Study On The Role And Mechanism Of Indisulam In The Radiation Sensitivity Of Human Cervical Cancer HeLa Cells

Cervical cancer is a common tumor in women,which seriously affects women’s reproductive health.Radiotherapy is the main treatment method.The radiation resistance of cervical cancer is a very difficult scientific problem in the field of radiation therapy,so how to enhance the sensitivity of cervical cancer cells to radiotherapy is very important.Recently,the pre-m RNA splicing patterns change plays a key role in cancer and its treatment.Therefore,using the drug targeting the process may be an effective method to improve the sensitivity of cancer cells to radiation.Indisulam（N（3-chloro-7-indolyl）-1,4-benzenedisulfonamide,E7070）,a splicing regulator of sulfonamides,has shown anti-tumor activity in many tumor cells,but its role in radiation sensitivity and mechanisms in cervical cancer cells remain unknown.We used splicing regulator sulfonamides indisulam to treat human cervical cancer HeLa cells and researched the function,mechanisms of indisulam in HeLa cells.We discussed the regulation of indisulam on proliferation,survival,cell cycle and apoptosis of HeLa cells from three regulation levels such as transcription,alternative splicing,protein,and further studied its influence on the radiation sensitivity of human cervical cancer HeLa cells.Our results showed that:（1）The proliferation of human cervical cancer HeLa cells was significantly inhibited with higher concentration and the prolongation of treatment time in the range of 0-5μM indisulam,and the survival of human cervical cancer HeLa cells was significantly reduced at high concentration.Sequencing analysis of HeLa cells treated with 5μM indisulam showed that differential genes or proteins were related to cycle arrest,apoptosis,alternative splicing regulation and post-translational modification.Further verification showed that indisulam could induce G1/S phase arrest of HeLa cells,which was related to inhibition of Cyclin E1 and cyclin dependent kinase CDK2.It was also found that indisulam could induce the splicing factor RBM39degradation through ubiquitin pathway,leading to the imbalance of splicing isomer expression of p73,and thus triggering mitochondrial pathway-mediated apoptosis of HeLa cells.In addition,indisulam could inhibit the growth of xenograft tumor of human cervical cancer HeLa cells.It is suggested that indisulam may be used as a candidate drug in the treatment of human cervical cancer.（2）Human cervical cancer HeLa cells were treated by low concentration of indisulam combined with radiation.The study indicated that the proliferation and clonal survival rate of human cervical cancer HeLa cells treated by 1.25n M indisulam combined with 2Gy X ray were lower than those treated by X ray alone,and it could significantly induce the cycle arrest of HeLa cells and further promote the incidence of apoptosis.In addition,1.25n M indisulam combined with 2Gy X-ray could significantly inhibit the expression of splicing factor RBM39 in cervical cancer HeLa,and up-regulate the pro-apoptotic protein TAp73,down-regulate apoptosis suppression factorΔNp73 expression,which showed that p73 splicing isomers might be related to apoptosis induced by indisulam and radiation.In the same way,1.25n M indisulam combined with 2Gy ¹²C⁶⁺ion beam irradiation could significantly suppress the survival of human cervical cancer HeLa cells,induce the cycle arrest and apoptosis of HeLa cells,and also could down-regulate the expression of alternative splicing factor RBM39.The results showed that the low concentration of indisulam combined with X-ray or¹²C⁶⁺ion beam radiation could enhance the radiation sensitivity of HeLa cells,suggesting that indisulam might be used as a new type of radiotherapy sensitization drug for cervical cancer and applied in clinic.