Oleanolic acid inhibits the proliferation of Hela cells in cervical cancer by regulating the ACSL4 ferroptosis signaling pathway

Background: Cervical cancer continues to be the leading cause of cancer deaths among women worldwide. Oleanolic acid (OA) is a naturally occurring substance found in the leaves, fruits, and rhizomes of plants that has anti-cancer activity. Methods: We used tumor-bearing mice as the animal model and Hela cell as cell models. Western blot was used for detecting the expression of proteins in ferroptosis related proteins acyl-CoA synthase long-chain family member 4 (ACSL4), ferritin heavy chain (FTH1), transferrin receptor (TfR1) and glutathione peroxidase 4 (GPX4) in vivo and in vitro. MTT and EdU was for the detection of the viability of Hela cells. Results: In vivo experiments showed that OA signicantly reduced the size and mass of cervical cancer tumors. In vitro experiments showed that OA signicantly reduced the viability and proliferation capacity of Hela cells. In both in vivo and in vitro assays, OA increased the level of oxidative stress and Fe 2+ content, and increased the expression of ferroptosis related proteins. We found high expression of ACSL4 in both xenograft models and cervical carcinoma cells. Meanwhile, knockdown of ACSL4 expression using shRNA in cervical cancer cells signicantly increased cell viability and proliferation. In addition, decreased ROS levels and GPX4 were detected in ACSL4 knockdown cervical cancer cells, suggesting that ACSL4 inhibition may contribute to the reduction of ferroptosis within Hela cells and thus improve Hela cell survival. Conclusion: Promotion of ACSL4 dependent ferroptosis through OA may be an effective approach to treat cervical cancer.


Introduction
Cervical cancer is one of the most common tumors in women and is the leading cause of cancerous death in women [1]. Most patients with cervical cancer receive radiation therapy and adjuvant chemotherapy, which can signi cantly improve patient survival [2]. However, excessive radioresistance, chemical resistance, and tumor metastasis pose problems for treatment, and the biology of cervical cancer and the underlying molecular mechanisms have not been proven. Therefore, exploring the signaling mechanisms will provide an effective reference for the treatment of cervical cancer.
Ferroptosis is a regulated form of cell death, which means it can be attenuated or accelerated by speci c genetic or pharmacological manipulations [3]. With the increased understanding of ferroptosis, it has been found that ferroptosis plays an important role in cancers such as kidney and liver cancer, where it can inhibit tumor growth in vivo and kill tumor cells in vitro [4,5]. Therefore, the induction of tumor ferroptosis by small molecules has become an available strategy for the treatment of cancer. In addition, the alterations of expression in tumor genes affect ferroptosis in tumor and many studies have demonstrated that the acyl-CoA synthase long chain family member 4 (ACSL4) gene regulates ferroptosis [6][7][8]. Yuan et al., 2016 [6]demonstrate that ACSL4 contributes to the accumulation of lipid intermediates during hypertrophy disease, and oxidative stress is essential for ferroptosis.
Oleanolic acid (OA) is a pentacyclic triterpene naturally distributed in the leaves, fruits and seeds of plants [9]. OA has been shown to have several biological activities, such as antioxidant [10], antiin ammatory [11], and antitumor activities [12]. Although the antitumor activity of OA has been reported, its speci c role in cervical cancer is unknown.
In this study, we hypothesized that OA would not only reduce Hela tumor invasion in mice, but also inhibit Hela cell growth and proliferation. We examined the regulation of oxidative stress, Fe 2+ levels and ferroptosis related proteins. In addition, we examined the changes in the survival and proliferation capacity of human cervical cancer cells and the expression of ferroptosis related proteins after ACSL4 inhibition.

Animal modeling and grouping
The animal experiments were conducted in accordance with the accepted standards of animal care. This study was approved by the Xuzhou City Hospital of Chinese Medicine Animal Care and Use Committee.
The nude mice were kept under spece c pathogen free (SPF) conditions for one week, and 5×10 7 Hela cells were injected under the left axillary skin after acclimatization. The tumors of Hela cell-inoculated mice were measured every 3 days after modeling, and tumor diameter ≥ 0.5 cm was considered successful. The tumor-bearing mice were randomly divided into control group (n= 6), 40 mg/kg OA group (n= 6) and 80 mg/kg OA group (n= 6). All groups underwent subcutaneous injection modeling, and control mice received saline intraperitoneal injections, while 40 mg/kg OA group and 80 mg/kg OA group received 40 mg/kg oleanolic acid and 80 mg/kg oleanolic acid intraperitoneal lasted 15 days. Tumors were measured and weighed every 3 days, and tumor volume (V; mm 3 ) was calculated according to the following formula: V = Major axis × Minor axis × height × 0.52. After disposing of nude mice, tumors were removed and weighed.

Histopathological analysis
The tumors were xed in 4% paraformaldehyde solution for 24 hours, dehydrated, embedded in para n, and then serially sectioned (4 μm) with a microtome (Histocore Biocut, Thermo Fisher Scienti c, USA). The samples were then stained with hematoxylin and eosin (HE) and then dehydrated twice. Sections were sealed with glass and placed under a microscope (Olympus, Japan) to observe the morphology of the cells.

Determination of iron content, MDA and GSH
The iron content of the different groups was analyzed using an iron assay kit (ab83366, Abcam, UK) according to the manufacturer's instructions. The absorbance was measured at 593 nm using a micro spectrophotometer (Nanodrop, Thermo). Oxygen radicals act on the unsaturated fatty acids of lipids to produce lipid peroxide; the latter is gradually broken down into a complex series of compounds, such as malondialdehyde (MDA), so oxidation can be detected by measuring the level of MDA. The content of MDA was measured using Malondialdehyde (MDA) content detection kit (BC0025, Solarbio, China) and the absorbance of the supernatant was determined at 532 nm. Reduced glutathione (GSH) is the main antioxidant sulfhydryl substance in cells, and plays an important role in antioxidation, protein sulfhydryl protection and amino acid transport across membranes. The Micro Reduced Glutathione (GSH) Assay Kit (BC1175, Solarbio, China) was used to measure the absorbance at 412 nm to detect the GSH activity in samples.

Determination of reactive oxygen species (ROS) in vivo and in vitro
Tumor tissues were rapidly frozen and sectioned, and sections were incubated in 5 μM ROS uorescent

Statistical analysis
The statistics were performed using GraphPad Prism 8 . All data are presented as mean ± standard deviation (SD) of three independent experiments. T-test was used to compare the data between the two groups, ANOVA was used to compare data among three groups. Results were considered to be statistically signi cant for values * P<0.05, **P<0.01.

OA suppresses the growth of tumor
The chemical structural formula of OA is shown in Figure 1A. To verify whether OA could be used as a potential drug for cervical cancer, we tested the effect of inhibition of OA on cervical cancer tumor growth using a mouse xenograft model. Intraperitoneal injection-treated nude mice in 40 mg/kg or 80 mg/kg OA groups formed smaller tumors compared to the control group ( Figure 1B). Moreover, the volume and mass of tumor were also signi cantly reduced (p<0.05, Figure 1C and D). As shown in Figure 1E, tumor pathology sections of HE veri ed that OA treatment of tumor-bearing mice resulted in massive in ltration of tumor cells. Overall, these ndings con rmed that OA has the ability to suppress cervical cancer tumors.

OA increases the accumulation of iron and ROS in tumor
Fe 2+ was signi cantly accumulated in the tumor tissues of OA-treated tumor-bearing mice compared to the control group (Figure 2A). In Figure 2B, the MDA activities of 40 mg/kg OA and 80 mg/kg OA groups were higher than that of control group signi cantly (p<0.05). On the contrary, the GSH contents in the 40 mg/kg OA and 80 mg/kg OA groups were lower than that in the control group observably (p<0.05, Figure  2C). Since lipid peroxidation is a feature of rust disease, we next measured ROS content in tumor tissues [15]. The brightness of the ROS-DHE probe was signi cantly higher in the 40 mg/kg OA and 80 mg/kg OA groups than in the control group (p<0.05, Figure 2D and E).

OA regulates the expression of ferroptosis related proteins in tumor
As several studies have reported that ferroptosis regulates tumors, experiments were performed to test whether OA could mediate the ferroptosis signaling pathway. For this purpose, the translation levels of key parameters in the ferroptosis signaling pathway were analyzed ( Figure 3A and B). Under OA-treated conditions, the expressions of ACSL4 and TfR1 were stronger in the 80 mg/kg group than that in the 40 mg/kg group, whereas the trend was reversed for FTH1 and GPX4. Similarly, the expression levels of these proteins in Hela cells showed the same trend as in tumors ( Figure 5C and D).

OA treatment inhibits the viability of Hela cell and increases the accumulation of iron and ROS
After treatment with OA (0, 5, 10, 20 μM), the viability of Hela cells decreased with increasing concentration of OA ( Figure 4A). The contents of Fe 2+ and MDA increased with increasing OA concentration, while GSH decreased with increasing OA concentration ( Figure 4B-D). At the same time, the content of intracellular ROS increased with the increase of OA concentration ( Figure 4E). These results indicated that the addition of OA coincubation decreased Hela cell viability and increased the level of intracellular oxidative stress.

OA inhibits Hela cell proliferation in a dose-dependent manner
To elucidate the effect of OA on cell proliferation, Hela cells were treated with different concentrations of OA (0, 5, 10 and 20 μM) and subjected to EdU and Hoechst33342 assays (Fig. 5A) and analyzed (Fig. 5B). EdU can be doped into the DNA of proliferating cells, and after capturing images with uorescence microscopy, the total number of EdU cells was counted and expressed relative to the EdU cell counts of Hoechst33342 cells. The proliferative capacity of Hela cells decreased signi cantly with increasing OA concentration (p<0.05, Fig. 5A and B).
Silencing of ACSL4 decreases the sensitivity of Hela cells to OA As shown in Figure 6A, si-ACSL4 successfully blocked the expression of ACSL4. In Figure 6B, cell viability increased after ACSL4 was inhibited. At the same time, the proportion of newly proliferating cells was down-regulated as seen by EdU proliferation analysis (Fig. 6C and D), and the intracellular ROS content was also down-regulated by ow cytometry analysis (Fig. 6E). ACSL4 knockdown reduced OA-induced ACSL4 high expression and signi cantly increased OA-induced GPX4 expression ( Figure 6 F and G). The results suggest that the OA-induced ferroptosis in Hela cells can be reduced by inhibiting the expression of ACSL4.

Discussion
Cervical cancer is one of the most common tumors in women and is clinically treated with radiotherapy and chemotherapy. However, the use of these methods is limited due to metastasis and resistance of the tumor. Therefore, exploring the signaling mechanisms of cervical cancer will provide an effective reference for treatment. Abnormal accumulation of iron, ROS or both is associated with various diseases of cancer. The induction of ferroptosis is an approach to suppressing tumor growth [16,17], but oxidative stress-induced ferroptosis has not yet been linked to the pathology of cervical cancer. In this study, we elucidated the regulatory role of ACSL4 in ferroptosis and its mechanisms in cervical cancer cells.
Namely, OA increased intracellular ROS levels by regulating expressions of ferroptpsis related proteins, which lead to ferroptosis. Further si-ACSL4 experiments showed that ferroptosis was ameliorated after ACSL4 was silenced. These new ndings are shown in Figure 7.
Ferrroptosis is an iron-dependent form of cell death caused primarily by the peroxidation of phospholipids (PLs) containing polyunsaturated fatty acids (PUFAs) [8]. ACSL4 is a long-chain fatty acidcoenzyme A ligase that is required for PUFA-PLs biosynthesis, which in turn lead to elevated lipid peroxidation and ferroptosis [18,19]. Yuan et al. 2016 [6] found that ACSL4 contributes to the accumulation of lipid intermediates during ferroptosis and identi ed ACSL4 as a biomarker and contributor to ferroptosis. OA is an excellent natural anticancer agent, and recent studies have shown that OA can inhibit the growth of leukemia, non-small cell lung cancer (NSCLC) cell lines and other malignant tumors [20][21][22] . In both in vivo and in vitro experiments, results showed that ACSL4 expression levels and ROS accumulation were further increased were further increased in OA-treated tumor tissues and Hela cells, indicating an increase in the intensity of ferroptosis. GPX4 could use GSH to convert lipid peroxides (PUFA-PL-OOH) into non-toxic lipid alcohol (PUFA-PL-OH), which removes the accumulation of lipid peroxides in cells, thereby reducing lipid peroxidation and inhibiting ferroptosis [8,23].
Our results also suggested that inhibition of ACSL4 reduces the occurrence of ferroptosis. Since ACSL4 regulates lipid peroxidation, suppression of ACSL4 limits erastininduced lipotoxicity in ferroptosis, which is consistent with previous studies [24,25]. The process of ferroptosis is dynamic and involves iron metabolism in addition to lipid peroxidation [17]. Iron, which can alternate between the insoluble oxidized Fe 3+ form and the reduced Fe 2+ pro-oxidant form, can induce membrane lipid peroxidation by generating soluble free radicals, highly reactive oxygen species (ROS) and hydroxide ions (OH -) through the Fenton reaction [26]. The transferrin (Tf) pathway is important for the acquisition of iron by cells, and Tf-Fe 3+ delivers iron to cells by binding to TfR1 on the cell surface and is released as Fe 2+ via divalent metal transporter 1 (DMT1) [27]. Ferritin is the major intracellular iron storage protein, containing both ferritin heavy chain (FTH1) and ferritin light chain (FTL1). However, only FTH1 is able to convert Fe 2+ to Fe 3+ for storage in the ferritin mineral nucleus through its ferritin peroxidase activity to prevent Fenton reactions from occurring [28]. In our study, as ACSL4 levels increased, TfR1 expression signi cantly increased and FTH1 expression decreased. OA can effectively upregulated ferroptosis by promoting the expression ACSL4. After silencing of ACSL4, Hela cells decreased their sensitivity to OA. Thus, OA is targeted to promote ACSL4-mediated ferroptosis, thereby inhibiting the growth and proliferation of cervical cancer cells.

Conclusion
OA is a natural activator of ACSL4 and can modulate multiple levels and targets simultaneously. Notably, ACSL4-mediated ferroptosis activation reduced the size of cervical cancer tumors, decreased the viability of Hela cells, and enhanced their anticancer effects in cervical cancer. This supports that OA can be targeted to promote ACSL4 activation of ferroptosis as a potential anticancer agent in cervical cancer. The datasets used and/or analysed during the current study are available from the corresponding author on reasonable request.
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