Study On The Antitumor Effect Of The Activated PPARΓ And Retinoid Receptors On Human Osteosarcoma

AimOsteosarcoma (OS) is the most common non-hematological malignant tumor of bone in children and adults. Without systemic treatment, few patients with osteosarcoma achieve long term disease free status, even the optimal local treatment is taken. Clinical management of OS faces numerous challenges, including adverse effects associated with chemotherapies, chemoresistance, recurrence, and pulmonary metastasis. In this project, we focused on trying differentiation therapy to OS with activating PPARγ, RARαand RXRα.Methods and procedureWe used cell proliferation test, PCR, Western blot, recombinant adevovirus, reporter construct and histochemistry technique to elucidate the synergistic anti-proliferation effect on human osteosarcoma when PPARγand retinoids receptors were activated.1. We checked the mRNA and protein expression for PPARγand different subtype receptors of RAR and RXR in primary human osteosarcoma and osteosarcoma cell lines, in order to make sure these nuclear receptors were available in osteosarcoma cells.2. We tested the anti-proliferation effects of Tro, ATRA, 9CRA and Tro combinated with ATRA or 9CRA on human osteosarcoma.3. We constructed the recombinant RARα, RXRαadenovirus with AdEasy system and tested the function of the recombinant adenovirus. We tested the anti-proliferation effects of exogenous overexpress nuclear receptors from the recombinant adenovirus or exogenous overexpress of the nuclear receptors combinated with corresponding nuclear receptor agonists on human osteosarcoma.4. We infected the osteosarcoma cells with recombinant adenovirus and then injected the cells to proximal tibia for ectopic tumorigencity test in nude mice. Track the tumor with Xenogen system, sacrifice the animals at the end of test, and retrieve the tumor mass for proliferation, apoptosis and differentiation test with histochemistry technique.5. Test the effects of Tro, ATRA and 9CRA on osteogenic differentiation in Mesenchymal stem cell and osteosarcoma cell lines, the alkaline phosphatase expression in primary osteosarcoma and osteosarcoma cell lines, and the tumorigenicity of primary osteosarcoma.Results1. The expressions of PPARγand different type of RAR and RXR were detectalbe in all tested cells.2. RARαand RXRαrecombinant adenovirus were sucessfully constructed, and the receptors expressed by which could be activated by the corresponding agonists and regulated the target gene expressions.3. Tro, ATRA and 9CRA, as well as exogenous overexpress PPARγ, RARαand RXRαcombinated with corresponding agonists, could inhibited the proliferation of osteosarcoma.4. There was a synergistic anti-proliferation effect in combination of Tro with ATRA or 9CRA; similar results were observed if there existed co-overexpressions of PPARγ2 with RARαor RXRαin vivo and in vitro test. Activation of the nuclear receptors induced the apoptosis and differentiation in osteosarcoma cells.5. Tro,ATRA and 9CRA induced the osteogenic differentiation of Mesenchymal stem cell and there was a apparently synergistic effect in combination Tro with ATRA or 9CRA, although Tro had very little effect on osteogenic differentiation. Similar results were found in osteosarcoma.ConclusionsActivated PPARγ, RXRαand RARαcan inhibit the growth of osteosarcoma and there exist synergistic anti-proliferation effects on osteosarcoma when co-activations of PPARγ2 with RXRαor RARαoccur. This effect may partly result from the osteogenic differentiation induced by the nuclear receptor agonists.