Ribozyme-mediated down-regulation of survivin expression sensitizes human melanoma cells to topotecan in vitro and in vivo
Marzia Pennati1,
Mara Binda1,
Michelandrea De Cesare1,
Graziella Pratesi1,
Marco Folini1,
Lorenzo Citti2,
Maria Grazia Daidone1,
Franco Zunino1 and
Nadia Zaffaroni1,3
1 Department of Experimental Oncology, Istituto Nazionale per lo Studio e la Cura dei Tumori, I-20133 Milan and 2 Istituto di Fisiologia Clinica-CNR, Pisa, Italy
3 To whom correspondence should be addressed Email: nadia.zaffaroni{at}istitutotumori.mi.it
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Abstract
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The ability of melanoma cells to evade engagement of apoptosis plays a significant role in their resistance to chemotherapy. In an attempt to lower the apoptotic threshold of melanoma cells as a possible strategy to increase their drug sensitivity, we generated a hammerhead ribozyme to down-regulate the expression of the anti-apoptotic protein survivin. The JR8 human melanoma cell line was stably transfected with the active ribozyme RZsurv (targeting the 3' end of the GUC294 triplet in the exon 3 of the survivin mRNA) or the catalytically inactive ribozyme mutRZsurv (carrying a mutation in the catalytic core of RZsurv). Two polyclonal cell populations expressing the active (JR8/RZsurv) or the mutant (JR8/mutRZsurv) ribozyme were selected for the study. JR8/RZsurv cells were characterized by a markedly lower survivin protein level than JR8 parental cells, whereas a negligible reduction in survivin expression was observed in JR8/mutRZsurv cells. JR8/RZsurv cells showed a significantly increased sensitivity to the topoisomerase-I inhibitor topotecan (as detected by clonogenic cell survival) compared with JR8/mutRZsurv cells. Moreover, the extent of drug-induced apoptosis (in terms of percentage of apoptotic nuclei and level of caspase-9 and caspase-3 catalytic activity) was significantly greater in JR8/RZsurv than in JR8/mutRZsurv cells. Finally, an increased antitumor activity of oral topotecan was observed in JR8/RZsurv cells grown as xenograft tumors in athymic nude mice compared with JR8/mutRZsurv cells. These results demonstrate that attenuation of survivin expression renders human melanoma cells more susceptible to topotecan-induced apoptosis and more responsive to in vivo treatment, and support the concept that survivin is an attractive target for new therapeutic interventions in melanoma.
Abbreviations: DOTAP, N-(1-(2,3 dioleoyloxy)propyl)-N,N,N-trimethylammoniummethyl sulfate; IAP, inhibitors of apoptosis protein
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Introduction
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Cutaneous melanoma is the most aggressive form of skin tumors and its incidence is increasing more rapidly than that of any other cancer (1). In contrast to localized melanoma, which has a good prognosis after adequate surgery, disseminated disease is characterized by a very poor clinical outcome, which is not modified by conventional anticancer treatments (2). Although not yet fully elucidated, the basis of melanoma resistance to chemotherapy seems to rely on dysregulation of apoptosis (3,4). In fact, it has been shown that cells within melanoma lesions are characterized by an inherently low level of spontaneous apoptosis (5). Moreover, defects at multiple levels of the two major apoptotic pathways have been found in melanoma cells, including altered death receptor signaling (6), increased levels of anti-apoptotic proteins belonging to the Bcl-2 family such as Bcl-2, Bcl-xL and Mcl-1 (7), inactivation of the apoptosis effector Apaf-1 (8), and over-expression of members of the inhibitors of apoptosis protein (IAP) family such as ML-IAP and survivin (9,10).
From the increasing evidence that dysregulation of apoptosis contributes to in vivo drug resistance of tumor cells comes confidence that new therapeutic approaches aimed at correcting the dysfunctional apoptotic programme could improve the treatment outcome of patients with chemotherapy refractory tumors such as melanoma. In this context, it has been demonstrated that down-regulation of Bcl-2 and Mcl-1 by antisense oligonucleotides sensitized human melanoma to dacarbazine in cell culture as well as in SCID mouse xenotransplantation models (11,12). Moreover, in the clinical setting, sensitization of malignant melanoma to dacarbazine by concomitant Bcl-2 antisense therapy was confirmed in a phase III study in patients with Bcl-2-expressing advanced melanoma (13). Again, reduction of Bcl-xL protein expression by a specific antisense oligonucleotide significantly increased the in vitro sensitivity of melanoma cells to cisplatin (14). Using a different approach dealing with the recovery of Apaf-1 expression by treatment with the demethylating agent 5'-aza-2'-deoxycytidine, Soengas et al. (8) were able to enhance the in vitro sensitivity of Apaf-1-negative melanoma cells to doxorubicin.
The possibility of modulating the chemosensitivity of melanoma and other tumor cell types by targeting survivin has been also actively pursued in the last few years. The protein is a structurally unique member of the IAP family, which acts as a cell survival factor since it is involved in the control of mitotic progression and inhibition of apoptosis (15) and is selectively expressed in most common human tumors (16) including melanoma (10). It has been demonstrated by Grossman et al. (17) that the forced expression of a phosphorylation-defective survivin mutant (Thr34
Ala) in melanoma cell lines, besides triggering apoptosis, enhanced the level of programmed cell death induced by in vitro treatment with cisplatin. Such findings have been successively confirmed by our group using a ribozyme-mediated approach to inhibit survivin expression in a different melanoma cell line (18). Specifically, we demonstrated that melanoma cells engineered to express hammerhead ribozymes targeting different portions of survivin mRNA displayed an enhanced apoptotic response to cisplatin compared with cells transfected with a mutant, catalytically inactive ribozyme. A number of additional studies carried out in experimental models of other tumor types showed that targeting of survivin by antisense oligonucleotides, dominant negative mutants and cdc2 kinase inhibitors resulted in an increased sensitivity to anticancer agents including taxol (19,20), etoposide (21), doxorubicin (22) and 5-fluorouracil (23).
As no information is available regarding the relevance of survivin expression in the response of tumor cells to topoisomerase-I inhibitors, we proposed to assess the effect of ribozyme-mediated survivin inhibition on the response of human melanoma cells to topotecan. We found that attenuation of survivin expression increased the in vitro cytotoxic activity of the topoisomerase-I inhibitor as a consequence of an enhanced caspase-9-dependent apoptotic response of melanoma cells and also increased the antitumor activity of topotecan in a mouse xenograft model.
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Materials and methods
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Drugs and cell line
Topotecan (S-9-dimethyl-10-hydroxycamptothecin hydrochloride; Smith-Kline Beecham Pharmaceuticals, King of Prussia, PA) was dissolved in sterile water, cisplatin (Bristol-Myers, Evansville, IL) in saline solution, and temozolomide (Schering Plough, Madison, NJ) in dimethylsulfoxide and then in a saline solution immediately before use.
The JR8 human melanoma cell line was maintained in the logarithmic growth phase at 37°C in a 5% CO2 humidified atmosphere using RPMI 1640 (BioWhittaker, Verviers, Belgium) supplemented with 10% fetal calf serum, 2 mM L-glutamine and 0.25% gentamycin.
Synthesis of ribozymes and in vitro cleavage reaction
A hammerhead ribozyme addressed to the GUC294 triplet located in exon 3 of the survivin mRNA was used in the study. The ribozyme sequence, 5'-CAACCGCUGAUGAGGCCGAAAGGCCGAAACGAAUG-3' (RZsurv), was synthesized by in vitro transcription. A mutant ribozyme (mutRZsurv) was also designed and synthesized by deleting the G12 from the catalytic core (as indicated by the circled letter in Figure 1A) in order to obtain a negative control. Single-stranded synthetic DNA oligonucleotides encoding active and mutant ribozymes were obtained from MWG (Biotech AG, Ebersberg, Germany). The oligonucleotide sequences were the following: RZ+ (5'-AGCTTCAACCGCTGATGAGGCCGAAAGGCCGAAACGAATGT-3'), RZ (5'-CTAGACATTCGTTTCGGCCTTTCGGCCTCATCAGCGGTTGA-3'), mutRZ+ (5'-AGCTTCAACCGCTGATGAGGCCGAAAGGCCAAACGAATGT-3') and mutRZ (5'-CTAGACATTCGTTTGGCCTTTCGGCCTCATCAGCGGTTGA-3'). Annealing of the complementary oligonucleotides produced two fragments with HindIII and XbaI protruding ends. Each fragment was inserted into the pRc/CMV vector (Invitrogen, San Diego, CA), which had been digested previously with HindIII and XbaI restriction enzymes. The presence and the correct orientation of the inserts was verified by DNA sequencing (AmpliCycleTM, Perkin Elmer-Roche Molecular System, Branchburg, NJ). The resulting plasmids were named pRcRZsurv and pRcmutRZsurv. To obtain the active and the mutant ribozyme, both plasmids were linearized with XbaI and used as templates for in vitro transcription, as described previously (24).

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Fig. 1. (A) Schematic representation of survivin mRNA and base pairing of the ribozyme with its RNA target sequence. The cleavage site is indicated by an arrow. (B) In vitro cleavage of the 362-nt synthetic 32P-RNA substrate (10 nM) by RZsurv into the cleavage products (310 and 52-nt). The synthetic 32P-substrate was incubated with 101000 nM RZsurv or 1001000 nM mutRZsurv for 120 min at 37°C (BL: 32P-substrate RNA incubated without the ribozyme). The reaction products were resolved on 5% polyacrylamide/7 M urea gel and visualized by autoradiography.
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The pCI survivin plasmid (24), digested previously with StyI restriction enzyme, was used for in vitro synthesis of the specific RNA substrate. The linear template was transcribed in vitro in the presence of [
-32P]cytidine triphosphate (10 µCi/µl, 800 Ci/mmol, Amersham, Buckinghamshire, UK), using the Riboprobe in vitro transcription system (Promega, Italia srl, Milan, Italy) according to the manufacturer's instructions. 32P-substrate RNA (10 nM) was subsequently mixed with increasing concentrations of both ribozymes in the presence of 50 mM MgCl2, and incubated at 37°C for 120 min. Products of the cleavage reaction were resolved on 5% polyacrylamide/7 M urea gel and the results were quantified by densitometric analysis (ImageQuanT software, Molecular Dynamics, Sunnyvale, CA).
Transfection of cells with the ribozyme expression vectors
DOTAP {N-[1-(2,3 dioleoyloxy)propyl]-N,N,N-trimethylammoniummethyl sulfate, Boehringer Mannheim, Mannheim, Germany}-mediated transfection of JR8 cells was performed according to the manufacturer's instructions. Briefly, cells were transfected with 5 µg of pRcRZsurv vector (or pRcmutRZsurv vector) that had been complexed with 30 µg of DOTAP. Twenty-four hours after transfection, the culture medium containing the DOTAP/DNA mixture was replaced by a selection medium containing G418 to a final concentration of 2 mg/ml. Two polyclonal cell populations (one expressing the active ribozyme, JR8/RZsurv cells, and one expressing the mutant ribozyme, JR8/mutRZsurv cells) were selected for the study.
Western immunoblotting
Total cellular lysates were separated on a 15% sodium dodecylsulfatepolyacrylamide gel and transferred to nitrocellulose. The filters were blocked in phosphate-buffered saline with 5% skim milk and then incubated overnight with the primary antibodies to survivin (Novus Biologicals, Littleton, UK). The filters were then incubated with the secondary peroxidase-linked whole antibodies (Amersham Biosciences Europe GmbH, Freiburg, Germany). Bound antibodies were detected using the enhanced chemioluminescence western blotting detection system (Amersham Biosciences). An anti-proliferating cell nuclear antigen (PCNA) monoclonal antibody (Santa Cruz Biotechnology, Santa Cruz, CA) was used on each blot to ensure equal loading of protein on the gel. Results were quantified by densitometric analysis.
Clonogenic assay
Cells were plated at appropriate concentrations in plastic dishes, allowed to attach for 24 h and then exposed to 1.010 ng/ml topotecan for 24 h, 0.110 µg/ml temozolomide for 24 h or 0.110 µg/ml cisplatin for 1 h. At the end of treatment, cells were washed with phosphate-buffered saline and incubated at 37°C in a 5% CO2 humidified atmosphere for 10 days. Colonies consisting of at least 50 cells were stained with crystal violet in 70% v/v ethanol/water and counted under the microscope. Each experimental point was run four times. The colony-forming efficiency was calculated from the number of colonies counted and the number of morphologically intact single cells seeded. The in vitro effect of drugs was expressed in terms of IC50, i.e. the concentration able to inhibit clonogenic cell survival by 50%.
Apoptosis analysis
Cells exposed to 10 ng/ml topotecan for 24 h were incubated at 37°C for 72 h and then scored for nuclear morphology of apoptosis (chromatin condensation, DNA fragmentation) by labeling with a solution containing 50 µg/ml propidium iodide, 50 mg/ml RNAse and 0.05% Nonidet P-40. After staining, the slides were examined using fluorescence microscopy. The percentage of cells with an apoptotic nuclear morphology was determined by scoring at least 500 cells in each sample.
Caspase-9 and caspase-3 catalytic activity was determined at 72 h after drug exposure by means of the Caspase-9/Mch6 Fluorometric Protease Assay Kit (MBL) and the Caspase-3 Assay Kit (Pharmingen, San Diego, CA), respectively. Total protein and the specific fluorogenic substrate (leu-glu-his-asp-7-amino-4-trifluoromethylcoumarin, LEHD-AFC, for caspase-9 and N-acetyl-Asp-Glu-Val-Asp-aldehyde-7-amino-4-methylcoumarin, Ac-DEVD-AMC, for caspase-3) were mixed for 1 h at 37°C. In the assay for caspase-3 activity, a negative control was obtained by incubating each sample in the presence of the caspase inhibitor Ac-DEVD-CHO. Hydrolysis of the specific substrates for caspase-9 and caspase-3 was monitored by spectrofluorometry at 505 nm and 440 nm, respectively.
In vivo studies
Female athymic Swiss nude mice (910 weeks old; Charles River, Calco, Italy) were used. Mice were kept in laminar flow rooms at constant temperature and humidity with free access to food and water. Experimental protocols were approved by the Ethics Committee for Animal Experimentation of the Istituto Nazionale Tumori, Milan, according to the United Kingdom Coordinating Committee on Cancer Research Guidelines (25). Cells from in vitro cultures of JR8, JR8/mutRZsurv or JR8/RZsurv were intramuscularly injected into the right hind leg of mice (107 cells/mouse). For each cell line, half of the group (58 mice) was left untreated and half was treated with topotecan. Topotecan was dissolved in sterile, distilled water and delivered in a volume of 10 ml/kg body wt. The drug was administered orally at the dose of 10 mg/kg every seventh day for four times (q7dx4), starting the day after cell injection. Mice were inspected daily to assess tumor take, i.e. the ratio between the number of growing tumors over the total number of tumor-injected mice. Tumor growth was followed by biweekly measurements of tumor diameters with a Vernier caliper. Tumor volume (TV) was calculated according to the formula: TV (mm3) = d2 x D/2 where d and D are the shortest and the longest diameter, respectively. Growth curves designed plotting mean TV versus time.
Reverse transcriptasePCR analysis of ribozyme expression
Total RNA isolated from tumors originating from JR8/RZsurv and JR8/mutRZsurv cells xenotransplanted into nude mice was reverse-transcribed using a GeneAmp RNA PCR core kit (Perkin Elmer) according to the manufacturer's instructions. To analyze ribozyme (RZsurv and mutRZsurv) expression the resultant cDNA was amplified using T7 and SP6 primers and by performing 30 cycles of PCR (at 95°C for 1 min, 47°C for 1 min and 72°C for 1 min), followed by a 7-min extension step at 72°C. PCR products were verified by agarose gel electrophoresis. Vectors pRcRZsurv and pRcmutRZsurv were used during PCR amplification as control for the correct fragment size.
Statistical analysis
Student's t test was used to analyze the differences between mutant and active ribozyme-expressing cells in terms of cell surviving fraction and in vitro catalytic activity of caspase-9 and caspase-3. The Fisher exact text was used to compare tumor take between animal groups. All tests were two-sided. P values <0.05 were considered statistically significant.
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Results
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In vitro catalytic activity of the anti-survivin ribozyme
The catalytic potential of the RZsurv ribozyme was assessed as the ability to cleave an internally 32P-labeled synthetic RNA substrate obtained by cloning a portion of the human survivin mRNA. The size of the substrate was 362-nt, and the cleavage reaction (the cleavage site is indicated by the arrow in Figure 1A) was expected to produce two fragments of 310 and 52 nt, respectively. Experiments performed by incubating 10 nM of labeled RNA substrate with increasing concentrations (from 10 to 1000 nM) of the RZsurv ribozyme for 120 min at 37°C (Figure 1B) resulted in a decrease of the full-length substrate and accumulation of the expected cleavage products. The efficiency of cleavage was dependent on the substrate/ribozyme ratio, with the cleavage products being detectable at a ratio of 1:10. As expected, the mutRZsurv ribozyme, carrying a mutation (i.e. deletion of G12) in the catalytic core of the RZsurv ribozyme, did not induce any cleavage in the synthetic RNA (Figure 1B).
Ribozyme-mediated attenuation of survivin expression in JR8 melanoma cells
To evaluate the effect of the ribozyme in intact cells, the JR8 human melanoma cell line inherently over-expressing survivin was transfected with pRcRZsurv and pRcmutRZsurv vectors containing the sequence coding for the active RZsurv ribozyme and the catalytically inactive mutRZsurv ribozyme, respectively. The transfectants were treated with G418 for 1 month, and the G418-resistant clones were selected and screened for survivin expression by western blotting. To rule out the possibility that attenuation of survivin expression was simply due to clonal divergence, we used two polyclonal populations of transfectants proven to endogenously express the active (JR8/RZsurv cells) or the mutant (JR8/mutRZsurv cells) ribozyme (data not shown). JR8/RZsurv cells were characterized by a markedly lower survivin protein level (60 ± 8%) than JR8 parental cells, as assessed by western blot analysis in four independent cultures. Conversely, a very modest reduction (13 ± 7%) in survivin expression was observed in mutRZsurv cells (Figure 2).

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Fig. 2. Survivin expression in melanoma cell clones. (A) A representative western blotting experiment illustrating the expression of survivin protein in JR8, JR8/RZsurv and JR8/mutRZsurv cells is shown. PCNA was used as control for loading. (B) Densitometric quantification of band intensities for survivin. Data represent mean values ± SD of three independent experiments.
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Attenuation of survivin expression did not appreciably affect the growth potential of melanoma cells. In fact, only a modest increase in the doubling time of JR8/RZsurv cells compared with JR8 and JR8/mutRZsurv cells was observed (29 ± 3 versus 24 ± 3 and 25 ± 4 h, respectively).
Survivin inhibition sensitizes JR8 melanoma cells to topotecan-induced apoptosis
To determine whether the level of survivin expression is associated with the chemosensitivity of melanoma cells, JR8/RZsurv and JR8/mutRZsurv cell lines were analyzed for their clonogenic cell survival profiles after exposure to different concentrations of the topoisomerase-I inhibitor topotecan for 24 h (Figure 3). Transfection with the active ribozyme sequence did not appreciably modify the plating efficiency of JR8/RZsurv cells compared with JR8/mutRZsurv cells (10.2 ± 1.5 and 13.4 ± 2.1%, respectively). A dose-dependent reduction in cell survival was observed in both cell lines following topotecan exposure. However, JR8/RZsurv cells showed enhanced sensitivity to the drug compared with JR8/mutRZsurv cells, as indicated by the significantly (P < 0.01) lower concentration required to inhibit clonogenic cell survival by 50% (IC50: 1.86 ± 0.44 versus 6.76 ± 0.53 ng/ml). In parallel, we assessed the susceptibility of melanoma cells as a function of the level of survivin expression to other DNA-interacting agents currently used in the treatment of melanoma patients. Specifically, we observed an increased sensitivity of JR8/RZsurv cells compared with cells expressing the mutant ribozyme to a 1-h cisplatin exposure, as indicated by the significantly (P < 0.05) lower IC50 (1.36 ± 0.40 versus 2.9 ± 0.30 µg/ml). Conversely, JR8/RZsurv and JR8/mutRZsurv cells showed a comparable sensitivity to a 24-h exposure to temozolomide (IC50: 5.81 ± 0.39 versus 6.29 ± 0.29 µg/ml).

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Fig. 3. Clonogenic cell survival curves obtained after exposure of JR8/RZsurv (filled circle) and JR8/mutRZsurv (filled square) cells to topotecan for 24 h. After treatment, cells were incubated at 37°C for 10 days. Colonies consisting of at least 50 cells were stained with crystal violet and counted under the microscope. Data represent mean values ± SD of three independent experiments.
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To investigate whether the increased cytotoxic activity of topotecan observed in JR8/RZsurv cells was mediated by an enhanced susceptibility of cells to undergo programmed cell death as a consequence of survivin inhibition, we examined the induction of apoptosis in the two cell lines. Specifically, the number of cells with apoptotic nuclei was determined by fluorescence microscopy 72 h after a 24-h exposure to 10 ng/ml topotecan (Figure 4A). Spontaneous apoptosis was observed in a small fraction of cells in both cell lines (0.5 ± 0.2 and 1.0 ± 0.2% of the overall cell population in JR8/mutRZsurv and JR8/RZsurv cells, respectively). Conversely, a significantly (P < 0.01) higher apoptotic response to topotecan was appreciable in JR8/RZsurv than in JR8/mutRZsurv cells (26.4 ± 2.6 versus 9.8 ± 1.3%) (Figure 4B).

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Fig. 4. (A) Propidium iodide staining of apoptotic JR8/RZsurv cells treated with topotecan. (B) The percentage of cells with an apoptotic morphology with respect to the overall population as assessed by fluorescence microscopy in untreated cells (empty column) and in cells exposed to topotecan for 72 h (black column). Data represent mean values ± SD of three independent experiments.
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To obtain mechanistic insights into the apoptotic pathway activated by lowering survivin expression, we evaluated the in vitro catalytic activity of caspase-9 and caspase-3 in the two melanoma cell lines. JR8/RZsurv and JR8/mutRZsurv cells were characterized by a similar basal level of caspase activity as assessed by hydrolysis of specific fluorogenic substrates (Figure 5A and B). Treatment with topotecan increased the activation of caspase-9 and caspase-3 in both cell lines. However, the catalytic activity of both enzymes was significantly (P < 0.05) higher (2- and 3-fold in the case of caspase-9 and caspase-3, respectively) in the JR8/RZsurv than in the JR8/mutRZsurv drug-treated cells (Figure 5A and B).

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Fig. 5. Caspase activation in JR8/RZsurv and JR8/mutRZsurv cells treated with topotecan. (A) Caspase-9 catalytic activity was determined by hydrolysis of the fluorogenic substrate LEHD-AFC in untreated cells (empty column) and in topotecan-treated cells (gray column). Data are expressed as relative fluorescence units (r.f.u.) and represent mean values ± SD of three independent experiments. (B) Caspase-3 catalytic activity was determined by hydrolysis of the fluorogenic substrate Ac-DEVD-AMC in untreated cells (empty column) and in topotecan-treated cells in the absence (gray column) or presence (black column) of the caspase inhibitor (Ac-DEVD-CHO). Data represent mean values ± SD of three independent experiments.
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Survivin inhibition enhances the antitumor activity of topotecan in vivo
The effect of survivin inhibition on the antitumor activity of topotecan was studied in athymic nude mice after injection of melanoma cells expressing the active or the mutant ribozyme. The endogenous presence of the ribozymes in tumors grown in the animal was confirmed by RTPCR (Figure 6). The drug (10 mg/kg) was administered orally every seventh day for four times (q7dx4), starting from the day after cell injection. Although JR8/RZsurv cells retained a reduced expression of survivin compared with JR8/mutRZsurv cells after xenografting in mouse (data not shown), survivin inhibition did not appreciably modify tumor take or growth in untreated animals. Specifically, at day 12 after cell injection, tumors were present in five of seven and three of eight mice receiving JR8/mutRZsurv and JR8/RZsurv cells, respectively (Table I), and tumor growth curves of the two groups of mice were similar and superimposable to that observed for JR8 parental cells (Figure 7). In JR8/mutRZsurv tumors, topotecan treatment initially retarded tumor growth (Figure 8; Table I). In fact, at day 12 the tumor was present in only one of five mice. However, at later intervals, tumors grew in most of the treated mice (4/5), and no difference was evident in tumor take or volume between treated and untreated mice. In contrast, topotecan was very effective in increasing the latency and reducing the take rate and growth of JR8/RZsurv tumors. In fact, at day 49 after cell injection, tumors had not yet appeared in topotecan-treated mice, whereas they were present in five of eight control mice (P = 0.0128). Again, at day 63, when control mice were killed for tumor burden, tumors were present in only three of eight treated mice. Topotecan-treated mice were kept under observation for >100 days, but tumor take did not increase over 50% (4/8 at day 104).

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Fig. 6. Ribozyme expression in tumors grown in nude mice. Ribozyme expression was detected by RTPCR in tissue samples obtained from tumors originating from JR8 cells transfected with the active ribozyme (JR8/RZsurv) or with the mutant ribozyme (JR8/mutRZsurv). Lanes pRcRZsurv and pRcmutRZsurv indicate plasmids used as controls for the size of each fragment during PCR amplification.
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Table I. Effects of oral topotecan on tumorigenicity (tumor takea) of JR8/mutRZsurv and JR8/RZsurv melanoma cells in nude mice
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Fig. 7. Growth curves of JR8, JR8/mutRZsurv and JR8/RZsurv melanoma cells in athymic nude mice. Cells were intramuscularly injected into the right hind leg of mice (107 cells/mouse) at day 0. Tumor volumes were calculated as described in Materials and Methods. JR8 (circle), JR8/mutRZsurv (filled circle) and JR8/RZsurv (square) cells.
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Fig. 8. Effect of ribozyme-mediated survivin inhibition on the anti-tumorigenic activity of oral topotecan in melanoma xenografts. Cells were intramuscularly injected into the right hind leg of mice (107 cells/mouse) at day 0. Treatment consisted of 10 mg/kg, q7dx4 from day 1. Tumor volumes were calculated as described in Materials and methods. Control JR8/mutRZsurv (circle), topotecan-treated JR8/mutRZsurv (filled circle), control JR8/RZsurv (square) and topotecan-treated JR8/Rzsurv cells (filled square).
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Discussion
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In the present study we demonstrated that specific inhibition of survivin expression enhances the sensitivity of human melanoma cells to the topoisomerase I inhibitor topotecan both in vitro and in vivo. To inhibit survivin we chose an approach based on the use of a hammerhead ribozyme since, due to its specific endoribonuclease activity, this small RNA molecule is able not only to specifically recognize its target RNA through complementary base pairing but also to cleave the substrate leading to its degradation and permanent inactivation. Moreover, the typical catalytic cycle of a ribozyme offers a considerable advantage with respect to the action mechanism of conventional antisense oligonucleotides (26). Specifically, one molecule of hammerhead ribozyme can cleave several RNA target molecules, which implies that, theoretically, very low concentrations are required to obtain a significant biological effect. In fact, the efficacy of the ribozyme-mediated approach in inhibiting the expression of several cancer-related genes has been clearly demonstrated in a number of studies carried out on experimental human tumor models (27). Specifically, here we describe the possibility to markedly attenuate the expression level of survivin in the JR8 human melanoma cell line, which inherently over-expresses the anti-apoptotic protein, by the use of a hammerhead ribozyme targeting a consensus sequence in exon 3 within survivin mRNA. This inhibitory effect was lost after inactivation of the catalytic core of the ribozyme (in JR8/mutRZsurv cells), which strongly suggests specific cleavage activity of the anti-survivin ribozyme not only in the cell-free-system but also in intact cells. Active-ribozyme transfectant cells showed enhanced sensitivity to topotecan with respect to cells transfected with the mutant ribozyme, as demonstrated by a reduced clonogenic cell survival and a significantly lower IC50 value. Such an increased susceptibility to drug treatment as a consequence of survivin down-regulation was not observed for all DNA-interacting agents tested. Although the molecular basis for this putative selectivity is presently unknown, it is plausible that different signals are activated in response to DNA damage, depending on the type of genotoxic lesion, with a variable impact on the survivin pathway.
In vitro sensitization to topotecan was paralleled by restoration of the susceptibility of JR8 cells expressing the active ribozyme to programmed cell death induced by the topoisomerase I inhibitor. Such an enhanced apoptotic response was mediated by an increased activation of caspase-9 and caspase-3, which is consistent with the recognized function of survivin to counteract mitochondrial-death pathway (28). However, it is still unclear whether survivin inhibits caspases through direct binding or indirectly, thereby requiring intermediate proteins (29). Specifically, a possible direct interaction of survivin with caspase-9 was reported by O'Connor et al. (30), whereas, more recently, Song et al. (31) suggested an alternative model for indirect inhibition of caspases by survivin based on its ability to physically interact with SMAC/Diablo. This mitochondrial factor, which is released into the cytosol in response to apoptotic stimuli, is known to bind to some IAPs, including XIAP, cIAP1,CIAP2 and livin, thus preventing them to inhibit caspases (32,33). Accordingly to Song's model, the capability of survivin to sequester SMAC would allow other IAPs to block caspases without being antagonized.
When tested in a human melanoma xenograft, ribozyme-mediated inhibition of survivin expression resulted in increased antitumorigenic activity of topotecan, as demonstrated by the significantly delayed tumor establishment. Based on our in vitro findings indicating an enhanced susceptibility to topotecan-induced apoptosis of JR8 cells expressing the active ribozyme, it is conceivable to hypothesize that such an increased apoptotic response also occurred in vivo. The delayed growth in nude mice of tumor cells containing the active ribozyme after topotecan treatment reflected the survival of a limited number of cells that escaped drug-induced apoptosis, presumably as a consequence of a heterogeneous expression of survivin. Our findings corroborate and extend previous results indicating the possibility to target survivin pathway to obtain an improved response to apoptosis-inducing anticancer drugs in in vivo tumor models. In this context, Yamamoto et al. (23) showed that down-regulation of survivin by forced expression of its natural antisense, the effector cell protease receptor-1 (34), resulted in enhanced sensitivity to cisplatin and 5-fluorouracil of the HT29 colon adenocarcinoma cell line transplanted into nude mice. O'Connor et al. (20) observed that sequential inhibition of p34cdc2 by purvalanol Athat blocks p34cdc2 phosphorylation on Thr34, which seems to be essential for the cytoprotective function of survivin (35)enhanced the antitumor activity of taxol in an MCF-7 breast cancer xenograft. Conversely to that reported in the aforementioned and other studies (17,19,23), in which down-regulation of survivin expression was sufficient to suppress de novo tumor formation and/or inhibit the growth of established tumors in immunodeficient mice as a consequence of an enhanced spontaneous apoptosis and a reduced proliferative potential, we did not observe any effect of survivin inhibition on the tumorigenicity of melanoma cells in the absence of other stimuli. It could be hypothesized that the concomitant over-expression of other anti-apoptotic factors, such as Bcl-2 and Bcl-xL (24), and cell survival factors in JR8 cells might produce a cytoprotective effect and contribute to prevent programmed cell death in this tumor model. However, it should be stressed that in JR8/RZsurv cells, survivin expression was attenuated but not completely abrogated. Inhibition below a certain threshold is possibly insufficient to abrogate the anti-apoptotic effect as well as to efficiently interfere with the major survivin function of preserving the mitotic apparatus and allowing normal cell cycle progression, as indicated by the lack of spontaneous apoptosis observed in JR8/RZsurv cells, which were also characterized by an in vitro proliferative potential similar to that of parental cells. Interestingly, and in keeping with this hypothesis, when we transduced the DU145 human prostate cancer cell line with a retroviral vector carrying the catalytic sequence of a hammerhead ribozyme targeting the CUA110 triplet in exon 1 of survivin mRNA, we obtained a polyclonal cell population that, as a consequence of the almost complete suppression of survivin expression, became polyploid, showed a significantly longer in vitro doubling time than parental cells, underwent spontaneous apoptosis and was completely unable to establish tumor after injection in athymic nude mice (36).
Overall, results of the present study indicate that down-regulation of survivin pathways is effective in enhancing the sensitivity of melanoma cells to topotecan both in vitro and in vivo and suggest, for the first time, a critical role of survivin in modulating the cellular response to a topoisomerase I inhibitor. This finding has potential clinical implications since it could provide a rational basis for the design of combined therapies, including survivin inhibitors, to enhance the responsiveness of melanoma to camptothecins. Such a chemosensitizing approach could be even better exploited for combination therapies with new camptothecin analogues, such as the 7-oxyimino methyl derivatives, which have already demonstrated a superior antitumor activity, compared with topotecan, in human tumor xenografts of different histological types including melanoma (37). However, considering the presence of other anti-apoptotic factors besides survivin in human melanomas (3), it is presumable that approaches based on the simultaneous targeting of different factors in different cell survival pathways could be preferably used to obtain an enhancement of tumor sensitivity to agents available in the clinical setting.
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Acknowledgments
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The study was supported by grants from the Italian Health Ministry (RF 02/171 and 02/184), National Research Council, Strategic Project Oncology, FIRB-MIUR (RBNE01X3NB), Fondazione Cariplo and Compagnia San Paolo. Marzia Pennati was supported by a fellowship by FIRC.
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Received October 15, 2003;
accepted January 26, 2004.