Effects Of Photodynamic Synergists Combined With FECH-siRNA On Sensitivity Of Ovarian Cancer Cells To Fluorescence Imaging

ObjectiveThis study is to explore whether siRNA combined single photodynamic synergistenhance5-aminolevulinic acid (ALA)-photodynamic diagnosis (PDD)sensitivity based on usingeach of them alone,and reduce ALA dose to avoid the side effects of ALA,which may establishthe experimental foundationforfurther application ofmultiple photodynamic synergists combinedwith siRNA or not.Methods1Humanovarian fibroblastHOF cells were isolatedfrom ovrian tissue by collagenase typeⅠ.Human ovarian carcinoma SK-OV-3cells and HOF cells were cultured routinely.2The optimum incubation time and the minimum concentration of ALA for SK-OV-3cellswere detected by fluorescence microscopy.3Ferrochelatase (FECH)-siRNAs were transfectedinto SK-OV-3cells by Lipofectamine RNAiMAX. The FECHgene knockdown in SK-OV-3cells at mRNA and protein levels wasdetectedby RT-qPCR and western blot, respectively.Fluorescence microscopy was used todetect protoporphyrin Ⅸ(PpⅨ)fluorescence of SK-OV-3cells and HOF cells aftersiRNAtransfection.4The optimum incubation conditions and the differences of PpⅨ fluorescence inSK-OV-3cells and HOF cells treated with deferoxamine (DFO), EthylenediaminetetraaceticAcid Calcium Disodium Salt Hydrate(EDTA-Na2Ca) ordimethyl sulfoxide(DMSO)combined with ALA were detected by fluorescence microscopy. The effects of siRNAcombined with singlephotodynamic synergiston PpⅨaccumulation andfluorescenceenhancement in SK-OV-3cells were also investigated by fluorescencemicroscopy.Results10.3mM ALAcould induce PpⅨ fluorescence in SK-OV-3cells. The fluorescencestrengthened within0~6h (P<0.05),plateauedat6~9h (P>0.05),and weakened after9h (P<0.05).2PpⅨ fluorescence of SK-OV-3cells was a little stronger than HOF cells’ afterincubatedwith0.3mM ALA for6h (P<0.01).3After FECH-siRNA transfection, FECHmRNA decreased to (0.05±0.01) times (P<0.01),and FECH protein decreased to (0.20±0.07) times.After combination with ALA, PpⅨfluorescence was significantly enhanced in SK-OV-3cells(P<0.05), but not changedin HOFcells (P>0.05).4PpⅨ fluorescence in SK-OV-3cellscould not be enhancedby DFOindependently (P>0.05),but could be enhancedby DFOcombined with ALA (P<0.01). The fluorescence didn’tchange when the concentrations of DFO were5~30mM(P>0.05), and it was the strongestat5h (P<0.05).5EDTA-Na2Ca could not enhance PpⅨ fluorescence in SK-OV-3cells independently (P>0.05), but could enhanced the fluorescence when combined with ALA (P<0.01). Thefluorescence was positively correlatedwith the concentration of EDTA-Na2Cafrom0mM to125mM (P<0.05),but didn’t significantlychange from2h to5h (P>0.05).6DMSO could not enhance PpⅨ fluorescence in SK-OV-3cells independently (P>0.05),butcould enhance the fluorescence when combined with ALA (P<0.01). The optimumconcentration of DMSO was6%(P<0.05). The fluorescence was the strongest at3h(P<0.05).7DFO, EDTA-Na2Ca or DMSO combined with ALA could not enhanced PpⅨfluorescencein HOF cells (P>0.05), but could enhance the fluorescence in SK-OV-3cells.All the enhancement of them inSK-OV-3cells had no significant difference(P>0.05),but was obviously higher than that in HOF cells (P<0.01).8EDTA-Na2Ca or DMSO combining with siRNA could enhance stronger PpⅨfluorescencein SK-OV-3cells than each of them alone (P<0.05). There was nosignificantdifference between the effects of DFO combined with siRNA and siRNAtransfection alone (P>0.05).Conclusion1The optimum incubation time of ALA on SK-OV-3cells was6h,and the minimum concentration was0.3mM. PpⅨ fluorescence in SK-OV-3cellsunder these conditionswasstronger than that in HOF cells.2After FECH-siRNA transfection mediated by Lipofectamine RNAiMAX, the mRNA andprotein expressionof FECHin SK-OV-3cells couldbe significantly inhibited.PpⅨfluorescence in SK-OV-3cells was enhanced by combination of siRNA transfection andlow-dose ALA,but thatin HOF cells.3DFO, EDTA-Na2Ca or DMSO combined with ALA could enhance PpⅨ fluorescenceinSK-OV-3cells. But none of them could enhance the fluorescencein HOF cells.4EDTA-Na2Caor DMSO combined with siRNA showed asynergismwith ALA. But there wasno synergism between DFO combined with siRNA and ALA.5This research demonstrates that PpⅨ fluorescence can be enhanced more effectively bycombining the methodsfor different targets. Thesemethods can enlarge the difference offluorescence between ovarian cancer cells and normal cells,thusenhancing the targetabilityof ALAto ovarian cancer cells and reducing the damage to normal cells.