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Cancers 2018, 10, 144 Cancers 2018, 10, x FOR PEER REVIEW Cancers 2018, 10, x FOR PEER REVIEW 2.2. TMZ Increases Viral Infection and Ad E1A Gene Expression in Human TNBC Cells 5 of 15 5 of 15 5 of 15 Human TNBC cell lines were infected with OAdmCherry alone or in combination with TMZ or a vehicle control dimethyl sulfoxide (DMSO). At 24 h post infection, mCherry expression was visualized by fluorescence microscopy (Figure 2A). OAdmCherry-infected HCC1937 and MDA-MB- 231 cells displayed 2% and 15% mCherry-positive cells, respectively. Treatment with DMSO slightly increased mCherry expression to 5% and 22%, respectively. In contrast, a greater mCherry expression was observed in OAdmCherry/TMZ-treated cells, increasing to 21% and 50%, respectively (Figure 2B). These results suggest that TMZ increases OAdmCherry infection as early as 24 h post treatment with TMZ. To further validate adenovirus infection, the expression of Ad E1A, a key component of Ad replication machinery, was evaluated by Western blot assay. Similarly to the results observed for mCherry expression, Ad E1A expression levels were modest in cells infected with OAdmCherry alone or in combination with DMSO, whereas OAdmCherry/TMZ-treated cells exhibited greater levels of adenovirus (Ad) early region 1A (E1A) expression (Figure 2C). 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 Figure 2. Effect of temozolomide (TMZ) treatment on virus infection and adenovirus early region Figure 2. Effect of temozolomide (TMZ) treatment on virus infection and adenovirus early region 1A 1A (E(E1T1AoA)f)uerxtphrerssvsiaolnidiniantheuhTmuMmanZant’sritaprbliepi-lnlietey-gntaeotgivfaetcibivlrietabstrteectahsnetcaecrdan(eTncNeorBvC(irT)uNcseBellCns:t)r(yAce)ilnlHst:ou(mTANa)nBHTCuNcmBeCallnsc,eTtlhlNsewBHeCCrecCe1ll9s37 cellilninfeectwedawsitnhfeocntceodlytwicitahdeannovAirduGsmFPChaelorrnye(OoArdinmcCohmerbryin)atiaonmuwltiitphliDcitMyoSfOin(fdecrtuiognvcoenhcicenletrcaotinontrol) or were infected with oncolytic adenovirus mCherry (OAdmCherry) at a multiplicity of infection TMZof.2A.5ta2l4onhepoorsint icnomfebctinioanti,oGnwFPithexTpMrZesosriovnehwicalesdvimisuetahlyizlesudlfboyxidfleu(oDrMesScOen).cEexmpriecsrsoiosncopfmyC(Fhiegruryre 3A). concentration of 2.5 alone or in combination with TMZ or vehicle dimethyl sulfoxide (DMSO). was evaluated by fluorescence microscopy. Scale: 200 μm. (B) Percentage of mCherry-positive cells AdGFP-infected HCC1937 cells displayed 12% GFP-positive cells. Treatment with DMSO slightly Expression of mCherry was evaluated by fluorescence microscopy. Scale: 200 μm. (B) Percentage of calculated relative to number of cells in the field. Results represent the mean of three repeated increased GFP expression to 18%, whereas a greater GFP expression was observed in TMZ-treated mCherry-positive cells calculated relative to number of cells in the field. Results represent the mean measurements ± standard deviation (SD; error bars) (* p < 0.05). (C) Expression of Ad E1A was cells, increasing to 45% (Figure 3B). These results confirm that TMZ could facilitate adenovirus entry of three repeated measurements ± standard deviation (SD; error bars) (* p < 0.05). (C) Expression of evaluated by Western blot assay at 24 h post treatment. Actin was used as a loading control. into TNBC cells. This result suggests that TMZ may represent a useful chemotherapeutic drug to Ad E1A was evaluated by Western blot assay at 24 h post treatment. Actin was used as a loading A representative assay is shown from three performed. increase adenovirus infection in those cancer cells that exhibit poor infectability. control. A representative assay is shown from three performed. Figure 3. Temozolomide (TMZ) facilitates adenovirus entry into triple-negative breast cancer (TNBC) Figure 3. Temozolomide (TMZ) facilitates adenovirus entry into triple-negative breast cancer (TNBC) Figure 3. Temozolomide (TMZ) facilitates adenovirus entry into triple-negative breast cancer (TNBC) cells: (A) HCC1937 cells were infected with adenovirus expressing green fluorescent protein (AdGFP) cells: (A) HCC1937 cells were infected with adenovirus expressing green fluorescent protein (AdGFP) cells: (Aat)aHmCuClt1ip9l3ic7itcyeollfsinwfecrteioinfceocntecednwtraithioandoefn5oavloirnuesoerxinprceosmsibningagtiroenenwfitlhuoTrMesZce(0n.4t pmrMot)eoinr v(AehdicGleFP) at a multiplicity of infection concentration of 5 alone or in combination with TMZ (0.4 mM) or vehicle at a multiplicity of infection concentration of 5 alone or in combination with TMZ (0.4 mM) or vehicle dimethyl sulfoxide (DMSO). Expression of GFP was evaluated by fluorescence microscopy. Scale: 200 dimethyl sulfoxide (DMSO). Expression of GFP was evaluated by fluorescence microscopy. Scale: μm. (B) Percentage of GFP-positive cells calculated relative to number of cells in the field. Results dimet2h0y0 lμsmu.lf(oBx)iPderc(eDnMtagSeOo)f. EGxFpPr-peossitoinveocfeGllsFcPalwcualsatevdarleulatievde btoynfulumobrerscoef nceclelsminictrhoesficeolpdy. .RSecsaulets: 200 μm. (B) Percentage of GFP-positive cells calculated relative to number of cells in the field. Results represent the mean of three repeated measurements ± standard deviation (SD; error bars) (* p < 0.05). represent the mean of three repeated measurements ± standard deviation (SD; error bars) (* p < 0.05). represent the mean of three repeated measurements ± standard deviation (SD; error bars) (* p < 0.05). 2.4. TMZ Enhances OAd-Mediated Oncolytic Cell Death Partly as a Result of Increased Virus Replication 2.4. TMZ Enhances OAd-Mediated Oncolytic Cell Death Partly as a Result of Increased Virus Replication 2.4. TMZ EHnhuamnacnesTONABdC-MceelldliiantesdwOenrceoilnyfteictCedellwDitehatehithPeartAlydGasFaPRoersOulAtdomf ICnchreerarsyedasVdireuscsrRibepdliacbatoivone Human TNBC cell lines were infected with either AdGFP or OAdmCherry as described above alone or in combination with TMZ or vehicle control DMSO. At 72 h post treatment, crystal violet alone or in combination with TMZ or vehicle control DMSO. At 72 h post treatment, crystal violet Human TNBC cell lines were infected with either AdGFP or OAdmCherry as described above staining (Figure 3A) showed that the combination of OAdmCherry and TMZ induced greater CPE in staining (Figure 3A) showed that the combination of OAdmCherry and TMZ induced greater alone or in combination with TMZ or vehicle control DMSO. At 72 h post treatment, crystal violet both HCC1937 and MDA-MB-231 cell lines (23% and 42% cell viability) as compared with either staining (Figure 3A) showed that the combination of OAdmCherry and TMZ induced greater CPE in OAdmCherry alone (80% and 78% cell viability) or TMZ alone (75% and 87% cell viability) (Figure both H4BC)C. 1A9d3G7FaPndidMnDoAt i-nMduBc-e23c1ytcoetollxliciniteysa(l2o3n%e oarnidn 4co2m%bcinealltiovniawbiiltihtyT)MasZc(oFmigpuraere4dA,wB)i.thTMeiZther OAdminCchrearsreydalOoAndem(8C0h%eraryndvi7r8u%s pcreoldluvciatiboinlitayp)pororxTimMaZtelaylo1n0e-fo(7ld5%inabnodth87c%ellcleilnlevsiacobmiliptyar)e(dFitgoure 4B). AdGFP did not induce cytotoxicity alone or in combination with TMZ (Figure 4A,B). TMZ increased OAdmCherry virus production approximately 10-fold in both cell lines compared toPDF Image | Temozolomide Enhances Triple-Negative Breast Cancer Virotherapy
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