Experimental Study Of Anti-tumor Effects Of Adenovirus Mediated Antisense C-myc Gene And Caffeine On The Osteosarcoma Cells

Section 1:Construction of antisense c-myc recombinant adenovirus and itsanti-tumor effect on osteosarcoma cell lines MG-63 and U2OS.Objective: The protooncogene c-myc plays an important role in regulating cell proliferation.it is often amplified and overexpressed in osteosarcoma cells,moreover,lt can promote cell transformation and induce tumor metastatic features. We constructed the recombinant adenovirus encoding antisense c-myc gene fragment and investigated its effect on osteosarcoma cells with different P53 gene type. Methods: The 750bp second exon of c-myc was inserted in a reverse direction into adenovirus pshutter-CMV vector.then the vector was cotransformed into the BJ5183 cells with pAdEasy-1. The recombinant adenovirus plasmids were amplified in XL10-Gold and packed in AD-293 cells.and the recombinant adenovirus ( Ad-Asc-myc ) were identified.Two human osteosarcoma cell lines MG-63(P53-deficient), U2OS(P53-wild type) were infected by the Ad-Asc-myc in vitro respectively.The degree of cell proliferation Inhibition was evaluated by cell growth curve.Transcription and expression of c-myc protein,bcl-2,bax,E2F-1gene in osteosarcoma cells were examined by Western blot and RT-PCR.Cell apoptosis and cell cycle change were observed by Acridine Orange staining and Flow Cytometry(FCM). Results: Ad-Asc-myc encoding antisense c-myc fragment was obtained with the titer of 2×10~9pfu/ml. The proliferation of both osteosarcoma cell lines were significantly inhibited by Ad-Asc-myc, while U2OS(P53- wild type) was more susceptible to Ad-Asc-myc induced growth inhibition than MG-63(P53-deficient).The expression of c-myc protein were downregulated in both cell lines 48h after Ad-Asc-myc infection,and expression level of bcl-2 gene were downregulated, expression level of bax gene were upregulated.while there were no change in theexpression of E2F-1. The apoptosis of infected cells was induced.Cell cycle of MG-63 cells arrested in G2/M phase and U2OS cells arrested in G1 phase. Conclusion: Ad-Asc-myc could inhibit proliferation of osteosarcoma cells and induce apoptosis through both P53-dependent and P53-independent pathways.Section 2:Adenovirus mediated antisense C-myc gene on the chemotherapysensitivity of osteosarcoma cells to cisplatin.Objective C-myc is an oncogene and a transcriptional activator implicated in the control of cell proliferation.differentiation and transformation, in osteosarcoma.overexpression of c-myc can promote cell transformation and induce a more aggressive phenotype and metastatic feature, In addition,overexpression of c-myc in some cancers has been reported to induce resistance in response to antineoplastic agents.To construct the recombinant adenovirus encoding antisense c-myc fragment and to investigate its effect on the chemotherapy sensitivity of osteosarcoma MG-63 cells to cisplatin. Methods The recombinant adenovirus (Ad-Asc-myc) encoding antisense c-myc fragment was constructed by cloning c-myc cDNA of about 750 base pairs in a reverse direction into adenovirus vector, then undergoing recombination,amplifying and being complemented in vivo.the osteosarcoma MG-63 cells were transfected by the Ad-Asc-myc in vitro,and Wright staining.Western Blot,MTT,Flow Cytometry(FCM) were used to study cell morphology.expression of c-myc protein.tumor cell proliferation in vitro.apoptosis and cell cycle change. Results Ad-Asc-myc encoding antisense c-myc fragment was obtained with the titer of 2x109pfu/ml. Ad-Asc-myc down-regulated the expression of c-myc protein after transfectedMG-63 cells for 48h, combined with the treatment of 2.0\ 5.0ug/ml cisplatin for 2h can inhibited tumor cells proliferation in vitro by33.4%and 54.2% respectively, which were significantly difference compared with control recombinant adenovirus( Ad-LacZ)groups(P<0.05). FCM analysis showed that Ad-Asc-myc can induce apoptosis of transfected cells.which was enhanced by the treatment of cisplatin. cell cycle analysis showed that obvious G2/M phase arrest in transfected cells.Conclusion: Ad-Asc-myc increased the chemotherapy sensitivity of osteosarcoma MG-63 cells to cisplatin as well as induced apoptosis.Section 3:Enhancement effect of adenovirus mediated antisense C-myc gene andcaffeine on the chemotherapy of osteosarcoma cells to cisplatin.Objective: The cancer biology field has showed that overexpression of oncogenes.with or without functional loss of tumor suppressor genes.is responsible for the progression of human malignancies through a multistep processes, antisense technology can reduce a certain gene expression.Caffeine has enhancement effect on chemotherapy of osteosarcoma cell to cisplatin, we constructed the recombinant adenovirus (Ad-Asc-myc) encoding antisense c-myc fragment and investigated its effect on the in vitro sensitivity of osteosarcoma MG-63 cells to cisplatin. Methods: The recombinant adenovirus (Ad-Asc-myc) encoding antisense c-myc fragment was constructed by cloning c-myc cDNA of about 750 base pairs in a reverse direction into adenovirus vector, then undergoing recombination,amplifying and being complemented in vivo. Ad-Asc-myc and caffeine was used respectively or together to co-operate with cisplatin to treat the osteosarcoma MG-63 cells in vitro ,and Western Blot.MTT.Flow Cytometry(FCM),electron microscope were used to evaluate expression ofc-myc protein.tumor cell proliferation in vitro.apoptosis and cell cycle analysis. Results: Ad-Asc-myc encoding antisense c-myc fragment was obtained with the titer of 2x109pfu/ml. Ad-Asc-myc down-regulated the expression of c-myc protein after transfected MG-63 cells for 48h,and induced tumor cells apoptosis , inhibited tumor cells proliferation in vitro. Ad-Asc-myc or caffeine can enhance the effects of 2.0, 5.0ug/ml cisplatin on MG-63 cells.moreover, Ad-Asc-myc combined with caffeine significantly enhance the effects of cisplatin on MG-63 cells, not only on cisplatin-induced apoptosis.but also on tumor cells proliferation repression in vitro . The apoptosis-associated genes expression level of bcl-2 gene were downregulated, expression level of bax gene were upregulated.while there were no change in the expression of E2F-1. FCM analysis showed that cisplatin treatment induced a block in S phase, and caffeine reversed this block and speeded up the progression of cells out of the S phase;Ad-Asc-myc can induced obvious G^Mphase arrest in transfected cells. Conclusion: Ad-Asc-myc combined with caffeine enhance apoptosis-induced and chemotherapy effects of osteosarcoma MG-63 cells to cisplatin .