Prostate cancer (PCa), with an increasing incidence in China,1-3 is one of the most common malignant tumors in the western world. Since the first observation in 1941, androgen deprivation therapy (ADT) became the major therapeutic option for advanced PCa.4,5 ADT induces massive apoptosis of cancer cells and clinically leads to tumor regression in most of the cases. However, after a remission period of several years, the PCa usually progresses to a state of hormone-refractory prostate cancer (HRPC) in spite of the low androgen levels.6,7 Although HRPC tumors could grow without androgen, most of the HRPC cells continue to express androgen receptor (AR).8,9 However, the role of AR in HRPC still remains unclear.
PC3, isolated from PCa bone metastasis, is an androgen independent human PCa cell line10 and widely used to study HRPC or even to study AR role in HRPC by transfection of functional human AR cDNA11 for its lack of expression of AR protein.12 But most of the reports chose virus promoter to drive the AR expression in PC3 cells.8,13-15 Because of the strong potency of the virus promoter, the AR expression pattern and level might not represent the physiological conditions. And the in vitro studies of cancer cells growth and invasion might not adequately represent the behavior of PCa cells in vivo.
In the present study, we tried to design the research model closer to the real physiological conditions, and hope to examine the AR functions in PC3 cell line expressed by physiological control. we used a PC3 stable cell line that expressed human AR driven by natural human AR promoter (PC3-AR9)16 to study the AR role in PC3 proliferation and metastasis ability by both in vitro experiments and in vivo prostate orthotopic xenograft model.
The PC3-V, PC3-AR9 and LNCap cells were kindly gifted from Dr. Chawn shang Chang (George Whipple Lab for Cancer Research, University of Rochester, NY, USA). All of the cells were maintained at 37°C in RPMI 1640 supplemented with 10% fetal calf serum (FCS) or charcoal-deprived FCS (CDS-FCS), 100 U/ml penicillin, and 100 μg/ml streptomycin under 5% CO2.
The PC3-V, PC3AR9 and LNCap cells were cultured in 10% FCS to 90% confluence, then lysed in radioimmunoprecipitation assay (RIPA) lysis buffer, separated on 8% SDS-PAGE gel, and transferred to polyvinylidene difluoride (PVDF) membrane. After blocked by 5% nonfat milk in buffer, the membrane was incubated in the primary antibody, then in the secondary antibody conjugated by alkaline phosphatase (AP), after that developed in AP conjugate substrate kit (BIO-RAD, USA). The anti-AR (N20), anti-tubulin primary antibody, AP-conjugated secondary antibodies were also from Santa Cruz Biotechnology (USA).
Transient transfection and reporter gene assay
The cells plated in 48-well plates were cultured in RPMI 1640 with 10% CDS-FCS before transfection. Mouse mammary tumor virus (MMTV) plasmids at 0.5 μg per well were transfected into cells using SuperFect transfection kit (Qiangen, USA). Thymidine kinase-renilla luciferase vector (TK-PRL) 5 ng (Promega, USA) was used in each well for the internal control. The medium was changed 3 hours after transfection, and cells were treated with 1 nmol/L dihydrotestosterone (DHT), 10 nmol/L DHT or ethanol (ETOH) control for 16 hours.
Then the cells were lysed, and the luciferase activity was analyzed using dual-luciferase reporter assay system (Promega) according to the manufacturer's instructions.
PC3-V and PC3-AR9 cells were seeded in 24-well plates and incubated with RPMI 1640 with 10% CDS-FCS in 37°C and 5% CO2 incubator with medium changed every 48 hours. After 24 hours, cells were treated with 1 nmol/L DHT or ETOH control. The plates were stained with MTT (Sigma, USA) at days 0, 2, 4, 6 after treating with DHT or ETOH. The absorbency was read at a test wave length of 595 nm.
Soft agar assay
One percent base agar (DNA grade) 0.5 ml was put into each well of the six-well plate. After cooling down, melt 1% agar in microwave, cooling to 40°C in a water bath. Then mixed the agar, 3×RPMI 1640 medium and cell suspension by a ratio of 1:1:1, and come to a cell concentration of 104 cells/ml. After coagulation, 2 ml RPMI 1640 supplemented with 10% FCS, and replaced medium every 3 days were added to supply the nutrition.
After 2-week culture in 37°C incubator, the colonies were stained by 1 mg/ml INT (Sigma) solution.
Wound healing assay
Cells were seeded in a 6-cm dish and cultured to confluency. The cell monolayer was then scraped firmly with a plastic pipette tip then was put back to 37°C incubator. The “wounded” areas were photographed by a phase contrast microscopy in the time points 0, 12, 30, 45 and 56 hours after scraping.
In vitroinvasion assay
In serum-free RPMI 1640 medium, 5×104 cells were added to the cell culture inserts and RPMI 1640 medium containing 10% FCS was added to the bottom chamber.
The cells were then incubated for 22 hours at 37°C, and the upper chamber was removed. The cells on the bottom of the upper chambers were stained with 1% toluidine blue, and the number of cells was counted under a microscope. The invasion ratio was the ratio of the cells invading through Matrigel insert membrane to the cells migrating through control insert membrane.
Mouse prostate orthotopic xenograft model
The PC3-V and PC3-AR9 cells (1×106) were suspended in 50 μl of Matrigel (Becton Dickinson Biosciences, USA). After anesthesia, the abdomens of 12-week-old athymic nude mice were surgically opened in sterile environments. Cells were injected directly into anterior prostate by 25-gauge needle, and the abdomens were closed by silk sutures. After 12 weeks, the nude mice were sacrificed. The xenograft primary tumors and metastatic tumors were weighed and embedded in paraffin for further analyses.
Immunohistochemical (IHC) staining
Mice xenograft tissue samples were fixed in 10% formalin and embedded in paraffin. After routine rehydration, antigen retrieval and blocking, the sections were incubated in the primary antibody at 4°C overnight. The primary antibody was recognized by the biotinylated secondary antibody and visualized by vectastain avidin-biotin complex peroxidase system (ABC kit) as well as peroxidase substrate 3,3′-diaminobenzidine kit (DAB kit). The primary rabbit anti-AR (N20) antibody was from Santa Cruz Biotechnology, and the primary rabbit anti-PCNA antibody was from Abcam Company, USA. The biotinylated secondary antibody, ABC kit and DAB kit were all from VECTOR Laboratories, USA.
Apoptotic cells were detected by terminal deoxynucleotidyl transferase-mediated biotinylated deoxyuridine triphos-phate nickel end labeling (TUNEL) assay as recommended by the manufacturer (Roche, USA). Briefly, paraffin-embedded tissue was deparaffinized, digested with proteinase K (20 μg/ml) for 15 minutes at room temperature, then labeled for 60 minutes at room temperature and mounted in 4,6′-diamidino-2-phenylindole-2 HCL (DAPI).
Numerical data were presented as mean ± standard deviation (SD). Statistical analysis between groups was performed by the two-side Student's t test with SAS 6.0.
P values less than 0.05 were considered statistically significant.
Expression level and transcriptional function of AR in PC3-AR9 cells
Firstly, the AR expression level in PC3-V and PC3-AR9 cells were checked. As expected, there was no AR protein expression in PC3-V cells, and a 110-kD AR band was detected in PC3-AR9 cells (Figure 1A). LNCap cells (another AR positive cell line) are positive control (Figure 1A). To confirm the AR in pC3-AR9 cells functional, we used reporter gene assay (MMTV-LUC). When treated with 1 nmol/L or 10 nmol/L DHT, the luciferase activity increased dramatically in PC3-AR9 cells (P <0.01), but no increase in PC3-V cells (Figure 1B).
AR functioned as a suppressor in PC3 cellsin vitro
The MTT assay was used to detect the AR role in PC3 cells growth. PC3-AR9 cells grew much slower than PC3-V cells with or without DHT treatment (Figure 2A). DHT had no effect on PC3-V cells growth, but could mildly promote PC3-AR9 cells growth.
MTT assay could only reflect the cell growth ability in attachment conditions. For the tumor cells, one of the properties differing from normal cells is the anchor-independent growth. A colony formation assay was performed to evaluate the AR role in PC3 cells anchor-independent growth. After 2-week culture in soft agar, many colonies formed in PC3-V cells, but almost no colony formed in PC3-AR9 cells (Figure 2B).
To test the migration ability, a wound healing assay was performed. The cells in culture dishes were scraped with a plastic pipette tip to generate the same size of “wound” area. Then the pictures were taken at different time point to monitor the “wound healing” ability (Figure 2C). At the time points of 12 and 30 hours, the “wound” area in PC3-AR9 cells was bigger than PC3-V cells. At 45 hour, the PC3-AR9 cells still had obvious “wound”, but PC3-V cells' wounds were almost healed. The “wound” of PC3-AR9 cells were healed at 56 hour (data not shown), 11 hours later than PC3-V cells.
A chamber invasion assay was then performe to detect the invasion ability of PC3 cells. After 22-hour culture in the Boyden chambers, more PC3-V cells invaded through the Matrigel membrane than PC3-AR9 cells. The invasion ratio of PC3-V cells was 45% while PC3-AR9 only 24% (P <0.01, Figure 2D).
AR suppressed the PC3 cells growth and metastasis abilitiesin vivo
To generate the in vivo prostate tumor model, we orthotopically implanted the PC3-V and PC3-AR9 cells into the anterior prostate of athymic nude mice. Twelve weeks later, we sacrificed the mice to harvest the primary and metastasis tumors. PC3-V cells formed bigger primary and metastasis tumors than PC3-AR9 cells (Figure 3A). The average primary tumor weight of PC3-V was 0.67 g, but in PC3-AR9 it was only 0.31 g (P <0.01). For the metastasis, the average weight of total meta-lymph nodes in the PC3-V group was 0.12 g, but in the PC3-AR9 group it was only 0.03 g (P <0.01).
To confirm the PC3-AR9 cells could express stably AR in vivo, IHC staining of human AR with the antibody N20 was presented (Figure 3B). AR, which mainly located in nucleus, could presented in most cells of the PC3-AR9 xenograft tissue. But in PC3-V xenograft tissue, only few cells were positive staining, which may possibely be stromal cells. IHC staining for proliferating cell nuclear antigen (PCNA) was performed in the primary xenograft tumors. There were more PCNA positive cells in PC3-V tumors than in PC3-AR9 tumors (Figure 3C). The proliferation index was 0.45 in PC3-V tumors, but only 0.24 in PC3-AR9 tumors (P <0.01). There were more apoptotic cells in PC3-AR9 tumors than in PC3-V tumors (Figure 3D); the apoptosis index was 0.0143 in PC3-V tumors, but 0.0243 in PC3-AR9 tumors (P <0.05).
AR is important in the development and progression of PCa,8 but the role of AR in HRPC is still not well-known. Many of cell lines were used to study the PCa, but the results were often controversial.12,17 PC3 cell line was widely used to study the hormone-refractory PCa,18 or even to study AR's role in PCa by transfection of human AR cDNA.11 But in most of the reports, viral promoter were uesd to drive the expression of human AR.13-15 The viral promoter is always very strong, for example, the AR expression level is much higher in PC3-AR2 cells (AR expression driven by cytomegalovirus promoter) than in PC3-AR9 cells. DHT could inhibit the growth of PC3-AR2 cells dramatically but wildly promote the growth of PC3-AR9 cells.16 The artificial control of AR expression might not represent the actural AR function under physiological and cellular conditions. In this work, we chose the PC3-AR9 cell line, which AR expression was controlled by human natural AR promoter. The cells could modify the AR expression according to its physiological needs.16
The extracellular conditions of in vitro cell culture were different from in vivo. The in vitro assays might not reflect the real physiological status accurately. Many mouse xenograft models19 were used to study the PCa.
For the cell line xenograft model, three paterns were widely used: the subcutaneous model, the sub-renal capsule model and the orthotopic model.20-23 Each model had its advantages and disadvantages.20 Only the orthotopic model could mimic the real prostate conditions, including the hormone level and the surrounding prostate stromal cells. The orthotopic model was also a good way to study the metastasis of PCa.20 The in vitro growth of PC3-AR9 was slightly increased in the presence of 1 nmol/L DHT. However, by using the orthotopic xenograft model, we found that PC3-AR9 formed smaller primary tumors than PC3-V cells. The possible explanation for these contrasting in vivo and in vitro findings might be that the in vivo observation is in a relatively long-term condition, and primary tumor growth could be influenced by the prostate microenvironments as well as surrounding stromal cells, whereas in vitro cell culture is under a simplified, non-physiological condition. Our results suggest that the in vivo tumor cell growth might lead to more accurate assessment than the short term in vitro growth assay.
By MTT assay, we found that PC3-AR9 grew much slower than PC3-V cells with or without treatment of DHT. It is interesting that DHT could mildly promote the growth of PC3-AR9 cells. After transfection of functional human AR driven by its own promoter, the PC3-AR9 cells might adapt the new survival conditions with AR, which indicated that the androgen effect was not always equal to the AR effect, and the un-activated AR might have functions. Scaccianoce et al24 also had the similar reports in DU145 cells. They transfected functional human AR to DU145 cells and found that untreated DU145-AR cells showed a lower proliferation rate than mock transfected cells, but responded to testosterone treatment. The anchor-independent growth ability is also an important property of tumor cells, we performed soft agar assay and found AR could almost deprive the anchor-independent growth ability of PC3-AR9 cells. Metastasis, the biggest difference between malignant and benign tumors, often made the tumor to be incurable and lethal. Migration and invasion were two key steps of metastasis. By wound healing assay and chamber invasion assay, we found that AR could decrease the migration and invasion ability of PC3 cells dramatically. By using an orthotopic xenograft mouse model, we found that the PC3-AR9 cells formed much smaller primary and metastastic tumors in vivo. The proliferation index of the PC3-AR9 primary tumors was much lower than PC3-V tumors.
On the basis of the above mentioned results and analyses, we could come to a conclusion that the AR functioned as a tumor suppressor in PC3 cells both in vitro and in vivo. These are consistent with the clinical findings that ADT was only initially effective at blocking the tumor growth, but eventually failed, or even promoted the tumor progression at later stages. These observations might challenge the classic concept that AR is a stimulator in PCa, and contribute to the research and therapy of the clinical HRPC.
1. Ian LH, Li H, Yang Y, Ho CF. Comparisons of the incidence and pathological characteristics of prostate cancer
between Chinese and Portuguese in Macau. Chin Med J 2008; 121: 292-294.
2. Na YQ. Are we ready for prostate cancer
? Chin Med J 2008; 121: 291.
3. Ho SF, Lao HF, Li K, Tse MK. Clinical results of radical prostatectomy for patients with prostate cancer
in Macau. Chin Med J 2008; 121: 295-298.
4. Miyamoto H, Messing EM, Chang C. Androgen deprivation therapy for prostate cancer
: current status and future prospects. Prostate 2004; 61: 332-353.
5. Yang Y. Treatment of the positive surgical margin following radical prostatectomy. Chin Med J 2008; 121: 375-379.
6. Hu MQ, Na YQ. Metabolism of adrenal androgen and its impacts on prostate cancer
after castration. Chin Med J 2008; 121: 369-374.
7. Chen CD, Welsbie DS, Tran C, Baek SH, Chen R, Vessella R, et al. Molecular determinants of resistance to antiandrogen therapy. Nat Med 2004; 10: 33-39.
8. Heinlein CA, Chang C. Androgen receptor
in prostate cancer
. Endocr Rev 2004; 25: 276-308.
9. Edwards J, Krishna NS, Grigor KM, Bartlett JM. Androgen receptor
gene amplification and protein expression in hormone refractory prostate cancer
. Br J Cancer 2003; 89: 552-556.
10. Kaighn ME, Narayan KS, Ohnuki Y, Lechner JF, Jones LW. Establishment and characterization of a human prostatic carcinoma cell line (PC-3). Invest Urol 1979; 17: 16-23.
11. Xu XF, Zhou SW, Zhang X, Ye ZQ, Zhang JH, Ma X, et al. Prostate androgen-regulated gene: a novel potential target for androgen-independent prostate cancer
therapy. Asian J Androl 2006; 8: 455-462.
12. Yu SQ, Lai KP, Xia SJ, Chang HC, Chang C, Yeh S. The diverse and contrasting effects of using human prostate cancer
cell lines to study androgen receptor
roles in prostate cancer
. Asian J Androl 2009; 11: 39-48.
13. Diallo JS, Peant B, Lessard L, Delvoye N, Le Page C, Mes-Masson AM, et al. An androgen-independent androgen receptor
function protects from inositol hexakisphosphate toxicity in the PC3/PC3 (AR) prostate cancer
cell lines. Prostate 2006; 66: 1245-1256.
14. Yuan S, Trachtenberg J, Mills GB, Brown TJ, Xu F, Keating A. Androgen-induced inhibition of cell proliferation in an androgen-insensitive prostate cancer
cell line (PC-3) transfected with a human androgen receptor
complementary DNA. Cancer Res 1993; 53: 1304-1311.
15. Heisler LE, Evangelou A, Lew AM, Trachtenberg J, Elsholtz HP, Brown TJ. Androgen-dependent cell cycle arrest and apoptotic death in PC-3 prostatic cell cultures expressing a full-length human androgen receptor
. Mol Cell Endocrinol 1997; 126: 59-73.
16. Altuwaijri S, Wu CC, Niu YJ, Mizokami A, Chang HC, Chang C. Expression of human AR cDNA driven by its own promoter results in mild promotion, but not suppression, of growth in human prostate cancer
PC-3 cells. Asian J Androl 2007; 9: 181-188.
17. Jiang J, Wang LF, Fang YH, Jin FS, Jin WS. Proliferative response of human prostate cancer
cell to hormone inhibited by androgen receptor
antisense RNA. Chin Med J 2004; 117: 684-688.
18. Liu RL, Zhang ZH, Zhao WM, Wang M, Qi SY, Li J, et al. Expression of nucleostemin in prostate cancer
and its effect on the proliferation of PC-3 cells. Chin Med J 2008; 121: 299-304.
19. van Weerden WM, Romijn JC. Use of nude mouse xenograft models in prostate cancer
research. Prostate 2000; 43: 263-271.
20. Sato N, Gleave ME, Bruchovsky N, Rennie PS, Beraldi E, Sullivan LD. A metastatic and androgen-sensitive human prostate cancer
model using intraprostatic inoculation of LNCaP cells in SCID mice. Cancer Res 1997; 57: 1584-1589.
21. Tuomela JM, Valta MP, Vaananen K, Harkonen PL. Alendronate decreases orthotopic PC-3 prostate tumor growth and metastasis to prostate-draining lymph nodes in nude mice. BMC Cancer 2008; 8: 81.
22. Liu D, Hornsby PJ. Fibroblast stimulation of blood vessel development and cancer cell invasion in a subrenal capsule xenograft model: stress-induced premature senescence does not increase effect. Neoplasia 2007; 9: 418-426.
23. Zhang Y, Ma Y, Lu HP, Gao JH, Liang CS, Liu CZ, et al. Inhibition of human prostate cancer
xenograft growth by 125
I labeled triple-helix forming oligonucleotide directed against androgen receptor
. Chin Med J 2008; 121: 2284-2289.
24. Scaccianoce E, Festuccia C, Dondi D, Guerini V, Bologna M, Motta M, et al. Characterization of prostate cancer
DU145 cells expressing the recombinant androgen receptor
. Oncol Res 2003; 14: 101-112.