Figure 1.
Mitostatin expression in prostate cancer cell lines.
A: Western blot analysis: Mitostatin is differentially expressed in human prostate cancer cell lines. Protein loading was confirmed by reprobing the membrane with antibody to β-actin. B: Western blots showing the expression of Mitostatin in PC3, DU145 and LNCaP cell lines utilized in this study.
Figure 2.
Mitostatin expression affects colony formation of prostate cancer cells.
Mitostatin over-expression in prostate cancer derived cell lines [PC3, A; DU145, B; and LNCaP, C] decreased the ability to form colonies after 15 days, while cells with depletion of endogenous Mitostatin protein showed a similar (DU145 M2) or higher (PC3 M2) number of colonies compared to parental cells. Columns, average per dish from triplicate experiments; bars, SEM. * P<0.05; ** P<0.01.
Figure 3.
Mitostatin inhibits in vitro migration of prostate cancer cells.
A and B: Mitostatin over-expressing clones showed reduced migratory properties compared to parental cells. Migration was assessed through migration (A) and the wound healing assays (B). Cells with down-regulation of the endogenous Mitostatin protein (PC3 M2, PC3 Mitostatin shRNA, and DU145 M2) showed an increased malignant behavior. Data are expressed as percentage of migration versus parental cells. Columns, representative images of triplicates from three independent experiments; bars, SEM. * P<0.05; ** P<0.01.
Figure 4.
Mitostatin inhibits in vitro invasion and adhesive properties to laminin of prostate cancer cells.
A: Mitostatin inhibits invasive properties of prostate cancer cells. Cells with down-regulation of the endogenous Mitostatin protein (PC3 M2, PC3 Mitostatin shRNA, and DU145 M2) showed an increased malignant behavior. B: Mitostatin over-expression affects cell ability to attach to laminin. Data are expressed as percentage of migration versus parental cells. Columns, representative images of triplicates from three independent experiments; bars, SEM. * P<0.05; ** P<0.01.
Figure 5.
Mitostatin affects anchorage-independent growth of prostate cancer cells.
A: Data from triplicate cultures of parental PC3 and LNCaP, and clones PC3 V5, PC3 B2, PC3 M2, LNCaP V5, LNCaP B1A, LNCaP B3A, and LNCaP A3A plated in soft-agar generating colonies larger than 0.2 mm in diameter. Clones over-expressing Mitostatin showed a decreased ability to form colonies, while the Mitostatin down-regulated clone, PC3 M2, showed a higher number of colonies compared to parental cells. Colonies were counted after 21 days. * P<0.05; ** P<0.01 B: Representative colonies of LNCaP parental cells (left), LNCaP B1A (middle), and LNCaP A3A (right) are shown after 21 days (magnification ×200).
Figure 6.
Effects of Mitostatin over-expression on the LNCaP cell tumorigenicity in nude mice.
A: Xenografts established by sub cutaneous injection of LNCaP, LNCaP V5, LNCaP B1A and LNCaP B3A cells in athymic (BALB/c nu/nu) were grown for 65 days. B: Tumor volume in animals injected with cells over-expressing Mitostatin was markedly decreased compared to tumors in animals injected with control cells. The graph illustrates the distribution in tumor sizes formed in BALB/c nu/nu mice through 65 days. *: P<0.05. C, D: Immunohistochemical and immunoblot analysis of Mitostatin expression in LNCaP and derivative tumor xenografts. Immunohistochemistry was performed using both anti-V5 (upper panels) and anti-Mitostatin (lower panels) antibodies. Scale bar: 50 µm.
Figure 7.
Mitostatin is down-regulated in human prostate cancers.
A: Relative fold of Mitostatin mRNA levels in normal prostatic tissue samples (N, black), and Gleason 7 prostate cancer samples (K, bright gray). Mitostatin showed a consistent down-regulation in cancer tissues (P = 0.029). Columns, representative images of triplicates; bars, SD. B–G: Immunohistochemical detection of Mitostatin in human prostate. Mitostatin protein is localized in the cytoplasm of normal (B) and atrophic (C) prostatic glands. Higher levels of Mitostatin protein were observed in PIN lesions (D) and in some tumors (E). F: In addition to a weak staining, Mitostatin was also detected in the nuclei in few cases of adenocarcinoma. G: Mitostatin expression is present in an atrophic gland (black arrow) in contrast with the surrounding negative neoplastic glands. Scale bar: 50 µm.