Efficient inhibition of murine breast cancer growth and metastasis by gene transferred mouse survivin Thr34-->Ala mutant

Abstract

Background: Metastasis in breast cancer is a vital concern in treatment because most women with primary breast cancer have micrometastases to distant sites at diagnosis. As a member of the inhibitor of apoptosis protein (IAP) family, survivin has been proposed as an attractive target for new anticancer interventions. In this study, we investigated the role of the plasmid encoding the phosphorylation-defective mouse survivin threonine 34-->alanine mutant (Msurvivin T34A plasmid) in suppressing both murine primary breast carcinomas and pulmonary metastases.

Methods: In vitro study, induction of apoptosis by Msurvivin T34A plasmid complexed with cationic liposome (DOTAP/Chol) was examined by PI staining fluorescence microscopy and flow cytometric analysis. The anti-tumor and anti-metastases activity of Msurvivin T34A plasmid complexed with cationic liposome (DOTAP/Chol) was evaluated in female BALB/c mice bearing 4T1 s.c. tumors. Mice were treated twice weekly with i.v. administration of Msurvivin T34A plasmid complexed with cationic liposome (DOTAP/Chol), PORF-9 null plasmid complexed with cationic liposome (DOTAP/Chol), 0.9% NaCl solution for 4 weeks. Tumor volume was observed. After sacrificed, tumor net weight was measured and Lung metastatic nodules of each group were counted. Assessment of apoptotic cells by TUNEL assay was conducted in tumor tissue. Microvessel density within tumor tissue was determined by CD31 immunohistochemistry. Alginate-encapsulated tumor cells test was conducted to evaluate the effect on angiogenesis. By experiment of cytotoxicity T lymphocytes, we test whether Msurvivin T34A plasmid complexed with cationic liposome (DOTAP/Chol) can induce specific cell immune response.

Results: Administration of Msurvivin T34A plasmid complexed with cationic liposome (DOTAP/Chol) resulted in significant inhibition in the growth and metastases of 4T1 tumor model. These anti-tumor and anti-metastases responses were associated with triggering the apoptosis of tumor cells directly, inhibiting angiogenesis and inducing specific cellular immune response.

Conclusion: The present findings suggest that the Msurvivin T34A plasmid complexed with cationic liposome may provide an effective approach to inhibit the growth and metastases of a highly metastatic mouse breast cancer model with minimal side effects.

Figure 1

Fluorescence microscopic appearance of propidium iodide (PI)-stained nuclei. 4T1 cells were treated with Msurvivin T34A plasmid(2 μg per well)for 48 hr. (a) untransfected (b)transfected with PORF-9 null plasmid(c)transfected with Msurvivin T34A plasmid, reduction of cell volume, condensation of nuclear, nuclear fragmentation, and apoptotic bodies.

Figure 2

Representative DNA fluorescence histograms of PI-stained cells. 4T1 cells were treated with Msurvivin T34A plasmid(2 μg per well)for 48 hr.(a)untreated,(b)treated with PORF-9 null plasmid,(c)treated with Msurvivin T34A plasmid, with(a) 2%,(b)8.6% and(d) 43% sub-G1 cells(apoptotic cells), respectively, as assessed by flow cytometry.

Figure 3

Anti-tumor effects of Msurvivin T34A plasmid:lipo complexes versus normal saline, PORF-9 null plasmid:lipo complexes. The tumor-bearing mice in each group received the corresponding treatment as detailed in "Materials and Methods". Data were presented as means ± SE. Each group contained 10 mice.(A) Tumor volume was shown up to 38. The differences between Msurvivin T34A plasmid:lipo and control groups were significant (P < 0.05) starting day 27.(B)Tumor weight was measured after each mice from every group was sacrificed. Compared with control groups, the Msurvivin T34A plasmid treated mice showed significant difference. (P < 0.01)

Figure 4

The anti-metastases effect of Msurvivin T34A plasmid:lipo complexes. (a) NS group, (b) PORF-9 null plasmid group, (c) Msurvivin T34A plasmid group. Compared with Msurvivin T34A plasmid group, a large number of lung metastatic nodules and structural destruction of pulmonary alveoli can be observed in NS and PORF-9 null plasmid group.

Figure 5

Inhibition of intratumoral angiogenesis assayed by CD31 staining of microvessel. Vascularization within tumors was detected by an antibody to CD 31 and vascular density was quantified by counting the number of microvessel per high power field (×400). (A) Shown are representative sections from each group. (a)NS group, (b) PORF-9 null plasmid group, (c) Msurvivin T34A plasmid group. The arrows point to the necrosis. (b) CD31 positive microvessel in each group. Values were expressed as means ± SE (5 high power fields/slide).

Figure 6

Terminal deoxynucleotidyltransferase-mediated dUTP nick-end labeling staining of tumor tissues. Sections after treatment were stained by TUNEL analysis to detect apoptotic cells. Percent apoptosis was determined by counting the number of apoptotic cells and dividing by the total number of cells in the field (5 high power fields/slide). (A) Representative Field from each group. (a) NS group, (b) PORF-9 null plasmid group, (c) Msurvivin T34A plasmid group. (B) Percent apoptosis in each group. Values were expressed as means ± SE. Msurvivin T34A plasmid:lipo complexes increased the percent apoptosis in breast tumor sections relative to either normal saline or PORF-9 null plasmid:lipo complexes. (p < 0.05, respectively).

Figure 7

Msurvivin T34A plasmid induced apoptosis of murine endothelial (MS1) cells in vitro. MS1 cells were treated with Msurvivin T34A plasmid(2 μg per well)for 48 hr.(a)untreated,(b)treated with PORF-9 null plasmid,(c)treated with Msurvivin T34A plasmid, with(a) 6.9%,(b) 18.9%and(d) 76.1% sub-G1 cells(apoptotic cells), respectively, as assessed by flow cytometry.

Figure 8

Vascularization of alginate implants. (A) Representative alginate beads from each group.(a)NS group,(b)PORF-9 plasmid null plasmid group,(c)Msurvivin T34A plasmid group.(B) FITC-dextran uptake of beads from Msurvivin T34A plasmid treated mice showed a significant decrease compared with control groups(P < 0.05). The data were expressed as means ± S.E.

Figure 9

Msurvivin T34A plasmid induces a specific cellular immune response. Msurvivin T34A plasmid showed increased cytotoxicity against survivin-positive target cells while the other groups did not induce specific cytotoxicity reaction. (P < 0.05) The data were expressed as means ± S.E. of triplicate samples from one representative experiment.