Study On Apoptosis Mechanism Of Human Colorectal Cancer SW620 Cells Through ROS-mediated By Pentoxifylline

Objective:Colorectal cancer is one of the malignant tumor in the gastrointestinal tract.The global cancer statistics in 2020 reflected that the incidence and mortality in China accounted for 12.2% and 9.5%,respectively.In addition,colorectal cancer is becoming younger due to unhealthy lifestyle.The current treatment is still based on surgical resection and chemotherapy,but it has many disadvantages,such as high cost of treatment,high recurrence and side effects,function damage and complications.Compared with surgical resection and chemotherapy,targeted therapy has less side effects and higher pertinence,but it is also restricted of clinical treatment because of resistance.Therefore,there is an urgent to develop new therapeutic drugs with cheap,efficient and low toxic.Pentoxifylline(PTX),a methylxanthine derivate extracted from cocoa beans,is well known for the treatment of cerebral blood circulation disorders,peripheral vascular diseases,sudden deafness,intermittent claudication and other diseases.Recently,it has been proved to have anticancer activity,which can inhibit the proliferation of a variety of cancer cells.However,the molecular mechanism in colorectal cancer is not clear.Thus,in this study,SW620 colorectal cancer cells were used to study the mechanism of PTX induced apoptosis,which provides a theoretical basis for PTX possible is a potential drug,and also provides a new strategy for the clinical treatment of colorectal cancer.Methods:SW620 cells were stimulated with different concentrations of PTX.1.The morphological changes of SW620 cells were observed by microscope.The effect of PTX on cell proliferation of SW620 cells was determimned by MTT assay.2.SW620 cells were stained by Annexin V-FITC/PI,PI single staining and DCFH-DA probe respectively.The rate of apoptotic,cycle distribution and intracellular ROS were measured by flow cytometry.DAPI staining were used to measure apoptosis after PTX incubation.3.Westren blot(WB)was used to masure the protein content of cell apoptosis,cell cycle and autophagy-related proteins in SW620.4.SW620 cells were pretreated by NAC(N-acetyl-L-cysteine,ROS scavenge)and 3-MA(3-Methyladenine,autophagy inhibitor),and the effects of intracellular ROS on cell proliferation,apoptosis,autophagy and AKT/GSK-3β signaling pathway were detected by MTT and WB assays.Results:1.MTT assay revealed that PTX inhibited the proliferation of human colorectal cancer cells in a concentration-dependent manner,and observed through microscopethe,the morphology of SW620 cells changed significantly with the increase of PTX concentration.2.Flow cytometry results suggested that PTX mediated G0/G1 phase arrest in SW620 cells.WB results also showed that PTX upregulated the expression levels of p21 and downregulated cyclin D1 protein.3.Flow cytometry certified that PTX induced cell apoptosis in SW620.With the increase of PTX concentration,DAPI staining observed that the blue fluorescence became stronger and the number increased.4.The results of flow cytometry indicated that PTX promoted the intracellular ROS in a concentration dependent manner.Additionally,MTT assay results suggested that NAC pretreatment increased the viability of SW620 cell and decreased the inhibition of PTX on SW620 cell proliferation.5.According to the WB results,PTX induced the expression of Cleaved-PARP protein,controlled AKT and GSK-3β phosphorylation,thereby inhibited AKT/GSK-3β signaling pathway.After NAC treatment,p-AKT and p-GSK-3β protein can be expressed in SW620 cells,but total AKT and GSK-3β had no significant change.6.WB assay demonstrated that with the increase of PTX concentration,the protein expressions of LC3B-Ⅱ and Beclin-1 were both rised,while its were down after NAC pretreatment.MTT analysis of pretreatment with 3-MA,indicated that attenuated the inhibitory effect of PTX on SW620 cell proliferation.Conclusions:According to the above results,it can be proved that PTX could resist proliferation and promote apoptos in SW620 cells.The first,PTX induced SW620 cells to arrests in G0/G1 phase by up-regulating p21 and down-regulating Cyclin D1 protein,thereby inhibiting the cell proliferation.The second,PTX can induce the increase of intracellular ROS,through intracellular ROS mediated the phosphorylation of AKT and GSK-3β,then controlling AKT/GSK-3β signaling pathway.In addition,though intracellular ROS up-regulating LC3B-Ⅱ and beclin-1 protein,activating autophagy and eventually promoting SW620 cell apoptosis.In conclusion,PTX not only has been used in clinical treatment to improve cerebral circulation,anti-inflammatory,but also can be used as a potential lead compound,which provides a new strategy for the treatment of colorectal cancer.