| Backgound and Objective:Adenovirus is a kind of liner double-stranded DNA virus. The human adenovirus family consists of 57 known serotypes. All serotypes were divided into seven subgroups according to their hemagglutination properties, oncogenic potential, genotyping and phylogenetic analyses. Serotype 35 (Ad35) belongs to the subgroup B and uses human CD46 as a cellular receptor.Monoclonal antibodies are novel approaches for cancer therapy and is developing rapidly. Cetuximab is a chimeric monoclonal antibody which targets epidermal growth factor receptor (EGFR). By binding to EGFR, Cetuximab could induce antibody dependent cellular cytotoxity (ADCC) and complement dependent cytotoxity (CDC) to inhibit tumor growth.Membrane cofactor protein (CD46) is a membrane bond complement inhibitor wildly expressed in nearly all human tissues except erythrocyte. It is involved in inactivation of C3b and C4b deposited on host as the cofactor of the serine protease factor I. Many types of tumor might upregulate CD46 expression to escape from complement attack. It is reported that ovarian cancer tissues and ovarian cancer cell lines could upregulate CD46 to restrict CDC triggered by Cetuximab.Ad35K++ is a kind of Ad35 fiber knob protein mutants. And it has an increased affinity to human CD46 than wild type Ad35 fiber knob protein. In vitro experiment showed that preincubation with Ad35K++ could induce CD46 degradation and internalization.In summary, we would like explore the interaction between CD46 on ovarian cancer and Ad35K++. To investigate whether Ad35K++ could enhance CDC triggered by Cetuximab to ovarian cancer, and to explore possible mechanisms.Methods:1. Ovarian cancer cell lines were cultured in a humidified atmosphere at 37℃ in5% CO2.2. EGFR levels were quantified by flow cytometry.3. Flow cytometry was used to measure CD46 levels of ovarian cancer cells. Ad35K++ was added to a concentration at 20μg/ml each well and cells were incubated with Ad35K++ for 48 h,24 h,8 h,1 h and 15 min.4. Cell viability was measured by the colorimetric MTT array.5. Determination of apoptosis by flow cytometry.6. Expression of Bax, Bcl-2, c-caspase3, c-PARP was detected by Western blotting.Results:1. Flow cytometry studies showed that the expression of EGFR level on the surface of SK-OV-3 was significantly higher than that of A2780.2. Incubation with Ad35K++ could strongly inhibit CD46 level of SK-OV-3 cells. The CD46 level was the lowest at 8h.3. Ad35K++ could sensitize ovarian cancer cells to CDC triggered by Cetuximab.4. Ad35K++ has effectively improved complement mediated apoptosis triggered by Cetuximab.5. The group which was incubated with Ad35K++ followed by Cetuximab and NHS has an increased expression of Bax, c-caspase3 and c-PARP, but a decreased expression of Bcl-2.Conclusion:Ad35K++ could enhance antitumor effect of Cetuximab by removing CD46 from SK-OV-3 cells. After incubation with Ad35K++ for 8h, the overall inhibition rate of SK-OV-3 cells treated with Cetuximab and NHS was significantly higher than compare groups. Furthermore, Ad35K++ alone did not exert any cytotoxity to ovarian cancer cells which means that Ad35K++ shows good safety. More cells went into apoptosis for complement attack after pre-incubation of Ad35K++ followed by Cetuximab and NHS which indicates that Ad35K++ could reinforce complement mediate apoptosis triggered by Cetuximab. The protein levels of apoptosis, including Bax, C-caspase3 and C-PARP have been significantly augmented through pre-incubation of Ad35K++, but protein level of anti-apoptosis protein Bcl-2 has been decreased. These findings suggested that an activation of mitochondria-mediated apoptotic pathway might be responsible for the improvement of complement mediated apoptosis. In summary, our experiments showed that Ad35K++ could be a new and effective approach to enhance cetuximab efficiency in treating ovarian cancer with good safety. |
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