Wnt/β-catenin Signalling Pathway Is Involved In Regulating Carbon Ions To Overcome The Radioresistance Of Quiescent HeLa Cells

The presence of quiescent cancer cell populations is one of the reasons for tumor radiation resistance,and their proliferative activity when triggered can lead to cancer recurrence and metastasis.Conventional radiotherapy is mainly based on low-LETγ-rays,electron beams or X-ray,which have limited lethality to quiescent cancer cells.In recent years,the continuous development of heavy ion cancer treatment technology has brought more hope to overcome the radiation resistance of cancer cells and improve the curative level of tumor radiotherapy.However,the effectiveness and mechanism of heavy ions in overcoming the radio-resistance of quiescent cancer cells is currently unknown.In this study,human cervical cancer HeLa cells were studied and DNA damage was used as an entry point to firstly investigate the biological effects of proliferating and quiescent HeLa cells exposed to X-ray and carbon ion beam irradiation.Quiescent HeLa cells have strong radio-resistance and DNA damage repair ability.We found that the cell activity and clonal survival rate of quiescent HeLa cells were significantly higher than those of proliferating cells after X-ray or carbon ion beam irradiation under the same dose conditions,and the irradiation induced lessγ-H2AX foci formation,while the damage peak was shifted back,indicating that quiescent cancer cells have stronger radiation resistance.In addition,the expression of DNA damage repair factors in quiescent HeLa cells was also significantly higher than that in proliferating cells,suggesting that quiescent cancer cells have stronger DNA damage repair ability.Meanwhile,the expression analysis of key regulatory genes of the DNA repair pathway revealed that the DNA damage repair pathways of proliferating and quiescent HeLa cells were different after carbon ion beam irradiation,and we hypothesized that proliferating HeLa cells mainly relied on the NHEJ pathway,while quiescent HeLa cells mainly dependent on the HR pathway.Carbon ion beam can effectively eliminate quiescent HeLa cells.We observed a stronger inhibitory effect of carbon ion beam radiation on the proliferation and clonogenic ability of quiescent HeLa cells compared to X-ray,indicating that quiescent cells are more sensitive to carbon ion radiation.The carbon ion beam can directly kill quiescent HeLa cells by inducing complex DNA damage.The fractions of the activity released（FAR）induced by the two kinds of radiation was in the order of carbon ion beam/proliferating cells>carbon ion beam/quiescent cells>X-ray/proliferating cells>X-ray/quiescent cells.Meanwhile,the results showed that carbon ion beam irradiation induced more focal points of bothγ-H2AX and 53BP1 at each different time point,and the carbon ion-induced focal points lasted longer,indicating that carbon ions can induce more severe DNA double-strand break damage.Furthermore,we found that carbon ion beam significantly induced cycle re-entry in quiescent HeLa cells,thereby increasing their sensitivity to radiation.At 24 h after carbon ion beam irradiation,we could observe a significant G₂/M phase arrest in quiescent cells.When DNA damage is too much to be repaired,an apoptotic program is initiated to remove the damaged cells.Our results suggest that carbon ion beam may regulate apoptosis in quiescent HeLa cells by activating the mitochondrial apoptotic pathway.The high expression ofβ-catenin in cervical cancer suggests thatβ-catenin may have an important role in the development of cervical cancer.To investigate whether the Wnt/β-catenin pathway is involved in regulating the radiosensitivity of tumor cells,we conducted a study on the effects of carbon ion beam alone or in combination with the Wnt/β-catenin pathway inhibitor XAV939 or the Wnt/β-catenin pathway agonist HLY78 on the radiosensitivity of HeLa cells.The results showed that the Wnt signaling pathway inhibitor XAV939 could enhance the radiosensitivity of HeLa cells by promoting DNA damage,G₂/M phase arrest and apoptosis induced by carbon ion beam radiation,while the Wnt pathway agonist HLY78 could protect HeLa cells from damage by alleviating the above effects induced by carbon-ion beam.It is suggested that the classica Wnt signaling pathway is involved in regulating the radiosensitivity of human cervical cancer HeLa cells,and it may be a promising molecular target for cervical cancer therapy.In addition,we further explored the role of the classical Wnt signaling pathway in overcoming the radiation resistance of quiescent HeLa cells by heavy ion beam and analyzed its possible molecular mechanism.First,we found that the silenced Wnt signaling pathway may be a necessary factor to maintain the dormant state of quiescent cancer cells.We could reduce the likelihood of tumor recurrence and metastasis by suppressing the activation of Wnt//β-catenin pathway to keep quiescent cancer cells in a permanent dormant state.Secondly,we observed that inhibition of the Wnt/β-catenin signaling pathway could improve the radioresistance of quiescent HeLa cells to carbon ion beam by alleviating carbon ion beam-induced DSB damage,improving DNA damage repair,maintaining quiescent depth,and inhibiting IR-induced apoptosis,and therefore we hypothesized that the classical Wnt signaling pathway was involved in carbon ion beam overcoming the radioresistance of quiescent HeLa cells.