Network pharmacology and experimental methods to explore the mechanism of Guizhi Fuling capsule in overcoming cisplatin-resistant ovarian cancer

doi:10.21203/rs.3.rs-4832576/v1

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Network pharmacology and experimental methods to explore the mechanism of Guizhi Fuling capsule in overcoming cisplatin-resistant ovarian cancer

https://doi.org/10.21203/rs.3.rs-4832576/v1

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Objective

Ovarian cancer is a common gynecological malignant tumor, and its incidence ranks high among female malignant tumors. Based on network pharmacology and bioinformatics analysis, the mechanism of GFC in the treatment of ovarian cancer was explored by in vitro and in vivo experiments.

Methods: Firstly, the common target genes of ovarian cancer and the effective chemical components of GFC were screened, and network pharmacology was used to identify the potential targets of GFC in the treatment of ovarian cancer. Then, the prediction results of network pharmacology were verified by cell and animal experiments. Finally, the use of LC-MS/MS of GFC representative compounds and the main effective component is analyzed.

Results: Effective chemical components of GFC can target 265 potential sites in ovarian cancer. Gene enrichment analysis has revealed that the dysregulation of glycolysis metabolism and its associated pathways are promising targets for treating ovarian cancer. Experimental results have demonstrated that cisplatin resistance in ovarian cancer is correlated with elevated glycolysis levels. When combined with cisplatin, GFC consistently suppresses glycolysis levels and the activity of its regulatory enzymes in A2780/DDP cells, while initially increasing and subsequently decreasing oxidative phosphorylation levels. Oxidative phosphorylation of ATP activating ROS caused mitochondrial autophagy and apoptosis. Animal studies have further confirmed that the combination treatment of GFC and cisplatin effectively inhibits tumor growth, as well as glycolysis and oxidative phosphorylation in mice.

Conclusions: GFC combined with cisplatin can rebalance energy metabolism and promote the apoptosis of ovarian cancer cells.

According to statistics, the incidence of ovarian cancer ranks the third in the female reproductive system malignant tumors, and its mortality is second only to breast cancer and cervical cancer. Globally, the incidence of ovarian cancer in developed countries is 9.1 per 100,000, while the incidence in developing countries is 5.0 per 100,000^[1]. At present, the increase of drug resistance in ovarian cancer seriously affects the treatment effect of ovarian cancer. There are many reasons for the occurrence of ovarian cancer drug resistance, such as individual differences in drug sensitivity, gene mutations, poor body immunity, cancer cell metastasis and so on^{[2, 3]}. Therefore, accurate treatment plans should be formulated according to the specific conditions of patients in the treatment process to avoid the occurrence of drug resistance and improve the treatment effect and survival rate of patients.

Otto Warburg, a German biochemist, proposed that the way of glucose metabolism in tumor cells has changed from oxidative phosphorylation to aerobic glycolysis, namely the Warburg effect^[4]. Tumor cells choose glycolysis as the main way of energy production. On the one hand, through glycolysis, they can obtain intermediate metabolites for the synthesis of fat, protein and nucleic acid to meet their active synthesis needs^[5]. Secondly, lactic acid produced by glycolysis can provide an acidic environment for the growth and invasion of tumor cells, which is conducive to the survival and spread of tumor cells^[6]. Therefore, understanding the relationship between tumors and oxidative phosphorylation and glycolysis will help us to understand the metabolic characteristics of tumors more deeply, and provide new ideas and methods for the diagnosis and treatment of tumors.

There is mounting evidence that the application of network pharmacology and bioinformatics can accurately predict the interaction between active chemical components in herbal medicines and specific disease targets. Guizhi Fuling capsule (GFC) is a traditional Chinese medicine formula composed of cassia twig, Poria cocos, Mony peel, red peony root and peach kernel. Studies have shown that GFC has anti-inflammatory and anti-tumor effects^[7–9]. However, the anti-tumor mechanism of GFC is not fully understood, and there are still some limitations and challenges in clinical application. The aim of this study is to explore the molecular mechanism of GFC in reversing cisplatin resistance of ovarian cancer, and to lay a theoretical foundation for clinical application.

Cell lines and cell culture

Human ovarian cancer cell line A2780 and cisplatin-resistant ovarian cancer cell line A2780/DDP were purchased from Beijing Dingguo Changsheng Biotechnology Co., Ltd. (Beijing, China). The cells were grown in DMEM/high glucose (Hyclone, USA) with 10% fetal bovine serum (Hyclone, Logan, Utah, USA), 100 U/mL penicillin, and 100 mg/mL streptomycin (Genview, Australia), and they were incubated at 37℃ and 5% CO₂ .

Reagents and antibodies

The Guizhi Fuling capsule (GFC) is a proprietary compound Chinese medicine formulated by Jiangsu Kangyuan Pharmaceutical Co., Ltd. (Lianyungang, Jiangsu, China)comprising Guizhi, Mudanpi, Taoren, Baishao, and Fuling in an equimolar ratio. The concentration of GFC crude drug is 0.31g/capsule. Cisplatin was procured from Sigma Aldrich Company (St. Louis, MO, USA). Primary antibodies included HIF-1α, HK2, PKM2, LDHA, ATP1, ATP5, Caspase 3/p17/p19, Caspase 9/p35, Bcl2, BAX, P glycoprotein, MRP, BCRP were obtained from Proteintech Company (Wuhan, Hubei, China). HRP-Goat anti-rabbit recombinant secondary antibody (H + L) was obtained from Proteintech Company (Wuhan, Hubei, China).

Screening of ovarian cancer and GFC targets

Utilizing the GeneCards database collection (https://www.genecards.org/) of targets related to ovarian cancer. Utilizing TCMSP (http://tcmspw.com/tcmsp.php) and ETCM (http://www.tcmip.cn/ETCM/index.php) for the exploration of the chemical composition and targets of GFC.

Target gene enrichment and network pharmacological analysis

A Venn diagram was constructed, and the overlapping region was identified as a potential target for GFC treatment of ovarian cancer. CFC and ovarian cancer were found to have common targets for Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis, with all screenings showing p < 0.05. TCMSP filters the active components of GFC corresponding to common target genes and constructs a network pharmacological map, and the filtration criteria are: oral bioavailability (OB) ≥ 30%, drug similarity (DL) ≥ 0.18.

Cell viability assay

Parental A2780 cells or resistant A2780/DDP cells were seeded into 96-well plates at a density of 5000 cells/well. After 16 hours of culture, the culture medium was removed. The cells were then treated with different concentrations of cisplatin (ranging from 0 to 300 µM) or GFC solution (ranging from 0 to 300 mg/mL) for 24 hours. Subsequently, 10 µL of MTT solution (Solarbio, Beijing, China) was added to each well and incubated at 37 ℃ for 4 hours. The absorbance was measured using a microplate reader set to a wavelength of 570 nm.

Cell proliferation was detected by EdU

Cell slides were initially placed in 24-well plates, and either parental A2780 cells or drug-resistant A2780/DDP cells at a density of 2×10⁴ cells/well were seeded into the plates. After 16 hours of culture, the culture medium was removed. The cells were then treated with drug-free culture medium for 24 hours followed by treatment with the drug for another 24 hours. Subsequently, 300 µL of EdU-labeled culture medium (Beyotime, Shanghai, China) was added and incubated at 37 ºC in the dark for 2 hours. The slides were then gently washed once with PBS using sterile forceps. Following this, the cells were fixed with 4% paraformaldehyde for 30 minutes and permeabilized with Triton X-100 solution (0.1%) for an additional 5 minutes at room temperature. Nuclei staining was achieved by adding 300 µL of Hoechst33342 solution and incubating in the dark at room temperature for a further period of time lasting up to approximately thirty minutes. Finally, experimental results were captured using a fluorescence microscope.

Glucose, pyruvate and lactic acid assay

After 24 hours of incubation, A2780 cells, drug-resistant A2780/DDP cells, drug-free A2780/DDP cells, and drug-treated A2780/DDP cells were collected with a total cell count of 5×10⁶. The cells were lysed using low temperature ultrasonic waves and subsequently boiled in water for 10 minutes. The resulting cell lysate was then centrifuged at 8000 g for 10 minutes at room temperature, and the supernatant was retained. Following the instructions provided with the glucose, pyruvate, and lactic acid detection kit (mlbio, Shanghai, China), detection reagents were added to each sample. Subsequently, the absorbance value of each sample was measured at wavelengths of 505nm, 520nm, and 570nm using an enzyme label reader.

Mitochondrial transhydrogenase 2 (TH2) enzyme activity assay

A total of 5×10⁶ cells were homogenized using an ice-bath homogenizer, and the homogenate was then centrifuged at 600 g for 5 minutes at 4°C. The supernatant was removed and subjected to a further centrifugation at 110000 g for 10 minutes at 4°C. Subsequently, the resulting supernatant was combined with the reaction reagent (Sangon Biotecand, Shanghai, China) incubated in a water bath at 37°C for 5 minutes. The absorbance was measured using a microplate reader set to a wavelength of 375 nm.

ATP content assay

The protocol was carried out in accordance with the instructions of the ATP content detection kit (mlbio, Shanghai, China). A total of 5×10⁶ cells were collected and added to 1 mL of acidic extract. The cells were then disrupted using an ice bath homogenizer. The homogenate was subsequently centrifuged at 8000g for 10 minutes at 4℃. The resulting supernatant was combined with 1mL of alkaline extract, thoroughly mixed, and then subjected to another round of centrifugation at 8000g for 10 minutes at 4℃. The wavelength for enzyme label measurement was set to 700nm.

Hexokinase, pyruvate kinase and lactate dehydrogenase enzyme activities assay

A total of 5×10⁶ cells were added to 1mL of the extract, and the cells were disrupted by sonication and centrifuged at 8000 g for 10 min at 4℃. The supernatant was mixed with the reaction reagent (mlbio, Shanghai, China), and the wavelength of the microplate reader was adjusted to 340nm, 340nm and 450nm for measurement.

Rreactive oxygen species assay

A2780/DDP cells were treated for 24 hours in both the drug-free control group and the drug experimental group. After treatment, cells were collected following pancreatic enzyme digestion. The cell concentration was adjusted to 1×10⁷/mL using DCFH-DA solution (final concentration of 10µmol/L). Subsequently, the cells were incubated in a 37 ºC incubator for 20 minutes and then washed three times with serum-free cell culture solution to ensure complete removal of any DCFH-DA that had not entered the cells. The intensity of fluorescence before and after stimulation was measured using a fluorescent enzyme label under conditions set at an excitation wavelength of 488nm and an emission wavelength of 525nm.

Cell apoptosis was detected by Annexin V-PE/7-AAD

After the treatment of drug-free and drug-free experimental group with A2780/DDP cells for 24 h, the amount of pancreatic enzyme digestion was 4×10⁵ cells. The cells were washed twice with pre-cooled PBS, centrifuged at 4℃ for 5 min at 300 g, and then the supernatant was discarded and added with 100 µl Binding Buffer. Annexin 5µl V-PE and 5µl 7-AAD staining solution (Beyotime, Shanghai, China) were added, incubated for 10 min at room temperature and protected from light, and detected by flow cytometry.

Cell apoptosis was detected by TUNEL assay

Cell climbs were initially placed in 24-well plates, followed by seeding of 2×10⁵ A2780/DDP cells in each well. After 16 hours of adherent culture, the cells in both the control group and the drug experimental groups were treated for 24 hours. The slides were then gently grabbed with sterile forceps and washed once with PBS. Subsequently, the cells were fixed with 4% paraformaldehyde for 30 minutes, followed by incubation with a Triton X−100 solution (0.1%) for 5 minutes at room temperature to permeabilize the cells. Next, 50µL of TUNEL detection solution (Beyotime, Shanghai, China) was added and incubated at 37ºC in the dark for 60 minutes, after which the experimental results were captured using a fluorescence microscope.

Chemical constituents of GFC based on LC-MS/MS analysis

The GFC powder was dissolved in 50% aqueous methanol, and the supernatant was retained after centrifugation. Using Q Exactive Orbitrap high resolution mass spectrometry to mass spectrum data collection, specific parameters setting is: The positive ion and negative ion modes were scanned separately. The ion source voltage was 3.2kV, Capillary temp was 320 ° C, Aux gas heater temp was 350 ° C, Sheath gas flow rate was 40L/min. The Aux gas flow rate was 15L/min.

Western blot

The parental A2780 cells, drug-resistant A2780/DDP cells, A2780/DDP cells without drug treatment, and A2780/DDP cells treated with drug were lysed using RIPA solution to extract protein. Equal amounts of protein samples from each group were loaded for SDS-PAGE electrophoresis at 80V for 25 min and then at 120V for 50 min. The protein bands in the SDS-PAGE gel were transferred to a PVDF membrane at 25V and 1.3A for 14 minutes. The PVDF membranes containing proteins were washed with TBST, followed by overnight blocking with a 5% skim milk solution, a two-hour incubation with the primary antibody, and finally a one-hour incubation with the secondary antibody. Subsequently, the ECL solution (Vazyme, Shanghai, China)was used to develop the imaging.

Statistical Analysis

The experimental data were all repeated at least three times and expressed as mean ± SD, and all of the datas were analyzed with SPSS 19.0. The differences in cell cycle and proliferation, glycolytic parameters and oxidative phosphorylation between parental A2780 cells and resistant A2780/DDP cells were evaluated by two-tailed Student's t test. In A2780/DDP cells, the differences of cell cycle and proliferation, apoptosis, glycolysis and oxidative phosphorylation in different experimental groups were evaluated by Analysis of variance (ANOVA). P value lower than 0.05 was considered significant.

GFC treatment of ovarian cancer target prediction and build network pharmacology

A total of 10392 ovarian cancer targets were obtained from the GeneCards database. There are 414 targets for obtaining the chemical composition of GFC from the TCMSP and ETCM databases. As shown in Fig. 1-A, the targets for GFC and ovarian cancer have 265 intersections. Enrichment analysis was performed on the targets of GFC in the treatment of ovarian cancer. GO and KEGG correlation analysis showed that the top 20 enriched items (P < 0.05), Mainly includes the Glycolysis and Gluconeogenesis, Pyruvate metabolism, Carbon metabolism, Starch and sucrose metabolism, etc. (Fig. 1-B and C). Furthermore, a network pharmacological map of GFC for the treatment of ovarian cancer was constructed (Fig. 1-D).

Cisplatin resistance in ovarian cancer caused by glycolysis

The imbalance between glycolysis and oxidative phosphorylation is a ubiquitous feature of cancer cells. As shown in Fig. 2-A to F, the glycolysis level and the expression levels of key enzymes in the glycolysis pathway in cisplatin-resistant A2780/DDP cells were significantly higher than those in parental A2780 cells (p < 0.05), but there was no significant difference in oxidative phosphorylation level and oxidative phosphorylation regulatory enzymes between the two cells (p > 0.05). In addition, the sensitivity of A2780 cells and A2780/DDP cells to cisplatin and GFC was determined. Both cisplatin and GFC inhibited the growth of A2780 and A2780/DDP cells in a concentration-dependent manner. The IC₅₀ of cisplatin for A2780 cells and A2780/DDP cells was 22.60 ± 0.14µM and 39.48 ± 0.36µM, respectively (Fig. 3-G and I). The IC₅₀ of GFC on A2780 cells and A2780/DDP cells was 0.60 ± 0.14mg/mL and 1.04 ± 0.31mg/mL (Fig. 2-H and J). Cisplatin combined with GFC at IC₅ concentrations had no effect on A2780/DDP cell proliferation (Fig. 2-K).

GFC combined with cisplatin regulated glycolysis and oxidative phosphorylation levels in A2780/DDP cells

The changes of glycolysis and oxidative phosphorylation in A2780/DDP cells treated with GFC alone, cisplatin alone, or GFC combined with cisplatin were measured. As shown in Fig. 3-A to F, the combined treatment of the two drugs continuously inhibited glucose consumption, pyruvate production, lactate accumulation, and the activities of their related regulatory enzymes (p < 0.05). As shown in Fig. 3-G, the protein expression levels of HK2, PKM2 and LDHA in A2780/DDP cells were significantly decreased after 24 hours of drug treatment (p < 0.05). However, as shown in Fig. 3-H and I, the ATP content and mitochondrial transhydrogenase 2 (TH2) in A2780/DDP cells increased at first and then decreased after drug combined intervention. The expressions of ATP1 and ATP5 protein in A2780 / DDP cells were significantly inhibited by GFC alone or combined with drug intervention (p < 0.05), and the inhibitory effect of combined drug intervention was better than that of GFC alone.

GFC combined with cisplatin activates ROS to induce mitophagy in A2780/DDP cells

Firstly, we found that GFC combined with cisplatin could promote a significant increase in ROS level in A2780/DDP cells (p < 0.01) (Fig. 4-A). In addition, we found that the combined treatment of GFC and cisplatin could activate the NF-kB/TGF-β1/PINK1/Parkin signaling pathway in A2780/DDP cells (Fig. 4-B). The NF-kB/TGF-β1/PINK1/Parkin signaling pathway is closely related to mitochondrial damage. In A2780/DDP cells, the presence of mitochondrial vesicles, autophagosomes and autolysosomes was detected by fluorescence and transmission electron microscopy (Fig. 4-C and D).

GFC combined with cisplatin reversed cisplatin resistance by inducing apoptosis in A2780/DDP cells

Tunel fluorescence staining showed that GFC combined with cisplatin could significantly promote the apoptosis of A2780/DDP cells (p < 0.01) (Fig. 5-A). In addition, flow cytometry showed that the combined treatment of GFC and cisplatin significantly promoted the sum of early and late apoptosis of A2780/DDP cells (p < 0.01) (Fig. 5-B). We tested the apoptosis regulation related protein expression level, the results showed that combination of drugs intervention can promote A2780 / DDP cells Casepase 3/p19/p17, Casepase 9 /p37/p35, and the expression of BAX protein, inhibition of Bcl2 protein expression (Fig. 5-C). Compared with cisplatin alone, GFC combined with cisplatin significantly increased the content of cisplatin in A2780/DDP cells (p < 0.01) (Fig. 5-D). Finally, the expression levels of drug resistance regulatory proteins in A2780/DDP cells were detected, and the results showed that the combined treatment of GFC and cisplatin significantly inhibited the expression of P-gp, MRP and BCRP proteins (p < 0.05) (Fig. 5-E).

GFC combined with cisplatin significantly inhibited tumor energy metabolism and growth in vivo

A2780/DDP xenograft mouse model was established. As shown in Fig. 6-A, when the mice with transplanted tumors were treated with GFC and cisplatin for 14 days, the tumor size in the armpit of the mice was significantly smaller than that of the drug treatment group alone. The body weight of the mice with transplanted tumors in the combined drug treatment group was also significantly higher than that in the drug treatment group alone (p < 0.01). As shown in Fig. 6-B, the tumor volume of the combined drug treatment group was significantly reduced from day 7 (p < 0.01). The results of HE staining showed that the density of tumor tissue, the degree of cell arrangement disorder, the number of cells and nuclear atypia were decreased after combined treatment (Fig. 6-C). The effect of GFC combined with cisplatin on glycolysis and oxidative phosphorylation levels in A2780/DDP cells showed that the combined drug treatment group could inhibit the expression of HIF-1α, HK2, PKM2, ATP1 and ATP5 proteins (Fig. 6-D and E). The contents of ATP and lactate in tumor tissues were inhibited in the combined treatment group (p < 0.05) (Fig. 6-F and G).

Chemical composition identification of GFC

TIC scans in the positive and negative ion modes are shown in Figs. 7-A and B, respectively. With mzcloud and mzVault TCM characteristic peak identification of natural products database. As shown in Table 1, according to the relative content of chemical composition, the top ten chemical constituents were Paeoniflorin (18.769%), Amygdalin (17.971%) and Gallic acid (9.197%), Albiflorin (8.195%), Sucrose (5.809%), Pyrogallol (5.535%), Citric Acid (4.841%) and Benzoylpaeoniflorin (3.167%), Cinnamaldehyde (3.122%) and Coumarin (2.790%).

Table 1

Chemical composition information of GFC
Name	Molecular Formula	Mass Deviation (ppm)	Molecular weight	Retention time (min)	Matching score	Peak area	Relative content (%)
Paeoniflorin	C23 H28 O11	-1.24	480.16257	20.453	95	16964529909	18.769
Amygdalin	C20 H27 N O11	-1.04	457.15793	18.98	92	16243272815	17.971
Gallic acid	C7 H6 O5	-1.32	170.0213	6.436	91.9	8313014507	9.197
Albiflorin	C23 H28 O11	-0.98	480.16269	20.073	90.6	7406859492	8.195
Sucrose	C12 H22 O11	-1.06	342.11585	1.538	93	5250208001	5.809
Pyrogallol	C6 H6 O3	-1.91	126.03145	7.387	78.2	5002810521	5.535
Citric acid	C6 H8 O7	-0.62	192.02688	1.637	88.8	4375690189	4.841
Benzoylpaeoniflorin	C30 H32 O12	-0.95	584.18882	26.315	94.8	2862092830	3.167
Cinnamaldehyde	C9 H8 O	-0.37	132.05747	27.149	90.4	2821466370	3.122
Coumarin	C9 H6 O2	-0.34	146.03673	24.04	85.5	2522063014	2.790
Pachymic acid	C33 H52 O5	-0.96	528.38097	46.207	88.8	2296343941	2.541
Oxypaeoniflorin	C23 H28 O12	-1.2	496.15748	18.679	91	2201121535	2.435
Xanthoxyline	C10 H12 O4	-0.65	196.07343	22.879	78.6	1796256850	1.987
1,2,3,4,6-Pentagalloylglucose	C41 H32 O26	-1.33	940.11693	24.066	92.2	1391808836	1.540
2-Pyrrolidinecarboxylic acid	C5 H9 N O2	-0.02	115.06333	1.529	82.8	1280302707	1.417
5-Hydroxymethylfurfural	C6 H6 O3	-0.14	126.03168	1.47	81.5	1174344010	1.299
Adenosine	C10 H13 N5 O4	-0.78	267.09654	9.49	90.1	1002608539	1.109
L-Valine	C5 H11 N O2	-0.37	117.07894	1.532	74.2	853038631.1	0.944
Poricoic acid A	C31 H46 O5	-0.86	498.33409	40.846	90.4	602135988.1	0.666
3-O-Acetyl-1α-hydroxytrametenolic acid	C32 H50 O5	-1.12	514.36525	45.424	79.4	598630694.7	0.662

There is a close relationship between tumors and oxidative phosphorylation and glycolysis. Oxidative phosphorylation is the main way in which cells produce ATP through oxidative reactions, which is an efficient way to generate energy. However, in tumor cells, the mode of glucose metabolism is changed from oxidative phosphorylation to aerobic glycolysis^{[10, 11]}. Aerobic glycolysis, a way in which cells produce energy by breaking down glucose under hypoxic conditions, produces a smaller amount of ATP but at a faster rate. Energy production and growth of tumor cells can be inhibited by inhibiting key enzymes of glycolysis, such as hexokinase 2 (HK2) or lactate dehydrogenase A (LDHA)^{[12, 13]}. In addition, a deeper understanding of tumor metabolism, by activating oxidative phosphorylation or restoring mitochondrial function, offers the possibility to develop new cancer therapies.

The molecular mechanism of drug resistance in ovarian cancer is a complex process involving changes in multiple aspects. For example, the abnormalities of signaling pathways and metabolic enzymes, epithelial-mesenchymal transition (EMT), abnormal expression of miRNA, the role of cancer stem cells (CSC), and changes in the immune microenvironment can cause drug resistance of ovarian cancer^[14–16]. Recent studies have found that the balance between oxidative phosphorylation and glycolysis plays a crucial role in the development and drug resistance of ovarian cancer. Tumor necrosis factor receptor-associated protein 1 (TRAP1) is a mitochondrial chaperone protein from Hsp90 family^[17]. Up-regulation of TRAP1 expression increased aerobic glycolysis in ovarian cancer cells^[18]. When TRAP1 expression is low or silenced, the oxygen consumption of ovarian cancer cells increases, leading to a high dependence on oxidative phosphorylation and is associated with platinum resistance^[19]. Abnormal activation of PI3K/AKT/mTOR pathway plays a key role in the formation of drug resistance in ovarian cancer^[20]. The key enzymes in glucose metabolism of ovarian cancer cells, such as hexokinase, 6-phosphofructokinase and lactate dehydrogenase, can promote the growth of ovarian cancer cells and reduce the sensitivity to chemotherapy drugs^[21–23]. When ovarian cancer cells are attacked by chemotherapeutic drugs, they will increase glycolysis and glutamine metabolism to produce more energy and reducing power, thus resisting the pressure of drugs. In this study, the cisplatin sensitive ovarian cancer cell line A2780 and cisplatin resistance cell line A2780/DDP were used as research objects. The results showed that compared with A2780 cells, the activities of glucose metabolism regulatory enzymes such as hexokinase (HK), pyruvate kinase (PK) and lactate dehydrogenase (LDH) were increased in A2780/DDP cells, leading to increased glycolysis level. However, there was no significant difference in oxidative phosphorylation between A2780 and DDP cells. The increased glycolysis level in A2780/DDP cells is closely related to the cisplatin resistance phenotype.

GFC is a traditional Chinese medicine widely used in clinical practice to treat gynecological inflammatory diseases. Recent studies have shown that GFC can inhibit the growth of tumors and improve the sensitivity of chemotherapy drugs.In this study, we identified GFC through database searches and identified 265 common targets for ovarian cancer. Subsequently, we constructed a network pharmacology model. Functional enrichment analysis of these common targets revealed that the dysregulation of glycolysis is a key factor in GFC treatment for ovarian cancer. We predicted that glycolysis would be the key point in the treatment of ovarian cancer by GFC, and found that the abnormality of glycolysis was a significant feature of cisplatin resistance in ovarian cancer. Therefore, we tested whether GFC could also exert inhibitory effect on cisplatin-resistant ovarian cancer. ELISA and Western blot were used to confirm that GFC combined with cisplatin could reduce the level of glycolysis by inhibiting the enzyme activity and protein expression of glucose metabolism regulatory enzymes in A2780/DDP cells. In addition, the ATP content of A2780/DDP cells increased at first and then decreased during the treatment of GFC combined with cisplatin. Further studies showed that elevated ATP could induce ROS, activate the NF-κB/TGF-β1/PINK1/Parkin signaling pathway, and promote mitophagy in A2780/DDP cells. We found that GFC combined with cisplatin could promote the apoptosis of A2780/DDP cells as detected by TUNEL and flow cytometry. Finally, we carried out in vivo experiments to verify that GFC combined with cisplatin could inhibit the tumor growth, glucose metabolism and oxidative phosphorylase expression in tumor-bearing mice.

In summary, the relationship between tumors and oxidative phosphorylation and glycolysis is a complex and worthy area of further study. By regulating the expression of related genes or key enzymes in energy metabolism pathways, the balance of oxidative phosphorylation and glycolysis of tumor cells can be reshuffled, thereby enhancing their sensitivity to chemotherapy drugs. By in-depth study of the mechanisms and effects of this metabolic shift, we can better understand tumor growth and development and develop more effective treatments.

This study showed that imbalance of glycolysis and oxidative phosphorylation is a prominent feature of cisplatin resistant ovarian cancer cell line A2780/DDP. GFC combined with cisplatin can reshape the balance of glycolysis and oxidative phosphorylation, induce ROS production, and promote mitophagy and apoptosis in A2780/DDP cells. GFC improve the curative effect of cisplatin for the treatment provides a theoretical basis for cisplatin resistance ovarian cancer.

Authors Declarations

Ethical Approval and Consent to participate

This study was conducted at the School of Life Sciences and Health at Northeastern University in accordance with the Declaration of Helsinki and approved by the local Council of Governance (no. NEU-EC-2023A079S). Their care during research on animals follows ARRIVE guidelines and is conducted in accordance with the National Institutes of Health Guidelines for the Care and Use of Laboratory Animals (NIH Publication No. 8023, as revised in 1978).

Patient consent for publication

Not applicable.

Availability of data and materials

The datasets during and/or analysed during the current study available from the corresponding author on reasonable request.

Competing interests

The authors declare that they have no competing interests.

Funding

This work was financially supported by the Liaoning Province Applied Basic Research Program (Grant 2022JH2/101300039), Shenyang Science and Technology Plan (22-321-33-08), and Shenyang Bureau of Science and Technology (2023 Special Project to Assist China Medical University in high-quality development 23-506-3-01-10). The above funds were used to carry out the research related consumables and reagents, detection, carrier synthesis and publication costs.

Authors' contributions

DL and DL data analysis, manuscript writing. MF, LY and CX cell experimental manipulation and data collation. LF, ZJ and LE data collection and statistical analysis. ZY project resource provision and management. SY experimental design, manuscript writing, experimental operation and project management.All authors read and approved the final manuscript.

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Network pharmacology and experimental methods to explore the mechanism of Guizhi Fuling capsule in overcoming cisplatin-resistant ovarian cancer

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Abstract

Figures

Introduction

Materials and methods

Cell lines and cell culture

Reagents and antibodies

Screening of ovarian cancer and GFC targets

Target gene enrichment and network pharmacological analysis

Cell viability assay

Glucose, pyruvate and lactic acid assay

Mitochondrial transhydrogenase 2 (TH2) enzyme activity assay

Hexokinase, pyruvate kinase and lactate dehydrogenase enzyme activities assay

Rreactive oxygen species assay

Statistical Analysis

Results

GFC treatment of ovarian cancer target prediction and build network pharmacology

Cisplatin resistance in ovarian cancer caused by glycolysis

GFC combined with cisplatin regulated glycolysis and oxidative phosphorylation levels in A2780/DDP cells

GFC combined with cisplatin activates ROS to induce mitophagy in A2780/DDP cells

GFC combined with cisplatin reversed cisplatin resistance by inducing apoptosis in A2780/DDP cells

GFC combined with cisplatin significantly inhibited tumor energy metabolism and growth in vivo

Chemical composition identification of GFC

Discussion

Conclusion

Declarations

References

Additional Declarations

Supplementary Files

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