| Autophagy is an evolutionarily conserved intracellular self-catabolic process for maintaining cell homoeostasis by turnover of cellular components. It functions as an endogenous clearing system safeguarding cell integrity, by degrading and recycling long-lived proteins and damaged organelles(such as endoplasmic reticulum, Golgi apparatus and mitochondria) by lysosomal /vacuolar processes. Autophagy can be triggered by diverse stimuli, such as starvation, reactive oxygen species(ROS), pathogens, hypoxia and ionizing radiation(IR), and it has been reported to be an important mechanism, especially in the development of cancers. Autophagy can either play a pro-survival role and deteriorate the cancer therapeutic outcome, or work as programmed cell death to ameliorate overall anti-tumor efficacy, depending on cellular context, strength and duration of stress-inducing signals. It is a trend to promote the anti-tumor efficiency by targeting the autophagy; therefore, it is beneficial for individual therapy to clarify the functions and mechanisms of autophagy in cancer treatment.ATM(Ataxia-telangiectasia mutated) is a serine/threonine protein kinase and a member of the phosphoinositide 3-kinase-related protein kinas e(PI3K) family. The consensus of the ATM phosphorylation motif is hydrophobic-X-hydrophobic- [S/T]-Q. The most important role of ATM is to be the first responder to DNA double strand breaks(DSBs), and to phosphorylate the downstream substrates involved in DNA repair and cell cycle regulation. Interestingly, ATM has recently been shown to regulate autophagy in response to genotoxic and oxidative stimulation. However, functions of ATM in IR-induced autophagy and the underlying mechanisms have remained unclear.Recently, MAPK14(mitogen-activated protein kinase 14) has been reported to share a phosphorylation site with ATM/ATR, at TQ263, after DNA damage by IR. MAPK14 belongs to stress-induced protein kinase and serine/threonine protein kinase,and it could be activated by multiple stimuli. MAPK14 plays an important role in numerous processes including survival, differentiation and cell proliferation. According to many reports, MAPK14 has a dual role in autophagy, both as positive and negative regulator. On the one hand, prolonged inactivation of MAPK14 triggers AMPK-dependent nuclear localization of Fox O3 A and subsequent activation of its target genes, consequently leading to autophagy. On the other hand, activation of MAPK14 induces autophagy by transcriptional regulation of autophagy-related genes. However, up to now, the regulation of ATM to MAPK14 and its specific role in IR-induced autophagy has not been exactly clarified.Objective:To explore the roles of ATM in IR-induced autophagy and further identify whether ATM regulates IR-induced autophagy via MAPK14, hoping to provide the theoretical basis for its application in future cancer treatment.Methods:(1)human cervical cancer cells Hela and human lung cancer cells H1299 were used in this study;(2) the knockdown cell models of ATM or MAPK14 were established by genetic engineering;(3) X-irradiation conditions were as follows: voltage 180 k V, current 18.0 m A, filtration plate 0.25 mm Cu and 1.0 mm Al, distance between target and X source 60 cm, dose rate 0.40 Gy/min;(4) CCK8(cell counting Kit-8) assay and colony formation assay were chosen for cell survival detection and radiosensitivity measurement;(5) MDC staining and GFP-LC3 relocalization was used for autophagy analysis;(6) Western blot was conducted to detect protein expression;(7) Co-immunoprecipitation were implemented to detect protein interaction;(8) Flow cytometry was used for apoptosis analysis.Results:1. IR induced autophagy and ATM phosphorylation at Ser1981 site.In both Hela cell and H1299 cell, the MAPLC3-II/MAPLC3-I ratio increased in a dose-dependent manner(0Gy,2Gy,4Gy and 8Gy)as well as in a time-dependent manner(4h,8h,16 h and 24h), reached its peak at 16-24 h after 8Gy IR. The cells with GFP-LC3 exhibited a significant increase of punctuate cytoplasmic structures(autophagic vesicles) when treated with IR as compared with the mock group. Thepercentage of MDC-positive cells promoted from 17.32% to 54.77% and from 7.83% to 25.93% after 8Gy IR treatment in Hela and H1299 cells respectively. Above data indicated that autophagy was induced by IR in Hela and H1299 cells. At the same time, compared with the sham-irradiation group, the phosphorylation of ATM at Ser 1981 site was observed to arrive at its peak level 30 min after radiation and went back to the basal level 4h.2. Inhibition of ATM reduced expression of MAPK14 and phosphorylation of MAPKAPK2.The silencing of MAPK14 had no effects on the expression of ATM, but the the silencing of ATM decreased the expression of MAPK14 significantly. Similarily, MAPK14 sh RNA decreased the expression of its downstream targeted gene MAPKAPK2, which suggested that MAPK14 might locate downstream of ATM. In addition, KU55933 inhibited the phosphorylation of ATM effectively as well as the phosphorylation of MAPKAPK2, which indicated the function of ATM in MAPK14-MAPKAPK2 pathway.3. Loss of ATM or MAPK14 resulted in hypersensitivity to IR.Hela cells and H1299 cells were pretreated with KU55933 of 100 n M and 700 n M 2h before different doses of IR treatment, and then the colony formation were conducted to detect the radiosensitivity. Compared with DMSO treated cells, KU55933 decreased the D0 value(3.59 vs 3.02 and 2.21 vs 1.59).In cell model group, compared with Psuper group, the value of D0 decreased significantly(Hela-ATMRi:2.77 vs 2.13; Hela-MAPK14Ri: 2.77 vs 2.19; H1299-ATMRi:2.07 vs 1.44; H1299-MAPK14Ri:2.07 vs 1.52), suggesting that ATM as well as MAPK14 may participate into the regulation of radiosensitivity.4. Loss of ATM or MAPK14 had no effect on IR-induced apoptosis.The percentages of apoptotic cells increased after IR in Psuper cells, ATMRi cells and MAPK14 Ri cells respectively( 8.61% vs12.5%, 8.57% vs 14.08%, 9.26% vs13.88%). But there was no significance of the increase degree within three groups, indicating that neither ATM nor MAPK14 had function on IR-induced apoptosis. In addition, the similar results were observed in H1299 cells, which was(6.1% vs 10.2%,5.1% vs 11.8%,5.3% vs 9.8%).5. Inhibition of ATM and MAPK14 reduced IR-induced autophagy in Hela cell.KU55933 treatment: the MAPLC3-II/MAPLC3-I ratio increased after radiation in a dose-dependent manner in DMSO-treated group(0Gy:1.00, 2Gy:1.46, 4Gy:1.36, 8Gy:1.93), while after the pretreatment of KU55933(100n M), the MAPLC3-II/MAPLC3-I ratio showed no change(0Gy:1.92, 2Gy:2.05, 4Gy:2.08, 8Gy:1.40). Compared with DMSO treated cells, MAPLC3-II/MAPLC3-I ratio in KU55933 only treated cells increased significantly(1.00 vs 1.92), but after 8 Gy IR, MAPLC3-II/MAPLC3-I ratio was lower than cells without IR(1.92 vs 1.40). Immunofluorescence results showed a similar result in Hela cells with GFP-LC3 expression. The percentage of puncture-containing cells increased by 124% in control cells, and increased only by 34.2% in cells with KU55933 pretreatment. MDC staining showed same result(202% vs 35.72%). Three results above confirmed that the inhibition of ATM by KU55933 could repress the IR-induced autophagy in Hela cells.Silencing treatment: the MAPLC3-II/MAPLC3-I ratio was observed to increase to 2.71 times as control after IR in Psuper cell, but ATMRi and MAPK14 Ri themselves blocked the conversion of MAPLC3-I to MAPLC3-II, which was 0.81 vs 0.75 and 1.19 vs 0.91 after and before IR. Flow cytometry results showed that IR increased autophagy by 272% in control cells, but only by 53.18% or 24.76% in ATMRi and MAPK14 Ri cells respectively, suggesting that KU55933, silence of either ATM or MAPK14 could repress IR-induced autophagy.6. Inhibition of ATM and MAPK14 reduced IR-induced autophagy in H1299 cell.KU55933 treatment: similar with Hela cell, the percentage of puncture-containing H1299 cells increased by 129% in control cells, and increased only by 29.22% in cells with KU55933(700n M) pretreatment. MDC staining showed same result(231% vs 24%), confirming that the inhibition of ATM by KU55933 could repress the IR-induced autophagy in H1299 cells.Silencing treatment: compared with the mock group, the MAPLC3-II/MAPLC3-I ratio in Psuper cell, ATMRi and MAPK14 Ri were 1.00 vs 2.07, 0.91 vs 0.84 and 2.30 vs 0.65 respectively. In addition, MDC staining results showed that the increse percentage of positive MDC stained cells after IR was 136%, 27% and-55%. Above further suggested the silence of ATM inhibited IR-induce autophagy. silence of MAPK14 promoted the basal autophagy level at the same time inhibited the IR-induced autophagy.7. Inhibition of ATM and MAPK14 blokage the IR induced effect on m TOR pathway.In Hela cells, western blotting analysis showed a significant decrease by 46% in Akt expression, by 59% in phospho-m TOR expression and by 58% in phospho-p S6 K expression in cells treated with IR only. After treatment with 100 n M KU55933, cells only showed a slight decrease in Akt, phospho-m TOR and phospho-p S6 K expression, namely 36%, 28%, and 21%, respectively. At the same time, the decrease in phosphorylation of P70S6 K by m TOR was not observed in ATMRi(0.43 vs 0.34)and MAPK14 Ri cells(0.32 vs 0.33), whereas it was detected in wild type cells(1.00 vs 0.43). These results suggested that IR caused a down-regulation of m TOR function, and this could be decreased by KU55933,silence of ATM and silence of MAPK14.In H1299 cells, western blotting demonstrated significant reduction in phospho-m TOR, phospho-p S6 K and Akt(by 84%, 70% and 50%, respectively) after IR treatment. After combination treatment of 700 n M KU55933 with IR, cells showed slight reduction in phospho-m TOR, phosphop S6 K expression and Akt(by 40%, 38% and 10% respectively). A direct role for ATM and MAPK14 in suppression of m TOR was further demonstrated in ATMRi,whereas Psuper cells significantly repressed m TORC1 in response to IR. MAPK14 Ri cells expressed remarkable reduction in m TOR level first then reversed to elevate after IR treatment, consistent with changes in MAPLC3. These results illustrate that IR caused down-regulation of m TOR signalling which could be inhibited by KU55933, silence of ATM and silence of MAPK14. These results above further demonstrated that ATM and MAPK14 regulate IR-induced autophagy via m TOR pathways.8. The effect of ATM inhibition and MAPK14 inhibition on Beclin 1/PI3 KIII complex.In Hela cell, Beclin1 bond to PI3 KIII in Psuper cells, and the interaction was significantly increased by 1.63 times after IR treatment. Interestingly, in ATMRi and MAPK14 Ri cells, Beclin 1/PI3 KIII interaction decreased significantly from 2.93 to1.36 and from 1.99 to 1.09 respectively after IR treatment. In addition, the interaction of Beclin 1/ATM was observed in Psuper cells but disappeared after knock down of ATM or MAPK14. Also, the interaction of MAPK14/Bcl-2 and MAPK14/Beclin1 were found to increase with IR treatment, but significantly decreased and did not change after knock down of ATM or MAPK14 respectively. Those results together suggested that both ATM and MAPK14 participated in the process of autophagy via partially affecting Beclin 1/PI3 KIII complexes.Beclin1 bound to PI3 KIII in H1299 cells, and the interaction significantly increased by 29.05 times after IR, however, this interaction did not change by IR after KU55933 pre-treatment. Furthermore, the interaction of Beclin1/p-MAPKAPK2 and Beclin1/MAPKAPK2 were detected in Psuper cells, and neither of them changed remarkably after IR treatment; however, both interactions were impaired by IR treatment in ATMRi cells and MAPK14 Ri cells. The above-mentioned results suggest that ATM and MAPK14 might play roles in Beclin1/PI3 KIII complex, which could explain the function of ATM and MAPK14 on IR-induced autophagy.Conclusions:1. IR induced autophagy and activation of ATM in Hela cells and H1299 cells.2. MAPK14 was one of substrates of ATM.3. IR-induced autophagy could be inhibited by silencing of ATM-MAPK14.4. Inhibition of ATM-MAPK14 blocked the IR-induced effect on m TOR pathway, and partialy decreased IR-induced autophagy.5. Inhibition of ATM-MAPK14 affected the Beclin1/PI3 KIII complex and then decreased IR-induced autophagy..6. ATM-MAPK14 regulated the IR-induced autophagy through m TOR pathway and Beclin1/PI3 KIII complex, and finally affected the radiosensitivity. |
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