Photodynamic Therapy Of Cholangiocarcinoma With A New Photosensitizer HCE6

Backgrounds and ObjectiveCholangiocarcinoma(CC)worldwide is the second commonest primary liver tumour after hepatocellular carcinoma(HCC),and it is also the most common biliary malignancy.It grows from the epithelium of intra-or extra-hepatic bile ducts and accounts for 3% of gastrointestinal tumors.With the improvements in the technique of endoscopy and the people’s knowledge about this disease,the incidence and mortality rates for CC appear to be increasing steeply and steadily across the world over the past few decades.CC has a poor prognosis and a low survival rate.The most common risk factors of CC include: primary sclerosing cholangitis(with or without ulcerative colitis),cirrhosis of any cause and chronic viral hepatitis B or C.The most typically common symptom of CC is the features of biliary obstruction,such as jaundice,pale stool,dark urine and pruritus and so on.Surgery is the only curative treatment for patients with CC but it is usually at the advanced stage on the first time of diagnose because of the lack of effective measures of diagnosis(CT,MRI,and blood test).More than 70% of patients are unresectable at diagnosis.Also the surgery has a risk of high rate of complications and recurrence.Therefore,palliation of the symptoms from biliary obstruction has a significant importance for the patients with CC,especially for the unresectable patients.Endoscopic biliary stent placement for patients with unresectable CC is widely accepted and is considered as an effective palliation therapy,with metal stents preferred over plastic stents in patients with long patency.It can provide benefits in terms of symptomatic improvement and quality of life.However,this endoscopic technique also faces the challenge of re-obstruction of the stents and it can not provide benefits on the survival time.Other traditional therapies such as chemotherapy and radiotherapy also have poor results for CC.So looking for new treatments are desirable for CC.PDT is widely used in a variety of solid tumors(bladder cancer,esophageal cancer,prostate cancer,head and neck neoplasms,and so on)as a minimally invasive method,which consists of three essential components: photosensitizer,light,and oxygen.In many clinical trials and trials in vivo and in vitro,PDT has shown encouraging results in reducing biliary obstruction,improving the quality of life and increasing survival rates for patients with advanced cholangiocarcinoma.But PDT is also associated with side effects: severe cutaneous phototoxicity and prolonged time refraining from sunlight.To avoid these adverse effects,new photosensitizers with higher efficacy and lower side effect are needed for this technology.Therefore,the aim of our study is to evaluate the effect and potential mechanisms of a new photosensitizer 2-(1-Hexyloxyethyl)-2-devinyl chlorin e6 trisodium salt(HCE6)mediated PDT in treating human cholangiocarcinoma.MethodsHuman cholangiocarcinoma cell lines FRH-0201 and QBC-939 were cultured for this study.The cells were cultured in Dulbecco’s modified Eagle’s medium(DMEM,Gibco,Life Technologies,Inc.)supplemented with 10% fetal bovine serum(FBS,Hyclone,USA).The cells in the log phase of growth at 70?90% confluency were seeded onto a 96-well microplate at a density of 1′104 cells/well.After an overnight incubation,the cells were incubated with variable concentrations of HCE6(0.5,1,2,4,8 ug/ml,respectively)at 37?C for 24 hours without exposure to any light,and then illuminated with different laser doses(2.5,5,10,15 J/cm2,respectively)at 652 nm.After different treatments,a cell counting kit 8(CCK8)assay was used to evaluate the cell viability of cholangiocarcinoma cells and its growth inhibition ratio was calculated.We evaluated the relationship between the efficacy of HCE6 mediated PDT with concentration of HCE6 or dose of laser light.In order to evaluate the effect of the incubation time of HCE6 prior to and following light exposure on the phototoxicity of HCE6?mediated PDT,the FRH-0201 cells were incubated with HCE6 for 3h,6h,12 h,and 24 h,respectively,prior to the light exposure.Subsequent to being exposed to light at a dose of 10J/cm2(8 cm-diameter illumination),the cells were cultured in the dark for a further 24 h.Then the cell viability was also determined using a CCK8 assay.In order to compare the efficacy of HCE6 mediated PDT with HiPorfin mediated PDT or Photofrin mediated PDT in human cholangiocarcinoma cells,the cells cultured in the 96-well microplate were divided into three groups as follows:(1)HCE6 mediated PDT group;(2)HiPorfin mediated PDT group;(3)Photofrin mediated PDT group.The FRH-0201 cells and QBC-939 cells were incubated with 2μg/ml of HCE6 or HiPorfin or Photofrin for 24 h in the dark prior to being exposed to a laser light at 665 nm or 630 nm(10 J/cm2).Then the inhibition rate was evaluated by a CCK8 assay.We also evaluated the apoptosis of the cholangiocarcinoma cells by an AO-EB staining assay.Flow cytometry assay was applied to quantify the amount of cells in apoptosis with an Annexin V-FITC/PI kit.The proteins of the FRH-0201 cells were collected and we evaluated the expression of apoptosis protein caspase-3 and caspase-8 by western blot analysis.All values were analyzed statistically with the SPSS 21.0 software.Statistical significance was established at a P-value<0.05 All the assays were repeated three times.ResultsAccording to the CCK8 assay,HCE6 mediated PDT could significantly inhibit the proliferation of FRH0201、QBC939 cells in a concentration-dependent and dose-dependent manner.The inhibitory effect of PDT on cell proliferation elevated along with the increase in the concentration of HCE6 and dose of laser light.When the concentration of HCE6 was 2ug/ml and light irradiation was 10J/cm2,the relative growth inhibition rate was 83.6% for FRH-0201 and 84% for QBC939 respectively.And with the concentration of photosensitizer and the laser energy increased,the inhibition rate was not significantly different from that.As for the optimal incubation time of HCE6,the inhibition rate following a 6-h,12-h,24 h incubation period was higher than that with a 3-h incubation period(p<0.05).And there was no significant difference in the survival fraction of FRH-0201 and QBC-939 between the 6-h,12-h and 48-h incubation period(p>0.05).The inhibition effect of HCE6 mediated PDT was higher than HiPorfin mediated PDT or Photofrin mediated PDT with the same photosensitizer concentration of 2ug/ml(p<0.01).After treating with HCE6 mediated PDT,AO-EB staining assay showed a significant higher apoptosis rate in the HCE6 mediated PDT group than the control group.According to the flow cytometry assay,the apoptosis rate of the HCE6 mediated PDT group was higher than the control group,indicating that HCE6 mediated PDT could significantly induced the apoptosis of FRH-0201 cells.Western blotting analysis demonstrated that the expression of apoptosis protein caspase-3 and caspase-8 were increased after HCE6 mediated PDT,but there was no significant difference between the other three groups(control group,drug only group,light only group).ConclusionOur study demonstrated that HCE6 may be a promising photosensitizer with a higher efficacy and a shorter incubation time of photosensitizer than HiPorfin and Photofrin for PDT therapy.HCE6 mediated PDT showed an encouraging result for treating human cholangiocarcinoma by inducing the apoptosis of the cholangiocarcinoma cells and increasing the expression of apoptosis protein caspase-3 and caspase-8.