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Logo of nihpaAbout Author manuscriptsSubmit a manuscriptHHS Public Access; Author Manuscript; Accepted for publication in peer reviewed journal;
 
Cancer Gene Ther. Author manuscript; available in PMC 2014 February 6.
Published in final edited form as:
PMCID: PMC3915357
NIHMSID: NIHMS536775

Enhanced tumor suppression in vitro and in vivo by co-expression of survivin-specific siRNA and wild-type p53 protein

Abstract

The development of malignant prostate cancer involves multiple genetic alterations. For example, alterations in both survivin and p53 are reported to have crucial roles in prostate cancer progression. However, little is known regarding the interrelationships between p53 and survivin in prostate cancer. Our data demonstrate that the expression of survivin is inversely correlated with that of wtp53 protein (rs=0.548) in prostate cancer and in normal prostate tissues. We have developed a therapeutic strategy, in which two antitumor factors, small interfering RNA-survivin and p53 protein, are co-expressed from the same plasmid, and have examined their effects on the growth of PC3, an androgen-independent prostate cancer cell line. When p53 was expressed along with a survivin-specific short hairpin RNA (shRNA), tumor cell proliferation was significantly suppressed and apoptosis occurred. In addition, this combination also abrogated the expression of downstream target molecules such as cyclin-dependent kinase 4 and c-Myc, while enhancing the expression of GRIM19. These changes in gene expression occurred distinctly in the presence of survivin-shRNA/wtp53 compared with control or single treatment groups. Intratumoral injection of the co-expressed construct inhibited the growth and survival of tumor xenografts in a nude mouse model. These studies revealed evidence of an interaction between p53 and survivin proteins plus a complex signaling network operating downstream of the wtp53-survivin pathway that actively controls tumor cell proliferation, survival and apoptosis.

Keywords: prostate cancer, p53, survivin, siRNA

Introduction

Prostate cancer is a leading cause of cancer death in men, with an estimated 28 660 deaths in 2008; an estimated 192 280 new cases will occur in the United States during 2009.1 When the disease is confined to the prostate, it can be cured by radical prostatectomy or radiation therapy. However, there are no curative options for locally advanced, recurrent or metastatic complications of prostate cancer.2 There is, therefore, a need to investigate alternative treatment strategies to improve these outcomes. Cell growth and cell death are usually determined by a balance between the activities of oncogenes and tumor-suppressor genes. P53 is a well-recognized tumor suppressor. Inactivating mutations in p53 are frequently observed in several cancers; mutations in p53 occur in approximately 70% of hormone-refractory prostate cancer.3 Thus, restoration of wild-type p53 (wtp53) function in tumors is an attractive therapeutic strategy.4,5 However, in the face of several activated oncogenes in tumors, restoration of p53 alone may be insufficient for reducing growth of the tumor. Therefore, it may also be worthwhile to inactivate the oncogenic protein expression while augmenting the wtp53 driven antitumor activity. One major problem with advanced tumors is their resistance to cytototoxic therapy via apoptosis. Survivin, a member of the inhibitor of apoptosis protein family has been found to exert anti-apoptotic effects and is overexpressed in the majority of human tumors, including prostate cancer.6,7 Accumulating evidence suggests that survivin has an important role in the survival of hormone-refractory prostate cancer. Activation and abnormal expression of survivin in prostate cancer decrease apoptosis,8 which results in the generation and progression of the cancer. Survivin overexpression and loss of wtp53 function occurs in many cancers. However, little is known regarding the interrelationships between these proteins in the regulation of prostate cancer growth. It has been previously reported that inhibition of survivin expression using RNA interference (RNAi) suppressed the growth of androgen-independent prostate cancer cells,9,10 whereas transient overexpression of p53 induced multi-nucleation and resulted in suppression of tumor cell growth as well.11,12

In this study, we have developed a pre-clinical model for studying the effects of co-expressed p53 protein and survivin-specific small interfering RNA (siRNA) on PC3, a p53/AR prostate cancer cell line, which is a representative model of late stage hormone refractory disease. Our data suggest that survivin may bind to wtp53 protein and alter its conformation, function and half-life. We show here that RNAi-mediated inactivation of survivin in combination with a restoration of wtp53 is an effective strategy for suppressing tumor growth. The abrogation of survivin combined with a concurrent expression of p53 protein not only inhibited proliferation strongly but also induced apoptosis thereby reducing tumorigenicity.

Materials and methods

Immunohistochemical analyses of p53 and survivin in patient normal tissue versus prostate cancer specimens

We used 26 samples of normal prostate tissue obtained from nontumorous areas of prostatectomy specimens. Another 30 samples of cancerous tissue were derived from radical prostatectomy specimens from patients with organ-confined disease with no previous therapy. Tumors were collected from Second Hospital of Inner Mongolia University for the Nationalities and Prostate Disease Prevention and Treatment Research Center (Jilin University, China), with the informed consent of the patients under an institutionally approved protocol. After formalin fixation and paraffin embedding, the tissue sections were stained with 20 μgml−1 of rabbit anti-p53 or anti-survivin antibodies (Santa Cruz Biotech, Inc., Santa Cruz, CA). Immunohistochemical staining was performed using Vectastain Elite ABC peroxidase kits (Vector Labs, Burlingame, CA). wtp53 is very labile, and normally difficult to detect by immunohistochemistry, whereas strong immunohistochemical staining of p53 has been reported to reflect the presence of stable, nonfunctional mutant p53 protein.13 Alternatively, this immunohistochemically detectable p53 may be a conformationally altered nonmutant protein that is functionally inactive and has an increased half-life. Therefore, samples exhibiting strong immunohistochemical staining were defined as expressing altered p53 (that is, a conformationally stabilized p53), as previously reported.14,15 Stained tissues were screened independently by two investigators and classified according to staining intensity (− or +). Positive expression consisted of brown particles in the nucleus or cytoplasm. We considered the tissue to be positive (+) if the staining intensity was moderate to strong in >10% of cells examined. Weakly stained (<10% of total cells examined) or non-immunoreactive cells were considered negative (−). Positive–negative reactions were determined after counting cells in five independent high-power fields (400×) of each sample.

Construction of a plasmid vector capable of co-expressing siRNA survivin and p53

As shown in Figure 1, we constructed a series of expression plasmids that contain, individually or in combination, short hairpin RNA (shRNA) specific to survivin and p53 expression elements. The p53 coding region with a Kozak sequence was amplified by PCR from the pQE40-p53 plasmid (provided by Dr Xichun Liu, Department of Pathophysiology, Jilin University) as template using the following primers: forward: 5′-GGGGTACCGCCACCATGGAGGAGCCGCAGTCAG-3′; reverse: 5′-GGAATTCTCAGTCTGAGTCAGGCCCTT CTG-3′. The underlined nucleotides in these primers correspond to the KpnI and EcoRI recognition sites, respectively. The resultant PCR product was cleaved with Kpn1 and EcoR1 and inserted into pcDNA3.1(+) (Invitrogen, Carlsbad, CA) under the control of the cytomegalovirus promoter to generate pcDNA3.1-p53(Pp53). A 585-bp fragment, containing an shRNA specific for survivin (Sh-survivin) under the control of U6 promoter was PCR amplified from the plasmid pGCsiliencer1.0-U6 (provided by Dr Xichun Liu) using the following primers: forward: 5′-GCAGATCTTGCTTCG CGATGTACGGGCC-3′; reverse: 5′-CGTCGCGAGGG CTATGAACTAATGACCC-3′. The underlined nucleotides in these primers correspond to the BglII and NruI sites, respectively. This cleaved fragment was sub-cloned into the BglII and NruI sites of the Pp53 vector to generate the plasmid pcDNA3.1-p53/U6 Sh-survivin, which expresses both siRNA-survivin and the p53 protein, from the same plasmid, and was designated as PSh-sur-p53. The shRNA produced from this plasmid targets base coordinates 394 to 412 of survivin mRNA (based on Genbank accession no: NM001168). All recombinant plasmids were verified by DNA sequencing (Shenggong Bioengineering Ltd, Shanghai, China).

Figure 1
Expression vector inserts used in this study. The U6 promoter directs the expression of survivin-specific shRNA and the cytomegalovirus (CMV) promoter drives the expression of p53 protein.

Cell culture and transfection

Human prostate cancer cell line PC3 was grown in Iscove’s modification of Dulbecco’s medium (Hyclone, South Logan, UT) supplemented with 10% fetal bovine serum (Gibco/Invitrogen, Paisley, UK). Transfections were performed using the Lipofectamine 2000 reagent (Invitrogen). After transfection, cells were used for proliferation assays, cell cycle detection, reverse transcription-PCR (RT-PCR) and immunoblot analyses. Transfection efficiency was determined using flow cytometry. Cells transfected with the scrambled shRNA vector, which has no significant homology to mouse or human shRNA gene sequences, was used as a control.

Cell proliferation assay

Approximately 6×103 PC3 cells per well were transfected with the plasmids of interest, and cell growth was evaluated after 48 and 72 h using the MTT (3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide) assay. MTT (5mg ml−1) was added to the wells, and the plates were incubated for 4 h at 37 °C. The MTT reaction was stopped by adding of dimethyl sulphoxide (150 μl per well). Well contents were mixed for 10 min vigorously before measuring absorbance at 570nm (A570) in a multiwell plate reader. Cell growth inhibition was calculated using the following formula:

equation M1

A570e and A570c correspond to the absorbance obtained with the experimental and control groups, respectively.

Reverse transcription-PCR

For RT-PCR analysis after 48 h of transfection, cells were collected and total RNA was extracted using the Trizol Reagent (Invitrogen). Approximately, 5 μg of total RNA (purified after DNAse I treatment) from each sample was converted to complementary DNA using a commercially available RT-PCR kit (Promega, Madison, WI). The resultant complementary DNAs were used in PCR reactions with gene-specific primers and the products were analyzed using gel electrophoresis.

Immunoblot analyses

Cells were lysed using a buffer as described previously.16 After centrifugation at 15 000 g for 30 min, the supernatant was collected. 30–50 μg of cell extracts were separated on 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to polyvinylidene difluoride membranes (Millipore Corp., Bedford, MA). Membranes were blocked for 1 h with 5% nonfat dry milk in TBST (Tris-buffered saline with 0.1% Tween-20) and subsequently incubated overnight at 4 °C with the indicated primary antibodies diluted in TBST. After three TBST washes, ECL reagents (Amersham Pharmacia Biotech, Amersham, UK) were used for detecting protein bands. The following antibodies (Santa Cruz Biotech, Inc.) were used: anti-p53 (sc-6243), anti-survivin (sc-10811), anti-cyclin-dependent kinase 4 (CDK4) (sc-260), anti-β-actin (sc-130656). Anti-GRIM19 antibodies were kindly provided by Dr Jiadi Hu (University MD Dental School, Baltimore, MD).17 Band intensities were determined densitometrically and the results are shown as relative expression. Each experiment was conducted three separate times in duplicate and the results represent the mean±s.e. (n=3).

Flow cytometry

The procedure was performed as described previously,16 Cells (1×106) were collected and washed with cold phosphate-buffered saline (PBS) containing EDTA (4 mmol l−1). Cells were fixed in 900 μl of 70% cold ethanol, collected by centrifugation and washed once again with PBS containing EDTA (4 mmol l−1). Cells were re-suspended in PBS (containing acetic acid 4 mmol l−1, and propidium iodide 20 ml l−1, 0.2% Triton X-100, 40 mg l−1 RNase A), and incubated for at least 30 min at 4 °C. The cells were then subjected to flow cytometry (FACScan, Becton Dickinson, Franklin Lakes, NJ), and data were analyzed using Cell Quest software.

Tumor xenografts

Athymic nude male mice (nu/nu; 6–8 weeks) were acquired from the Institute of Zoology, Chinese Academy of Sciences, Beijing and used in accordance with an institutionally approved Animal Care and Use protocol. To determine the effect of recombinant plasmids on tumor cell growth, PC3 cells (5×106 per 150 μl) were inoculated subcutaneously into the right flank of nude mice, and establishment of palpable tumors was determined. Tumors were measured using calipers every second day for 17 days. On reaching ~157.56±28.54mm3 (on day 17) tumor-bearing mice (n=5 per group) were divided into five groups. Mice in each of these groups were treated with: (1) mock transfection (PBS buffer alone); (2) scrambled shRNA-vector alone (Sh-scramble); (3) U6-survivin (Shsurvivin); (4) pcDNA3.1-p53 (Pp53); or (5) pcDNA3.1-p53/U6 Sh-survivin (PSh-sur-p53). As previously described,16 equal amounts of expression vectors (20 μg per mice) were prepared in 50 μl of PBS buffer and injected subcutaneously into the tumor by using a syringe fitted with a 27-gauge needle. Immediately after injection, tumors were pulsed with an electroporator ECM 830 (BTX, San Diego, CA). Pulses were delivered at a frequency of 1 s−1, 200Vcm−1, with a length of 20ms. This process was repeated on days 21 and 27. Mice were killed on day 35. Tumor growth was measured as wet weight, and tumor size was photographed. A portion of each tumor was fixed in formalin and embedded in paraffin for immunohistochemical analyses. The remaining tissue was snap frozen in liquid nitrogen immediately for RT-PCR, immunoblot and terminal deoxyuridyl transferase-mediated nick end labeling (TUNEL) assay (Roche, Penzberg, Germany).

Statistical analysis

Statistical comparisons were performed using analysis of variance to determine the significance of differences observed between different treatment groups. Values are expressed as mean±s.d. from at least three separate experiments in which each experiment had five samples per treatment group and differences were considered significant at a P-value of <0.05.

Results

p53 and survivin are overexpressed in primary human prostate cancers

To determine whether loss of p53 function and over-expression of survivin occur in human prostate cancers, we subjected normal (n=26) and prostate cancer (n=30) tissue samples to immunohistochemical analyses with p53- and surivivin-specific antibodies. The analyses showed a strong overexpression of both p53 and survivin proteins in prostate cancer tissues. This was in contrast to normal prostate tissues, which had very weak signals. The increased p53 protein expression might represent p53 conformational changes that increase p53 half-life or represent mtp53. wtp53 has a short half-life and cannot be detected using immunohistochemical assay.18 As summarized in Figure 2 and Table 1, the high p53 and survivin levels found in prostate tumor specimens were significantly different from the lower expression in normal prostate tissues (P<0.001). Further, the expression of survivin strongly correlated with the presence of stable p53 protein in prostate cancer tissue, as estimated by Spearman analysis (rs=0.548).

Figure 2
Immunohistochemical analysis of p53 and survivin expression in normal and prostatic cancer tissues (×200).
Table 1
Immunohistochemical analysis of survivin and p53 expression in normal prostatic tissue and prostatic cancer tissue

Efficient knockdown of siRNA survivin and enhanced p53 gene expression in human PC3 prostate cancer cells

As there is a strong correlation between the loss of p53 function and a rise in survivin levels in prostate cancer tissue, we next determined whether a restoration of their normal levels would suppress tumor cell growth. To this end, we constructed a plasmid that could simultaneously silence survivin mRNA and express wtp53 (Figure 1). With 61.4% transfection efficiency determined by flow cytometry (data not shown), we first tested its effect on survivin and p53 expression in a surrogate prostate tumor cell line PC3. As shown in Figure 3, analysis of the mRNA derived from Pp53, Sh-survivin and PSh-sur-p53 transfected cells using RT-PCR (Figures 3a and b) and immunoblot (Figures 3c and d) demonstrated a specific reduction in survivin mRNA and protein levels. P53 mRNA levels increased in the presence of specific Sh-survivin, Pp53 and PSh-sur-p53 relative to the scrambled shRNA-transfected cells or mock-transfected cells. However, p53 protein expression was decreased in cells transfected with Sh-survivin, relative to Pp53 or PShsur-p53 treatments.

Figure 3
(a) RT-PCR and (c) immunoblot analysis of p53 and survivin in PC3 cells transfected with recombinant plasmids. Quantification of p53 and survivin mRNA (b) and protein levels (d), respectively, from three separate experiments and normalized to mRNA of ...

Co-expression of survivin shRNA and wtp53 inhibits cell proliferation and induces apoptosis

To assess the potential effects of RNAi-mediated silencing of survivin combined with p53 protein overexpression on cell proliferation and survival, a MTT assay was performed 48 and 72 h after transfection with different plasmids. A dramatic reduction of PC3 cell proliferation was observed on co-expression of p53 and survivin shRNA (Figure 4). Although survivin shRNA or wtp53 overexpression each had an inhibitory effect on the proliferation of cells, compared with the scramble control and non-treated control groups, they were not as efficient as PSh-sur-p53. After transfection, the percentage of viable cells in the presence of Pp53, Sh-survivin and PSh-sur-p53 were approximately 58.5, 56.8 and 40.3% compared with the empty vector-transfected control after 48 h, respectively. At 72 h, these percentages decreased further to 42, 43.5 and 26.4%, in the presence of Pp53, Sh-survivin and PSh-sur-p53, respectively. Thus, co-expression of shRNA-survivin and p53 dramatically suppressed cell growth.

Figure 4
Growth inhibitory ratios for PC3 cells transfected with different plasmids, averaged for three separate experiments. *P<0.05, **P<0.01 versus mock or Sh-scramble controls; #P<0.05 versus Pp53 or Sh-survivin groups.

Sh-survivin/Pp53 suppresses cell growth by increasing the accumulation of cells in the G1 phase

To determine whether growth inhibition was associated with specific accumulation of cells in a specific phase of the division cycle, a flowcytometric analysis of cell cycle distribution was performed after transfecting different plasmids (described above) into PC3 cells. P53- or Sh-survivin-treated cells arrested in G0/G1 phase, with a decline of G2/M peak. PSh-sur-p53 caused a significant reduction in the number of cells in S phase and a corresponding increase in the number of cells in G0/G1 phase compared with other groups (Table 2).

Table 2
Cell cycle analysis in transfected PC-3 cells

PSh-sur-p53-induced significant changes in GRIM-19 expression

Our recent studies have shown a loss of GRIM-19 expression in a number of human tumors including prostate cancer.19 GRIM19 is a pro-apoptotic gene product encoded by a gene located in the 19p13.2 region of the human chromosome essential for prostate tumor suppression.17,20 We hypothesized that expression of shRNA-survivin and wtp53 would change the cellular GRIM-19 expression to promote growth arrest. Therefore, we checked to determine if these growth suppressive effects are a result of a change in GRIM-19 expression. As shown in Figures 5a and b, GRIM19 expression was significantly upregulated in Pp53 and PSh-sur-p53 groups compared with Sh-survivin and the two control groups. We also detected the oncogenes c-Myc and CDK4 in these transfected samples. As expected, transcripts for both c-Myc and CDK4 were significantly decreased in the PSh-sur-p53 group. This corresponded with similar trends observed at the protein level (Figures 5c and d).

Figure 5
Expression of GRIM19, c-Myc and CDK4 in PC3 cells transfected with different plasmids. (a) RT-PCR analysis of GRIM19, c-Myc and CDK4 mRNAs. (c) Immunoblot analysis for GRIM19, CDK4. (b, d) Quantification of the images in (a) and (c). *P<0.05,** ...

Knockdown of survivin by RNAi and enhanced p53 expression inhibit downstream signaling and tumorigenesis in nude mice

To evaluate the effects of co-expressed Sh-survivin and p53 on prostate tumor growth in vivo, we used a nude mouse tumor xenograft model. Animals were killed on day 35 and tumor weights were determined. Sh-survivin or Pp53 alone reduced tumor volume relative to the two control groups. The difference in tumor weight between both Pp53 and Sh-survivin was not statistically significant (P>0.05). The group treated with PSh-sur-p53 had marked tumor growth suppression compared with the scrambled siRNA control (P<0.01) (Figure 6e).

Figure 6
(a) TUNEL assay for PC3 xenografts treated with recombinant plasmids. (×200) (b, c) Immunohistochemical analysis of survivin and p53 expression for PC3 xenografts treated with recombinant plasmids (×200). (d) Images and (e) weight of PC3 ...

The tumor volume of mice treated with buffer or empty vector control was 947.38±90.50mm3 on day 35. The tumor volume in mice treated with scrambled siRNA control was 905.09±92.15mm3, and that of the group treated with survivin siRNA was 609.8± 65.57mm3, the group treated with Pp53 was 564.83± 64.16 and PSh-sur-p53 was 286.25±40.25mm3 (Figure 6d).

To determine whether the transfected plasmids were actively expressed in tumor cells, we examined p53 or survivin transcripts using RT-PCR and immunoblot. As shown in Supplementary Figures S1a and b, levels of survivin transcripts in Pp53-, Sh-survivin- and PSh-surp53-treated tumors were significantly lower compared with groups treated with scramble and empty vector. In contrast, p53-specific mRNA expression was increased in the Pp53, Sh-survivin and PSh-sur-p53 groups. The immunoblot analyses were generally consistent with the RT-PCR data, except for the Sh-survivin-transfected group in which p53 levels showed no remarkable changes compared with the control groups (Supplementary Figures S1c and d).

To determine the potential mechanism of tumor growth inhibition in vivo, PC3 tumors treated with PSh-sur-p53, survivin siRNA, Pp53, scrambled siRNA control or mock control were excised and analyzed by TUNEL and immunohistochemical staining. The experiments showed that PSh-sur-p53-treated tumors had undergone massive apoptosis with necrotic tissue. Transfection with either Sh-survivin or Pp53 also triggered apoptosis to some extent in PC3 cells (Figure 6a). These samples stained weakly for p53 and survivin, compared with the scrambled siRNA controls (Figures 6b and c). These data showed that co-expressed Sh-survivin and p53 injected into the tumor, exerts strong antitumor effects via an induction of apoptosis in tumor xenografts.

We also determined c-Myc, CDK4, and GRIM19 mRNA and protein levels in tumors. Consistent with in vitro observations (Figure 5), the expressions of c-Myc and CDK4 in tumors treated with PSh-sur-p53 were significantly lower compared with those that received Sh-survivin, scramble or mock controls. Moreover, a rise in GRIM19 expression in tumors treated with either Pp53 or PSh-sur-p53 suggests that GRIM19 may be a downstream target of p53 (Figures 7a and b). Also, immunoblot analysis revealed that the levels of GRIM19 (Figures 7c and d) increased and CDK4 decreased in the Pp53 and shRNA survivin groups, while expression of those proteins were altered the greatest in the PSh-sur-p53 treatment group.

Figure 7
(a) RT-PCR analysis of GRIM-19, CDK4 and c-Myc mRNA in PC3 xenografts. (b) Immunoblot analyses for GRIM19, CDK4 in PC-3 xenografts. (c, d) Quantification of mRNA and protein in (a, b) by densitometric assessment. *P<0.05, **P<0.01 versus ...

Discussion

Aberrant expression of p53 and survivin were frequently found in advanced prostate cancers.2124 Tumors harboring p53 gene mutations showed greater survivin gene expression.25 It has been reported that increased survivin expression is associated with accumulation of apparent mutant p53 in gastric cancer and pancreatic carcinoma, as assayed by immunohistochemical staining.26,27 Also, clinical studies have shown that the expression of survivin and p53 were found to be significantly related (P=0.037), and 70% (27/39) of patients diagnosed with primary glioblastoma multiforme showed coordinated expression of p53 and survivin.28 In contrast, Tanaka et al.29 demonstrated no clear relationship between mutant p53 protein and survivin expression in breast cancer tissues. Additionally, seven cases with no p53 mutations nonetheless showed increased survivin expression in breast cancer.25 Moreover, transfection of embryonic fibroblasts with a temperature-sensitive p53 mutant did not abolish survivin gene expression. Positive p53 immunostaining has been reported to be related to p53 mutations (or conformational alterations) that increase the half-life of the p53 protein, leading to its accumulation.13,30,31 Therefore, in this study we refer to immunodetectable p53 as altered p53. Our study showed a correlation (rs=0.548) between increased levels of p53 and survivin proteins expressed in prostate tumors (Figure 1) (Table 1). Our data, considered together with data from the above studies, suggest that p53 may be directly altered conformationally in the presence of survivin. Further, this conformationally altered p53 may have a longer half-life and may be functionally inactive, but not necessarily a mutant. These data indicate that the presence of immunodetectable p53 along with increased survivin protein expression might serve as a prognostic marker for certain cancers.

RNAi is a potent tool for silencing the function of specific genes in tumors.32,33 Many gene products involved in carcinogenesis have already been considered as targets for RNAi. Although survivin is expressed below the level of detection in normal prostate epithelial cells, it is found to be very abundant in prostate cancer cells.34 To develop a more effective therapeutic strategy, we have combined plasmid-based RNAi against survivin with simultaneous restoration of wtp53. We hypothesized that this strategy may exert a synergistic suppression on tumor growth.

By using a chromatin immunoprecipitation method, Hoffman et al.35 have suggested that wtp53 normally represses transcription of the survivin gene. This study demonstrated that PC3 tumors undergo a significantly stronger growth inhibition after treatment with PSh-sur-p53 compared with the other constructs, indicating that wtp53 alone may not be enough to repress survivin gene expression, especially if survivin alters the conformation and functionality of wtp53. As shown by MTT assay, co-expression of Sh-survivin and wtp53 suppresses the growth of prostate cancer cells more effectively than that of either agent alone (Figure 4). This explanation is further confirmed by flowcytometry. Investigation of cell cycle distribution has shown that the PSh-sur-p53 treatment causes an approximate threefold reduction in the number of cells in S phase (from 39 to 12.7%) and a corresponding increase in the number of cells in G0/G1 phase, thereby preventing cell growth (Table 2). It has been reported previously that transfection with shRNA directed against survivin resulted in an accumulation of cells in the G0/G1 phase, and concomitant loss of G2/M phase cells.3638 Our results are consistent with those reports. There were fewer cells in G2/M phase on transfection with either wtp53 or Sh-survivin (Table 2). These data indicate that on treatment with PSh-sur-p53, PC3 cells induced molecular changes in cell cycle distribution with the majority of cells arrested in G0/G1 phase. As survivin is part of the centromere,3941 knock-down of survivin may result in a loss of mitotic spindle formation and hence a loss of G2/M cells.

In this study, an unexpected finding was that despite a remarkable inhibition in tumor proliferation, the rate of apoptosis in the Pp53, Sh-survivin and PSh-sur-p53 treatments was not significantly different; co-expressed PSh-sur-p53 did appear to have a slightly higher apoptosis index as detected by TUNEL staining (Figure 6a) and Annexin V-FITC kit (data not shown). A recent report found that survivin has a nuclear export signal and that in cancer cells the anti-apoptotic and mitotic roles of survivin can be separated through mutation of its nuclear export signal, which abrogates the cytoprotective activity of the protein but still allows mitosis to proceed.42,43 Alternatively, the plethora of p53-dependent genes are not trans-activated simultaneously. Instead, they are expressed in a cell type- and physiologic state-specific manner, that is, apoptosis versus cell cycle arrest.

Furthermore, we have shown that PSh-sur-p53-induced changes in several growth-associated genes, GRIM19, c-Myc and CDK4. GRIM19 is a newly characterized death-regulatory protein whose inactivation leads to a growth advantage of cells in the presence of interferon/RA.20 Our study showed that GRIM-19 expression was increased significantly in the Pp53 and PSh-sur-p53 groups compared with other groups (Figures 5 and and7).7). These data suggest that GRIM-19 might act in a p53-dependent manner to execute anti-proliferative and pro-apoptotic functions. That p53 can upregulate GRIM-19 is a novel observation. It is known that p53 activation is associated with c-Myc downregulation in some cells.44 The c-Myc oncoprotein is a transcription factor that promotes cell growth and proliferation, as well as apoptosis under certain conditions. Deregulated c-Myc can induce aberrant proliferation, loss of terminal differentiation, abrogation of DNA damage-induced cell cycle arrest, genomic instability and oncogenesis.45 Transcripts encoding c-Myc and cell cycle kinase 4 (CDK4) were significantly decreased in the PSh-sur-p53 group when compared with transfection with either Pp53 or Sh-survivin alone (Figures 5 and and7).7). In fact, c-Myc mRNA levels remained the same and CDK4 was only slightly downregulated after Pp53 and Sh-survivin treatment. We surmise that blocking survivin or overexpressing wtp53 alone may not sufficiently affect the downstream signaling molecules CDK4 and c-Myc genes. Thus, PSh-sur-p53 treatment is able to exert its antiproliferative effects through downregulation of c-Myc and CDK4 specifically when compared with the other groups. In addition, c-Myc, a target of signal transducer and activator of transcription-3, is upregulated by constitutive activation of signal transducer and activator of transcription-3 signaling that contributes to oncogenesis.16,46 Furthermore, we have shown that GRIM19 suppressed constitutive signal transducer and activator of transcription-3-induced cellular transformation by downregulating the genes involved in cell proliferation and apoptosis.19 Therefore, a mechanism involving p53-GRIM19-signal transducer and activator of transcription-3-c-Myc could be envisaged based on our observations.

In summary, the combined treatment strategy based on PSh-sur-p53 has been shown to be more effective than either single treatment alone. Our study presents a novel therapeutic approach potentially combining Sh-survivin and wtp53 expression for use in anti-prostate cancer therapy. In addition, GRIM19, c-Myc and CDK4, have distinct roles in anti-proliferative effects in vitro and in vivo.

Supplementary Material

Spplementary fig.1

Supp. Figure legend

Acknowledgments

This work was supported by ‘The Research Fund for the Doctoral Program of Higher Education China’ in 2007. Grant 20070183012. We thank Dr Ruijuan Gao, Tyler Bassett and Suqin Pan for providing valuable help and technical support. DVK is supported by the NIH grants CA105005 and CA78282.

Footnotes

Conflict of interest

The authors declare no conflict of interest.

Supplementary Information accompanies the paper on Cancer Gene Therapy website (http://www.nature.com/cgt)

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