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Tumor Biology |
Department of Biochemistry and Molecular Biology [Y. G., N. K.], Division of Urology [N. K.], and the Cancer Center [N. K.], University of Maryland School of Medicine, Baltimore, Maryland 21201
ABSTRACT
Previous studies (Y. Guo and N. Kyprianou, Cell Growth Diff., 9: 185–193, 1998) have demonstrated that overexpression of transforming growth factor (TGF) ß type II receptor (TßRII) gene in human prostate cancer cells LNCaP, which are refractory to TGF-ß1 and lack TßRII receptor expression, can restore TGF-ß1 sensitivity and suppress in vitro tumorigenic growth by inhibiting cell proliferation. In the present study, we investigated the effect of TßRII receptor overexpression in LNCaP cells on apoptosis induction and tumorigenicity. The ability of LNCaP cells that overexpress TßRII to undergo apoptosis in response to TGF-ß1 was examined by DNA fragmentation and terminal transferase-mediated dUTP-biotin end labeling analysis. To explore the potential apoptotic nature of TGF-ß1-mediated antitumor effect against human prostate cancer cells, the expression of apoptotic proteins bcl-2 and bax was examined by Western blot analyses. The significance of caspase 1 in TGF-ß1-mediated apoptosis was also determined by examining the expression and activation of caspase 1 by reverse transcription-PCR and Western blot analyses, respectively. Comparative analysis of tumorigenicity of the parental LNCaP and TßRII-overexpressing clones in severely combined immunodeficient mice revealed a significant suppression of tumor growth in TßRII transfectant clones compared with parental LNCaP cells and neomycin-control clones (P < 0.05). A significantly higher incidence of endogenous apoptosis was observed in TßRII clone-61-derived tumor compared with the parental LNCaP tumors. This induction of apoptosis in the LNCaP tumors with restored TGF-ß1 signaling was associated with decreased bcl-2 expression, increased bax, and caspase-1 immunoreactivty. Moreover, an increased expression of the cyclin-dependent kinase inhibitor p27Kip1 was detected in TßRII-overexpressing tumors compared with the parental tumors. LNCaP TßRII transfectant cells exhibited a marked induction of apoptosis, paralleled with a decreased bcl-2 expression in response to TGF-ß1 treatment in vitro. This TGF-ß1-mediated apoptosis induction in TßRII transfectant cells was significantly protected by the caspase-1 inhibitor (zVAD-fmk) in a dose-dependent manner. Furthermore, a significant temporal induction of caspase-1 mRNA and protein expression was detected in TßRII cells in response to TGF-ß1 treatment. Our findings suggest that restoration of TGF-ß1 signaling suppresses tumorigenicity of human prostate cancer cells by inducing apoptosis, potentially via a caspase-1-mediated pathway.
INTRODUCTION
Homeostasis in the prostate gland is maintained by a balance between cell death and cell proliferation (1 , 2) . Both cellular processes are tightly controlled by androgens and an array of positive and negative growth factors (3 , 4) . Autocrine production of growth stimulatory factors or altered expression or responsiveness to growth inhibitory factors in the prostate gland may potentially deregulate cell proliferation and/or apoptosis, which ultimately results in prostate cancer development and progression (5 , 6) . TGF3 -ß1 was originally implicated as a physiological regulator of prostate growth from evidence that indicated that the activation of apoptosis in the rat ventral prostate in response to castration-induced androgen withdrawal was associated with induced expression of both the TGF-ß1 ligand and its receptors (7 , 8) . More recent studies (9 , 10) suggest that TGF-ß1 can directly induce apoptosis in normal and malignant prostatic cells, which points to a potential involvement of the TGF-ß1-mediated apoptotic pathway in prostate tumorigenesis.
TGF-ß is a multifunctional polypeptide growth factor that elicits its diverse biological roles by interaction with two major types of cell surface receptors, TßRI and TßRII, which are serine/threonine kinases (11 , 12) . TßRII receptor is the primary receptor target for TGF-ß1, and the initiation of TGF-ß-mediated signal transduction cascade is caused by the selective binding of TßRII receptor to the TGF-ß ligand (13) . Once bound to a TßRII receptor, TGF-ß1 can be recognized by the TßRI receptor that is recruited into the ternary complex and phosphorylated by TßRII at serine and threonine residues. Subsequently, phosphorylated TßRI receptor can transduce the signal to the downstream components to elicit diverse cellular effects (14) . The direct involvement of both TGF-ß receptors in the TGF-ß-mediated signal transduction pathway implies that the alteration of functional TßRI and/or TßRII receptor expression may potentially contribute to the disruption of the TGF-ß signaling pathway. Although both TGF-ß receptor components are required for TGF-ß signal transduction, recent studies have demonstrated loss, or mutational alteration of TßRII receptor in several human malignancies including breast cancer (15) , T-cell lymphomas (16) , colon cancer (17) , and head and neck squamous carcinoma (18) .
Studies by this laboratory as well as by others (19, 20, 21, 22) demonstrated a decreased expression of both TßRI and TßRII receptors in human prostatic adenocarcinoma compared with the normal prostate, which indicates that deregulation of TGF-ß signaling pathway may be causally involved in prostate tumorigenesis. Moreover, our recent in vitro studies demonstrated that overexpression of TßRII gene in human prostate cancer cells LNCaP, which are resistant to TGF-ß1, restores TGF-ß1 sensitivity and suppresses tumorigenicity (21) . The restoration of antiproliferative activity of TGF-ß by overexpression of TßRII in LNCaP cells was associated with the significant induction of key cell cycle regulators, such as p21Cip1 and p27Kip1 (21) .
Although TGF-ß has been implicated in apoptosis induction in a variety of cell types, including myeloid leukemia cells (23) , primary endometrial cells (24) , gastric carcinoma cells (25) , colon adenoma cells (26) , primary hepatocytes (27) , and hepatoma cells (28) , the underlying molecular mechanisms by which TGF-ß1 induces apo-ptosis remain largely undefined.
bcl-2 and bax proteins are potent regulators of apoptosis in many cellular systems (29 , 30) . It is believed that the antagonistic effect of bcl-2 and bax on apoptosis is regulated by the formation of heterodimers, with the ratio of bcl-2:bax ultimately determining the susceptibility of a cell to undergo apoptosis (31 , 32) . There is strong evidence to suggest that there is a potential involvement of bcl-2 and bax proteins in the induction of apoptosis in prostate cancer cells in response to diverse programmed cell death signals (33, 34, 35) ; however, it is not fully understood whether these two apoptotic regulators have any significance in TGF-ß1-mediated apoptosis.
Caspases have been recently suggested to play a critical role in the regulation of apoptosis. Thus far, 10 members of mammalian caspases have been identified, named caspase 1–10 (36) . Caspases are synthesized as proenzymes that are activated by proteolysis to form an active heterotetramer. The active caspases have a cysteine protease activity and can selectively cleave substrate after an aspartate residue (36) . More importantly, overexpresion of ICE (caspase 1) or many other caspase family members have been found to induce apoptosis in mammalian and insect cells (37) . Also, a variety of caspase inhibitors, such as the cowpox virus protein CrmA as well as certain peptide methyl ketones and peptide aldehydes, were found to prevent apoptosis in a number of biological systems by binding to the active-site cysteine of caspases (38 , 39) . Significantly enough, several caspases such as ICE and caspase 3 have recently been implicated in TGF-ß-induced apoptotic pathways in rat hepatoma cells and human hepatoma cells, respectively (40 , 41) .
In the present study, we investigated whether restoration of TGF-ß1 signaling pathway by overexpression of TßRII receptor in human prostate cancer cells leads to the activation of apoptosis as a molecular mechanism that results in the suppression of prostate tumorigenic growth. The present findings indicate that overexpression of TßRII receptor in human prostate cancer cells suppresses tumorigenicity by inducing apoptosis via a caspase-1-mediated pathway.
MATERIALS AND METHODS
In Vivo Tumorigenicity in SCID Mice.
The parental LNCaP cells, neomycin-control clone, and TßRII transfectant clones were inoculated s.c. (5 x 106 cells/site) in the presence of Matrigel (Collaborative Res. Inc., Bedford, MA) into SCID male mice (4 weeks old, 5 animals/group). Control animals were inoculated with Matrigel alone. Mice were maintained in a pathogen-free environment and were monitored for tumor growth and tumor size weekly until day 40 when mice were killed. Tumor volume (V) was determined using the equation V = (L x W2) x 0.5 in which V = volume, L= length, and W = width.
Immunohistochemical Analysis.
Six weeks after postinoculation, tumor-bearing animals were killed, and tumors were surgically excised. Xenograft tumor tissue specimens were fixed in 10% formalin, embedded in paraffin, and cut into 6-µm sections. Expression of bax, TßRII, p27Kip1, and caspase-1 proteins was determined using antihuman rabbit polyclonal antibodies obtained from Santa Cruz Biotechnology (Santa Cruz, CA). bcl-2 immunoreactivity was detected using the antibody from DAKO Corporation (Carpinteria, CA). The immunoperoxidase procedure was conducted as described previously (19)
. The color reaction was developed using diaminobenzidine, and sections were counterstained with hemotoxylin. Negative control slides consisted of excluding the primary antibody but retaining all of the other steps.
In Situ Detection of Apoptosis.
Detection of apoptosis in situ was performed in paraffin-embedded sections using the ApoTag Kit (Oncor, Gaithersburg, MD), based on the TUNEL assay as described previously (42)
. Sections of rat ventral prostate after castration were used as biologically positive controls (43)
. Negative controls consisted of consecutive sections of each case in which the terminal deoxynucleotidyltransferase (TdT) enzyme was omitted. Sections were counterstained with methyl green.
Quantitative Analysis of Staining.
Cells were counted in four fields randomly selected at x400. Quantitation of immunoreactivity was performed by two independent observers (Y. G. and N. K.). The counting of immunoreactive cells was based on the distribution of positive cells in four different fields within the same section, and the percentage of positive immunoreactivity was expressed as the percentage of the number of stained cells over the total number of cells, as described previously (19)
.
Cell Viability Assay.
LNCaP TßRII clone 61 transfectant cells were first plated at 4 x 105/well in 6-well plates. At 70% confluency, cells were treated with 5 ng/ml TGF-ß1 (R&D Systems, Minneapolis, MN). Caspase-1 inhibitor, zVAD-fmk (Stratagene, La Jolla, CA) was added to the culture media at increasing concentrations (0, 10, 20, 50, and 100 µM). Cell death was determined after 2 and 5 days of simultaneous exposure to the inhibitor and TGF-ß, respectively, using the trypan-blue-exclusion cell viability assay.
RT-PCR Analysis.
RNA was extracted from the prostate cancer cells as described previously (19
, 21)
. RT-PCR was performed using 1 µg of total cellular RNA and the Ribo Clone cDNA synthesis kit (Promega Corp., Madison, WI) in a Sratagene thermal cycler (La Jolla, CA). Human ICE-ß primers were obtained from Stratagene and the sequences were as follows: (a) sense, 5'-ATCCGTTCCATGGGTGAAGGTACA-3'; and (b) antisense, 5'-CAAATGCCTCCAGCTCTGTAATCA-3'. The primers for the human GAPDH were obtained from Clontch (Palo Alto, CA), and the sequences were as follows: (a) sense, 5'-TGAAGGTCGGAGTCAACGGATTTGGT-3'; and (b) antisense, 5'-CATGTGGGCCATGAGGTCCACCAC-3'. The conditions used for the RT-PCR performance were as follows: 95°C for 5 min; 60°C for 2 min, 72°C for 2 min (35 cycles); and 72°C for 10 min (1 cycle), final extension. The integrity of the RNA used for RT was confirmed using the GAPDH synthesis as a positive control reaction. The amplified RT-PCR products were electrophoretically analyzed through 1% agarose gels visualized by ethidium bromide staining and photographed under UV illumination.
Western Blot Analysis.
Total cell lysates prepared as described previously (36)
, were subjected to electrophoretic analysis through 12.5% SDS-PAGE gel (60 µg protein/well). After electrophoresis, proteins were electrotransferred to nitrocellulose membranes (Hybond C; Amersham Int. Arlington Heights, IL). Complete transfer was evaluated using prestained protein standards (Bio-Rad, Melville, NY). After blocking in 5% nonfat milk in Tris-buffered saline + Tween-20, the membrane was incubated with the respective primary antibody. A mouse monoclonal antibody against the human bcl-2 obtained from DAKO (Glostrup, Denmark) was used for detection of bcl-2 expression. Expression of bax and caspase-1 proteins in response to TGF-ß1 treatment was determined using primary rabbit polyclonal antibodies against each specific protein from Santa Cruz Biotechnology (Santa Cruz, CA). Each antibody was used at a concentration of 1 µg/ml (1 h, room temperature). Membranes were subsequently incubated with the biotinylated secondary antibody (1:2000) for 30 min, and color detection was obtained using the biotin-avidin horseradish peroxidase complex (ABC Kit; Santa Cruz Biotechnology) for the detection of antibody complexes with diaminobenzidine.
DNA Fragmentation Assay.
Cells, plated at a density of 5 x 106 in T-75 flasks, were exposed to TGF-ß1 at a concentration of 5 ng/ml. After 3 and 5 days of treatment, cells were trypsinized and pellets were washed and lysed by the addition of lysis buffer [0.1 M NaCl, 50 mM Tris (pH 8.0), 0.5% SDS, and 25 mM EDTA). After the incubation with proteinase K for 4 h at 48°C, nucleosomal DNA was extracted with phenol/chloroform, precipitated with ethanol, and resolved in TE buffer (10 mM Tris and 1 mM EDTA). Samples consisting of equal amounts of nucleosomal DNA (5 µg) were electrophoresed through 1.8% agarose gel containing 1 mg/ml ethidium bromide and visualized under UV illumination.
Statistical Analysis.
Statistical analysis was conducted using Student’s t test for analysis of significance between the different values. Values were expressed as the mean ± SE, and they were considered significant at a P of less than 0.05.
RESULTS
Our previous studies demonstrated that the restoration of TGF-ß1 sensitivity and signaling in human prostate cancer cells results in the suppression of tumorigenic growth of LNCaP cells in vitro (21)
. To determine whether overexpression of TßRII might suppress prostate tumorigenicity in vivo, SCID mice were inoculated with parental LNCaP cells, neomycin-control clone, and three selected TßRII transfectant clones: 8, 13, and 61. As shown in Fig. 1
, there was a significant amount of growth suppression of LNCaP TßRII-derived tumors compared with those derived from parental LNCaP and neomycin-control cells (P < 0.05). To confirm that LNCaP TßRII tumors express high levels of TßRII receptor protein, an immunohistochemical analysis was conducted using paraffin-embedded sections of prostatic tumor xenografts. Fig. 2
indicated a representative H&E staining of the parental LNCaP (a) and TßRII transfectant clone (b). A significantly higher expression of TßRII protein was found in TßRII clone 61-derived tumors compared with parental LNCaP tumors (Fig. 2, c and d)
. Quantitative analysis of the data shown in Table 1
reinforces the in vivo overexpression of the TßRII receptor in the LNCaP clone 61-derived tumor.
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and summarized in Table 1
, a significantly higher number (approximately 100-fold) of TUNEL-positive apoptotic cells was observed in tumors derived from TßRII clone 61 compared with parental cells (24.5% versus 1.28%, respectively).
To gain a mechanistic insight into the TGF-ß1-mediated apoptotic pathway in human prostate cancer cells, the potential involvement of key apoptotic regulators such as caspases, bcl-2, and bax proteins was investigated, both in vitro in response to exogenous TGF-ß1 and in the LNCaP-derived tumors growing in vivo. The results summarized on Table 1
indicate increased expression of bax (75.5% versus 51.9%), and caspase 1 (73% versus 38%) and a parallel reduction in bcl-2 expression levels (8.7% versus 26.1%), respectively, in TßRII clone 61-derived tumors compared with parental tumors.
The expression of the cyclin-dependent kinase inhibitor, p27Kip1 was also examined from tumor sections derived from parental LNCaP cells and TßRII clone 61 by immunohistochemical analysis. A significant increase in the expression of p27Kip1 (73.5%) was observed in tumors derived from TßRII clone 61 compared with parental LNCaP tumors (25.3%; Fig. 2
and Table 1
).
To further confirm the in vivo induction of apoptosis in the LNCaP TßRII tumors, DNA fragmentation was assessed in parental cells and TßRII clone 61 transfectants in response to exogenous TGF-ß1 treatment (5 ng/ml). Fig. 3
indicates the enhanced appearance of nucleosomal ladder in TßRII clone 61 after 3 and 5 days of TGF-ß1 treatment, whereas no DNA laddering was detected in the parental LNCaP cells. Western blot analysis revealed a significant down-regulation of bcl-2 protein expression in TßRII clone 61 cells in response to TGF-ß1 treatment, whereas no significant changes were detected in bax protein levels during the treatment time course (Fig. 4)
.
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, a significant dose-dependent protection from TGF-ß1-mediated cell death by the caspase-1 inhibitor, zVAD-fmk, was observed in TßRII clone 61 transfectants treated with TGF-ß1 for 2 days. Significantly enough, this rescue from apoptosis was maintained with longer-term exposure (5 days) to the caspase-1 inhibitor (Fig. 5B)
. Quantitative analysis of apoptotic cells revealed a significant inhibition of TGF-ß1-mediated apoptosis by the treatment of caspase-1 inhibitor (zVAD-fmk) in a dose-dependent manner, as shown in Fig. 5C
.
|
. This up-regulation of caspase-1 mRNA expression was maintained for 3 days posttreatment and it was temporally correlated with the kinetics of apoptosis induction (Fig. 3)
. Furthermore, Western blot analysis shown in Fig. 7
revealed a similar course of activation of caspase-1 protein in response to exogenous TGF-ß. Increase in the p10 subunit was detected after 18 h of TGF-ß treatment.
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We have demonstrated previously (21) that overexpression of TßRII receptor in human prostate cancer cells, LNCaP, restores TGF-ß1 signaling mechanism and TGF-ß sensitivity. In the present study, we demonstrated that TGF-ß1 treatment of TßRII transfectants resulted in a significant induction of apoptosis. A significantly higher incidence of apoptosis was also detected in vivo in prostatic tumors derived from TßRII cells compared with those derived from parental LNCaP cells. The increased expression of a cyclin-dependent kinase inhibitor, p27Kip1, observed in prostatic tumors derived from TßRII clone 61 compared with that from parental LNCaP cells, is consistent with our previous in vitro observations that TGF-ß1 treatment of TßRII clone leads to cell cycle G1 arrest via induction of p27Kip1 (21) . These findings imply that overexpression of TßRII in human prostate cancer cells suppresses their tumorigenic behavior by mediating the induction of apoptosis and inhibition of cell proliferation.
TGF-ß1 treatment of TßRII clone 61 caused a significant induction of bax protein expression and down-regulation of bcl-2 expression compared with parental LNCaP cells. This observation directly correlates with the in vivo significantly higher expression of bax and lower bcl-2 protein expression in TßRII-derived prostatic tumors, which suggests that up-regulation of bax and down-regulation of bcl-2 protein expression is potentially involved in TGF-ß-mediated apoptosis in human prostate cancer cells. Treatment with caspase-1 inhibitor in TßRII clone 61 caused a significant protection of TGF-ß1-mediated apoptosis in a time- and dose-dependent manner. This finding is consistent with previous studies that reported that the treatment of caspase-1 inhibitor causes a significant inhibition of TGF-ß1-mediated apoptosis in rat and human hepatoma cells (40 , 41) . Our findings demonstrated a significant induction of expression of caspase 1 after 18 h of TGF-ß1 treatment, persistent for 3 days at both the mRNA and protein level. Finally, the activation of caspase 1 in TGF-ß1-mediated apoptosis in human prostate cancer cells was also investigated by testing the expression of an active form (p10 subunit) of caspase 1. It is known that caspase 1 is a cysteine protease that cleaves inactive pro-interleukin-1ß to generate the active proinflammatory cytokine interleukin-1ß (44) . Caspase 1 was first isolated from the human monocytic cell line THP1 as an enzyme composed of Mr 10,000 (p10) and Mr 20,000 (p20) subunits. It is expressed in many tissues as an inactive Mr 45,000 precursor protein (p45), from which the active enzyme subunits are derived by an autocatalytic cleavage process (45) . We found a significant induction of expression of p10 subunit of caspase 1 in TßRII clone 61 after 6 h of TGF-ß1 treatment, an increase that was persistent for 3 days. These findings strongly suggest that caspase 1 is potentially involved in TGF-ß1-mediated apoptosis in human prostate cancer cells.
Caspases can be classified into two major families by the lengths of their NH2-terminal prodomains (37) . Caspases 1, 2, 4, 5, 8, and 10, which have long prodomains, are implicated in targeting and regulating apoptosis. Caspases 3, 6, 7, and 9—with short prodomains—seem to play essential roles in the execution of apoptosis by operating at the downstream end of the DNA repair enzyme poly(ADP-ribose) polymerase whose cleavage is essential for the induction of apoptosis (37) . Whether or not other caspases may play potential roles in TGF-ß1-mediated apoptosis in human prostate cancer cells warrants further investigation.
In conclusion, this study provides the first evidence that overexpression of TßRII in human prostate cancer cells inhibits tumorigenic growth via induction of apoptosis. The apoptotic profile of these tumors involves induction of ICE-ß (caspase 1), down-regulation of bcl-2, and up-regulation of bax proteins. These findings provide a novel mechanistic insight into TGF-ß1-mediated apoptosis in human prostate cancer cells.
FOOTNOTES
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
1 This study was supported by an NIH National Institute of Diabetes and Digestive and Kidney Diseases R01 Grant (DK 53525-02). 
2 To whom requests for reprints should be addressed, at Division of Urology, De-partment of Surgery, University of Maryland Medical Center, 22 South Greene Street, Baltimore, MD 21201. Phone: (410) 706-7549; Fax: (410) 706-0311; E-mail: NKYPRIANOU{at}SURGERY1.UMARYLAND EDU.
3 The abbreviations used are: TGF, transforming growth factor; TßRI, transforming growth factor ß type I receptor; TßRII, transforming growth factor ß type II receptor; SCID, severely combined immunodeficient; TUNEL, terminal transferase-mediated dUTP-biotin end labeling; RT, reverse transcription; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; ICE-ß, interleukin-converting enzyme-ß.
Received 9/16/98. Accepted 1/15/99.
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X174 molecular marker; Lane 2, negative control (cDNA omitted); Lanes 3, control, without TGF-ß1 treatment; Lanes 4–10, cells treated with TGF-ß1 treatment for 3, 6, 9, 18, 24, 48, and 72 h, respectively. The size of the RT-PCR products is shown on the right.