Experimental Study Of Effects Of Insulin-like Growth Factors On Human Osteosarcoma

BackgroundIt is known to all that most malignant tumors of human usually result from the mutation process of multi-genes, so the risk of tumor gets higher along with the increase of age. However, there exist some malignant tumors such as Osteosarcoma have their specific peak age range, which is always inconsistent with the theory of gene mutation. Recently, accumulated evidence shows these tumors which have a younger trend are often associated with the autocrine and paracrine mechanism of many kinds of growth factors.Osteosarcoma is the most common primary malignant tumor of bones, which has a peak incidence in adolescence and usually occurs at the metaphysic of long bones with a rapid bone turnover Surgery combined with multiagent chemotherapy has resulted in significantly improved survival of patients with osteosarcoma. However, improvements in survival have reached a plateau during the past twenty years. Thus, novel agents the act via different mechanisms are need in order to achieve further improvement in the survival rate in clinical. It has been found recently the development of many tumors depend on the autocrine and paracrine mechanism of growth factors and their receptors. During the growth of Osteosarcoma, many growth factors are usually over-expressed. The heights of the adolescences with osteosarcoma are usually higher than the ones of the same age.During the adolescence, there is not only a sexual hormone peak, but also a growth hormone climax. However, the growth hormone does not have a direct effect on the development of the musculoskeletal system, but through some factors which are called The Insulin like Growth Factors, including IGF-1and IGF-2. Thus, scientists assume whether the IGFs play a certain role in the bone malignant tumors? Along with IGF was discovered, some other factors related have been found gradually, which is ultimately forming a growth factor family-----IGF family.Until now, the members indentified in the IGF family include:two receptors (IGF-1R and IGF-2R), two ligands(IGF-1and IGF-2), six binding proteins(IGFBP1-6) and four relate polypeptides(IGFRP1-4).IGF-Ⅰ is a70amino acid peptide and its gene is located on chromosome12. IGF-Ⅱ is a67amino acid peptide and its gene is located on chromosome11close to H19, a paternally imprinted gene. Although IGFs in the circulation is mainly produced by the liver, they can be synthesized by almost tissue in the body, exerting biological effects on most cell types. The ubiquity of sites of production and action has led to the concept that these peptides act by autocrine and paracrine as well as by endocrine mechanisms.IGFs play an important role in the growth, development and homeostasis of the human being, but their half-life are short. Six IGFBPs in the circulation have effectively dragged on the influences of IGFs, binding them and transferring them to the specific targeted tissues. IGFBPs also regulate the bioactivity of the IGF receptors and ligands. The balance between the free IGFs and binding proteins determines the destiny of the cells.Serum levels ofIGF-Ⅰ are age-dependent; they increase slowly from birth to pubertal peak and thereafter decline with age. The serum concentration of IGF-Ⅱ peptide (400-600ng/ml) is higher than that of IGF-Ⅰ (100-200ng/ml) at all ages. It remains stable after puberty and is not regulated by GH [14]. Despite its presence at higher concentration in the circulation, IGF-Ⅱis believed to play a less important role than IGF-Ⅰ in postnatal growth.The effects of IGFs are mediated by their receptors IGF-IR and IGF-ⅡR. IGF-IR is a tetramer consisting of two identical extracellular a-subunits (conferring ligand binding specificity) and two identical trans-membrane β-subunits (possessing tyrosine kinase activity). IGF-IR resembles the IR, with which it shares60%homology. It is a protein tyrosine kinase receptor that is involved in the proliferation and differentiation of normal cells during development, with little metabolism impact. Mouse embryos lacking IGF-IR have lung, neurologic, skin and bone defects and are variably viable after birth. IGF-IR is also implicated in malignant progression and establishment of the transformed phenotype for a variety of cancer cells. Knock-out mouse embryo cells deficient for this receptor are refractory to transformation induced by viruses, oncogenes and other over-expressed GF receptors. In contrast to other tyrosine kinase receptors, such as EGF receptor (EGF-R) family members, IGF-IR requires ligand binding to trigger the appropriate downstream pathways. IGF-IR over-expression alone is insufficient to cause receptor activation. IGF-IIR, also called the IGF-Ⅱ/mannose-6phosphate (M6P) receptor, is a monomer that has no intrinsic tyrosine kinase activity. It does not bind insulin but binds IGF-Ⅱ, whose major function is degradation of IGF-Ⅱ. Loss of IGF-ⅡR is also correlated with increased IGF-Ⅱinitiated IGF-IR activation and increased proliferation, which has been found in many tumors. It has been commonly known that IGF-ⅡR is a tumor-suppression factor that plays an important role in the development of tumors.The study about the IGF family in the osteosarcoma was started twenty years ago. Due to the limitation of the understanding and the technology at that time, the research is not deep enough, without any meaningful conclusion. Recently, however, along with the certain effects of IGF-IR targeted therapy which had been show in the Ewing’s sarcoma, scientists have renewed their interests of the IGF research.There has been much research about the IGF family in the osteosarcoma, so far. Studies show that IGF-1is a potent mitogen for human osteosarcoma, inhibiting the GH-IGF-1-axis can effectively inhibit the growth of osteosarcoma. IGF-1and IGF-2are the richest growth factors in the bone, especially IGF-2, which can be produced by osteoblast cells in vitro and can induce the osteoblast cells to proliferate and differentiate.Sofia et al. reported that the serum levels of IGF-1and IGFBP-3in osteosarcoma patients are lower than that in the normal control, while concentration of IGF-2is increased notably, which is related with a bad outcome. After the tumor was removed, the concentration level of IGF-1and IGF-2returned to the normal. Then they inhibited the IGF-2level in the cell-culture medium, the growth of osteosarcoma MG-63was notably inhibited. In most tissues of the body, IGF-2was in a congenital hibernating state, but was activated in the tumor.The result that mRNA of IGF-2was highly expressed in the cells and tissues of osteosarcoma show IGF-2is also activated in the osteosarcoma. These evidence points out that IGF-2may play a more important role in OS cells.Wang et al.found in the normal bone tissue, IGF-1R was not expressed, but over-expressed in the osteosarcoma tissue. Furthermore, the over-expression was closely related with the metastasis and the outcome, lowering the expression can inhibit the growth of OS, promote apoptosis and enhance the sensitivity of radical therapy and chemotherapy. The OS cell line HOS58was cultured in vitro and the proliferation potency was related with IGF-1R, decreasing with IGF-1R lowering down. OS cell, which was derived from the transformed pro-osteo cells, also secretes IGF-1. The autocrine cycle IGF-1/IGF-1R was related with the growth of OS. However, the IGF-1R targeted therapy shows limited effects in the osteosarcoma therapy.So far, the research about the IGF family in osteosarcoma was much, but the results were not completely uniform. Our research intends to systemically study the effects of the IGF family in osteosarcoma development. The members of the IGF family were detected in the osteosarcoma tissues and the relation between the expression of these factors and the outcome of OS patients was analyzed in order to manifest the IGF influence on osteosarcoma. The research is not only beneficial for us to know the mechanism of osteosarcoma, but also offers a new train of thought and theory basis.Objective1. Ascertain the main IGF legend in the osteosarcoma;2. Study the relationship between the members of the IGF family and tumor type, stage, outcome;3. Detect the expression of IGF members on the surface of common osteosarcoma cells;4. Study and testify the IGF members influence on osteosarcoma.Method1. Detect the expression of IGF-1,IGF-2,IGF-1R and IGF-2R in the osteosarcoma tissues by immunohistochemistry;2. Analyze the relationship between these members and clinic pathologic characteristics and treatment outcomes by the statistical method;3. Culture the common osteosarcoma cell lines MG-63and Sao-2and detect the expression of IGF receptors;4. Study the effects of a-IR3on osteosarcoma cells by con-culture to offer a theory basis for osteosarcoma targeted therapy.Results 1. the IGF family indeed plays an important role in the microenvironment of osteosarcomaThe four members of the IGF family has been detected in most OS tissues, respectively IGF-Ⅰ (46/49) and IGF-Ⅱ (44/49), IGF-IR(47/49) and IGF-IIR(44/49). They were mainly detected in the cytoplasm of tumor cells, few on the cytomembranes. However, following the new score system,26/49OS tissue was IGF-Ⅰ strong positive but only2samples was IGF-Ⅱ strong positive, suggesting IGF-Ⅰ may play a greater role than IGF-Ⅱ in the local osteosarcoma tissues.2. The level of IGF-IR protein expression was associated with the malignance degree of osteosarcomaBecause both the IGF-Ⅰ and IGF-Ⅱ take actions through the same receptor---IGF-IR, the association of IGF-IR staining with clinicaopathological factors of osteosarcoma patients has been analyzed. Based on the pathological types and the imaging data, the specimens have been divided into the low malignance group and the high malignance group. The level of IGF-IR protein expression was closely associated with the malignance degree of osteosarcoma (P=0.012). However, there was no correlation between IGF-IR expression and other clinic pathological factors of patients including gender, age, anatomic location, surgical stage (P=0.159,0.145,0.903,0.28respectively). Higher expression of IGF-IR protein was more frequently found in high grade osteosarcoma patients.3. The level of IGF-IR protein expression was closely associated with the survival of osteosarcoma patientsThe samples were divided into two expression groups as described previously. An intensity score of≥8was used to classify tumor patients with high-IGF-IR expression group (n=27), and<8intensity score was used to classify tumor patients with low-IGF-IR expression group (n=22).To determine the effect of the status of IGF-IR protein expression on survival of osteosarcoma patients, Kaplan-Meier analysis was performed. From the log-rank test, we showed that the over survival time was significantly different between high IGF-IR expression group and low IGF-IR expression group (P=0.032). Patients with low IGF-IR expression had better survival than those with high IGF-IR expression.4. Stronger positive reaction for IGF-IR was detected in MG-63cellsBoth the MG-63OS cells and Saos-2OS cells show the IGF-IR positive IGF-IR also mainly expressed in the cytoplasm of the OS cells. The positive reaction for IGF-IR was distinctly different between MG-63cells and Saos-2OS cells. Compared with Saos-2cell, a stronger positive reaction for IGF-IR was detected in MG-63cells. In contrast, IGF-IIR was few detected in the two kinds of OS cells.5. A certain concentration should be reached to show a-IR3effects of inhibiting on MG-63cells and the effects were enhanced with the prolongation of treatment time.To further study the effects of IGF-IR on the MG-63, a well known anti-IGF-IR molecular antibody a-IR3was used. CCK-8assay was used to investigate the proliferation rates of the MG-63cells treated with various concentrations of a-IR3for different periods of time. In my experiment,4different concentrations of a-IR3was added (0.1ug/ml,0.5ug/ml,1ug/ml,2ug/ml) at four time points. Unexpectedly, having being treated with a-IR3at final concentration of0.1,0.5,1ug/mL the inhabitation effects were not manifest. However, treated with a-IR3at final concentration of2ug/ml, the effects were obvious. GI of the different time points groups was99.51%,67.13%,58.03%and55.01%, respectively (P<0.05). These results indicate that a certain concentration should be reached to show a-IR3effects of inhibiting on MG-63cells and the effects were enhanced with the prolongation of treatment time.ConclusionOur data confirm that the autocrine and paracrine mechanism of the IGF family indeed exist in the microenvironment of osteosarcoma. IGF-Ⅰ may be the main ligand in the local osteosarcoma tissues. The IGF-IR expression on OS cells is correlated with development of osteosarcoma and the patient outcome. Thus, targeting IGF-IR will be a potential therapeutically strategy for osteosarcoma. It is likely to become a potential therapeutic target either alone, or in combination with other targeted molecular or chemotherapeutic agents.