Allicin inhibits the biological activities of cervical cancer cells by suppressing circEIF4G2

Abstract Allicin is a safe herbal extract believed to have antitumor effects, which, however, remain unclear. The aim of the present work was to discuss Allicin antitumor effects on cervical cancer using cell experiments. Using Hela and Siha to our research objectives in our study, first step, difference concentration of Allicin (20, 40, and 80 μM) treated Hela and Siha cell lines, and next step, discuss circEIF4G2 effects in Allicin antitumor effects in Hela and Siha cell lines; the cell proliferation and EdU‐positive cell number by CCK‐8 and EdU staining; cell apoptosis rate by flow cytometry; invasion cell number by transwell assay; wound healing rate by wound healing assay; and relative mRNA and protein levels using qRT‐PCR and WB assay. With Allicin supplement, the cell proliferation and EdU‐positive cell number were significantly depressed with cell apoptosis rate significantly increasing; invasion cell number and wound healing rate significantly suppressed with circEIF4G2 mRNA expression significantly down‐regulation (p < .05, respectively). However, there was no significant difference among Allicin, si‐circEIF4G2, and Allicin+si‐circEIF4G2 in cell biological activities including cell proliferation, apoptosis, invasion and migration, and relative gene and protein expression. Allicin depresses biological activities of cervical cancer cells through down‐regulating circEIF4G2/HOXA1/AKT/mTOR.

sequence conservation, stable expression, and tissue specificity (Chen et al., 2021;Su et al., 2019;Zhang & Dai, 2022).An increasing number of studies have shown that circRNAs have a potential application value in markers for cancer diagnosis and therapeutic targets (Chen et al., 2019;Yu & Liu, 2019).As a member of circRNAs, circEIF4G2 effectively inhibited biological activities of CC cells after knockdown in our previous study (Mao et al., 2019).
In the present study, we wanted to explore whether allicin depresses the biological activities of CC cells (Hela and Siha, which are human papillomavirus-positive cell lines) and whether it exerts its effect by down-regulating circEIF4G2 expression.

| MATERIAL S AND ME THODS
Main reagents: Allicin (95% purity) from Nanjing Zelang Medical Technology Co., Ltd.We prepared different allicin working solutions at 20, 40, and 80 μM concentrations by using the DMEM/F-12 medium.Furthermore, cell apoptosis, cell counting kit-8, and two-step reverse transcription polymerase chain reaction assay kits purchased from Nanjing KeyGEN BioTech Co., Ltd., Beyotime Institute of Biotechnology in Jiangsu and Beijing Tiangen BioTech Co., Ltd., respectively.Primers and internal reference (glyceraldehyde 3-phosphate dehydrogenase [GAPDH]) were synthesized from Sangon Biotech Co., Ltd., Shanghai.HOXA1, AKT, and primary anti-mTOR antibodies were all purchased from Abcam, and si-negative control (NC) and si-circEIF4G2 were designed and synthesized from Nanjing KeyGEN BioTech Co., Ltd.

| Cell culture
HeLa and Siha cells were preserved in our laboratory.The culture system was DMEM/F-12, which contained 10% fetal bovine serum.The cells were cultured at 37°C and 5% CO 2 , with medium replacement once a day and cell passage once every 2-3 days.Cells at the logarithmic growth phase were then collected for subsequent experiments.

| Cell transfection
HeLa and Siha (2 × 10 5 ) cells were transfected with si-NC or si-circEIF4G2 by using Lipofectamine® 3000 according to grouping requirements depending on the manufacturer's protocol.After 48 h culture, the cells were collected for the next experiment.

| Cell grouping
The cells were grouped into the following: NC (routinely treated group), dimethyl sulfoxide (DMSO) (culture medium supplemented with DMSO), and allicin-L, allicin-M, and allicin-H (culture medium supplemented with allicin dissolved in DMSO at final concentrations of 20, 40, and 80 μM, respectively).We also divided the cells as follows: si-NC (cells transfected with si-NC using Lipofectamine® 3000), si-circEIF4G2 (cells transfected with si-circEIF4G2 using Lipofectamine® 3000), allicin (culture medium supplemented with allicin dissolved in DMSO at a final concentration of 80 μM), and allicin+si-circEIF4G2 (cells transfected with si-circEIF4G2 by Lipofectamine® 3000 and treated with allicin at a final concentration of 80 μM).After receiving the corresponding treatment for 48 h, the cells in each group were collected for subsequent experiments.

| Cell proliferation by CCK-8 assay
After undergoing the corresponding treatment for 48 h, the cells in each group were added with 10 μL of CCK-8 stock solution.Following a continuous cell culture for 1 h, we measured the absorbance value (OD value) in each well by using a microplate reader and then calculated the cell proliferation inhibition rate.This experiment was repeated thrice.

| Gene expression detection by quantitative RT-PCR (qRT-PPCR)
After 48 h of culture, cells were collected from the incubator and washed twice using phosphate-buffered saline solution (PBS).
After adding 1 mL of cell lysate TRIzol in each well, we extracted total RNA to determine RNA concentration and purity by using a microspectrophotometer.Then, cDNA was reverse-transcribed and synthesized, followed by amplification.Table 1 shows the primer sequences.The reaction system consisted of the following: SYBR® Premix Ex Taq TM II, 5 μL; upstream primer, 0.25 μL; downstream primer, 0.25 μL; ROX, 0.2 μL; and cDNA, 2.5 μL (total volume: 10 μL).Amplification conditions were as follows: pre-denaturation at 95°C for 30 s, denaturation at 95°C for 30 s, annealing at 57°C for 10 s, and extension at 72°C for 15 s (40 cycles).GAPDH was used as an internal reference, and the relative expression of the target gene was calculated by the 2 −ΔΔCt method.

| Flow cytometry
After the corresponding treatment for 48 h, cells were added into 0.25% pancreatin for digestion, centrifuged at 2000 r/min for 5 min, resuspended with 0.5 mL 2% PBS, and fixed with 1.5 mL of 100% ethanol on the ice at 4°C overnight.After centrifugation at 1000 r/ min for 5 min, cells were washed with 3 mL of 2% PBS, followed by a water bath in 100 μL of RNase A at 37°C for 30 min and uniform staining with 400 μL of propidium iodide at 4°C in the dark for 30 min.Thereafter, we determined apoptosis by flow cytometry and recorded red fluorescence at 488 nm excitation wavelength.Data were analyzed using CellQuest.

| EdU staining
Cells from each group were seeded into a 96-well plate at 1 × 10 5 cells/well and cultured overnight.Afterward, each well was added with 100 μL of culture medium containing 50 μmol/L EdU for incubation for 2 h and then washed twice with PBS for 5 min each time.After fixing and staining, we observed these cells under a fluorescence microscope.

| Cell invasion capability by transwell assay
We applied a layer of Matrigel matrix above a semipermeable membrane, filled the lower chamber with culture medium containing trace supplements, inserted it with a transwell insert, and added treated cell suspension (1 × 10 5 cells) to the upper chamber.Subsequently, the transwell system was incubated in a cell incubator for 48 h.After cell suspension removal, the upper chamber was washed using PBS.In the lower chamber, we removed the culture medium, added 800 μL of methanol, which was fixed at room temperature for 30 min, and then washed thrice using PBS.
Afterward, this chamber was placed in a solution premixed with 0.1% crystal violet for reaction at room temperature for 30 min.
After being washed three times using PBS, the cells were counted and photographed under an inverted microscope.

| Wound healing assay
After the corresponding treatment for 48 h, the migration capability of cells in each group was assessed by wound healing assay.Cells in the logarithmic growth phase were collected from each group, scratched perpendicular to a cell culture plate using a pipette tip, and then washed thrice using PBS.At 0 and 48 h, we photographed and measured the width of the wound, respectively.

| Western blotting (WB) assay
Relevant protein levels in the cells of each group were evaluated using the WB assay.After adding protease inhibitors, we quantified the proteins through a bicinchoninic acid assay.Subsequently, we added and boiled sample buffer for 5 min and separated the proteins by using SDS-PAGE, transferred them to a PVDF membrane, and blocked them in 5% skim milk for 1 h.Afterward, primary antibodies were added for incubation overnight.After washing the membrane thrice, we added secondary antibodies, followed by incubation for 1 h, membrane washing three times, and finally, enhanced chemiluminescence development.The gray value was analyzed using Gel-Pr32.

| Statistical methods
Expressed as mean ± SD, data were analyzed and processed using SPSS 22.0.We used the F test and LSD-t test for multigroup and pairwise comparisons of measurement data, respectively.A p-value <.05 was considered statistically significant.

| Allicin on Hela and Siha cell apoptosis
According to flow cytometry results, the apoptotic rate in DMSO group presented no significant difference (p > .05, Figure 2),

| Allicin on migration capability of Hela and Siha cells
Wound healing assay results revealed the wound healing rate did not significantly differ between the DMSO and NC groups (p > .05,

| Effect of allicin on circEIF4G2 gene expression
According to qRT-PCR results, circEIF4G2 gene level in DMSO group exhibited no significant difference (p > .05, Figure 5), suggesting that DMSO has no effect on the circEIF4G2 gene level in Hela and Siha cells.After allicin treatment, circEIF4G2 gene expression was reduced significantly in allicin-L, allicin-M, and allicin-H groups (p < .05,p < .01 or p < .001, Figure 5).Therefore, allicin-L, allicin-M, and allicin-H decreased the circEIF4G2 gene expression in Hela and Siha cell lines, with best effect noted in allicin-H.

| Effect of circEIF4G2 on CC cell proliferation inhibited by allicin
According to CCK-8 assay and EdU staining results, the cell proliferation rate (p < .001, Figure 6a,b) and EdU-positive cell count (p < .001, Figure 6c,d) decreased significantly in si-circEIF4G2, allicin, and allicin+si-circEIF4G2 groups.However, both showed no significant differences between these three groups (p > .05, Figure 6).Therefore, allicin might regulate circEIF4G2 to reduce CC cell proliferation.respectively, Figure 7a,b).However, it was not significantly different between such groups (p > .05, Figure 7).Therefore, allicin might regulate circEIF4G2 to increase CC cell apoptosis.

| Role of circEIF4G2 in CC cells' invasive capability inhibited by allicin
Transwell assay demonstrated that invasive cell number decreased significantly in si-circEIF4G2, allicin, and allicin+si-circEIF4G2 groups (each group: p < .001, Figure 8a,b).However, it was not significantly different between these groups (p > .05, Figure 8).
Thus, allicin might regulate circEIF4G2 to reduce CC cell invasion ability.

| Role of circEIF4G2 in CC cell's migration capability inhibited by allicin
Based on the wound healing assay, wound healing rate decreased significantly in the si-circEIF4G2, allicin, and allicin+si-circEIF4G2 groups (each group: p < .001, Figure 9a,b).However, it showed no significant differences between such groups (p > .05, Figure 9).
Therefore, allicin might regulate circEIF4G2 to decrease CC cell migration ability.
However, they did not show significant differences between these groups (p > .05, Figure 10).

| DISCUSS ION
This study may be the first to use allicin at different concentra-  (Liu et al., 2018;Xie et al., 2022).Allicin is an herb extract that was previously reported to have antitumor effects against lung cancer (Bai et al., 2023), but its effects on CC are still unclear.Present study revealed allicin significantly reduced invasion and metastasis rates of Hela and Siha cells, with excellent inhibitory effects at high concentrations.Allicin, which is the main active ingredient of garlic, has various effects, including antitumor, antibacterial, anti-free radical, anti-inflammatory, and regulatory effects on the body's immune system (Zhou et al., 2022).It also has a certain inhibitory effect on the invasion and metastasis of various tumor cells, such as liver cancer cells (Alamir & Patil, 2021).Allicin has been confirmed to increase the rupture of liver cancer cell nuclei and promote apoptosis (Chu et al., 2013;Zou et al., 2016).In our study, the proliferation, invasion, and migration capabilities of Hela and Siha cells were all significantly inhibited after allicin treatment, while their apoptotic rates increased significantly.In addition, high-dose allicin presented the best inhibitory effect.Further detection revealed that the expression of the circEIF4G2 gene was inhibited significantly.
Increasing evidence suggests that circRNA disorders are related to the occurrence and development of cancers and other diseases (Dragomir & Calin, 2018;Li et al., 2018).Compared with linear RNAs, circRNAs exhibit higher stability because of their closed-loop structure and resistance to RNase R (Zhang et al., 2021).Some circRNA levels in various human cells present tissue and disease specificity.They are also increased in brain tissues, especially in synapses.
Owing to advances in circRNA microarrays and bioinformatics analysis, many new circRNAs and their functions have been identified.For example, Chen et al. (Fu et al., 2017) conducted a circRNA microarray analysis and found that the hsa_cir_0128298 levels increased in hepatocellular carcinoma (HCC) tissues, suggesting that this circRNA can be used as a new diagnostic and prognostic biomarker for HCC.
Wang et al. (Wang & Li, 2018) also proved that circ_0067934 is an oncogene and that its expression correlates with a poor prognosis in patients with non-small-cell lung cancer.Furthermore, our previous work (Mao et al., 2019) revealed that circEIF4G2 has a promoting effect on CC.The present study then confirmed that reducing the expression of circEIF4G2 can effectively mitigate CC cells' biological activities.In addition, the transfection of si-circEIF4G2 into Hela and Siha cells significantly reduced the activities of these cells, but when this transfection was combined with allicin intervention, the anticancer effect was not enhanced.Therefore, allicin may exert its anticancer effect by inhibiting circEIF4G2.
The abnormally high expression of the HOXA1/AKT/mTOR signaling pathway may be one of the key factors leading to tumor occurrence and development (Ma et al., 2022;Zhang et al., 2019).
In this study, circEIF4G2 was decreased in Hela and Siha cells, CC cell activities were inhibited, and the HOXA1/AKT/mTOR signaling pathway was significantly inhibited.Based on previous research results, circEIF4G2 has been confirmed to regulate HOXA1 (Mao et al., 2019).
In conclusion, allicin can inhibit CC cells' biological activities by Allicin+si-circEIF4G2, The cell transfected with si-circEIF4G2 and treated with 80 μ M Allicin; si-circEIF4G2, The cells were transfected with si-circEIF4G2; si-NC, The cells were transfected with si-NC (negative control).### p < .001,compared with si-NC.

|
Allicin on Hela and Siha cell proliferation CCK-8 assay and EdU staining showed no significant difference in cell proliferation rate or EdU-positive cell count in the DMSO group (DMSO's concentration is 0.25%) (p > .05, Figure 1), indicating that DMSO is nontoxic to Hela and Siha cells.After allicin treatment, the cell proliferation rate (Figure 1a,b) and EdU-positive cell count (Figure 1c,d) reduced significantly in the allicin-L, allicin-M, and allicin-H groups (p < .05,p < .01,or p < .001, Figure 1a,d).Taken together, 0.25% DMSO concentration was safe for Hela and Siha cell lines, and allicin-L, allicin-M, and allicin-H decreased Hela and Siha cell proliferation, with allicin-H showing the best effect.
Figure 2a,b).Overall, 0.25% DMSO concentration was safe for Hela and Siha cell lines, and allicin-L, allicin-M, and allicin-H increased apoptosis of Hela and Siha cells, with allicin-H exhibiting best effect.

Flow
cytometry revealed apoptotic rate increased significantly in si-circEIF4G2, allicin, and allicin+si-circEIF4G2 groups (p < .001,F I G U R E 3 Effects of allicin on invasion capability of Hela and Siha cells.Allicin-H, The cells were treated with 80 μ M Allicin; Allicin-L, The cells were treated with 20 μ M Allicin; Allicin-M, The cells were treated with 40 μ M Allicin; DMSO, The cells were treated with DMSO; NC, The cells were treated with normal.(a) Invasion Hela cell number by transwell assay (×100).(b) Invasion Siha cell number by transwell assay (×100).*p < .05;**p < .01;***p < .001,compared with NC group.

F I G U R E 4
Effects of Allicin on migration capability of Hela and Siha cells.Allicin-H, The cells were treated with 80 μ M Allicin; Allicin-L, The cells were treated with 20 μ M Allicin; Allicin-M, The cells were treated with 40 μ M Allicin; DMSO, The cells were treated with DMSO; NC, The cells were treated with normal.(a) Wound healing rate in different Hela cell groups (×100).(b) Wound healing rate in different Siha cell groups (×100).*p < .05;**p < .01;***p < .001,compared with NC group.F I G U R E 5 Effect of Allicin on circEIF4G2 gene expression.Allicin-H, The cells were treated with 80 μ M Allicin; Allicin-L, The cells were treated with 20 μ M Allicin; Allicin-M, The cells were treated with 40 μ M Allicin; DMSO, The cells were treated with DMSO; NC, The cells were treated with normal.*p < .05;**p < .01;***p < .001,compared with NC group.F I G U R E 6 Effect of circEIF4G2 on Allicin inhibiting proliferation of CC cells.(a) Cell proliferation in different Hela cell groups.(b) Cell proliferation in different Siha cell groups.(c) EdU-positive Hela cell number (×100).(d) EdU-positive Siha cell number (×100).Allicin, The cells were treated with 80 μ M Allicin; Allicin+si-circEIF4G2, The cell transfected with si-circEIF4G2 and treated with 80 μ M Allicin; si-circEIF4G2, The cells were transfected with si-circEIF4G2; si-NC, The cells were transfected with si-NC (negative control); ### p < .001,compared with si-NC.F I G U R E 7 Legend on next page.F I G U R E 7 Effect of circEIF4G2 on Allicin promoting apoptosis of CC cells.(a) Cell apoptosis rate in different Hela cell groups.(b) Cell apoptosis rate in different Siha cell groups.Allicin, The cells were treated with 80 μM Allicin; Allicin+si-circEIF4G2, The cell transfected with si-circEIF4G2 and treated with 80 μM Allicin; si-circEIF4G2, The cells were transfected with si-circEIF4G2; si-NC, The cells were transfected with si-NC (negative control); ### p < .001,compared with si-NC.F I G U R E 8 Role of circEIF4G2 in Allicin inhibiting invasive capability of CC cells.(a) Invasion Hela cell number by transwell assay (×100).(b) Invasion Siha cell number by transwell assay (×100).Allicin, The cells were treated with 80 μ M Allicin; Allicin+si-circEIF4G2, The cell transfected with si-circEIF4G2 and treated with 80 μ M Allicin; si-circEIF4G2, The cells were transfected with si-circEIF4G2; si-NC, The cells were transfected with si-NC (negative control).### p < .001,compared with si-NC.
tions to suppress Hela and Siha cells and detect proliferation rates in different treatment groups.Results showed allicin treatment significantly inhibited proliferation of Hela and Siha cells, especially at high concentrations.Excessive cell proliferation can promote tumor growth, distant metastasis, and local invasion, which are the main limiting factors in the clinical treatment of CC.Reducing the invasion and metastasis rates of tumor cells can improve clinical efficacy

F
Role of circEIF4G2 in Allicin inhibiting migration capability of CC cells.(a) Wound healing rate in different Hela cell groups (×100).(b) Wound healing rate in different Siha cell groups (×100).Allicin, The cells were treated with 80 μ M Allicin; Allicin+si-circEIF4G2, The cell transfected with si-circEIF4G2 and treated with 80 μ M Allicin; si-circEIF4G2, The cells were transfected with si-circEIF4G2; si-NC, The cells were transfected with si-NC (negative control).### p < .001,compared with si-NC.
Formal analysis (equal); funding acquisition (equal); investigation (equal); software (equal); supervision (equal); validation (equal).Xu Rui: Funding acquisition (equal); software (equal); supervision (equal); validation (equal).Li Yuan: Investigation (equal); F I G U R E 1 0 Relative mRNA expression by qRT-PCR assay.(a) Relative gene expression in different Hela cell groups.(b) Relative gene expression in different Siha cell groups.Allicin, The cells were treated with 80 μ M Allicin; Allicin+si-circEIF4G2, The cell transfected with si-circEIF4G2 and treated with 80 μ M Allicin; si-circEIF4G2, The cells were transfected with si-circEIF4G2; si-NC, The cells were transfected with si-NC (negative control).### p < .001,compared with si-NC.F I G U R E 11 Relative proteins expression by WB assay.(a) Relative protein expression in different Hela cell groups.(b) Relative protein expression in different Siha cell groups.Allicin, The cells were treated with 80 μ M Allicin;