IGF-binding protein-3 (IGFBP-3) has been shown to induce apoptosis in an insulin-like growth factor (IGF)-independent manner in various cell systems, however, the underlying molecular mechanisms remain unknown. In the present study, we showed that IGFBP-3 significantly enhanced interleukin-24 (IL-24)-induced cell death in prostate cancer (PC) cell lines
Insulin-like growth factor (IGF)-binding protein-3 (IGFBP-3) is one of the six characterized binding partners of IGF-I, and is the most abundant IGFBP found in circulation (
Interleukin (IL)-24 was originally identified as melanoma differentiation-associated gene-7 (mda-7), which is a cytokine tumor suppressor located in a cluster on chromosome 1q32 encoding IL-10, -19 and -20 (
This tumor cell-specific growth-inhibitory effect has also been observed in multiple animal models
The mammalian target of rapamycin (mTOR) is a highly conserved serine/threonine kinase that regulates cell growth, cell cycle progression and metabolism. The PI3K/AKT signaling pathway activates mTOR, which in turn directly phosphorylates ribosome protein S6 kinase 1 (S6K) and eIF4E-binding protein 1, both of which are important in control of protein translation initiation (
IGFBP-3 is differentially expressed across normal prostate tissue types (
LNCap, PC-3, P69 and HEK293 cell lines were obtained from the American Type Culture Collection (ATCC; Manassas, VA, USA) and cultured in the recommended growth medium (Invitrogen, Carlsbad, CA, USA) at 5% CO2, 37°C. HEK293 cells transfected with, and stably expressing the pcDNA3 expression vector containing IGFBP-3 (IGFBP-3 cDNA was constructed by our laboratory). HEK293/IGFBP-3, and a vector control (HEK293/pcDNA3-control) were grown in 10% fetal bovine serum (FBS) Dulbecco's modified Eagle's medium (DMEM) supplemented with 200 mg/ml G418 for selection. Soluble recombinant human IGFBP-3 and IL-24 were purchased from PeproTech, USA.
Cells were seeded in triplicate flasks and grown under standard conditions until ~70% confluency. Cells were grown for 24 h in a serum-free medium (SFM) to remove IGFBP-3 from the serum and then grown for up to 24 h in SFM supplemented with or without IL-24 (0.2, 0.4 and 0.8
Cell viability was measured by mitochondrial conversion of 3-(4,5-dimethylthiazolyl-2)-2,5-diphenyltetrazolium bromide (MTT) to a colored product. Following treatment with drugs, MTT was added. Cells were then solubilized in dimethylsulphoxide (DMSO). The amount of converted MTT was determined by measuring absorbance at 570 nm.
Full-length Mcl-1 or IGFBP-3 cDNA was cloned into the
LNCap cells were transfected with an IGFBP-3 overexpression vector or Mcl-1 siRNAs for 24 h. The cells were reseeded in 60-mm plates and colony formation was monitored over the subsequent 10 days.
Cells were seeded in duplicate flasks and grown under standard conditions until ~70% confluency, and then grown for 24 h in SFM to remove IGFBP-3 from the serum. The cells were then grown for up to 24 h in SFM supplemented with IL-24 (0.2–0.8
Cells were first incubated in SFM for 24 h, and then in SFM plus an anti-IGF-IR antibody (Santa Cruz Biotechnology) or an anti-IGFBP-3 antiserum at a content level of 5
Subconfluent monolayers were grown in SFM for 24 h. The cells were then grown in SFM for 24 h, with or without IL-24. The media were then harvested, centrifuged to remove floating cells and stored at −70°C. The number of attached cells was quantified using a haemocytometer. Proteins from conditioned medium were concentrated using 'Microsep' 10 K centrifuge columns (Gelman Laboratory). The levels of IGFBP-3 were assessed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) immunoblotting and the IGFBP-3 protein was detected by anti-IGFBP-3 antiserum (polyclonal from Diagnostic System Laboratory, USA) using an enhanced chemiluminescence (ECL) system (KPL, USA).
Cell lysates and immunoprecipitates were separated by SDS-PAGE and transferred to nitrocellulose membranes, followed by immunoblotting with the specified primary antibodies and horseradish peroxidase-conjugated secondary antibodies. Immunoreactive bands were visualized with SuperSignal West Pico Chemiluminescent Substrates (Thermo Scientific Pierce, Rockford, IL, USA).
Recombinant adenoviruses (pAd/CMV/IGFBP-3 or pAd/CMV/IL-24) contained IGFBP-3 or IL-24 between the cytomegalovirus (CMV) promoter and the polyadenylation signal (SV40pA). These adenoviral vectors were propagated in HEK293 cells (Invitrogen) using a Stratagene MBS mammalian transfection kit (La Jolla, CA, USA) with a modified calcium phosphate transfection protocol. The transfected cells were incubated at 37°C for 7 days, harvested and subjected to 4 cycles of freezing (dry ice and alcohol)/thawing in a 37°C water bath. The cell lysates were centrifuged at 12,000 × g and 4°C for 10 min; then the supernatant (primary virus stock) was transferred to a fresh screw-cap mini-centrifuge tube and stored at −80°C. Recombinant adenoviruses were further amplified using the same procedure; the cell lysates were centrifuged with cesium chloride step gradients at 60,000 × g and 4°C for 2 h, allowing the viruses to separate from the defective particles and empty capsids. The recovered virus bands were dialyzed 3 times against phosphate-buffered saline (PBS). The viruses Ad5.IGFBP-3 or Ad5.IL-24 were aliquoted in a buffer containing 10 mmol/l Tris-HCl (pH 8.1), 10 mmol/l MgCl2 and 10% v/v glycerol and then stored at −80°C.
Four-week-old athymic nude mice were obtained from the Animal Research Center, Shanghai Laboratory Animal Research Center, China. All procedures were performed in accordance with an animal protocol approved by the First Affiliated Hospital of Medical School, Xi'an Jiaotong University, and by the local Experimental Ethics Committee. All efforts were made to minimize animal suffering and reduce the number of animals used. Tumors were established by subcutaneous injection of 5×106 LNcap cells into the right flank of the mice. When a tumor grew to the size of ~50 mm3, the host mouse was randomly assigned to one of the 3 groups (each n=6): control (DMSO dissolved in 50
All data represent the mean of 3 separate experiments. Each experiment was carried out in duplicate or triplicate parallel flasks. Each experiment was repeated 3 times, and results are presented as the mean of the 3 separate experiments. Statistical analysis was carried out using SPSS for Windows statistical software (release 10.0.5; SPSS, Inc., Chicago, IL, USA). Analysis of variance (ANOVA) was used to determine differences among means. Pair-wise comparisons were made using Tukey's post hoc test for multiple comparisons.
Differential sensitivity to IL-24 was observed in PC cell lines. Whereas the LNCap and PC-3 cell lines were sensitive to the inhibition of cell proliferation, the P69 cell line was not sensitive to the drug treatment. However, as experiments on the effects of IGFBP-3 were carried out in SFM, we initially sought to verify that the differential sensitivity to IL-24-induced cell proliferation inhibition was retained under serum-free conditions. Therefore, both LNCap, PC-3 and P69 cells were treated with IL-24 (0.2, 0.4 and 0.8
As the pro-apoptotic IGFBP-3 protein improves the sensitivity of cancer cell lines to the induction of apoptosis (
The IGF-1R blocking antibody αIR3 (5
A recent study shows that PC cell lines express high levels of Mcl-1, and that suppression of Mcl-1 induces significant cell death (
In the present study, we have shown that IGFBP-3 potentiates IL-24-induced apoptosis in PC cell lines. Adding to the former findings that IGFBP-3 enhanced apoptosis by suppressing certain survival signalling pathway. We then investigated the mechanism by which IGFBP-3 overexpression suppresses Mcl-1 protein. Initially, mTOR activity was assessed in cells treated with IGFBP3-M. IGFBP3-M suppressed mTOR activity, assessed on basis of weakened phosphorylation of mTOR and its effectors (S6K and S6) in LNCap cells (
mTOR activity can be suppressed upon energy deprivation, as depleted ATP levels trigger AMPK activation (
To investigate the effects of IGFBP-3 and IL-24 on cell growth
IGF-binding protein-3 (IGFBP-3) is a pro-apoptotic protein previously shown to potentiate apoptotic cell death in a number of systems. In the present study, we showed that in human prostate cancer (PC) cells IGFBP-3 promotes IL-24-induced apoptosis. This important finding suggests that IGFBP-3 may be used to improve IL-24-induced apoptosis in cancer cells without affecting the survival of non-tumorigenic cells.
Apoptosis is induced by both extrinsic and intrinsic pathways initiated by the activation of death receptors and stress-inducing stimuli (
The mTOR pathway plays multiple roles in cell growth, proliferation and survival. In the present study, we determined whether mTOR and its downstream factors are involved in IL-24 sensitization induced by IGFBP-3 overexpression. Unexpectedly, IGFBP-3 overexpression led to reduced levels of phosphorylated mTOR, as well as its downstream effector S6K. Downregulation of mTOR and S6K using siRNA made cells more sensitive to IL-24. Importantly, we showed that knockdown of either mTOR or S6K downregulates Mcl-1 protein levels. These results indicated that Mcl-1 is a downstream target of mTOR and S6K. Furthermore, Mcl-1 may be regulated at the translational level, as the main function of mTOR is to control translation via direct regulation of S6K and 4E-BP. This assumption is supported by several studies in the literature that Mcl-1 is a potential downstream target of eIF4E, which contains a unique G+C-rich 5′ untranslated region among the anti-apoptotic Bcl-2 members (
Surprisingly, overexpression of IGFBP-3 led to a marked increase in the ADP/ATP ratio within cells, indicating a decrease in ATP content. AMPK is a central cellular energy-sensing system that actively participates in the metabolism-cancer interaction via regulation of the mTOR pathway. AMPK activation directly phosphorylates and activates TSC2 by enhancing its GAP activity, leading to inhibition of mTOR signaling (
In summary, through inhibiting mTOR activation, IGFBP-3 increases the sensitivity of cancer cells to IL-24-induced apoptosis and may also be important in both the prevention of PC, and as a potential adjuvant for a number of other therapeutic regimens used in the treatment of metastatic prostate cancer.
The present study was supported by the National Science Foundation of China (grant nos. 30901500, 81072107 and 81372736), and the Science and Technology Program of Shaanxi Province (grant no. 2009JQ4002).
Prostate cancer cell lines are sensitive to IL-24-induced apoptosis. (A) Cell proliferation 24 h after treatment with IL-24 (0.2, 0.4 or 0.8
IGFBP-3 promotes IL-24-induced apoptosis in human prostate cancer cell lines. LNCap, PC-3 and P69 cells treated with 0.8
IGFBP-3 promotes IL-24-induced apoptosis in the presence of an IGF-1 receptor blocking antibody or IGFBP-3 antibody. Results shown represent the mean ± SD (n=3). Significant differences are denoted by **P<0.01. IGFBP-3, IGF-binding protein-3; IL-24, interleukin-24.
Mcl-1 is essential for survival of prostate cancer cells. (A) Survival of LNCap and PC-3 cells 24 (b) or 48 h (a) after transfection with siRNA targeting Mcl-1 or a control siRNA. (B) LNCap cells were transfected with siCon or Mcl-1 siRNA for 24 h. (C) LNCap cells were transfected with empty vector control (pcDNA3), Mcl-1 (pcDNA3/Mcl-1) or IGFBP3-M for 24 h, followed by treatment with 0.8
Overexpression of IGFBP-3 inhibits mTOR-mediated Mcl-1 downregulation. (A) LNCap cells were transfected with IGFBP-3 plasmid or control vector for 30 h. (B) LNCap cells were transfected with control or mTOR siRNA for 30 h. (C) LNCap cells were transfected with control or mTOR siRNA for 20 h, followed by treatment with 0.8
The Mcl-1 decrease, induced by IGFBP-3 overexpression, is mediated by S6K downregulation. (A) LNCap cells were transfected with control or S6K siRNA for 30 h. (B and C) LNCap cells were transfected with control or S6K siRNA for 20 h, followed by treatment with 0.8
IGFBP-3 overexpression leads to mTOR inhibition through AMPK. (A and B) LNCap cells were transfected with control, IGFBP-3 or Mcl-1 siRNA for 48 h. ADP/ATP ratios were determined using the EnzyLight ADP/ATP ratio assay kit. Data are expressed as the mean of 3 independent experiments and error bars reflect SD. (C) LNCap cells were transfected with control, IGFBP-3 or AMPKα1/2 siRNA for 48 h. The indicated protein levels were measured using western blot analysis (B and C) and blots shown are representative of 3 independent experiments. IGFBP-3, IGF-binding protein-3; IL-24, interleukin-24; AMPK, adenosine monophosphate-activated protein kinase.
Tumor growth and gene expression