- Email: [email protected]
Inhibition of Prostate Cancer Using RNA Interference-directed Knockdown of Platelet-derived Growth Factor Receptor
Basic and Translational Science Inhibition of Prostate Cancer Using RNA Interference-directed Knockdown of Platelet-derived Growth Factor Receptor Yong Hyun Park, Su Yeon Seo, Minju Ha, Ja Hyeon Ku, Hyeon Hoe Kim, and Cheol Kwak OBJECTIVES METHODS
RESULTS
CONCLUSIONS
To determine whether platelet-derived growth factor receptor (PDGFR) plays a role in the tumorigenicity of prostate cancer cells. PC3 prostate cancer cells were transfected with small interfering (si)PDGFR-␣ and siPDGFR-, constructed according to the conventional small interfering RNA design standard. Reverse transcriptase polymerase chain reaction, Western blot analysis, and cell growth were studied to determine the characteristics of PDGFR-␣ and PDGFR- in vitro. The prostate cancer xenograft model was established to investigate whether knockout of PDGFR-␣ and PDGFR- decreases prostate cancer tumor growth in vivo. The experimental groups were defined as group 1 (PC3 cells only), group 2 (PC3 cells transfected with small interfering green fluorescent protein), group 3 (PC3 cells transfected with siPDGFR-␣), group 4 (PC3 cells transfected with siPDGFR-), and group 5 (PC3 cells transfected with siPDGFR-␣ and siPDGFR-). Western blot analysis revealed that siPDGFR-␣ and siPDGFR- significantly blocked PDGFR-␣ and PDGFR- protein expression. After 48 hours of transfection of the PC3 cells with siPDGFR-␣ and siPDGFR-, the relative fractions of viable cells were reduced to 47.7% (P ⫽ .007) and 38.5% (P ⫽ .010). In vivo, mice treated with siPDGFR-␣ or siPDGFR- and siPDGFR-␣ plus siPDGFR- had significant tumor cell growth arrest compared with the mice in groups 1 and 2 (P ⫽ .001). In addition, a significant reduction in the microvessel density was observed in tumors from the mice treated with siPDGFR-␣ or siPDGFR- and siPDGFR-␣ plus siPDGFR- (P ⬍ .001). The results of the present study suggest that siPDGFR-␣ and siPDGFR- might inhibit prostate cancer cell growth by the suppression of angiogenesis. UROLOGY 77: 1509.e9 –1509.e15, 2011. © 2011 Elsevier Inc.
P
rostate cancer is the most common cancer in men in the United States and the second leading cause of cancer-related death, accounting for 9% of all cancer-related deaths in men.1,2 The progression of prostate cancer from hormone-sensitive to hormone-refractory disease is a major challenge to clinical management. Investigations to improve the understanding of the biologic mechanisms associated with hormone-refractory prostate cancer are ongoing, because this could provide insight into the process of tumor suppression. Several growth factor receptors with intrinsic tyrosine kinase activity have been implicated in the development and progression of cancer. Growth factor This research was supported by the Seoul National University Research Fund (grant 04-2006-014-0). From the Department of Urology, Seoul National University College of Medicine, Seoul, Korea; and Seoul National University School of Biological Sciences and Center for National Creative Research, Seoul, Korea Reprint requests: Cheol Kwak, M.D., Ph.D., Department of Urology, Seoul National University College of Medicine, 101 Daehak-no, Jongno-gu, Seoul 110-744 Korea. E-mail: [email protected] Submitted: September 16, 2010, accepted (with revisions): January 25, 2011
© 2011 Elsevier Inc. All Rights Reserved
receptor-driven tumorigenicity might be related to increased proliferation, suppression of apoptosis, and activation of processes favoring metastatic spread. Consequently, growth factor receptors have become a reasonable target for therapeutic intervention.3 Platelet-derived growth factor (PDGF) is a 30-kd protein consisting of disulfide bond homodimers or heterodimers of the A and B chains. Although normal PDGF function is critical for normal embryonal development and homeostasis, overactivity of the PDGF/ PDGF receptor (PDGFR) axis has been implicated in several disorders characterized by excessive cell growth. These include fibrotic conditions, plaque formation in atherosclerosis, and certain malignancies.4 To date, only a few studies have examined the consequences of PDGFR manipulation in experimental prostate cancer cell systems. In the present study, to determine whether PDGFR plays a role in the tumorigenicity of prostate cancer cells, small interfering RNA (siRNA) were used to selectively inhibit the expression of PDGFR-␣ and - in PC3 prostate cancer 0090-4295/11/$36.00 1509.e9 doi:10.1016/j.urology.2011.01.050
cells to determine the association of PDGFR with the prostate cancer.
MATERIAL AND METHODS Cell Lines and Culture The human normal prostate cell line RWPE1 and prostate cancer cell lines LNCaP and PC3 were purchased from the American Type Culture Collection (Manassas, VA) and grown in Roswell Park Memorial Institute-1640 medium supplemented with 10% heat-inactivated fetal bovine serum, 50 U/mL of penicillin, and 50 g/mL of streptomycin in a humidified atmosphere with 5% carbon dioxide at 37°C.
RNA Extraction and Reverse TranscriptasePolymerase Chain Reaction Total cellular RNA was isolated with Trizol reagent (Invitrogen, Carlsbad, CA) according to the manufacturer’s instructions. cDNA was synthesized from 1 g of total RNA using the SuperScript First-Strand synthesis system (Invitrogen) with the oligodT primer. Prepared cDNA samples were amplified and analyzed using polymerase chain reaction (PCR). The primers used were as follows: forward primer 5=-CAT CGT GGA GGA TGA TGA TTC TG-3= and reverse primer 5=-GCT CTT CAC AGC ATC TTC ATT TTG-3= for PDGFR-␣; forward primer 5=-AGC TCT ACA GCA ATG CTC TGC C-3= and reverse primer 5=-GGC TGT CAC AGG AGA TGG TTG-3= for PDGFR-. Glyceraldehyde 3-phosphate dehydrogenase was used as an internal control for each reaction. The PCR products were analyzed by electrophoresis in a 1.5% agarose gel and visualized using ultraviolet fluorescence after staining with ethidium bromide.
Western Blot Analysis The cells were homogenized in lysis buffer containing a protease inhibitor cocktail (Sigma-Aldrich, St. Louis, MO). Cell lysates containing 50 g of protein were electrophoresed on 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to a polyvinylidene difluoride membrane. The membranes were blocked with 5% nonfat milk, followed by incubation overnight with the appropriate primary antibody at 4°C. The membranes were then washed 3 times and incubated with goat anti-rabbit antibody (Cell Signaling Technology, Danvers, MA) or horse antimouse antibody (Cell Signaling Technology) conjugated with horseradish peroxidase for 1 hour and visualized using the Amersham ECL Western blotting detection reagents (GE Healthcare, Chalfont St. Giles, Buckinghamshire, United Kingdom). The primary antibodies used were rabbit anti-PDGFR-␣ monoclonal antibody (1:1000, Cell Signaling Technology), rabbit anti-PDGFR- monoclonal antibody (1: 1000, Cell Signaling Technology), and mouse anti-glyceraldehyde 3-phosphate dehydrogenase monoclonal antibody (1: 2000, Sigma-Aldrich).
siRNA Treatment of Cells siRNA targeted to PDGFR-␣ (siPDGFR-␣1, 5=-UAU AAU GGC AGA AUC AUC ATT-3= and siPDGFR-␣2, 5=-UAC AAU AGU AUA AUG GCC ATT-3=) and PDGFR- (siPDGFR-1, 5=-UUG ACG GCC ACU UUC AUC GTT-3= and siPDGFR-2, 5=-UGU CAC AGG AGA UGG UUG ATT-3=) were synthesized and annealed, as described previously.5 A control RNAi construct for the green fluorescent 1509.e10
Figure 1. Expression of PDGFR. (A) Endogenous mRNA expression of PDGFR. PC3 cells showed stronger expression of PDGFR-␣ and similar expression of PDGFR- compared with RWPE1. (B) Densitometry analysis revealed 99.2% reduction in PDGFR-␣ mRNA expression in siPDGFR␣1-treated cells and 96.9% reduction in PDGFR- mRNA expression in siPDGFR-2-treated cells. (C) Western blot analysis showed decrease in PDGFR expression in PC3 cells treated with siPDGFR compared with control, Lipofectamine, and siGFP. protein (GFP) gene, siGFP (Samchully Pharm, Seoul, Korea), was also used. The PC3 cells were seeded 24 hours before siRNA treatment to allow adherent cell growth. The cultures were incubated for 4 hours with 5 L of 20 M siRNA molecules precomplexed in 2 mL of Opti-MEM medium with 5 L of Lipofectamine 2000 (Invitrogen) according to the manufacturer’s protocol. After incubation, the incubation medium was replaced by complete medium, and the cells were cultivated under standard conditions.
Cell Proliferation Assay The PC3 cells were plated in 96-well plates at a density of 5 ⫻ 103 cells/well. At 24 and 48 hours after transfection, 20 L of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (Sigma-Aldrich) stock solution (5 mg/mL) was added to 200 L of medium in each well, and the plates were incubated at 37°C for 4 hours. Subsequently, 150 L of dimethyl sulfoxide was added to each well. The plate was shaken on a rotary platform at room temperature for 10 minutes, and the absorbance at a wavelength of 490 nm was measured with a microplate reader using a test wavelength of 490 nm. UROLOGY 77 (6), 2011
Figure 2. Inhibition of PDGFR-␣ and PDGFR- expression inhibited growth of PC3 cells. (A) After 24 hours of siPDGFR-␣1 and siPDGFR-␣2 transfection. (B) After 48 hours of siPDGFR-␣1 and siPDGFR-␣2 transfection. (C) After 24 hours of siPDGFR-1 and siPDGFR-2 transfection. (D) After 48 hours of siPDGFR-1 and siPDGFR-2 transfection. Columns represent mean (n ⫽ 3); bars, standard error. *P ⬍ .05, statistically significant compared with control.
In Vivo Effects of siPDGFR on Prostate Cancer Cell Growth in Xenograft Nude Mice Specific pathogen-free athymic nude mice (age 4-5 weeks) were purchased from Central Lab Animal (Seoul, Korea). They were allowed to acclimatize for 1 week before initiating the experiment. They were maintained in a 12-hour light/12-hour dark cycle under pathogen-free conditions and fed with standard diet and water ad libitum. The Institutional Animal Care and Use Committee of Seoul National University Hospital reviewed and approved all procedures. The experimental groups were defined as group 1 (2 ⫻ 106 PC3 cells only, n ⫽ 5), group 2 (2 ⫻ 106 PC3 cells transfected with control siRNA, n ⫽ 5), group 3 (2 ⫻ 106 PC3 cells transfected with siPDGFR-␣, n ⫽ 5), group 4 (2 ⫻ 106 PC3 cells transfected with siPDGFR-, n ⫽ 5), and group 5 (2 ⫻ 106 PC3 cells transfected with siPDGFR-␣ and siPDGFR-, n ⫽ 5). Cell viability was assessed using trypan blue dye, and same number of viable cells was inoculated in each group immediately after transfection. Tumor growth was measured every 3 days with a caliper, and the body weight and tumor diameters were recorded. The tumor volume was calculated using the formula: volume (mm3) ⫽ 0.5 ab2, where a and b are the 2 maximum diameters. At 4 weeks after injection, the mice were killed, and the tumors were harvested.
Immunohistochemistry of CD31 and Microvessel Density Assessment The tumors were fixed in 10% neutral buffered formalin and embedded in paraffin. Tissue sections (4 m) were dewaxed UROLOGY 77 (6), 2011
and placed in high pH antigen retrieval solution (Dako, Carpinteria, CA) heated to 121°C for 4 minutes, and then at 90°C for an additional 10 minutes. After blocking endogenous peroxidase activity, monoclonal rabbit antibody to CD31 (Santa Cruz Biotechnology, Santa Cruz, CA) diluted 1:100 in antibody diluent (Dako) was added to each slide and allowed to incubate at room temperature for 1 hour. After washing off the unbound primary antibody, the sections were treated with commercial biotinylated secondary anti-immunoglobulin, followed by avidin coupled to biotinylated horseradish peroxidase, at room temperature, according to the manufacturer’s instructions (Dako). The immunohistochemical reactions were developed with diaminobenzidine as the chromogenic peroxidase substrate. The sections were lightly counterstained with Mayer’s hematoxylin after immunohistochemistry. The sections without primary antibody served as the negative controls. Microvessel density was evaluated according to the criteria of Gasparini and Harris6 by a single observer who was unaware of the treatment group. Using low-power light microscopy (⫻40), the tissue sections were screened; the 5 areas with the most intense neovascularization were selected for additional study. The microvessel counts of these areas were performed under high-power light microscopy (⫻200). To reduce the observerrelated variation, the counting of the microvessels was performed using a computer image analyzer. The mean microvessel 1509.e11
significantly blocked PDGFR-␣ and PDGFR- mRNA expression. Furthermore, densitometry analysis revealed a reduction to 0.8% ⫾ 0.2% in PDGFR-␣ mRNA expression in the siPDGFR-␣1 treated cells compared with the control, and a significant difference was observed in the comparisons with the siPDGFR-␣2- or siGFP-treated cells (P ⬍ .001). In addition, PDGFR- mRNA expression in the siPDGFR-2-treated cells was reduced to 3.1% ⫾ 0.6% compared with the control (P ⫽ .004). No significant difference was observed in the comparisons with the siPDGFR-1-treated cells (P ⫽ .836). To further confirm the findings associated with siPDGFR, Western blot analysis was performed (Fig. 1C). After normalization with glyceraldehyde 3-phosphate dehydrogenase, the expression levels of PDGFR-␣ and PDGFR- in siPDGFR-␣- and siPDGFR--treated cells were found to be significantly lower than in the control, Lipofectamine-treated, and siGFP-treated cells, consistent with the results from reverse transcriptase-PCR.
Figure 3. In vivo effects of siPDGFR in prostate cancer xenograft model. (A) No significant difference in mean body weight found among groups during experimental period. (B) Significant reduction in tumor volume observed in mice treated with siPDGFR-␣, siPDGFR-, and siPDGFR-␣ plus siPDGFR- at 2 weeks (P ⬍ .001), 3 weeks (P ⬍ .001), and 4 weeks (P ⬍ .001) compared with mice in control group.
count of the 5 most vascular areas was defined as the microvessel density, expressed as the absolute number of microvessels/ 0.74 mm2 (⫻200 field).
Statistical Analysis All in vitro experiments were performed ⱖ3 times, and the reported results are from representative experiments. Intergroup comparisons were analyzed using the Kruskal-Wallis test and the Statistical Package for Social Sciences, version 17.0, statistical software (SPSS, Chicago, IL). A value of P ⬍ .05 was considered statistically significant.
RESULTS PDGFR mRNA Expression in Prostate Cancer Cell Lines To study the endogenous expression of PDGFR in prostate cancer, the expression of PDGFR-␣ and PDGFR- was first examined in the RWPE1, LNCaP, and PC3 cell lines. As shown in Figure 1A, PC3 showed stronger expression of PDGFR-␣ and was similar to the expression of PDGFR- compared with RWPE1. However, LNCaP showed no expression of PDGFR-␣. Thus, the PC3 cell line was used for subsequent experiments. Effect of siRNA on PDGFR Expression As shown in Figure 1B, semiquantitative reverse transcriptase-PCR revealed that siPDGFR-␣ and siPDGFR- 1509.e12
Effects of siPDGFR on PC3 Cell Viability As shown in Figure 2, the siPDGFR-␣1- or siPDGFR␣2-treated cells exhibited a significant decline in the relative fraction of viable cells. In contrast, the relative fraction of viable cells in the Lipofectaminetreated or siGFP-treated cells was unaffected compared with the control cells. Compared with the control, Lipofectamine-treated, and siGFP-treated cells, the relative fraction of viable cells in siPDGFR-␣1- and siPDGFR-␣2-treated cells was reduced to 47.7% and 67.2% at 24 hours (P ⫽ .007) and 61.9% and 87.8% at 48 hours (P ⫽ .015), respectively. Similar to siPDGFR-␣, siPDGFR-1 and siPDGFR-2 decreased the relative fraction of viable cells by 83.3% and 47.2% at 24 hours (P ⫽ .010) and 77.4% and 38.5% at 48 hours (P ⫽ .009), respectively. Therefore, all subsequent in vivo experiments were performed using siPDGFR-␣1 and siPDGFR-2.
RNA Interference-Mediated Inhibition of PDGFR in Prostate Cancer Xenograft No significant difference was seen in the mean body weight among the 5 groups during the experimental period (Fig. 3A, P ⫽ 865). Figure 3B shows the significant antitumor effects of siPDGFR-␣ and siPDGFR- compared with the controls. The mice in groups 3, 4, and 5 had significant tumor growth arrest compared with the mice in groups 1 and 2 (88.8, 71.5, 42.9, 19.4, and 14.4 mm3 for groups 1, 2, 3, 4, and 5, respectively; P ⫽ .001). Compared with groups 1 and 2, the tumors in groups 3, 4, and 5 were characterized by increased necrotic areas and fewer microvessels (Fig. 4). A significant reduction in the microvessel density was observed in the tumors from groups 3, 4, and 5 (47.7, 45.8, 14.3, 7.2, and 3.0 in groups 1 through 5, respectively, P ⬍ .001). UROLOGY 77 (6), 2011
Figure 4. Microscopic changes and microvessel density in xenografted tumors. (A) Hematoxylin-eosin staining showed minimal necrotic cells in (A1) group 1 and (A2) group 2. In contrast, tumors in (A3) group 3, (A4) group 4, and (A5) group 5 showed large areas of necrosis. (B) Histograph corresponding to number of cells showing immunoreactivity against CD31 in each group.
COMMENT The physiologic role of the PDGF isoforms and their receptors during embryonal development and wound healing has been well studied using knockout of genes for PDGF isoforms and receptors in mice.7-10 PDGF isoforms exert their biologic effects UROLOGY 77 (6), 2011
by binding to structurally similar PDGF-␣ and PDGF- tyrosine kinase receptors.11 It is well known that uncontrolled activation of these receptors ultimately promotes a variety of cellular processes, including proliferation, migration, and survival. Thus, overexpression 1509.e13
of the PDGFs and their receptors has been associated with many types of cancers, such as glioblastoma,12,13 ovary cancer,14 and prostate cancer.15-19 Some investigators have reported that PDGFR is expressed in high fractions of prostate cancer by immunohistochemistry.15-17 Ko et al16 reported that PDGFR expression was detected in 88% of primary prostate cancer samples and 80% of metastatic bone marrow samples; another study supported this finding.15,17 However, a review of 5 published prostate expression studies, using microarray analysis of 100 clinically localized prostate cancers, showed that PDGFR expression was upregulated considerably in small populations of prostate cancer, 5% of clinically localized prostate cancer and 16% of metastatic prostate cancer, and no significant association between PDGFR expression and prognostic factors, including preoperative prostate-specific antigen level, Gleason score, and tumor stage, was identified.20 This result might have been due to the inherent limitations of immunohistochemical studies. Also, selection bias might have played a significant role, and is a major issue in molecular epidemiologic studies that rely on tumor tissue because the availability of tissue blocks could be a function of the underlying disease, hospital diagnostic practices, pathology laboratory preservation procedures, storage protocols, and the study population.21 In addition, the differences in PDGFR expression could have resulted in part from the different cutoff levels used for grading PDGFR expression in each study. Moreover, although immunohistochemical studies have confirmed coexpression of ligands and receptors, in human tumor tissues, their functional significance still remains unclear. Few in vitro and in vivo studies have been done on the association between PDGFR and prostate cancer. Kim et al18 reported that targeting the phosphorylation of PDGFR in tumor-associated endothelial cells using imatinib, with or without paclitaxel, led to regression of multidrug-resistant prostate cancer and to inhibition of lymph node metastases. In addition, Kimura et al19 reported that imatinib improved the outcome of radioimmunotherapy in a mouse model of prostate cancer by reducing the tumor interstitial fluid pressure, with the subsequent increase of 131ICC49 uptake into the tumor and inhibition of hypoxia-inducible factor-1␣, resulting in improved tumor radiosensitivity. Imatinib mesylate inhibits tyrosine kinases encoded by the bcr-abl oncogene, as well as the receptor tyrosine kinases encoded by c-kit and PDGFR.22 Thus, the major caveat of these studies was the use of tyrosine kinase inhibitors with incompletely characterized specificity. Therefore, in the present study, the consequences of PDGFR knockout, in human prostate cancer cells, using siRNA specific for PDGFR-␣ and PDGFR- was investigated. RNA interference is a recently developed technique used to silence proteins in a sequence-specific manner by inhibiting mRNA and consequently reducing protein expression. siRNA transfection is always a transient 1509.e14
event because the introduced siRNA lacks the ability to replicate itself and quickly disappears owing to degradation and dilution by cell division.23 Once siRNA disappears in the transfected cells, the target gene quickly recovers to its normal state. It is well known that the levels of the targeted gene in siRNA-treated cells have typically recovered by 5-7 days after siRNA transfection (ie, after 7-10 rounds of cell division).24 In the present study, PDGFR knockdown was detected at 24 hours after siRNA transfection (Fig. 2). However, the effects of the knockdown decreased within 2-3 days in vitro (Fig. 3) and by 3 weeks in vivo (Fig. 4). Using the siRNA specific for PDGFR-␣ and PDGFR-, the results of the present study showed that PDGFR knockdown caused cell death. This appeared to occur by the inhibition of angiogenesis. Although both PDGFR-␣ and PDGFR- mediate strong mitogenic signals, some investigators have reported that PDGFR- induces more potent transforming signals than the signals transduced by PDGFR-␣.25-28 Yu et al25 suggested that activation of PDGFR-␣ might transduce both positive and negative signaling for cell transformation, and PDGFR- might mainly induce positive signaling for cell transformation. However, no study has demonstrated whether PDGFR-␣ or PDGFR- plays a more important role in experimental prostate cancer cell systems. The present study is the first to show that inhibition of PDGFR- had superior antitumor activity compared with siPDGFR-␣. Furthermore, the results of our study have provided in vivo evidence that prostate cancer tumorigenesis is associated with PDGFR expression.
CONCLUSIONS The results of the present study have provided evidence suggesting a critical and specific role for PDGFR-␣ and PDGFR- in the PC3 prostate cancer cell line viability and proliferation. siPDGFR-␣ and siPDGFR- successfully inhibited the expression of PDGFR-␣ and PDGFR-, and the inhibition of PDGFR-␣ and  might have resulted in the inhibition of prostate cancer growth by the suppression of angiogenesis. References 1. U.S. Preventive Services Task Force. Screening for prostate cancer: U.S. Preventive Services Task Force recommendation statement. Ann Intern Med 2008;149:185-191. 2. Espey DK, Wu XC, Swan J, et al. Annual report to the nation on the status of cancer, 1975-2004, featuring cancer in American Indians and Alaska Natives. Cancer. 2007;110:2119-2152. 3. Shawver LK, Slamon D, Ullrich A. Smart drugs: tyrosine kinase inhibitors in cancer therapy. Cancer Cell. 2002;1:117-123. 4. Pietras K, Rubin K, Sjoblom T, et al. Inhibition of PDGF receptor signaling in tumor stroma enhances antitumor effect of chemotherapy. Cancer Res. 2002;62:5476-5484. 5. Reynolds A, Leake D, Boese Q, et al. Rational siRNA design for RNA interference. Nat Biotechnol. 2004;22:326-330. 6. Gasparini G, Harris AL. Clinical importance of the determination of tumor angiogenesis in breast carcinoma: much more than a new prognostic tool. J Clin Oncol. 1995;13:765-782.
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7. Betsholtz C. Insight into the physiological functions of PDGF through genetic studies in mice. Cytokine Growth Factor Rev. 2004; 15:215-228. 8. Robson MC, Phillips LG, Thomason A, et al. Platelet-derived growth factor BB for the treatment of chronic pressure ulcers. Lancet. 1992;339:23-25. 9. Ding H, Wu X, Bostrom H, et al. A specific requirement for PDGF-C in palate formation and PDGFR-alpha signaling. Nat Genet. 2004;36:1111-1116. 10. Soriano P. Abnormal kidney development and hematological disorders in PDGF beta-receptor mutant mice. Genes Dev. 1994;8: 1888-1896. 11. Rosenkranz S, Kazlauskas A. Evidence for distinct signaling properties and biological responses induced by the PDGF receptor alpha and beta subtypes. Growth Factors. 1999;16:201-216. 12. Hagerstrand D, Hesselager G, Achterberg S, et al. Characterization of an imatinib-sensitive subset of high-grade human glioma cultures. Oncogene. 2006;25:4913-4922. 13. Hesselager G, Uhrbom L, Westermark B, et al. Complementary effects of platelet-derived growth factor autocrine stimulation and p53 or Ink4a-Arf deletion in a mouse glioma model. Cancer Res. 2003;63:4305-4309. 14. Ostman A. PDGF receptors-mediators of autocrine tumor growth and regulators of tumor vasculature and stroma. Cytokine Growth Factor Rev. 2004;15:275-286. 15. Fudge K, Wang CY, Stearns ME. Immunohistochemistry analysis of platelet-derived growth factor A and B chains and platelet-derived growth factor alpha and beta receptor expression in benign prostatic hyperplasias and Gleason-graded human prostate adenocarcinomas. Mod Pathol. 1994;7:549-554. 16. Ko YJ, Small EJ, Kabbinavar F, et al. A multi-institutional phase II study of SU101, a platelet-derived growth factor receptor inhibitor, for patients with hormone-refractory prostate cancer. Clin Cancer Res. 2001;7:800-805. 17. Chott A, Sun Z, Morganstern D, et al. Tyrosine kinases expressed in vivo by human prostate cancer bone marrow metastases and loss
UROLOGY 77 (6), 2011
18.
19.
20.
21.
22. 23. 24. 25.
26.
27.
28.
of the type 1 insulin-like growth factor receptor. Am J Pathol. 1999;155:1271-1279. Kim SJ, Uehara H, Yazici S, et al. Targeting platelet-derived growth factor receptor on endothelial cells of multidrug-resistant prostate cancer. J Natl Cancer Inst. 2006;98:783-793. Kimura Y, Inoue K, Abe M, et al. PDGFRbeta and HIF-1alpha inhibition with imatinib and radioimmunotherapy of experimental prostate cancer. Cancer Biol Ther. 2007;6:1763-1772. Hofer MD, Fecko A, Shen R, et al. Expression of the plateletderived growth factor receptor in prostate cancer and treatment implications with tyrosine kinase inhibitors. Neoplasia. 2004;6: 503-512. Hoppin JA, Tolbert PE, Taylor JA, et al. Potential for selection bias with tumor tissue retrieval in molecular epidemiology studies. Ann Epidemiol. 2002;12:1-6. Homsi J, Daud AI. Spectrum of activity and mechanism of action of VEGF/PDGF inhibitors. Cancer Control. 2007;14:285-294. Zhou D, He QS, Wang C, et al. RNA interference and potential applications. Curr Top Med Chem. 2006;6:901-911. Tuschl T. Expanding small RNA interference. Nat Biotechnol. 2002;20:446-448. Yu J, Deuel TF, Kim HR. Platelet-derived growth factor (PDGF) receptor-alpha activates c-Jun NH2-terminal kinase-1 and antagonizes PDGF receptor-beta-induced phenotypic transformation. J Biol Chem. 2000;275:19076-19082. Kim HR, Upadhyay S, Korsmeyer S, et al. Platelet-derived growth factor (PDGF) B and A homodimers transform murine fibroblasts depending on the genetic background of the cell. J Biol Chem. 1994;269:30604-30608. Frisch SM, Vuori K, Kelaita D, et al. A role for Jun-N-terminal kinase in anoikis; suppression by bcl-2 and crmA. J Cell Biol. 1996;135:1377-1382. Bejcek BE, Hoffman RM, Lipps D, et al. The v-sis oncogene product but not platelet-derived growth factor (PDGF) A homodimers activate PDGF alpha and beta receptors intracellularly and initiate cellular transformation. J Biol Chem. 1992;267: 3289-3293.
1509.e15
RESULTS
CONCLUSIONS
To determine whether platelet-derived growth factor receptor (PDGFR) plays a role in the tumorigenicity of prostate cancer cells. PC3 prostate cancer cells were transfected with small interfering (si)PDGFR-␣ and siPDGFR-, constructed according to the conventional small interfering RNA design standard. Reverse transcriptase polymerase chain reaction, Western blot analysis, and cell growth were studied to determine the characteristics of PDGFR-␣ and PDGFR- in vitro. The prostate cancer xenograft model was established to investigate whether knockout of PDGFR-␣ and PDGFR- decreases prostate cancer tumor growth in vivo. The experimental groups were defined as group 1 (PC3 cells only), group 2 (PC3 cells transfected with small interfering green fluorescent protein), group 3 (PC3 cells transfected with siPDGFR-␣), group 4 (PC3 cells transfected with siPDGFR-), and group 5 (PC3 cells transfected with siPDGFR-␣ and siPDGFR-). Western blot analysis revealed that siPDGFR-␣ and siPDGFR- significantly blocked PDGFR-␣ and PDGFR- protein expression. After 48 hours of transfection of the PC3 cells with siPDGFR-␣ and siPDGFR-, the relative fractions of viable cells were reduced to 47.7% (P ⫽ .007) and 38.5% (P ⫽ .010). In vivo, mice treated with siPDGFR-␣ or siPDGFR- and siPDGFR-␣ plus siPDGFR- had significant tumor cell growth arrest compared with the mice in groups 1 and 2 (P ⫽ .001). In addition, a significant reduction in the microvessel density was observed in tumors from the mice treated with siPDGFR-␣ or siPDGFR- and siPDGFR-␣ plus siPDGFR- (P ⬍ .001). The results of the present study suggest that siPDGFR-␣ and siPDGFR- might inhibit prostate cancer cell growth by the suppression of angiogenesis. UROLOGY 77: 1509.e9 –1509.e15, 2011. © 2011 Elsevier Inc.
P
rostate cancer is the most common cancer in men in the United States and the second leading cause of cancer-related death, accounting for 9% of all cancer-related deaths in men.1,2 The progression of prostate cancer from hormone-sensitive to hormone-refractory disease is a major challenge to clinical management. Investigations to improve the understanding of the biologic mechanisms associated with hormone-refractory prostate cancer are ongoing, because this could provide insight into the process of tumor suppression. Several growth factor receptors with intrinsic tyrosine kinase activity have been implicated in the development and progression of cancer. Growth factor This research was supported by the Seoul National University Research Fund (grant 04-2006-014-0). From the Department of Urology, Seoul National University College of Medicine, Seoul, Korea; and Seoul National University School of Biological Sciences and Center for National Creative Research, Seoul, Korea Reprint requests: Cheol Kwak, M.D., Ph.D., Department of Urology, Seoul National University College of Medicine, 101 Daehak-no, Jongno-gu, Seoul 110-744 Korea. E-mail: [email protected] Submitted: September 16, 2010, accepted (with revisions): January 25, 2011
© 2011 Elsevier Inc. All Rights Reserved
receptor-driven tumorigenicity might be related to increased proliferation, suppression of apoptosis, and activation of processes favoring metastatic spread. Consequently, growth factor receptors have become a reasonable target for therapeutic intervention.3 Platelet-derived growth factor (PDGF) is a 30-kd protein consisting of disulfide bond homodimers or heterodimers of the A and B chains. Although normal PDGF function is critical for normal embryonal development and homeostasis, overactivity of the PDGF/ PDGF receptor (PDGFR) axis has been implicated in several disorders characterized by excessive cell growth. These include fibrotic conditions, plaque formation in atherosclerosis, and certain malignancies.4 To date, only a few studies have examined the consequences of PDGFR manipulation in experimental prostate cancer cell systems. In the present study, to determine whether PDGFR plays a role in the tumorigenicity of prostate cancer cells, small interfering RNA (siRNA) were used to selectively inhibit the expression of PDGFR-␣ and - in PC3 prostate cancer 0090-4295/11/$36.00 1509.e9 doi:10.1016/j.urology.2011.01.050
cells to determine the association of PDGFR with the prostate cancer.
MATERIAL AND METHODS Cell Lines and Culture The human normal prostate cell line RWPE1 and prostate cancer cell lines LNCaP and PC3 were purchased from the American Type Culture Collection (Manassas, VA) and grown in Roswell Park Memorial Institute-1640 medium supplemented with 10% heat-inactivated fetal bovine serum, 50 U/mL of penicillin, and 50 g/mL of streptomycin in a humidified atmosphere with 5% carbon dioxide at 37°C.
RNA Extraction and Reverse TranscriptasePolymerase Chain Reaction Total cellular RNA was isolated with Trizol reagent (Invitrogen, Carlsbad, CA) according to the manufacturer’s instructions. cDNA was synthesized from 1 g of total RNA using the SuperScript First-Strand synthesis system (Invitrogen) with the oligodT primer. Prepared cDNA samples were amplified and analyzed using polymerase chain reaction (PCR). The primers used were as follows: forward primer 5=-CAT CGT GGA GGA TGA TGA TTC TG-3= and reverse primer 5=-GCT CTT CAC AGC ATC TTC ATT TTG-3= for PDGFR-␣; forward primer 5=-AGC TCT ACA GCA ATG CTC TGC C-3= and reverse primer 5=-GGC TGT CAC AGG AGA TGG TTG-3= for PDGFR-. Glyceraldehyde 3-phosphate dehydrogenase was used as an internal control for each reaction. The PCR products were analyzed by electrophoresis in a 1.5% agarose gel and visualized using ultraviolet fluorescence after staining with ethidium bromide.
Western Blot Analysis The cells were homogenized in lysis buffer containing a protease inhibitor cocktail (Sigma-Aldrich, St. Louis, MO). Cell lysates containing 50 g of protein were electrophoresed on 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to a polyvinylidene difluoride membrane. The membranes were blocked with 5% nonfat milk, followed by incubation overnight with the appropriate primary antibody at 4°C. The membranes were then washed 3 times and incubated with goat anti-rabbit antibody (Cell Signaling Technology, Danvers, MA) or horse antimouse antibody (Cell Signaling Technology) conjugated with horseradish peroxidase for 1 hour and visualized using the Amersham ECL Western blotting detection reagents (GE Healthcare, Chalfont St. Giles, Buckinghamshire, United Kingdom). The primary antibodies used were rabbit anti-PDGFR-␣ monoclonal antibody (1:1000, Cell Signaling Technology), rabbit anti-PDGFR- monoclonal antibody (1: 1000, Cell Signaling Technology), and mouse anti-glyceraldehyde 3-phosphate dehydrogenase monoclonal antibody (1: 2000, Sigma-Aldrich).
siRNA Treatment of Cells siRNA targeted to PDGFR-␣ (siPDGFR-␣1, 5=-UAU AAU GGC AGA AUC AUC ATT-3= and siPDGFR-␣2, 5=-UAC AAU AGU AUA AUG GCC ATT-3=) and PDGFR- (siPDGFR-1, 5=-UUG ACG GCC ACU UUC AUC GTT-3= and siPDGFR-2, 5=-UGU CAC AGG AGA UGG UUG ATT-3=) were synthesized and annealed, as described previously.5 A control RNAi construct for the green fluorescent 1509.e10
Figure 1. Expression of PDGFR. (A) Endogenous mRNA expression of PDGFR. PC3 cells showed stronger expression of PDGFR-␣ and similar expression of PDGFR- compared with RWPE1. (B) Densitometry analysis revealed 99.2% reduction in PDGFR-␣ mRNA expression in siPDGFR␣1-treated cells and 96.9% reduction in PDGFR- mRNA expression in siPDGFR-2-treated cells. (C) Western blot analysis showed decrease in PDGFR expression in PC3 cells treated with siPDGFR compared with control, Lipofectamine, and siGFP. protein (GFP) gene, siGFP (Samchully Pharm, Seoul, Korea), was also used. The PC3 cells were seeded 24 hours before siRNA treatment to allow adherent cell growth. The cultures were incubated for 4 hours with 5 L of 20 M siRNA molecules precomplexed in 2 mL of Opti-MEM medium with 5 L of Lipofectamine 2000 (Invitrogen) according to the manufacturer’s protocol. After incubation, the incubation medium was replaced by complete medium, and the cells were cultivated under standard conditions.
Cell Proliferation Assay The PC3 cells were plated in 96-well plates at a density of 5 ⫻ 103 cells/well. At 24 and 48 hours after transfection, 20 L of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (Sigma-Aldrich) stock solution (5 mg/mL) was added to 200 L of medium in each well, and the plates were incubated at 37°C for 4 hours. Subsequently, 150 L of dimethyl sulfoxide was added to each well. The plate was shaken on a rotary platform at room temperature for 10 minutes, and the absorbance at a wavelength of 490 nm was measured with a microplate reader using a test wavelength of 490 nm. UROLOGY 77 (6), 2011
Figure 2. Inhibition of PDGFR-␣ and PDGFR- expression inhibited growth of PC3 cells. (A) After 24 hours of siPDGFR-␣1 and siPDGFR-␣2 transfection. (B) After 48 hours of siPDGFR-␣1 and siPDGFR-␣2 transfection. (C) After 24 hours of siPDGFR-1 and siPDGFR-2 transfection. (D) After 48 hours of siPDGFR-1 and siPDGFR-2 transfection. Columns represent mean (n ⫽ 3); bars, standard error. *P ⬍ .05, statistically significant compared with control.
In Vivo Effects of siPDGFR on Prostate Cancer Cell Growth in Xenograft Nude Mice Specific pathogen-free athymic nude mice (age 4-5 weeks) were purchased from Central Lab Animal (Seoul, Korea). They were allowed to acclimatize for 1 week before initiating the experiment. They were maintained in a 12-hour light/12-hour dark cycle under pathogen-free conditions and fed with standard diet and water ad libitum. The Institutional Animal Care and Use Committee of Seoul National University Hospital reviewed and approved all procedures. The experimental groups were defined as group 1 (2 ⫻ 106 PC3 cells only, n ⫽ 5), group 2 (2 ⫻ 106 PC3 cells transfected with control siRNA, n ⫽ 5), group 3 (2 ⫻ 106 PC3 cells transfected with siPDGFR-␣, n ⫽ 5), group 4 (2 ⫻ 106 PC3 cells transfected with siPDGFR-, n ⫽ 5), and group 5 (2 ⫻ 106 PC3 cells transfected with siPDGFR-␣ and siPDGFR-, n ⫽ 5). Cell viability was assessed using trypan blue dye, and same number of viable cells was inoculated in each group immediately after transfection. Tumor growth was measured every 3 days with a caliper, and the body weight and tumor diameters were recorded. The tumor volume was calculated using the formula: volume (mm3) ⫽ 0.5 ab2, where a and b are the 2 maximum diameters. At 4 weeks after injection, the mice were killed, and the tumors were harvested.
Immunohistochemistry of CD31 and Microvessel Density Assessment The tumors were fixed in 10% neutral buffered formalin and embedded in paraffin. Tissue sections (4 m) were dewaxed UROLOGY 77 (6), 2011
and placed in high pH antigen retrieval solution (Dako, Carpinteria, CA) heated to 121°C for 4 minutes, and then at 90°C for an additional 10 minutes. After blocking endogenous peroxidase activity, monoclonal rabbit antibody to CD31 (Santa Cruz Biotechnology, Santa Cruz, CA) diluted 1:100 in antibody diluent (Dako) was added to each slide and allowed to incubate at room temperature for 1 hour. After washing off the unbound primary antibody, the sections were treated with commercial biotinylated secondary anti-immunoglobulin, followed by avidin coupled to biotinylated horseradish peroxidase, at room temperature, according to the manufacturer’s instructions (Dako). The immunohistochemical reactions were developed with diaminobenzidine as the chromogenic peroxidase substrate. The sections were lightly counterstained with Mayer’s hematoxylin after immunohistochemistry. The sections without primary antibody served as the negative controls. Microvessel density was evaluated according to the criteria of Gasparini and Harris6 by a single observer who was unaware of the treatment group. Using low-power light microscopy (⫻40), the tissue sections were screened; the 5 areas with the most intense neovascularization were selected for additional study. The microvessel counts of these areas were performed under high-power light microscopy (⫻200). To reduce the observerrelated variation, the counting of the microvessels was performed using a computer image analyzer. The mean microvessel 1509.e11
significantly blocked PDGFR-␣ and PDGFR- mRNA expression. Furthermore, densitometry analysis revealed a reduction to 0.8% ⫾ 0.2% in PDGFR-␣ mRNA expression in the siPDGFR-␣1 treated cells compared with the control, and a significant difference was observed in the comparisons with the siPDGFR-␣2- or siGFP-treated cells (P ⬍ .001). In addition, PDGFR- mRNA expression in the siPDGFR-2-treated cells was reduced to 3.1% ⫾ 0.6% compared with the control (P ⫽ .004). No significant difference was observed in the comparisons with the siPDGFR-1-treated cells (P ⫽ .836). To further confirm the findings associated with siPDGFR, Western blot analysis was performed (Fig. 1C). After normalization with glyceraldehyde 3-phosphate dehydrogenase, the expression levels of PDGFR-␣ and PDGFR- in siPDGFR-␣- and siPDGFR--treated cells were found to be significantly lower than in the control, Lipofectamine-treated, and siGFP-treated cells, consistent with the results from reverse transcriptase-PCR.
Figure 3. In vivo effects of siPDGFR in prostate cancer xenograft model. (A) No significant difference in mean body weight found among groups during experimental period. (B) Significant reduction in tumor volume observed in mice treated with siPDGFR-␣, siPDGFR-, and siPDGFR-␣ plus siPDGFR- at 2 weeks (P ⬍ .001), 3 weeks (P ⬍ .001), and 4 weeks (P ⬍ .001) compared with mice in control group.
count of the 5 most vascular areas was defined as the microvessel density, expressed as the absolute number of microvessels/ 0.74 mm2 (⫻200 field).
Statistical Analysis All in vitro experiments were performed ⱖ3 times, and the reported results are from representative experiments. Intergroup comparisons were analyzed using the Kruskal-Wallis test and the Statistical Package for Social Sciences, version 17.0, statistical software (SPSS, Chicago, IL). A value of P ⬍ .05 was considered statistically significant.
RESULTS PDGFR mRNA Expression in Prostate Cancer Cell Lines To study the endogenous expression of PDGFR in prostate cancer, the expression of PDGFR-␣ and PDGFR- was first examined in the RWPE1, LNCaP, and PC3 cell lines. As shown in Figure 1A, PC3 showed stronger expression of PDGFR-␣ and was similar to the expression of PDGFR- compared with RWPE1. However, LNCaP showed no expression of PDGFR-␣. Thus, the PC3 cell line was used for subsequent experiments. Effect of siRNA on PDGFR Expression As shown in Figure 1B, semiquantitative reverse transcriptase-PCR revealed that siPDGFR-␣ and siPDGFR- 1509.e12
Effects of siPDGFR on PC3 Cell Viability As shown in Figure 2, the siPDGFR-␣1- or siPDGFR␣2-treated cells exhibited a significant decline in the relative fraction of viable cells. In contrast, the relative fraction of viable cells in the Lipofectaminetreated or siGFP-treated cells was unaffected compared with the control cells. Compared with the control, Lipofectamine-treated, and siGFP-treated cells, the relative fraction of viable cells in siPDGFR-␣1- and siPDGFR-␣2-treated cells was reduced to 47.7% and 67.2% at 24 hours (P ⫽ .007) and 61.9% and 87.8% at 48 hours (P ⫽ .015), respectively. Similar to siPDGFR-␣, siPDGFR-1 and siPDGFR-2 decreased the relative fraction of viable cells by 83.3% and 47.2% at 24 hours (P ⫽ .010) and 77.4% and 38.5% at 48 hours (P ⫽ .009), respectively. Therefore, all subsequent in vivo experiments were performed using siPDGFR-␣1 and siPDGFR-2.
RNA Interference-Mediated Inhibition of PDGFR in Prostate Cancer Xenograft No significant difference was seen in the mean body weight among the 5 groups during the experimental period (Fig. 3A, P ⫽ 865). Figure 3B shows the significant antitumor effects of siPDGFR-␣ and siPDGFR- compared with the controls. The mice in groups 3, 4, and 5 had significant tumor growth arrest compared with the mice in groups 1 and 2 (88.8, 71.5, 42.9, 19.4, and 14.4 mm3 for groups 1, 2, 3, 4, and 5, respectively; P ⫽ .001). Compared with groups 1 and 2, the tumors in groups 3, 4, and 5 were characterized by increased necrotic areas and fewer microvessels (Fig. 4). A significant reduction in the microvessel density was observed in the tumors from groups 3, 4, and 5 (47.7, 45.8, 14.3, 7.2, and 3.0 in groups 1 through 5, respectively, P ⬍ .001). UROLOGY 77 (6), 2011
Figure 4. Microscopic changes and microvessel density in xenografted tumors. (A) Hematoxylin-eosin staining showed minimal necrotic cells in (A1) group 1 and (A2) group 2. In contrast, tumors in (A3) group 3, (A4) group 4, and (A5) group 5 showed large areas of necrosis. (B) Histograph corresponding to number of cells showing immunoreactivity against CD31 in each group.
COMMENT The physiologic role of the PDGF isoforms and their receptors during embryonal development and wound healing has been well studied using knockout of genes for PDGF isoforms and receptors in mice.7-10 PDGF isoforms exert their biologic effects UROLOGY 77 (6), 2011
by binding to structurally similar PDGF-␣ and PDGF- tyrosine kinase receptors.11 It is well known that uncontrolled activation of these receptors ultimately promotes a variety of cellular processes, including proliferation, migration, and survival. Thus, overexpression 1509.e13
of the PDGFs and their receptors has been associated with many types of cancers, such as glioblastoma,12,13 ovary cancer,14 and prostate cancer.15-19 Some investigators have reported that PDGFR is expressed in high fractions of prostate cancer by immunohistochemistry.15-17 Ko et al16 reported that PDGFR expression was detected in 88% of primary prostate cancer samples and 80% of metastatic bone marrow samples; another study supported this finding.15,17 However, a review of 5 published prostate expression studies, using microarray analysis of 100 clinically localized prostate cancers, showed that PDGFR expression was upregulated considerably in small populations of prostate cancer, 5% of clinically localized prostate cancer and 16% of metastatic prostate cancer, and no significant association between PDGFR expression and prognostic factors, including preoperative prostate-specific antigen level, Gleason score, and tumor stage, was identified.20 This result might have been due to the inherent limitations of immunohistochemical studies. Also, selection bias might have played a significant role, and is a major issue in molecular epidemiologic studies that rely on tumor tissue because the availability of tissue blocks could be a function of the underlying disease, hospital diagnostic practices, pathology laboratory preservation procedures, storage protocols, and the study population.21 In addition, the differences in PDGFR expression could have resulted in part from the different cutoff levels used for grading PDGFR expression in each study. Moreover, although immunohistochemical studies have confirmed coexpression of ligands and receptors, in human tumor tissues, their functional significance still remains unclear. Few in vitro and in vivo studies have been done on the association between PDGFR and prostate cancer. Kim et al18 reported that targeting the phosphorylation of PDGFR in tumor-associated endothelial cells using imatinib, with or without paclitaxel, led to regression of multidrug-resistant prostate cancer and to inhibition of lymph node metastases. In addition, Kimura et al19 reported that imatinib improved the outcome of radioimmunotherapy in a mouse model of prostate cancer by reducing the tumor interstitial fluid pressure, with the subsequent increase of 131ICC49 uptake into the tumor and inhibition of hypoxia-inducible factor-1␣, resulting in improved tumor radiosensitivity. Imatinib mesylate inhibits tyrosine kinases encoded by the bcr-abl oncogene, as well as the receptor tyrosine kinases encoded by c-kit and PDGFR.22 Thus, the major caveat of these studies was the use of tyrosine kinase inhibitors with incompletely characterized specificity. Therefore, in the present study, the consequences of PDGFR knockout, in human prostate cancer cells, using siRNA specific for PDGFR-␣ and PDGFR- was investigated. RNA interference is a recently developed technique used to silence proteins in a sequence-specific manner by inhibiting mRNA and consequently reducing protein expression. siRNA transfection is always a transient 1509.e14
event because the introduced siRNA lacks the ability to replicate itself and quickly disappears owing to degradation and dilution by cell division.23 Once siRNA disappears in the transfected cells, the target gene quickly recovers to its normal state. It is well known that the levels of the targeted gene in siRNA-treated cells have typically recovered by 5-7 days after siRNA transfection (ie, after 7-10 rounds of cell division).24 In the present study, PDGFR knockdown was detected at 24 hours after siRNA transfection (Fig. 2). However, the effects of the knockdown decreased within 2-3 days in vitro (Fig. 3) and by 3 weeks in vivo (Fig. 4). Using the siRNA specific for PDGFR-␣ and PDGFR-, the results of the present study showed that PDGFR knockdown caused cell death. This appeared to occur by the inhibition of angiogenesis. Although both PDGFR-␣ and PDGFR- mediate strong mitogenic signals, some investigators have reported that PDGFR- induces more potent transforming signals than the signals transduced by PDGFR-␣.25-28 Yu et al25 suggested that activation of PDGFR-␣ might transduce both positive and negative signaling for cell transformation, and PDGFR- might mainly induce positive signaling for cell transformation. However, no study has demonstrated whether PDGFR-␣ or PDGFR- plays a more important role in experimental prostate cancer cell systems. The present study is the first to show that inhibition of PDGFR- had superior antitumor activity compared with siPDGFR-␣. Furthermore, the results of our study have provided in vivo evidence that prostate cancer tumorigenesis is associated with PDGFR expression.
CONCLUSIONS The results of the present study have provided evidence suggesting a critical and specific role for PDGFR-␣ and PDGFR- in the PC3 prostate cancer cell line viability and proliferation. siPDGFR-␣ and siPDGFR- successfully inhibited the expression of PDGFR-␣ and PDGFR-, and the inhibition of PDGFR-␣ and  might have resulted in the inhibition of prostate cancer growth by the suppression of angiogenesis. References 1. U.S. Preventive Services Task Force. Screening for prostate cancer: U.S. Preventive Services Task Force recommendation statement. Ann Intern Med 2008;149:185-191. 2. Espey DK, Wu XC, Swan J, et al. Annual report to the nation on the status of cancer, 1975-2004, featuring cancer in American Indians and Alaska Natives. Cancer. 2007;110:2119-2152. 3. Shawver LK, Slamon D, Ullrich A. Smart drugs: tyrosine kinase inhibitors in cancer therapy. Cancer Cell. 2002;1:117-123. 4. Pietras K, Rubin K, Sjoblom T, et al. Inhibition of PDGF receptor signaling in tumor stroma enhances antitumor effect of chemotherapy. Cancer Res. 2002;62:5476-5484. 5. Reynolds A, Leake D, Boese Q, et al. Rational siRNA design for RNA interference. Nat Biotechnol. 2004;22:326-330. 6. Gasparini G, Harris AL. Clinical importance of the determination of tumor angiogenesis in breast carcinoma: much more than a new prognostic tool. J Clin Oncol. 1995;13:765-782.
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7. Betsholtz C. Insight into the physiological functions of PDGF through genetic studies in mice. Cytokine Growth Factor Rev. 2004; 15:215-228. 8. Robson MC, Phillips LG, Thomason A, et al. Platelet-derived growth factor BB for the treatment of chronic pressure ulcers. Lancet. 1992;339:23-25. 9. Ding H, Wu X, Bostrom H, et al. A specific requirement for PDGF-C in palate formation and PDGFR-alpha signaling. Nat Genet. 2004;36:1111-1116. 10. Soriano P. Abnormal kidney development and hematological disorders in PDGF beta-receptor mutant mice. Genes Dev. 1994;8: 1888-1896. 11. Rosenkranz S, Kazlauskas A. Evidence for distinct signaling properties and biological responses induced by the PDGF receptor alpha and beta subtypes. Growth Factors. 1999;16:201-216. 12. Hagerstrand D, Hesselager G, Achterberg S, et al. Characterization of an imatinib-sensitive subset of high-grade human glioma cultures. Oncogene. 2006;25:4913-4922. 13. Hesselager G, Uhrbom L, Westermark B, et al. Complementary effects of platelet-derived growth factor autocrine stimulation and p53 or Ink4a-Arf deletion in a mouse glioma model. Cancer Res. 2003;63:4305-4309. 14. Ostman A. PDGF receptors-mediators of autocrine tumor growth and regulators of tumor vasculature and stroma. Cytokine Growth Factor Rev. 2004;15:275-286. 15. Fudge K, Wang CY, Stearns ME. Immunohistochemistry analysis of platelet-derived growth factor A and B chains and platelet-derived growth factor alpha and beta receptor expression in benign prostatic hyperplasias and Gleason-graded human prostate adenocarcinomas. Mod Pathol. 1994;7:549-554. 16. Ko YJ, Small EJ, Kabbinavar F, et al. A multi-institutional phase II study of SU101, a platelet-derived growth factor receptor inhibitor, for patients with hormone-refractory prostate cancer. Clin Cancer Res. 2001;7:800-805. 17. Chott A, Sun Z, Morganstern D, et al. Tyrosine kinases expressed in vivo by human prostate cancer bone marrow metastases and loss
UROLOGY 77 (6), 2011
18.
19.
20.
21.
22. 23. 24. 25.
26.
27.
28.
of the type 1 insulin-like growth factor receptor. Am J Pathol. 1999;155:1271-1279. Kim SJ, Uehara H, Yazici S, et al. Targeting platelet-derived growth factor receptor on endothelial cells of multidrug-resistant prostate cancer. J Natl Cancer Inst. 2006;98:783-793. Kimura Y, Inoue K, Abe M, et al. PDGFRbeta and HIF-1alpha inhibition with imatinib and radioimmunotherapy of experimental prostate cancer. Cancer Biol Ther. 2007;6:1763-1772. Hofer MD, Fecko A, Shen R, et al. Expression of the plateletderived growth factor receptor in prostate cancer and treatment implications with tyrosine kinase inhibitors. Neoplasia. 2004;6: 503-512. Hoppin JA, Tolbert PE, Taylor JA, et al. Potential for selection bias with tumor tissue retrieval in molecular epidemiology studies. Ann Epidemiol. 2002;12:1-6. Homsi J, Daud AI. Spectrum of activity and mechanism of action of VEGF/PDGF inhibitors. Cancer Control. 2007;14:285-294. Zhou D, He QS, Wang C, et al. RNA interference and potential applications. Curr Top Med Chem. 2006;6:901-911. Tuschl T. Expanding small RNA interference. Nat Biotechnol. 2002;20:446-448. Yu J, Deuel TF, Kim HR. Platelet-derived growth factor (PDGF) receptor-alpha activates c-Jun NH2-terminal kinase-1 and antagonizes PDGF receptor-beta-induced phenotypic transformation. J Biol Chem. 2000;275:19076-19082. Kim HR, Upadhyay S, Korsmeyer S, et al. Platelet-derived growth factor (PDGF) B and A homodimers transform murine fibroblasts depending on the genetic background of the cell. J Biol Chem. 1994;269:30604-30608. Frisch SM, Vuori K, Kelaita D, et al. A role for Jun-N-terminal kinase in anoikis; suppression by bcl-2 and crmA. J Cell Biol. 1996;135:1377-1382. Bejcek BE, Hoffman RM, Lipps D, et al. The v-sis oncogene product but not platelet-derived growth factor (PDGF) A homodimers activate PDGF alpha and beta receptors intracellularly and initiate cellular transformation. J Biol Chem. 1992;267: 3289-3293.
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