synergistic suppression of prostatic cancer cells by co

JPET #180364

Synergistic Suppression of Prostatic Cancer Cells by Co-expression of both

Mdm2-siRNA and Wide-type P53 Gene in vitro and in vivo

Kun Ji, Bo Wang, Yue-ting Shao, Ling Zhang, Ya-nan Liu, Chen Shao, Xiao-jie Li, Xin Li,

Jia-di Hu, Xue-jian Zhao, De-qi Xu, Yang Li, Lu Cai

Department of Pathophysiology, Prostate Diseases Prevention and Treatment Research Center,

Norman Bethune College of Medicine, Jilin University, Changchun, P. R. China (K.J., B.W.,

Y.S., L.Z., Y.L., C.S., X.L., X.L., X.Z., Y.L.); Department of Pathophysiology, College of

Basic Medicine, Shenyang Medical College, Shenyang, P. R. China (K.J.); Department of

Pathology, Inner Mongolia Forestry General Hospital, Yakeshi, Inner Mongolia Autonomous

Region, P. R. China (B.W.); Department of Pediatrics and Radiation Oncology, the University

of Louisville, Louisville, KY, USA (B.W., L.C.); Department of Oncology and Diagnostic

Sciences, University of Maryland Dental School, Baltimore, MD, USA (J.H.); Laboratory of

Enteric and Sexually Transmitted Diseases, Center for Biologics Evaluation and Research,

Food and Drug Administration, Bethesda, Maryland, USA (D.X.)

JPET Fast Forward. Published on March 28, 2011 as DOI:10.1124/jpet.111.180364

Copyright 2011 by the American Society for Pharmacology and Experimental Therapeutics.

This article has not been copyedited and formatted. The final version may differ from this version.JPET Fast Forward. Published on March 28, 2011 as DOI: 10.1124/jpet.111.180364

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Running title: Synergistic suppression of prostate cancer by mdm2-siRNA/p53

Address correspondence to: Dr. Yang Li. Department of Pathophysiology, Prostate Diseases

Prevention and Treatment Research Center, Norman Bethune College of Medicine, Jilin

University, Changchun 130021 China or Dr. De-Qi Xu, MD 20892, USA. Phone:

+86-431-85619485. E-mail: [email protected]

The number of text pages, number of tables, figures, and references

Text pages: 35

Tables: 1

Figures: 8

References: 40

The number of words

Abstract: 249

Introduction: 740

Discussion: 1,311

ABBREVIATIONS: MDM2, Murine double minute 2; wt p53, wild-type p53; mt p53,

mutated-type p53; PI, propidium iodide; PBS, phosphate-buffered saline; MTT, 3-(4,

5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide; GFP, green fluorescent

protein; AI, apoptotic index.

Section options: Chemotherapy, Antibiotics, and Gene Therapy


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ABSTRACT

The objective was to evaluate the cell growth and death effects in human prostate cancer cells

by inhibiting Murine Double Minute 2 (MDM2) expression with overexpressing wild-type

(wt) p53 gene. Prostate PC-3 tumor cells were transfected with a plasmid containing either

mdm2-siRNA (Si-mdm2), wt p53 gene (Pp53) alone or both (Pmp53) using lipofectamine in

vitro and attenuated Salmonella in vivo. Cell growth, apoptosis, and the expression of related

genes and proteins were examined in vitro and in vivo by flow cytometry and Western

blotting assays. We demonstrated that human prostate tumors had the increased expressions of

MDM2 and mutant p53 proteins. Transfection of the PC-3 cells with Pmp53 plasmid in vitro

offered a significant inhibition of cell growth and an increase in apoptotic cell death compared

to the Si-mdm2 or Pp53 group. These effects were associated with up-regulation of p21 and

down-regulation of HIF-1α expression in Pmp53 transfected cells. To validate the in vitro

findings, the nude mice implanted with PC-3 cells were treated with attenuated salmonella

carrying the plasmids, which showed that Pmp53 plasmid significantly inhibited the tumor

growth rate in vivo compared to Si-mdm2 or Pp53 plasmid alone. Tumor tissues from the

mice treated with Pmp53 plasmid showed an increased expression of p21 and decreased

expression of HIF-1α proteins, with an increased apoptotic effect. These results suggest that

knockdown of mdm2 expression by its specific siRNA with an overexpression of wt p53 gene

offers a synergistic inhibition of prostate cancer cell growth in vitro and in vivo.


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Introduction

Prostate cancer is the most common cancer in men (Kamangar et al., 2006), and an

efficient therapy for this cancer remains an urgent need. Cell growth and death are usually

determined by a balance between oncogenes and tumor-suppressor genes. P53 plays an

essential regulatory role in the development and homeostasis of cells and tissues (Vousden

and Lane, 2007). P53 inactivation caused by its negative regulators such as Murine Double

Minute 2 (MDM2) contributes to the development of a large number of human cancers.

MDM2 is an E3 ubiquitin ligase that directly binds to the p53 for its ubiquitylation (Tao and

Levine, 1999). Tumors with over-expression of MDM2 were resistant to p53 gene therapy

(Wiman, 2006). Reportedly prostate tumors with MDM2 knockdown were sensitive to

radiation therapy both in vitro and in vivo (Mu et al., 2004a, 2004b, 2008; Stoyanova et al.,

2007). Therefore, the anti-tumor strategy to interfere with the p53–MDM2 feedback loop

holds a promise to reinstate the p53 tumor suppressing pathway (Wade and Wahl, 2009).

Several agents such as Nutlin-3a that disrupts MDM2-p53 interaction inhibit the growth

of certain tumors, but are less effective in the other tumors (Patton et al., 2006; Wade et al.,

2006). In addition, these agents also lack selectivity for the tumor cells. Therefore, gene

therapy selectively targeting to the tumor cells with little effect to normal tissue is an

interesting and urgent tool to improve the therapeutic efficacy (Lee et al., 2004). For instance,

gastric cancer cell lines carrying wt p53 gene were infected with adenovirus expressing

RPL23 (Ad-RPL23) to stabilize wt p53 by inhibiting MDM2-mediated p53 degradation. The

adenovirus delivery of RPL23 significantly inhibited the proliferation of gastric cancer cells

with wt p53 (Zhang et al., 2010).

In the above study, the successful restoration of p53 function needs the presence of


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endogenous wt p53 in the tumors. However, inactivation of p53 activity often occurs in the

tumors due to its deletions, mutations, protein destruction or protein sequestration (Bernal et

al., 2010). Thus, reinstatement of wt p53 function in the tumors with mutated (mt)

endogenous p53 is specially daunting (Bernal et al., 2010). A few studies have explored for

the forced expression of wt p53 gene with MDM2 negative regulator genes such as FHIT

(Nishizaki et al., 2004) or FUS1 (Deng et al., 2007) to negatively inhibit MDM2 that in turn

up-regulates the exogenous wt p53 function in the cancer cells. However, expression of single

MDM2 negative regulator either FHIT or FUS1 to down-regulate MDM2’s p53 suppression

may be not ideal since which of these MDM2 negative regulators is the most effective in

suppressing MDM2 function and whether these MDM2 negative regulators have other

functions except for suppressing MDM2 function remains uncertain. Therefore, to directly

down-regulate MDM2 by its specific siRNA in the tumors cells with a forced-expression of

exogenous wt p53 may be a better approach.

In addition, the transfection approaches (adenovirus and nanoparticle), used in the above

studies (Deng et al., 2007; Nishizaki et al., 2004), are also not tumor cell selective. Reportedly

salmonella would infect and preferentially accumulate within the tumor xenograft in vivo,

with a tumor/normal tissue ratio of approximately 1,000:1 (Pawelek et al., 1997). Attenuated

salmonella Typhimurium Ty21a (S. Typhi Ty21a) is a facultative anaerobic bacterium and

capable of replicating preferentially in tumor cells and has been explored for developing

therapeutic proteins to tumors (Cryz et al., 1993; Kimura et al., 2010).

In the present study, therefore, we have made plasmids either containing mdm2-siRNA

or human wt p53 (Pmp53) alone or containing both. The effects on the tumor cell growth and

apoptotic death by transfection with Pmp53 in the prostate cancer cells in vitro and in vivo


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were compared with that by transfection with either mdm2-siRNA or wt p53 alone. The

unique features of the present study included (1) that we have co-transfected, for the first time,

mdm2-siRNA to directly down-regulate MDM2 expression with human wt p53 gene in the

prostate cancer cells in vitro and in vivo; (2) for the in vivo study, we have used, for the first

time, S. Typhi Ty21a to carry various plasmids predominantly transfected into prostate tumor

xenografts in a nude tumor-bearing mouse model. This application of the S. Typhi Ty2la

bacteria carrying plasmid containing both mdm2-siRNA and wt p53 will ensure the

transfected cells to express wt p53 with efficient inactivation of its negative regulator MDM2

expression no matter the tumor cells with or without the presence of wt p53.


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Materials and Methods

Construction of a Plasmid Vector Capable of Co-expressing mdm2 siRNA and wt

p53. By a method used in our previous studies (Zhang et al., 2007), we constructed a series

of expression plasmids that contain siRNA specific to mdm2 and/or wt p53 expression

element. The pcDNA3.1-p53 (Pp53) plasmid containing wt p53 coding region was generated

as described before (Shao et al., 2010). A siRNA with the sequence of

GACACTTATACTATGAAAG (Genbank accession number NM002392) was selected for

specifically targeting to the fragment corresponding nucleotides 152 to 171 of MDM2 mRNA.

The oligonucleotide contains a sense strand of 19 nucleotides followed by a short spacer (loop

sequence: TTCAAGAGA), an antisense strand and a five Ts terminator. A scrambled siRNA

was used as a negative control. Double-stranded DNA oligonucleotides were cloned into

pGCsilencerU6/ Neo/GFP, which also expresses a green fluorescent protein (GFP) gene (Jikai

Chemical, Inc., Zhejiang, China), to generate plasmids Si-mdm2 and Si-scramble. A 585bp

fragment, containing Si-mdm2 under the control of the U6 promoter was PCR amplified from

the plasmid Si-mdm2 using the following primers: forward =

5'-GCAGATCTTGCTTCGCGATGTACGGGCC-3'; reverse =

5'-GCAGATCTTGCTTCGCGATGTACGGGCC-3'. The underlined nucleotides in these

primers correspond to the BglII and NruI restriction sites, respectively. This fragment was

sub-cloned into the BglII and NruI sites of the Pp53 vector to generate the plasmid

pcDNA3.1- p53/U6 Si-mdm2, which can express both mdm2-siRNA and p53 protein from

the same plasmid and was designated as Psi-mdm2-p53 (shortly, Pmp53). All recombinant

plasmids were verified by sequencing (Shenggong Bioengineering, Shanghai, China).

Human Prostate Tissues. We obtained 10 samples of normal prostate tissues from


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nontumor areas of prostatectomy specimens, and 19 samples of cancer tissues from radical

prostatectomy specimens of patients with organ-confined tumor without previous therapy.

Both prostate normal and tumor tissues (Gleason score=9) were collected from Inner

Mongolia Forestry General Hospital (Yakeshi, China) and Prostate Disease Prevention and

Treatment Research Center (Jilin University, China), with the informed consent of the patients

under an institutionally approved protocol.

Cell Culture and Transfection. PC-3 cells were cultured in IMDM medium

supplemented with 10% fetal bovine serum (FBS), penicillin-streptomycin, and L-glutamine,

as described previously (Mu et al., 2005). Cells were transfected with various plasmids using

the Lipofectamine TM 2000 reagent (Invitrogen, Carlsbad, CA, USA) and 1% FBS for

various times before analysis of mRNA and protein levels, apoptosis, and cell proliferation. In

combination treatments, plasmids-transfected cells were incubated for an additional 48-72 h

before measurement.

Cell Proliferation Assay. Approximately 6×103 PC-3 cells per well were transfected

with the plasmids of interest, and cell growth was evaluated after 48 and 72 h using the

4,5-dimethylthiazol-2-yl-2,5-diphenyltetrazolium bromide (MTT) assay following the

instruction provided by the Kit.

Reverse Transcription-PCR. For RT-PCR analysis at 48 h after transfection, cells were

collected and total RNA was extracted using the Trizol Reagent (Invitrogen, Carlsbad, CA,

USA). 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, USA). The resultant complementary DNAs were used in PCR

reactions with the gene-specific primers of interest (Table 1), and the optimal conditions of


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PCR reaction, including cycles, annealing times and primer concentrations for each gene were

obtained by performing series of pre-experiments (Data not shown). The PCR products at the

optimal conditions for each gene of interest were separated by agarose gel electrophoresis and

visualized by ethidium bromide staining.

Flow Cytometry. PC-3 cells were incubated with plasmids for 48, 72 h, harvested and

counted, and 1 × 106 cells were resuspended in 100 μl phosphate-buffered saline (PBS). Then,

5 μl of propidium iodide (PI, Beckman, USA) was added, incubated for 30 min at room

temperature in dark. Then the cells were subjected to flow cytometry to measure the apoptosis

rate (%) with a Beckman Coulter Epics-XL-MCL cytometer (California, USA).

Xenograft Models. The models of PC-3 xenografts were established using athymic

nude male mice (nu/nu; 6–8 weeks) that were acquired from the Institute of Zoology, Chinese

Academy of Sciences, Beijing and used in accordance with an Animal Care and Use protocol

approved by Jilin University. Cultured cells were washed with and resuspended in serum-free

media. The suspension (5 × 106 cells in 150 μl per mouse) was inoculated subcutaneously into

the right flank of nude mice. The sizes of tumors in these mice were measured starting from

10 days after cell injection until 21 days using calipers. These tumor-bearing mice were then

randomly divided into five groups (6 mice per group): (1) mock transfection with PBS buffer

as the normal control; (2) scrambled siRNA-vector alone (Si-scramble) as the vector control;

(3) pcDNA3.1-p53 (Pp53) as wt p53 transfection alone; (4) U6-mdm2 (Si-mdm2) as mdm2

siRNA transfection alone; (5) pcDNA3.1-p53/U6 Si-mdm2 (PSi-mdm2-p53, Pmp53) as the

combined transfection. Mice in each of these groups were inoculated bacteria with different

plasmids (108 cfu/50 µl) per mouse via intratumoral injection by using a syringe fitted with a

27-gauge needle. At 4 days after treatment, tumors were measured using calipers every 4 days


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for 32 days and the data were plotted using Kaplan-Meier method to analyze the tumor

growth curves. In addition, the tumor xenografts were also measured for the wet weight and

size when mice were sacrificed.

To ensure the tumor-preferable distribution of the bacteria, an additional pilot study was

performed before the above animal experiment. Tissue samples from the tumor, liver, lung,

spleen, heart and kidney of three tumor-bearing mice treated with bacteria carrying

Si-scramble at day 1 (24 h), 2, 3, 5, and day 10 after bacteria treatment were used for bacterial

distribution analysis. Tissues were weighed and then 100 mg of each tissue in 3 ml of PBS

were excised, minced, and homogenized. Then the homogenized tissues were plated onto

Luria-Bertani agar containing ampicillin (100 mg/ml) in triplicate, 24 h later bacterium

colony count was evaluated as the colony-forming unit (CFU).

Immunohistochemic Staining. Each paraffin-embedded tissue section at 4 μm in

thickness was deparaffinized, hydrated, and incubated in 3% H2O2 and microwaved for 10

min to block endogenous peroxidase activity. After formalin fixation and paraffin embedding,

the tissue sections were incubated with the primary antibodies [anti-mt-p53, wt-p53,

anti-mdm2 antibodies, from Santa Cruz Biotech Inc. (Santa Cruz, CA, USA), or anti-PCNA

anti-body, from Beijing Biosynthesis Biotechnology Co. LTD (Beijing, China)] for 60 min,

washed with PBS, and then were incubated with the biotinylated secondary antibody. Finally

the sections were stained with 3, 3'-diaminobenzidine (DAB) and counterstained with

hematoxylin. Serum blocking solution in place of the primary antibody was used as a negative

control. Stained tissues were screened independently by two investigators and classified

according to staining intensity (－or＋). We considered the tissue to be positive (＋) if the

staining intensity was moderate to strong in＞10% of cells examined. Weakly stained (＜10%


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of total cells examined) or non-immunoreactive cells were considered negative (－). Positive

or negative reaction was determined after counting cells in five random high-power fields of

each sample.

Western Blot Analysis. The protein levels of MDM2, wt p53, p21 (Cyclin-dependent

kinase inhibitor 1A), cdk4, cyclin-D1, hypophosphorylated retinoblastoma (pRb), E2F1,

hypoxia-inducible factor 1alpha (HIF-1α) and β-actin in cultured cells and xenografts were

analyzed by Western blotting as described previously (Zhang et al., 2008). The monoclonal

antibodies against MDM2, wt p53, p21, pRb, cyclin-D1, and HIF-1α were obtained from

Oncogene Research Products (Boston, MA, USA). Anti-E2F1 monoclonal antibody was

purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Anti-β-actin monoclonal

antibody was obtained from Sigma (St Louis, MO, USA).

TUNEL Staining of Apoptotic Cells. The tissue sections of xenografts from the nude

mice were dewaxed and hydrated, and then were subjected to TUNEL staining following the

instruction provided by TUNEL kit (Promega, Madison, WI, USA). Four fields (400 x) were

randomly chosen and analyzed, the apoptotic index (AI) was defined as follows: AI (%) = 100

× apoptotic cells/total tumor cells.

Transmission Electron Microscopy. Tissue blocks (~1 mm3) were fixed in 2.5%

glutaraldehyde (in 0.1 M sodium cacodylate, pH 7.4) for 2 h and stained by 1% OsO4 at 4 °C

for 2 h and 0.9% OsO4 and 0.3% K4Fe (CN)6 at 4 °C for 2 h. Sections in 80 nm thick were

deposited on carbon and Formvar-coated, 200-mesh, nickel grids (Electron Microscopy

Sciences, MA,USA) and were stained with 3% uranyl acetate and Reynolds lead citrate for

visualization under a 120 kV JEM-1200EX transmission electron microscope.

Statistical Analysis. For in vitro study, three separate experiments were done. For in


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vivo study, six animals were included. Data are expressed as the mean ± SD. One-way

ANOVA was used to determine if differences exist and if so, a post hoc Tukey’s test was used

for the difference analysis between groups, with Origin 7.5 laboratory data analysis and

graphing software. Statistical significance was considered at p < 0.05.


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Results

MDM2 and mt p53 are Over-expressed in Primary Human Prostate Cancers.

Immunohistochemical analysis of the mt p53 and MDM2 protein expression in human

prostate tissues (Fig. 1A) showed that MDM2 expression mainly localized in the nucleus in

the prostate tumors and was detectable in 68.4% of the primary prostate cancers (13/19) while

mt p53 protein was detected in 52.6% of the primary prostate cancers (10/19). Semi-

quantitative analysis of each protein, as described in the Materials and Methods, was

presented in Fig. 1B. This study confirms the increased expression of mt p53 and MDM2

proteins in the human prostate cancer tissues compared to normal prostate tissues.

Effect on the Gene and Protein Expressions of MDM2 and wt p53 in Human PC-3

Prostate Cancer Cells by Transfection with both mdm2 siRNA and wt 53. The expression

of mdm2 and wt p53 genes and proteins in the PC-3 cells transfected with different plasmids

(illustration of the constructions of these plasmids are shown in Fig. 2A) was examined by

RT-PCR and Western blot analysis, respectively. As expected, the mdm2-mRNA and protein

expressions were significantly decreased in the PC-3 cells transfected with Si-mdm2 and

Pmp53 compared to the cells transfected with mock and Si-scramble (Fig. 2B, C). In contrast,

the wt p53 mRNA and protein expressions were significantly increased in the PC-3 cells

transfected with Pp53 and Pmp53 and slightly increased in the PC-3 cells transfected with

Si-mdm2 compared to the cells transfected with mock and Si-scramble (Fig. 2B,C). The

results suggest the effectiveness of the transfected plasmids on the target genes.

Effects on the Cell Proliferation and Apoptotic Death by Co-expression of

mdm2-siRNA and wt p53 Gene in the Prostate Cancer Cells. The potential effects on cell

proliferation and death by treatment with Pmp53 were compared to those with Pp53 and


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Si-mdm2 by two assays: MTT and apoptotic cell death. MTT assay showed that treatments

with Pp53, Si-mdm2 and Pmp53 resulted in a significant inhibition of the PC-3 cell

proliferation at both 24 and 72 h after transfection, with a maximal inhibition by Pmp53

treatment (Fig. 3A).

For apoptotic cell death, flow cytometry (FCM) was employed to detect the fluorescent

intensity after tumor cells were double-stained with Annexin V and PI at 72 h transfection.

Representative raw FCM data are shown in Fig. 3B, in which the early (Annexin V positive

only) and the late (Annexin V and PI both positive) apoptotic cells are distributed in the Q4

and Q2 regions, respectively, and necrotic cells (PI positive only) are in the Q1 region. It is

noted that there was a few necrotic cells and there was also no significant difference for the

incidence of necrotic cells among the groups. Compared to necrotic cells, there was a

significantly increased portion of apoptotic cells among the tumors cells of each group.

Quantitative analysis based in FCM showed that the apoptotic death was significantly

increased in the cells transfected with Pp53 or Si-mdm2 plasmid compared to the cells

transfected with mock or Si-scramble, but was the most evident in the cells treated with

Pmp53 plasmid among the all groups (Fig. 3B, C).

Effects of Co-expression mdm2-siRNA and wt p53 Gene on the Expression of

CDK4, Cyclin-D1 and HIF-1α Genes in the Prostate Cancer Cells. To explore the possible

mechanisms underlying the induction of PC-3 cell death by co-expression of mdm2-siRNA

and wt p53 gene, gene and protein expressions of p21, CDK4, cyclin-D1 and HIF-1α were

examined with RT-PCR (Fig. 4A) and Western blot (Fig. 4B) assays. It is shown that p21

mRNA and protein levels were low in mock and Si-scramble group, but significantly

increased in the cells transfected with Pp53 or Pmp53 plasmid (Fig. 4 A, B). In contrast,


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expressions of CDK4, cyclin-D1 and HIF-1α were significantly evident in the cells treated

with either mock or Si-scramble, but significantly decreased in the cells treated with Pp53,

Si-mdm2 and Pmp53 plasmids (Fig. 4).

Preferable Transfection of the S. Typhi Ty21a Carrying Plasmids into Xenograft

PC-3 Tumors and Efficient Expressions of MDM2 and wt p53 Proteins. The models of

PC-3 xenografts were established as described in the Materials and Methods. Athymic nude

male mice (nu/nu; 6–8 weeks) were implanted with PC-3 tumor cell suspension at 5 × 106

cells in 150 μl per mouse at the upper back of the mice. The sizes of tumors were measured

daily starting from 10 days after cell implantation. At 21 days of post-implantation, the

tumor-bearing mice were inoculated with bacteria carrying the specific plasmid for each

group via intratumorally injection with 108 cfu/50 µl per mouse.

To ensure the S. Typhi Ty21a carrying these plasmids preferentially localized in the

tumor tissue, we have monitored the kinetics of bacterium distribution in the xenograft tumor

and different organs of the tumor-bearing mice with bacterium injection at specified

post-injection times (Fig. 5A). At 24 h (1 day) after injection, bacteria could be found in the

tumors and organs (Data not shown). The bacteria were significantly increased in the tumors

and decreased in the liver and other tissues at 3 days after administration (Fig. 5A). At 10

days after bacterium injection, the bacterium accumulation was observed predominantly in the

tumor tissue, very less in the liver (Fig. 5A), and undetectable in other organs (Data not

shown). Quantitative analysis of the bacteria by CFU, as described in the Materials and

Methods and presented in Fig. 5B, C, confirms the predominantly distribution of these

bacteria in the tumor tissue.

To determine if the transfected plasmids were efficiently expressed in tumor cells, the


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expressions of MDM2 and wt p53 proteins were examined by Western blotting. As shown in

Fig. 6A, MDM2 expression was significantly low in the tumors of mice treated with Si-mdm2

and Pmp53, but not changed in the tumors of mice treated with Pp53, as compared to that in

the tumors of mice treated with Si-scramble or mock. This result suggests that the successful

transfection of bacteria with mdm2-siRNA in vivo efficiently suppress mdm2 expression.

Expression of wt p53 was increased significantly in the tumors of mice treated with Pmp53

and Pp53 and only slightly in the tumors of mice treated with Si-mdm2, as compared to that

in the tumors of mice treated with Si-scramble or mock (Fig. 6A). This confirms the

successful transfection of the bacteria with wt p53 gene to efficiently express the wt p53

protein. Furthermore, immunohistochemical staining for MDM2 and wt p53 was

representatively provided in Fig. 6B, which confirmed that the expression of MDM2 was

decreased and expression of wt p53 was increased in the tumor cells treated with Pmp53

compared to the tumors cells treated with Si-scramble.

Suppression of the Prostate Tumor Xenograft Growth by Co-expression of Mdm2-

siRNA and wt p53. The potential therapeutic effect on the prostate xenograft growth was

examined in the tumor-bearing mice treated with the bacteria carrying plasmid containing

both mdm2-siRNA and wt p53 gene (Fig. 7). Dynamic tumor growth that was monitored from

the 4th day until the 32nd day after bacteria treatment (Fig. 7A) showed that sizes of the tumor

xenografts in the mice treated with mock or Si-scramble increased slowly from day 4 to day

16 and quickly from day 16 until day 32, while tumor xenografts in the mice treated with

Pmp53 grew very slowly thorough the whole period, and also significantly smaller than those

in the mice treated with either Pp53 or Si-mdm2. When tumor-bearing mice treated with

different plasmids were sacrificed, tumor xenografts were removed for measuring tumor sizes


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and weights. Fig. 7B showed that the individual tumor xenografts from the mice treated with

Pmp53 plasmid were significantly smaller than those from mice treated with Si-scramble. Fig.

7C showed that tumor weights were significantly decreased in the tumor-bearing mice treated

with Pp53, Si-mdm2 or Pmp53, compared to the mice treated with mock or Si-scramble

plasmid. However, the inhibitory effect on tumor growth was more evident in the mice treated

with Pmp53 than that in the mice treated with either Pp53 or Si-mdm2 alone (Fig. 7A, C).

Effects of Co-expression of mdm2-siRNA and wt p53 on Tumor Cell Death and

Expression of the p53-MDM2 Downstream Signaling Targets in the Prostate Tumor

Xenografts. Histological examination with H&E staining revealed a few focal areas of

necrosis cells along with tissue disorganization in Pp53, Si-mdm2 and Pmp53 treatment

groups (Fig. 8A). With TUNEL staining (Fig. 8B) apoptotic cells around the necrotic area

were observed. The apoptotic cells showed chromatin condensation, chromatin crescent, and

nucleus fragmentation, under the examination with transmission electron microscope (Fig.

8C). Although the incidence of apoptotic cells was increased in the tumor tissues from all the

mice treated with Pp53, Si-mdm2 and Pmp53, the most highest incidence of the apoptotic

cells was seen in the tumors from the mice treated with Pmp53 (Fig. 8B, C).

Tumor cell proliferation was also analyzed by immunohistochemical staining for PCNA

that acts a marker for cells in early G1 phase and S phase of the cell cycle. Treatment with

Pmp53 plasmid caused the maximal inhibition of cell proliferation since there were

significantly decreased numbers of PCNA positive cells compared to other groups (Fig. 8D).

To determine the potential mechanism of tumor growth inhibition in vivo, p21 protein

expression was examined by Western blotting assay (Fig. 8E), p21 protein expression was

very low in the Si-scramble group but significantly increased in the groups treated with Pp53


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and Pmp53. In contrast, the protein expressions of E2F-1, pRb, and HIF-1α were significantly

decreased in the groups treated with Pp53, Si-mdm2, and Pmp53 groups compared to the

Si-scramble treated group (Fig. 8E).


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Discussion

Widespread use of prostate-specific antigen (PSA) and digital rectal examination

screenings has led to earlier diagnosis, but there remains lack of a significantly efficient

therapeutic approach (Derweesh et al., 2004). Gene therapy remains to be considered as a

potential to providing an efficient therapeutic approach (Freytag et al., 2007).

From human prostate cancer samples, we demonstrated significant increases in mt p53

and MDM2 expressions compared to the normal prostate tissues (Fig. 1). These results

indicated that increased expressions of MDM2 and mt p53 may play important roles in the

formation of prostate cancer. The tumor suppressor p53 is a powerful anti-tumor molecule

that is often inactivated by over-expression of its negative mediators. Down-regulation of p53

results in the prevention of p53-mediated apoptosis and cell cycle arrest (Momand et al.,

1992). MDM2 inhibits p53 transcriptional activity, favors its nuclear export and stimulates its

degradation. Therefore, inhibiting the p53–MDM2 interaction is a promising approach for

activating p53 in the tumor cells with the presence of endogenous wt p53 (Chene, 2003;

Gottifredi and Prives, 2001).

However, p53 is also frequently inactivated by mutations or deletions in cancer cells.

Isaacs et al. reported for the first time of p53 gene mutations in prostate cancer cells, and other

studies also confirmed the high incidence of mt p53 in prostate cancer (Hainaut and Hollstein,

2000; Isaacs et al., 1991). The results from our own observation that about 52.6 % of human

prostate cancers showed the presence of mt p53 (Fig. 1 B) also supports these previous studies.

Under such condition, only inactivation of MDM2 would not be able to restore p53 function.

To solve this issue, forced expression of both FHIT and wt p53 by adenoviral vector in

non-small cell lung carcinoma (NSCLC) cells was found to result in a synergistic inhibition of


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the tumor cell proliferation in vitro and growth in nude mice (Nishizaki et al., 2004). This is

because FHIT can inactivate MDM2 and interrupt the association of MDM2 with p53, leading

to p53 stabilization. A similar study reported that co-expression of FUS1 and p53 by

nanoparticle-mediated gene transfer significantly and synergistically inhibited NSCLC cell

growth and induced apoptosis (Deng et al., 2007). They found that the observed synergistic

tumor suppression by FUS1 and p53 concurred with the FUS1-mediated down-regulation of

MDM2 expression. Although these findings showed that combinational expressions of a gene

to inhibit MDM2 function with exogenous wt p53 gene in the tumor cells with mt p53 may be a

gene therapeutic strategy, it maybe not ideal since which of these MDM2 negative regulators

is the most effective in suppressing MDM2 function and whether these MDM2 negative

regulators have other functions except for suppressing MDM2 function remains uncertain.

The novel finding in the present study thus is that we have demonstrated, for the first

time, that the plasmid containing both mdm2-siRNA to directly down-regulate mdm2

expression and function, and wt p53 gene can synergistically inhibit the growth of the prostate

tumor cells in vitro and in vivo. MTT assay and flow cytometric analysis of apoptotic cells

showed the maximal inhibition of PC-3 cell growth and induction of apoptotic cell death in

the cells transfected with both mdm2-siRNA and wt p53 (Pmp53 group) compared to the

group treated with either mdm2-siRNA or wt p53 alone (Fig. 3). Treatment of PC-3

tumor-bearing mice with Pmp53 plasmid also provided a maximal inhibition of the tumor

xenograft growth, shown by the smallest tumor volume and weight among the five groups

(Fig. 6). Both in vitro and in vivo studies clearly showed the advantage of transfection of the

cells or xenografts in the tumor-bearing mice with the plasmid containing both mdm2-siRNA

and wt p53. Under this case, forced-expression of wt p53 can be optimally expressed with


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efficient inactivation of mdm2 by its siRNA (Fig. 6).

In addition, MDM2 is known to interact with other regulatory proteins, such as pRb

(Xiao et al., 1995) and E2F-1 (Martin et al., 1995) independent of p53. In the present study,

we investigated the expression levels of these genes and proteins, such as p21, CDK4,

cyclinD1, HIF-1α, pRb and E2F-1. We demonstrated that treatment with Pmp53 plasmid

increased the expression of p21 and inhibited E2F-1, pRb, HIF-1α expression in certain extent

(Fig. 4, 8).

Cell cycle arrest takes place when there is a block in cell-cycle division. It is known that

p53 arrests the cell cycle by stimulating the expression of p21 that blocks DNA replication by

inhibiting PCNA activity and also mediates growth arrest by inhibiting the action of G1

cyclin-dependent kinases (CDKs). Inhibition of CDKs results in an inhibition of both G1-to-S

and G2-to-mitosis transitions by causing hypophosphorylation of Rb and preventing the

release of E2F (Zheng et al., 2006). Studies by Hsieh et al. and Kowalik et al. Separately

confirmed that overexpression of MDM2 limited the native E2F-1’s ability to induce

p53-dependent apoptosis (Hsieh et al., 1997; Kowalik et al., 1998). We also showed that

restoration of wt p53 function resulted in up-regulation of p21, which may be responsible for

the down-regulation of its other downstream genes such as pRb and E2F-1 expression (Fig.

8E).

Yamakuchi et al. showed that p53 is able to inhibit HIF-1 by inducing microRNA-107,

leading to a suppression of tumor angiogenesis and tumor growth (Yamakuchi et al., 2010).

The transcription factor HIF-1 is composed of the subunits HIF-1α and HIF-1β, which are

basic helix-loop-helix DNA binding proteins. The HIF-1α subunit is overexpressed in a

variety of human cancers. To confirm this finding, we also demonstrated the inhibitory


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expression of HIF-1α by up-regulation of p53 in the prostate tumor cells in vitro (Fig. 3) and

in vivo (Fig. 8). Based on the above information, therefore, we assumed that the

synergistically inhibitive effects of co-expression of mdm2-siRNA and wt p53 on prostate

tumor cells observed in the present study is most likely through two facts: (1) inhibition of

HIF-1α activity by up-regulation of wt p53 expression would impair hypoxic signaling,

resulting in an inhibition of tumor growth, angiogenesis, and vessel maturation (Stoeltzing et

al., 2004); (2) Up-regulation of wt p53 in the tumor tissues enhances the spontaneous

apoptotic cell death (Deng et al., 2007).

Most human and rodent solid tumors contain a substantial fraction of cells that are

hypoxic. Tumor hypoxia occurs because of the stochastic and slow development of the

vasculature during tumor growth. S. Typhi Ty21a have an excellent safe profile (Thamm et al.,

2005), a propensity homing to tumors (Clairmont et al., 2000). In the present study, the

strategy is that when the S. Typhi Ty21a carrying the plasmids of interest were infected the

tumor cells, the tissues with overexpression of HIF-1α is in favor of the growth of these

bacteria and expression of the genes of interest to develop therapeutic action. It should be

mentioned that although we have ensured the tumor-specific and efficient transfection of the

bacteria containing target genes by intratumoral injection of these bacteria, it does not means

that we have to intratumorally give it since a few studies have reported the effectiveness when

it was given by intravenously (Zhang et al., 2007; Zhao et al., 2005).

In summary, in the present study, we demonstrated that human prostate tumors exhibited

increased expressions of MDM2 and mutant p53 proteins. Transfection of the PC-3 cells with

both mdm2-siRNA and wt 53 in vitro and in vivo significantly increased the tumor cell growth

and increased apoptotic cell death compared to knockdown of mdm2 or forced expression of


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wt p53 alone. These effects were associated with up-regulating p21 and down-regulating

HIF-1α expression. These results suggest that knockdown of mdm2 expression by its specific

siRNA with an overexpression of wt p53 gene offers a synergistic inhibition of prostate

cancer cell growth in vitro and in vivo. This strategy offered a significantly synergistic

inhibition of the tumor cell growth and is the better than use of the plasmid containing either

mdm2-siRNA or wt p53 alone.


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Acknowledge:

We are grateful to Dr. Yi Tan for his assistance of the figure preparation and Mrs. Shuqin

Pan for her assistance of pathological technology.

Authorship Contributions:

Participated in research design: Kun Ji, Yue-ting Shao, Xue-jian Zhao, De-Qi Xu, Yang Li

and Lu Cai

Conducted experiments: Kun Ji, Bo Wang, Ya-nan Liu, Chen Shao, Xiao-jie Li and Xin Li

Contributed new reagents or analytic tools: Kun Ji, Bo Wang, Yue-ting Shao, Ling Zhang and

Jia-di Hu

Performed data analysis: Kun Ji, Bo Wang, De-Qi Xu and Yang Li

Wrote or contributed to the writing of the manuscript: Kun Ji, Bo Wang, Ling Zhang, De-Qi

Xu, Yang Li and Lu Cai

Others: Kun Ji, Yue-ting Shao and Yang Li acquired funding for the research.


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Footnotes:

This work was supported by Research Fund for the Doctoral Program of Higher Education of

China [Grants 20070183012] and Scientific and technological development plan project in

Jilin Province [Grants 200705360].

K.J. and B.W. contributed equally to this work.


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FIGURE LEGENDS

Fig. 1. Expression of Mdm2 and mt p53 in human prostate tissues. Human benign and

cancer tissue (Gleason score 9) sections were immunostained with antibodies against

MDM2 and mt p53, respectively (A, ×200). Semi-quantitative analysis for the

expression percent of positive staining based on the methods described in the

Materials and Methods are provided (B).

Fig. 2. Expression of Mdm2 and wt p53 in PC-3 cells transfected with various plasmids.

Constructions of various plasmids are illustrated (A). Expression of mdm2 and wt

p53 at mRNA (B) and protein (C) levels were examined with RT-PCR and Western

blotting assays, respectively. Data shown were means ± SD of three separate

experiments. ap<0.05 vs mock or Si-scramble group; bp<0.05 vs Pp53 group; cp<0.05

vs Si-mdm2 group.

Fig. 3. Effects of transfection with various plasmids on cell growth. (A) Growth

inhibitory effects were evaluated with MTT assays for the PC-3 cells transfected with

different plasmids, and the data were presented as the ratio to control (mock). Data

shown were means ± SD of three separate experiments. ap<0.05 vs mock or

Si-scramble group; bp<0.05 vs Pp53 group; cp<0.05 vs Si-mdm2 group. (B) The raw

data of flow cytometry with Annexin V and PI staining for detecting cell death are

representatively shown from each group. Q1: necrotic cells; Q2: late apoptotic cells;

Q3: normal cells; Q4: early apoptotic cells. (C) The average data of flow cytometry

are presented for the results at 72 h after treatment. ap<0.05 vs Si-scramble group;


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bp<0.05 vs Pp53 group; cp<0.05 vs Si-mdm2 group.

Fig. 4. Effects of transfection with various plasmids on the expression of cell

cycle-related protein in the PC-3 cells. Gene and protein expressions of p21, CDK4,

cyclin-D1, and HIF-1α were examined with RT-PCR (A) and Western blotting assays

(B), respectively. ap<0.05 vs mock or Si-scramble group; bp<0.05 vs Pp53 group;

cp<0.05 vs Si-mdm2 group.

Fig. 5. Assessment of tumor-preferable distribution of the bacteria. Tissue samples from

the tumor xenografts, liver, lung, spleen, heart and kidney of three tumor-bearing

mice treated with bacteria carrying Si-scramble at different times after bacteria

treatment were used for bacterial distribution analysis. Tissue (100 mg) either from

tumors or each organ was in 3 ml of PBS and homogenized well, and the

homogenized tissue was plated onto Luria-Bertani agar. Representative images of the

plates planted with different tissues at 3 day (72 h) and 10 day (10d) after bacterial

injection are given in panel A. T: tumor xenograft; Li: liver; Lu: lung; S: spleen; H:

heart; K: kidney. Panels B and C are the quantitative analysis of the bacterium counts

by CFU/g tissue at day 3 (B) and day (C) after bacterial injection. ap<0.05 vs any one

of the other groups.

Fig. 6. Protein expression of Mdm2 and wt p53 in the PC3 tumor xenografts treated

with various plasmids. Western blotting analysis (A) and immunohistochemical

staining (B, x 200) were performed for MDM2 and wt p53 protein expressions in the

PC-3 tumor xenografts from the tumor-bearing mice treated with different plasmids


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at the termination of the study. ap<0.05 vs mock or Si-scramble group; bp<0.05 vs

Pp53 group; cp<0.05 vs Si-mdm2 group.

Fig. 7. Inhibition of tumor growth in vivo by treatments with various plasmids. Tumor

growth cures over the 32-day period were made based on the tumor sizes measured

very fourth day for the tumor-bearing mice with different plasmid treatments (A).

Images of tumors from Si-scramble and Pmp53 treatments were collected when these

tumor-bearing mice were sacrificed at the 32nd after treatment (B). Tumor wet

weights were measured when the animals were killed at the 32nd after treatment (C).

ap<0.05 vs mock or Si-scramble group; bp<0.05 vs Pp53 group; cp<0.05 vs Si-mdm2

group.

Fig. 8. Effects of various plasmid treatments on the induction of apoptotic cell death

and the expression of cell cycle arrest-related gene and proteins in the tumor

xenografts. H&E staining (A, x 200), TUNEL fluorescent staining (B, x 200),

electron microscopic analysis (C, x 6000) and immunohistochemical staining for

PCNA (D, x 200) were performed for the tumor cells treated with different plasmids

in tumor-bearing mice. E: Western blotting assay for the protein expressions of P21,

E2F-1, pRb, and HIF-1α in the PC-3 tumor xenografts at the 32nd day after treatments

with different plasmids. ap<0.05 vs mock or Si-scramble group; bp<0.05 vs Pp53

group; cp<0.05 vs Si-mdm2 group.


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Table 1. Primers and the optimal PCR conditions

Primer Sequence (5’-3’) Product

Size (bp)

Annealing

Temperature

(°C)

No. of

cycles

GAPDH

Sense: 5’-GGGTGATGCTGGTGCTGAGTATGT-3’

Antisense: 5’-AAGAATGGGAGTTGCTGTTGAAGTC-3’

700 57 30

MDM2

Sense: 5’-GAAAGCCAAACTGGAAAACTCA-3’

Antisense: 5’-ACATGTAAAGCAGGCCATAAGA-3’

364 58 30

P53

Sense: 5'-CCTCCTCAGCATCTTATCCG-3'

Antisense: 5'-CACAAACACGCACCTCAAA-3'

259 62 30

P21

Sense: 5'- AGCTCAATGGACTGGAAGGG - 3'

Antisense: 5'-GAGCTGGAAGGTGTTTGGGG - 3'

485 55 35

CDK4

Sense: 5'-CGTGAGGTGGCTTTACTGAG-3'

Antisense: 5'- CTTGATCGTTTCGGCTGG-3

267 58 28

Cyclin-D1

Sense: 5'-TGGATGCTGGAGGTCTGCGAG-3'

Antisense : 5'-GGCTTCGATCTGCTCCTGGC-3'

355 60 28

HIF-1α

Sense: 5'- TGGAGGCGGCGAGAACAT -3'

Antisense: 5' - CTCAGTAGCATCAGCACGAGGG -3'

168 58 20


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A

B

Normal

Cancer0

20

40

60

Md

m2

exp

ress

ion

per

cen

t 13/19

0/10

Normal

Cancer0

20

40

10/19

mt-

p53

exp

ress

ion

per

cen

t

0/10

Fig.1


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A

GAPDH

mdm2

wt-p53

β-actin

Wt-p53

Mdm2

B C

U6 Si-scrambleSi-scramble

CMV p53Pp53

U6 Si-mdm2Si-mdm2

U6 Si-mdm2Psi-mdm2-p53 CMV p53

Mdm2 wt-p530.0

0.3

0.6

0.9

1.2a,c

b

a

a,b,c

a,b

Op

tica

l den

sity

(%) mock

Si-scramble Pp53 Si-mdm2 Pmp53

a

Mdm2 wt-p530.0

0.3

0.6

0.9

1.2

a,b,c

b

a

a,b,c

a,b

a

Op

tica

l den

sity

(%) mock


Fig.2


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B

A

C

48h 72h0

20

40

60

80

100

a,b,c

aa

a,b,c

aG

row

th r

atio

(%)

mock Si-scramble Pp53 Si-mdm2 Ppmp53a

72h0

10

20

30a,b,c

a

Ap

op

tosi

s ra

te(%

) Si-scram b le P p53 S i-m dm 2 P m p 53

a

.

Fig.3


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Cyclin-D1

β-actin

HIF-1α

p21

CDK4

B

p21

cyclin-D1

cdk4

HIF-1α

GAPDH

p21 cdk4 cyclin-D1 HIF-1a0.0

0.2

0.4

0.6

0.8

a,c

a,b

a

a a a

a,c

a,b

ab

a,c

Op

tica

l den

sity

(%)

mock Si-scramble Pp53 Si-mdm2 Pmp53

a

p21 CDK4 Cyclin-D1 HIF-1a0.0

0.2

0.4

0.6

aa

a

aaaa a

a

a

a

Op

tica

l den

sity

(%) mock


a

Fig.4


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T/72h Li/72h Lu/72h S/72h

H/72h K/72h T/10d T/10d

A

B C

Tumor

Liver

050

100150200250300350

CF

U/g

a

Tumor

Liver

Lung

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