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Cancers 2018, 10, 144 4 of 15 2.2. TMZ Increases Viral Infection and Ad E1A Gene Expression in Human TNBC Cells Cancers 2018, 10, x FOR PEER REVIEW 4 of 15 Human TNBC cell lines were infected with OAdmCherry alone or in combination with TMZ or a 2.2. TMZ Increases Viral Infection and Ad E1A Gene Expression in Human TNBC Cells vehicle control dimethyl sulfoxide (DMSO). At 24 h post infection, mCherry expression was visualized Human TNBC cell lines were infected with OAdmCherry alone or in combination with TMZ or by fluorescence microscopy (Figure 2A). OAdmCherry-infected HCC1937 and MDA-MB-231 cells a vehicle control dimethyl sulfoxide (DMSO). At 24 h post infection, mCherry expression was displayed 2% and 15% mCherry-positive cells, respectively. Treatment with DMSO slightly increased visualized by fluorescence microscopy (Figure 2A). OAdmCherry-infected HCC1937 and MDA-MB- mCherry expression to 5% and 22%, respectively. In contrast, a greater mCherry expression was 231 cells displayed 2% and 15% mCherry-positive cells, respectively. Treatment with DMSO slightly observed in OAdmCherry/TMZ-treated cells, increasing to 21% and 50%, respectively (Figure 2B). increased mCherry expression to 5% and 22%, respectively. In contrast, a greater mCherry expression These results suggest that TMZ increases OAdmCherry infection as early as 24 h post treatment with was observed in OAdmCherry/TMZ-treated cells, increasing to 21% and 50%, respectively (Figure TMZ. To further validate adenovirus infection, the expression of Ad E1A, a key component of Ad 2B). These results suggest that TMZ increases OAdmCherry infection as early as 24 h post treatment replication machinery, was evaluated by Western blot assay. Similarly to the results observed for with TMZ. To further validate adenovirus infection, the expression of Ad E1A, a key component of mCherry expression, Ad E1A expression levels were modest in cells infected with OAdmCherry alone Ad replication machinery, was evaluated by Western blot assay. Similarly to the results observed for or in combination with DMSO, whereas OAdmCherry/TMZ-treated cells exhibited greater levels of mCherry expression, Ad E1A expression levels were modest in cells infected with OAdmCherry adenovirus (Ad) early region 1A (E1A) expression (Figure 2C). These results suggest that TMZ has the alone or in combination with DMSO, whereas OAdmCherry/TMZ-treated cells exhibited greater abillietyvetlos ionfcaredaesneovOirAuds (iAnfde)cteiaornlyarnedgiAond1EA1A(Ee1xAp)rexspsiroesnsionnT(NFiBgCurece2llCs).. These results suggest that TMZ has the ability to increase OAd infection and Ad E1A expression in TNBC cells. 2.3. TMZ Facilitates Adenovirus Entry into Human TNBC Cells 2.3. TMZ Facilitates Adenovirus Entry into Human TNBC Cells To further validate TMZ’s ability to facilitate the adenovirus entry into TNBC cells, the HCC1937 cell line TwoafsuirnthferctveadlidwaittehTaMnZA’sdaGbiFliPtyatlofnaeciolirtaitne cthoemabdienaotvioirnusweintthryDiMntoSOTN(dBCrucgelvlse,hthicelHe cCoCn1t9r3o7l) or cell line was infected with an AdGFP alone or in combination with DMSO (drug vehicle control) or TMZ. At 24 h post infection, GFP expression was visualized by fluorescence microscopy (Figure 3A). TMZ. At 24 h post infection, GFP expression was visualized by fluorescence microscopy (Figure 3A). AdGFP-infected HCC1937 cells displayed 12% GFP-positive cells. Treatment with DMSO slightly AdGFP-infected HCC1937 cells displayed 12% GFP-positive cells. Treatment with DMSO slightly increased GFP expression to 18%, whereas a greater GFP expression was observed in TMZ-treated increased GFP expression to 18%, whereas a greater GFP expression was observed in TMZ-treated cells, increasing to 45% (Figure 3B). These results confirm that TMZ could facilitate adenovirus entry cells, increasing to 45% (Figure 3B). These results confirm that TMZ could facilitate adenovirus entry into TNBC cells. This result suggests that TMZ may represent a useful chemotherapeutic drug to into TNBC cells. This result suggests that TMZ may represent a useful chemotherapeutic drug to increase adenovirus infection in those cancer cells that exhibit poor infectability. increase adenovirus infection in those cancer cells that exhibit poor infectability. Figure 2. Cont.PDF Image | Temozolomide Enhances Triple-Negative Breast Cancer Virotherapy
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