Experimental Study On Targeted Killing Of Colorectal Cancer Stem Cells Through Notch Signaling Pathway By CD133-Pc

ObjectiveTo study the effect of photodynamic therapy on the biological characteristics of human colorectal cancer stem cell-like cells(CRC-CSCs)by CD133-Pc(CD 133 polypeptide-phthalocyanine conjugate)in vitro,and to explore the molecular mechanism.Methods1.Enrichment and characterization of CRC-CSCs.CD133+colorectal cancer cells were isolated by immune-magnetic bead based cell sorting.CRC-CSCs were enriched by suspension culture using stem cell conditioned medium.The expression of the CRC-CSCs marker CD 133 was detected by flow cytometry.The self-renewal ability of CRC-CSCs was verified by limiting dilution assay in vitro.The levels of stermness associated proteins were detected by Western blot analysis and immunofluorescence.The tumorigenicity of CRC-CSCs was verified by subcutaneous transplanted tumor model in nude mice.2.Observation of endocytosis and subcellular localization.Parental colorectal cancer cells and CRC-CSCs were incubated with CD133-Pc and organelles-specific fluorescent probes were used to localize the mitochondrion,the lysosome,or the ER.The endocytosis or subcellular localization of CD133-Pc was observed by fluorescence microscopy or laser-scanning confocal microscopy.3.Study on photodynamic therapy in vitro.Immortalized colorectal epithelial cells,parental colorectal cancer cells or CRC-CSCs were treated with different concentrations of CD133-Pc under normoxia condition.The cell viability was detected by Alamar Blue assay.The mammoshphere formation and self-renewal ability of parental colorectal cancer cells and CRC-CSCs was determined by suspension culture using stem cell conditioned medium.The changes of protein levels in related pathways were detected by Western blot analysis.The level of ROS was detected by flow cytometry after incubation with DCFH-DA.Apoptosis was determined by flow cytometry after Anexin V/PI staining.The role of ROS and autophagy in photodynamic therapy was verified by co-treatment with ROS and autophagy inhibitors.4.Overexpression of Notchl The lentiviral vector carrying Notch1 was used to infect CRC-CSCs.The stable Notchl-overexpressing CRC-CSCs cell line was constructed by Puromycin screening.Results1.CRC-CSCs were enriched from HT29 and SW620 by magnetic bead-based cell sorting and ultra-low adhesion suspension culture.The positive rates of CD133 in HT29 and SW620 were 86.1%and 98.6%as evaluated by flow cytometry.The CD133,c-Myc,Nanog,and Oct4 protein levels were increased in CD133+ HT29 and SW620 cells.The results of tumor transplantation in nude mice were confirmed that CRC-CSCs had two orders of magnitude higher tumorigenicity than parental cells.2.The endocytosis efficiency of CD133-Pc was higher in CRC-CSCs(or cell with high expression of CD 133),and there was obvious accumulation of CD 133-Pc in the mitochondrion,lysosomes and endoplasmic reticulum.3.With the increase of CD133-Pc concentration,the viability of parental cells and CRC-CSCs was decreased.However,the inhibitory effect of CD133-Pc PDT on colorectal cancer cells was greater than that on immortalized colorectal epithelial cells.The inhibitory effect of CD133-Pc is greater than that on the traditional photosensitizer(Photosan and Pc).The inhibitory effect of CD133-Pc PDT on CRC-CSCs was increased with the enhancement of laser intensity.4.The mammoshphere formation ability and self-renewal of parental Colorectal cancer cells and CRC-CSCs was inhibited by CD 133-Pc PDT.The protein levels of Notchl and Nanog were reducted in CRC-CSCs after treatment with CD 133-Pc PDT.Over-expression of Notchl in CRC-CSCs resulted in resistance to CD133-Pc PDT.5.After CD133-Pc PDT of CRC-CSCs the level of intracellular ROS was increased with the increase in the concentation of CD133-Pc.In addition,the protein levels of Nrf 2 and Keap1 were increased with the increase in the the concentration of CD133-Pc.The inhibitory effect of CD133-Pc PDT on the viability of CRC-CSCs could be reversed by ROS inhibitor.6.After CD133-Pc PDT of CRC-CSCs the rate of apoptotic cells was increased with the increase in the concentation of CD 133-Pc.7.After photodynamic treatment of CRC-CSCs with different concentrations of CD133-Pc,autophagy of CRC-CSCs were promoted.The inhibitory effect of CD 133-Pc PDT on the viability of CRC-CSCs could be reversed by autophagy inhibitor.Conclusions1.The CRC-CSCs were enriched by magnetic bead-based cell sorting;CD 133+ cells possess characteristics of cancer stem cells.2.CRC-CSCs were killed by CD133-Pc PDT with much higher efficiency than PDT with traditional photosensitizers in vitro.3.The mechanism of the damage effect of CD133-Pc PDT is include CD 133-Pc endocytosis followed by ROS production after laser irradiation,which results in inhibition of the Nocth signaling pathway and inhibit the stemness of CRC-CSCs and eventual induction of programmed cell death.