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Cancers 2018, 10, 144 11 of 15 In conclusion, this study provides the experimental evidence showing that TMZ can be used to enhance oncolytic virotherapy in TNBC cells, which may represent an alternative approach to destroy TNBC tumors in patients with resistance to chemotherapy. Most importantly, human TNBC cells were efficiently destroyed by the combined therapy of OAd with TMZ. In addition, these chemovirotherapies may allow for the use of less-toxic doses to achieve therapeutic efficacy and prime the immune system to reduce the chances of cancer recurrences. 4. Materials and Methods 4.1. Cell Lines and Culture Conditions Human embryonic kidney cell line (HEK-293) (Cat# CRL-1573), human TNBC HCC1937 (Cat# CRL-2336) and MDA-MB-231 cells (Cat # HTB-26), and murine TNBC 4T1 cells (Cat# CRL-2539) were purchased from the American Type Culture Collection (ATCC) (Manassas, VA, USA). HCC1937 and 4T1 cells were grown in RPMI-1640 medium (Cat# 10-040-CV). MDA-MB-231 and HEK-293 cells were grown in Dulbecco’s Modified Eagle’s Medium (DMEM) (Cat# 10-013-CV). All media were supplemented as previously described [38]. All cell culture reagents were obtained from Corning Cellgro (Manassas, VA, USA). 4.2. Adenoviral Vectors and Drugs A replication-deficient adenoviral vector expressing green fluorescent protein (AdGFP) under regulation of a cytomegalovirus (CMV) promoter was used as a negative control for virus replication as previously described [38]. The conditionally replicating adenovirus expressing mCherry red fluorescent protein on the capsid was constructed by homologous recombination in Escherichia coli (BJ5183 strain) using the fiber gene modified AdEasy-1 backbone vector AdEz-F5/3 (Ad5∆E1/∆E3-F5/3) and a modified pShuttle vector pSl∆24-pIX-mCherry. This shuttle vector contained the mCherry coding sequence inserted downstream from the Ad5 minor capsid pIX gene to generate a C-terminal pIX fusion and a 24-basepair deletion in the Ad5 E1A gene coding sequence (∆24) [39]. TMZ stock solution of 50 mM was prepared in DMSO and stored at −20 ◦C. The final volume of TMZ and vehicle control DMSO added to the cell cultures was less than 1%. All drugs were purchased from Sigma-Aldrich (St. Louis, MO, USA). 4.3. Single and Combined Therapies A total of 2.5 × 104 cells were plated in a 24-well plate and treated 24 h later with the indicated therapy. Viral infection was performed at an indicated MOI concentration, whereas TMZ treatment was performed at an indicated millimolar (mM) concentration. OAdmCherry-mediated CPE was evaluated at 72 h post infection by crystal violet staining. Suspended cells were removed by aspiration; the remaining adherent cells were then fixed with 3.7% formaldehyde for 3 min at room temperature. The excess formaldehyde was washed with Phosphate-buffered saline (PBS); the cells were then stained using 1% crystal violet at room temperature for 3 min. Excess crystal violet was washed away with PBS. Plates were then scanned using an HP Scanjet 4070 scanner (HP, Palo Alto, CA, USA). The remaining crystal violet was then solubilized with a 2% sodium dodecyl sulfate (SDS) solution, and the sample absorbances were measured at 590 nm using a Synergy HT Multi-Mode Microplate Reader (Bio-Tek, Winooski, VT, USA). The absorbance (OD) values of each treatment were then normalized to mock-treated cells converting each sample OD into the cell viability percentage (%) according to the following formula: cell viability % = (OD of treated cells/OD of mock-treated cells) × 100%, as described previously [40]. The expression of mCherry was assessed 24 h after OAd infection using a Leica DM1000 fluorescence microscope with an N2.1 filter at 587 and 610 nm for excitation and emission, respectively. Cell viability was assessed 72 h after TMZ treatment by measuring the conversion of tetrazolium salt 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium (MTT) to formazan, as describedPDF Image | Temozolomide Enhances Triple-Negative Breast Cancer Virotherapy
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