| Background and objectiveMalignant tumor is considered as one of the most serious disease threatening the health of mankind. The traditional major therapy includes surgery excision, chemotherapy, radiotherapy and etc. As the development of molecular biology and virology, the reseach on gene-viro therapy become very popular in recent years. Chemo-gene-viral therapy in the treatment of cancer has already been reported, and clinical trials have showed enhanced and even synergistic antitumor activity when conditionally replicating adenoviruses (CRAds) were used in combination with chemotheraphy, which paves a new way for cancer treatment.Here we construct a newly designed oncolytic adenovirus, which utilizes Adenovirusserotype 5 (Ad5) as backbones, and make use of the shaft and knob of serotype 11 (Ad11) as well as tail of Ad5 to make up a chimerical virus which is deficient of E1B55KD region. This virus overcome the limitation that are necessarily dependent on high expression of CAR on cancer cell surface, and specially target on P53 deficient or any mutant cancer cell, while have no cytotoxic effect on those normal cells. The virus replicate, proliferate in the cancer cell, activate apoptosis and its following pathways, which lead to oncolysis, the released progeny virions continue to infect the peripheral cancer cells and ultimately decimate all the cancer cell existed. Cisplatin has been in clinical use for several decades, and is still the most effective adjuvant for a variety of cancers. It is a platinum metal complexe, which fuctions like alkylating agent. Cisplatin kills the tumor cells by targeting at the DNA and interfering with its replication. Since the conditionally replicating adenoviruses SG511 and cisplatin works in different ways, we combined the two agents together to investigate if they work synergistically in the treatment of cancer.MethodWe first constructed the adenovirus SG511, SG511-EGFP, and studied the infection and replication ability of SG511 in tumor cell lines Hela and HT-29. Utilizing the adenovirus vector SG511-EGFP, which carrying green fluorescent protein (GFP), to transfect the tumor cell Hela, HT-29, and make use of flurescence microscope to observe the effect of transfection. Take advantage of flow cytometer quantitation to determine the expression of GFP in different cancer cell. The growth inhibition of SG511 or/and cisplatin on two cell lines were determined by crystal violet staining and MTT assay. Evaluating the combination effect in different tumor cell lines with the use of Chou-Talalay method. Then apoptosis induced by the reagents was detected by DAPI staining and flow cytometry analysis. Westernblot was applied to anaylize the apoptosis-related protein: PARP, caspase-3, caspase 8, caspase-9, Bcl-2, Mcl-1 etc. Furthermore, negative effects on normal cells were evaluated in the normal cell line L02 by MTT and crystal violet staining.Result1. A significant portion of malignant tumor cells was permissive to SG511-EGFP infection with a dose-dependent manner. 2. Both SG511 and cisplatin showed dose-response cytotoxicity for Hela and HT-29, when combined together, it showed synergistic cytotoxicity.3. As to cell morphology, the numbers of broken and necrosed cells increased in the combination group and bright nuclear fragmentation was identified by DAPI staining.4. The combination group induced the apotosis with enhanced activity of caspase-3,-8,-9 and parp.5. Much lower levels of Bcl-2, Mcl-1, and Bid were expressed in the combination group compared to treatment of cells with either SG511 or cisplatin alone.6. Combined use of cisplatin and oncolytic virus SG511 had only a little greater cytotoxic effect compared with cisplatin alone.Conclusion1. Oncolytic adenovirus SG511 can transfect tumor cell line like Hela, HT29 with a dose-dependent manner and has a relatively high efficiency.2. SG511 and cisplatin could induce apoptosis synergistically in solid tumor cell lines.3. SG511 is an tumor-specific replicative adenovirus for virotherapy of cancer, and had only a little greater cytotoxic effect in combination with cisplatin...
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