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Cancers 2018, 10, 144 7 of 15 2.5. Combination of OAdmCherry with TMZ Alters Autophagy in Human TNBC Cells Because TMZ is known to induce autophagy, we next investigated whether the combination of OAdmCherry with TMZ could enhance autophagy induction over that of either agent independently. HCC1937 and MDA-MB-231 cell lines were transfected with plasmid Enhanced Green Fluorescent Protein-Microtubule-associated protein 1A/1B-light chain 3 (pEGFP-LC3) followed by either TMZ or OAdmCherry alone or in combination. The formation of cytoplasmic punctate GFP fluorescence was then observed. The conversion of cytoplasmic diffuse GFP-LC3-1 to membrane-associated GFP-LC3-II formed punctate patterns, indicating LC3-II incorporation into the autophagosomes. This formation of punctate was observed 48 h after treatment (Figure 5A). TMZ-treated HCC1937 and MDA-MB-231 cells displayed an accumulation of the fluorescent punctate pattern. HCC1937 cells showed 6 dots per cell and MDA-MB-231 showed 13 dots per cell on average. OAdmCherry-infected HCC1937 and MDA-MB-231 cells displayed a similar effect; these cells showed five and eight dots per cell on average, respectively. Greater fluorescent punctate pattern accumulation was observed with the combined treatment. HCC1937 cells showed 22 dots per cell and MDA-MB-231 cells showed 85 dots per cell on average (Figure 5B). Next, the conversion of LC3-I to LC3-II, an autophagy marker [28], was evaluated. Western blot analysis revealed two reactive LC3 species: an upper band corresponding to LC3-I (19 kDa) and a lower band corresponding to LC3-II (17 kDa). In both TNBC cell lines, a marked accumulation of LC3-II was observed with the combined treatment as compared to untreated and TMZ- or OAdmCherry-treated cells (Figure 5C). These results suggest that the combination of OAdmCherry and TMZ alters autophagy in TNBC cell lines more so than either treatment alone. 2.6. TMZ Increases OAdmCherry Infectivity and Ad E1A Expression, and Combined Therapy Strongly Inhibits Clonogenic Survival in Mouse TNBC Cells Because the mouse TNBC 4T1 cell line represents an animal stage IV human breast cancer, we further investigated whether the combined therapy of OAd with TMZ could be effective in this cell line. Previously we found that TMZ treatment facilitated AdGFP entry into 4T1 cells [27]. We first evaluated OAdmCherry-mediated CPE and TMZ-induced cytotoxicity in 4T1 cells. Mouse TNBC 4T1 cells were infected with OAdmCherry or AdGFP at increasing concentrations of MOI. At 72 h post infection, crystal violet staining revealed that OAdmCherry-mediated CPE increased in a virus-dose-dependent manner. In contrast, AdGFP-treated cells did not induce CPE even at the highest MOI concentration of 100 (Figure 6A). The 4T1 cells displayed greater resistance to OAdmCherry-mediated CPE as compared with human TNBC cell lines (Figure 1A). However, 4T1 cells responded relatively efficiently to OAdmCherry-mediated CPE. For example, OAdmCherry at a MOI concentration of 10 reduced cell viability to 66% (Figure 6B). Next, TMZ-mediated cytotoxicity was determined. 4T1 cells were treated at increasing TMZ concentrations. At 72 h post treatment, a MTT assay revealed that cell viability decreased in a dose-dependent manner. TMZ at 0.4 mM decreased cell viability by 67% in 4T1 cells (Figure 6C). We then investigated whether TMZ enhances viral replication and spread. The 4T1 cells were infected with OAdmCherry alone or in combination with TMZ or DMSO. At 72 h post infection, mCherry expression was visualized by fluorescence microscopy (Figure 5D). OAdmCherry-infected 4T1 cells displayed 20% mCherry-positive cells. Treatment with DMSO slightly increased mCherry expression to 25%. In contrast, the combined treatment with TMZ significantly increased mCherry expression to 65% (Figure 5E). To further assess the effect of TMZ upon adenovirus (Ad) replication, Ad E1A protein expression was evaluated by Western blot assay. Similarly to the results observed for mCherry expression, Ad E1A expression levels were modest in both OAdmCherry alone or in combination with DMSO, whereas TMZ-treated cells exhibited greater levels of Ad E1A expression (Figure 5F). These results suggest that TMZ has the ability to increase OAd replication in murine 4T1 TNBC cells. Finally, the therapeutic effect of the combination of OAdmCherry with TMZ was evaluated in vitro; single colonies were visualized by crystal violet staining (Figure 5G). The clonogenic survival assay revealed that in 4T1 cells, TMZ-alone and OAdmCherry-alone induced 45% and 65% survival, respectively, whereas the combined therapy (OAdmCherry/TMZ) resultedPDF Image | Temozolomide Enhances Triple-Negative Breast Cancer Virotherapy
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