A cell-permeable dominant-negative survivin protein induces apoptosis and sensitizes prostate cancer cells to TNF-α therapy
© Cheung et al; licensee BioMed Central Ltd. 2010
Received: 5 August 2010
Accepted: 1 October 2010
Published: 1 October 2010
The Erratum to this article has been published in Cancer Cell International 2010 10:43
Survivin is a member of the inhibitor-of-apoptosis (IAP) family which is widely expressed by many different cancers. Overexpression of survivin is associated with drug resistance in cancer cells, and reduced patient survival after chemotherapy and radiotherapy. Agents that antagonize the function of survivin hold promise for treating many forms of cancer. The purpose of this study was to investigate whether a cell-permeable dominant-negative survivin protein would demonstrate bioactivity against prostate and cervical cancer cells grown in three dimensional culture.
A dominant-negative survivin (C84A) protein fused to the cell penetrating peptide poly-arginine (R9) was expressed in E. coli and purified by affinity chromatography. Western blot analysis revealed that dNSurR9-C84A penetrated into 3D-cultured HeLa and DU145 cancer cells, and a cell viability assay revealed it induced cancer cell death. It increased the activities of caspase-9 and caspase-3, and rendered DU145 cells sensitive to TNF-α via by a mechanism involving activation of caspase-8.
The results demonstrate that antagonism of survivin function triggers the apoptosis of prostate and cervical cancer cells grown in 3D culture. It renders cancer cells sensitive to the proapoptotic affects of TNF-α, suggesting that survivin blocks the extrinsic pathway of apoptosis. Combination of the biologically active dNSurR9-C84A protein or other survivin antagonists with TNF-α therapy warrants consideration as an approach to cancer therapy.
Survivin is a member of the inhibitors of apoptosis (IAP) family. Overexpression of survivin renders cancer cells resistant to anti-cancer therapy including chemotherapy and radiation therapy [1–5]. It causes oral cancer cells to be resistant to the anti-mitotic compounds vincristine and colchicine, such that down-regulation of survivin restores their drug sensitivity . Overexpression of survivin inhibited the tamoxifen and cisplatin-induced apoptosis of human breast and gastric cancer cells [3, 5]. It enhanced the repair of DNA double-strand breaks in radiation-treated oral cancer cells by upregulating the molecular sensor of DNA damage, Ku70 . The level of survivin expression was inversely related to the degree of apoptosis, and positively related to the risk of local tumor recurrence in rectal cancer patients treated with radiotherapy . Patients with gastric tumors that express low levels of survivin appear to have a longer mean survival time after cisplatin treatment than patients with high levels of expression . Survivin expression is associated with the metastasis of human prostate cancer to bone . Thus, survivin plays an important role in tumorigenesis and tumor metastasis, and where levels of survivin expression serve as an indicator of therapeutic effectiveness.
At the molecular level, survivin is bifunctional in that it is a suppressor of apoptosis and plays a central role in cell division. A study using surface plasmon resonance spectroscopy and immunoprecipitation analysis showed that a recombinant survivin protein was able to bind directly to both caspase-3 and caspase-7 with nanomolar affinity . Targeting of survivin by siRNA induces the activation of caspase-9 and caspase-3 in various cancer cells [8, 9]. It appears to be mitochondrial survivin rather than cytosolic survivin that inhibits apoptosis through interference with caspases [8, 10, 11]. Survivin also plays a role in inhibiting the caspase-independent apoptosis of cancer cells . Translocation of the apoptosis-inducing factor (AIF) from the cytoplasm to the nucleus is a molecular indicator of the caspase-independent apoptosis of cells. Down-regulation of survivin by siRNA induces the translocation of AIF from the cytoplasm to the nucleus in various cancer cells .
Progress in the development of survivin inhibitors has been slow despite the fact that survivin plays multiple roles in cancer cell survival, and renders cancers insensitive to chemotherapy. In the past ten years only a few small molecule inhibitors of survivin have been developed and only one survivin inhibitor, YM155, has reached clinical trials [13–17]. Therefore, it is of interest to identify novel macromolecular inhibitors of survivin, and to explore their clinical utility. The 3D-structure of survivin has been determined by x-ray crystallography, which together with the gene sequence reveals that the 16.5 kDa survivin protein monomer comprises an N-terminal Zn2+-binding baculovirus IAP repeat (BIR) domain consisting of a three-stranded anti-parallel β-sheet surrounded by four small α helices that is linked to a 65 A° amphipathic C-terminal α-helix [18–20]. Survivin exists as a dimer and has an extensive dimerization interface along a hydrophobic surface on the BIR domain of each survivin monomer. Mutagenesis studies have shown that the BIR domain plays a key role in the anti-apoptotic function of survivin. Thus, point mutations such as C84A in the BIR domain prevent requisite dimerization of survivin, producing a dominant-negative mutant that interferes with the anti-apoptotic function of native survivin [21–23]. A Thr34 residue is located at the amino-terminal end of helix II of the BIR, surrounded by a sequence that matches the consensus phosphorylation site S/T-P-X-R for the mitotic kinase complex, p34cdc2-cyclin B1. Mutation of Thr34 to Ala (T34A) removes the phosphorylation site and prevents survivin from binding to activated caspase-9, creating a dominant-negative survivin molecule that disrupts cell division and induces apoptosis [24–26]. These and other dominant-negative forms of survivin are macromolecular inhibitors that have potential utility in the treatment of cancer.
Here we created a cell-permeable dominant-negative C84A survivin protein and investigated its biological activity against cancer cells grown in 3D culture.
Production of a cell-permeable dominant-negative C84A survivin protein
A preliminary study revealed that dNSurR9-C84A was able to penetrate into human DU145 prostate cancer cells in two dimensional (2D) cultures within 30 min of addition of the protein (Additional file 1) . Here, the ability of dNSurR9-C84A to penetrate into 3D cultured cancer cells grown in semi-solid media containing Matrigel™was examined. DU145 prostate cancer and HeLa epithelial cervical cancer cells grown as 3D cultures (Figure 1D) were incubated with either PBS control or dNSurR9-C84A (20 μg/mL) for 1 to 5 h, and the cytosolic fraction of the cells was subjected to Western blotting. Cytosolic appearance of dNSurR9-C84A in DU145 and HeLa cells was observed as early as 1 to 2 h after addition of dNSurR9-C84A (Figure 1E).
dNSurR9-C84A reduces the viability of DU145 and HeLa cancer cells
dNSurR9-C84A increases the activity of caspases-3 and 9 in a time-dependent fashion
dNSurR9-C84A restores cell sensitivity to TNF-α via activation of caspase-8
The protein expression profiles of 2D and 3D-cultured cancer cells are different . In comparison to 2D-cultured cells, the behavior of cells cultured under 3D conditions is more reminiscent of that of cells growing in situ. Hence 3D cell cultures represent an improved in vitro system to model the behavior of potential therapeutic drugs. Here we used a 3D cell culture system to test the ability of the survivin antagonist dNSurR9-C84A to kill prostate and cervical cancer cells. This is the first report of the production and characterization of a "cell-permeable" recombinant form of survivin in which cysteine at position 84 in the zinc-coordination site in the BIR domain has been substituted with alanine. The architecture of the BIR domain is disturbed by mutation of the C84 residue to alanine, and the ability of survivin to dimerize and interfere with caspases is inhibited [21–23]. Herein, we revealed that dNSurR9-C84A was able to kill 3D-cultured prostate and cervical cancer cells, where killing was associated with increased levels of caspase-3, and with DNA fragmentation in the case of DU145 cells. Further, dNSurR9-C84A rendered prostate cancer cells sensitive to the proapoptotic effects of TNF-α by potentiating the upregulation of caspase-8 activity, suggesting that survivin inhibits the extrinsic pathway of apoptosis. In accord, melatonin has been shown to reduce both survivin and Bcl-2 protein levels and subsequently increase the sensitivity of human PC3 prostate cancer cells to TNF-α . Further, adhesion of the aggressive prostate cancer cell line PC3 to fibronectin results in upregulation of survivin and protects the cells from apoptosis induced by TNF-α . Adenoviral expression of dominant negative T34A survivin counteracted the ability of fibronectin to protect the cells from undergoing apoptosis, whereas wild-type survivin protected non-adherent cells from TNF-α-induced apoptosis.
It remains to be determined whether survivin inhibits TNF-α signaling through direct interaction with caspase-8 or via an indirect interaction, but at least one study indicates a direct interaction is unlikely . Survivin is believed to be primarily involved in the intrinsic apoptosis pathway. Biochemical and structural analysis revealed that survivin physically binds to caspase-3 and caspase-7 and inhibits their activities [8, 10]. Therefore, it is possible that survivin interferes with the TNF-α simulated apoptosis through both indirect regulation of the caspase-8 activity and direct regulation of caspase-3 activity.
Our laboratory has previously shown that intratumoral delivery of a plasmid expressing dNSur-C84A causes tumour apoptosis in an animal model of lymphoma . However, gene therapy clinical trials are hindered by immune responses that can lead to overwhelming inflammation and the death of patients . It is pertinent to devise other treatment forms where gene therapy proves to be problematic. Macromolecular protein approaches targeting tumour survival factors, as exemplified here with dNSurR9-C84A may hold promise for therapy.
In conclusion, a cell-permeable form of the dominant-negative (C84A) survivin protein has been successfully produced, and demonstrated to exert biological activity in being able to kill prostate and cervical cancer cells. Importantly, it was discovered that dNSurR9-C84A antagonizes survivin's ability to suppress TNF-α signaling, rendering prostate cancer cells susceptible to the proapoptotic effects of TNF-α. dNSurR9-C84A is a both a novel tool for probing the function of survivin, and a potential therapeutic agent for augmenting cancer therapy.
Materials and methods
Cell Lines, antibodies and reagents
The cell lines DU145 (human prostate carcinoma), and HeLa (cervical epithelial carcinoma) were purchased from the American Type Culture Collection (ATCC, Manassas, VA). DU145 cells were cultured in a mixture of Matrigel™(BD Biosciences, San Jose, CA) and RPMI-1640 (Gibco, Grand Island, NY) in a 1:1 ratio. HeLa cells were cultured in a mixture of Matrigel and Dulbecco's modified Eagle medium (DMEM) (Gibco, Grand Island, NY) in a 1:1 ratio. Both the latter media were supplemented with 10% foetal bovine serum, penicillin (100 U/mL), streptomycin (100 μg/mL) and L-glutamine (0.29 mg/mL). Cells were cultured in the semi-solid medium for five days to allow the formation of three dimensional cellular spheres. Antibodies used in this study included a goat anti-GST antibody (Amersham Biosciences, Freiburg, Germany) and a rabbit anti-human/mouse survivin antibody (Alpha Diagnostic, San Antonio, TX).
Construction of a dominant-negative survivin expression vector
A dominant-negative cell-permeable form of survivin (dNSurR9-C84A) comprising 9 N-terminal arginine residues (R9, cell-permeable peptide carrier) fused to the C84A dominant-negative survivin mutant was constructed for the study. Briefly, the cDNA of a pcDNA3 expression plasmid encoding dominant-negative survivin which contains the entire coding region of mouse survivin (nucleotides 75-583; GenBank accession No. NM_009689) and a T-to-G substitution at nucleotide 354 that changes the cysteine residue at amino acid 84 in the extreme C-terminal region of the BIR domain to an alanine was amplified using the sense primer 5'- GGGGATCCATGCGACGACGACGACGACGACGACGACGAGGAGCTCCGGCGCTGCCCCAG-3' (encodes R9) and the antisense primer 5'-GGGATCCTTAGGCAGCCAGC-3'. The resulting PCR product was subcloned into pGEM-T (Promega Corp., Madison, WI), excised by digestion with BamH I, and cloned into the expression vector pGEX-2T (Pharmacia Biotech, Piscataway, NJ) to give the vector pGEX-2T/dNSurR9-C84A. The integrity of the pGEX-2T/dNSurR9-C84A vector was confirmed by DNA sequence analysis.
Production of a recombinant dominant-negative survivin protein dNSurR9-C84A
The dNSurR9-C84A expression vector was transformed into DH5α bacteria, and the transformants were cultured at 37°C in LB medium containing 100 mg/L of ampicillin. When the OD600 nm reached 0.7, protein expression was induced with IPTG (0.7 mM) (Invitrogen, Carlsbad, CA) at 30°C for 3 h. The bacteria were pelleted and lysed in STE buffer containing 0.1 mg/ml of lysozyme, 10 mM dithiothreitol and 0.7% sarkosyl, and sonicated for 30 sec. The GST-tagged recombinant dNSurR9-C84A protein was purified by glutathione-Sepharose chromatography, and dialyzed twice against PBS.
SDS-PAGE and Western blotting
Cells were lysed with lysis buffer (10 mM Tris, 1 mM EDTA, 1 mM DTT, 60 mM KCl, 0.5% NP-40 and Complete Protease Inhibitor Cocktail Tablet from Roche, Germany, containing a mixture of protease inhibitors), and proteins were resolved on 12% polyacrylamide SDS gels under reducing conditions. The gels were either stained with Coomassie blue or proteins electrophoretically transferred to Hybond C Extra nitrocellulose membranes (Amersham Life Science, Amersham, UK). The membranes were blocked overnight at 4°C with 5% non-fat milk powder, incubated with primary antibodies for 90 min at RT, and then with a horseradish peroxidase-conjugated secondary antibody (Sigma Chemical Co., St Louis, MD). Immunoreactivity was detected by Enhanced Chemiluminescence (ECL) (Amersham International, Buckingham, UK) and autoradiography.
Cell viability assays
Cells (4 × 103) were cultured in a semi-solid culture medium comprised of Matrigel™and RPMI for five days to allow the formation of three dimensional cellular spheres. The cellular spheres were treated with test reagents for 36 h. Dispase (BD Biosciences, San Jose, CA), a bacillus-derived neutral metalloprotease, was used to recover cells cultured in the Matrigel™. Live cells were resuspended in PBS and cell viability was analyzed with the CellTiter96 MTS cell proliferation assay kit (Promega Corp., Madison, WI) with measurements being made on a 96-well plate reader (Bio-Tek Instruments Inc.).
Measurement of caspase activity
The activity of caspase-3 in cell lysates was determined with the Apo-ONE® Homogeneous Caspase-3/-7 apoptosis detection kit (Promega Crop., Madison, WI). Caspase-8 activity was determined with the Caspase-8 Fluorometric Protease Assay Kit (Biovision, Mountain View, CA). Caspase-9 activity was determined with the Caspase-9 Fluorometric Protease Assay Kit (Biovision, Mountain View, CA).
Measurement of DNA fragmentation
Cells were stained with the TUNEL reagent (In-Situ Apoptosis Detection kit, Roche Diagnostic, Mannheim, Germany) to detect DNA fragmentation, counter-stained with propidium iodide (PI), and examined by fluorescence microscopy.
The Student's t-test was used with p < 0.05 indicating a statistically significant difference.
baculovirus IAP repeat
poly-arginine tagged dominant-negative survivin protein
inhibitor of apoptosis protein
transferase (TdT)-mediated dUTP-biotin nick end labelling.
Antonio Cheung was supported by a University of Auckland International Doctoral Scholarship. We are grateful for support from the Maurice and Phyllis Paykel Trust, and Auckland Uniservices Ltd.
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