| Radiation therapy is an effective method for clinical treatment of cervical cancer, but the unsatisfactory radiosensitivity usually leads to poor prognosis. With the development of molecular biology and the research on mechanisms of radioresistance, certain genes have been found to affect the radiosensitivity of tumor cells, consequently influence the radiation-induced death in tumor cells. Many studies show that radiation can induce autophagy which functions as not only an adaptive response to protect tumor cells against adverse environment, but also the typeⅡprogrammed cell death to prevent tumor growth.Heat shock protein90α(HSP90α) is a kind of molecular chaperone that can protect protein spatial structure and guarantee the normal function of the protein molecules. Inhibition of the activity of HSP90α can lead to its client proteins degration. Deoxycytidine kinase(d CK) is a rate limiting enzyme critical for phosphorylation of endogenous deoxynucleosides for DNA synthesis. The study has confirmed that specific block of d CK expression by si RNA can enhance the radiosensitivity of cervical cancer. In this study, we focused on the roles of HSP90α/d CK in radiation-induced autophagic cell death in Hela cells. ObjectiveThis study is aiming to explore the effect of HSP90α/d CK on the radiation-induced autophagic death in Hela cells so as to find novel targets for the radiotherapy in cervical cancer. Methods(1)Cell lines: Human cervical cancer cell line Hela was used in the study;(2)Irradiation condition: X-320 ix RAD irradiator was used and the irradiation conditions were as follows, voltage 180 k V, current 20.0 m A, filtration plate 2.0 mm Al, distance between target and X source 70 cm, dose rate 1.0 Gy/min;(3)Calcium phosphate transfection method was used to create HSP90αRi、d CKRi and AKTRi models, liposomes transfection method was used to create over expression of AKT cell model;(4)Protein expression was analyzed by Western blot;(5)Autophagy was analyzed by FACS assay;(6)Cells viability was evaluated by MTT method;(7)The interaction of proteins was analyzed by immunofluorescence staining and Co-IP;(8)SPSS software and Student t-test was used for statistical analysis, p<0.05 was considered the difference was statistically significant. Results1. IR induced autophagic death and decreased HSP90α expression in Hela cellsCompared with sham-irradiation group, the expression of P62 and HSP90α decreased and the ratio of LC3Ⅱ/LC3Ⅰincreased at 24 h after 8 Gy. FACS assay showed autophagy was higher than sham-irradiation group at 24 h after 8 Gy treatment(P<0.05). MTT assary showed that cells viability was lower than sham-irradiation group at 72 h after 8 Gy treatment, but CQ treatment obviously inhibit the radiation-induced cell death(P<0.05).2. Effects of HSP90α on the radiation-induced autophagic death in Hela cellsHSP90αRi cell model was established.(1)Compared with control p SUPER group, the expression of P62 decreased and the ratio of LC3Ⅱ/ LC3Ⅰ increased in HSP90αRi group. Compared with sham-irradiation group, after 8 Gy treatment the expression of P62 decreased 19.58% in control p SUPER group and 97.58%in HSP90αRi group. Compared with sham-irradiation group, after 8 Gy treatment the ratio of LC3Ⅱ/ LC3Ⅰ increased 23.38% in control p SUPER group and 47.04%in HSP90αRi group.(2)FACS assay showed that 8 Gy treatment increased autophagy by 43.40% in control p SUPER group and 55.92% in HSP90αRi group(P<0.05).(3)MTT assary showed that 8 Gy treatment decreased cells viability by 19.29% in control p SUPER group and 31.24% in HSP90αRi group(P<0.05). These results indicate that HSP90α silence obviously promoted the radiation-induced autophagic death in Hela cells.3. Effects of d CK on the radiation-induced autophagic death in Hela cellsd CKRi cell model was established.(1)Compared with control p SUPER group, the expression of P62 decreased and the ratio of LC3Ⅱ/ LC3Ⅰ increased in d CKRi group. Compared with sham-irradiation group, after 8 Gy treatment the expression of P62 decreased by 38.60% in control p SUPER group and 41.11%in d CKRi group. Compared with sham-irradiation group, after 8 Gy treatment the ratio of LC3Ⅱ/ LC3Ⅰ increased by 22.24% in control p SUPER group and 23.74%in d CKRi group.(2)FACS assay showed that 8 Gy treatment increased autophagy by 21.23% in control p SUPER group and 60.21% in d CKRi group(P<0.05).(3)MTT assary showed that 8 Gy treatment decreased cells viability by 19.29% in control p SUPER group and 28.83% in d CKRi group(P<0.05). These results indicate that d CK silence obviously promoted the radiation-induced autophagic death in Hela cells.4. HSP90α interacted with d CK maintaining the expression of AKT in Hela cellsImmunofluorescent staining revealed co-localization of HSP90α and d CK. Co-IP revealed HSP90α interacted with d CK and 8 Gy treatments decreased their interaction. HSP90α silence obviously decreased the expression and the activity of d CK. HSP90α colocalizated with AKT. Both HSP90α silence and d CK silence obviously decreased the expression of AKT. These results indicate that d CK is likely to be a client protein of HSP90α, and they collectively decreased the expression of AKT.5. Effects of d CK on the radiation-induced autophagic death in Hela cellsAKTRi cell model and AKT re-store cell model were established.(1)Compared with control p SUPER group, the expression of P62 decreased and the ratio of LC3Ⅱ/ LC3Ⅰ increased in AKTRi group. Compared with sham-irradiation group, after 8 Gy treatment the expression of P62 decreased by 51.49%in control p SUPER group and 59.36%in AKTRi group. Compared with sham-irradiation group, after 8 Gy treatment the ratio of LC3Ⅱ/ LC3Ⅰ increased by 29.79% in control p SUPER group and 58.98%in AKTRi group. After overexpression of AKT, as compared with control Vector group, the expression of P62 increased and the ratio of LC3Ⅱ/ LC3Ⅰ decreased in AKT overexpression group. Compared with sham-irradiation group, after 8 Gy treatment the expression of P62 decreased by 21.21%in control Vector group and 8.35%in AKT overexpression group. Compared with sham-irradiation group, after 8 Gy treatment the ratio of LC3Ⅱ/ LC3Ⅰincreased by 45.60% in control Vector group and 5.88%in AKT overexpression group.(2)MTT assary showed that 8 Gy treatment decreased cells viability by 23.65% in control p SUPER group and 46.21% in AKTRi group. After overexpression of AKT, 8 Gy treatment decreased cells viability by 30.21% in control vector group and 13.73% in AKT overexpression group(P<0.05). These results indicate that AKT silence obviously promoted the radiation-induced autophagic death and AKT overexpression inhibited the radiation-induced autophagic death in Hela cells. Conclusion1. IR induced autophagic death and decreased HSP90α expression in Hela cells2. d CK was likely to be a client protein of HSP90α, its stability and function needed the maintenance of HSP90α.3. HSP90α/d CK may regulate the radiation-induced autophagic death by maining the expression of AKT. |
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