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Endocrinology Vol. 138, No. 3 1145-1150
Copyright © 1997 by The Endocrine Society

Articles

The Effects of Growth Factors Associated with Osteoblasts on Prostate Carcinoma Proliferation and Chemotaxis: Implications for the Development of Metastatic Disease1

Candace K. Ritchie, Laura R. Andrews, Kris G. Thomas, Donald J. Tindall and Lorraine A. Fitzpatrick

Endocrine Research Unit, Department of Internal Medicine and the Department of Biochemistry and Molecular Biology, Mayo Clinic and Mayo Foundation, Rochester Minnesota 55905

Address all correspondence and requests for reprints to: Lorraine A. Fitzpatrick, M.D., 5–164 West Joseph, Mayo Clinic and Mayo Foundation, Rochester, Minnesota 55905.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The extensive mortality and morbidity associated with prostate cancer is caused by the high prevalence of metastatic disease at the time of diagnosis. The area most frequently involved in metastatic prostate cancer is the skeleton. Unlike other cancers, which metastasize to bone and destroy the bone matrix, prostate cancer is unique in that it is osteogenic, resulting in the formation of dense, sclerotic bone with high levels of osteoblastic activity. We proposed that factors produced by bone cells may be responsible for the development of prostate carcinoma metastasis. We studied the effects of these growth factors on prostate cell proliferation by [3H]thymidine incorporation and chemotaxis by the double-filter chamber method. Three prostate carcinoma cell lines were studied, LNCaP (androgen responsive) and PC-3 and DU-145 (androgen unresponsive). The bone-associated growth factors tested were: insulin-like growth factors I and II (IGF-I, IGF-II), transforming growth factor ß, interleukin (IL)-1ß, IL-6, and tumor necrosis factor {alpha} (TNF-{alpha}). IGF-I and IGF-II significantly increased proliferation in all three cell lines, whereas IL-6, TNF-{alpha}, and IL-1ß significantly decreased proliferation. Transforming growth factor ß induced a biphasic response in proliferation in DU-145 and PC-3 cells and produced no response on LNCaP cells. Increased cell chemotaxis occurred in the presence of IGF-I and IGF-II, and decreased cell chemotaxis occurred with the addition of TNF-{alpha} and IL-1ß. These data indicate that growth factors produced by bone cells alter prostate carcinoma cell proliferation and chemotaxis and suggest that modulations of the production of these factors may be a potential therapeutic intervention in deterring the metastasis of prostate carcinoma to bone.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
IN THE United States, prostate cancer is the most commonly diagnosed cancer and is the second cause of death caused by cancer in men. With the increase in the aging population, it is predicted that the number of deaths caused by prostate cancer will increase up to 50% by the yr 2015 (1). The high mortality and morbidity associated with prostate carcinoma is caused predominantly by the fact that most patients have metastatic disease at the time of diagnosis. The metastasis of prostate cancer can occur to many organs, including liver, lymph nodes, lung, and bone, but the skeleton is the most common site for metastasis and occurs in 70–80% of patients with metastatic prostate carcinoma (2). The molecular and cellular mechanisms for the growth and metastasis of prostate cancer are poorly understood (3).

Skeletal metastases of prostate cancer cause sclerotic or osteoblastic deposits at the sites of tumor involvement. These metastases are associated with high levels of osteoblastic activity; formation of dense, sclerotic bone; and increased production of prostatic acid phosphatase (4). The characteristics of metastatic prostate cancer to bone are unique compared with the osteolytic phenotype of skeletal metastases caused by other cancers.

Clinical observations of cancer patients have suggested that certain tumors produce metastasis to specific organs. In 1889, Paget (5) analyzed 735 autopsy records of women with breast cancer. He discovered a nonrandom pattern of visceral metastases to the liver. He proposed the seed-and-soil theory of cancer metastasis. Certain tumor cells (the seed) have a specific affinity for the milieu of certain organs (the soil). Metastasis results only when the seed and soil are matched. In this decade, work by Fidler and colleagues (6) has verified and extended this theory.

Many factors have been proposed or studied to determine the mechanism(s) responsible for the involvement of bone in metastatic prostate cancer. What is it in the soil of bone that is attracting the prostate carcinoma cell seeds? Why does prostate cancer specifically metastasize to bone? Is there something in the bone microenvironment that attracts prostate cells? It is plausible that bone produces a factor or factors that attract prostate cells? Many growth factors are produced by osteoblasts and are incorporated into the bone matrix or are present in the microenvironment (7, 8). These growth factors can stimulate or inhibit the proliferation and differentiation of osteoblasts and osteoblast progenitors. These factors include transforming growth factor (TGF)-ß (9, 10), insulin-like growth factors I and II [IGF-I and IGF-II (11, 12)], interleukin (IL)-6 (13), IL-1ß (14), and tumor necrosis factor {alpha} [TNF-{alpha} (15)].

In this study, we evaluated the effects of polypeptide growth factors that are secreted by osteoblasts or stored in bone matrix on the proliferation and metastasis of prostate cancer cells. Two of the cell lines studied are not responsive to androgens and may represent a later stage of tumor. Use of these cell lines allowed us to investigate models of early (androgen responsive) and late (androgen unresponsive) stages of prostate carcinoma. We determined that growth factors and cytokines produced by osteoblasts can alter the proliferation and chemotaxis of prostate carcinoma cells.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Reagents
LNCaP, DU-145, and PC-3 prostate carcinoma cell lines were purchased from American Type Tissue Collection, (Rockville, MD). RPMI-1640 media, IGF-I, IGF-II, TGF-ß1, IL-6, IL-1ß, and tumor necrosis factor {alpha} (TNF-{alpha}) were obtained from Sigma Chemical Co. (St. Louis, MO) [Methyl-3H]thymidine (88.8 Ci/mM) was obtained from DuPont (Wilmington, DE). FBS was purchased from Hyclone (Logan, UT). Bio-Rad Protein Dye reagent was obtained from Bio-Rad (Richmond, CA). Opti-fluor scintillation cocktail was obtained from Packard Chemical Co. (Meriden, CT). Penicillin-streptomycin and trypsin-EDTA were purchased from GIBCO (Grand Island, NY).

Cell culture methods
To study the effects of growth factors on growth and metastasis of prostate cells, three prostate carcinoma cell lines were used: the androgen unresponsive cell lines, DU-145 and PC-3, and the androgen responsive cell line, LNCaP.

LNCaP (16), DU-145 (17), and PC-3 (18) are prostatic adenocarcinoma cell lines derived from a lymph node metastasis, metastatic prostate adenocarcinoma, and a primary prostatic tumor, respectively. Cells were grown and maintained in RPMI 1640 medium supplemented with 10% FBS in 95%O2/5%CO2 atmosphere. Cells were passaged by treatment with trypsin-EDTA and seeded in 75-cm2 flasks at initial densities ranging from 103–105 cells/cm2. The culture medium was changed every 3–4 days. For cell proliferation and protein determination assays, the cells were plated in 24-well plates at initial densities ranging from 30,000–40,000 cells/well. After allowing for cell attachment, the cells were rinsed, and the medium was changed to serum-free RPMI-1640. PC-3 and DU-145 cells were incubated 48 h in serum-free medium before the addition of effectors. LNCaP cells were incubated 24 h in serum-free media to reduce problems with detachment of the cells from the culture plate. Cell viability by trypan blue exclusion was not altered after serum deprivation at these time points. Effectors were added to the cultures in serum-free media for an additional 24 h. The concentrations of IGFs and TGF-ß used were comparable with concentrations that elicited responses in osteoblast-like cells. Each concentration of effector was tested in quadruplicate.

Determination of DNA synthesis and protein concentration
Eighteen hours after the addition of effectors, 1 µCi [3H] thymidine was added to each well. After a 6-h pulse, DNA synthesis was determined by measurement of the incorporation of [methyl-3H]thymidine, as previously described (19). To normalize data, all experiments were expressed as the percent vehicle-treated control of thymidine incorporation adjusted for protein concentration (cpm/µg protein). Protein was determined on the same cell extracts as thymidine incorporation (20). Control DU-145 cells average CPM/ml ranged from 700,000–800,000; PC-3 averaged 350,000–400,000 CPM/ml; and LNCaP ranged from 180,000–200,000 CPM/ml.

Chemotaxis
Chemotaxis assays were performed by the Boyden double-chamber method (21, 22). The effectors were placed in serum-free RPMI 1640 in the bottom of the chamber. A 0.45-µm nitrocellulose filter was placed in contact with the effector. A 5.0-µm polycarbonate filter was placed upon the nitrocellulose filter, and the cells, in serum-free media, were placed on the top (polycarbonate) filter. Chambers were incubated 4 h in a humidified incubator (95% O2/5% C02.) Each experiment consisted of 6 chambers (three chambers for the effector, three for a vehicle-treated control). Membranes were fixed with isopropanol, stained with hematoxylin, and mounted. The slides were randomized and coded during blinded analysis. Chemotatic activity was quantified as the number of cells located on the nitrocellulose membrane as viewed at 200x magnification. Twenty random fields were read for each membrane.

Statistical analyses
For cell proliferation studies, a one-way ANOVA with post hoc tests (Bonferroni adjustment) was used to determine the significance of various treatments vs. the vehicle-treated control values. Unless otherwise stated, each concentration of effector was studied in three separate studies of quadruplicate wells. For studies of chemotaxis, an unpaired Student’s t test was used to determine statistical differences between effector-treated and vehicle-treated control groups.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
IGF-I was added to the three prostate cell lines at concentrations ranging from 1–100 ng/ml. The proliferation of all three cell lines was significantly increased by treatment with 100 ng/ml IGF-I (Fig. 1Go). Proliferation of DU-145 cells increased to 151.9 ± 9.3%, PC-3 cells to 150.0 ± 9.4% and LNCaP cells to 138.1 ± 2.9% compared with vehicle-treated control (P < 0.05). The addition of IGF-II to prostate carcinoma cells elicited significant increases in cell proliferation (Fig. 2Go). With the DU-145 and PC-3 cells, significant increases in proliferation occurred at 10 ng/ml IGF-II. Proliferation of DU-145 cells was increased 131.4 ± 6.9%, and proliferation of PC-3 cells increased 222.3 ± 75.5% compared with vehicle-treated controls (P < 0.05). Proliferation of LNCaP cells was increased to 162.9 ± 10.9% of control values at 100 ng/ml IGF-II (P < 0.05).



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  		Figure 1. Dose response of IGF-I on proliferation of prostate carcinoma cells. DU-145 (n = 3), PC-3 (n = 3), and LNCaP (n = 3) cells were incubated for 24 h in the presence of 0, 1.0, 10, and 100 ng/ml IGF-I. Results (mean ± SEM) expressed as the percent vehicle-treated control of [3H]thymidine/µg protein. *, P < 0.05 as compared with vehicle-treated cells by ANOVA.

 


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  		Figure 2. Dose response of IGF-II on proliferation of prostate carcinoma cells. DU-145 (n = 4), PC-3 (n = 3), and LNCaP (n = 3) cells were incubated for 24 h in the presence of 0, 1.0, 10, and 100 ng/ml IGF-II. Results (mean ± SEM) expressed as the percent vehicle-treated control of [3H]thymidine/µg protein. *, P < 0.05 as compared with vehicle-treated cells by ANOVA.

 
To test the chemoattractive properties of these factors, chemotaxis assays were performed. IGF-I and IGF-II increased cell chemotaxis at 100 ng/ml (Fig. 3Go, A and B; P < 0.05). The ability of these factors to increase proliferation and chemotaxis suggests that they may aid in producing metastatic lesions.



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  		Figure 3. Effect of IGF-I and IGF-II on prostate carcinoma cell chemotaxis. Prostate carcinoma cells were incubated with 100 ng/ml IGF-I (panel A) or 100 ng/ml IGF-II (panel B), and chemotaxis was assessed by the Boyden chamber method. *, P < 0.05; **, P < 0.01 as compared with vehicle-treated control by Student’s t test. Results expressed as mean ± SEM, n = 2.

 
TGF-ß1 induced biphasic responses in proliferation in the androgen unresponsive cell lines, DU-145 and PC-3, and caused no significant change in thymidine incorporation in the LNCaP cells (Fig. 4Go). The proliferation of DU-145 cells was significantly increased in the presence of 0.001 and 0.01 ng/ml TGF-ß1 (123.0 ± 5.7 and 127.5 ± 9.5% of control, P < 0.05) and significantly decreased at concentrations of 0.1 to 10 ng/ml TGF-ß1 (73.5 ± 6.5%, 47.5 ± 2.8%, and 30.0 ± 1.0% of control, P < 0.05). Similarly, proliferation increased in PC-3 cells in a dose-dependent manner in response to TGF-ß1, which became significant at a concentration of 0.1 ng/ml (138 ± 14.4% of vehicle-treated control) followed by a significant decrease in proliferation at 1 and 10 ng/ml TGF-ß1 (83.5 ± 0.5% of control at 10 ng/ml). LNCaP cells do not have cell surface receptors for TGF-ß1 (23) and were unresponsive.



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  		Figure 4. Dose response of TGF-ß on proliferation of prostate carcinoma cells. DU-145 (n = 3), PC-3 (n = 3), and LNCaP (n = 3) cells were incubated for 24 h in the presence of 0, 0.001, 0.01, 0.1, 1.0, and 10 ng/ml TGF-ß. Results (mean ± SEM) expressed as the percent vehicle-treated control of [3H]thymidine/µg protein. *, P < 0.05 as compared with vehicle-treated cells by ANOVA.

 
All three cell lines were treated with IL-6 at concentrations ranging from 0.2–200 pg/ml (Fig. 5Go). The proliferation of DU-145 and PC-3 cell lines was significantly decreased at 0.2 pg/ml IL-6 (DU-145, 71.6 ± 6.9% of vehicle treated-control; PC-3, 72.9 ± 7.0% of vehicle-treated control; P < 0.05); proliferation of LNCaP cells was not inhibited until the addition of a 100-fold higher dose of IL-6. At a concentration of 20 pg/ml of IL-6, LNCaP cell proliferation was 81.9 ± 4.0% of vehicle-treated control (P < 0.05).



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  		Figure 5. Dose response of IL-6 on proliferation of prostate carcinoma cells. DU-145 (n = 4), PC-3 (n = 3), and LNCaP (n = 4) cells were incubated for 24 h in the presence of 0, 0.2, 2, 20, and 200 pg/ml IL-6. Results (mean ± SEM) expressed as the percent vehicle-treated control of[3H]thymidine/µg protein. *, P < 0.05 as compared with vehicle-treated cells by ANOVA.

 
IL-1ß treatment caused significantly decreased proliferation in all three cell lines, although the effect was more pronounced in the LNCaP cells. At 5 IU IL-1ß, DU-145 cells were inhibited to 80.4 ± 5.3% (P < 0.05), PC-3 cells were inhibited to 88.3 ± 0.2% (P < 0.05), and LNCaP cells were inhibited to 36.5 ± 7.5% compared with vehicle-treated control cells (P < 0.001) (Fig. 6Go).



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  		Figure 6. Dose response of IL-1ß on proliferation of prostate carcinoma cells. DU-145 (n = 3), PC-3 (n = 4), and LNCaP (n = 3) cells were incubated for 24 h in the presence of 0, 2.5, 5, 10, and 20 IU IL-1ß. Results (mean ± SEM) expressed as percent vehicle-treated control of [3H]thymidine/µg protein. *, P < 0.05; ***, P < 0.001 as compared with vehicle-treated cells by ANOVA.

 
TNF-{alpha} significantly decreased cell proliferation in a dose-dependent fashion in all three cell lines (Fig. 7Go). At 10 ng/ml TNF-{alpha}, proliferation of all three cell lines were significantly decreased; proliferation of DU-145 was reduced to 61.6 ± 9.5%, PC-3 to 70.0 ± 9.3%, and LNCaP cells were inhibited to 70.6 ± 8.0% of vehicle-treated control (P < 0.05). The inhibition of DNA synthesis in LNCaP cells by TNF-{alpha} was pronounced at the highest concentration assayed and reduced to 44.4 ± 4.1% of vehicle-treated control.



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  		Figure 7. Dose response of TNF-{alpha} on proliferation of prostate carcinoma cells. DU-145 (n = 4), PC-3 (n = 4), and LNCaP (n = 3) cells were incubated for 24 h in the presence of 0, 1.0, 10, and 100 ng/ml TNF-{alpha}. Results (mean ± SEM) expressed as percent vehicle-treated control of [3H]thymidine/µg protein. *, P < 0.05 as compared with vehicle-treated cells by ANOVA.

 
TNF{alpha} and IL-1ß are cytokines that effect the growth of osteoblasts. TNF-{alpha} (100 ng/ml) and IL-1ß (10 IU) decreased cell migration. The effect was more pronounced with IL-1ß (Fig. 8Go, A and B).



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  		Figure 8. Effect of TNF-{alpha} and IL-1ß on prostate carcinoma cell chemotaxis. Prostate carcinoma cells were incubated with 100 ng/ml TNF-{alpha} (panel A) or 10 IU IL-1ß (panel B), and chemotaxis was assessed by the Boyden chamber method. *P < 0.05; **, P < 0.01 as compared with vehicle-treated control by Student’s t test. Results expressed as mean ± SEM, n = 2.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Prostate carcinoma cells preferentially metastasize to bone, suggesting that there is some factor or factors in bone that attract prostate carcinoma cells to the skeleton. What factor or factors create a fertile soil for the metastasis of prostate to bone is unknown. We have investigated several bone-derived growth factors to determine their effects on the growth and chemotaxis of prostate cells. The IGFs are potent mitogens that stimulate the growth of prostate cells (24). IGFs in the circulation have a high affinity for binding proteins (IGFBPs), which modulate the anabolic effects of IGFs. Cohen and colleagues (25) reported that the serum level of IGFBP-2 is increased in patients with prostate cancer. This suggests that the IGFs may be major determinants in the growth and proliferation of tumor cells. In this study, IGF-I and IGF-II had a stimulatory effect on prostate cell proliferation and cell migration. It is not surprising that these mitogenic growth factors stimulated cell proliferation in all of the prostate cell lines tested. All three of the cell lines tested in this study have IGF receptors. Previous studies suggested that LNCaP cells were not responsive to IGF-I (26). However, under the conditions of this study, cell proliferation was increased significantly at a dose of IGF-I known to have mitogenic effects on bone (27). Pietrzowski and co-workers (28) determined that IGF-I analogues inhibited prostate cell proliferation in the DU-145 cell line, presumably by competitively binding to the receptor. More recently, Figueroa et al. (29) concluded that IGFBPs inhibit DU-145 cell growth and attachment. All three cell lines in our study (DU-145, PC-3, and LNCaP) recently have been shown to produce and secrete one or more IGFBPs (30, 31). Under normal conditions, proteases, such as cathespin D or urokinase, hydrolyze the IGFBPs, thus allowing for the mitogenic effect of the IGFs. Because bone is an organ abundant in IGFs, the correlation between the stimulation of cell proliferation and the increase in cell migration suggests that growth factors, such as the IGFs, can increase prostate cell metastasis in two ways: first, by increasing cell number (proliferation); and second, by attracting the prostate carcinoma cells to the bone (chemotaxis).

TGF-{alpha} and TGF-ß have antithetical effects on prostate cells. TGF-{alpha} is secreted by both androgen responsive and androgen unresponsive cell lines (32, 33). TGF-{alpha} binds to and activates the EGF receptor in prostate cells lines, resulting in increased cell proliferation in LNCaP cells but has no effect on androgen unresponsive cells (34). TGF-ß is detectable in human benign and malignant prostate tissue (35). The androgen unresponsive cell lines, DU-145 and PC-3, possess TGF-ß receptors, secrete latent TGF-ß, and are growth inhibited by exogenous TGF-ß. TGF-ß receptors are not detectable on the LNCaP cell line (23). The effect of proliferation in response to TGF-ß1 in the androgen unresponsive cells is consistent with the effect of TFG-ß1 on other cell types (33). Our data suggest that low doses of TGF-ß1 could result in increased cellular proliferation in the prostate carcinoma cell lines, DU-145 and PC-3. Bone is a rich source of TGF-ß, and it is possible that the low concentrations of activated TGF-ß present during the bone remodeling cycle enhance the proliferation of prostate cancer cells.

The cytokines, such as the ILs and tumor necrosis factor, have a great potential for the inhibition of cancer growth. Tjota and co-workers (36) studied the effects of the combination of IL-2 and lymphokine-activated killer cells on rat prostate cell tumors. This combined therapy resulted in prevention of prostate tumor metastases to lung, a retardation of primary tumor growth, regression of established pulmonary metastases, and increased survival of the treated rats as compared with the untreated controls or those groups treated with IL-2 or lymphokine-activated killer cells alone. Furthermore, prostate cancer cells have receptors for IL-6 and TNF-{alpha}, and both compounds inhibited cancer cell proliferation in earlier studies, suggesting that these receptors may be useful therapeutically (37, 38). In our study, IL-6, TNF-{alpha}, and IL-1ß significantly inhibited the proliferation of androgen responsive (LNCaP) and androgen unresponsive cells (DU-145 and PC-3). TNF-{alpha} and IL-1ß individually decreased cell chemotaxis in all three cell lines (39). Sherwood and co-workers (40) administered TNF-{alpha} into nude mice bearing PC-3 tumors and produced significant inhibition of the primary tumor growth. Our studies confirm that TNF and other cytokines inhibit prostate cancer growth and motility at the cellular level.

The contribution of growth factors and cytokines to the growth and metastasis of prostate cancer is, without doubt, multifactorial. The role of the skeleton and growth factors/hormones associated with bone growth and differentiation is poorly defined. Stimulatory growth factors, such as the IGFs and TGF-ß, have been implicated in a causative role in enhancing the metastasis of prostate cancer. Possible therapeutic models for decreasing the growth and spread of cancer include inactive derivatives and/or antibodies raised to these growth factors. Conversely, the ILs and related compounds may have a direct benefit in causing regression of not only the primary tumor but also the spontaneous metastasis of prostate carcinoma.


   Footnotes
 
1 This work was supported in part by USPHS Grants CA-58225, AR-08304, and the Cap Cure Foundation. Presented in part at The American Society for Cell Biology Meeting, New Orleans, LA, 1993. Back

Received July 18, 1996.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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