Signal Transduction Pathways Mediated by Secreted and Non-Secreted Forms of Intact Insulin-Like Growth Factor Binding Protein-3 (IGFBP-3) and Its 1-97 N-Terminal Fragment in PC-3 Human Prostate Cancer Cells

doi:10.4236/jct.2013.48152

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Journal of Cancer Therapy, 2013, 4, 1290-1297

http://dx.doi.org/10.4236/jct.2013.48152 Published Online October 2013 (http://www.scirp.org/journal/jct)

Signal Transduction Pathways Mediated by Secreted and

Non-Secreted Forms of Intact Insulin-Like Growth Factor

Binding Protein-3 (IGFBP-3) and Its 1-97 N-Terminal

Fragment in PC-3 Human Prostate Cancer Cells*

Hanief Mohammad Shahjee1#, Benjamin Kefas2, Nisan Bhattacharyya1, Mohamed K. Radwan3

1Diabetes Branch, NIDDK, National Institutes of Health, Bethesda, USA; 2Division of Neuro-Oncology, Neurology Department,

University of Virginia Health System, Charlottesville, USA; 3Department of Biochemistry and Biophysics, University of Rochester

Medical Center, Rochester, USA.

Email: #[email protected]

Received August 6th, 2013; revised September 3rd, 2013; accepted September 10th, 2013

tion License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly

cited.

ABSTRACT

Our previous results indicated that both the secreted and the intracellular form of full length and 1-97 N-terminal frag-

ment of IGFBP-3 induce apoptosis in PC-3 human prostate cancer cells in an IGF-dependent and independent manner.

This study was undertaken to delineate possible down-stream signaling pathways that are involved in this process. In-

tact IGFBP-3 and its N-terminal 1-97 fragments with or without a signal propeptide were fused to YFP and expressed in

PC-3 human prostate cancer cells. In some cases, the putative IGF-binding site was presented in full length IGFBP-3

and its N-terminal fragment was also mutated. Extent of apoptosis was quantified using FACS. Up-regulation of total

Stat-1 and activation of phospho-Stat-1 were shown by western blot. TGF-β signal was measured by luciferase reporter

assay. Results from inhibitor studies indicated that both the Caspase 8 and caspase 9 pathways are involved in IGFBP-3

(non-secreted form) which induced apoptosis in PC-3 cells. Exogenous addition of IGFBP-3 to PC-3 cells increased

Stat-1 protein expression/tyrosine phosphorylation. Interestingly, results also showed that knockdown of Stat-1 by

siRNA potentiated the IGFBP-3 induced apoptosis in PC-3 cells. In addition, both full-length IGFBP-3 and its 1-97 N-

terminal fragments inhibited TGF-β signaling in these cells. This is the first report that compares the signal transduction

pathways involved in apoptotic pathways mediated by IGFBP-3 in PC-3 human prostate cancer cells. Non-secreted

form of full length IGFBP-3 and its N-terminal fragments induced apoptosis in PC-3 cells via activation of caspase 8

and caspase 9. Although, only non-secreted form of IGFBP-3 is involved in inducing apoptosis in PC-3 cells via cas-

pase 8 and caspase 9 activation pathways but both secreted and non-secreted forms of IGFBP-3 are involved in modu-

lating Stat-1 and TGF-β pathways to induce apoptotic actions in PC-3 cells. Non-secreted intact IGFBP-3 and its

N-terminal fragments induced apoptosis in PC-3 cells via activation of caspase 8 and caspase 9 pathways. Modulation

in STAT-1 and TGF-β pathways may also be important for IGFBP-3 induced apoptosis in PC-3 cells in general. These

studies clearly demonstrate that secreted and non-secreted FL and 1-97 N-terminal fragments induce apoptosis in PC-3

cells by regulating different mechanistic pathways.

Keywords: N-Terminal Fragment; Apoptosis; Caspases; Human Prostate Cancer Cells

1. Introduction

Insulin-like growth factor binding protein-3 (IGFBP-3),

the most abundant protein of the six IGFBPs, is an im-

portant modulator of insulin-like growth factor (IGF) bio-

logical activity. IGFBP-3 binds to IGF-I and IGF-II with

high affinities and restricts their bioavailability by form-

ing stable ternary complexes with an acid-labile subunit.

These complexes have been reported to be stable in plas-

ma [1]. These complexes play major roles by preventing

the IGFs from activating IGF-I receptors on target cells

*This work was supported by an intramural grant from NIDDK, NIH.

Authors’ contributions: HMS carried out all experiments for these

studies. NB supervised the research and inter-pretation of the data was

carried out by HMS and NB. HMS, BK, NB and MKR made the final

draft for the paper.

#Corresponding author.

Signal Transduction Pathways Mediated by Secreted and Non-Secreted Forms of Intact Insulin-Like Growth Factor

Binding Protein-3 (IGFBP-3) and Its 1-97 N-Terminal Fragment in PC-3 Human Prostate Cancer Cells

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and stimulating cell proliferation and survival [2,3]. Sev-

eral reports have indicated that IGFBP-3 induces cell

death and inhibits cell proliferation in various cell types,

including prostate cancer cells (PC-3 cells), and it’s in-

dependent of its IGF-I binding [4-6]. IGFBP-3 is a se-

creted protein but it can be internalized to the nucleus

when it’s added to cells. Other reports have indicated that

the presence of IGFBP-3 in the cytoplasm [5,7] or in the

mitochondria [8] can induce cell death. Although the

IGFBP-3 receptor has not been identified, several signal

transduction mechanisms responsible for biological ac-

tions of IGFBP-3 have been implicated [9-12]. IGFBP-

3’s availability to cells and tissues is also regulated by

proteolysis. Proteolysis of IGFBP-3 protein was initially

demonstrated in serum of pregnant women, where it cir-

culates primarily in low molecular weight forms [13].

These fragments bind to IGF with lower affinities, there-

by increasing the limited availability of IGF to target re-

ceptors. Interestingly, various IGFBP-3 fragments have

been reported to mediate direct stimulatory or inhibitory

actions at the target cells [14].

Results from our previous study have demonstrated

that when full-length IGFBP-3 is added exogenously to

PC-3 cells, it can be processed into a small N-terminal

fragment (amino acids 1-97). Our results also demon-

strated that expression of 1-97 N-terminal IGFBP-3

fragments induces apoptosis in these cells [15]. Irrespec-

tive of the presence of a signal peptide, expression of

either the wild-type intact IGFBP-3 and N-terminal 1-97

fragment or an IGF-nonbinding mutant 6m (mutations in

six IGF-binding amino acid residues) fusion proteins

caused a loss of cell viability indicative of apoptosis.

However, the signal transduction mechanisms specific to

either the full-length or the 1-97 N-terminal fragment of

IGFBP-3 have not been studied.

In this study, not only we confirmed the involvement

of secreted as well as non-secreted forms of IGFBP-3 in

inducing apoptosis in PC-3 cells, but also we studied the

possible signaling pathways modulated by these various

forms of IGFBP-3 during the process. We examined the

induction of apoptosis in the presence or absence of cas-

pase-8 and -9 inhibitors. Surprisingly, we found that in-

tracellular form of intact and 1-97 N-terminal fragments

of IGFBP-3 (wild-type or mutant) could induce apoptosis

in a caspase-8 and -9 dependent manner in comparison to

secreted form of IGFBP-3. Our results also demonstrate

that exogenous addition of IGFBP-3 up-regulates Stat-1

protein levels and its tyrosine phosphorylation in a time-

dependent fashion. To better understand the role of

STAT-1 in IGFBP-3 induced apoptosis in PC-3 cells, we

knocked down the Stat-1 expression by using STAT-1

siRNA technique in PC-3 cells. In contrast to a previous

report [10], our results (Figure 1) showed that STAT1

siRNA treatment further potentiates IGFBP-3 (secreted

(a)

(b)

Figure 1. siRNA knockdown of Stat-1 protein in PC-3 cells.

(a) Stat-1 protein expression levels were reduced after

transfection with Stat-1 siRNA. PC-3 cells were transfected

with no DNA (Untreated; Lane 1), scrambled siRNA (Lane

2), non-target (NT) siRNA (lane 3), and Stat-1 siRNA (Lane

4) for 24, 48, and 72 h. Whole cell extracts were prepared

and western blot analyses were performed to examine the

Stat-1 protein expression levels by using an anti-human

Stat-1 antibody. Stat-1 protein expression level is decreased

after 48 and 72 h of transfection. Blot is representative re-

sult of three independent experiments. Transfection of Stat-

1 siRNA potentiates IGFBP-3-mediated apoptosis in PC-3

cells. (b) PC-3 cells were first transfected with either scram-

bled (open bars) or Stat-1 siRNA (black bars) for 24 h and

subsequently with various constructs expressing the full-

length and/or N-terminal fragments of IGFBP-3 protein for

another 48 h (total 72 h). Results from FACS analyses are

shown. Percentages of non-viable cells are indicated. Each

experiment was performed in duplicate on three separate

occasions. Data is expressed as mean ± SEM.

and non-secreted), which induced apoptosis in these cells,

thereby suggesting that Stat-1 may be an important target

molecule for IGFBP-3, which induced PC-3 cell death. In

addition, when we transfected a PC-3 stable cell line ex-

pressing CAGA-TGF-β reporter luciferase gene with vari-

ous IGFBP-3 constructs (Figure 2), we observed that

both secreted as well as non-secreted forms of IGFBP-3

constructs inhibited TGF-β signal in these cells, thereby

providing evidence that IGFBP-3 also exploits the TGF-β

signaling pathways for its apoptotic action in PC-3 cells.

2. Materials and Methods

2.1. Materials

The caspase 8-selective inhibitor Z-IETD-fmk, the cas-

pase 9-selective inhibitor Z-LEHD-fmk, and annexinV-

Signal Transduction Pathways Mediated by Secreted and Non-Secreted Forms of Intact Insulin-Like Growth Factor

Binding Protein-3 (IGFBP-3) and Its 1-97 N-Terminal Fragment in PC-3 Human Prostate Cancer Cells

1292

Figure 2. Transient transfection of IGFBP-3 constructs

reduces TGFβ signaling in PC-3 cells. PC-3 cell-line stably

expressing (CAGA) 12 tkluc (a TGFβ-responsive promoter

construct) was used for this experiment. These cells were

transfected with the indicated IGFBP-3 constructs and were

subsequently treated with TGFβ (10 ng/ml) or carrier for 24

h. Dual luciferase (renilla luciferase was used as the trans-

fection control) assays were performed. Fold activation

(luciferase activity) values (+/−TGFβ added) obtained for

each sample are shown. There is decreased TGFβ signaling

in cells transfected with constructs that are expressing in-

tracellular full-length and N-terminal 1-97 IGFBP-3. Each

experiment was repeated three times in triplicates. Data is

expressed as mean ± SEM.

APC were purchased from BD Pharmingen (San Diego,

CA). Recombinant glycosylated human IGFBP-3 was ob-

tained from R & D Systems (Minneapolis, MN). Lipo-

fectamine plus transfection reagent was obtained from Invi-

trogen (Carlsbad, CA). TGF-β and mouse β-actin anti-

body were purchased from Sigma Aldrich (St Louis, MO).

Stat-1 siRNA was purchased from Dharmacon (Lafayette,

CO). Rabbit anti-human antibodies against total and ty-

rosine phosphorylated (Y701) Stat-1 proteins were ob-

tained from Cell Signaling Technology (Danvers, MA).

2.2. Transfection

PC-3 cells (1.5 × 105 cells/6-well) were grown to 70% -

80% confluence in F12K medium containing 10% fetal

bovine serum (FBS). Cells were transfected (37˚C, 3 h)

in 1 ml of serum-free medium containing 500 ng of dif-

ferent plasmid constructs using Lipofectamine Plus (In-

vitrogen). The cells were recovered by adding 1.7 ml of

F12K medium containing 0.3 ml of FBS and were incu-

bated at 37˚C for 48 h unless otherwise specified.

2.3. Whole Cell Extract Preparation

PC-3 cells were incubated with noted concentrations of

recombinant IGFBP-3 (R & D Systems, Minneapolis,

MN) for indicated periods. After incubation, cells were

washed with phosphate-buffered saline (PBS) and were

lysed with 100 µl per 20-µl cellpellet of whole cell ex-

tract buffer (10 mM HEPES, pH 7.4, 10% glycerol, 250

mM NaCl, 1 mM EDTA, 0.1% Nonidet P-40, 1 mM di-

thiothreitol, and 1× protease inhibitor mixture) at 4˚C C

for 20 min. Lysates were vortexedat high speed for 15 s

and placed on ice for 5 - 10 min. After centrifugation

(12,000 rpm, 5 min, 4˚C), whole cell extracts (superna-

tants) were stored at −70˚C.

2.4. Western Blot

Protein concentrations were measured using the DC pro-

tein assay (Bio-Rad). Proteins were fractionated using

4% - 20% SDS-PAGE (Bio-Rad) under reducing condi-

tions. For whole cell extracts, 50 µg of protein was load-

ed per lane. Specific proteins were identified by western

blotting using Rabbit anti-Stat-1 antibody or Rabbit pStat-

1 antibody (Cell Signaling Technology, Danvers, MA).

Equal loading of the protein was confirmed by using

mouse anti-human β-actin antibody (Sigma, St. Louis, MO).

Protein bands were detected by using SuperSignal West

Pico Chemiluminescent Substrate (Pierce, Rockford, IL).

2.5. siRNA Transfection

PC-3 cells were plated in antibiotic-free F12K medium

(1.0 × 105 cells/ml for 12-well plate) for transfection.

Cells were transfected with either Stat-1, non-targetor

scrambled siRNA following a procedure provided by the

supplier (Dharmacon). Cells were incubated for 24 - 96 h

for protein analysis. For cell-death analysis experiments,

cells were first transfected with STAT-1 siRNA or

scrambled siRNA for 24 h and then were transfected with

the indicated IGFBP-3 constructs in serum-free media for

3 h and incubated in serum containing media for 48 h for

FACS analysis.

2.6. Fluorescence Activated Cell Sorting

(FACS) Analysis

After transfecting PC-3 cells with IGFBP-3 constructs

for 3 hrs, 20 µM each of caspase 8 and caspase 9 inhibi-

tor was added before cells were incubated at 37˚C in se-

rum containing media for recovery. For analysis of apop-

tosis, both attached and floating cells were collected. The

cell pellets were washed with 1× binding buffer (10 mM

HEPES, pH 7.4, 140 mM NaCl, 2.5 mM CaCl2) (BD

Pharmingen, San Diego, CA) and were stained using 300

- 500 µl of binding buffer containing 5 µl of annexin

V-APC at room temperature for 15 - 30 min. Cells were

analyzed using a CyAnLX flow cytometer equipped with

Summit software (DakoCytomation, Fort Collins, CO).

2.7. Luciferase Reporter Assay

PC-3 cells were transfected with the TGF-β-responsive

reporter construct (CAGA) 12 tkluc plasmid DNA for 3

hrs. Cells were recovered in serum-containing media and

cells were incubated for overnight at 37˚C. Selective

marker G418 was added and cells that stably expressed

this construct were selected after 3 weeks of incubation

with G418. One of the stable cell (CAGA 11), was tran-

Signal Transduction Pathways Mediated by Secreted and Non-Secreted Forms of Intact Insulin-Like Growth Factor

Binding Protein-3 (IGFBP-3) and Its 1-97 N-Terminal Fragment in PC-3 Human Prostate Cancer Cells

1293

siently transfected with YFPN1, PrewtFL, Prewt (1-97),

Pre6m (1-97), WtFL, Wt (1-97), and 6m (1-97) along

with Renilla luciferase construct (transfection control) for

3 h followed by the recovery in 10% serum containing

F12K media for 24 h. Cells were washed and TGF-β (10

ng/ml) was added to fresh serum-free media overnight at

37˚C. Cells were washed, lysed and luciferase assay was

performed using a dual luciferase assay system (Promega,

Madison, WI). Luciferase activities were normalized by

using renilla luciferase amounts present in each extract.

2.8. Statistical Analysis

All data were presented as means ± standard deviation

(SD). The significance of the difference between mean

values was determined by an analysis of variance with p

< 0.05 considered significant

3. Results

3.1. Addition of Inhibitors Specific for Caspase-8

and Caspase-9 Reduces IGFBP-3-Induced

Apoptosis in PC-3 Cells

Results from our previous study demonstrated that the

expression of various IGFBP-3 fusion protein constructs

induced apoptosis in PC-3 cells. [5,15]. In order to un-

derstand the mechanism(s) by which full-length IGFBP-

3 and its N-terminal 1-97 fragment induce apoptosis, we

transfected PC-3 cells with various IGFBP-3 constructs

with or without a signal peptide. Apoptosis was deter-

mined in untreated cells (red bars) and in the presence of

20 μm of caspase-8 (IETD; yellow bars) and 20 μm of

caspase-9 (LEHD, green bars) specific inhibitors. The

number of apoptotic cells was determined by Annexin-V

staining (FACS). Results indicated that the addition of

either caspase 8 or 9 inhibitor significantly inhibited

IGFBP-3-induced apoptosis in PC-3 cells (Figure 3).

These results clearly shows that Intracellular form of full

length and 1-97 N-terminal fragments of WT and 6m

IGFBP-3 induce apoptosis in PC-3 cancer cells using

both caspase-8 and caspase-9 pathway which is not the

case with secreted form of Intact and 1-97 N-terminal

fragment of IGFBP-3. These results also confirm that

IGFBP-3 does not need to depend on the availability of

IGF or be secreted to induce apoptosis in PC-3 cells and

interestingly secreted form of IGFBP-3 is not involved in

inducing apoptosis via caspase 8 and caspase 9 activation

pathways.

3.2. Addition of Exogenous IGFBP-3 Increases

the Expression of Stat-1 Protein Level and

Its Tyrosine Phosphorylation (Y701)

in PC-3 Cells

A previous report [10] has shown that the addition of

Figure 3. IGFBP-3 induced apoptosis in PC-3 cells is inhib-

ited by inhibitors specific for Caspase-8 and Caspase-9.

PC-3 cells were transfected with plasmid constructs direct-

ing the expression of either the full-length IGFBP-3 or its

N-terminal 1-97 fragments. Cells were subsequently treated

with either DMSO (vehicle; Red bars) or 10 μM IETD

(caspase-8 inhibitor; yellow bars) and LEHD (caspase-9

inhibitor; green bars) for 48 h. Results from FACS analyses

(Annexin-V+ cells) are shown. Percentages of non-viable

cells are indicated. Figure 3 showed significant inhibition of

IGFBP-3 induced apoptosis in PC-3 cells in the presence of

caspase-8 and/or caspase-9 inhibitors. Experiments were

performed in triplicate twice and error bars represent mean

± SEM.

IGFBP-3 induces apoptosis and differentiation via STAT-

1 in rat chondroprogenitor cells. In order to investigate

the roles of STAT-1 signaling pathways in IGFBP-3 in-

duced apoptosis in PC-3 cells, we incubated the cells

with different concentrations of exogenous recombinant

IGFBP-3. Expression of total Stat-1 levels in PC-3 cells

were increased in a dose-dependent manner, peaking

after 50 ng/ml of IGFBP-3 (Figure 4(a) ) addition. There

was also a transient increase in tyrosine-phosphorylated

Stat-1 (pY701 STAT-1) levels within 10 min of incuba-

tion with 50 ng of IGFBP-3 (Figure 4(b)). These results

demonstrate that addition of IGFBP-3 can elevate the

expression levels as well as pY701 Stat-1 in PC-3 cells

indicating the involvement of Stat-1 signaling pathways.

3.3. Introduction of Stat-1 siRNA Potentiates the

IGFBP-3-Induced Apoptosis in PC-3 Cells

In order to better understand the roles of STAT-1 signal

pathways in IGFBP-3 induced apoptosis in PC-3 cells,

we first tested the effectiveness of a siRNA to block the

expression of Stat-1 in these cells. Western blot analysis

showed marked decrease in Stat-1 protein levels after 48

and 72 h of siRNA transfection in PC-3 cells (lanes

marked as Stat-1siRNA) compared to cells with no

siRNA treatment or transfected either with a scrambled

siRNA or a non-target siRNA (Figure 1(a)).There was

also a significant decrease in Stat-1 mRNA levels after

24 hrs of siRNA transfection in PC-3 cells (data not shown

Signal Transduction Pathways Mediated by Secreted and Non-Secreted Forms of Intact Insulin-Like Growth Factor

Binding Protein-3 (IGFBP-3) and Its 1-97 N-Terminal Fragment in PC-3 Human Prostate Cancer Cells

1294

(a)

(b)

Figure 4. Addition of exogenous IGFBP-3 induced Stat-1

protein expression as well as its tyrosine phosphorylation

(pY701) in PC-3 cells. (a) PC-3 cells were treated with ex-

ogenous recombinant IGFBP-3 (in ng/ml are indicated be-

low each lane) for 1 h. Whole cell extracts were prepared

and a western blot analysis was performed. Anti-human

Stat-1 antibody was used to examine the Stat-1 protein (91

kDa) (upper panel) and β-actin (42 kDa) (lower panel) ex-

pression levels in each sample. There is a dose-dependent

increase of Stat-1 protein expression level in the presence of

exogenously added recombinant IGFBP-3. This data is a

representative result of three independent experiments. (b)

IGFBP-3 treatment causes transient increase in Stat-1 tyro-

sine phosphorylation (pY701) PC-3 cells were treated with

50 ng/ml of IGFBP-3 for indicated periods of time. Whole

cell extracts were prepared and western blot was performed

by using an antibody against pY701 STAT-1(upper panel;

indicated by a 91 kDa) (upper panel) and unmodified

STAT-1 (middle panel, 91 kDA). The experiment was re-

peated three times. The blot was stripped and was reused to

detect for the β-actin (lower panel; indicated by a 42 kDa

protein) level using an anti-human β-actin antibody and

that served as the loading control. Blot is a representative

result of three independent experiments.

shown) Secondly, cell viability was determined in PC

cells transfected first with either STAT-1 siRNA (black

bars) or a scrambled siRNA (white bars) and then with

full length and various 1-97 N-terminal IGFBP-3 con-

structs. FACS analysis results showed that the IGFBP-

3-induced apoptosis was potentiated in cells transfected

with Stat-1 siRNA (Figure 1(b)), a result that is in con-

tradiction with a previous observation [10] but this report

confirms that Stat-1 plays an important role in IGFBP-3

induced apoptosis in PC-3 cells.

3.4. Expression of Intracellular IGFBP-3

Constructs Inhibits TGF-β SIgnaling

in PC-3 Cells

TGF-β has been implicated in several cellular processes

including apoptosis and proliferation [16,17]. In order to

determine and delineate the involvements of TGF-β sig-

naling pathways, we compared the effects of intact

IGFBP-3 or its N-terminal 1-97-IGFBP-3-YFP fragments

in PC-3 cells. First, several PC-3 cell-lines that stably ex-

pressed (CAGA) 12 tkluc construct, a TGF-β-responsive

luciferase reporter system, were created. One of these

clonal cell-lines was selected for transfection with vari-

ous constructs (WTFL IGFBP-3/1-97 N-terminal frag-

ment and 6m FL IGFBP-3/ 1-97 N-terminal fragment)

that express IGFBP-3 with or without its signal peptide.

Extent of TGF-β signals were measured by luciferase

output with without the addition of TGF-β. There was

significant inhibitions of TGF-β-mediated (CAGA) 12

luciferase activities in cells transfected with YFP-WT-

FL-IGFBP-3, YFP-WT-1-97-IGFBP-3, or Pre-WT-1-97-

IGFBP-3-YFP in comparison to cells transfected with

YFP empty vector or untransfected control (Figure 2).

Similar results were observed with YFP-6m-1-97-IGFBP-

3 and Pre-6m-1-97-IGFBP-3-YFP in which the IGF-bind-

ing site had been mutated, indicating that the inhibition

of TGF-β signaling occurs through an IGF-independent

mechanism. Interestingly, cells transfected with Pre-WT-

FL-IGFBP-3-YFP had a little effect. Whole cell extracts

and media collected from these transfected cells were

examined and equivalent expression of IGFBP-3 fusion

protein was observed in each sample (data not shown).

Full-length IGFBP-3 as well as its 1-97 N-terminal frag-

ment that expressed only intracellular protein inhibited

TGF-β signaling in PC-3 cells. This indicates that TGF-β

signaling pathways are involved in cellular actions of

IGFBP-3.

4. Discussion

IGFBP-3, the most abundant IGFBP in human serum,

acts not only as a carrier of IGFs prolonging their half-

lives in circulation, but also functions as a modulator of

IGF activity by regulating their availability to interact

with IGF receptors [18]. However, there is increasing

evidence that IGFBP-3 may have its own biological ac-

tions. For example, IGFBP-3 can inhibit cell proliferation

in an IGF-independent manner [4,19,20]. Conceptually,

IGFBP-3 can exert its actions on target cells by: 1) in-

ducing apoptosis, 2) regulating cell cycle and prolifera-

tion, and 3) inducing cross-talk with major signal trans-

duction pathways. Mechanisms for inducing apoptosis, in

part, have been demonstrated by studies showing that

IGFBP-3 increases the ratio of proapoptotic to antiapop-

totic proteins in breast cancer cells [21]. As a regulator of

the cell cycle, IGFBP-3 has been shown to modulate the

induction of the cyclin-dependent kinase inhibitory pro-

tein p21/WAF/CIP1 in LNCaP prostate cancer cells [22].

Previous studies demonstrated that caspase-8 can acti-

vate caspase-3 and -7 directly and/or through triggering

the activation of caspase-9 by the release of mitochon-

Signal Transduction Pathways Mediated by Secreted and Non-Secreted Forms of Intact Insulin-Like Growth Factor

Binding Protein-3 (IGFBP-3) and Its 1-97 N-Terminal Fragment in PC-3 Human Prostate Cancer Cells

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drial cytochrome c [23]. Being consistent with our pre-

vious studies [5], current data suggest that non-secreted

IGFBP-3 induced apoptosis in PC-3 cells, is mediated by

caspase-8 as well as the caspase-9 pathways. Our results

indicate that apoptosis induced by non-secreted IGFBP-3

as compared to secreted form could be blocked after ad-

dition of caspase-8 and -9 inhibitors, indicating that both

initiator caspase pathways are involved in this process.

Several studies have demonstrated that full-length

IGFBP-3 can induce various signaling pathways [6,17,24,

25]. In this regard, PC-3 cells were transfected with dif-

ferent IGFBP-3 constructs and then treated with inhibi-

tors specific for PI-3K and p38 MAPK. Results from

FACS analyses indicated that there was little or no differ-

ence in the extent of cell death (data not shown), indicat-

ing that the PI-3K and p38 MAPK signaling pathways

may not be involved in IGFBP-3 mediated cell death in

PC-3 cells.

Another signaling molecule (Stat-1) has been impli-

cated in IGFBP-3 induced apoptosis in rat chondropro-

genitor cells [10], this study led us to demonstrate the

role of Stat-1 in IGFBP-3 induced apoptosis in PC-3 hu-

man prostate cancer cells. Dose response experiment show-

ed increased Stat-1 protein expression at higher concen-

trations of exogenous IGFBP-3. Tyrosine phosphoryla-

tion of Stat-1 was also enhanced at higher concentrations

of exogenous IGFBP-3 in a time dependent manner (Fig-

ures 2(a) and (b)). In contrast to this previous report, our

results indicated that STAT-1 knock-down potentiated

the apoptotic effects of various IGFBP-3 constructs in

PC-3 human prostate cancer cells (Figures 3(a) and (b)).

The data suggest that Stat-1 protein might be playing a

protective role in PC-3 cells and the data also suggest

that the involvement of STAT-1 in apoptosis and prolif-

eration might be cell type dependent.

IGFBP-3 has shown to interact with transforming

growth factor-β (TGF-β) cell surface receptors. In mink

lung epithelial cells and other cell-types, IGFBP-3 binds

to TGFβ-receptor typeV (TGFβ-RV) [12]. An IGFBP-3

mediated inhibitory effect on growth involving this par-

ticular receptor has been proposed, but mechanisms of

action have not been fully elucidated. In this pathway,

IGFBP-3 also binds to and activates intracellular signal-

ing via the TGF-β RII and TGF-β RI heteromeric com-

plex [24,25]. Addition of IGFBP-3 activated Smad2 pho-

sphorylation and inhibited cell growth in these cells.

Studies have also indicated that the addition of TGF-β

can potentiate IGFBP-3-induced cell death in PC-3 cells

(6). In that regard, our results demonstrated that the ex-

pression of IGFBP-3 constructs could significantly in-

hibit TGF-β signaling (Figure 2) in PC-3 cells. This im-

plies that IGFBP-3 might interfere with TGF-β signaling

pathways. It is also interesting to note that this inhibition

was more prominent in cells where IGFBP-3 is expressed

intra-cellularly. Results from our previous studies [5,15]

clearly indicated that the IGFBP-3-YFP fusion proteins

expressed from Pre wt 1-97, Pre6m 1-97 WtFL, Wt 1-97,

and 6 m 1-97 constructs are present predominantly inside

the cells whereas Prewt FL protein was present outside in

the media. How these intracellular proteins can interfere

with TGF-β signals is not clear yet. Although we did not

measure differential Smad activation levels in these cells,

our data clearly suggest that TGF-β signaling pathways

are playing an important role in IGFBP-3 induced bio-

logical actions in these cells. Future studies would be

needed to elucidate the roles of IGFBP-3 in TGF-β sig-

naling by comparing the gene and protein expression

profile in these cells. Our data indicate that full length

IGFBP-3 and its N-terminal fragments induced the apo-

ptosis of PC-3 cells through the modulations of caspases

8, 9. STAT-1 and TGF-β signal pathways are also in-

volved. It also suggests the modulation of STAT-1 and

TGF-β pathways as possible therapy for prostate cancer.

5. Acknowledgements

We gratefully acknowledge Drs. William Chong (NIDCR,

NIH) and Maria Mochin-Peters (University of Maryland,

Baltimore) for comments and suggestions.

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