The combination of Angelica sinensis polysaccharide and cisplatin promotes ferroptosis in cisplatin-resistant ovarian cancer cells by regulating GPX4, thereby reversing their resistance to cisplatin

doi:10.21203/rs.3.rs-3312243/v1

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Research Article

The combination of Angelica sinensis polysaccharide and cisplatin promotes ferroptosis in cisplatin-resistant ovarian cancer cells by regulating GPX4, thereby reversing their resistance to cisplatin

https://doi.org/10.21203/rs.3.rs-3312243/v1

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Background

Angelica sinensis (Oliv.) Diels has been used for centuries in Chinese traditional medicine to treat gynecological ailments. Numerous studies indicate that Angelica sinensis polysaccharide (ASP), an extract from Angelica sinensis, can inhibit various forms of cancer. Nevertheless, the therapeutic potential of ASP for treating ovarian cancer remains largely unexplored.

Methods

The study investigated cell proliferation and invasion, as well as the effects of a combination of ASP and DDP after tumor formation in nude mice. Tumor size was monitored, and HE staining was conducted on tissue samples. The identification of key gene GPX4 was performed via RNA-seq and bioinformatic analysis. GPX4 was overexpressed using lentivirus transfection, and its expression was evaluated via RT-qPCR and western blot. Additionally, the ferroptosis of cells was assessed through the measurement of Fe²⁺, malondialdehyde (MDA), superoxide dismutase (SOD), and glutathione (GSH).

Results

The results indicated a synergistic effect of ASP combined with DDP, leading to better inhibition of proliferation and invasion of SKOV3/DDP cells. Similarly, ASP combined with DDP demonstrated tumor proliferation inhibition in tumor-forming nude mice, while maintaining good safety. Bioinformatics analysis of 843 differentially expressed genes (DEGs) revealed that the key gene GPX4 played a significant role in the mechanism of action. Furthermore, the expression of GPX4 was inhibited by ASP combined with DDP, which resulted in SKOV3/DDP inhibition of proliferation and invasion. The study also demonstrated that ASP combined with DDP led to increased levels of Fe²⁺ and MDA, while decreasing levels of GSH and SOD, suggesting the promotion ferroptosis of SKOV3/DDP cells.

Conclusions

The combination of ASP and DDP has the ability to inhibit the proliferation and invasion of SKOV3/DDP cells. Additionally, inhibiting GPX4 expression in SKOV3/DDP cells promotes ferroptosis.

ovarian cancer

traditional Chinese medicine

angelica sinensis polysaccharide (ASP)

GPX4

ferroptosis

Ovarian cancer is among the top three malignancies affecting women[1]. Ovarian cancer is often diagnosed at a later stage due to the absence of typical early symptoms[2]. Cytoreductive surgery and postoperative platinum-based chemotherapy remain the standard treatments for ovarian cancer[1]. While medical technology has advanced and initial treatment outcomes for ovarian cancer have improved, many patients eventually develop chemotherapy resistance, leading to tumor recurrence and metastasis. Cisplatin (DDP) is the representative drug that patients tend to develop resistance to[3]. Hence, investigating the signaling pathways and targeted drugs tied to multi-drug resistance in ovarian cancer and, subsequently, addressing the phenomenon of multi-drug resistance, is critical for enhancing patient survival rates[4].

Angelica sinensis (Oliv.) Diels is a classic Chinese medicine prescription that has been utilized for the treatment of various gynecological diseases for thousands of years in China[5]. Angelica sinensis polysaccharide (ASP) is a major extracted component of Angelica sinensis[6]. ASP enhances the immune function of the body and acts as an immunomodulator[7], and possesses antioxidant abilities that help protect the liver[8], heart[9], and kidney[10]. Recent studies have found that ASP exhibits an antitumor effect and may aid in treating lung, liver, breast[11], and cervical cancers[12].In addition, in order to make ASP have a higher therapeutic effect on tumors, researchers analyzed ASP using 1H-NMR and FT-IR spectroscopy and created a hypoxia-responsive nano-drug delivery system relying on ASP[13, 14]. However, the effect of ASP on ovarian cancer has not been reported.

In this study, we investigated the in vitro effects of ASP on SKOV3 ovarian cancer cells, as well as cisplatin-resistant SKOV3/DDP cells. Our experiments demonstrated that ASP combined with DDP can effectively reverse the resistance of SKOV3/DDP cells to DDP. We also verified the efficacy of ASP combined with DDP against tumors in nude mice, as well as evaluated the safety of ASP in vivo. Furthermore, we utilized RNA-seq data to identify differentially expressed genes (DEGs) and analyzed them to identify key genes. The effects of these key genes on cell function were subsequently verified through experiments.

Cell culture

SKOV3 and SKOV3/DDP cells were purchased from Shanghai Fuyu Biotechnology Co., LTD. McCoy '5a (Tianhang Bio, Hangzhou, China) with 10% Fetal Bovine Serum (FBS) (Service Bio, Wuhan, China)was cultured in an incubator with 37 ℃ and 5% CO₂. In addition, SKOV3/DDP cells need to be added 0.5ug/ml DDP (Meilun Bio Dalian, China) to maintain DDP resistance.

Cell viability and combination index (CI)

The SKOV3 and SKOV3/DDP cells of the DDP group were subjected to varying concentrations of cisplatin (0 µg/mL, 1.5 µg/mL, 3 µg/mL, 6 µg/mL, 12 µg/mL, 24 µg/mL) and cultured in a 37℃, 5% CO₂ incubator. Meanwhile, for the ASP group, SKOV3 and SKOV3/DDP cells were treated with different concentrations of ASP (Batch Number: C23A6Y1, Yuanye Bio, Shanghai, China) (0 µg/mL, 50 µg/mL, 100 µg/mL, 150 µg/mL, 200 µg/mL, 250 µg/mL, 300 µg/mL) and also cultured in a 37℃, 5% CO₂ incubator for 48 hours. In the ASP combined with DDP group, SKOV3/DDP cells were subjected to 10 µg/mL DDP concentration, followed by the addition of different concentrations (0 µg/mL, 50 µg/mL, 100 µg/mL, 150 µg/mL, 200 µg/mL, 250 µg/mL, 300 µg/mL) of ASP in each group. The cell viability assay was conducted using CCK8 (Wanlei Bio, Shenyang, China). Furthermore, we used CompuSyn software (CompuSyn Inc.) to determine the Combination index (CI). The study results indicated the following: CI = 0.85 to 0.9 representing slight synergism; CI = 0.7 to 0.85 representing moderate synergism; CI = 0.3 to 0.7 representing synergism; CI = 0.1 to 0.3 representing strong synergism; and CI < 0.1 representing very strong synergism.

In vivo xenograft assays

The study utilized female BALB/c nude mice obtained from Cavens Laboratory Animal Co., Ltd. The mice, which were 4 weeks old, were split into ten mice per group. To induce tumor formation, SKOV3/DDP cells were dissolved in 20 µL of phosphate-buffered saline (PBS) and then subcutaneously injected into the armpits of the mice. The growth of the tumors to 100 mm³ was set as day 1. For the Vehicle (Veh) group, the nude mice were intraperitoneally injected with 50 mL/kg/d PBS. Meanwhile, the DDP group was intraperitoneally injected with 4 mg/kg DDP on day 1 and 8 [15]. Following Hu Zhuang et al.'s research on restraining breast tumor growth through the intraperitoneal injection of 0.2 mg/kg/d ASP in nude mice [16], the ASP group mice were also intraperitoneally injected with 0.2 mg/kg/d ASP. The tumors of the mice in each group were monitored every three days to determine the tumor volume (Fig. 3A).After 21 days, the mice were euthanized, and the tumors were collected for analysis (Fig. 3B). The animal experiment was conducted following the Guidelines for the Care and Use of Laboratory Animals and approved by the Ethics Committee of Harbin Medical University (certificate number: KY2023-37)..

Histological analysis

We took tissue extraction and embedding sections from the liver, kidney, and heart of the killed nude mice. Hematoxylin (Solarbio, Shanghai, China) and eosin Y (Sangon, Shanghai, China) were used for staining. Histological sections were taken with a microscope (OLYMPUS, Japan).

Cell invasion assays

The Transwells (Labselect, Hefei, China) were mixed with Matrigel (Corning, USA) and placed into a 24-well plate, which was then placed in a 37℃ incubator for 2 hours to allow the Matrigel to solidify. Next, 5×10⁴ cells per well were added to the upper chamber and 800 µl of culture medium, containing 10% FBS, was added to the lower chamber. After 24 hours, the transwells were removed, washed twice with PBS, fixed with 4% neutral paraformaldehyde (Aladdin, Shanghai, China) for 20 minutes, and stained with 0.1% crystal violet (Amresco, USA) for 5 minutes. Finally, cells on the outer surface of the membrane were observed with a microscope and the number of cells passing through the permeable membrane in the visual field was randomly counted.

RNA-seq analysis

SKOV3/DDP cells were treated with either DDP or ASP combined with DDP. RNA was then extracted from both groups of cells using TRIzol (TaKaRa Bio, Dalian, China). The experiment was repeated three times using independent biological samples. The high-throughput sequencing platform NovaSeq 6000 by Illumina was utilized to sequence the samples. Raw data were filtered and assessed for quality. The clean reads were mapped to the human genome (GENCODE download, GRCh38) for Inter-sample expression level and principal component analysis. The DESeq2 R package was employed to analyze the raw data, and the differential expression threshold for DEGs was set to P value < 0.05, and |log₂FoldChange| > 1. DEGs were subjected to GO and KEGG analyses using David (v2022q2, https://david-d.ncifcrf.gov/).

Lentivirus infection

The sequence of GPX4 gene (NM_001039848.4, GENERAL Bio, Chuzhou, China) was linked to the pLVX-IRES-puro vector (Fenghui Bio, Changsha, China) and transfected into 293T cells. After 48 hours, the lentivirus supernatant was collected and used to transfect SKOV3/DDP cells for 12 hours. Subsequently, the cells were screened with puromycin for 7 days to select for GPX4 overexpression. The overexpression of GPX4 was confirmed using RT-qPCR and Western blotting.

RT-qPCR

RNA extracted from SKOV3/DDP cells in each group was obtained via TRIzol (TaKaRa Bio, Dalian, China). Then, mRNA was reverse-transcribed into cDNA with the BeyoRT™ II cDNA First Strand Synthesis Kit (Beyotime, Shanghai, China). GAPDH was used as the internal parameter. The reaction system consisted of 1 µL cDNA and 10 µL SYBR Green Master Mix (Solarbio, Beijing, China), 0.5 µL of upstream and downstream primers each (GENERAL Bio, Chuzhou, China), and 8 µL ddH₂O. The reaction conditions included an initial denaturation of 95°C for 5 min, denaturation of 95°C for 10 s, annealing of 60°C for 10 s, extension of 72°C for 1 min (40 cycles), and final extension of 1 min 30s at 72°C. The primer sequences used were GPX4 F, GAAGCAGGAGCCAGGGAGT and GPX4 R, CGCAGCCGTTCTTGTCG; β-actin F, GGCACCCAGCACAATGAA and β-actin R, TAGAAGCATTTGCGGTGG.

Western blot

Each group was lysed with cell lysis solution (Wanlei Bio, Shenyang, China). The BCA protein concentration assay kit (Wanleibio, Shenyang, China) was used to quantify the protein concentration of each group of cells. The proteins were isolated by 14% SDS-polyacrylamide gel electrophoresis (SDS-PAGE) (Wanlei Bio, Shenyang, China) and transferred to polyvinylidene fluoride (PVDF) membranes (Millipore, Billerica, MA, USA). PVDF membrane was sealed with 5% skim milk for 1 hour and incubated overnight with primary anti-GPX4 (Abclonal, Wuhan, China) and anti-β-actin (Wanlei Bio, Shenyang, China) at 4℃. Sheep Anti-Rabbit IgG-HRP (Wanlei Bio, Shenyang, China) was added and incubated at 37℃ for 45min. Finally, ECL (Wanlei Bio, Shenyang, China) was added for exposure scanning, and Gel-Pro-Analyzer software was used to analyze the optical density of the strips.

Ferroptosis determination

Fe²⁺, MDA, SOD, and GSH are indicators of ferroptosis. To measure the contents of these indicators, we used Ferrous Iron Colorimetric Assay Kit (Elabscience, Wuhan, China), MDA Colorimetric Assay Kit (Wanlei Bio, Shenyang, China), Total Superoxide Dismutase Activity Assay Kit (Wanlei Bio, Shenyang, China), and Reduced Glutathione Colorimetric Assay Kit (Wanlei Bio, Shenyang, China) following the manufacturer's instructions. We measured the absorbance and calculated the concentrations of Fe²⁺, MDA, SOD, and GSH in each cell group.

Statistical analysis

We used GraphPad Prism 8.0 software to analyze the results of the experiments. The student t-test was used to compare the differences between two groups, while ANOVA was used to compare between three or more groups. Statistical significance was set at P < 0.05.

ASP and DDP can inhibit the proliferation of SKOV3 and SKOV3/DDP

SKOV3 and SKOV3/DDP cells were treated with DDP (1.5µg/ml, 3 µg/ml, 6µg/ml, 12µg/ml, 24 µg/ml), and cell viability was measured 48 hours later. The results are shown below, SKOV3 groups: compared with the vehicle group, the viability of each group was 81.95%±5.37, 57.70%±7.66, 43.28%±7.09, 35.22%±4.07, 24.56%±3.03; SKOV3/DDP groups: Compared with the vehicle group, the viability of each group was 94.07%±4.36, 88.15%±10.89, 79.20%±9.10, 66.82%±7.92, 53.08%±7.58(Fig. 1A). The viability of each group decreased with increasing DDP concentration when compared to the vehicle group. Similarly, SKOV3/DDP groups showed a decrease in viability with increasing DDP concentration. Student t-test was used to compare the proliferative activity of DDP against SKOV3 and SKOV3/DDP cells, showing significant differences in sensitivity to DDP (P<0.01). We also treated SKOV3 and SKOV3/DDP cells with ASP (50 µg/mL, 100 µg/mL, 150 µg/mL, 200 µg/mL, 250 µg/mL, 300µg/mL), and cell viability was measured 48 hours later. SKOV3 group: Compared with the vehicle group, the viability of each group was 98.95%±8.59, 97.03%±7.57, 94.96%±13.33, 90.37%±6.49, 82.54%±3.83, 76.33%±3.56. SKOV3/DDP group: Compared with the vehicle group, the viability of each group was 98.76%±8.13, 97.61%±6.73, 96.37%±6.56, 90.72%±5.05, 87.86%±0.64, 83.92%±1.41(Fig. 1B).Both SKOV3 and SKOV3/DDP groups showed a decrease in viability with increasing ASP concentration. Overall, ASP inhibited the proliferation of SKOV3 and SKOV3/DDP cells, with increasing inhibition at higher concentrations of ASP.

ASP combined with DDP increases the sensitivity of SKOV3/DDP to DDP

To assess the efficacy of the combination of ASP and DDP on SKOV3/DDP cells, we calculated the combination index (CI) of ASP and DDP. Each group was supplemented with 10 µg/mL of DDP and varying concentrations of ASP. After 48 hours, cell viability was examined and compared to that of the control group. Cell viability decreased with increasing concentrations of ASP, with values of 75.74%±6.76, 70.39%±6.77, 59.96%±8.83, 56.90%±2.91, 51.95%±3.88, 47.47%±3.93, and 38.33%±4.87% (Fig. 2A). Using CompuSyn software, we calculated the CI for each group and obtained values of 1.06, 0.73, 0.71, 0.65, 0.60, and 0.47 (Fig. 2B). These results indicate that ASP and DDP exhibit a synergistic effect, except for ASP at a concentration of 50 µg/mL.

Effect of ASP combined with DDP on SKOV3/DDP in vivo, and the evaluation of safety

The tumor size and growth rate in nude mice were lower in the ASP combined with DDP group than in the other groups. ASP combined with DDP showed higher tumor inhibition rates (52.05%, 48.93%, and 73.99%) than ASP group, DDP group, and vehicle group (Fig. 3C). We collected liver, kidney, and heart tissue from the mice and used HE staining to assess the safety of ASP and DDP treatment in vivo. The results indicated that ASP combined with DDP had no significant effect on the morphology of the liver, kidney, or heart, suggesting that it is a safe treatment option (Fig. 3D).

Effects of ASP combined with DDP on SKOV3/DDP cell invasion

The effects of the ASP group, the DDP group, and ASP combined with DDP group on SKOV3/DDP cell invasion were investigated using the Transwell method. The combined ASP and DDP group demonstrated lower average cell counts passing through the permeable membrane of the Transwell compared to the ASP and DDP groups. The ASP combined with DDP showed a stronger inhibition of SKOV3/DDP cell invasion (Fig. 4A&B).

The cell's transcriptome was sequenced to identify key genes

To investigate whether the combination of ASP and DDP can regulate key genes in SKOV3/DDP transcription process, we conducted transcriptome sequencing on two groups: ASP combined with DDP and DDP alone. First, the correlation analysis (Fig. 5A) and principal component analysis (PCA) (Fig. 5B) indicate that the obtained data is reliable and there are significant differences between the data of the DDP group and the ASP combined with DDP group. Next, using the defined threshold, we obtained 843 DEGs(Fig. 5C), among which 405 down-regulated genes and 438 up-regulated genes existed (Fig. 5D) (Table.1s).We then analyzed these DEGs by GO and KEGG. The GO enrichment analysis showed that Molecular Function (MF) was mainly enriched in structural constituent of chromatin, structural constituent of ribosome, and protein heterodimerization activity. Biological Processes (BP) were mainly concentrated in cytoplasmic translation, nucleosome assembly, and translation. Cellular Component (CC) was mainly concentrated in nucleosome and mitochondrial inner membrane (Fig. 6A&B) (Table.2s). KEGG enrichment analysis showed that DEGs were mainly concentrated in oxidative phosphorylation, tumor transcriptional regulation, and other pathways (Fig. 6C&D) (Table.3s). Previous studies by Xiaodong Wu et al. have found that the expression level of GPX4 in SKOV3/DDP cells is higher than in SKOV3 cells and that its increase is associated with the prognosis and drug resistance of patients[17]. After conducting a comprehensive analysis on both log₂ Fold Change ranking and P-value ranking, as well as reviewing relevant publications, it is postulated that GPX4 is a key gene involved in the regulation of SKOV3/DDP cells. Consequently, our subsequent experiment will be centered on the exploration of GPX4.

RTqPCR and western blot analysis of key genes GPX4

We treated SKOV3/DDP cells with ASP, DDP, and a combination of ASP and DDP. GPX4 expression was detected via both RT-qPCR (Fig. 7A) and western blot (Fig. 7B&C). Our results showed that GPX4 was significantly down-regulated in the group treated with a combination of ASP and DDP. Furthermore, to investigate the role of GPX4 in SKVO3/DDP cells, we generated stable SKVO3/DDP cells that overexpress GPX4, referred to as SKOV3/DDP-GPX4 cells. We then compared the expression of GPX4 in SKOV3/DDP, SKOV3/DDP-NC (negative control group), and SKOV3/DDP-GPX4 cells using RT-qPCR (Fig. 7D) and western blot (Fig. 7E&F). The data suggested that GPX4 was highly expressed in SKOV3/DDP-GPX4 cells.

ASP combined with DDP inhibited SKOV3/DDP proliferation and invasion by inhibiting GPX4

The control group, SKOV3/DDP-NC group, and SKOV3/DDP-GPX4 group were treated with Veh, DDP, ASP combined with DDP, respectively, and the cell proliferation of each group was detected using CCK8. The results showed that the cell viability of the control group, SKOV3/DDP-NC group, and SKOV3/DDP-GPX4 group treated with ASP combined with DDP was significantly lower than that of the group treated with DDP alone (Fig. 8A). Similarly, transwell assay was performed to assess the cell invasion ability, and it was observed that the cell invasion ability of the control group, SKOV3/DDP-NC group, and SKOV3/DDP-GPX4 group treated with ASP combined with DDP was significantly lower than that of the group treated with DDP alone (Fig. 8B&C). These results indicate that ASP combined with DDP can effectively inhibit cell proliferation and invasion by suppressing GPX4.

ASP combined with DDP can cause ferroptosis of SKOV3/DDP by inhibiting GPX4 expression of SKOV3/DDP

To investigate how ASP combined with DDP facilitates ferroptosis in SKOV3/DDP cells, we conducted experiments to determine the levels of Fe²⁺, MDA, SOD, and GSH. The control group, SKOV3/DDP-NC group, and SKOV3/DDP-GPX4 group were treated with Veh, DDP, and ASP combined with DDP. We observed a significant increase in Fe²⁺ and MDA content in all groups following the treatment of ASP combined with DDP (Fig. 9A&B). Conversely, the levels of SOD and GSH were significantly reduced in all groups treated with ASP combined with DDP (Fig. 9C&D).

At present, drug resistance remains a significant cause of poor prognosis in individuals with cancer[18]. Research has indicated that drug resistance in tumors is connected to the tumor microenvironment, and traditional Chinese medicine may be able to reverse drug resistance by regulating the tumor microenvironment [19]. ASP possesses multiple functions, such as anti-tumor capabilities, the regulation of cell microenvironment, and the treatment of gynecological diseases[6, 20]. Thus, we hypothesize that ASP can reverse the drug resistance of ovarian cancer by regulating its microenvironment or substance metabolism. Initially, we conducted a verification experiment on SKOV3 and the sensitivity of SKOV3/DDP cells to DDP. The experiment demonstrated that the sensitivity of SKOV3/DDP cells to DDP was lower than that of SKOV3 cells. We also conducted experiments on whether ASP has an inhibitory effect on SKOV3 and SKOV3/DDP cells. The results indicated that ASP had an inhibitory effect on SKOV3 but had a poor inhibitory effect on SKOV3/DDP cells. To better understand whether the combination of two drugs has a synergistic effect, we calculated the combination index (CI) values of the two drugs[21]. The results showed that the two drugs had excellent synergistic effects. Additionally, we performed in vivo testing with the drugs. After subcutaneously forming tumors in nude mice, we intraperitoneally injected the drugs and periodically measured the condition of the tumors in the mice. The results showed that the combination of ASP and DDP had an excellent tumor inhibition effect. Furthermore, evaluating drug safety is crucial when considering drug clinical applications [22]. It has been reported that ASP has antioxidant protection on cardiac cells [23] and reduce liver fibrosis induced by ccl₄[24].The liver, heart, and kidney conditions were evaluated in each group of nude mice, and no differences were found among them, demonstrating that ASP combined with DDP had good safety. Tumor invasion is related to drug resistance [25], so the effects of ASP, DDP, and ASP combined with DDP on SKOV3/DDP invasion were investigated. The results revealed that ASP combined with DDP could better inhibit SKOV3/DDP invasion. Next, we explored the specific mechanism by which ASP combined with DDP changes the resistance of SKOV3/DDP cells to DDP.

Comparison of the RNA-seq data between the DDP group and the ASP combined with DDP group revealed the presence of numerous DEGs involved in transcription. GO [26] and KEGG[27] analyses of the DEGs showed enrichment in metabolic processes and cellular transcriptional regulation. Specifically, cancer cells undergo different metabolic processes from normal cells[28], with increased glycolysis demonstrated by the Warburg effect [29] and a dependence on rapid tricarboxylic acid (TCA) cycling to sustain proliferation [30]. The Warburg effect has also been linked to drug resistance [31]. Additionally, drug resistance in ovarian cancer has been associated with cellular transcriptional regulation [32]. In conclusion, the analysis suggests a relationship between the combination of ASP with DDP and the regulation of drug resistance in SKOV3/DDP cells.

We sequenced differentially expressed genes based on their log₂ fold change and p-value, and reviewed the relevant literature. According to previous studies, GPX4 expression is higher in SKOV3/DDP cells compared to SKOV3 cells, and inhibition of GPX4 can reduce drug resistance in resistant cells[17]. Therefore, we hypothesized that downregulation of GPX4, caused by the combination of ASP and DDP, is responsible for the decreased resistance of SKOV3/DDP cells.

In subsequent experiments, SKOV3/DDP cells were stably transfected with lentivirus to overexpress GPX4, and relevant experiments were conducted. The results showed that overexpressing GPX4 in SKOV3/DDP cells increased their resistance to DDP, while combining ASP with DDP reversed DDP resistance.

Studies have shown that GPX4 is a crucial gene involved in regulating ferroptosis in cells [33]. Ferroptosis is a complex biological process characterized by a toxic accumulation of lipid peroxides on cell membranes[34, 35]. It has also been associated with tumor occurrence and development [36]. Specifically, Wang Y et al. demonstrated that the regulation of ferroptosis can affect the sensitivity of platinum-resistant ovarian cells to DDP[37]. The presence of Fe²⁺[38]and MDA[39], which are products of membrane lipid peroxidation, plays a crucial role in detecting ferroptosis. Moreover, SOD [40] and GSH [41] are also used as diagnostic criteria for ferroptosis in cells. Experimental results revealed that the combination of ASP and DDP treatment led to a significant increase in Fe²⁺ and MDA levels, while SOD and GSH levels were significantly decreased. These changes indirectly indicate a reduction in GPX4 expression and an exacerbation of ferroptosis in SKOV3/DDP cells.(Fig. 10).

Because ASP is predominantly composed of glucose (Glc), galactose (Gal), arabinose (Ara), rhamnose (Rha), fucose (Fuc), xylose (Xyl), and galacturonic acid (GalUA)[6], we will conduct further investigations on the monosaccharide components in ASP in order to examine their impact on tumor. Additionally, our study reveals an interesting finding from the GO analysis, indicating an enrichment of the Cellular Component (CC) in the mitochondrial inner membrane. It is well-known that cellular ferroptosis induces alterations in mitochondria [42]. Mitochondria, being instrumental in cysteine-deprivation induced (CDI) ferroptosis, do not, however, play a significant role in inhibiting GPX4-induced ferroptosis[43]. As a result, we question whether ASP combined with DDP possesses an alternative mechanism for regulating the ferroptosis of SKOV3/DDP cells, aside from its influence on GPX4. Moreover, it is worth noting that GPX4 expression is frequently controlled by Non-coding RNA [44]. We hypothesize that the combination of ASP and DDP can regulate the GPX4 expression in SKOV3/DDP through non-coding RNA, thereby promoting an increase in SKOV/DDP ferroptosis. This aspect will also be one of our focal points of investigation.

Due to the intricate mechanism of ferroptosis in tumors, additional experiments will be conducted to determine the specific mechanism by which ASP combined with DDP inhibits GPX4 expression.

We discovered that ASP combined with DDP can regulate the transcription process of SKOV3/DDP, and it can inhibit the expression of the key gene GPX4 to promote the ferroptosis of SKOV3/DDP. This finding provides a theoretical basis for the clinical application of ASP.

ASP Angelica Sinensis Polysaccharide

DDP cisplatin

MDA malondialdehyde

SOD superoxide dismutase

GSH Glutathione

HE Hematoxylin-eosin

GPX4 Recombinant Glutathione Peroxidase 4

RNA-seq RNA-sequencing

DEGs Differentially Expressed Genes

CCK8 Cell Counting Kit-8

PBS Phosphate Buffered Saline

FBS Fetal Bovine Serum

CI Combination Index

PVDF polyvinylidene fluoride

Veh Vehicle group

GO Gene Ontology

KEGG Kyoto Encyclopedia of Genes and Genomes

MF Molecular Function

BP Biological Processes

CC Cellular Component

MDA Malondialdehyde

CDI cysteine-deprivation induced

Acknowledgements

Not applicable.

Author Contributions

Experiment Design: Yaoxian Wang Weikang Guo. Experiment Operation: Weikang Guo, Fei

Lei, Ruxin Zheng, Xinyao Zhao,Yunshun Tong.Data Statistics: Yuze Gu, Mengdi Yang, Yunshun Tong. Article Written: Weikang Guo,Wanyue Wang.

Disclosure statement

The authors declare no competing interests.

Funding

WU JIEPING Medical Foundation Special Fund for Clinical Research (No.320.6750.2023-05-4).

Data availability

All datasets generated for this study are included in the article/ supplementary material, further inquiries can be directed to the corresponding authors.

Ethics approval and consent to participate

The animal study was reviewed and approved by the Ethics Committee of Harbin Medical University(certificate number: KY2023-37).

Consent for publication

Manuscript is approved by all authors for publication.

Competing interests

The authors declare no competing interests.

Author details

1Department of Gynecology, Harbin Medical University Cancer Hospital, Harbin,150081, China

2. School of Basic Medical Sciences, Qiqihar Medical University, Qiqihar,161006,China

3. School of Science, Department of Biological Sciences, Xi’an Jiaotong-Liverpool University, Suzhou, 215123, China

Bray F, Ferlay J, Soerjomataram I, Siegel RL, Torre LA, Jemal A: Global cancer statistics 2018: GLOBOCAN estimates of incidence and mortality worldwide for 36 cancers in 185 countries. CA Cancer J Clin 2018, 68(6):394-424.
Stewart C, Ralyea C, Lockwood S: Ovarian Cancer: An Integrated Review. Semin Oncol Nurs 2019, 35(2):151-156.
Buechel M, Herzog TJ, Westin SN, Coleman RL, Monk BJ, Moore KN: Treatment of patients with recurrent epithelial ovarian cancer for whom platinum is still an option. Ann Oncol 2019, 30(5):721-732.
Munoz-Galvan S, Carnero A: Targeting Cancer Stem Cells to Overcome Therapy Resistance in Ovarian Cancer. Cells 2020, 9(6).
Wei WL, Zeng R, Gu CM, Qu Y, Huang LF: Angelica sinensis in China-A review of botanical profile, ethnopharmacology, phytochemistry and chemical analysis. J Ethnopharmacol 2016, 190:116-141.
Nai J, Zhang C, Shao H, Li B, Li H, Gao L, Dai M, Zhu L, Sheng H: Extraction, structure, pharmacological activities and drug carrier applications of Angelica sinensis polysaccharide. Int J Biol Macromol 2021, 183:2337-2353.
Chen Y, Duan JA, Qian D, Guo J, Song B, Yang M: Assessment and comparison of immunoregulatory activity of four hydrosoluble fractions of Angelica sinensisin vitro on the peritoneal macrophages in ICR mice. Int Immunopharmacol 2010, 10(4):422-430.
Cao P, Sun J, Sullivan MA, Huang X, Wang H, Zhang Y, Wang N, Wang K: Angelica sinensis polysaccharide protects against acetaminophen-induced acute liver injury and cell death by suppressing oxidative stress and hepatic apoptosis in vivo and in vitro. Int J Biol Macromol 2018, 111:1133-1139.
Song X, Kong J, Song J, Pan R, Wang L: Angelica sinensis Polysaccharide Alleviates Myocardial Fibrosis and Oxidative Stress in the Heart of Hypertensive Rats. Comput Math Methods Med 2021, 2021:6710006.
Sui Y, Liu W, Tian W, Li XQ, Cao W: A branched arabinoglucan from Angelica sinensis ameliorates diabetic renal damage in rats. Phytother Res 2019, 33(3):818-831.
Zhang Y, Zhou T, Wang H, Cui Z, Cheng F, Wang KP: Structural characterization and in vitro antitumor activity of an acidic polysaccharide from Angelica sinensis (Oliv.) Diels. Carbohydr Polym 2016, 147:401-408.
Cao W, Li XQ, Wang X, Fan HT, Zhang XN, Hou Y, Liu SB, Mei QB: A novel polysaccharide, isolated from Angelica sinensis (Oliv.) Diels induces the apoptosis of cervical cancer HeLa cells through an intrinsic apoptotic pathway. Phytomedicine 2010, 17(8-9):598-605.
Fang L, Lin H, Wu Z, Wang Z, Fan X, Cheng Z, Hou X, Chen D: In vitro/vivo evaluation of novel mitochondrial targeting charge-reversal polysaccharide-based antitumor nanoparticle. Carbohydr Polym 2020, 234:115930.
Liu X, Wu Z, Guo C, Guo H, Su Y, Chen Q, Sun C, Liu Q, Chen D, Mu H: Hypoxia responsive nano-drug delivery system based on angelica polysaccharide for liver cancer therapy. Drug Deliv 2022, 29(1):138-148.
Lu Y, Pan Q, Gao W, Pu Y, He B: Reversal of cisplatin chemotherapy resistance by glutathione-resistant copper-based nanomedicine via cuproptosis. J Mater Chem B 2022, 10(33):6296-6306.
Zhou WJ, Wang S, Hu Z, Zhou ZY, Song CJ: Angelica sinensis polysaccharides promotes apoptosis in human breast cancer cells via CREB-regulated caspase-3 activation. Biochem Biophys Res Commun 2015, 467(3):562-569.
Wu X, Shen S, Qin J, Fei W, Fan F, Gu J, Shen T, Zhang T, Cheng X: High co-expression of SLC7A11 and GPX4 as a predictor of platinum resistance and poor prognosis in patients with epithelial ovarian cancer. BJOG 2022, 129 Suppl 2:40-49.
Chatterjee S, Damle SG, Sharma AK: Mechanisms of resistance against cancer therapeutic drugs. Curr Pharm Biotechnol 2014, 15(12):1105-1112.
Cui Q, Wang JQ, Assaraf YG, Ren L, Gupta P, Wei L, Ashby CR, Jr., Yang DH, Chen ZS: Modulating ROS to overcome multidrug resistance in cancer. Drug Resist Updat 2018, 41:1-25.
Chen XP, Li W, Xiao XF, Zhang LL, Liu CX: Phytochemical and pharmacological studies on Radix Angelica sinensis. Chin J Nat Med 2013, 11(6):577-587.
Chou TC: Theoretical basis, experimental design, and computerized simulation of synergism and antagonism in drug combination studies. Pharmacol Rev 2006, 58(3):621-681.
Mansi L, Thiery-Vuillemin A, Nguyen T, Bazan F, Calcagno F, Rocquain J, Demarchi M, Villanueva C, Maurina T, Pivot X: Safety profile of new anticancer drugs. Expert Opin Drug Saf 2010, 9(2):301-317.
Niu X, Zhang J, Ling C, Bai M, Peng Y, Sun S, Li Y, Zhang Z: Polysaccharide from Angelica sinensis protects H9c2 cells against oxidative injury and endoplasmic reticulum stress by activating the ATF6 pathway. J Int Med Res 2018, 46(5):1717-1733.
Wang K, Wang J, Song M, Wang H, Xia N, Zhang Y: Angelica sinensis polysaccharide attenuates CCl(4)-induced liver fibrosis via the IL-22/STAT3 pathway. Int J Biol Macromol 2020, 162:273-283.
Liang Y, McDonnell S, Clynes M: Examining the relationship between cancer invasion/metastasis and drug resistance. Curr Cancer Drug Targets 2002, 2(3):257-277.
Ashburner M, Ball CA, Blake JA, Botstein D, Butler H, Cherry JM, Davis AP, Dolinski K, Dwight SS, Eppig JT et al: Gene ontology: tool for the unification of biology. The Gene Ontology Consortium. Nat Genet 2000, 25(1):25-29.
Kanehisa M, Goto S: KEGG: kyoto encyclopedia of genes and genomes. Nucleic Acids Res 2000, 28(1):27-30.
Xiong Y, Wang L, Feng Y, Li X, Jia L, Han Y: Flexibility in metabolism bestows tenacious viability on cancer. Life Sci 2018, 208:20-25.
Liu C, Jin Y, Fan Z: The Mechanism of Warburg Effect-Induced Chemoresistance in Cancer. Front Oncol 2021, 11:698023.
Eniafe J, Jiang S: The functional roles of TCA cycle metabolites in cancer. Oncogene 2021, 40(19):3351-3363.
Bhattacharya B, Mohd Omar MF, Soong R: The Warburg effect and drug resistance. Br J Pharmacol 2016, 173(6):970-979.
Richardson A, Kaye SB: Drug resistance in ovarian cancer: the emerging importance of gene transcription and spatio-temporal regulation of resistance. Drug Resist Updat 2005, 8(5):311-321.
Seibt TM, Proneth B, Conrad M: Role of GPX4 in ferroptosis and its pharmacological implication. Free Radic Biol Med 2019, 133:144-152.
Hassannia B, Vandenabeele P, Vanden Berghe T: Targeting Ferroptosis to Iron Out Cancer. Cancer Cell 2019, 35(6):830-849.
Mou Y, Wang J, Wu J, He D, Zhang C, Duan C, Li B: Ferroptosis, a new form of cell death: opportunities and challenges in cancer. J Hematol Oncol 2019, 12(1):34.
Lei G, Zhuang L, Gan B: Targeting ferroptosis as a vulnerability in cancer. Nat Rev Cancer 2022, 22(7):381-396.
Wang Y, Zhao G, Condello S, Huang H, Cardenas H, Tanner EJ, Wei J, Ji Y, Li J, Tan Y et al: Frizzled-7 Identifies Platinum-Tolerant Ovarian Cancer Cells Susceptible to Ferroptosis. Cancer Res 2021, 81(2):384-399.
Dixon SJ, Lemberg KM, Lamprecht MR, Skouta R, Zaitsev EM, Gleason CE, Patel DN, Bauer AJ, Cantley AM, Yang WS et al: Ferroptosis: an iron-dependent form of nonapoptotic cell death. Cell 2012, 149(5):1060-1072.
Ursini F, Bosello Travain V, Cozza G, Miotto G, Roveri A, Toppo S, Maiorino M: A white paper on Phospholipid Hydroperoxide Glutathione Peroxidase (GPx4) forty years later. Free Radic Biol Med 2022, 188:117-133.
Song JX, An JR, Chen Q, Yang XY, Jia CL, Xu S, Zhao YS, Ji ES: Liraglutide attenuates hepatic iron levels and ferroptosis in db/db mice. Bioengineered 2022, 13(4):8334-8348.
Ursini F, Maiorino M: Lipid peroxidation and ferroptosis: The role of GSH and GPx4. Free Radic Biol Med 2020, 152:175-185.
Wang H, Liu C, Zhao Y, Gao G: Mitochondria regulation in ferroptosis. Eur J Cell Biol 2020, 99(1):151058.
Fernandez-Acosta R, Hassannia B, Caroen J, Wiernicki B, Alvarez-Alminaque D, Verstraeten B, Van der Eycken J, Vandenabeele P, Vanden Berghe T, Pardo-Andreu GL: Molecular Mechanisms of Nemorosone-Induced Ferroptosis in Cancer Cells. Cells 2023, 12(5).
He GN, Bao NR, Wang S, Xi M, Zhang TH, Chen FS: Ketamine Induces Ferroptosis of Liver Cancer Cells by Targeting lncRNA PVT1/miR-214-3p/GPX4. Drug Des Devel Ther 2021, 15:3965-3978.

No competing interests reported.

Table1s.xlsx
Supplementary Information Additional fle : Table S1. DEGs from DDP group and the ASP combined with DDP group transcriptional sequencing.
Table2s.xlsx
Table S2. The result of GO analysis
Table3s.xlsx
Table S2. The result of GO analysis

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The combination of Angelica sinensis polysaccharide and cisplatin promotes ferroptosis in cisplatin-resistant ovarian cancer cells by regulating GPX4, thereby reversing their resistance to cisplatin

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Abstract

Figures

Introduction

Materials and methods

Cell culture

Cell viability and combination index (CI)

In vivo xenograft assays

Histological analysis

Cell invasion assays

RNA-seq analysis

Lentivirus infection

RT-qPCR

Western blot

Ferroptosis determination

Statistical analysis

Results

ASP and DDP can inhibit the proliferation of SKOV3 and SKOV3/DDP

ASP combined with DDP increases the sensitivity of SKOV3/DDP to DDP

Effect of ASP combined with DDP on SKOV3/DDP in vivo, and the evaluation of safety

Effects of ASP combined with DDP on SKOV3/DDP cell invasion

The cell's transcriptome was sequenced to identify key genes

RTqPCR and western blot analysis of key genes GPX4

ASP combined with DDP inhibited SKOV3/DDP proliferation and invasion by inhibiting GPX4

ASP combined with DDP can cause ferroptosis of SKOV3/DDP by inhibiting GPX4 expression of SKOV3/DDP

Discussion

Conclusions

Abbreviations

Declarations

References

Additional Declarations

Supplementary Files

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