The antiapoptotic gene survivin is highly expressed in human chondrosarcoma and promotes drug resistance in chondrosarcoma cells in vitro

Abstract

Background: Chondrosarcoma is virtually resistant to chemotherapy and radiation therapy. Survivin, the smallest member of the inhibitor of apoptosis protein family, is a critical factor for tumor progression and resistance to conventional therapeutic approaches in a wide range of malignancies. However, the role of survivin in chondrosarcoma has not been well studied. We examined the importance of survivin gene expression in chondrosarcoma and analysed its influences on proliferation, apoptosis and resistance to chemotherapy in vitro.

Methods: Resected chondrosarcoma specimens from which paraffin-embedded tissues could be extracted were available from 12 patients. In vitro experiments were performed in human chondrosarcoma cell lines SW1353 and Hs819.T. Immunohistochemistry, immunoblot, quantitative PCR, RNA interference, gene-overexpression and analyses of cell proliferation and apoptosis were performed.

Results: Expression of survivin protein was detected in all chondrosarcoma specimens analyzed, while undetectable in adult human cartilage. RNA interference targeting survivin resulted in a G2/M-arrest of the cell cycle and led to increased rates of apoptosis in chondrosarcoma cells in vitro. Overexpression of survivin resulted in pronounced resistance to doxorubicin treatment.

Conclusions: These findings indicate that survivin plays a role in the pathogenesis and pronounced chemoresistance of high grade chondrosarcoma. Survivin antagonizing therapeutic strategies may lead to new treatment options in unresectable and metastasized chondrosarcoma.

Figure 1

Survivin expression in human chondrosarcoma. Immunohistochemistry and immunoblot for survivin (red staining) from human chondrosarcoma specimens. A: Low-power image of human high-grade chondrosarcoma displays strong cellular expression of survivin protein. B: High-power magnification reveals the predominantly cytoplasmic staining, although strong nuclear signals are detectable. C and D: Other specimen of a grade III chondrosarcoma stained with monoclonal antibody, shows a similar pattern of staining. E: Strong survivin signal in a tumor cell displaying a mitotic figure (arrow). F: To verify the expression of survivin in human chondrosarcoma, immunoblots were performed from 3 high grade chondrosarcoma lysates (Patient Nr. 5, 7, 10). As control for the correct molecular weight, in vitro-transcribed and -translated (IVTT) recombinant survivin protein, derived from the full-length human cDNA was loaded. Furthermore, lysates from adult human cartilage served as a negative control. Total protein loaded was 1 μg for recombinant human survivin, 60 μg for chondrosarcoma and cartilage lysates. For A, B and E the polyclonal rabbit anti-survivin antibody AF886 was used. For C and D the monoclonal mouse anti-survivin antibody clone 32.1 was used. Original magnifications: 200× (A and C) and 400× (B and D) and 600× (E).

Figure 2

Survivin subcellular localization in human chondrosarcoma cells in vitro. Immunofluorescence localization of survivin using chondrosarcoma cells (SW1353) cultured on glass slides (A,D,G) and 4,6-diamidino-2-phenylindole-staining (DAPI) of the identical positions (B,E,H). Overlay of both stainings (C,F,I). A-C: The top row clearly shows the heterogeneous subcellular distribution from predominant cytoplasmic (lower cell) in the majority of the cell population to mixed cytoplasmic-nuclear in a smaller fraction of cells (upper cell). D-F: In a premitotic cell, survivin localizes to the mitotic spindle apparatus (arrow). Of note, here survivin signal appears stronger compared to the surrounding non-mitotic cells. G-I: In late telophase the mid-body (arrow) stains positive for survivin protein. Original magnifications: 400× (A-I)

Figure 3

Suppression of survivin expression by transfection of siRNA. RNA interference was performed in SW1353 and Hs 819.T, either for GFP as control or for survivin. A: A pronounced decrease of survivin protein levels was measured by immunoblotting in SW1353 and Hs819.T. B: Quantitative real time PCR confirmed the subtotal suppression of survivin expression in SW1353 (left) and Hs819.T (right).

Figure 4

Influence of survivin knockdown on proliferation and cell viability distribution of chondrosarcoma cells. The influences of RNA interference with survivin gene expression on cell viability and proliferation were measured by employing the MTT - assay (A) and by measuring BrdU incorporation (B). A: Cell viability was analyzed by MTT assay. Knockdown of survivin was performed at 0 hours and repeated at 48 hours in SW1353 (left) and Hs819.T (right). Significant reduction of viable cells compared to the untransfected control were seen in SW1353 after 48 hours and in Hs819.T at 72 hours.. Knockdown of GFP resulted in no significant alterations (Data not shown). The error bars represent +/-SEM. B: Proliferation of chondrosarcoma cell lines SW1353 and Hs819.T was measured by BrdU incorporation and subsequent detection employing a ELISA chemiluminescence immunoassay. Knock down was performed 24 hours prior to the incubation with BrdU. Knockdown of GFP resulted in no significant alterations. The error bars represent +/-SEM. A: Original results of one representative experiment are shown. B: Original results of three representative experiments are shown. P values less than 0.05 were considered significant (*).

Figure 5

Effects of survivin knock down on cell cycle distribution in chondrosarcoma cells. SW1353 were transfected with siRNA targeting survivin and cell cycle distribution was determined by PI staining and FACS analysis after 24 hours. Both attached and detached cells were collected for the FACS analysis. GFP was transfected as control. Original dot blots and measured gates (left) and resulting histograms (right) are shown. The second peak of the resulting histogram represents the G2/M-phase fraction. The original results of one representative experiment are shown.

Figure 6

Influence of survivin knockdown on apoptotic rate of chondrosarcoma cells. Influences of RNA interference against survivin on programmed cell death of SW1353 (A and B) and Hs819.T (C and D) were measured by caspase 3/7 activity (A and C) and by analysing the sub-G_0/1-phase fraction by using the fluorescence-activated cell sorting-propidium iodide staining method (B and D). Survivin knockdown resulted in moderate elevations of the indicators of apoptotic activity in SW 1353 (A and B, left) but not in Hs819.T (C and D). Transfection of GFP had no significant effects on apoptosis. Pronounced elevations of apoptotic markers were seen when the cells were stressed with doxorubicin 5 μM over 24 hours (A - D, right). The cytotoxic treatment resulted in a substantial increase of caspase 3/7 activity and fraction of apoptotic cells. Suppression of survivin sensitized the cells to doxorubicin treatment and further increased the apoptotic activity significantly in both cell lines. Again, transfection of GFP siRNA was used as a control. The error bars represent +/-SEM. P values less than 0.05 were considered significant (*). The original results of one representative experiment are shown.

Figure 7

Influence of survivin overexpression on proliferation and apoptosis of chondrosarcoma cells in vitro. A: Overexpression of human full length survivin by transfection of plasmid DNA led to a significant increase of protein level, as measured by immunoblot. Empty vector pcDNA3 was transfected as control. B: MTT - analysis over 5 days after transfection showed no influences on the proliferative activity of SW1353. C and D: Overexpression of survivin resulted in no alterations of spontaneous apoptotic rate as measured by caspase 3/7 activity (C) and by analysing the sub-G_0/1-phase fraction by using the fluorescence-activated cell sorting-propidium iodide staining method (D). When SW1353 cells were exposed for 24 hours to doxorubicin (5 μM) (right) the apoptotic fraction and caspase activity of not transfected and pcDNA3 transfected cells increased markedly. Transfection of survivin resulted in significantly reduced rates of apoptosis after cytotoxic treatment. The error bars represent +/-SEM. P values less than 0.05 were considered significant (*). The original results of one representative experiment are shown.