Involvement Of Autophagic Cell Death In Mitotic Catastrophe Synergistically Induced By Cisplatin And ATR Inhibition

Cisplatin is a platinum-containing chemotherapeutic drug,which is widely used in the treatment of various solid tumors such as ovarian cancer,prostate cancer,lung cancer,and nasopharyngeal cancer.Because patients often develop drug resistance that leads to relapse,there is an urgent need to improve the clinical protocols of cisplatin treatment.The cross-linking reaction between cisplatin and DNA strands leads to single-strand DNA break（SSB）.The ATR-Chk1 pathway activated by ss DNA can activate DNA damage response,and the ATR-Chk1 pathway can also activate cell apoptosis,becoming an important mechanism for cisplatin to kill cells.On the other hand,ATR-Chk1 can activate cell cycle checkpoints to cause cell cycle arrest,and at the same time up-regulate DNA repair activity,stabilize and restart collapsed DNA replication forks,etc.,and promote cell survival.Therefore,the signal pathway activated by DDR is one of the main mechanisms of cisplatin resistance.In view of the fact that the ATR-Chk1 pathway activated by ss DNA is the main DDR pathway upregulated by cisplatin,inhibiting ATR may improve the efficacy of cisplatin and significantly reduce cisplatin resistance.Currently,multiple clinical trials are testing the therapeutic effect of cisplatin combined with ATR inhibitors on various malignant tumors.In the case of DNA damage,inhibiting ATR will cause the G₂/M cell cycle checkpoint and DNA repair and DNA replication fork restart to be inhibited,causing cells to enter mitosis with damaged or unreplicated DNA.Cells with DNA damage and genomic defects cannot meet the Spindle Assembly Checkpoint（SAC）and other reasons and experience long-term mitotic arrest,leading to mitotic catastrophe,which in turn activates the cell death mechanism.However,the mode and regulation mechanism of cell death under mitotic catastrophe are still unclear.Therefore,this study used the mitotic disaster caused by cisplatin combined with the ATR inhibitor VE822 as a model to explore the role of autophagic cell death and apoptosis in the mitotic catastrophe.First,we tested the sensitivity of various cancer cell lines to cisplatin by MTT experiment.The results showed that the human colorectal cancer cell line SW480 and breast cancer cell line MDA-MB-231 were the most sensitive to cisplatin,with an IC₅₀value of 26.39μM and 73μM,respectively.Subsequently,SW480 and MDA-MB-231cancer cells were treated with 5μM cisplatin,a concentration that was much lower than the IC₅₀,for 24 and 48 h.No significant inhibitory effect was seen,however,cell cycle analysis showed significant G₂ arrest,suggesting that activation of the G₂/M cell cycle checkpoint and DNA repair mechanisms by DDR protected the cancer cells from the cytotoxicity of 5μM cisplatin.Strikingly,inclusion of non-cytotoxic 1μM VE822,an ATR inhibitor,significantly sensitized the SW480 and MDA-MB-231 cancer cells to 5μM cisplatin,as shown by both MTT and colony formation experiments.The combination of non-cytotoxic 5μM cisplatin and 1μM VE822 significantly inhibited the survival of the two types of cancer cells.The combination index（CI）of cisplatin and VE822 calculated by MTT experiment was below 1,demonstrating significant synergy between the two drugs.Microscopic observation after DAPI staining revealed that the combination of cisplatin and VE822 significantly increased the number of micronuclei and grape-shaped nuclei,suggesting that the combination of the two drugs caused severe mitotic catastrophe.When a mitotic catastrophe occurs,a large amount of cellular DNA enters the cytoplasm,thereby activating cGAS-STING pathway.It has been reported in the literature that activated STING can activate autophagy.Western Blot detection found that the combination of cisplatin and VE822 caused strong and time-dependent autophagy.sh RNA-mediated knockdown of cGAS or STING could prevent autophagy,which proved that the autophagy caused by the combination of cisplatin and VE822was mediated by the cGAS-STING signaling pathway.The traditional view is that under starvation or stress,autophagy is a self-protection mechanism that promote cell survival.Next,we used the autophagy inhibitor Bafilomycin A1（Baf-A1）or knockdown of cGAS and STING to inhibit autophagy.The MTT experiment proved that the inhibition of autophagy lead to a significant increase in cell survival,indicating that under the cisplatin and VE822-mediated mitotic catastrophe,autophagic cell death was the main mode of cell death.Similar to apoptosis,the cell membrane will also turn over in the late stage of autophagic cell death,and cells with Annexin V staining will appear,making it indistinguishable from apoptosis.Flow cytometry analysis found that inhibition of autophagy,but not apoptosis by the caspase3 inhibitor Z-VAD-FMK,significantly reduced the number of Annexin V-positive cells,further proving that autophagy,not apoptosis,was the main mechanism of cell death under mitotic catastrophe,and that inhibition of apoptosis did not affect the occurrence of autophagic cell death.In summary,our results showed that cisplatin caused DNA single-strand breaks and the ATR inhibitor VE822 abolished the G₂/M cell cycle checkpoint to block the G₂arrest caused by cisplatin.Therefore,the combination of cisplatin and VE822 resulted in mitotic catastrophe to generate a large amount of cytoplasmic DNA,which activated the cGAS-STING signaling pathway,leading to autophagic cell death.Under the cisplatin and VE822-induced mitosis catastrophe model,autophagy,but not apoptosis,was the mode of cell death.Therefore,this study used the ATR inhibitor VE822combined with the small molecule chemotherapeutic drug cisplatin to establish a cancer cell mitotic catastrophe model and clarified the mode and molecular mechanism of cell death,thus providing valuable information for research on the induction of cell death and drug resistance.