HCE6,a Novel Photosensitizer,Exhibited Anti-tumor Effects In Vitro With Photodynamic Therapy In FRH0201 Human Cholangiocarcinoma Cells

AimHCE6 was a novel second-generation water-soluble photosensitizer.To study the spectral characteristic of HCE6 and investigate the HCE6-mediated photodynamic therapy?HCE6-PDT?cytotoxic effect in human cholangiocarcinoma FRH0201 cells and the preliminary mechanism,as well as the internal relation between apoptosis and autophagy,for the theoretical basis of diagnosis and treatment of cholangiocarcinoma.MethodPart1:Uv/fluorescence spectrophotometer were made to detect HCE6 in 50 mM Tris-HCl-NaCl buffer solution.The absorption spectra and emission spectra of HCE6 were also determined while solution acidity?pH?of Tris-HCl-NaCl buffer solution and photosensitizer concentration were adjusted.Part2:The uptake of HCE6 were monitored by flow cytometry.The phototoxicity of HCE6 and IC₅₀ was studied by CCK-8 assay.The dark toxicity of HCE6?light intensity and the IC₅₀ of other tumor cells and normal calls were also studied by CCK-8 assay.The morphological variationwasmonitored by inverted microscope,DAPI staining and AO/EB double staining.Reactive oxygen species?ROS?was measured by 2',7'-Dichlorofluorescin diacetate staining.Annexin V/Propidium Iodide double staining were detected for apoptosis by flow cytometry.The activation of apoptosis-associated protein or autophagy marker protein were evaluated by western blotting.Before HCE6-PDT treatment,some of the cells were pretreated with chloroquine?CQ?.ResultPart1:There are three distinct peaks at 398,499,649 nm in the absorption spectra,respectively,which the maximum peak was at 398 nm.The fluorescence emission spectra showed that DVDMS fluorescence emission was at 652 nm.Part2:Studies demonstrated maximal uptake of DVDMS occurred within 4 h.Activation of DVDMS resulted in significant increases in ROS production.HCE6-PDT demonstrated the ability to inhibit FRH0201 cell viability in an HCE6 concentrationand light dose-dependent manner.Additionally,HCE6-PDT could also induce apoptosis,autophagy,necrosis of FRH0201 cell.Pretreatment with the autophagy inhibitor CQ showed the potential in reducing the apoptosis rate induced by HCE6-PDT in FRH0201cells.Conclusion?1?HCE6 could be used as Photodynamic diagnostic reagent.?2?HCE6-PDT had a significant cytotoxic effect in FRH0201 cells,which was dose-dependent and light-dependent.?3?HCE6-PDT-induced apoptosis and autophagy in FRH0201.?4?HCE6-PDT induced autophagy,which further promoted the apoptosis in FRH0201 cells.