Insulin-like growth factor (IGF) merupakan factor pertumbuhan berupa polipeptida yang memiliki homologi fungsional dengan insulin.1 IGF has a wide range of metabolic and developmental functions, including embryogenesis and postnatal organogenesis.2
THE IGF PATHWAY
The IGF system involves complex regulatory network comprising IGF-1 and IGF-2 ligands, six specific high-affinity binding proteins (IGFBP-1 to IGFBP-6) and IGFBP proteases (IGFBP-prs), and IGF-1 and IGF-2 cell surface receptors (IGF-1R and IGF-2R).3 The half-life and bioavailability of IGF-1 and IGF-2 in circulation varies depending on the affinity and specificity of the IGFBPs in the serum.4 IGFBP-3, the most critical of the binding protein, binds to 70% to 80% of the IGF-1.5 IGFBP-3 seems to have a non-IGF mediated anti-proliferative and pro-apoptotic action resulting from its association with cell surface proteins or receptors.10
Most of the actions of IGF-1 and IGF-2 are mediated by high-affinity ligand binding to IGF-1R, although recent evidence suggests that actions of IGF-2 are also mediated through the high-affinity binding with an insulin receptor (IR) isoform-insulin receptor exon 11 isoform (IR-A).11 Upon binding of the IGFs to IGF-1R, the receptor's intrinsic tyrosine kinase is activated, resulting in the phosphorylation of the insulin receptor substrates (IRSs).11-13 The tyrosine-phosphorylated IRS activates phosphatidylinositol-3kinase (PI3K), which catalyzes the conversion of phosphatidylinositol biphosphate (PIP2) to phosphatidylinositol triphosphate (PIP3). V-Akt murine thymoma viral oncogene homolog (Akt) is activated by PIP3. Activated Akt results in a cascade of phosphorylation events in the cytosol, resulting in inactivation of key proteins (Bcl-2 antagonist of cell death, caspase 9, and forkhead transcription factor family) involved in apoptosis.14 The activation of IGF-1R also modulates the voltage-gated calcium channels, causing transient increase in the intracellular Ca2+ level and thereby regulating the nuclear transcription factor, cAMP response element-binding protein (CREB).
THE insulin-like growth factors were discovered on the basis of their ability to stimulate cartilage sulfation and to replace the "sulfation factor activity" of GH, as determined using an in vivo assay, in an in vitro test system (1). The biological significance of this finding was quickly expanded beyond the study of cartilage sulfation to include stimulation of DNA synthesis (2), proteoglycan synthesis (3), glycosaminoglycan synthesis (4), and protein synthesis (5). Most of these studies used tissue preparations such as isolated diaphragm, cartilage, or epididymal fat pads to study biologicalactivity. In recognition of its generalized pleiotypic actions, in the early 1970's sulfation factor was renamed somatomedin (mediator of the effects of somatotropin) and was included in the emerging classification of broad spectrum growth factors along with platelet derived growth factor, fibroblast growth factor, and epidermal growth factor (6). During the period in which the biological actions of sulfat
The role of IGFBP-3 as a growth-inhibitory protein has been previously demonstrated by us and others in various cell types (9,12-17). Initially, IGFBP-3 was thought to inhibit growth by binding to IGFs and sequestering them from their receptor. Later, the cell growth-inhibitory effect of IGFBP-3 was suggested to also be IGF-independent and to involve cell growth arrest (14, 15). Recently, this inhibitory effect of IGFBP-3 was suggested to be mediated by interaction with a putative IGFBP-3 receptor. Although the IGF-independent growth-inhibitory role of IGFBP-3 has been recently investigated, an apoptosis-inducing role for IGFBP-3 has not been previously determined. This is the first demonstration of IGFBP-3 as a cell death-promoting agent.
Partial blocking of IGFBP-3-induced apoptosis by IGF suggests two possibilities. First, the presence of IGF may prevent the cells from undergoing apoptotic changes through the IGF receptor-mediated cell survival pathway. Second, some of the IGFBP-3 would not be available to induce apoptosis through its own receptors, since it formed IGF•IGFBP-3 complexes. Furthermore, the inability of IGF to fully block IGFBP-3-induced apoptosis even at a 5-fold higher molar concentrations supports the notion that the pathway of IGFBP-3-induced apoptosis may not always involve IGF and IGF receptor. In addition, since IGF analogues that do not bind IGFBP-3 did not reverse the IGFBP-3 effect at all, this further suggests that IGFBP-3 induces apoptosis via an IGF-independent pathway through an IGFBP-3 receptor. This IGFBP-3 cell surface receptor has been first proposed in Hs578T breast cancer cells by affinity cross-linking of125I-IGFBP-3 to cell membrane and cell lysate extracts (18). In this study, we have shown that PC-3 cells also bind IGFBP-3 and that several potential IGFBP-3 receptors exists in PC-3 cells.
IGFs have been shown to protect cells from undergoing apoptosis through an IGF receptor-mediated cell survival pathway (35-38). Both the effects of decreases in the number of IGF receptors causing massive apoptosis and the overexpression of IGF receptors protecting cells from apoptosis have been demonstrated in vivo (35). The roles of IGFs and the IGF receptors as autocrine survival factors (36) and as protective agents that prevent apoptosis induced by other agents such as etoposide have been shown extensively (37). Mutant versions of p53 protein, commonly associated with malignant states, have been shown to derepress the IGF receptor promoter, with ensuing mitogenic activation by locally produced or circulating IGFs (38). All of the above mentioned studies indicate the important role of IGFs and IGF receptors in preventing cells from undergoing apoptosis through a cell survival pathway. We demonstrated here an alternate pathway for the induction of apoptosis that is independent of these apoptosis-protecting agents. By demonstrating IGFBP-3-induced apoptosis in the IGF receptor-negative (R(−)) murine fibroblast cell line, we proved our hypothesis that IGFBP-3 may induce apoptosis independently of the IGF receptor-mediated survival pathway. Therefore, the ratio of free IGFs and IGFBP-3 will regulate cell growth not only by balancing the rate of cell proliferation and cell growth arrest, but also by regulating the rate at which the cells might be induced to undergo apoptosis.
The apoptosis-inducing effect of IGFBP-3 in R(−) cells provides ample evidence to suggest that similar IGF receptor-independent pathways are present in PC-3 cells and possibly in other cell lines. Treatment with IGF-I partially decreased the incidence of apoptosis in IGFBP-3-overexpressing cells but did not have any effect on R(−) cells, suggesting that the partial suppression of apoptosis by IGF is through the formation of IGF•IGFBP-3 complexes. Similar to the results found in PC-3 cells, IGFBP-3-neutralizing antibodies partially decreased the degree of apoptosis in IGFBP-3-overexpressing R(−) cells. In PC-3 cells, IGF-I partially blocked IGFBP-3-induced apoptosis, but the IGF analogue, which binds to the IGF receptor and not to IGFBP-3, was unable to block IGFBP-3-induced apoptosis. These observations not only suggest the involvement of an IGF-independent pathway, but they also demonstrate that IGFBP-3 must be free of IGF to be able to bind to its receptor and initiate its effect on cell death and that the activation of the IGF receptor does not protect cells from IGFBP-3-induced apoptosis.
The expression of the cell growth-inhibitory IGFBP-3 has been shown to be induced by various apoptosis-inducing agents, such as TGF-β1 (20-22), retinoids (21), TNF-α (23), and the tumor suppressor gene p53 (24). IGFBP-3 has been previously shown to mediate the growth-inhibitory effect of both retinoic acid and TGF-β1 (20, 21). However, the mechanism by which the IGFBP-3 reduces the cell number, under these conditions, is not known. In this work, we have demonstrated that IGFBP-3 mediates the growth-inhibitory effect of TGF-β1 by inducing apoptosis. This may apply to other agents that have not yet been investigated.
The PC-3 cells are p53-negative (39) and have the machinery to express low levels of IGFBP-3 (8) under serum-free conditions. TGF-β1 is a potent growth inhibitor of epithelial cells and has been shown to induce apoptosis and down-regulate Bcl-2 expression (40, 41). The dramatic elevation of the 44-kDa IGFBP-3 protein within 12 h of TGF-β1 treatment and the significant effect of TGF-β1 on apoptosis that was observed about 18–24 h after treatment suggest that the TGF-β1-induced elevation of IGFBP-3 protein in the conditioned media is the primary signal that activated apoptosis in this cell line. Blocking TGF-β1-induced apoptosis at the IGFBP-3 transcriptional level confirmed the role of IGFBP-3 as the mediator of TGF-β1-induced apoptosis in PC-3 cells. Co-treatment with IGFBP-3 antisense (but not sense) thiolated oligonucleotide and TGF-β1 verified the role of IGFBP-3 in the TGF-β1-induced apoptosis. Furthermore, neutralization of IGFBP-3 action in TGF-β1-treated cells with IGFBP-3-neutralizing antibodies (but not control IgG) confirmed that IGFBP-3 must be secreted and allowed to bind to its receptor to initiate apoptosis. The latter observation also confirms that the TGF-β1-mediated increase in IGFBP-3 transcription must pass through steps such as IGFBP-3 secretion and the binding of this protein to its receptor to initiate apoptosis.
Inappropriate expression of genes involved in cell proliferation has been shown to alter regulation of apoptosis. Both Bcl-2, which promotes cell survival, and Bax, which promotes cell death, have been implicated as major mediators in the control of apoptotic pathways, and it has been suggested that the ratio of Bcl-2 to Bax controls the relative susceptibility of cells to death stimuli. TGF-β1, retinoic acid, TNF-α, and p53 are known to induce apoptosis by regulating Bcl-2 and Bax expression (40-47). Since all of these apoptosis-inducing agents also induce IGFBP-3 expression, we anticipate that IGFBP-3-induced apoptosis may also involve regulation of the Bcl-2:Bax ratio. In addition, the expression of ICE or ICE-like proteases that are final mediators of the apoptosis pathway is involved in the mechanism of action of IGFBP-3 as well as the above agents.
The role of IGFBP-3 in mediating p53 effects was proposed when p53 was demonstrated to activate the IGFBP-3 promotor (24). Recently, it has been shown that mutants of p53 that have lost the ability to activate IGFBP-3 and Bax expression but maintained their activation of the cyclin-dependent kinase inhibitor p21 are able to induce cell cycle arrest but are unable to induce apoptosis (48). Furthermore, a p53 mutant that activates Bax expression but only partially activates the IGFBP-3 promotor is only partially effective in inducing apoptosis (49). Thus, a p53-dependent role of IGFBP-3 has been previously demonstrated. By demonstrating IGFBP-3-induced apoptosis in PC-3 cells that lack the p53 gene, we have demonstrated that IGFBP-3 can also induce apoptosis in a p53-independent fashion.
We present a hypothesis based on the results from this study and other previous reports from this and other groups in the diagrammatic representation shown in Fig. 9. We propose that the independent and interdependent effects of IGFs and IGFBPs on the regulation of cell number involve two pathways that interact at several levels. IGFs mediate survival via the IGF receptor. IGFBP-3 is able to block this pathway by sequestering IGFs away from the IGF receptor. IGFBP-3 mediates apoptosis via its own receptors, while IGFs can prevent this effect by binding to IGFBP-3. Thus, IGFBP-3 can mediate cell death by both IGF-dependent and IGF-independent pathways.
Diagrammatic representation of IGF- and IGFBP-3-mediated regulation of cell growth and apoptosis. Details are given under "Discussion."
Both normal cell growth (50) and various pathologies associated with neoplastic cell proliferation, such as breast cancer (20, 26, 51, 52), prostate cancer, and benign prostatic hyperplasia (11), are also associated with altered expression of IGFs and IGFBPs. Earlier observations, however, did not directly demonstrate a role for IGFBP-3 in inducing apoptosis but provided ample evidence to suggest that IGFBP-3 is important in regulating cell number in such situations. Our data demonstrate that IGFBP-3 induces apoptosis at physiological concentrations and that IGFBP-3 may act through an IGF•IGF receptor-independent pathway. IGFBP-3 mediates the induction of apoptosis by TGF-β1 and may mediate similar actions of other growth-regulatory factors.
The Insulin-like growth factor binding protein also known as IGFBP serves as a carrier protein for Insulin-like growth factor 1 (IGF-1).
Approximately 98% of IGF-1 is always bound to one of 6 binding proteins (IGF-BP). IGFBP-3, the most abundant protein, accounts for 80% of all IGF binding. IGF-1 binds to IGFBP-3 in a 1:1 molar ratio. IGF-BP also binds to IGF-1 inside the liver, allowing growth hormone to continuously act upon the liver to produce more IGF-1. This is important because proliferating IGF-1 + IGF-BP complex allow growth of the femur and the muscle.
IGF binding proteins (IGFBPs) are 24 to 45 kDa proteins. All six IGFBPs share 50% homology with each other and have binding affinities for IGF-I and IGF-II at the same order of magnitude as the ligands have for the IGF-IR.
The IGFBPs help to lengthen the half-life of circulating IGFs in all tissues, including the prostate. Individual IGFBPs may act to enhance or attenuate IGF signaling depending on their physiological context (i.e. cell type).
Penjelasan penelitian quercetin-IGF-1:
Pengukuran IGF-1 dan IGFBP 3 menggunakan metode immunoradiometric assay (IRMA). Sensitivitas dan spesifisitas IRMA sama dengan ELISA. Namun, IRMA tidak terpengaruh factor-faktor stabilisator seperti ELISA (rcw3).
Statistic menggunakan metode ANOVA menunjukkan perbedaan yang signifikan (p<0.05). Pengukuran dilakukan di media sel PC-3 sehingga mekanisme neuroimunohumoral kurang berpengaruh.
Hasilnya menunjukkan bahwa quercetin menginduksi sekresi IGFBP-3 dan IGFBP-3 mengurangi jumlah ligan (IGF-1) yang tersedia untuk bereaksi dengan IGF-1R. IGFBP-3 sendiri juga dapat menginduksi apoptosis pada sel PC-3, serta mempotensiasi efek apoptotic factor lainnya seperti radiasi ionisasi dan agen kemoterapi.
Oleh karena itu, penelitian ini menunjukkan bahwa quercetin dapat menghambat pertumbuhan sel kanker prostate in vitro dan bersifat pro-apoptotik. Quercetin menginduksi apoptosis melalui down regulation IGF-1 dan up regulation IGFBP-3 pada sel PC-3.
IGFBP1 terutama terdapat di hepar. IGFBP2 terdeteksi dengan kuat di otak, hepar, otot, ginjal, usus, lambung, dan ekspresi yang lebih lemah terdapat di kulit, lien, ginjal (caput), dan ovarium. IGFBP3 hanya terdeteksi di hepar, ginjal, dan kulit. IGFBP4 dan IGFBP5 terdapat di semua jaringan kecuali lien. Ekspresi IGFBP6 terkuat terdapat di lien, otak, dan lambung (2399-1).
hasil penelitian lain:
Sebuah penelitian mengenai pengaruh quercetin terhadap VEGF (Vascular Endothelial Growth Factor) pada kultur sel granulose babi menunjukkan bahwa produksi VEGF oleh sel granulose babi dihambat secara signifikan ( ) oleh quercetin dengan konsentrasi 5 dan 50 g/ml. makin besar dosis quercetin, efek yang timbul makin bermakna ( ) (VEGF).
pemberian Lycopene pada pria dewasa sehat tidak memberikan efek terhadap konsentrasi IGF-1 (p=0.52) dan IGFBP-3 (p=0.55). namun terdapat hubungan antara perubahan konsentrasi lycopene dan perubahan kadar IGFBP-3 pada kelompok perlakuan (p=0.008). In conclusion, lycopene supplementation did not influence serum total IGF-I and IGFBP-3 concentrations in our randomized, placebo-controlled, double-blinded crossover trial in a population at greater risk of colorectal cancer. However, lycopene supplementation may decrease IGF-I bioavailability by increasing IGFBP-1 and -2 concentrations. Thus, it may provide a means of ultimately reducing colorectal cancer risk and potentially the risks of other major cancers such as prostate and premenopausal breast cancer. However, interindividual variation in IGFBP-1 and -2 effects was high, possibly complicated by differences in fasting duration and, consequently, insulin concentrations. Therefore, results must be confirmed in larger randomized intervention studies with control for the duration of fasting. (lycopene).