The Mechanism Of Action Of Chloroquine On The Proliferation And Apoptosis Of Cervical Cancer Cells

According to GLOBOCAN 2020,cervical cancer(CC)is the fourth most common cancer in women worldwide after breast,colorectal,and lung cancer,and the second most common cancer in developing countries.Early aggressive surgical treatment of cervical cancer combined with radiotherapy has a good prognosis,but patients with metastatic and recurrent cervical cancer still have a poor prognosis.Chloroquine(CQ),was originally found to be used primarily for the prevention and treatment of malaria.In addition,CQ is also considered a potential anticancer agent that can exert antitumor effects alone or in combination with chemotherapeutic agents.In our study,we found that CQ can effectively inhibit the proliferation of cervical cancer cells and induce their apoptosis.No study has been reported on the specific mechanism of action of chloroquine on cervical cancer cells.In this study,we investigated the effects of CQ on the proliferation,survival,autophagy and apoptosis of human cervical cancer He La cells based on network pharmacology and cell biology,and analyzed its possible biological mechanisms,in order to provide new clues for the clinical treatment of cervical cancer.In this study,the human cervical cancer cell line He La was selected as the cell model,and the Cell Counting Kit-8 method(CCK-8 method)was used to detect the effect of different concentrations of CQ groups on He La cell proliferation,the clone formation assay to detect the effect of He La cell colony formation after the action of CQ,and the wound healing assay to detect the effect of He La cell migration ability.Flow cytometry was used to detect the effect of CQ on apoptosis of He La cells.DCFH-DA reagent was used to detect the production of reactive oxygen species in He La cells.The expression levels of autophagy-related proteins(P62,Beclin1,LC3II)and apoptosis proteins(Bax,Bcl-2,PARP)in He La cells were detected by protein immunoblotting.Based on the network pharmacology,we investigated the potential mechanism of CQ action in cervical cancer,screened CQ and potential therapeutic targets in cervical cancer through the network database,and then constructed a drug-disease-target network and performed GO functional analysis and KEGG pathway analysis.Finally,protein immunoblotting assay was used to detect the expression of PI3K/AKT/MDM2 signaling pathway-related proteins in He La cells.The results showed that the proliferation inhibition rate of human cervical cancer He La cell line was calculated in vitro by culturing different concentrations of CQ on He La cells,and the quantitative efficacy curve was plotted.The experiments were divided into three groups:(1)control group(0 μM),(2)low concentration group(20 μM),and(3)high concentration group(75 μM).CQ inhibited the clone formation of He La cells compared to the control group(P<0.05).The results of wound healing assay showed that CQ could inhibit the invasive ability of He La cells(P<0.05).Flow cytometry assay showed that CQ induced apoptosis in He La cells compared with the control group(P<0.05).DCFH-DA staining assay revealed a large number of green fluorophores in the CQ-treated group compared with the control group,indicating that CQ treatment could lead to an increase in ROS levels.Protein immunoblotting assay(Western blot,WB)revealed that CQ increased the expression of autophagy signature proteins Beclin1 and LC3 II and decreased the expression of autophagy receptor protein P62 in He La cells compared with the control group(P<0.05);compared with the control group,apoptosis signature proteins Bax/Bcl-2,Cleaved-PARP expression levels were increased(P<0.05).The network pharmacology results showed that there were 7846 therapeutic targets for cervical cancer and 126 action targets for CQ,and a total of 100 targets were obtained after taking the intersection.Network visualization and PPI analysis of common targets were performed,and core targets such as TNF,SLC6A4,SLC6A2,and MDM2 were obtained.the results of GO enrichment analysis,biological functions included response to xenobiotic stimuli,response to drugs,intracellular signaling,etc.the results of KEGG pathway analysis were mainly related to PI3K-AKT signaling pathway,c AMP signaling pathway,etc.PI3K-AKT signaling pathway is a key signal for cell growth,proliferation and apoptosis,and the abnormal regulation of its through-loop may lead to tumorigenesis.Therefore,we further investigated the effect of CQ on PI3K/AKT pathway.WB results showed that CQ treatment significantly decreased the expression of phosphorylated PI3K/AKT/MDM2 protein compared with the control group(P<0.05).