Growth Inhibitory Effects And Mechanism Of ATRA Combined With IFN-β And GRIM-19and STAT3on Human Osteosarcoma Cell

BackgroudOsteosarcoma is the most common primary malignant tumor of bone, encompassing a class of osteoid-producing neoplasms that range in clinical behavior and responsiveness to therapeutic regimens. Primary bone sarcomas are uncommon malignant tumors representing2-3%of all new cases of malignancy, and1.6%of all cancer deaths in Chinese, respectively. Further improvement in survival of osteosarcoma patients needs to find well-validated prognostic markers and better therapeutic agents, which leads us to explore in greater depth the molecular and genetic events involved in osteosarcoma progression.Interferons inhibit cell growth by stimulating the synthesis of growth inhibitors or activating apoptosis. Although IFNs themselves strongly inhibit cell growth, many tumor cells are insensitive to IFN-induced growth arrest. In many tumor cells moderately sensitive to IFNs, pretreatment with all-trans retinoic acid (RA), a vitamin-A metabolite, enhances sensitivity to IFN-induced growth inhibition. However, the mechanisms that regulate IFN/RA-induced growth inhibition are poorly defined.Signal transducer and activator of transcription3also known as STAT3is a transcription factor which in humans is encoded by the STAT3gene. In benign cells, the STAT3signaling is under tight regulation so that the signal is transient. However, aberrant signaling by STAT3was frequently found in many human malignancies. Aberrant STAT3expression has been implicated as a key participant in tumor survival, proliferation, angiogenesis and metastasis. Aberrantly active STAT3promotes uncontrolled growth and survival through dysregulation of expression of downstream targeted genes including:And. Some researchers have reported that STAT3down-regulation could inhibit proliferation and induce apoptosis in many human tumor cells using small interfering RNA (siRNA) or decoy oligodeoxynucleotide methods. Thus, STAT3has the potential of becoming a therapeutic target for the treatment of human cancers.The GRIM-19gene codes for16kDa protein with no apparent sequence motif or domain identities with other proteins in the public databases. The GRIM-19gene maps to a locus on human chromosome19p13.2. The GRIM-19mRNAis induced by IFN-α/-β but not by RA alone. However, the combination of IFN/RA robustly induced gene expression. Ectopic expression of GRIM-19induces cell death and moderate levels of it sensitizes cells to IFN-β/RA-induced apoptosis. GRIM-19isolated as a cell death activator in a genetic screen used to define mechanisms involved in IFN-β and retinoic acid-induced cell death. Antisense ablation of GRIM-19caused resistance to cell death induced by IFN plus retinoic acid and conferred a growth advantage to cells. GRIM-19inhibits transcription driven by activation of STAT3, but not STAT1. It neither inhibits the ligand-induced activation of STAT3nor blocks its ability to bind to DNA. Mutational analysis indicates that the transactivation domain of STAT3, especially residue S727, is required for GRIM-19binding. Because constitutively active STAT3up-regulates antiapoptotic genes to promote tumor survival, its inhibition by GRIM-19also demonstrates an antioncogenic effect exerted by biological therapeutics.ObjectiveTo research growth inhibition of human osteosarcoma cell(MG-63)intervened by ATRA combined with IFN-P,and to research gene GRIM-19and proto-oncogene STAT3.and to study the relation between GRIM-19and STAT3,and to explore the possible site for osteosarcoma target therapy. Materials and methodsMTT colorimetric methods research growth inhibition of MG-63intervened by different concentration of ATRA/IFN-β; flow cytometry AnnexinV-FITC/PI methods research the condition of apoptosis; RT-PCR methods study amplification of gene GRIM-19and STAT3induced by ATRA combined with IFN-β or singly; Western blot methods study the expression of GRIM-19and STAT3of MG-63.ResultsATRA/IFN-β can inhibit the growth of MG-63;and this is associated with concentration and time.It is significant that both compare with other groups (P＜0.05).Either ATRA or IFN-β can induce apoptosis of MG-63,but both can greatly increase it.The expression of GRIM-19mRNA is increased significantly(1.62±0.095)(P＜0.01).While,the expression of STAT3mRNA decreased(0.12±0.032)(P<0.01).There is significant statistic difference that GRIM-19protein increases(l.85±0.060)(P＜0.01) and STAT3protein decreases(0.54±0.075)(P＜0.01)ConclusionATRA or IFN-β can inhibit proliferation of MG-63,and the effect of inhibition increases significantly used both. The mechanism may be that ATRA/IFN-β induce the high expression of GRIM-19,and the transactivation domain (TAD) of STAT3appears to be a direct target of GRIM-19,decr easing the expression of proto-oncogene STAT3. Because STAT3is activated constitutively by oncogenes and autocrine growth factors, GRIM-19may prove to be a novel antioncogenic protein.