Study On The Killing Effect Of MPDT On Colorectal Cancer And Its Mechanism In Vitro And Vivo

Colorectal cancer(CRC)is one of the main malignant tumors in human beings.In recent years,with the development of optical fiber,endoscopy and other technologies,photodynamic therapy(PDT)has been widely used in the treatment of colorectal cancer and other lacunar tumors.However,the rapid consumption of oxygen and photosensitizers in clinical treatment has led to a sharp decline in the efficacy of PDT.Metronomic photodynamic therapy(mPDT)has been proposed.In mPDT,both the photosensitizer and light are delivered continuously at low rates for extended periods of time to increase selective tumor cell killing through apoptosis.Objective:The purpose of this study is to explore the killing effect and mechanism of ALA-mPDT on human colorectal cancer cells both in vitro and in vivo,aiming at seeking a highly effective therapy for the treatment of colorectal cancer.Methods:1.In vitro,the human colorectal cancer cell line SW837 was treated with mPDT of different ALA concentrations(0.125,0.25,0.5,1,2mM),light power densities(0.2,0.4,0.8mW/cm2)and light energy densities(2.5,5J/cm2)respectively.The relative survival rate of the cells was detected by MTT analysis,comparing with the aPDT at the same dose;the Annexin V-FITC/PI double staining was used to detect the apoptosis;the Ad-GFP-LC3B kit was applied to monitor the autophagy of the cells at different time points after treatment;the ultrastructure of cells at 3h and 12h after treatment was examined by transmission electron microscope;and the expressions of proteins related to apoptosis and autophagy were detected by Western Blot.2.In vivo,We developed an mPDT suppository kit including ALA and LED suppositories and analyzed its killing effect on rectal tumors in rabbits.Methods:The ALA(10 wt%)suppository was prepared using ALA powder,type 36 semi-synthetic fatty acid glyceride,and azone.The LED suppository was constructed by encapsulating LED units and a circuit in transparent epoxy resin.VX2 cells were injected into the rectal submucosa of rabbits to establish a carcinoma model in situ.mPDT was applied when tumor grew to about 1cm3.The ALA suppository was inserted into the rectal cavity for 30 min of uptaking and had been activated for 1 h by the LED suppository at a power density of 20 mW/cm2.The mPDT process was repeated three times(once a day).MRI was used to monitor tumor growth,histopathology and TUNEL staining were performed at 14 days after mPDT.Results:1.We found that cell death was induced to a greater extent by mPDT than by the same dose of aPDT.The survival rates for mPDT vs.aPDT were 35.2%,32.4%,27.6%,31.6%vs.85.7%,71.1%,67.8%,42.1%after 3,6,12 and 24h PDT,respectively.2.ALA-mPDT induced earlier apoptosis.The apoptosis rates for mPDT vs.aPDT were 43.2%,47.3%,54.7%,50.3%vs.14.6%,17.6%,27.1%,53.2%at 3,6,12,and 24h post-PDT,respectively.The expression of cleaved caspase-3 and cleaved PARP in the mPDT treatment group was significantly higher at 3h and 6h compared with aPDT:2.45、5.83 times(3h)and 2.47,1.21 times(6h),respectively.3.ALA-mPDT induced earlier autophagy.mPDT induced a peak rate of autophagy of 20%at 3h,whereas aPDT induced two smaller peaks at 3h(14.1%)and 12h(15.8%).Advanced autophagosomes were more abundant in mPDT-than aPDT-treated cells and appeared earlier after mPDT(3h)than after aPDT(3-12 h).Western Blot results showed that the ratio of LC3-Ⅱ/Ⅰ at 3h was higher(2.15 times)after mPDT than aPDT.4.mPDT suppository kit was developed successfully.The ALA suppository tended to become soft in 30min at 37℃ and was localized in colorectal tumour after administration.The LED suppository can achieve a stable output with the electric power and the output power density were 0.2W and 20 mW/cm2 respectively.5.The overall response rate was 60%in the mPDT group using the kit.The tumor size was decreased up to about 50%at 7 days post-mPDT and almost eliminated at 14 days.HE staining showed that only 6.16%of the tumor tissue remained after mPDT treatment.TUNEL detection showed that the apoptosis rate was 18.9%.Conclusion:Both in vitro and in vivo experiments showed that ALA-mPDT significantly inhibited the proliferation of colorectal cancer cells.Moreover,mPDT caused more serious and earlier cell death through the mechanism of apoptosis and autophagy compared with aPDT,suggesting that mPDT may be a superior choice than aPDT for the treatment for human CRC.The suppository kit tends to be a revolutionary change for current.