Correlation of IGFBP-6 expression with apoptosis and migration of colorectal carcinoma cells.

Colorectal cancer (CRC) is one of the most common malignant tumors in digestive tract. Previous study found close correlation between insulin-like growth factor binding proteins (IGFBPs) and occurrence of multiple tumors. This study aims to analyze the effects of IGFBP6 on the apoptosis and migration of tumor cells, and to investigate underlying mechanism. HCT-116 or SW480 cell was cultured with 1.0 mg/l, 10 mg/l and 100 mg/l IGFBP-6. MTT assay was employed to test the proliferation activity of tumor cells after differential treatment. The cell cycle of tumor cells was detected by flow cytometry, while Transwell assay was used to quantify the invasion and migration of tumor cells after IGFBP-6 intervention. In experimental group with IGFPB-6 application, the proliferation rate of HCG-116 or SW480 cells was gradually decreased with higher concentrations of IGFBP-6 (p< 0.05). The ratio of cells at G0/G1 phase was increased while S phase and G2/M phase ratio were all decreased with IGFPB-6. With further elevated concentration of IGFPB-6, there was more potency of higher G0/G1 ratio and lower S phase or G2/M phase (p< 0.05). Both invasion and migration ability of HCT-116 or SW480 cells in experimental group were decreased. With elevated IGFBP-6 concentration, cell invasion and migration were further weakened (p< 0.05).IGFBP-6 could inhibit invasion and migration of colorectal carcinoma cells possibly via inhibiting proliferation activity and arresting cell cycle of HCT-116 or SW480 cells.

Insulin-like growth factor-6 (IGFBP-6) stimulates neutrophil oxidative burst, degranulation and chemotaxis.

The aim of this study was to understand whether insulin-like growth factor-binding protein-6 (IGFBP-6) has functional effects on neutrophils, in particular when they cross epithelium during inflammation. We found that IGFBP-6 increased ROS production (cytofluorimetry), degranulation of primary and tertiary granules (ELISA) and transmigration through the epithelial monolayer. No priming by IGFBP-6 on neutrophils stimulated by either PMA or fMLP was observed. IGFBP-6 is an agonist of neutrophils' functions, most likely when these cells have been already activated by other stimuli.

Autoregulation of insulin-like growth factor 2 and insulin-like growth factor-binding protein 6 in periodontal ligament cells in vitro.

The insulin-like growth factor (IGF) system plays an important role in tissue development and presumably also governs pathophysiology of the periodontal ligament (PDL). It has been the aim of this study to elucidate the specific expression pattern of IGF2 and IGFBP6 in PDL cells and to determine whether PDL cells feature autoregulatory mechanisms upon exposure to these IGF components. Human PDL cells (n=6) were exposed to IGF2 (100 ng/ml), IGFBP6 (450 ng/ml, 675 ng/ml, 1125 ng/ml) or a combination of 100 ng/ml IGF2 and 675 ng/ml IGFBP6 for 1, 3 or 5d. qRT-PCR was run for IGF2, IGFBP6, Ki67, ALP, osteocalcin. Immunocytochemical quantification was performed for IGF2 and IGFBP6. Results showed a time-dependent increase in IGF2 and IGFBP6 gene expression, as opposed to a general decrease at the protein level. At the transcriptional and protein level, challenge with IGF2 and IGFBP6 dampened the expression of both molecules at all time points investigated. Only in the case of IGF2 did combined treatment with IGF2 and IGFBP6 contrarily increased protein expression in both nuclear and cytoplasmatic structures compared to the vehicle treated controls. Analyses of PDL cell proliferation and differentiation revealed Ki67 downregulation by IGF2 and IGFBP6 alone or in combination. Beyond this, the osteogenic differentiation potential of PDL cells was suppressed as ALP and osteocalcin expression was reduced. Our results indicate that IGF2 and IGFBP6 appear to govern various regulatory feedback mechanisms in PDL cells. Thus, the functional properties of these molecules in oral structures are presumably self-controlled under impact of different biological processes such as expression levels of these IGF components, cell proliferation and differentiation.

Contributions of the N- and C-terminal domains of IGF binding protein-6 to IGF binding.

Insulin-like growth factors IGF-I and IGF -II are important mediators of growth. A family of six high affinity IGF binding proteins (IGFBPs) modulate IGF action. IGFBPs have three domains, of which the N- and C-domains are involved in high affinity IGF binding. IGFBP-6 is unique in its 20-100-fold IGF-II binding specificity over IGF-I. The aim of this study was to determine the contributions of the N- and C-domains of IGFBP-6 to its IGF binding properties. We confirmed that differential dissociation kinetics are responsible for the IGF-II binding preference of IGFBP-6. The N-domain has rapid association kinetics, similar to full-length IGFBP-6, but both IGF-I and -II dissociate rapidly from this domain, thereby reducing its binding affinity for IGF-II approximately 50-fold. However, the N-domain binds IGF-I and -II with similar affinities and it has a similar IGF-I binding affinity to full-length IGFBP-6. This suggests that the C-domain confers the IGF-II binding preference of IGFBP-6; indeed, IGF-I bound inconsistently with very low affinity to the C-domain. Coincubation studies showed that isolated N- and C-domains of IGFBP-6 do not strongly cooperate to enhance IGF binding. The results of the binding studies are supported by the effects of the IGFBP-6 domains on IGF-induced colon cancer cell proliferation; the N-domain inhibited IGF-II induced proliferation with approximately 20-fold lower potency than IGFBP-6 and it was equipotent in inhibiting IGF-I- and IGF-II-induced proliferation. Coincubation of C-domain had no additional effect on N-domain-induced inhibition of proliferation. In conclusion, both the N- and C-domains of IGFBP-6 are involved in IGF binding, the C-domain is responsible for the IGF-II binding preference of IGFBP-6 and intact IGFBP-6 is necessary for high affinity IGF binding.

Effect of an insulin-like growth factor binding protein fusion protein on thymidine incorporation in neuroblastoma and rhabdomyosarcoma cell lines.

A fusion protein, FP 6/3, composed of IGF binding protein (IGFBP)-6 and IGFBP-3 was synthesized where the complete sequences of each binding protein were fused together into a single chimeric protein. The orientation of this fusion protein's structure has the N terminus of IGFBP-3 fused to the C terminus of IGFBP-6, leaving the key binding areas of each open. FP 6/3 bound to cells via its IGFBP-3 component and retained the increased affinity for IGF-II via its IGFBP-6 component. The effect of FP 6/3 on growth was determined in cell lines from both neuroblastoma and rhabdomyosarcoma, where IGF-II is an autocrine growth factor. In studies using FP 6/3, IGFBP-3, or IGFBP-6, a growth inhibition effect was shown for all when present under coincubation conditions with IGF-II. However, with transient exposure, FP 6/3 was the only IGFBP that retained this growth-inhibition property. Under transient exposure conditions, FP 6/3 was found to be effective when exposure was limited to as few as 10 min and concentrations were as low as 1 nm. These findings with FP 6/3 suggest that it potentially could lead be used as therapy against cancers in which IGF-II is an autocrine growth factor because it brings an inhibition action directly to tumor cells.

Insulin-like growth factor binding protein-6 and CCI-779, an ester analogue of rapamycin, additively inhibit rhabdomyosarcoma growth.

Rhabdomyosarcomas secrete high levels of insulin-like growth factor-II, suggesting this autocrine growth factor plays a major role in the unregulated growth of this childhood cancer. Treatment of Rh30 rhabdomyosarcoma cells with insulin-like growth factor binding protein-6 (IGFBP-6; 1000 ng/ml), which binds insulin-like growth factor-II with high affinity, inhibited growth in vitro (p < 0.001). Co-incubation of cells with rapamycin (1.56 ng/ml), an inhibitor of p70 S6 kinase, and IGFBP-6 (200 ng/ml) resulted in a significant reduction in Rh30 cell number compared to rapamycin or IGFBP-6 alone (p < 0.05 for both). Co-treatment of Rh30 cells with CCI-779 (5 and 50 ng/ml), an ester analogue of rapamycin, and IGFBP-6 (200 or 1000 ng/ml) also inhibited growth in vitro relative to CCI-779 alone (p < 0.01 and p < 0.001, respectively). In a nude mouse model, xenografts of Rh30 cells transfected with a recombinant vector encoding IGFBP-6 (phBP6-E3) showed delayed growth relative to vector control xenografts (27 days vs. 19 days to reach an average tumour volume of 0.5 cm (3); p < 0.001). Treatment with CCI-779 (10 mg/kg) of mice inoculated with vector control xenografts, also delayed growth (to 31 days; p = 0.0055) relative to untreated mice with vector control xenografts. Co-treatment with CCI-779 (10 mg/kg) reduced phBP6-E3 transfected xenograft growth even further (to 45 days) compared to vector control xenografts (p < 0.001, day 33). CCI-779 thus acts additively with IGFBP-6 to reduce rhabdomyosarcoma growth both in vitro and in vivo.

Differential effects of insulin-like growth factor binding proteins-1, -2, -3, and -6 on cultured growth plate chondrocytes.

BACKGROUND: In children with chronic renal failure (CRF), impairment of longitudinal growth is in part due to excess amounts of circulating high-affinity insulin-like growth factor binding proteins (IGFBPs) that might decrease or prevent insulin-like growth factor (IGF) binding to its signaling receptor. However, it appears from the clinical studies that various IGFBPs may have contrasting effects on longitudinal growth. Because of the potential importance of the IGFBPs as modulators of longitudinal growth in pediatric CRF, the aim of the present study was to investigate the biological effects of IGFBP-1, -2, -3, and -6 on cultured growth plate chondrocytes that express the type 1 IGF receptor. METHODS: The effects of exogenous IGFBPs on IGF-independent and IGF-dependent proliferation of rat growth plate chondrocytes in primary culture were investigated. Proliferation was assessed by colony formation of agarose-stabilized long-term suspension cultures and by the [3H]thymidine assay. The effects of IGFBPs on IGF-I binding and the binding of IGFBPs to chondrocytes were assessed by binding studies with radiolabeled proteins in monolayer culture. RESULTS: Intact IGFBP-1, IGFBP-2 and IGFBP-6 inhibited in equimolar concentration the IGF-I- and IGF-II-stimulated DNA synthesis and cell proliferation, whereas the biological activity of IGFBP-3 was complex. It had an IGF-independent antiproliferative effect and also inhibited IGF-dependent chondrocyte proliferation under coincubation conditions, whereas under preincubation conditions IGFBP-3 enhanced IGF-I-responsiveness. Studies on the mechanism by which IGFBP-3 potentiated IGF activity demonstrated that under preincubation conditions IGFBP-3 is capable to associate with the cell membrane and to facilitate IGF-I cell surface binding. CONCLUSIONS: Intact IGFBP-1, IGFBP-2 and IGFBP-6 act exclusively as growth inhibitors on IGF-dependent proliferation of growth plate chondrocytes. IGFBP-3, however, can either inhibit IGF-independent and IGF-dependent cell proliferation, or enhance IGF responsiveness of chondrocytes dependent on the temporal relationship to the IGF exposure.

Insulin-like growth factor (IGF)-binding protein-6 inhibits IGF-II-induced but not basal proliferation and adhesion of LIM 1215 colon cancer cells.

IGF-II is an autocrine growth factor for many colon cancer cells. This study aimed to determine the role of IGF-II in proliferation and adhesion of LIM 1215 colon cancer cells. RT-PCR demonstrated expression of IGF-I and IGF-II mRNA. Addition of IGF-I or -II increased monolayer proliferation in a dose-dependent manner. Although addition of IGFBP-6 had no effect on basal proliferation, coincubation of IGFBP-6 decreased IGF-II but not IGF-I-induced proliferation. Colony formation in agar was increased by IGF-II, an effect inhibited by coincubation with IGFBP-6. IGFBP-6 alone significantly decreased colony formation. Preincubation of cells with IGF-II increased adhesion to type IV collagen, fibronectin and laminin. IGFBP-6 had no effect on basal cell adhesion but completely inhibited the effects of IGF-II. LIM 1215 colon cancer cells are therefore IGF-responsive but IGF-II is not a major autocrine factor for these cells in monolayer, suggesting heterogeneity between colon carcinoma cell lines with respect to the role of the IGF system.

Effect of IGFBP-derived peptides on incorporation of(35)SO(4)into proteoglycans.

18 amino acid peptides from the C-terminal region of IGFBP-3, -5 (P3, P5), increased the incorporation of(35)SO(4)into proteoglycans in endothelial cells with greater stimulation in large vessel than microvessel cells. The homologous region of IGFBP-6 (P6) also stimulated sulfate uptake, but less potently than P3 and P5. P6 variants were synthesized with one or two amino acids changed to the basic amino acid in the equivalent position of P3. The P6 variants with one additional basic amino acid behaved similarly to P6. The P6 mutant with two altered amino acids was equipotent to P3. P3F, a scrambled version of P3 was less effective than P3. P3, P5, P6, P3F and all P6 variants all stimulated glucose uptake, which occurred only in microvessel cells. P1, P2, P4, and equimolar intact IGFBP-3 stimulated neither glucose uptake nor sulfate incorporation. Thus, C-terminal basic portions of IGFBP-3, -5 and -6 alter two specific functions of endothelial cells with sufficient differences to suggest mediation by distinct mechanisms.

Mitogenic and antiapoptotic effects of insulin-like growth factor binding protein-6 in the human osteoblastic osteosarcoma cell line Saos-2/B-10.

Insulin-like growth factor (IGF) I is a potent mitogen for human osteosarcoma cells such as the Saos-2/B-10 cell line. IGF binding proteins (IGFBPs) prevent stimulation of DNA synthesis by IGFs. In contrast to recombinant human (rh) IGFBP-2, -3, -4, and -5, 10-100 nM rhIGFBP-6 stimulated [(3)H]thymidine incorporation into DNA and multiplication of Saos-2/B-10 cells. Upon withdrawal of serum, 30 nM IGFBP-6 also decreased apoptosis (within 4 h) and increased protein content and sodium-dependent phosphate uptake (within 24 h), but less potently than IGF I. (125)I-labeled rhIGFBP-6 did not bind to the cells, and cold IGFBP-6 did not affect (125)I-labeled IGF I binding. Production of IGF I, IGF II, and IGFBP-6 by the cells or significant degradation of rhIGFBP-6 could not be detected within 24 h of incubation. Thus, among the rhIGFBPs tested, rhIGFBP-6 is unique in stimulating osteosarcoma cell growth. Furthermore, it has an antiapoptotic effect.

Insulin-like growth factor-binding protein-6 produced by human PC-3 prostate cancer cells: isolation, characterization and its biological action.

The PC-3 human prostatic carcinoma cell line has been extensively used as a model for studies on the regulation of prostate tumor cell proliferation. Because of the importance of IGF-binding proteins (IGFBPs) in the control of IGF activities that regulate cell proliferation in normal and malignant cell types, we undertook studies to characterize the IGFBPs produced by PC-3 prostate tumor cells in culture. We previously found, using an IGF-I affinity column for purification and a polyethylene glycol (PEG) precipitation assay for IGFBP detection, that PC-3 cells in culture produced a single predominant IGFBP, IGFBP-4, which inhibits IGF activities. We now present evidence that PC-3 cells also produce IGFBP-6 in abundant quantity; in the previous study this was apparently not detected in the IGF-I-bound fraction with the PEG precipitation and Western ligand blot assays. In the current study, IGF-II affinity purification of IGFBPs produced by PC-3 cells, followed by C8 HPLC reverse-phase chromatography using a shallow acetonitrile gradient, produced two major protein peaks. N-terminal amino acid sequence of peak 1 was identical to that of IGFBP-6 while that of peak 2 was identical to that of IGFBP-4. Characterization of purified IGFBP-6 from PC-3 cells revealed properties which are distinct from other IGFBPs. PEG did not precipitate the complex of 125I-IGF-II/IGFBP-6 while it precipitated the complexes between 125I-IGF-II and other IGFBPs. Indeed, IGFBP-6 decreased the amount of 125I-IGF-II tracer in the PEG precipitate in a dose-dependent manner. Also, the binding of IGFBP-6 with 125I-IGF-II was poor in Western ligand blots compared with other IGFBPs. In studies on IGFBP-6 actions, IGFBP-6 completely inhibited IGF-II-induced [3H]thymidine incorporation in MC3T3-E1 mouse osteoblast cells while it had only minimal inhibitory effects on IGF-I-induced [3H]thymidine incorporation. This differential effect is associated with the fact that IGFBP-6 has greater affinity for IGF-II than IGF-I. The results of this study indicated that (1) Western ligand blotting is not sensitive for identification of IGFBP-6, (2) the unique behavior of IGFBP-6 in the PEG assay system necessitates the use of charcoal adsorption procedure for IGFBP-6 activity detection and (3) PC-3 cells should provide a useful model system for studying regulation of IGFBP-6 expression and the role of IGFBP-6 in modulating IGF actions.

Roles of insulin-like growth factor (IGF) receptors and IGF-binding proteins in IGF-II-induced proliferation and differentiation of L6A1 rat myoblasts.

Insulin-like growth factor II (IGF-II) stimulates the proliferation and differentiation of rat myoblasts. Previous studies suggest that these response are mediated by the IGF-I receptor, but the IGF-II/mannose 6-phosphate receptor was recently implicated in differentiation of mouse myoblasts. L6A1 myoblasts synthesize IGF-binding protein-4 (IGFBP-4), IGFBP-5, and IGFBP-6, which modulate IGF action. We studied the roles of IGF receptors and IGFBPs in L6A1 myoblast proliferation and differentiation by comparing the effects of IGF-II and a number of IGF-II mutants with decreased affinities for IGF receptors and/or IGFBPs. IGF-II induced concentration-dependent proliferation with a maximum increase of 47%; half-maximal proliferation was seen with approximately 50 ng/ml. [Arg54, Arg55]IGF-II bound to the IGF-I receptor with slightly lower affinity than IGF-II, did not bind to the IGF-II/mannose 6-phosphate receptor, and bound to IGFBPs secreted by myoblasts with approximately 16-fold decreased affinity. It induced proliferation with equal potency to IGF-II. [Leu27]IGF-II, which did not bind to the IGF-I receptor but bound to the IGF-II/mannose 6-phosphate receptor and IGFBPs with slightly lower affinity than IGF-II, had a markedly impaired proliferative effect, inducing proliferation only at high concentrations. [Thr48, Ser49, Ile50]IGF-II, which bound to the IGF-I receptor with slightly lower affinity than IGF-II but did not substantially bind to the IGF-II/mannose 6-phosphate receptor or IGFBPs, induced proliferation with approximately 5-fold greater potency than IGF-II. The order of potency in inducing myoblast differentiation was the same, although there was less difference in the relative potencies of IGF-II and mutants. Coincubation of recombinant human (rh) IGFBP-6 in molar excess with IGF-II inhibited myoblast proliferation and differentiation. rhIGFBP-6 was slightly less potent did not inhibit proliferation or proliferation or differentiation induced by [Thr48,Ser49,Ile50]IGF-II. These results suggest that 1) IGF-II-induced proliferation and differentiation of L6A1 myoblasts are predominantly mediated by the IGF-I receptor; 2) the IGF-II/mannose 6-phosphate receptor is not required for these actions of IGF-II; 3) nevertheless, the IGF-II/mannose 6-phosphate receptor may be capable of mediating these actions; and 4) IGFBPs secreted by myoblasts inhibit IGF actions.