| Esophageal squamous cell carcinoma is a vary common malignant tumor of digestive system with the mortality rate ranking the six in the word.In China,more and more people suffer from this disease and about 15 million die from it.According to incomplete statistics,There are more than 30 million people worldwide are died from this disease every year.So it is essential for us to the study of this disease.Radiation is the most important and effective therapeutic method.Esophageal also belongs to the moderately sensitive tumors to radiotherapy.Although increasing the irradiation intensity could improve the therapeutic effect,the normal tissues might be also damaged and radioresistance might be another major challenge resulting in locoregional recurrence and metastasis.Therefore,how to improve the radiosensitivity of esophageal cancer cell has become a hotpot currently and problem to be solved.Telomerase is a ribonucleoprotein enzyme and high telomerase activity has been found in tumors,especially malignant tumor.So telomerase can be a effective treatment target.It has been extensively reported that targeting telomerase is effective for cancer and the most direct way is to inhabit the activity of telomerase.Imetelstat,a 13-mer oligonucleotide which is covalently modified with lipid extensions,competitively suppresses enzymatic activity of telomerase.Furthermore,it has been document that telomerase activity was remarkably elevated and inhibition of telomerase blocks proliferation of cancer cells both in vitro and in vivo,while there was no documents focusing squamous cell carcinoma although imetelstat has also applied clinically for multiple tumor types,including chronic lymphocytic leukemia,multiple myeloma,breast,lung and so on.So we need to conduct some experiment to verify radiosensitization of imetelstat and the underlying mechanism was studied through the investigation on key proteins in DSBs repair pathway,cell cycle and cell apoptosis of Kyse410 and Kyse520.To solve the problems above,we conduct the following experiment:Firstly,plate clone formation assay were performed to explore the effect of imetelstat on Kyse410 and Kyse520 which exposed to the varying degrees of radiation.We found that the survival rate of after exposure y-photons combined with the treatment of imetelstat was lower than controls.Secondly,we detected the effect of imetelstat and control on cell apoptosis and cell cycle of Kyse410 and Kyse520 cell with flow cytometry.It was found that imetelstat promotes cell apoptosis of Kyse410 and Kyse520 cell.However,imetelstae exhibited no changes in cell cycle.Thirdly,Western blot was used to explore the expression of related proteins which play roles in the DNA repair process,such as y-H2AX,p53,KU70,caspase3 and so on.We found that imetelstat inhibited DSBs repair system.The expression of y-H2AX and p53 was upregulated with the treatment of imetelstat.Finally,we injected Kyse520 cells into nude mice to induce the formtion of tumor.When the size of tumor reach approximately 50 mm3,we added imtetelstat or control combined with irradiation.We found that tumor isolated from mice in the imetelstat+radiation group showed serious pathological changes than other group.In conclusion,the accumulated findings suggested that imetelstat effectively promoted cell apoptosis in vivo and in vitro.In addition,imetelstat also inhibited Kyse520 in vivo.The expression of y-H2AX,p53 and caspase3 related to DNA repair signaling and apoptosis were regulated by imetelstat.This study supported the potential of imetelstat as a radiosensitizer for esophageal squamous cell carcinoma through inducing cell apoptosis and imetelstat might serve as a potential novel therapeutic strategy. |
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