Angiotensin-converting enzyme 2 (ACE2) and neuropilin 1, a vascular endothelial growth factor (VEGF) receptor, were identified to bind to the SARS-CoV-2 spike receptor-binding domain (spike RBD). In silico analysis based on 3D structure, multiple sequence alignment, and molecular docking of second domain of soluble Flt-1 (sFlt-1) and spike RBD revealed structural similarities, sequence homology, and protein-protein interaction. Interaction and binding of recombinant spike RBD (rspike RBD) and recombinant sFlt-1 (rsFlt-1) in vitro induced a conformational change, as revealed by spectrofluorimetric data, with increased fluorescence intensity in emission spectra as compared to either of the proteins alone. Results on ELISA confirmed the binding and cross-reactivity of rspike-RBD and rsFlt-1 as determined by using either specific antibodies towards each protein or immunized human serum. We found that polyclonal or monoclonal anti-spike RBD antibodies can recognize either rsFlt-1 or rspike RBD, showing cross-reactivity for the two proteins in a dose-dependent binding response. Recognition of bound rspike RBD or rsFlt-1 by anti-Flt-1 or anti-spike RBD antibodies, respectively, as observed by immunoblotting, further confirmed interaction between the two proteins. Immunoprecipitation and immunoblot analysis demonstrated the identification of rspike RBD binding to the Flt-1 receptor on A549 cells. Further, the binding of rspike RBD to Flt-1 receptor was shown using immunofluorescence on 2D-culture or 3D-spheroid of MDA-MB-231 cells, which over-express Flt-1 receptor. Together, our study concludes that the Flt-1 receptor is a novel binding partner for SARS-CoV-2 spike RBD.
Introduction: 2-dimensional (2D) cell culture is commonly used for the evaluation of anticancer drugs, which is incapable of simulating the three dimensions (3D) microenvironment of the original tumors, therefore, a new pre-clinical platform for drug screening is urgently needed. Material and Methods MDA-MB231 cells were grown either as 2D- monolayers or 3-dimensional (3D) spheroids and treated with 5-FU or doxorubicin. Cytotoxicity assays were performed using trypan blue exclusion dye for 2D- monolayers and 3D spheroids. We have evaluated the efficacy of 5-fluorouracil (5-FU) and doxorubicin at different concentrations on the growth, size of the spheroids, induction of cytotoxicity and cell death over 10 days. Shapiro-Wilk test was used for normal distribution and two-way ANOVA for multiple group comparisons. Results 2D- and 3D cultures responded with significant sensitivity toward the cytotoxic effect of 5-FU and doxorubicin. However, 3D spheroids were less sensitive to either of these drugs. The 3D spheroids exhibited significant variations in morphometric parameters in response to either 5-FU or doxorubicin treatment. The efficacy of doxorubicin was 1000-fold more effective than 5-FU over 10 days growth period of tumor spheroids. When compared to control spheroids both 5-FU and doxorubicin showed the presence of dead cells in the core of the spheroids as measured by live/dead assay. Discussion and Conclusion: Results indicated that 3D spheroid culture recapitulates in vivo tumor microenvironment and reflects the concentration of chemotherapeutic drugs required for effective treatment in cancer patients. Spheroid analysis using AnaSP/ReViSP could be further upgraded to include fluorescent stains within the workflow.
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