Mechanism Of MMB Inducing G2/M Block In Colorectal Cancer Cells Via ATM-WEE1 Pathway

Colorectal cancer(CRC),also known as colorectal cancer,is a clinically common gastrointestinal malignant tumor,usually related to dietary changes and environmental factors and has a high morbidity and mortality.According to the cancer data statistics of 165 countries in 2018,the incidence and mortality of colorectal cancer in malignant tumors are among the top three in the world.The treatment of colorectal cancer is mainly surgery,radiotherapy and chemotherapy Commonly.Among them,fluorouracil,irinotecan and oxaliplatin,which are common chemotherapeutics,can predict hepatotoxicity.Therefore,the development of a highly effective and low toxicity drug for colorectal cancer is urgent.DNA stores the genetic information that organisms depend on for survival and reproduction which is very important to cells,so maintaining the integrity of DNA is the basis.When cancer cells are damaged by reactive oxygen species,chemicals,radiation and other external factors,it is easy to induce abnormal gene replication.If the abnormal gene replication is not repaired,it may damage the integrity of genome and lead to gene mutation.DNA damage types include point mutation,deletion,insertion,inversion or transposition,double bond break.When DNA double bond breaks,ataxia telangiectasia mutant ATM will recognize its breaking point and transmit DNA damage signal through its own Ser1981 phosphorylation.Cell DNA up-regulates repair pathway related proteins and initiates DNA repair.Cell cycle regulatory protein cascade regulates the process of cell cycle,thus cell cycle is blocked and mitosis is stopped,thus preventing the further expansion of damaged DNA information.Resveratrol,a plant hormone,has significant pharmacological activities in anti-tumor,anti-inflammatory and anti-oxidative damage.Resveratrol can induce apoptosis,necrosis,autophagy and cycle arrest of cancer cells.In the previous study,we found that resveratrol analogues(E-3-((4-methoxyphenylimino)methyl)benzene-1,2-diol(MMB)significantly inhibited the growth of colorectal cancer in vivo and in vitro,which has potential medicinal development value.Therefore,the mechanism of MMB blocking colorectal cancer was studied from apoptosis,necrosis,iron death,cycle,DNA damage,autophagy and so on.All the contents of this project are the first study,the results will provide new choice and reference for colorectal cancer treatment and resveratrol analogues research and developmentIn this study,we investigated the inhibitory effect of MMB on the growth of colorectal cancer in vitro and in vivo and further studied the effects of MMB on apoptosis,autophagy,DNA damage and cell cycle arrest of colorectal cancer cells to determine the exact pharmacological mechanism of MMB on the growth of colorectal cancer cellsWe will carry out the research through the following methods:1.MTT and plate clone crystal violet staining were used to determine the inhibition of MMB on colorectal cancer cell viability at the cell level.2 The toxicity of MMB intervention on healthy nude mice was determined by weight change,organ coefficient,tissue staining and blood biochemical analysis.3.The tumor bearing model of colorectal cancer in nude mice was established by subcutaneous injection.The inhibition effect of MMB intervention on the growth of colorectal cancer was verified by the changes of body weight,tumor volume and tumor weight,and organ coefficient.4.Annexin V-FITC/PI double staining was used to determine the effect of MMB intervention on the apoptosis of colorectal cancer cells,and further study the effect of MMB on the ferroptosis,necrosis and apoptosis of iron with small molecular inhibitors such as iron death,necrosis and apoptosis.5.PI staining,Western blot,RT-PCR,ATM and WEE1 small molecule inhibitor intervention were used to determine the effect of MMB intervention on ATM mediated periodic blocking signal pathway.We have the following results:1.MMB significantly inhibited the activities of DLD-1,SW480 and HCT116 cells in vitro.We used MTT method to study the effects of MMB on the cell viability of three colorectal cancer cells at the concentrations of 0,6.25,12.5,25,50,100 and 200 μM respectively.The IC50 of MMB on DLD-1,SW480 and HCT116 cells was 52.61,74.20 and 59.95 respectively.It was further confirmed that MMB inhibited the activity of colorectal cancer cells.2.MMB has no significant toxicity to healthy nude mice.BALB/C nude female mice were selected as experimental animals.The toxic effect of MMB on healthy nude mice in vivo was studied after 21 days of treatment with 1 mg/kg/3d MMB via caudal vein.The results showed that MMB intervention had no significant effect on the body weight of nude mice compared with the control group.There was no significant difference in the determination of alanine aminotransferase,glutamic oxaloacetylase,creatinine and urea nitrogen.There was no significant difference in the organ coefficients of heart,liver,spleen,lung,kidney and HE staining of tissue sections The above results showed that MMB had no significant toxicity to healthy nude mice.3.MMB significantly inhibited the growth of colorectal cancer bearing tissues.In order to study the effect of MMB on the growth of tumor bearing nude mice in vivo,we established a subcutaneous tumor bearing model,which was treated with 1 mg/kg/3d MMB via tail vein for 21 days.The results of tumor bearing experiment show that compared with the normal saline control group,MMB intervention did not significantly affect the body weight of nude mice,and there was no significant difference in organ index of heart,liver,spleen,lung and kidney.The tumor volume and mass of nude mice were significantly reduced.The HE staining results of tumor tissue sections showed that after MMB intervention,the tumor cells were characterized by atrophy of blood vessels,increase of nuclear volume and lack of cell division.The difference of tumor volume and mass between normal saline control group and MMB intervention group was statistically significant,indicating that MMB significantly inhibited the growth of colorectal cancer tissue.4.MMB did not inhibit the activity of rectal cancer cells mainly through apoptosis,necrosis,iron death etc.Annexin V-FITC/PI double staining was used to analyze the effect of MMB intervention on the apoptosis level of DLD-1,SW480 and HCT116 cells.The results showed that MMB did not cause significant apoptosis of three kinds of cells.Furthermore,Western blot confirmed that MMB did not significantly affect the expression of apoptosis related proteins BCL-2,Caspase 3,Caspase 8,Bax and PARP-1.We used Z-VAD-FMK(3 μM),Necrostatin 1(20 μM),Ferrostatin-1(2 μM)and MMB(gradient concentration)respectively to intervene DLD-1,SW480 and HCT116 cells for 48 h,and then measured the cell viability.The results showed that the inhibition of MMB on colorectal cancer cells was not significantly reversed.The results showed that MMB did not inhibit the activity of colorectal cancer cells through apoptosis,necrosis and iron death.5.MMB significantly induces G2/M arrest of colorectal cancer cells through ATM-WEE1 pathway.Flow cytometry analysis of PI staining showed that MMB significantly induced G2/M arrest in DLD-1,SW480 and HCT116 cells.Western blot showed that MMB significantly up-regulated the phosphorylation level of CDK1 Tyrl5 in G2/M phase,up-regulated the phosphorylation level of CDC25C Ser216 and the phosphorylation level of WEE1 Ser642 could increase the phosphorylation level of CDK1 Tyr15 and cause G2/M phase block.Further study confirmed that MMB significantly upregulated the phosphorylation of CDC25C Ser216 and WEE1 Ser642.ATM can up-regulate the phosphorylation level of CDC25C Ser216 by activating CHK1 and eventually lead to G2/M phase arrest.CGK733(10 μM),an ATM inhibitor,and MK1775(1 μM),a WEE1 inhibitor,significantly reduced the phosphorylation of CDK1 Tyr15,and reversed the MMB induced DLD-1,SW480,HCT116 cells G2/M arrest.These results indicate that MMB significantly induces G2/M arrest of colorectal cancer cells through ATM-WEE1 pathway.6.MMB induced autophagy of colorectal cancer cell line DLD-1.Western blot showed that MMB up-regulated LC3II expression,down-regulated p62 expression and increased ULK Ser317 phosphorylation level,which caused autophagy.At the same time,the phosphorylation level of AMPK increased and mTOR decreased.The results indicated that MMB could induce autophagy of colorectal cancer cells through ATM-AMPK pathwayWe come to the following conclusion:1.MMB inhibited the activities of DLD-1,SW480 and HCT116 cells in a dose-dependent manner.2.MMB significantly inhibited the growth of colorectal cancer in vivo.3 MMB induces G2/M arrest of colorectal cancer cell cycle through ATM-WEE1 pathway.4.MMB inhibition of colorectal cancer cell viability is related to its induction of autophagy.