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Keywords:

  • Survivin;
  • siRNA;
  • p53;
  • lung cancer;
  • Adriamycin

Abstract

  1. Top of page
  2. Abstract
  3. Material and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

Survivin is a member of the inhibitor of apoptosis protein (IAP) family that is specifically overexpressed in cancer tissues. p53 is one of the tumor suppressor genes; its induction in response to DNA damage causes apoptosis and correlates with drug sensitivity. To investigate the possible regulation of survivin by p53, we examined the level of survivin expression in lung cancer cell lines in response to adriamycin. Levels of survivin mRNA and protein in cell lines with wild-type p53 decreased dramatically after p53 induction, but no such reduction of survivin was observed in cell lines with mutated or null p53. Inhibition of wild-type p53 in A549 cells by small interfering (si) RNA significantly upregulated the expression of survivin. Survivin inhibition by siRNA in PC9 cells with mutated p53 significantly depressed cell proliferation. To investigate the sensitivity of cancer cells to adriamycin after inhibition of survivin, we depressed survivin expression using siRNA, and then added adriamycin at an IC50 dose. After a further 48 hr incubation with adriamycin, proliferation was significantly depressed in the cells treated with siRNA targeting survivin, in comparison with siRNA targeting scramble. Furthermore, both TUNEL and pro-caspase3 expression assay showed a significant increase in apoptosis after combined treatment with adriamycin and siRNA targeting survivin. Our results demonstrate that survivin is downregulated by p53, and that siRNA targeting of survivin increases cell sensitivity to adriamycin and promotes apoptosis. siRNA targeting of survivin could be potentially useful for increasing sensitivity to anticancer drugs, especially in drug-resistant cells with mutated p53. © 2005 Wiley-Liss, Inc.

The success of cancer treatment depends on the response to chemotherapeutic agents. However, malignancies often acquire resistance to drugs if they are used frequently. Inhibition of the apoptosis pathway is one of the factors that may be responsible for such drug resistance.1 Survivin is a member of the inhibitor of apoptosis protein (IAP) family that is specifically overexpressed in various cancers but not in normal adult tissues.2 Overexpression of survivin is correlated with poor prognosis in a number of tumor types, including lung cancer,3 colorectal cancer4 and gastric cancer.5 Like other mammalian IAPs (e.g., XIAP, c-IAP-1, c-IAP-2 and livin), survivin binds to caspase-3 and caspase-7.6 It has been suggested that survivin expression is regulated in a cell cycle-dependent manner.7 Survivin is maximally expressed in the G2/M phase and physically associates with mitotic spindle microtubules that regulate progression through mitosis. In contrast, survivin is definitively depressed in the G1 phase. p53 is one of the tumor suppressor genes, and it is frequently mutated in cancer tissue/cells.8 The crucial role of p53 is to maintain genetic stability through its participation in cell cycle checkpoints. After DNA damage induced by various cytotoxic agents, cells with wild-type p53 become preferentially arrested in the G0/G1 phase, after which they choose a path that results in either DNA repair or apoptosis. Apoptosis is closely linked to transcripts that are downregulated by p53. In contrast, mutation or deletion of p53 leads cells away from the apoptosis pathway, causing drug resistance.9 It is generally accepted that p53 functions as a transcriptional factor and transactivates some genes, resulting in cell growth modulation or death. For example, an elevated level of p21, the first product of p53 transactivation, results in underphosphorylation of the retinoblastoma (Rb) protein, which in turn sequesters the E2F transcription factor; as a result, the cell cycle is blocked in the G1 phase.10, 11 Additionally, some genes, such as stathmin or cdc2, could be negatively regulated by p53.12, 13 Previous reports suggest that p53 also downregulates the expression of survivin in some cell models and cancer cell lines.14, 15 More recent reports have shown that inhibition of survivin by anti-sense oligonucleotide blocks the cell proliferation of myeloid leukemic cells16 or lung cancer cells,17 although the mechanism of this transcriptional regulation is not fully understood and requires additional research.

From another viewpoint, inhibition of survivin might play a role in overcoming acquired drug resistance. It has not been clarified how DNA-damaging agents influence survivin expression and cause cell cycle arrest and apoptosis. One report has suggested that anti-sense targeting of survivin sensitizes lung cancer cells to chemotherapy.17 However, that study employed only 1 lung cancer cell line containing wild-type p53 and did not address the outcome that would be expected with mutated or deleted p53.

RNA interference (RNAi) is a mechanism whereby double-stranded RNA post-transcriptionally silences a specific gene. It has been reported that synthetic, double-stranded small-interfering RNA (siRNA) can effectively silence a gene through the RNAi mechanism.18 siRNA can be a novel tool for clarifying gene function in mammalian cells and may be applicable to gene-specific therapeutics.1 In our study, using siRNA, we aimed to sensitize lung cancer cell line to adriamycin. Our results suggest that siRNA targeting of survivin can inhibit cell growth and produce a combined anti-proliferative effect and apoptosis when combined with adriamycin, especially in cell lines containing mutated p53.

Material and methods

  1. Top of page
  2. Abstract
  3. Material and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

Drugs and cell lines

Adriamycin, obtained from Kyowa Hakko Kogyo Co. (Tokyo, Japan), was dissolved in distilled water and stored at −30°C until use. All cell lines used in our study were derived from patients with lung cancer. Lines NCI H226, H292, H358, H460, H522 and H1299 were obtained from the American Type Culture Collection (Manassas, VA). Lines A549, EBC-1, LK-2, Lu99, Lu99B, OBA-LK-1 and Sq-1 were provided by the Cell Resource Center for Biomedical Research, Institute of Development, Aging and Cancer, Tohoku University (Miyagi, Japan). SBC3, Lu65 and RERF-LC-KJ were obtained from the Japan Health Sciences Foundation (Tokyo, Japan). Lines PC9 and PC14 were kindly donated by Prof. Hayata, Tokyo Medical University (Tokyo, Japan). SBC3/ADM,20 a subline of SBC3 with approximately 8-fold stronger resistance to the growth-inhibitory effect of adriamycin, as determined by the MTT assay, was provided by Dr. Kiura, Okayama University (Okayama, Japan). Lu135 was provided by Riken Cell Bank (Tokyo, Japan). Ma46 was established in our laboratory from malignant effusion of an NSCLC patient. The cells were cultured in RPMI-1640 medium (Sigma Chemical Co., St. Louis, MO) supplemented with 10% fetal bovine serum under a humidified atmosphere of 5% CO2 and air at 37°C. All cell lines were discarded after 20 generations, and new lines were obtained from frozen stocks. Some cell lines were analyzed for their IC50 values using the MTT assay by incubating them with adriamycin for 72 hr.21 With regard to p53 status, NCI H226, H460, A549, SBC3, SBC3/ADM, Lu99 and Lu99B possess wild-type p53. EBC-1, PC9, LK2, Lu65, NCI H358, H522, H69, PC14, Lu135 and Lu65 possess mutated p53. NCI H1299 has deleted p53.22, 23, 24, 25, 26

Real-time RT-PCR

Total RNA was extracted from cells treated with adriamycin, siRNA or water using an RNeasy Mini Kit (Qiagen, Inc., Tokyo, Japan). For first-strand cDNA synthesis, 1 μg total RNA from a sample was added to components of the Super Script Preamplification System (Life Technologies, Inc., Gaithersburg, MD), as described in the user's manual. Real-Time PCR was performed using the Gene Amp 5700 Sequence Detection System (Perkin-Elmer), and mRNA expression was quantified. For this purpose, 1 μl cDNA was mixed with commercial reagents (TaqMan PCR Reagent Kit, Perkin-Elmer Biosystems), following the manufacturer's protocol. Survivin cDNA was amplified using a forward primer consisting of 5′-ATGGGTGCCCCGACGT-3′ and a reverse primer consisting of 5′-AATGTAGAGATGCGGTGGTCCTT-3′ and detected by a Taqman probe consisting of 5′- CCCCTGCCTGGCAGCCCTTTC-3′, each nucleotide corresponding to positions 50–65, 92–114 and 69–89 of the 1,619 bp survivin mRNA (GenBank NM001168). Relative quantification of gene expression was performed as described previously,27 using the housekeeping gene glyceraldehyde-3-phosphate (GAPDH) as an internal standard.

Western-blotting analysis

Cells treated with adriamycin, siRNA or water were harvested with trypsin/EDTA, and PBS-washed cell pellets were treated with HEPES lysate buffer (30 mM HEPES, 1% Triton X-100, 10% glycerol, 5 mM MgCl2, 25 mM NaF, 1 mM EDTA and 10 mM NaCl). Equal amounts of protein extracts were loaded onto sodium dodecyl sulfate-polyacrylamide gels and ran at 200 V for 45 min followed by transfer to nitrocellulose membranes at 100 V for 30 min. at room temperature. The membranes were probed with the following primary antibodies: affinity-purified rabbit anti-survivin antibody (R&D Systems, Inc., Minneapolis, MN), mouse monoclonal anti-p53 antibody (Santa Cruz Biotechnology, Inc., Santa Cruz, CA), rabbit anti-actin affinity isolated antibody (Sigma-Aldrich Co., St. Louis, MO) and mouse monoclonal anti-caspase3 antibody (Santa Cruz Biotechnology) at room temperature for 120 min. As secondary antibodies, goat anti-rabbit labeled with horseradish peroxidase (Amersham Biosciences, England) and sheep anti-mouse labeled with horseradish peroxidase (Santa Cruz Biotechnology) were used. Blots were developed using a chemiluminescence detection system (Perkin Elmer Life Sciences, Boston, MA).28

Flow cytometry

Cells were treated with adriamycin, harvested, washed with PBS, fixed with 70% methanol, washed with PBS and stained with propidium iodide solution (0.05 mg/ml propidium iodide, 0.1% Triton X-100, 0.1 mM EDTA and 0.05 mg/ml RNase A). Approximately 1 × 105 stained cells were analyzed by flow cytometry in a Becton Dickinson FACS calibur.28

siRNA transfection

The siRNA duplexes for survivin and p53 were synthesized by Dharmacon Research, Inc. (Lafayette, CO) using 2′-ACE protection chemistry. The siRNA targeting survivin corresponded to the coding region 206–404 relative to the start codon (GenBank NM001168). The siRNA targeting p53 corresponded to the coding region 775–793. BLAST searches of the human genome database were carried out to ensure the sequences would not target other gene transcripts. Cells in the exponential phase of growth were plated in 12-well tissue culture plate at 4 × 104 cells/well, grown for 24 hr and then transfected with 300 nM siRNA using oligofectamine and OPTI-MEM. Serum media (Invitrogen Life Technologies, Inc., Carlsbad, CA) were reduced according to the manufacturer's protocol. Gene silencing was examined with Western blotting 24–72 hr after transfection. Control cells were treated with siRNA duplex targeting scramble (Dharmacon). These studies were repeated 3 times and the data was presented as mean ± SE.

TUNEL assay

Cells were fixed in 4% paraformaldehyde (pH 7.4) and then stained and analyzed for apoptosis using an In Situ Cell Death Detection Kit, Fluorescein (Roche Diagnostics GmbH, Mannheim, Germany). Fixed cells were permeabilized using a mixture containing 0.1% sodium citrate and 0.1% TritonX100 and incubated with TUNEL reaction mixture containing terminal deoxynucleotidyltransferase and fluorescein-dUTP at 37°C for 60 min. Flow cytometric analysis using a FACS calibur was done to quantitate apoptosis.29

Cell viability analysis

Cells treated with adriamycin or transfected with siRNA duplex were washed with medium once and PBS twice, after staining with trypan blue.

Statistical analysis

All data are presented as mean ± SD or mean ± SE, and statistical analysis was done by Student's 2-tailed t-test (Stat View, SAS Institute, Inc.). Differences at p < 0.05 were considered significant.

Results

  1. Top of page
  2. Abstract
  3. Material and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

Survivin mRNA expression in lung cancer cell lines

The level of expression of survivin mRNA in the 22 human lung cancer cell lines was analyzed by TaqMan real-time PCR (Fig. 1). Normalization was performed using GAPDH as an internal control. Harvest and analysis of each cell line was repeated at least 3 times, and the mean and standard deviation for each cell lines is shown. All lung cancer cell lines expressed survivin mRNA, although the expression level varied. Among the 22 cell lines, the p53 status of 17 has been reported. The mean survivin expression of cells with wild-type p53, except for SBC3/ADM, tended to be less than that of cells with mutated or deleted p53 (p = 0.0192). Moreover SBC3/ADM, which is 8 times more adriamycin-resistant than SBC3 in terms of IC50, expressed about 3 times more survivin mRNA than did SBC3.

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Figure 1. Level of survivin mRNA in 22 lung cancer cell lines. (a) Cells were incubated in a 75 cm2 flask, harvested and analyzed using real-time PCR as described in Material and methods. All data were normalized relative to the concentration of mRNA for the housekeeping gene GAPDH and are presented as the mean ± SD for at least 3 independent experiments. p53 status is presented. (b) Comparison between SBC3 and SBC3/ADM, the adriamycin-resistant subline, is shown.

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Decrease of survivin expression after adriamycin exposure is dependent on functional p53 accumulation

To examine the p53 regulation of survivin expression, we monitored the expression of survivin protein in cells treated continuously with adriamycin at the IC50 dose by Western blotting (Fig. 2). Harvest, treatment and analysis of each cell line were repeated 3 times. The p53 phenotype of cell lines A549, NCI H460 and Lu99B has been reported previously as wild-type p53; PC9, PC14 and NCI H1299 possess mutant or deleted p53. In the cells with wild-type p53 (A549, H460 and Lu99B), p53 expression was induced 6 hr after adriamycin exposure and reached a peak level by 24 hr or later. Survivin protein expression was repressed for 72 hr after p53 accumulation (Fig. 2a). On the other hand, expression of survivin protein in cells with mutated or deleted p53 (PC9, PC14 and H1299) was not significantly decreased, and in fact appeared to be strongly increased in PC14 (Fig. 2b). Additionally, we analyzed survivin mRNA modification after adriamycin exposure using real-time PCR (Fig. 3). As was observed for the protein, the level of survivin mRNA showed a temporal decrease in all cell lines (A549, H460 and LU99B) containing wild-type p53. Repression of survivin mRNA in these cell lines started with accumulation of p53 during the first 6 hr (Fig. 3a). In contrast, in cell lines with mutated or deleted p53 (PC9, PC14 and H1299), survivin mRNA did not decrease throughout the period of adriamycin exposure. Furthermore, in cell line PC9, the level of survivin mRNA tended to increase (Fig. 3b).

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Figure 2. Expression of survivin and p53 protein in lung cancer cell lines with different p53 phenotypes following exposure to adriamycin for the indicated time. (a) Western-blotting analysis for expression of survivin and p53 in cell lines possessing wild-type p53, including A549, NCI H460 and LU99B. Each of the cell lines was incubated with adriamycin at the IC50 dose for the indicated time. Actin served as a control. Treatment, harvest and analysis were repeated 3 times. (c) Western-blotting analysis for expression of survivin and p53 in PC9 and PC14, possessing mutated p53, and in NCI H1299, possessing deleted p53. Each of the cell lines was incubated with adriamycin at the IC50 dose for the indicated time. Actin served as a control. Treatment, harvest and analysis were repeated 3 times. (b,d) Protein expression levels were presented as the mean±SD.

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Figure 3. Expression of survivin mRNA in lung cancer cell lines with different p53 phenotypes following exposure to adriamycin for the indicated time. Each of the cell lines with wild-type p53 (A549, NCI H460 and LU99B), mutated p53 (PC9 and PC14) or deleted p53 (NCI H1299) was incubated with adriamycin at the IC50 dose for the indicated time and analyzed by real-time PCR, as described in Material and methods. All data were normalized relative to the concentration of mRNA for the housekeeping gene GAPDH, and are presented as the mean ± SD for at least 3 independent experiments.

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Dependence of altered cell cycle distribution on p53 phenotype following exposure to adriamycin

In each of the cell lines treated with adriamycin, the cell cycle distribution was analyzed by flow cytometry (Fig. 4). It was found that the cell cycle distribution varied markedly depending on the p53 phenotype. That is, following exposure to adriamycin cells possessing wild-type p53 tended to show arrest in G1/S phase, whereas cells with mutated or deleted p53 became arrested in G2 phase. In cells containing wild-type p53, the G2/M peak tended to decline along with repression of survivin protein after 24 hr of adriamycin exposure, and the proportion of apoptotic cells (sub-G1) increased. On the other hand, in cells with mutated or deleted p53, the decline in the G2 peak was delayed in comparison with wild cells possessing wild-type p53, and only a small proportion of the cells became apoptotic after 24 hr of expression to adriamycin (Fig. 4).

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Figure 4. Cell cycle analysis of lung cancer cell lines with different p53 phenotypes after exposure to adriamycin. Each of the cell lines possessing wild-type p53 (A549, NCI H460 and LU99B), mutated p53 (PC9 and PC14) or deleted p53 (NCI H1299) was incubated with adriamycin at the IC50 dose for the indicated time and analyzed by flow cytometry as described in Material and methods.

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Inhibition of p53 using siRNA duplex, and resulting change in survivin expression

We examined whether wild-type p53 functionally regulates survivin, using the novel siRNA technique, which specifically inhibits p53. The siRNA duplex was designed to target coding region 775–793 after the start codon of p53. A549, a lung cancer cell line possessing wild-type p53, was transfected with siRNA duplex targeting p53, or scramble as a control, and the resulting levels of survivin expression were determined by Western blotting (Fig. 5a). All siRNA molecules have some intrinsic effect on treated cells. We compared cells treated with scrambled siRNA and cells treated with distilled water about p53 and survivin expression. In a result, there is not a significant difference between these. The siRNA duplex targeting p53 reduced p53 protein expression to 54% of the control level within 48 hr (Fig. 5b), and this was accompanied by an increase of survivin protein by as much as 2 times the control level (Fig. 5c).

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Figure 5. (a) Increasing survivin expression in A549 lung cancer cells possessing wild-type p53 as a result of p53 inhibition by siRNA duplex. A549 cells were treated with siRNA duplex targeting p53, scramble or distilled water and then 48 hr later, cell lysates were prepared from the siRNA-treated cells. (a) Expressions of p53, survivin and actin were analyzed by Western blotting. (b) The expression of p53 protein was analyzed densitometrically using a ChemiImager AlphaImager (ASTEC Co., Japan) and corrected relative to actin. (c) The expression of survivin protein was analyzed densitometrically using a ChemiImager AlphaImager and corrected relative to actin. All data are presented as the mean ± SD for at least 3 independent experiments. Statistical analysis was performed by Student's 2-tailed t-test. *p < 0.05 vs. cells treated with siRNA duplex targeting scramble.

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Inhibition of survivin expression by siRNA duplex inhibits cell proliferation and induces cell death

To evaluate the biological effect of survivin inhibition in lung cancer cell lines, transfection with siRNA duplex was performed. Cell line PC9, with mutated p53, was transfected with siRNA duplex targeting survivin or with that targeting scramble as a control. Scrambled siRNA did not have unspecific effect on survivin expression compared to distilled water in each point. It was found that expression of survivin protein was significantly repressed after transfection with anti-survivin, compared to the control (Fig. 6a,b). The level of survivin protein was reduced to 62% of the control within 48 hr and to 45% within 72 hr. We then counted the number of viable cells after siRNA transfection. As shown in Figure 6c, the repression of survivin had a direct effect on cell proliferation. At 48 hr post-siRNA, survivin repression significantly reduced the viable cell count to 45% of the scrambled siRNA treated cells (p < 0.05) and 47% of the control level at 72 hr (p < 0.05). Viable cell count of the scrambled siRNA treated cells was not different from distilled water treated cells in each point. In addition, apoptosis was induced to a greater extent by survivin repression, which is measured by the TUNEL assay (data not shown).

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Figure 6. Effects of siRNA targeting survivin on proliferation of PC9 lung cancer cells. PC9 cells were treated with siRNA duplex targeting surviving, scramble or distilled water. At the indicated time, the cells were harvested and assayed using the following procedure. (a) Expression of survivin and actin was analyzed by Western blotting, and actin was used as a control. (b) The expression of survivin protein was analyzed densitometrically using a ChemiImager AlphaImager, and corrected relative to actin. (c) Effect of siRNA targeting survivin (closed square), scramble (closed circle) or distilled water (closed triangle) on proliferation of PC9 cells. Cell proliferation was measured by counting the viable cells using trypan blue staining. All data are presented as the mean±S.E. for at least 3 independent experiments. Statistical analysis was performed by Student's 2-tailed t test. *p < 0.05 versus cells treated with siRNA duplex targeting scramble.

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Sensitization of lung cancer cell lines to adriamycin by siRNA targeting survivin

Based on the fact that cell lines with mutated or deleted p53 stably expressed survivin after exposure to adriamycin, we investigated the impact of survivin inhibition on adriamycin sensitivity in cells with mutated p53. Cell line PC9 possessing mutated p53 was transiently transfected with siRNA duplex targeting survivin, or with that targeting scramble as a control, for 48 hr. After the transfection, which significantly inhibited survivin expression, the medium was replaced and adriamycin at the IC50 dose, or water, was added. Adriamycin exposure was continued for 48 hr, and the cells were then harvested separately for Western blotting, viable cell assay, TUNEL assay and pro-caspase 3 assay. It was found that siRNA inhibited the expression of survivin by 57% at the start of adriamycin exposure and that survivin inhibition was weakened to 20% by 48 hr (data not shown). In terms of cell proliferation, anti-survivin siRNA duplex alone, adriamycin alone or a combination of both was significantly more repressive than anti-scramble siRNA followed by water, as a control (*p < 0.05, Fig. 7). That is, 48 hr after exposure to adriamycin or water, anti-survivin siRNA alone inhibited cell growth to 55% of the control, adriamycin alone reduced cell growth to 39%, and a combination of the 2r reduced cell growth to 21% of the control. Within 12 hr after exposure to adriamycin or water, exposure to anti-survivin siRNA or adriamycin alone did not significantly inhibit cell proliferation compared to the control; however the combination of the 2 significantly repressed cell proliferation to 44% of the control (*p < 0.05), and we compared anti-scrambled siRNA with distilled water followed by adriamycin or not. As a result, the scrambled siRNA effect on cell proliferation was small.

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Figure 7. Effects of siRNA duplex targeting of survivin on proliferation of PC9 lung cancer cells treated with adriamycin. PC9 cells were exposed to adriamycin or water after 48 hr transfection with siRNA duplex targeting surviving, scramble or distilled water. Open triangle:water after distilled water; open circle: water after transfection with siRNA duplex targeting scramble; open diamond: water after transfection with siRNA duplex targeting survivin; closed triangle: adriamycin after distilled water; closed circle: adriamycin after transfection with siRNA duplex targeting scramble; closed diamond: adriamycin after transfection with siRNA duplex targeting survivin. The data are presented as the mean±S.E. from 3 independent experiments. Statistical analysis was performed by Student's 2-tailed t-test. *p < 0.05 vs. cells treated with water after transfection with siRNA duplex targeting scramble. **p < 0.05 vs. other treatments.

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Induction of apoptosis in lung cancer cells by siRNA targeting survivin, and resulting sensitization to adriamycin

Additionally, we performed a TUNEL assay to evaluate apoptosis (Fig. 8). Cells were transfected with anti-scramble, anti-survivin siRNA duplex or distilled water for 48 hr and harvested for the assay 24 hr after exposure to adriamycin or water. Cells treated with water after anti-scramble were 5.1% TUNEL-positive, whereas cells treated with anti-survivin siRNA alone or adriamycin alone were 24.1% and 18.8% TUNEL-positive, respectively. Anti-survivin siRNA duplex induced significantly more apoptosis than that seen in the control (*p = 0.0298). Finally, the combination of anti-survivin siRNA duplex and adriamycin exposure resulted in 51.2% TUNEL-positivity, which was a significantly more potent effect than each of the other treatments (**p < 0.05). Intrinsic effect of scrambled siRNA on apoptosis was small, compared to cells treated with scrambled siRNA and cells treated with distilled water.

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Figure 8. Effects of siRNA targeting survivin on apoptosis of PC9 lung cancer cells treated with adriamycin, evaluated by TUNEL assay. PC9 cells were exposed to adriamycin or water for 24 hr after 48 hr transfection with duplex siRNA targeting surviving, scramble or distilled water. The data are presented as the mean±S.E. for 3 independent experiments. Statistical analysis was performed by Student's 2-tailed t-test, *p<0.05 vs. cells treated with anti-scrambled siRNA. **p<0.05 vs. cells treated with each of the other treatments.

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We additionally assessed procaspase-3 expressed in cells exposed to adriamycin after treatment with anti-scramble, anti-survivin siRNA duplex or distilled water (Fig. 9). It has already been reported that survivin potentially inhibits caspase-3 activation and inhibits apoptosis. The procaspase-3 level in the cells exposed to adriamycin and treated with anti-survivin siRNA decreased to 50% of the level in cells exposed to adriamycin followed by treatment with anti-scramble siRNA duplex. We treated distilled water to replace anti-scramble siRNA, and there is small effect on pro-caspase3 expression in anti-scrambled siRNA.

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Figure 9. Effects of siRNA targeting survivin on pro-caspase3 expression of PC9 lung cancer cells treated with adriamycin. PC9 cells were exposed to adriamycin for 24 hr after 48 hr transfection with duplex siRNA targeting survivin, scramble or distilled water, and each sample was analyzed by Western blotting. The data are presented as the mean ± S.E. for the 3 independent experiments. A representative blot is shown. Statistical analysis was performed by Student's 2-tailed t-test, *p < 0.05 vs. cells treated with other agents.

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Discussion

  1. Top of page
  2. Abstract
  3. Material and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

Survivin mRNA is expressed to various degrees in all of the 22 lung cancer cell lines used in our study. It has been reported that survivin mRNA is detectable in 85.5% of NSCLC tissue samples and that its expression level is correlated with poor prognosis.3 The mean survivin expression in 6 cell lines with wild-type p53, except for SBC3/ADM, tended to be low in comparison with the mean expression in 10 cell lines possessing mutant p53 (p = 0.019). There is no relationship between survivin expression and histology or origin of carcinoma (Table I). It has been reported that survivin expression is associated with accumulation of mutant p53 in gastric cancer and pancreatic carcinoma, assayed by immunohistochemical staining.30, 31 These data suggest that p53 might regulate survivin expression. In addition, after exposure to adriamycin, survivin expression show a transcriptional decrease following accumulation of wild-type p53. Adriamycin is generally classified as a topoisomerase II inhibitor that induces DNA double-strand breaks. The cellular response to DNA damage, which includes nuclear accumulation of p53, has been studied extensively using adriamycin. Thus, we used adriamycin in this study. In our study, p53 inhibition by siRNA duplex resulted in downregulation of survivin expression. The dependence of survivin repression on functional p53 has been investigated previously in a number of different cell models and cancer cell lines.14, 15 Although it is generally accepted that p53 activates a number of genes through direct interaction with their promoter DNA, the mechanism whereby p53 regulates survivin expression is still unclear.8 One possibility is that p53 might directly bind to the promoter of survivin and repress survivin transcription. In fact, a p53-binding motif is reported to exist within the promoter of survivin.14, 15 In contrast, Mirza et al.15 suggested that a p53-binding motif was not required for transcriptional repression of survivin. They suggested that chromatin deacetylation in the survivin promoter could contribute to p53-dependent repression of survivin gene expression. It is also possible that p53 might increase the level of another transcriptional regulator (e.g., p21) and indirectly downregulate survivin elsewhere downstream.11 In our study, both survivin and p53 expressions were low in 2 cell lines with wild-type p53 treated with adriamycin for 72 hr (Fig. 2a). It may be explained by indirect survivin regulation by another transcriptional factor. Z. Wang et al.32 previously showed that survivin post-translationally increased Mdm2 protein, and subsequently ubiquitination of p53, by blocking caspases that could cleave Mdm2 protein. We showed that p53 functionally repressed survivin expression. In our study, there is a possibility that survivin repression followed by adriamycin exposure might affect p53 accumulation in wild-type p53 cell lines. Survivin expression increased after adriamycin treatment in PC14 possessing mutant p53. Wall NR et al.33 also showed survivin protein increase in MCF7 following adriamycin treatment, and they suggested that survivin was phosphorylated by cdc2 and very little degraded by an ubiquitination-dependent mechanism.

Table I. Histology and Origin of Each Cell Line1
Cell LineHistologyOrigin
  • 1

    Ad: adenocarcinoma, Sq: squamous cell carcinoma, La: large cell carcinoma, Sm: small cell carcinoma, Metho: mesothelioma, Muc.: mucoepidermoid carcinoma, Prim.: primary, Lym.: lymph node, Effu.: effusion.

LU99LaPrim.
A549AdPrim.
EBC1SqPrim.
MA-46SqEffu.
RERF-LC-KJAdPrim.
OBALK1LaEffu.
Lu99BLaEffu.
PC9AdPrim.
SBC3SmPrim.
NCI-H292MucPrim.
LK-2SqPrim.
LU65LaPrim.
NCI-H358AdPrim.
PC14AdPrim.
Sq1SqPrim.
NCI-H226MethoEffu.
NCI-H460LaEffu.
NCI-H522AdPrim.
Lu 135SmPrim.
NCI-H1299LaLym.
NCI-H69SmPrim.

Investigation of cell cycle distribution after exposure to adriamycin has shown that cells possessing wild-type p53 tend to become arrested in G1 phase. In these cell lines, transcriptional p21 activation generally leads to G1 arrest. Additionally, we found G2/M phase repression and apoptosis progression accompanying repression of survivin protein. It has been reported previously that transfection with survivin anti-sense or dominant negative survivin gene resulted in accumulation of apoptotic cells and concomitant loss of G2/M phase cells.34, 35 Li et al.7 showed that cells transfected with a mutant survivin gene or survivin anti-sense appeared to show increased caspase3 activity when synchronized in G2/M phase but not in G1/S phase. We therefore analyzed the cell cycle distribution of cell lines possessing mutated or deleted p53. In contrast to cells with wild-type p53, these cells became arrested in G2/M phase. Thus, survivin retention in cells possessing mutant p53 might make them able to resist apoptosis at the G2/M checkpoint.

One critical point of our study was to investigate differences in the proliferation of cancer cells following survivin repression, with the expectation that survivin inhibition itself would have a potent anti-proliferation effect. In cells possessing mutated or deleted p53, survivin was stably expressed even after adriamycin exposure and cell cycle arrest at the G2/M phase, indicating an anti-apoptotic effect. Survivin inhibition by siRNA downstream of p53 induced cell apoptosis and enhanced the anti-proliferative effect. Survivin associates with microtubules of the mitotic spindle at the beginning of mitosis, and disruption of survivin-microtubule interactions increases caspase-3 activity.7 In order to inhibit survivin specifically, we used siRNA. This efficiently repressed survivin expression and inhibited cell proliferation in the absence of any cytotoxic stimulus. It has been reported that antisense targeting of survivin induces apoptosis in lung cancer cells. Using TUNEL assay, we also confirmed that anti-survivin siRNA duplex induced apoptosis.

Finally, survivin inhibition was found to sensitize PC9 to an anti-cancer agent. Exposure to Adriamycin after repression of survivin by siRNA significantly inhibited cell proliferation compared to cells exposed to either adriamycin alone or anti-survivin siRNA alone. Data obtained by the TUNEL assay confirmed that the difference in cell proliferation was based on apoptosis. In vitro binding experiments have indicated that survivin specifically binds to caspase-3 and -7, but not to caspase-8.6 We also identified repression of procaspase-3 (which means activation of caspase-3) in cells exposed to adriamycin after treatment with anti-survivin siRNA. Activation of caspase-3 by inhibition of survivin may thus promote sensitivity to adriamycin. In our study, the expression of survivin mRNA in SBC3/ADM cells was greater than that in the parental SBC cells (Fig. 1b), indicating that survivin expression is related to cell resistance to adriamycin. We identified survivin inhibition by siRNA in cells with mutated p53 sensitized to adriamycin. Combining transfection with a mutant survivin gene with exposure to adriamycin did not enhance apoptosis in HeLa cells and MCF-7 cells, which have wild-type p53, compared to a mutant survivin gene transfection alone or adriamycin alone.36 The combined effect of the two against apoptosis may be dependent on the character of each cell type, including p53 status or the compound targeting survivin. Additional studies will be needed to determine the combined effect of survivin inhibition and other drugs on other cell lines.

In conclusion, siRNA targeting survivin could be of potential value for increasing the sensitivity of cancer cells to anti-cancer drugs, especially drug-resistant cells that possess mutated p53.

Acknowledgements

  1. Top of page
  2. Abstract
  3. Material and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

We thank E. Hatashita, Y. Yamada, T. Wada and M. Nagasaka for experimental assistance. This investigation was selected for a Scholar-in-Training Award at the 95th Annual Meeting of the AACR.

References

  1. Top of page
  2. Abstract
  3. Material and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References
  • 1
    Brown JM, Bradly G. Wouters. Apoptosis, p53, and tumor cell sensitivity to anticancer agents. Cancer Res 1999: 59: 13919.
  • 2
    Ambrosini G, Adida C, Altieri DC. A novel anti-apoptosis gene, survivin, expressed in cancer and lymphoma. Nat Med 1997: 3: 91721.
  • 3
    Monzo M, Rosell R, Felip E, Astudillo J, Sanchez JJ, Maestre J, Martin C, Font A, Barnadas A, Abad A. A novel anti-apoptosis gene: re-expression of survivin messenger RNA as a prognosis marker in non-small-cell lung cancers. J Clin Oncol 1999: 17: 21004.
  • 4
    Kawasaki H, Altieri DC, Lu CD, Toyoda M, Tenjo T, Tanigawa N. Inhibition of apoptosis by survivin predicts shorter survival rates in colorectal cancer. Cancer Res 1998: 58: 50714.
  • 5
    Meng H, Lu CD, Sun YL, Dai DJ, Lee SW, Tanigawa N. Expression level of wild-type survivin in gastric cancer is an independent predictor of survival. World J Gastroenterol 2004: 10: 324550.
  • 6
    Tamm I, Wang Y, Sausville E, Scudiero DA, Vigna N, Oltersdorf T, Reed JC. IAP-family protein survivin inhibits caspase activity and apoptosis induced by Fas (CD95), Bax, caspases, and anticancer drugs. Cancer Res 1998: 58: 531520.
  • 7
    Li F, Ambrosini G, Chu EY, Plescia J, Tognin S, Marchisio PC, Altieri DC. Control of apoptosis and mitotic spindle checkpoint by survivin. Nature 1998: 396: 5804.
  • 8
    Ferrira CG, Tolis C, Giaconne G. p53 and chemosensitivity. Ann Oncol 1999: 10: 101121.
  • 9
    Lowe SW, Bodis S, McClatchey A, Remington L, Ruley HE, Fisher DE, Housman DE, Jacks T. p53 status and the efficacy of cancer therapy in vivo. Science 1994: 266: 80710.
  • 10
    El-Deiry WS, Tokino T, Velculescu VE, Levy DB, Parsons R, Trent JM, Lin D, Mercer WE, Kinzler KW, Vogelstein B. WAF1, a potential mediator of p53 tumor suppression. Cell 1993: 75: 81725.
  • 11
    Lohr K, Moritz C, Contente A, Dobbelstein M. p21/CDKN1A mediates negative regulation of transcription by p53. J Biol Chem 2003: 29: 3250716.
  • 12
    Ahn J, Murphy M, Kratowicz S, Wang A, Levine AJ, George DL. Down-regulation of the stathmin/Op18 and FKBP25 genes following p53 induction. Oncogene 1999: 18: 59548.
  • 13
    Yun J, Chae HD, Choy HE, Chung J, Yoo HS, Han MH, Shin DY. p53 negatively regulates cdc2 transcription via the CCAAT-binding NF-Y transcription factor. J Biol Chem 1999: 274: 2967782.
  • 14
    Hoffman WH, Biade S, Zilfou JT, Chen J, Murphy M. Transcriptional repression of the anti-apoptotic survivin gene by wild type p53. J Biol Chem 2002: 277: 324757.
  • 15
    Mirza A, McGuirk M, Hockenberry TN, Wu Q, Ashar H, Black S, Wen SF, Wang L, Kirschmeier P, Bishop WR, Nielsen LL, Pickett CB. et al. Human survivin is negatively regulated by wild-type p53 and participates in p53-dependent apoptotic pathway. Oncogene 2002: 21: 261322.
  • 16
    Carter BZ, Wang RY, Schober WD, Milella M, Chism D, Andreeff M. Targeting Survivin expression induces cell proliferation defect and subsequent cell death involving mitochondrial pathway in myeloid leukemic cells. Cell Cycle 2003: 2: 48893.
  • 17
    Olie RA, Simoes-Wust AP, Baumann B, Leech SH, Fabbro D, Stahel RA, Zangemeister-Wittke U. A novel antisense oligonucleotide targeting survivin expression induces apoptosis and sensitizes lung cancer cells to chemotherapy. Cancer Res 2000: 60: 28059.
  • 18
    Elbashir SM, Harborth J, Lendeckel W, Yalcin A, Weber K, Tuschl T. Duplexes of 21-nucleotide RNAs mediate RNA interference in cultured mammalian cells. Nature 2001: 411: 4948.
  • 19
    Brantl S. Antisense-RNA regulation and RNA interference. Biophysica Acta 2002: 1575: 1525.
  • 20
    Fukumoto H, Nishio K, Ohta S, Hanai N, Fukuoka K, Ohe Y, Sugihara K, Kodama T, Saija N. et al. Effect of chimeric antiganglioside GM2 antibody on ganglioside GM2-expressing human solid tumors in vivo. Int J Cancer 1999: 82: 75964.
  • 21
    Mosmann T. Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assays. J Immunol Methods 1983: 65: 5563.
  • 22
    Hensel CH, Xiang RH, Sakaguchi AY, Naylor SL. Use of the Single Strand Conformation Polymorphism Technique and PCR to Detect p53 Gene Mutations in Small Cell Lung Cancer. Oncogene 1991: 6: 106771.
  • 23
    Mitsudomi T, Steinberg SM, Nau MM, Carbone D, Damico D, Bodner S, Oie HK, Linnoila RI, Mulshine JL, Minna JD, Gazdar AF. p53 Gene mutations in non-small-cell lung cancer cell lines and their correlation with the presence of ras mutations and clinical features. Oncogene 1992: 7: 17180.
  • 24
    Kashii T, Mizushima Y, Monno S, Nakagawa K, Kobayashi M. Gene analysis of K-ras, H-ras, p53, and retinoblastoma susceptibility genes in human lung cancer cell lines by the polymerase chain Reaction/Single-Strand conformation polymorphism method. J Cancer Res Clin Oncol 1994: 120: 1438.
  • 25
    Jia LQ, Osada M, Ishioka C, Gamo M, Ikawa S, Suzuki T, Shimodaira H, Niitani T, Kudo T, Akiyama M, Kimura N, Matsuo M, et al. Screening the p53 status of human cell lines using a yeast functional assay. Mol Carcinogen 1997: 19: 24353.
  • 26
    Fujita T, Kiyama M, Tomizawa Y, Kohno T, Yokota J. Comprehensive analysis of p53 gene mutation characteristics in lung carcinoma with special reference to histological subtypes. Int J Oncol 1999: 15: 92734.
  • 27
    Yoshida M, Suzuki T, Komiya T, Hatashita E, Nishio K, Kazuhiko N, Nakagawa K, Fukuoka M. Induction of MRP5 and SMRP mRNA by adriamycin exposure and its overexpression in human lung cancer cells resistant to adriamycin. Int J Cancer 2001: 94: 4327.
  • 28
    Tamura K, Southwick EC, Kerns J, Rosi K, Carr BI, Wilcox C, Lazo JS. Cdc25 inhibition and cell cycle arrest by a synthetic thioalkyl vitamin K analogue. Cancer Res 2000: 60: 131725.
  • 29
    Sgonc R, Gruber J. Apoptosis detection: an overview. Exp Gerontol 1998: 33: 52533.
  • 30
    Lu CD, Altieri DC, Tanigawa N. Expression of a novel antiapoptosis gene, survivin, correlated with tumor cell apoptosis and p53 accumulation in gastric carcinomas. Cancer Res 1998: 58: 180812.
  • 31
    Sarela AI, Verbeke CS, Ramsdale J, Davies CL, Markham AF, Guillou PJ. Expression of survivin, a novel inhibitor of apoptosis and cell cycle regulatory protein, in pancreatic adenocarcinoma. Br J Cancer 2002: 86: 88692.
  • 32
    Wang Z, Fukuda S, Pelus LM. Survivin regulates the p53 tumor suppressor gene family. Oncogene 2004: 23: 814653.
  • 33
    Wall NR, O'Connor DS, Plescia J, Pommier Y, Altieri DC. Suppression of survivin phosphorylation on Thr34 by flavopiridol enhances tumor cell apoptosis. Cancer Res 2003: 63: 2305.
  • 34
    Ambrosini G, Adida C, Sirugo G, Altieri DC. Induction of apoptosis and inhibition of cell proliferation by survivin gene targeting. J Biol Chem 1998: 273: 1117782.
  • 35
    Grossman D, Kim PJ, Schechner JS, Altieri DC. Inhibition of melanoma tumor growth in vivo by survivin targeting. Proc Natl Acad Sci U S A 2001: 98: 63540.
  • 36
    Mehdi Mesri, Nathan R. Wall, Jia Li, Richard W. Kim, Dario C. Altieri. Cancer gene therapy using a suvivin mutant adenovirus. J Clin Inv 2001: 108: 98190.