Therapeutic effects of survivin dominant negative mutant in a mouse model of prostate cancer

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 effective therapeutics for prostate cancer. Mouse survivin T34A (mS-T34A) is a phosphorylation-defective 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 flow 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 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 effect on angiogenesis.

Results: Administration of Lip-mS resulted in significant 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.

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 Lip-mS transfected 293 cells (lane 1 5 μg, lane 2 2 μg). While no band was detected in null (lane 3) transfected or untreated (lane 4) 293 cells

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-mS-transfected cells but not the Lip-null-transfected and NS-treated cells. b Cellular apoptosis was further verified by flow cytometric analysis. The percentage of apoptotic cells with hypodiploid DNA content was 2% (NS), 9.3% (Lip-null) and 42% (Lip-mS), respectively

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 (filled triangle), Lip-null (opened diamond) and Lip-mS (filled square) every 2 days for 8 times. There was a significant difference 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

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 significant 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 quantified. The uptake of FITC-dextran from beads of Lip-mS-treated mice showed a significant decrease compared with control groups (P < 0.05)

Fig. 6

a H&E staining of tumor tissue. Increased necrotic and apoptotic tumor cells were observed in tumor from Lip-mS-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 fluorescent staining were defined as TUNEL-positive nuclei. c Percent of apoptosis in the Lip-mS groups markedly increased in comparison with controls (P < 0.05)