500 μmol/L tyrphostin 135Dumon K.R. Ishii H. Vecchione A. Trapasso F. Baldassarre G. Chakrani F. Druck T. Rosato E.F. Williams N.N. Baffa R. During M.J. Huebner K. Croce C.M. Fragile histidine triad expression delays tumor development and induces apoptosis in human pancreatic cancer.Cancer Res. 2001; 61: 4827-4836PubMed Google Scholar (Sigma Chemical Co.) for 2 hours before infected with 1 × 104 to 2 × 105 viral particles/cell of various viruses. Cells were processed as described20Tu S.P. Jiang X.H. Lin M.C. Cui J.T. Yang Y. Lum C.T. Zou B. Zhu Y.B. Jiang S.H. Wong W.M. Chan A.O. Yuen M.F. Lam S.K. Kung H.F. Wong B.C. Suppression of survivin expression inhibits in vivo tumorigenicity and angiogenesis in gastric cancer.Cancer Res. 2003; 63: 7724-7732PubMed Google Scholar and analyzed by flow cytometry (Coulter, Luton, United Kingdom) and fluorescence microscopy to determine the transduction efficiency, apoptosis, and cell cycle. The cell-cycle phase distribution was calculated from the resultant DNA histogram using Multicycle AV software (Phoenix Flow Systems, San Diego, CA). Cells with subdiploid DNA content were scored as apoptotic. Cell proliferation was measured by MTT assay.20Tu S.P. Jiang X.H. Lin M.C. Cui J.T. Yang Y. Lum C.T. Zou B. Zhu Y.B. Jiang S.H. Wong W.M. Chan A.O. Yuen M.F. Lam S.K. Kung H.F. Wong B.C. Suppression of survivin expression inhibits in vivo tumorigenicity and angiogenesis in gastric cancer.Cancer Res. 2003; 63: 7724-7732PubMed Google Scholar Caspase-3 activity was determined using the ApoAlert Caspase Colorimetric Assay Kit according to the manufacturer's instructions (Clontech, Palo Alto, CA). Cell extracts were prepared as previously described.36Wong B.C. Jiang X.H. Lin M.C. Tu S.P. Cui J.T. Jiang S.H. Wong W.M. Yuen M.F. Lam S.K. Kung H.F. Cyclooxygenase-2 inhibitor (SC-236) suppresses activator protein-1 through c-Jun NH2-terminal kinase.Gastroenterology. 2004; 126: 136-147Abstract Full Text Full Text PDF PubMed Scopus (49) Google Scholar Protein extracts were electrophoresed on 10% denaturing sodium dodecylsulfate gels and transferred to Immobilon-P membranes (Millipore, Bedford, MA). The blots were incubated with specific primary antibodies, followed by a peroxidase-conjugated second antibody (Santa Cruz Biochemical Co., Santa Cruz, CA) and visualized by enhanced chemiluminescence (ECL; Amersham, Piscataway, NJ). Survivin (2 μg/mL) polyclonal antibody was purchased from Alpha Diagnostic International Inc. (San Antonio, TX), and poly-(ADP-ribose)-polymerase (PARP), β-actin, caspase-3, and cytochrome c monoclonal antibodies were all purchased from Santa Cruz Biochemical Co. MPM-2 was purchased from Cell Signaling Co. (Beverly, MA). Cells were processed and analyzed using a Zeiss Axioscop fluorescence microscope as described previously.20Tu S.P. Jiang X.H. Lin M.C. Cui J.T. Yang Y. Lum C.T. Zou B. Zhu Y.B. Jiang S.H. Wong W.M. Chan A.O. Yuen M.F. Lam S.K. Kung H.F. Wong B.C. Suppression of survivin expression inhibits in vivo tumorigenicity and angiogenesis in gastric cancer.Cancer Res. 2003; 63: 7724-7732PubMed Google Scholar For AAV-mediated EGFP expression in colon cancer xenograft, mice were killed 4 days and 42 days after being injected with AAV-EGFP virus. Tumor tissues were mounted on positively charged slides (Fisher Scientific) and observed under a Zeiss Axioscop fluorescence microscope. Survivin expression in tumor tissues was detected using a DAKO LSAB+ Kit (DAKO, Carpenteria, CA) according to the manufacturer's instructions. Briefly, slides were boiled in 10 mmol/L citrate buffer, pH 6 (Bio Genex, San Ranmon, CA), for antigen retrieval and incubated with antisurvivin polyclonal antibody (1:100, Alpha Diagnostic International Inc.), followed by biotinylated anti-IgG antibody and streptAB-complex/HRP (DAKO). Sections were counterstained with hematoxylin and were independently evaluated by 2 blinded investigators. Survivin staining was recorded as the ratio of positively stained cells to all tumor cells in 5 random areas at 200-fold magnification. Apoptosis in xenograft tumors was determined by TUNEL staining using ApoAlert DNA Fragmentation Assay Kit (Cat No. K2024-1, Clontech Corp.).20Tu S.P. Jiang X.H. Lin M.C. Cui J.T. Yang Y. Lum C.T. Zou B. Zhu Y.B. Jiang S.H. Wong W.M. Chan A.O. Yuen M.F. Lam S.K. Kung H.F. Wong B.C. Suppression of survivin expression inhibits in vivo tumorigenicity and angiogenesis in gastric cancer.Cancer Res. 2003; 63: 7724-7732PubMed Google Scholar The percentage of apoptotic cells was assessed in 10 randomly selected fields viewed at 400× magnification. The apoptotic index (A/I) was calculated as number of apoptotic cells/total number of nucleated cells × 100%. Slides were incubated with rat monoclonal antimouse CD31 antibody (Pharmingen, San Diego, CA), followed by a Texas red fluorescently labeled anti-rat secondary antibody (1:200; Jackson ImmunoResearch Laboratory, Inc., West Grove, PA). Next, sections were stained with TUNEL (ApoAlert DNA Fragmentation Assay Kit, Clontech).20Tu S.P. Jiang X.H. Lin M.C. Cui J.T. Yang Y. Lum C.T. Zou B. Zhu Y.B. Jiang S.H. Wong W.M. Chan A.O. Yuen M.F. Lam S.K. Kung H.F. Wong B.C. Suppression of survivin expression inhibits in vivo tumorigenicity and angiogenesis in gastric cancer.Cancer Res. 2003; 63: 7724-7732PubMed Google Scholar The slides were examined under an immunofluorescent microscopy (Zeiss Plan-Neofluor; Carl Zeiss, Thornwood, NY). For quantification of endothelial cells, CD31-positive endothelial cells were identified by red fluorescence, and apoptotic cells exhibited nuclear green fluorescence. Endothelial cells undergoing apoptosis fluoresced yellow because of the overlapping green and red emissions. Microvessel density (MVD) was evaluated according to the previously described method.20Tu S.P. Jiang X.H. Lin M.C. Cui J.T. Yang Y. Lum C.T. Zou B. Zhu Y.B. Jiang S.H. Wong W.M. Chan A.O. Yuen M.F. Lam S.K. Kung H.F. Wong B.C. Suppression of survivin expression inhibits in vivo tumorigenicity and angiogenesis in gastric cancer.Cancer Res. 2003; 63: 7724-7732PubMed Google Scholar, 37Giatromanolaki A. Koukourakis M.I. Theodossiou D. Barbatis K. O'Byrne K. Harris A.L. Gatter K.C. Comparative evaluation of angiogenesis assessment with anti-factor-VIII and anti-CD31 immunostaining in non-small cell lung cancer.Clin Cancer Res. 1997; 3: 2485-2492PubMed Google Scholar, 38Baker C.H. Kedar D. McCarty M.F. Tsan R. Weber K.L. Bucana C.D. Fidler I.J. Blockade of epidermal growth factor receptor signaling on tumor cells and tumor-associated endothelial cells for therapy of human carcinomas.Am J Pathol. 2002; 161: 929-938Abstract Full Text Full Text PDF PubMed Scopus (136) Google Scholar Percentage of apoptotic endothelial cells was expressed as an average of the ratio of apoptotic endothelial cells to the total number of endothelial cells in 5 random 0.011 mm2 fields at 400× magnification. Female BALB/c nude mice, 5–6 weeks old, were bred in the Animal Laboratory Unit, The University of Hong Kong, Hong Kong. Colon cancer cells were transduced in vitro at 5 × 104 viral particles/cell with rAAV-Sur-Mut(Cys84Ala), rAAV-Sur(wt), and rAAV-EGFP or mock infected with PBS. Twelve hours after transduction, 2 × 106 SW1116 and Colo 205 viable cells were injected into the right and left flank of 5- to 6-week-old female nude mice, respectively, with 4 mice per group. All experiments were repeated at least twice. Tumor size was determined by measuring 2 perpendicular diameters with a caliper every 3 days.20Tu S.P. Jiang X.H. Lin M.C. Cui J.T. Yang Y. Lum C.T. Zou B. Zhu Y.B. Jiang S.H. Wong W.M. Chan A.O. Yuen M.F. Lam S.K. Kung H.F. Wong B.C. Suppression of survivin expression inhibits in vivo tumorigenicity and angiogenesis in gastric cancer.Cancer Res. 2003; 63: 7724-7732PubMed Google Scholar The protocol was approved by the Committee on the Use of Live Animals in Teaching and Research, University of Hong Kong, Hong Kong. Five- to 6-week-old female BALB/c nude mice were injected subcutaneously on the flanks with 2 × 106 exponentially growing SW1116 and Colo 205 cells. Tumors were allowed to grow to 100–150 mm3 (5–7 mm diameter). For local administration of rAAV, injection was given to 3 tumor sites with rAAV-Sur-Mut(Cys84Ala), rAAV-Sur(wt), or rAAV-EGFP at 5 × 1010 viral particles/site of injection or with PBS. Alternatively, mice were intraperitoneally (IP) injected with 50 mg/kg 5-fluorouracil (5-FU) daily for 5 days or a combination of rAAV virus and 5-FU. Tumor growth was measured weekly after injection. In other experiments, mice received an injection of uninfected exponentially growing 2 × 106 SW1116 cells. After 7 days, mice were given an injection of same dosage of rAAV virus and 5-FU (3 animals/group). After 4 days, animals were killed, and their total tumor burden was excised, fixed, and embedded. Data are reported as means ± SE. Statistically significant differences (P < .05) between groups were detected using unpaired t test, paired t test, and nonparametric tests, such as the Wilcoxon signed-rank test and the Mann-Whitney rank sum test, appropriate for the data. For categorical data, the χ2 test was used. To determine the efficiency of viral transduction using the rAAV vectors, we performed quantitative analysis in different human colon cancer cell lines (SW1116, Colo 205, HT-29). Cells were plated in triplicate at a density of 5 × 104 cells/well in 24-well culture plates and infected with rAAV-GFP (1 × 105 viral particles/cell). Seventy percent to 80% of HEK-293 cells were transduced by rAAV-EGFP as visualized in vitro by fluorescence microscopy and by the fraction of GFP-expressing cells measured by FACS analysis. The colon cancer cell lines were also efficiently transduced at rates ranging between 23% and 28%. Using a combination of rAAV and tyrphostin 1, the transduction rate was significantly enhanced to 48%–57% (Figure 1A and 1B). We first evaluated the in vitro effect of rAAV-Sur-Mut(Cys84Ala) on cell proliferation. MTT assays demonstrated that infection with rAAV-Sur-Mut(Cys84Ala) caused a dosage-dependent increase in cell death, compared with rAAV-Sur(wt), rAAV-EGFP, and mock-infected cells (Figure 1C). To exclude the possibility that preincubation with tyrphostin 1 affected cell survival, colon cells were infected with rAAV-EGFP or rAAV-Sur-Mut(Cys84Ala) with and without tyrphostin 1. Preincubation with tyrphostin 1, which led to accelerated transcription of the transgene, increased the percentage of dead cells after rAAV-Sur-Mut(Cys84Ala) transduction but did not affect mock, rAAV-Sur(wt), and rAAV-EGFP transduced cells (data not shown). To characterize further the proapoptotic effect of rAAV-Sur-Mut(Cys84Ala), we analyzed apoptosis induced by transduction with rAAV-Sur-Mut(Cys84Ala), rAAV-Sur(wt), and rAAV-EGFP or PBS (mock infection). Transduction of rAAV-Sur-Mut(Cys84Ala) induced apoptosis i
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