The Effect And Mechanism Of Alloimperatorin-Induced Apoptosis And Autophagy In Cervical Cancer Cells

Background: Cervical cancer is a global health problem that imposes a considerable economic and medical burden on society.Therefore,there is a need for concerted efforts to improve the treatment of cervical cancer.Although a variety of treatment options are currently available for cervical cancer,such as chemoradiotherapy and neoadjuvant or adjuvant chemotherapy,the proportion of local recurrence a nd distant metastasis is increasing,and the treatment of advanced inoperable cervical cancer remains a challenging problem.In addition,recurrent cervical cancer that is not suitable for radical treatment and new metastatic cervical cancer are considered incurable and have a poor prognosis.In recent years,the active components of natural herbal extracts have become a hot spot in the development of new chemotherapeutic drugs due to their advantages of multi-channel,multi-link,multi-target and low toxicity.At the same time,the mechanism of TCM treatment of tumor is physical and mental conditioning.It improves the immune capacity of the body,inhibits the rapid development of tumor and achieves the symbiosis between the human body and tumor.Based on our previous study,it was found that the monomeric Alloimperatorin extracted from the natural chinese herb of angelica dahuricae has certain anticancer activity.In this research background,further exploration of the anticancer mechanism of Alloimperatorin will lay a foundation for the development and research of new chemotherapy drugs for cervical cancer.Objective: The objectives of this study were as follows:(1)to investigate the anticancer activity of Alloimperatorin both in vivo and in vitro;(2)to examine the impact of Alloimperatorin on apoptosis in He La and Si Ha cells,specifically through the involvement of mitochondrial and death receptor pathways;(3)to assess the oxidative stress induced in He La and Si Ha cells by Alloimperatorin,and to exploe the relationship between reactive oxygen species generation and cell death;(4)to analyze the modulation of endoplasmic reticulum stress by Alloimperatorin,which occurs through oxidative stress and the induction of autophagy in He La and Si Ha cells via the GRP78/PERK/DDIT3 pathway;(5)to employ bioinformatics approaches to further elucidate the biological function,prognostic significance,and immunological value of HSPA5,the gene encoding endoplasmic reticulum stress protein GRP78,in cervical cancer.Methods:(1)CCK-8 cytotoxicity assay was used to detect the effects of different concentration of Alloimperatorin on the proliferation of cervical cancer cell lines of He La,Si Ha,MS-751,Caski and normal cervix Hcer Epic cells.High-content and highthroughput dynamic monitoring system,3D microsphere cells modeling and cells cloning assay were used to detect the effects of different concentration of Alloimperatorin on the proliferation of He La and Si Ha cells;(2)The effect of Alloimperatorin on the cell cycle and apoptosis of He La and Si Ha cells was detected by flow cytometry;(3)Cell scratch assay and high-throughput dynamic imaging system were used to detect the effects of Alloimperatorin on the migration ability of He La and Si Ha cells in cervical cancer;(4)The effects of Alloimperatorin on the expression of Cyclin B and CDC2 and matrix metalloproteins of MMP-2 and MMP-9 in He La and Si Ha cells were detected by Western Blot;(5)The tumor bearing model of He La cells in BALB/c nude mice was established to verify the inhibitory effect of Alloimperatorin on the growth of He La cells transplanted tumor in nude mice and the survival state of nude mice.Immunohistochemistry and Western Blot verified the effects of Alloimperatorin on Caspase-3,-8 and autophagy proteins of LC3,p62;(6)The apoptosis of He La and Si Ha cells and the changes of mitochondrial membrane potential induced by Alloimperatorin were detected by JC-10 fluorescent probe in a high-content and high-throughput imaging system.The apoptotic structure(apoptotic corpuscles)of He La and Si Ha cells induced by Alloimperatorin was detected by transmission electron microscopy.Real-time fluorescence quantitative PCR and Western Blot were used to verify the expression of core proteins and m RNA in the apoptosis pathway;(7)The production of reactive oxygen species(ROS)in He La and Si Ha cells induced by Alloimperatorin was detected by the reactive oxygen species(DCFH-DA)fluorescent probe combined with laser confocal detection.Immunofluorescence microscopy was used to detect the autophagy fluorescence of LC3 in He La and Si Ha cells induced by Alloimperatorin.The stable He La and Si Ha cells of m RFP-GFP-LC3 lentivirus were constructed and the autophagy flow of He La and Si Ha cells induced by Alloimperatorin was detected by confocal microscopy.The changes of autophagy structure(autophagosomes)of He La and Si Ha cells induced by Alloimperatorin were detected by fluoroscopic electron microscopy.The expressions of LC3,p62,ATG5,ATG12 and endoplasmic reticulum stress proteins of GRP78,PERK,e IF2α,DDIT3 were detected by Western Blot.The autophagy induction in He La and Si Ha cells was verified by si RNA GRP78 transfection experiment,which regulated by Alloimperatorin through GRP78/PERK/DDIT3 pathway;(8)The gene annotation file of GRP78 encoding gene HSPA5 was analyzed by bioinformatics,and the detailed information of HSPA5 gene was obtained from Gene Cards? database.The location information of HSPA5 gene in cells was obtained from HPA database.The expression of HSPA5 in pancarcinoma was analyzed by TCGA database,which confirmed the high expression of HSPA5 gene in cervical cancer;(9)Online database immunohistochemistry,q-PCR and Western Blot were used to verify the expression of HSPA5 gene in cervical cancer and its involvement in cell biological behavior,tumor immune infiltration,immune checkpoint,sensitive target drugs and clinical prognosis.Results:(1)Compared with the control group,Alloimperatorin inhibited the proliferation of He La,Si Ha,MS-751 and Caski cells at different concentrations,but had the most obvious inhibitory effect on He La and Si Ha cells and had no obvious inhibitory effect on normal cervical epithelial Hcer Epic cells.At the same time,it effectively inhibited the growth of He La cell subcutaneous transplantation tumor in nude mice and had no significant effect on the survival state of nude mice;(2)Compared with the control group,Alloimperatorin inhibited the migration of He La and Si Ha cells,blocked the cell cycle and inhibited the expression of matrix metalloproteins of MMP-2,MMP-9 and Cyclin B,CDC2;(3)Alloimperatorin decreased mitochondrial membrane potential of He La and Si Ha cells and induced apoptosis fluorescence of He La and Si Ha cells.The results of transmission electron microscope(TEM)showed that Alloimperatorin induced the apoptotic structure(apoptotic corpuscles)of He La and Si Ha cellls.Real-time quantitative PCR and Western Blot showed that Alloimperatorin induced the endogenous apoptotic proteins expression of Caspase-8,-9,Bax,Cyt-c and exogenous apoptotic proteins of Fas,FADD,Caspase-3 and down-regulated the expression of Bc L-2,PARP,Pro-caspase-3,Pro-caspase-8 and Pro-caspase-9;(4)In vivo immunohistochemical results confirmed that Alloimperatorin induced the expression of Caspase-3,-8 in tumor tissue;(5)Alloimperatorin induced oxidative stress of He La and Si Ha cells in cervical cancer and promoted the production of reactive oxygen species(ROS);(6)Compared with the control group,Alloimperatorin induced LC3 autophagy fluorescence in He La and Si Ha cells.Lentivirus transfection experiment of m RFP-GFP-LC3 confirmed that Alloimperatorin induced autophagy flow in He La and Si Ha cells;(7)Transmission electron microscopy(TEM)showed that Alloimperatorin induced autophagy structures(autophagosomes)in He La and Si Ha cells;(8)Western Blot results showed that Alloimperatorin induced the protein expression of LC3,ATG5,ATG12 and decreased the expression of p62 protein.Endoplasmic reticulum stress was induced by Alloimperatorin,which was accompanied by increased expression of endoplasmic reticulum stress proteins of GRP78,PERK,e IF2α and DDIT3.After addition of reactive oxygen species inhibitor(NAC),the expressions of LC3,ATG5,ATG12 and GRP78 were decreased,while the expression of p62 protein was increased.These results suggested that oxidative stress is involved in the regulation of endoplasmic reticulum stress-induced autophagy;(9)The results of si RNA transfection experiment showed that the expression of GRP78 in He La and Si Ha cells transfected with si RNAGRP78 was significantly lower than that in the control group,and the expressions of PERK,DDIT3 and LC3 in the Alloimperatorin treatment group were also decreased.These results indicated that GRP78/PERK/DDIT3 pathway is involved in autophagy regulated by Alloimperatorin;(10)Immunohistochemical results showed that Alloimperatorin induced the expression of autophagy protein LC3 and decreased the expression of p62 protein in the tumor tissue;(11)Bioinformatics analysis confirmed that the protein encoded by HSPA5 gene was a member of the heat shock protein 70(HSP70)family,which was located in the endoplasmic reticulum and located on chromosome 9q33.3.HSPA5 gene was mainly located in cytoplasm;(12)HSPA5 gene was highly expressed in cervical cancer and involved in regulating the biological function of cervical cancer;(13)HSPA5 gene was negatively correlated with the prognosis and overall survival of patients with cervical cancer.Involved in the regulation of multiple immune checkpoints and a variety of immune cell infiltration;(14)The expression level of HSPA5 gene was significantly negatively correlated with the methylation degree of cg14190817.HSAP5 is involved in the regulation of 6 tumor sensitive chemotherapy targeted drugs.Conclusion:(1)Alloimperatorin inducesed apoptosis of He La and Si Ha cells through mitochondrial and death receptor pathways,and thus plays an anticancer role in vivo;(2)Alloimperatorin regulated endoplasmic reticulum stress through oxidative stress and GRP78/PERK/DDIT3 pathway regulates autophagy of He La and Si Ha in cervical cancer;(3)GRP78 encoding gene HSPA5 is involved in the prognosis of cervical cancer,immune-related checkpoints,cellular immune invasion and methylation of cervical cancer,which is a potential clinical diagnostic marker of cervical cancer.