The effect and apoptosis mechanism of 6-methoxyflavone in HeLa cells

Abstract Introduction Tumour cell apoptosis is a crucial indicator for judging the antiproliferative effects of anti-cancer drugs. The detection of optical and macromolecular biomarkers is the most common method for assessing the level of apoptosis. We aimed to explore the anti-tumour mechanisms of 6-methoxyflavone. Materials and methods Three optical methods, including the percentage of apoptotic cells, cell morphology, and subcellular ultrastructure changes, were obtained using flow cytometry, inverted fluorescence microscopy, and transmission electron microscope imaging. The mRNA or protein expression of macromolecular biomarkers related to common apoptotic pathways was determined via polymerase chain reactions or western blot assays. The functional role of the core gene biomarker was investigated through overexpression, knockdown, and phosphorylation inhibitor (GSK2656157). Results Transcriptome sequencing and the optical biomarkers assays demonstrated that 6-methoxyflavone could induce apoptosis in HeLa cells. The expression of macromolecular biomarkers indicated that 6-methoxyflavone induced apoptosis through the PERK/EIF2α/ATF4/CHOP pathway. Phosphorylated PERK was identified as the core biomarker of this pathway. Both overexpression and GSK2656157 significantly altered the expression level of phosphorylated PERK in 6-methoxyflavone-treated HeLa cells. Discussion and conclusion Macromolecular biomarkers, such as phosphorylated PERK and phosphorylated EIF2α are of great significance for assessing the therapeutic effects of 6-methoxyflavone.


Introduction
Cervical cancer is a major threat to the health and well-being of adult women. The incidence (Bray et al. 2018), mortality (Bray et al. 2018), metastasis rate (Rong et al. 2019), and recurrence rate (Holloway and Lea 2019) of cervical cancer are still relatively high. Concerning the treatment of cervical adenocarcinoma, there are still some problems, such as the high cost of targeted preparations (Liu et al. 2021), drug and radiotherapy resistance (Suzuki et al. 2021), strong side effects of radiotherapy and chemotherapy (Okonogi et al. 2018), high probability of recurrence (Jung et al. 2017), metastasis (Zhou et al. 2021), lymphovascular infiltration (Saito et al. 2020), and poor prognosis (Jung et al. 2017). Therefore, the development of more effective treatment strategies for cervical cancer is critical. Cell proliferation assays of chemotherapeutic candidates have shown that 6methoxyflavone has anti-proliferative activity against HeLa cells (Kinjo et al. 2016). 6-Methoxyflavone can also significantly alleviate cisplatin-induced adverse effects (Shahid et al. 2017). Concurrently, 6-methoxyflavone has been shown to have broad anti-inflammatory (Wang et al. 2015) and immune-regulatory (So et al. 2014) effects. The above results indicate that 6-methoxyflavone has anti-cancer activity against HeLa cells, although its mechanism of action remains uncertain.
Furthermore, previous studies have shown that cell apoptosis plays a key role in cancer therapy (Carneiro and El-Deiry 2020) and is closely associated with efficacy assessment (Sch€ urmann et al. 2021), resistance reversal (Liu et al. 2020b), and sensitivity enhancement (Dey et al. 2021;Xie et al. 2021) of anticancer drugs. A retrospective single-center study of rectal cancer developed a novel apoptosis-based tumour regression grade to assess the efficacy of chemoradiotherapy (Ozaki et al. 2021). A multicolour fluorescent nanoprobe was used to observe the progression of tumour cell apoptosis to assess drug efficacy (Luan et al. 2018). Apoptosis-related phenomena were used as decisive factors in the evaluation of the efficacy of photodynamic therapy. Moreover, apoptosis can be used to assess disease severity (Chu et al. 2021).
In our study, we investigated the in vitro therapeutic effect of 6-methoxyflavone on cervical adenocarcinoma based on transcriptomics and relevant biomarkers of apoptosis. First, we identified the key biological processes of 6methoxyflavone exerting anti-tumour effects by transcriptome sequencing. RNA-seq analysis indicated that 6-methoxyflavone was significantly related to apoptosis. Subsequently, we screened three commonly used optical biomarkers and a variety of significant molecular biomarkers to evaluate the level of apoptosis and efficacy after 6-methoxyflavone intervention. The biomarker analyses indicated that 6-methoxyflavone induced apoptosis through the PERK/EIF2a/ATF4/CHOP pathway. Subsequently, overexpression, knockdown, inhibition of phosphorylation activity, and co-immunoprecipitation experiments further confirmed that the Thr982 phosphorylation activity of PERK was identified as the core biomarker of the pathway. Finally, this study aimed to provide new ideas for the clinical treatment of cervical cancer.

Materials and methods
Chemicals and cell culture 6-Methoxyflavone (purity ! 98%) was purchased from Weikeqi Biological Technology Co., Ltd. (Chengdu, China). Selective PERK phosphorylation inhibitor GSK2656157 was obtained from MedChem Express (New Jersey, USA) and dissolved in dimethyl sulfoxide (DMSO). HeLa, C33A, SiHa, and HaCaT human cell lines were purchased from the Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences (Beijing, China). Details of each cell line were presented in Table 1. There were no ethical issues involved in this study. C33A, SiHa, and HaCaT cells were cultured in modified Eagle's medium (HyClone, Logan, UT, USA) with 10% foetal bovine serum (FBS). HeLa cells were cultured in Dulbecco's modified Eagle's medium (HyClone, Logan, UT, USA) supplemented with 10% FBS. The cells were cultured at 37 C in a 5% CO 2 atmosphere.
The potential targets of 6-methoxyflavone The potential targets of 6-methoxyflavone were downloaded from four databases: Traditional Chinese Medicine Systems Pharmacology (TCMSP) (Ru et al. 2014), Traditional Chinese Medicine on Immuno-Oncology (TCMIO) (Liu et al. 2020a), HERB (Fang et al. 2021), and PubChem (Kim et al. 2016(Kim et al. , 2021 databases. TCMSP predicts targets by browsing the chemical-related targets sub-database. TCMIO predicts targets of immuno-oncology by browsing the ingredients section. HERB predicts targets by browsing ingredients related to gene target sections. Simultaneously, we obtained potential targets of 6-methoxyflavone from two columns of the PubChem database: chemical-gene co-occurrences in literature and bioassay results. In the bioassay results column, we only outputted the human targets. Finally, 130 potential targets of 6-methoxyflavone were identified.

Kyoto encyclopedia of genes and genomes (KEGG) pathways enrichment analysis of targets
The 130 targets of 6-methoxyflavone were used for KEGG (Kanehisa and Goto 2000;Kanehisa et al. 2017) pathway enrichment analysis of the DAVID 6.8 online database (Jiao et al. 2012). Then, we set the threshold of the false discovery rate to <0.001 to filter the pathways predicted by DAVID. Finally, we used GraphPad Prism version 8.0.1 for Windows (GraphPad Software, San Diego, California USA, www.graphpad.com) to visualize the enrichment pathways of targets.

Proliferation-related biomarker (cell activity) assays
The Enhanced Cell Counting kit-8 (CCK-8) (Beyotime, Shanghai, China) was used to detect the viability of HeLa, C33A, SiHa, and HaCaT cells. Cells were seeded and cultured in 96-well plates for 24 h. Then, the cell lines were treated with five concentrations of 6-methoxyflavone (20, 40, 80, 120, and 160 lM) for 24, 48, and 72 h. The HeLa cells were treated with seven concentrations of GSK2656157 (0.3125, 0.625,1.25, 2.5, 5, 10, and 20 lM) for 48 h. The control group was cultured with 0.16% DMSO. After that, 20 ll of fresh CCK-8 working solution was added and the cells were cultured for 1 h at 37 C. The optical density (OD) values were measured at 450 nm using a multifunctional microplate reader (Thermo Fisher Scientific, Madison, WI, USA). Graphpad Prism version 8.0.1 for Windows was used to calculate the half-maximal inhibitory concentration (IC50).

Eukaryotic transcriptome sequencing
Human HeLa cells were seeded in 6-cm dishes for 24 h and cultured with 0.16% DMSO and 65 lM 6-methoxyflavone for 48 h in an incubator. After 48 h, 1 ml RNAiso plus reagent (Takara, Dalian, China) was used to lyse cells and extract total RNA. The concentration and purity of RNA were confirmed using Qubit RNA assay kits and Qubit 2.0 Fluorometer (Life Technologies, Invitrogen division, Darmstadt, Germany). RNA integrity and genomic contamination were assessed using agarose gel electrophoresis. Transcriptome sequencing (RNAseq) assays were performed by Sangon Biotech Co., Ltd.
(Shanghai, China). After library construction and sample cluster generation, transcriptome sequencing was performed using an Illumina Hiseq TM platform (Illumina Inc, San Diego, California, USA). The relative expression of each gene was estimated using transcripts-per-million (TPM). The screening criteria for significantly differentially expressed genes (DEGs) were as follows: at least one of the two samples had a TPM value !5, the absolute value of log2FoldChange was >1, and q-Value was <0.05. The q-values were estimated by the false discovery rate (FDR) correction method. We then performed gene ontology (GO) and pathway classification enrichment analyses using the DAVID and KEGG databases. The screening criteria for significantly different items were pvalue <0.05. GraphPad Prism version 8.0.1 for Windows was used to visualize the significantly different items.

The assessments of three optical biomarkers of apoptosis
The single-cell suspensions were seeded and cultured in 6well plates for 24 h. After that, the cells were treated with five concentrations of 6-methoxyflavone (0.16% DMSO, 40, 65, 80, and 120 lM) for 48 h.

Bisbenzimide H 33342 labelled inverted fluorescence microscope imaging
The Hoechst 33342 staining dye solution kit (Beyotime Biotechnology, Jiangsu, China) was used to detect the ratio of apoptotic HeLa cells. Based on the manufacturer's instructions, 500 ll of fresh Hoechst 33342 solution was added and incubated for 30 min at 37 C. An inverted fluorescence microscope (Nikon Corp., Tokyo, Japan) was used to observe the morphology of the apoptotic HeLa cells.

Annexin V-fluorescein isothiocyanate (FITC) and propidium iodide (PI) labelled flow cytometry optical testing system
An annexin V-FITC/PI apoptosis detection kit (Meilunbio, Dalian, Liaoning, China) was used to measure the percentage of HeLa cells in the early and late apoptotic stages. The cells (5 Â 10 5 ) were carefully collected and suspended in a 200 ll binding buffer containing 10 ll of annexin V-FITC and 20 ll of PI. After 20 min of incubation, HeLa cells were analyzed by flow cytometry (BD Biosciences, San Jose, CA, USA).

Transmissive electron microscope (TEM) imaging
HeLa cells treated with 0.16% DMSO or 65 lM 6-methoxyflavone were collected and centrifuged at 3000 rpm for 10 min at 25 C. Cold 2.5% glutaraldehyde was used to fix for 48 h, followed by 1% osmium tetroxide for 1.5 h, followed by dehydration with 50, 70, 80, 90, 100% ethanol, and 100% acetone. Next, the cells were incubated with Epon812 epoxy resin and an equal volume of pure acetone for 2 h, embedded in 100% Epon812 resin, and cured at 35, 45, and 68 C for 24 h. Finally, the ultrathin section was completed using an ultra-thin microtome (Leica EM UC7, Wetzlar, Germany) and stained with uranyl acetate and lead citrate. The lead-stained sections were observed and photographed using a Transmission Electron Microscope (FEI Tecnai G2 Spirit Bio-TWIN, Hillsboro, USA).

The assessments of two types of macromolecular biomarkers of apoptosis
Real-time fluorescence quantitative polymerase chain reaction (PCR) Single-cell suspensions were seeded into 6-well plates for 24 h. HeLa cells were then treated with either 6-methoxyflavone (65 lM) or 0.16% DMSO for 48 h. Total RNA was extracted from 6-methoxyflavone-treated HeLa cells using RNAiso Plus reagent (Takara, Dalian, China). The complementary DNA was synthesized by genomic DNA eraser reaction and reverse transcription reaction using the PrimeScript TM RT reagent kit (Takara). Real-time PCR was performed using the TB Green V R Premix Ex Taq TM II kit (Takara). Glyceraldehyde 3phosphate dehydrogenase (GAPDH) and b-actin (Sangon Biotech, Shanghai, China) were used as housekeeping genes. The Livak method was used to analyze the relative quantitative real-time PCR data. The real-time PCR primer sequences are listed in Table 2.

Western blot analysis
HeLa cells were treated with either 6-methoxyflavone (65 lM) or 0.16% DMSO for 48 h, the cells were lysed for 20 min in cell lysis buffer (Beyotime) containing 1 mM phenylmethanesulfonyl fluoride (Beyotime). The lysate was centrifuged at 14,000 Â g for 5 min at 4 C. Total protein samples were obtained from the supernatants of HeLa cell lysates. Protein concentrations were detected using an enhanced bicinchoninic acid protein assay kit (Beyotime). Subsequently, protein lysates were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred onto polyvinylidene fluoride membranes (Merck Millipore, Burlington, MA, USA). After blocking with Tris-buffered saline-Tween 20 solutions containing 5% bovine serum albumin for 1 h at 25 C, the membranes were incubated with the primary antibodies overnight at 4 C. The primary antibodies used were: GAPDH, PERK (Sangon Biotech), phospho-PERK (Thr982), EIF2a, phospho-EIF2a (Ser51), ATF4, and CHOP rabbit antibodies (Beyotime). The membranes were incubated with horseradish peroxidase-conjugated anti-rabbit IgG (Sangon Biotech) for 1 h at 25 C. Finally, the protein bands were visually detected with an ultra-sensitive efficient chemiluminescence kit (Beyotime) using the Amersham Imager 680 system (GE Healthcare, Little Chalfont, Buckinghamshire, UK).
The functional role of the core gene biomarkers

Cell transfection
HeLa cells were seeded in 6-well plates for 24 h. First, the complete medium was changed to Opti-MEM reduced serum medium (Gibco, Burlington, Canada). Then, three EIF2AK3/ PERK small interfering RNAs (siRNA), GAPDH positive control siRNA, negative control siRNA (Genepharma, Shanghai, China), pcDNA empty vector (negative control), and human pcDNA3.1 3 Â Flag-hEIF2AK3/PERK vector (NM_004836.7) (Hanbio, Shanghai, China) were transfected into cells using Lipofectamine 2000 reagent (Invitrogen, Carlsbad, CA, USA) according to the manufacturer's instructions. After 6 h, the medium was replaced with a complete medium. After 48 h, a portion of the cells was digested with 0.25% trypsin buffer and collected by centrifugation at 1000 rpm for PCR analysis to determine transfection efficiency. Another part of the cells was re-plated for 24 h and treated with 0.16% DMSO and 65 lM 6-methoxyflavone for 48 h. Then, the cells were collected for PCR and western blot analyses. The siRNA oligo sequences are listed in Table 3.

PERK phosphorylation inhibitor assay
HeLa cells were seeded and cultured in 6-well plates for 24 h. Then, the cells were pre-treated with 0.08% DMSO and 1.25 lM GSK2656157 for 1 h. Subsequently, the cells were further treated with 0.08% DMSO and 65 lM 6-methoxyflavone for 48 h. The cells were digested and collected for western blot analyses.

Co-immunoprecipitation
HeLa cells were treated with 65 lM 6-methoxyflavone for 48 h. The cells were lysed using an immunoprecipitation cell lysis buffer (Beyotime). Protein A þ G magnetic beads (Beyotime) were washed with 1Â Tris-buffered saline in a magnetic separation rack three times and then incubated with primary antibody(anti-p-EIF2a) or normal rabbit IgG working solution (Beyotime) at 25 C for 1 h. Subsequently, cell lysates were incubated with magnetic beads conjugated with primary antibodies or normal rabbit IgG for 2 h at 25 C. After incubation, the magnetic beads were eluted in 1Â SDS-polyacrylamide gel electrophoresis loading buffer (Beyotime) at 95 C for 8 min. Finally, the supernatant was collected for western blot analysis.

Statistical analysis
Three independent biological replicates were used for each experiment. Statistical analysis was carried out using GraphPad Prism version 8.0.1 for Windows. All results were represented as means ± standard deviation (SD) and analyzed by paired t-test, Wilcoxon test, or one-way analysis of variance (ANOVA) with Dunnett's multiple comparisons tests. In inhibition assays of cell viability, IC50 values were calculated using non-linear regression (curve fit) analyses. Statistical significance was set at p < 0.05.

KEGG pathways enrichment analysis and targets network construction
We collected 34, 6, 36, and 91 potential targets of 6-methoxyflavone from the TCMSP, TCMIO, HERB, and PubChem databases, respectively. After duplicate removal, 130 potential  targets of 6-methoxyflavone were identified. KEGG pathway enrichment analyses showed that the targets were remarkably associated with 12 pathways, including pathways in cancer, neuroactive ligand-receptor interaction, calcium signalling pathway, etc. (p < 0.001) (Figure 1(A)). 6-Methoxyflavone was mainly related to pathways in cancer.

6-Methoxyflavone inhibits the cell viability of cervical cancer cell lines
We first tested cell viability using the CCK-8 assay. We treated HeLa, C33A, SiHa, and HaCaT cells with increasing concentrations of 6-methoxyflavone. We found that 6-methoxyflavone inhibited the viability of HeLa, C33A, SiHa, and HaCaT cells. Table 4 shows the IC50 values of 6-methoxyflavones in HeLa, C33A, SiHa, and HaCaT cells. The results revealed that HeLa cells are highly sensitive to 6-methoxyflavones. This result is consistent with that of a previous study on HeLa cells (Kinjo et al. 2016). Therefore, we chose HeLa cells for subsequent studies. For the subsequent experiments, HeLa cells were treated with 6-methoxyflavone (65 lM) for 48 h.

6-Methoxyflavone was significantly related to apoptotic biological processes
To reveal the anti-tumour mechanism of 6-methoxyflavone, we used RNA-seq to identify cancer-associated biological processes, molecular functions, KEGG pathways, and macromolecular biomarkers in HeLa cells treated with 6methoxyflavone.
Agarose gel electrophoresis showed that all six samples met the requirements for library construction (Figure 1(B)). Compared with the three normal control groups (0.16% DMSO), there were a total of 1365 significantly differentially expressed genes in the three 65 lM 6-methoxyflavonetreated groups. Among these 1365 genes, 538 were upregulated and 827 were downregulated (Figure 1(C,D)). These  genes are promising therapeutic targets and efficacy assessment biomarkers for 6-methoxyflavone. Subsequently, we performed GO and KEGG pathway analyses of these genes. We obtained 11 KEGG pathways, 157 biological processes, and 54 molecular functions. The KEGG enrichment results showed that 6-methoxyflavone was correlated with four cellular processes, three environmental information progresses, and four genetic information progresses (Figure 1(E)). The GO enrichment results indicated that 6-methoxyflavone was significantly related to eight apoptosis or death (Figure 1(F)), five cell proliferation or growth (Figure 1(G)), three endoplasmic reticulum stress (Figure 1(H)) biological processes, and five protein-binding and modification terms (Figure 1(I)) (p < 0.05). Based on the above analyses, we focussed on three items, namely the apoptotic process, phosphorylation, and PERK-mediated unfolded protein response.

6-Methoxyflavone induces HeLa cell apoptosis
The percentage of apoptotic cells, cell nucleus morphology, and subcellular ultrastructural changes were three important non-invasive optical biomarkers of apoptosis. Moreover, the subcellular ultrastructural change under transmission electron microscopy is the most classical and reliable method for evaluating apoptosis, and it is considered to be the gold standard for determining apoptosis. We observed the morphological changes in the nucleus and calculated the percentage of apoptotic cells in 6methoxyflavone-treated HeLa cells using Hoechst 33342 staining dye solution. As the concentration of 6-methoxyflavone increased, the nucleus became bright, condensed, irregular, and fragmented (Figure 2(A)). 6-Methoxyflavone significantly increased the ratio of apoptotic HeLa cells in a dose-dependent manner (Figure 2(B)).
We also detected the apoptotic rates of HeLa cells using the Annexin V-FITC/PI apoptosis detection kit. Flow cytometry analysis further showed that the percentage of 6-methoxyflavone-treated HeLa cells in the early and late apoptotic stages was significantly higher than that in the 0.16% DMSO group (Figure 2(C,D)).
TEM, which is the best technique to further analyze subcellular ultrastructural changes, was used to verify apoptosis  The relative expression of PERK, EIF2a, ATF4, and CHOP mRNAs in the PERK/EIF2a/ATF4/CHOP endocytoplasmic reticulum pathway. (E,F) The relative expression levels of the PERK/EIF2a/ATF4/CHOP pathway related proteins. Each assay was repeatedly performed in three times. Statistical analysis was carried out using paired t-test or Wilcoxon test. The differential expression of Caspase6 was analyzed by Wilcoxon test, and the other genes were analyzed by paired t-test. Ã p < 0.05. n.s.: not significant; p-PERK: phosphorylational PERK; p-EIF2a: phosphorylational EIF2a.
in this study. Ultrastructural changes in HeLa cells treated with 6-methoxyflavone (65 lM) and 0.16% DMSO were compared using transmission electron microscopy imaging. The TEM images of HeLa cells in the normal control group (0.16% DMSO) showed that the morphology and structure of the cell membrane, cytoplasm, organelles, and nucleus were normal (Figure 3(A)). However, when HeLa cells were treated with 6-methoxyflavone (65 lM) for 48 h, typical apoptotic morphological changes at various stages were observed in these cells, such as cytoplasmic vacuolation, formation of annular bodies, apoptotic bodies, cell budding, and cytoplasmic blebbing (Figure 3(B-F)). The results revealed that 6methoxyflavone significantly induced apoptosis in HeLa cells.

6-Methoxyflavone induces apoptosis through the PERK/ EIF2a/ATF4/CHOP pathway
As our results showed that 6-methoxyflavone induced HeLa cell apoptosis, we next investigated whether 6-methoxyflavone altered the mRNA and protein expression levels of the apoptotic pathway.
The mRNA expression of caspase3 in 6-methoxyflavonetreated HeLa cells was significantly higher than that in the 0.16% DMSO group, while the mRNA expression of Aparf1, caspase9, Fas, TNFR, and TRAF was significantly lower. Nevertheless, the mRNA expression of Bcl2, Bax, PARP, cas-pase7, FADD, TRADD, and caspase6 was not significantly altered by treatment with 6-methoxyflavone ( Figure 4(A,B)). Hence, we found that 6-methoxyflavone was unable to activate the mitochondrial or the death receptor pathway of apoptosis and only affected the mRNA expression of cas-pase3, Aparf1, caspase9, Fas, TNFR, and TRAF.
HeLa cells treated with 6-methoxyflavone significantly upregulated the mRNA and protein expression levels of EIF2S1/EIF2a/phosphorylational EIF2a (p-EIF2a), ATF4, and DDIT3/CHOP. Moreover, the mRNA and protein expression levels of EIF2AK3/PERK in 6-methoxyflavone-treated HeLa cells were significantly lower than those in the 0.16% DMSO group, while the protein expression levels of phosphorylated PERK (p-PERK) were significantly higher (Figure 4(D-F)). These results are consistent with the transcriptome sequencing results (Table 5). Resultantly, we found that 6-methoxyflavone induced apoptosis in HeLa cells via the PERK/EIF2a/ ATF4/CHOP pathway. EIF2AK3/PERK, especially the Thr982 phosphorylation of PERK, was the most significantly differentially expressed factor in this pathway.
The functional role of the core gene biomarkers In summary, we found that EIF2AK3/PERK was the core gene biomarker of 6-methoxyflavone-induced apoptosis in HeLa cells. To determine the specific functional role of EIF2AK3/ PERK, we performed gain-of-function and loss-of-function genetic manipulation and phosphorylation activity inhibition experiments.
We determined the efficiency of EIF2AK3 overexpression and knockdown using PCR and western blot analysis ( Figure  6(A-D)). Figure 5 shows the pcDNA3.1 3 Â Flag-EIF2AK3/PERK vector atlas. The intervention efficiency of three EIF2AK3/ PERK siRNAs and a human 3 Â Flag-hEIF2AK3/PERK vector met the requirements for genetic manipulation. Western blot experiments showed that both overexpression and GSK2656157 significantly altered the expression level of phosphorylated PERK in 6-methoxyflavone-treated HeLa cells, but knockdown experiments did not (Figure 6(C-E)). The expression levels of p-PERK, p-EIF2a, ATF4, and CHOP were significantly altered by GSK2656157, an inhibitor of PERK phosphorylation activity (Figure 6(E)). Figure 5(G) shows the inhibitory effect of GSK2656157 on HeLa cells using the CCK-8 assay. The IC50 value of GSK2656157 was 30.12 lM at 48 h. 1.25 lM GSK2656157 had a little inhibitory effect on HeLa cells (Figure 6(E,G)).
Moreover, co-immunoprecipitation assays of phosphorylated EIF2a further confirmed that there was an interaction between phosphorylated PERK and phosphorylated EIF2a (Figure 6(F)). Therefore, we believe that the Thr982 phosphorylation of PERK is the core mechanism by which 6-methoxyflavone exerts its pro-apoptotic effect. Thr982 phosphorylation activity of PERK is a core biomarker for evaluating the efficacy of 6-methoxyflavone. Other macromolecular biomarkers, such as p-EIF2a, ATF4, and CHOP are also important biomarkers for evaluating the efficacy of 6methoxyflavone.

Discussion
The Imperatae Rhizoma exhibits antioxidant (Zhou et al. 2013), anticancer (Li et al. 2020b), anti-complementary (Fu et al. 2010), anti-inflammatory (Zou et al. 2021), and immunological regulation (Lu and Huang 1996) activities. The compound 6-methoxyflavone extracted from Imperatae Rhizoma has exhibited anti-proliferative activity against HeLa cells (Kinjo et al. 2016). However, its anti-cancer effects and mechanisms in cervical cancer have not yet been clarified. Herein, we identified 130 potential targets of 6-methoxyflavone. KEGG pathway enrichment analyses of the targets showed that 6-methoxyflavone was mainly associated with pathways in cancer. Next, we tested the inhibitory effect of 6-methoxyflavone on HeLa, C33A, SiHa, and HaCaT cells using CCK-8 assays. The results revealed that 6-methoxyflavones are highly sensitive to HeLa cells. This result is consistent with that of a previous study on HeLa cells (Kinjo et al. 2016). Transcriptome sequencing identified that apoptosis was the key biological process of 6-methoxyflavone exerting antitumour effects. Subsequently, optical biomarker assays revealed that 6-methoxyflavone significantly induced apoptosis in HeLa cells. The apoptotic signalling pathways contain mitochondrial control (Burke 2017), death receptor (Jo et al. 2020;Sato et al. 2020), and endoplasmic reticulum pathways (Chern et al. 2019;Wang et al. 2020). We investigated whether 6methoxyflavone altered the mRNA and protein expression levels of significant molecular biomarkers of the apoptotic pathway.
At the mRNA expression level, 6-methoxyflavone was unable to activate the mitochondrial control pathway, death receptor pathway, and endoplasmic reticulum pathway of IRE1 and ATF6. However, the mRNA expression of EIF2AK3/ PERK, EIF2S1/EIF2a, ATF4, and DDIT3/CHOP was significantly altered by treatment with 6-methoxyflavone. Moreover, the results of western blot assays confirmed that 6-methoxyflavone significantly altered the protein expression levels of PERK, phosphorylated PERK, EIF2a, phosphorylational EIF2a, ATF4, and CHOP. In summary, significant molecular biomarker assays indicated that 6-methoxyflavone induced apoptosis in HeLa cells via the PERK/EIF2a/ATF4/ CHOP pathway.
The PERK/EIF2a/ATF4/CHOP pathway is highly related to the apoptosis of cervical cancer cells (Chitnis et al. 2012;Hiramatsu et al. 2020). First, endoplasmic reticulum stress triggers the activation and phosphorylation of PERK (Chitnis et al. 2012). Phosphorylation of PERK causes the activation and phosphorylation of EIF2a. Phosphorylation of EIF2a then upregulates the expression levels of ATF4 and CHOP (Hiramatsu et al. 2020). Finally, the overexpression of CHOP initiates the apoptosis-promoting program (Lee et al. 2018). In our study, 6-methoxyflavone triggered the activation and phosphorylation of PERK and EIF2a. Subsequently, phosphorylational EIF2a upregulated the expression levels of ATF4 and CHOP. Namely, 6-methoxyflavone induced cell apoptosis via the PERK/EIF2a/ATF4/CHOP pathway. Overexpression, knockdown, inhibition of phosphorylation activity, and coimmunoprecipitation experiments further confirmed that the  Each assay was performed from three independent experiments in sextuplicate (n ¼ 18). Statistical analysis was carried out using one-way analysis of variance (ANOVA) with Dunnett's multiple comparisons tests. Ã p < 0.05. n.s.: not significant; GSK: GSK2656157; 6MF: 6-methoxyflavone; p-PERK: phosphorylational PERK; p-EIF2a: phosphorylational EIF2a; DMSO: dimethyl sulfoxide.
Thr982 phosphorylation activity of PERK was identified as the core biomarker of the pathway. To summarize, this study suggests that 6-methoxyflavone is a potential drug for the treatment of cervical adenocarcinoma. Macromolecular biomarkers, such as p-PERK, p-EIF2a, ATF4, and CHOP are important biomarkers for evaluating the efficacy of 6methoxyflavone.

Conclusion
In this study, 6-methoxyflavone inhibited HeLa, C33A, SiHa, and HaCaT cell growth and induced HeLa cell apoptosis in a concentration-dependent manner. Moreover, 6-methoxyflavone can also significantly alter the mRNA and protein expression of EIF2AK3/PERK, EIF2S1/EIF2a, ATF4, and DDIT3/ CHOP. In conclusion, 6-methoxyflavone induces apoptosis in HeLa cells via the PERK/EIF2a/ATF4/CHOP pathway. These macromolecular biomarkers are of great significance for assessing the therapeutic effects of 6-methoxyflavone.