Qing-Re-Yi-Liu decoction suppresses the malignant behaviors of breast cancer cells by attenuating the MnSOD/CaMKII/AMPK signaling and Warburg effect

Qing-Re-Yi-Liu decoction suppresses the malignant behaviors of breast cancer cells by attenuating the MnSOD/CaMKII/AMPK signaling and Warburg effect | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Qing-Re-Yi-Liu decoction suppresses the malignant behaviors of breast cancer cells by attenuating the MnSOD/CaMKII/AMPK signaling and Warburg effect 哲 张, xin zhao, lian sun, jun wang, jing du, ying zhang, ying Yuan This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-3366517/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 5 You are reading this latest preprint version Abstract Background The Qing-Re-Yi-Liu decoction (QRYLD) is a clinical effective prescription for the treatment of breast cancer due to its activity of heat clearing and detoxification. Our preliminary studies have found that QRYLD can interfere with the Warburg effect of breast cancer cells, inhibiting the proliferation of breast cancer MCF-7 cells.The chemical components and molecular mechanisms underlying the actions of QRYLD in regulating the Warburg effect in breast cancer cells are still unclear. Methods The bioactive components of QRYLD aqueous extracts were detected by HPLC. The biological processes and signaling pathways in MCF-7 cells of QRYLD targets were measured with transcriptome analysis. The effect of QRYLD on the malignant behaviors of MCF-7 cells were analyzed by CCK-8 assay,transwell invasion assay, wound healing assay, apoptosis detection. The effect of QRYLD on glucose uptake, lactic acid production and Warburg effect in MCF-7 cells assessed by colorimetry and western blotting. The volumes of xenograft breast tumors and body weights of mice were measured, and the effect of QRYLD on the tumor tissues was assessed with immunohistochemistry. Results Here, we show that the QRYLD aqueous extracts contain several bioactive components. Analysis of transcriptomes indicated that QRYLD treatment altered the expression of many genes, such as manganese superoxide dismutase (MnSOD), that were involved in biological processes and signaling pathways, particularly for glucose metabolism in MCF-7 cells. Functionally, QRYLD treatment, like MnSOD silencing, inhibited the malignant behaviors of MCF-7 and enhanced their apoptosis while MnSOD over-expression had opposite effects. Furthermore, QRYLD treatment, like MnSOD silencing, limited glucose uptake and lactic acid production in MCF-7 cells, which were associated with a decrease in the relative levels of Glut-1, HIF-1α, c-Myc, HK-2, PFK-1, LDH-A, PKM-2, MnSOD, calmodulin dependent kinase II (CaMKII) and AMPK expression. Finally, treatment with QRYLD, like MnSOD silencing, significantly mitigated the growth of xenograft MCF-7 tumors in mice and reduced the expression of MnSOD, CaMkII and AMPK expression in the tumors. Conclusion These data suggest that QRYLD may target MnSOD to attenuate the MnSOD/CaMKII/AMPK signaling, leading to inhibition of the Warburg effect and malignant behaviors in MCF-7 cells. These findings may provide new insights into the pharmacological mechanisms underlying the actions of QRYLD in inhibiting the Warburg effect and malignant behaviors of breast cancer cells. QRYLD MnSOD malignant behaviors Warburg effect CaMKII/AMPK signaling Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Introduction Breast cancer is the most common malignant tumor in women worldwide. Currently, therapeutic strategies for the intervention of breast cancer include surgical resection of tumors, neoadjuvant and post-operative therapies, such as chemotherapy, target therapy, radiotherapy, immunotherapy and others. Traditional Chinese medicine (TCM) has been used for the treatment of breast cancer. There are many studies on the prevention and intervention of TCM for breast cancer. In the theory of TCM, the tumor development and progression are attributed to the accumulation of heat and toxin, and therapeutic strategies to clear heat and detoxify are important for the control of tumor growth. However, the pharmacological mechanisms underlying the actions of these TCMs in antitumor activity have not been clarified. Previous studies have shown that Scutellaria baicalensis and its bioactive ingredients have potent activities against the malignancy of breast cancer cells[ 1 ]and treatment with ethanol extracts of Andrographis paniculata inhibits the proliferation of breast cancer MCF-7 cells in a dose-dependent manner[ 2 ].Similarly, treatment with Coptidis rhizoma limits the growth of 4T1 breast cancer in mice by inducing their cell cycle arrest in G1 phase and suppressing DNA synthesis[ 3 ]while treatment with Radix isatidis attenuates the malignant behaviors of SK-BR-1 and MDA-MB-231 cells in a time-dependent manner by inducing cell cycle arrest in G2/M phases and apoptosis[ 4 ]. Furthermore, treatment with different doses of Pulsatilla saponin can inhibit the proliferation of MCF-7 cells in a dose-dependent manner[ 5 ]. Although many TCMs have been used in clinical practices for the intervention of breast cancer, the therapeutic efficacy of these TCMs is limited. Hence, discovery of new and safe TCMs, particularly for those with potent activities of heat clearing and detoxification is urgently needed. The Qing-Re-Yi-Liu decoction (QRYLD) is a clinical effective prescription for the treatment of breast cancer given its heat clearing and detoxification activities. Our preliminary studies have found that treatment with QRYLD inhibits the proliferation of breast cancer MCF-7 cells, which is associated with inhibition of the Warburg effect (aerobic glycolysis). The current studies aimed at investigating the pharmacological effect of QRYLD on the malignancy of MCF-7 breast tumors and their Warburg effect as well as the molecular mechanisms underlying the pharmacological action of QRYLD in breast cancer. Materials and Methods 1. QRYLD QRYLD is composed of Taraxacum officinala, Iphigenia indica, Forsythia suspensa, Cortex moutan, Hedyotis diffusa, Lonicera japonica, Prunella vulgaris and Lycii cortex root of T araxacum officinala L., Iphigenia indica L, Forsythia suspense L, Cortex moutan L, Hedyotis diffusa L, Lonicera japonica L, Prunella vulgaris L. and Lycii cortex L. , and the detailed components of ORYLD are shown in Table 1 (The plant name has been checked with www.worldfloraonline.org ). These medicinal herbs were obtained from the TCM Pharmacy of the First Affiliated Hospital of Xi'an Jiaotong University (Xi'an, Shaanxi, China) and were identified by experts in the Pharmacy Department. The QRYLD was soaked in 10 volumes of water and cooked at 100 ° C for 1 hour. The decoction was collected and the herbs were cooked with another 10 volumes of water for 40 minutes for collection of the decoction. The collected two decoctions were mixed, filtered through a filter, concentrated and dried using a spray dryer (Lemal, Changzhou, China). The dried ORYLD powder was dissolved in water and the chemical components in the aqueous ORYLD extracts were identified by HPLC using Agilent 1260 InfinityII liquid chromatography (Agilent Technologies, California, USA). Table 1 The composition of QRYLD and List of selected plant material traditionally use in diseases Family Species Chinese name Dosage(g) Part Lot No. Traditional use References Asteraceae Taraxacum L. Pugongying 30 herba 210601 anti-bacterial, anti-oxidant, anti-cancer, and anti-rheumatic activities [ 31 – 34 ] Colchicaceae Iphigenia indica L. Shancigu 30 caulis 210501 Anti-cancer, anti-inflammatory, pain relieving, blood pressure lowering, cough relieving, asthma relieving [ 35 – 37 ] Oleaceae Forsythia suspensa L. Lianqiao 15 fructus 210801 Anti-bacterial, anti-pyretic, anti-emetic, anti-inflammatory, diuretic, anti-liver injury, cardiotonic, diuretic, anti-hypertensive [ 38 – 41 ] Paeoniaceae Cortex Moutan L. Mudanpi 15 Radix bark 210701 Anti-bacterial, anti-inflammatory, anti- allergic, liver protective, hypoglycemic, anti-cancer [ 42 – 45 ] Rubiaceae Hedyotis diffusa L. Baihuasheshecao 30 herba 210601 Anti- inflammatory, anti-cancer, analgesic, liver and gallbladder protection [ 46 – 49 ] Caprifoliaceae Lonicera japonica L. Jinyinhua 15 Flos 210601 Anti-inflammatory, anti-pyretic, anti- endotoxin, hypolipidemic [ 50 – 53 ] Lamiaceae Prunella vulgaris L. Xiakucao 15 fructus 210801 Hypotension, hypoglycemic, anti- bacterial, anti-inflammatory, anti- allergic, and anti-viral [ 54 – 57 ] Canellaceae Lycii Cortex L. Digupi 15 Radix bark 210501 Anti-pyretic, anti-hypertensive, hypoglycemic, anti-cancer, and lipid-lowering [ 58 – 60 ] The plant name has been checked with http://www.theplantlist.org and www.worldfloraonline.org 2. Preparation of QRYLD-medicated serum Balb/c mice at 3–5 weeks of age were from the Laboratory Animal Center, Xi`an Jiaotong University Health Science Center and treated with 0.3 ml of QRYLD in saline at low-dose (LD, 5.6875g·kg-1·d-1), medium-dose (MD, 11.375g·kg-1·d-1) or high-dose (HD, 22.75g·kg-1·d-1), based on the ratio of body surface area of humans to mice by gavage daily for consecutive 7 days. Two hours after the last gavage, their blood samples were collected for preparation of serum samples. After being heated at 56°C for 30 minutes, the serum samples were filtered through a 0.45 µM filter and used as the ORYLD-medicated serum for cell culture. A 10% of medicated serum in RPMI-1640 medium was prepared (Solarbio, Beijing, China). 3. Cell culture Human breast cancer MCF-7 cells were obtained from Shanghai Institute of Cell Biology, China and cultured in RPMI-1640 medium (Solarbio) containing 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin at 37℃ in a 5% CO 2 incubator. 4. Transfection and transduction 293T cells were transfected with plasmids for lentivirus packaging and MnSOD expression or control GFP expression using lipofectamine 3000 to produce lent-MnSOD or control GFP virions by Jinsirui Biotechnology, Nanjing, China. Similarly, HEK293 cells were transfected with the adenovirus packaging plasmids and the plasmid for the expression of MnSOD-specific shRNA or control shRNA to generate Ad-sh-MnSOD or control Ad-sh virions by Jinsirui Biotechnology. MCF-7 cells were transduced with lentivirus for stable MnSOD over-expression at a MOI of 10 to generate Lent-MnSOD cells. The transduced cells were treated with 4 µg/ml of puromycin to induce stable expression. Furthermore, MCF-7 cells were infected with the Ad-sh-MnSOD or control Ad-sh at a MOI of 10 and treated with 2 µg/ml of puromycin to generate stable MnSOD silencing MCF-7 cells. The efficiency of MnSOD over-expression or silencing was examined by Western blot. 5. CCK-8 assay Wild-type, Ad-sh MnSOD, lent MnSOD MCF-7 cells (1×10 4 cells/well) were cultured in 96-well plates and treated with vehicle PBS, while the wild-type MCF-7 cells were treated with 10% of LD, MD, or HD of QRYLD-medicated serum, respectively. The proliferation of each group of cells was tested in triplicate for 24, 48 and 72 hours, respectively using CCK-8 assays. During the last 4-hour culture, individual wells were added with 10 µL of CCK-8 solution (Beyotime, Shanghai, China) and the viability of each group of cells was measured for the absorbance of each well at 450 nm using a microplate reader. 6. Transcriptome analysis MCF-7 cells were treated with vehicle as the control or MD of QRYLD-medicated serum for 48 hours. Their total RNA was extracted using Trizol (Life Technologies, California, USA) and the contained mRNAs were sorted using Oligotex mRNA sorting kit (Qiagen, Germany), per the manufacturer’s instructions. After qualification and quantification of mRNA in Illumina HiSeq TM 2000, mRNA samples from each group were reverse-transcribed into cDNA to generate the cDNA libraries using SMART-Seq V4 kit (Takara Bio, USA). The cDNA libraries were sequenced in NovaSeq 6000 machine (Illumina, USA). The differentially expressed genes (DEGs) were defined when a p-value of 2.0 or < 0.5. The biological nature of DEGs was analyzed using DAVID Bioinformatics Resources 6.7 and their biological processes or signaling pathways were analyzed by gene ontology (GO) and KEGG. In addition, the potential connections among these biological processes and signaling pathways of the DEGs were analyzed by the protein-protein interaction (PPI) using Cytoscape software. 7. Transwell invasion assay MCF-7 (4×10 4 cells/well) were treated with vehicle or MD of QRYLD-medicated serum for 48 hours. The cells, together with the same number of Ad-sh-MnSOD MCF-7, lent-MnSOD MCF-7 cells, were cultured into the upper chamber that had been coated with Matrigel (Thermo Fisher Scientific, Massachusetts, USA) and the bottom chamber was filled with 10% FBS medium. After being cultured for 24 hours, the cells on the upper chamber surface membrane were removed and the invaded cells on the upper chamber bottom surface were fixed in 4.0% paraformaldehyde and stained with 0.1% crystal violet solution for 10 minutes, followed by photoimaging under a Leica DMil inverted microscope (Leica, Wetzlar, Germany). The number of invaded cells was counted in a blinded manner. 8. Wound healing assay MCF-7 were treated with vehicle or MD of QRYLD-medicated serum for 48 hours. The cells, together with the same number of Ad-sh-MnSOD MCF-7, lent-MnSOD MCF-7 cells (1×10 6 cells/well) were cultured in 6-well plates. When the cells reached 95% confluency, the monolayer cells were wounded using a 10-µL sterile pipette tip. The wounded areas were photoimaged immediately after scratch and 48-hour culture. The wound healing ability of each group of cells was analyzed using ImageJ software. 9. Apoptosis Detection Ad-sh-MnSOD MCF-7, lent-MnSOD MCF-7, and MCF-7 cells that had been treated with vehicle saline or MD of QRYLD-medicated serum for 48 hours were harvested and stained with Annexin V-isothiocyanofluorescein (FITC) (Solarbio) and propidium iodide (PI) (Solarbio) for 10 minutes in the dark. The percentages of apoptotic cells were analyzed by flow cytometry and the data were analyzed by FlowJo software. 10. Western blotting Ad-sh-MnSOD MCF-7, lent-MnSOD MCF-7, and MCF-7 cells that had been treated with vehicle saline or MD of QRYLD-medicated serum for 48 hours were harvested and lyzed in lysis buffer, followed by centrifugation. After measurement of protein concentrations using a BCA kit, the cell lysates (30 µg/lane) were resolved in sodium dodecyl-sulfate polyacrylamide gel electrophoresis (SDS-PAGE) on 10% gels and transferred onto polyvinylidene difluoride (PVDF) membranes. After being blocked with 5% skimmed dry milk powder in TBST for 2 hours, the membranes were probed with primary antibodies against MnSOD, CaMKII, AMPK, HIF-1α, Glut-1, c-myc, HK-2, PFK-1, LDH-A, PKM-2 and β-actin (1:1000, Abiowell Biotechnology, Changsha, China) at 4℃ overnight, and reacted with horseradish peroxidase (HRP)-conjugated secondary antibodies (Abiowell Biotechnology), followed by developing with the enhanced chemiluminescent reagents. The data were analyzed by densitometric scanning using ImageJ software. 11. Glucose uptake and lactate levels MCF-7 cells were treated with vehicle saline or MD of QRYLD-medicated serum for 48 hours. The impact of QRYLD treatment on glucose uptake in those cells, together with the same number of Ad-sh-MnSOD MCF-7, lent-MnSOD MCF-7 was tested using the 2-deoxyglucose (2-DG)-based colorimetric glucose uptake assay kit (abcam), per the manufacturers’ protocol. Briefly, the cells were glucose-starved for 40 minutes, stimulated with insulin for 20 minutes and exposed to 1 mM 2-DG for 20 minutes, followed by washing. The cells were lyzed, frozen/thawed and heated at 80°C for 40 minutes. After neutralization, the samples were reacted with the reaction mixture, neutralized and reacted with the reaction B. The levels of 2-DG6P-related NADPH in each group of samples were measured for the absorbance at 412 nm in a microplate reader. The concentrations of glucose uptake were calculated, based on the standard curve established using the standard 2-DG6P provided by the manufacturer. In addition, the levels of lactic acid in the supernatants of cultured cells were measured by a fully automated biochemical analyzer (Solarbio). 12. Animal studies Female Balb/c nude mice were obtained from the Laboratory Animal Center, Xi`an Jiaotong University Health Science Center and housed in a specific pathogen-free (SPF) facility in our university. The mice were randomized and implanted with 3x10 6 MCF-7, Ad-sh-MnSOD MCF-7 or lent MnSOD MCF-7 cells in 100 µl of saline into their breast fat pads. Some mice with MCF-7 cells were treated intravenously with 11.375g kg − 1 d − 1 QRYLD in 0.3 ml of saline daily beginning on day 5 post inoculation for 10 consecutive days. Other groups of mice received the same volume of saline injection (n = 6 per group). The volumes of implanted tumors and body weights of mice were measured every other day for 4 weeks. The mice were euthanized and their tumor tissues were dissected, photoimaged and weighed. The tumor tissues were fixed in 10% formalin and paraffin-embedded. The tumor tissue sections (4 µm) were subjected to immunohistochemistry using primary antibodies against MnSOD (1:100), CaMKII (1:200), and AMPK (1:2000) (Abiowell Biotechnology), counterstained with hematoxylin, and analyzed using ImageJ software. All animal experiments were performed strictly following applicable national and institutional guidelines and were approved by the Ethics Committee of the First Affiliated Hospital of Xi'an Jiaotong University (No. 2017 Lunshenkezi No. 44, dated March 2, 2017). 13. Statistical analysis Data are shown as mean ± SEM and the difference among groups was analyzed by one-way ANOVA and post hoc Tukey’s test. The comparison between groups was performed using Student t -test. All statistical analyses were conducted using GraphPad Prism 8 software (GraphPad Software Company, California, USA). The difference was thought statistically significant when a P -value of < 0.05. Results 1. QRYLD aqueous extracts contain many bioactive components To understand the biological functions of QRYLD aqueous extracts, we prepared aqueous extracts of 3300 g QRYLD and achieved 410 g of QRYLD aqueous extracts in powder, leading to a powder recovery rate of approximately 55%. HPLC analyses revealed that there were six main chemical compounds at high contents (Fig. 1 ) and they were chlorogenic acid (133.21 ± 19.05 mg/g), caffeic acid (91.22 ± 11.38 mg/g), quercetin (39.88 ± 2.96mg/g), rutin (125.11 ± 16.29 mg/g), ferulic acid (189.53 ± 23.58 mg/g), and luteolin (62.39 ± 8.23 mg/g). Hence, the QRYLD aqueous extracts contained several chemical compounds with high bioactivity and their interaction may contribute to the functions of QRYLD. 2. QRYLD targets several biological processes and signaling pathways in breast cancer cells. Next, we explored whether QRYLD treatment could modulate the transcriptome profiles in MCF-7 cells. Following treatment with, or without, QRYLD-medicated serum for 48 hours, the transcriptomes in the untreated and QRYLD-treated MCF-7 cells were analyzed by RNA sequencing. The results indicated that there were 2918 DEGs, of them, 1693 genes were up-regulated while 1225 DEGs were down-regulated in the QRYLD-treated cells. It was notable that there were 293 DEGs belonging to oncogenes and tumor suppressor genes, and among them, 133 DEGs were down-regulated and 160 up-regulated in the QRYLD-treated cells (Fig. 2 A). Bioinformatic analyses predicted that these 293 DEGs were mainly involved in the metabolism-related signaling pathways or biological processes. The GO and KEGG analyses unveiled that the top 50 DEGs were directly related to the Warburg effect (Fig. 2 B). The connections among these targets and signal pathways predicted that these 293 DEGs were mainly involved in response to oxidative stress, cancer-related signaling pathways, glycogen synthesis and metabolism, pentose phosphate pathway (PPP), TCA cycle, glucose metabolism regulation, gluconeogenesis, glycolysis, and the MnSOD MAPK signaling (Fig. 2 C). Apparently, MnSOD may be a target of QRYLD in breast cancer. 3. QRYLD treatment limits the malignant behaviors of MCF-7 cells To understand the importance of MnSOD in the malignant behaviors of breast cancer cells, we generated stable MnSOD-silencing Ad-sh-MnSOD and MnSOD over-expressing Lent-MnSOD MCF-7 cells. Subsequently, wild-type MCF-7 cells were treated with vehicle or the LD, MD, or HD of QRYLD-medicated serum and their proliferation was tested by CCK-8 assays. The data indicated that compared with the control MCF-7 group, the proliferation activity of Ad-sh MnSOD MCF-7 cells was significantly reduced ( P < 0.01), while the proliferation activity of lent MnSOD MCF-7 cells was significantly enhanced ( P < 0.01). The proliferation activity of the QRYLD-treated MCF-7 cells (LD, MD, or HD of QRYLD) was significantly reduced ( P < 0.05, Fig. 3 A). Notably, treatment with MD of QRYLD-mediated serum achieved 50% inhibition of cell proliferation at 48 hours post treatment in MCF-7 cells. Accordingly, we used the MD of QRYLD-medicated serum for the subsequent experiments in vitro. Transwell invasion assays exhibited that the number of invaded cells in the QRYLD group was significantly reduced, relative to those in the control and MnSOD over-expression groups, but still slightly greater than those in the MnSOD silencing Ad-sh-MnSOD MCF-7 cells (Fig. 3 B). A similar pattern of wound healing ability was observed among the different groups of cells (Fig. 3 C). Thus, treatment with QRYLD, like MnSOD silencing, limited the malignant behaviors of MCF-7 cells in vitro. Flow cytometric analyses revealed that the percentages of apoptotic MCF-7 cells in the QRYLD group were significantly higher than those in the control and the MnSOD over-expressing lent-MnSOD MCF-7 cells, but lower than that in the MnSOD-silencing Ad-sh-MnSOD MCF-7 cells ( P < 0.05, P < 0.01, Fig. 3 D). These indicated that QRYLD treatment, like MnSOD silencing, promoted MCF-7 cell apoptosis, which might also contribute to its ability to limit the malignant behaviors of MCF-7 cells. 4. QRYLD treatment inhibits glucose uptake, lactic acid production and reduces the Warburg effect in MCF-7 cells by attenuating the MnSOD/CaMKII/AMPK signaling To further understand the pharmacological action of QRYLD, we tested the impact of QRYLD treatment on glucose uptake and lactic acid production in MCF-7 cells. Following treatment with MD of QRYLD-medicated serum for 48 hours, the levels of glucose uptake in the different groups of MCF-7 cells were measured by 2-DG based colorimetric assays. The results exhibited that the levels of glucose uptake in the QRYLD-treated MCF-7 cells were significantly less than that in the control and MnSOD over-expressing MCF-7 cells, but higher than that in the MnSOD silencing MCF-7 cells (Fig. 4 A) and a similar pattern of lactic acid levels in the supernatants of cultured cells was detected in the different groups of cells (Fig. 4 B). The decreased glucose uptake and lactic acid production evidenced that QRYLD treatment reduced the Warburg effect in MCF-7 cells. It is well known that several regulators are crucial for the Warburg effect in cancers. To explore how QRYLD treatment reduced the Warburg effect in MCF-7 cells, we tested the relative levels of HIF-1α, Glut-1, and C-Myc expression in the different groups of cells by Western blot. Similarly, the relative levels of HIF-1α, Glut-1, and C-Myc expression in the QRYLD-treated MCF-7 cells were significantly lower than that in the control and MnSOD over-expressing MCF-7 cells, but higher than that in the MnSOD-silencing MCF-7 cells (Fig. 4 C). A similar pattern of HK-2, PFK-1, LDH-A and PKM-2 expression was observed in the different groups of cells (Fig. 4 D). These data indicated that QRYLD treatment, like MnSOD silencing, inhibited the expression of these regulators and metabolic enzymes, contributing to its inhibition on the Warburg effect in MCF-7 cells. Given the importance of MnSOD in the pharmacological functions of QRYLD, we tested whether QRYLD treatment could modulate the MnSOD/CaMKII/AMPK signaling in MCF-7 cells by Western blot. As expected, the relative levels of MnSOD expression in the QRYLD-treated MCF-7 cells were significantly lower than that in the control and MnSOD over-expressing MCF-7 cells, but higher than that in the MnSOD silencing MCF-7 cells (Fig. 4 E). These demonstrated high efficiency of MnSOD over-expression and silencing and also suggest that QRYLD may target MnSOD expression in MCF-7 cells. A similar pattern of CaMKII and AMPK expression was observed in the different groups of cells. Collectively, these data provided novel evidence to demonstrate that QRYLD treatment, like MnSOD silencing, inhibited glucose uptake and lactic acid production to reduce the Warburg effect in MCF-7 cells by reducing the expression of several regulators and metabolic enzymes and attenuating the MnSOD/CaMKII/AMPK signaling 5. QRYLD treatment inhibits the growth of xenograft breast tumors in mice Finally, we tested the therapeutic effect of QRYLD treatment on the growth of xenograft breast cancer in mice. Following implantation with wild-type, MnSOD silencing or over-expressing MCF-7 cells, the mice with wild-type MCF-7 cells were randomized and treated intravenously with vehicle or QRYLD daily for 10 consecutive days. The dynamic growth of implanted tumors in the different groups of mice was monitored and the results revealed that compared with the control group, QRYLD treatment, like MnSOD silencing, significantly decreased the tumor volumes and weights in mice while MnSOD over-expression significantly increased the tumor volumes and weights in mice (Fig. 5 A and B). In contrast, there was no significant difference in body weights among the different groups of mice (Fig. 5 C). Immunohistochemistry displayed that compared with the control tumors, the tumors from the QRYLD-treated mice, like the MnSOD silencing tumors, exhibited a reduced level of MnSOD, CaMKII and AMPK expression in mice while the levels of MnSOD, CaMKII and AMPK expression in the MnSOD over-expressing tumors significantly increased (Fig. 5 D). Together, these data indicated that QRYLD treatment significantly inhibited the growth of implanted xenograft breast tumors in mice by attenuating the MnSOD/CaMKII/AMPK signaling. Discussion The SOD family is an important antioxidant system. Among them, MnSOD is a manganese-contained homotetramer in the mitochondrial matrix[ 6 – 10 ]. MnSOD expression is usually up-regulated in cancer cells, and can promote the release of hydrogen peroxide in mitochondria, and up-regulate the expression of CaMKII, which can phosphorylate and activate AMPK. AMPK is an important energy receptor in cells and can enhance glucose glycolysis and the Warburg effect[ 11 – 15 ]. The AMPK activation is also crucial for the cellular responses to stress, autophagy, survival and the growth of cancer cells[ 16 – 20 ]. Previous studies have shown that MnSOD over-expression up-regulates HIF-1α expression to regulate the cellular redox reactions and malignant behaviors of MDA-MB-231 cells in a hypoxic condition by eliminating superoxide anion[ 21 , 22 ].Accordingly, the MnSOD is a potent antioxidant to support the survival of cells under a hypoxic condition. MnSOD can shift oxidative phosphorylation into glycolysis in human triple negative breast cancer cells by transmitting the glucose signal and changing the oxygen transmission pathway[ 23 , 24 ]. In this study, we found that MnSOD over-expression enhanced glycolysis in MCF-7 cells by enhancing CaMKII and AMPK expression, and promoted the malignant behaviors, such as proliferation, migration, wound healing and invasion of MCF-7 cells. Conversely, MnSOD silencing dramatically decreased glucose uptake, lactic acid production, Glut-1 expression, the hallmarks of reduced levels of glycolysis and MnSOD silencing MCF-7 cells displayed less malignant aggressiveness in vitro, consistent with previous findings[ 26 – 27 ]. Our data were in disagreement with a previous observation that MnSOD deficiency does not significantly alter the levels of glycolysis in breast cancer cells[ 25 ]. The difference may stem from different breast cancer cell lines with varying metabolic state. Actually, MnSOD silencing promoted the occurrence of oxidative phosphorylation[ 28 ] In breast cancer cells, MnSOD may maintain a stable increase in hydrogen peroxide contents, thus maintaining the activation of AMPK and the transformation of intracellular metabolic mode into glycolysis while MnSOD silencing or inhibiting the activation of AMPK can reduce the growth vitality of cells[ 29 ]. Hence, the MnSOD/CaMKII/AMPK axis may be crucial for supporting the bioenergetics of cancer cells, and can promote the progression of breast cancer[ 30 ]. In our experiment, we found that MnSOD silencing also decreased the levels of CaMKII and AMPK expression in MCF-7 cells. Furthermore, MnSOD silencing also significantly reduced the expression levels of HK-2, PFK-1, LDH-A and PKM-2, the key metabolic enzymes in the Warburg effect process in MCF-7 cells. As a result, the MnSOD silenced MCF-7 cells exhibited less malignant aggressiveness in vitro and in vivo, which were associated with decreased levels of CaMKII and AMPK expression. In contrast, MnSOD over-expression also enhanced the expression of CaMKII, AMPK, key metabolic enzymes and critical regulators, such as HIF-1α, Glut-1and c-myc, in MCF7 cells, accompanied by enhanced malignant aggressiveness in vitro and in vivo. These paralleling data demonstrated that the MnSOD/CaMKII/AMPK axis was the key factor to regulate the progression of breast cancer and suggested that this axis may be served as a therapeutic target for developing therapies for the intervention of breast cancer. How did the QRYLD modulate the malignant behaviors of MCF-7 cells? We speculate that QRYLD treatment may attenuate the MnSOD/CaMKII/AMPK signaling to inhibit the Warburg effect and malignant behaviors of MCF-7 cells. Evidently, RNAseq analysis indicated that QRYLD treatment modulated the expression of many genes, including down-regulated MnSOD in MCF-7 cells. Bioinformatic analysis unveiled that the DEGs mainly participated in the metabolism-related signaling pathways, such as the MnSOD/MAPK signaling pathway, and biological processes, including the Warburg effect. Furthermore, QRYLD treatment significantly inhibited the malignant behaviors, such as the proliferation, migration and invasion of MCF-7 cells as well as the tumor growth in vivo. In addition, QRYLD treatment also decreased the expression levels of MnSOD, CaMKII and AMPK, the key metabolic enzymes in the Warburg effect process and critical regulators of HIF-1α, Glut-1and c-myc in MCF-7 cells as well as in the grown tumors in vivo. Interestingly, the therapeutic effects of QRYLD treatment were similar to that of MnSOD silencing in MCF-7 cells although they were less effective than the MnSOD silencing did. These data support the notion that QRYLD may suppress the expression of MnSOD and attenuate the MnSOD/CaMKII/AMPK signaling to inhibit the Warburg effect and malignant behaviors of MCF-7 cells. Our novel findings may provide new pharmacological mechanisms by which QRYLD treatment inhibits the malignancy of breast cancer(Fig. 6 ). In conclusion, our data indicated that the QRYLD contained several bioactive components and treatment with QRYLD significantly modulated the expression of many genes, including metabolism-related MnSOD. QRYLD treatment, like MnSOD silencing, significantly suppressed the malignant behaviors and the Warburg effect in MCF-7 cells as well as tumor growth in vivo, associated with inhibition of the MnSOD/CaMKII/AMPK signaling. Our findings may provide new insights into the pharmacological actions of QRYLD in inhibiting breast cancer growth and unveil new therapeutic targets for designing therapies for the intervention of breast cancer. Abbreviations QRYLD Qing-Re-Yi-Liu decoction MnSOD Manganese superoxide dismutase CaMKII Calmodulin dependent kinase II AMPK AMP-activated protein kinase Glut-1 Glucose transporter-1 HIF-1α Hypoxia-inducible factor 1α HK-2 Hexokinase-2 PFK-1 Phosphofructokinase-1 LDH-A Lactate Dehydrogenase-A PKM-2 Pyruvate kinase isozyme-2 Declarations Funding This work was funded by grants from the National Natural Science Foundation of China (no. 81703899), Shaanxi Provincial Natural Science Basic Research Program (no. 2017JM8068), Shaanxi Provincial Administration of Traditional Chinese Medicine Project(no.15-JC021), Xi'an Jiaotong University First Affiliated Hospital General Project(no. 2021ZXY-06), The second batch of provincial-level Chinese medicine young and middle-aged scientific and technological backbone projects (no. 2023-ZQNY-008). Conflicts of interest The authors declare no conflicts of interest regarding the publication of this paper. Author contributions Lianqing Sun provided the plant materials. Xinhan Zhao performed screening of extracts. Junxia Wang performed phytochemical analysis.Jing Du performed the vivo and vitro experiments. Ying Zhang and Ying Yuan conceived and supervised the study. Zhe Zhang drafted the manuscript. All authors read and approved the final manuscript. Availability of Data and Materials The data included in this investigation are available from the corresponding author. Ethics Approval and Consent to Participate All experimental protocols were implemented following the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health (NIH Publication No. 85-23, revised 1996). The materials and data are carried out in accordance with the Declaration of Helsinki. The study was approved by the Ethics Committee of the First Affiliated Hospital of Xi'an Jiaotong University (No. 2017 Lunshenkezi No. 44, dated March 2, 2017). References Yu P., Li JY., Luo YQ., Sun JY., Hu YF.,Lin B.,Meng XL., Xiang L., Mechanistic role of Scutellaria baicalensis georgi in breast cancer therapy, Am.J.Chin.Med. 51 (2023) 279-308. https://doi.org/10.1142/S0192415X23500155. Sholihah MM.,Indarto D.,Pramana TY., The inhibitory effect of Andrographis paniculata extract on proliferation of breast cancer cell line, Iop. Con. 546(2019) 20-29. Wang B., Xu SY., Liu JX., Li YF.,Xu HS., Han B.,Zhang J., Li XG., Ye XL., Total alkaloids of Coptidis rhizoma combined with exercise inhibits tumor growth of orthotopically transplanted 4T1 breast cancer mice by blocking cell cycle G_1/S transformation, Chin.J.Chin.Med. 44(2019) 1635-1641. Liu ZY.,Tang Y., Zhou RR., Shi XS., Zhang HM., Liu TF., Lian ZL., Shi XY., Bi-directional solid fermentation products of trametes robiniophila murr with Radix isatidis inhibit proliferation and metastasis of breast cancer cells, J.Chin.Med.Assoc. 81(2018) 520-530. Zou XJ., Hao YY., Hu YD., Xie SX., Bai W., Pulsatilla saponinA affects proliferation and radiosensitivity of breast cancer cells by regulating miR-24-3p/RNF2 expression, Chin.J.Path. 36(2020) 865-870. Kim NY., Ha IJ., Um JY., Kumar AP., Sethi G., Ahn KS., Loganic acid regulates the transition between epithelial and mesenchymal-like phenotypes by alleviating MnSOD expression in hepatocellular carcinoma cells, Life.Sci. 317(2023)1-12. https://doi.org/10.1016/j.lfs.2023.121458. Valentino RB., The structure-function relationships and physiological roles of MnSOD mutants, Bios.Rep. 42(2022) 1-12. https://doi.org/10.1042/BSR20220202. Al Haq AT., Tseng HY., Chen LM., Wang CC., Hsu HL., Targeting prooxidant MnSOD effect inhibits triple-negative breast cancer (TNBC) progression and M2 macrophage functions under the oncogenic stress, Cell.Death Dis.13(2022) 1-13. Liu SS., Hu JQ., Gu JF., Ni AM., Tang WH., Liu XY., Combined oncolytic adenovirus carrying MnSOD and mK5 genes both regulated by survivin promoter has a synergistic inhibitory effect on gastric cancer,Neoplasma. 69(2022) 36-48. Gius D.,.Lysine 68 acetylation directs MnSOD as a tetrameric detoxification complex versus a monomeric tumor promoter, Clin.Cancer.Res. 27(2021) 1-9. Quiros-Gonzalez I., Gonzalez-Menendez P., Alcon-Rodriguez S., Hevia, D. Sainz, RM.,Mayo JC., Androgen-dependent prostate cancer cells reprogram their metabolic signature upon Glut-1 upregulation by manganese superoxide dismutase,Free.Radi.Bio.Med. 165(2021) 1-12. https://doi.org/10.1016/j.freeradbiomed.2020.12.419. Gao YC., Zhu YM., Tran EL., Tokars V., Dean AE., Quan SH., Gius D., MnSOD Lysine 68 acetylation leads to cisplatin and doxorubicin resistance due to aberrant mitochondrial metabolism, Int.J.Biol.Sci. 17(2021) 1203-1216. Fu ZM., Cao XC., Liu LH., Cao XZ., Cui YH., Li X., Quan MF., Ren KQ., Chen A.,Xu C., Qiu YB., Chen XD., Wang Z., Cao JG., Genistein inhibits lung cancer cell stem-like characteristics by modulating MnSOD and FoxM1 expression, Oncol.Lett. 20(2020) 2506-2515. https://doi.org/10.3892/ol.2020.11802 Torrens-Mas M., Cordani M., Mullappilly N., Pacchiana R., Riganti C.,Palmieri M., Pons DG., Roca P., Oliver J., Donadelli M., Mutant p53 induces SIRT3/MnSOD axis to moderate ROS production in melanoma cells, Arch.Bio.Bio. 679(2020) 1-10. https://doi.org/10.1016/j.abb.2019.108219. Li QY., Sun Y., Liu BW., Li JB., Hao X., Ge WZ., Zhang XM., Bao SQ., Gong JM., Jiang ZH ., Qiu CJ., Zhao LQ., Zhao YP., Chen Y., Yang XJ., Ding YH., Wu ZZ., ACT001 modulates the NF-kappa B/MnSOD/ROS axis by targeting IKK beta to inhibit glioblastoma cell growth, J.Mol.Med. 98(2020) 263-277. Cao XC., Liu LH., Yuan Q., Li X., Cui YH., Ren KQ., Zou C., Chen A., Xu C., Qiu YB., Quan MF., Zhang JS., Cao JG., Chen XD., Isovitexin reduces carcinogenicity and stemness in hepatic carcinoma stem-like cells by modulating MnSOD and FoxM1, J.Exp.Clin.Cancer.Res. 38(2019) 1-18. https://doi.org/10.1186/s13046-019-1244-6. Zhu YM., Zou XH., Dean AE., O' Brien J., Gao YC., Tran EL., Park SH., Liu GX., Kieffer MB ., Jiang HY., Stauffer ME., Hart R., Quan SH., Satchell KJF., Horikoshi N., Bonini M., Gius D., Lysine 68 acetylation directs MnSOD as a tetrameric detoxification complex versus a monomeric tumor promoter, Nat.Commun. 10(2019) 1-15. https://doi.org/10.1038/s41467-019-10352-4. Fan JJ., Hsu WH., Hung HH., Zhang WJ., Lee YLA., Chen KC., Chu CY., Ko TP., Lee MT., Lin CW., Cheng CH., Reduction in MnSOD promotes the migration and invasion of squamous carcinoma cells, Int.J.Oncol. 54(2019) 1639-1650. https://doi.org/10.3892/ijo.2019.4750. Ma SJ., Fu A., Lim S., Chiew GGY., Luo KQ., MnSOD mediates shear stress-promoted tumor cell migration and adhesion, Free.Radi.Bio.Med. 129(2018) 46-58. https://doi.org/10.1016/j.freeradbiomed.2018.09.004. Liu F., Yuan Q., Cao XC., Zhang JL., Cao JG., Zhang JS., Xia LQ., Isovitexin suppresses stemness of lung cancer stem-Like cells through blockage of MnSOD/CaMKII/AMPK Signaling and glycolysis inhibition, Biomed.Res.Int. 21(2021) 1-15. https://doi.org/10.1155/2021/9972057. Jetawattana S., Venkataraman S., Domann F., Oberley L., A role for MnSOD and p53 in regulating HIF-1 alpha in breast cancer cell lines, Free.Radi.Bio.Med. 39(2005) 166-176. Jetawattana S., Venkataraman, S., Teoh MLT., Oberle, LW., Redox regulation of HIF-1 alpha by MnSOD via ROS, Free.Radi.Bio.Med. 41(2006) 110-119. He CX., Hart P., Fricano K., Vargas M., Thieraud K., de Abreu AL., Bonini M., Mangenese superoxide dismutase (MnSOD) promotes stem-like cell phenotypes in breast cancer, Faseb.J. 31(2017) 809-815. Al Haq AT., Tseng HY., Chen LM., Wang CC., Hsu HL., Targeting prooxidant MnSOD effect inhibits triple-negative breast cancer (TNBC) progression and M2 macrophage functions under the oncogenic stress, Cell.Death.Dis. 13(2022) 1-15. Hart PC., Fricano KA., Minshall R., Bonini M., Loss of caveolin-1 results in a MnSOD-dependent glycolytic shift in breast cancer, Free.Radi.Bio.Med. 53(2012) 43-53. https://doi.org/10.1016/j.freeradbiomed.2012.10.112. Ambrosone CB., Choi JY., Barlow W., Yeh IT., Hayes DF., Davis W., Rae JM., Nowak N., Conroy J., Li Y., Den Haese J., Lehman T., Hutchins LF., Livingston R., Ravdin P., Albain K., Pharmacogenetics and breast cancer treatment outcomes: results on oxidative stress related genotypes (MPO, MnSOD) from a southwest oncology group trial (S8897), Int.J.Biol.Marker. 22(2007) 61-69. Weydert CJ., Zhang Y., Sun W., Waugh TA., Teoh MLT., Andriinga KK., Aykin-Burns N., Spitz DR., Smith BJ., Oberley LW., Increased oxidative stress created by adenoviral MnSOD or CuZnSOD plus BCNU (1,3-bis(2-chloroethyl)-1-nitrosourea) inhibits breast cancer cell growth, Free.Radi.Bio.Med. 44(2008) 856-867. https://doi.org/10.1016/j.freeradbiomed.2007.11.009 Quiros-Gonzalez I., Gonzalez-Menendez P., Alcon-Rodriguez S., Hevia D., Sainz RM., Mayo JC., Androgen-dependent prostate cancer cells reprogram their metabolic signature upon Glut-1 upregulation by manganese superoxide dismutase (Mnsod/SOD2), Free.Radi.Bio.Med. 165(2021) 97-107. https://doi.org/10.1016/j.freeradbiomed.2020.12.419 Hart PC., Mao M., de Abreu ALP., Ansenberger-Fricano K., Ekoue DN., Ganini D., Kajdacsy-Balla A., Diamond AM., Minshall RD., Consolaro MEL., Santos JH., Bonini MG., MnSOD upregulation sustains the Warburg effect via mitochondrial ROS and AMPK-dependent signalling in cancer, Nat.Commun. 6(2015) 1-14. Ansenberger-Fricano K., Mao M., Green PD., Hall A., Kajdacsy-Balla A., Santos JA., Bonini MG., MnSOD regulation of AMPK-Mediated transition to glycolysis in breast cancer, Free.Radi.Bio.Med. 53(2012) 39-49. https://doi.org/10.1016/j.freeradbiomed.2012.10.100. Ye DY., Sun J., Li YQ., Evaluation of toxicity, bacteriostatic, analgesic, anti-inflammatory, and antipyretic activities of Huangqin-Honghua-Pugongying-Jinyinhua extract, VET,SCI.8(2021) 1-12. Min F., Xiao Z., Huaping S.,Yakong Z., Dandelion ( Taraxacum genus ): A review of chemical constituents and pharmacological effects, Mol. 28(2023) 1-31.https://doi.org/10.3390/molecules28135022. Zhang Y., Hu YF., Li W., Xu GY., Wang KR., Li L., Luo H., Zou L., Wu JS., Updates and advances on pharmacological properties of Taraxacum mongolicum Hand.-Mazz and its potential applications, Food Chem. 373(2022) 131-143. https://doi.org/10.1016/j.foodchem.2021.131380. Mehdi Sharifi-Rad,Thomas H. Roberts, Karl R. Matthews, Camila F. Bezerra, Maria Flaviana B. Morais-Braga, Henrique D.M. Coutinho, Farukh Sharopov, Bahare Salehi, Zubaida Yousaf, Majid Sharifi-Rad, María del Mar Contreras, Elena Maria Varoni, Deepa R. Verma, Marcello Iriti, Javad Sharifi-Rad, Ethnobotany of the genus Taraxacum —phytochemicals and antimicrobial activity, Phy.Res. 32(2018) 2131-2145. https://doi.org/10.1002/ptr.6157. Wang Y., Tang J., Sun P., Lin S., Zhang X.,.Effects of Iphigenia indica extract on proliferation and apoptosis of human colorectal cancer cell line SW480, Chin.J.Basic.Med. 27(2021) 1754-1758. Tang RP., Yang YH., Su HL., Huang GB.,Study on pollen viability of Iphigenia indica . Southwest.Chin.J.Agr.Sci. 22(2009) 153-155. Mukhopadhyay MJ, Mukhopadhyay S.,High frequency in vitro propagation through somatic embryogenesis of Iphigenia indica kunth et benth , an endangered medicinal and colchicines yielding herb of commercial interest, Cytologia. 73(2008) 97-103. Jun L., Liyao L., Zhen J., Jing C., Zuguo Z., Yi Z., Zhujin X., Zhen G., Antibacterial potential of Forsythia suspensa polysaccharide against resistant enterobacter cloacae with SHV-12 extended-spectrum β-lactamase, J.Cell.Mol.Med. 24(2020) 8763-8771. https://doi.org/10.1111/jcmm.15510. Zhao L., Li W., Dai SJ., Liu RX., Xie ZP., Zhang SM., Yue XD., Alkaloids bearing rare skeletons from Forsythia suspensa with anti-inflammatory and anti-viral activities in vitro, Phytochem. 186(2021) 1-12. https://doi.org/10.1016/j.phytochem.2021.112739. Shao SY., Zhang F., Yang YN., Feng ZM., Jiang JS., Zhang PC., Neuroprotective and anti-inflammatory phenylethanoid glycosides from the fruits of Forsythia suspense , Bioorg,Chem. 113(2021) 1-15. https://doi.org/10.1016/j.bioorg.2021.105025. Li W., Sun LT., Zhao L., Yue XD., Dai SJ., New C-9-monoterpenoid alkaloids featuring a rare skeleton with anti-inflammatory and antiviral activities from Forsythia suspense , Chem. Biodivers. 19(2022) 1-7. Wang R., Wang XC., Xia MQ., Yang LX., Cheng WK., Song QQ.,Combining network pharmacology with chromatographic fingerprinting and multicomponent quantitative analysis for the quality evaluation of Moutan cortex , Bio.Chr. 36(2022) 1-12.https://doi.org/10.1002/bmc.5434 Yang SC., Liu XY., He JY., Liu MH., Insight into seasonal change of phytochemicals, antioxidant, and anti-aging activities of root bark of paeonia suffruticosa (Cortex moutan) combined with multivariate statistical analysis, Mol. 26(2021) 1-19.https://doi.org/10.3390/molecules26206102. Zhang YC., Wu XY., Wang XH., Zeng Y., Liao YX., Zhang RZ., Zhai FQ., Zeng ZL., Grey relational analysis combined with network pharmacology to identify antioxidant components and uncover its mechanism from Moutan cortex , Front Pharmacol. 12(2021) 1-14. https://doi.org/10.3389/fphar.2021.748501 Zhou SJ., Lin HJ., Meng J., Discrimination and chemical composition quantitative model of Raw Moutan cortex and Moutan cortex carbon based on electronic nose, Math.Biosci.Eng. 19(2022) 9079-9097. Wu ZH., Yin B., You FM., Molecular mechanism of anti-colorectal cancer effect of Hedyotis diffusa willd and its extracts, Front Pharmacol. 13(2022) 1-10.https://doi.org/10.3389/fphar.2022.820474 Lu L.Effect of Hedyotis diffusa Polysaccharide on Apoptosis of Thyroid Cancer Cells, Pak.J.Zool. 54(2022) 1803-1810. https://dx.doi.org/10.17582/journal.pjz/20201101141148. Dong HP., Zhang Q., Hedyotis diffusa willd extract protects rat renal tubular epithelial cells from high glucose-induced injury by inhibiting PI3K/AKT signaling pathway, Scie. 48(2022) 532-537. Xu L, Li Y., Ji JJ., Lai YH., Chen J., Ding T., Li HC., Ding B., Ge WH., The anti-inflammatory effects of Hedyotis diffusa willd on SLE with STAT3 as a key target, J. Ethnopharmacol. 298(2022) 1-14. https://doi.org/10.1016/j.jep.2022.115597. Yang R., Lu Y., Hao H., Zhang M., Xuan J., Zhang YQ., Research progress on chemical constituents and pharmacological activities of iridoid glycosides in Lonicera japonica , Chin.J.Chin. 46(2021) 2746-2752. Jiang HF., Chen C., Jiang XY., Shen JL., Ling F., Li PF., Wang GX., Wang GX., Luteolin in Lonicera japonica inhibits the proliferation of white spot syndrome virus in the crayfish procambarus clarkia, Aquaculture. 550(2022) 1-10. https://doi.org/10.1016/j.aquaculture.2021.737852. Xu N., Du LH., Chen YC., Zhang JH., Zhu QF., Chen R., Peng GP., Wang QM., Yu HZ., Rao LQ., Lonicera japonica thunb as a promising antibacterial agent for bacillus cereus ATCC14579 based on network pharmacology, metabolomics, and in vitro experiments, Rsc.Adv. 13(2023) 15379-15390. https://doi.org/10.1039/D3RA00802A. Yang XP., Yu AQ., Hu WJ., Zhang ZJ., Ruan Y., Kuang HX., Wang M., Extraction, purification, structural characteristics, health benefits, and application of the polysaccharides from Lonicera japonica thumb, A.Rev.Mol. 28(2023) 1-11. https://doi.org/10.3390/molecules28124828. Dar Khursheed Ahmad, Senthilmurugan S, Venkatesan A, Antibacterial, antioxidant potential and functional group analysis of kashmir grown Prunella vulgaris L. root extract, Int.J.Bot.Stu. 7(2022) 19-25. Bai HT., Wang R., Li YL., Liang X., Zhang JH., Sun N., Yang J., Network pharmacology analysis, molecular docking, and in vitro verification reveal the action mechanism of Prunella vulgaris L . in treating breast vancer, Evid-Based.Compl.Alt. 12(2022) 1-19.https://doi.org/10.1155/2022/5481563 Dar KA., Senthilmurugan S., Ali S., Al-Sadoon MK., Paray BA., Studies on the isolation and identification of the antibacterial compound from Prunella vulgaris L . flower extract, J.King.Saud.Univ.Sci. 34(2022) 1-6. https://doi.org/10.1016/j.jksus.2022.102324. Kim J., Lee CG., Hwang S., Yun SH., Uprety LP., Oh KI., Singh S., Yoo J., Jeong H., Yong Y ., Anti-osteoarthritic effects of Prunella Vulgaris and Gentiana lutea in vitro and in vivo, Antioxidants. 12(2023) 47-57. https://doi.org/10.3390/antiox12010047. Chen HX., Olatunji OJ., Zhou YF., Anti-oxidative, anti-secretory and anti-inflammatory activities of the extract from the root bark of Lycium chinense ( Cortex Lycii ) against gastric ulcer in mice, J.Nat.Med-Tokyo. 70(2016) 610-619. Guo ZH., Yin H., Wu T., Wu SF., Liu LY., Zhang L., He YL., Zhang R., Liu N., Study on the mechanism of Cortex Lycii on lung cancer based on network pharmacology combined with experimental validation, J.Ethno. 293(2022) 1-10. https://doi.org/10.1016/j.jep.2022.115280. Zhao Sl., Yuan Z., Zhu YR., Ding HQ., Zheng XJ., Zhang JZ., Liu DL., Mechanism of hypolipidemic effect of Lycii cortex based on network pharmacology and molecular docking, Chin. Trad.Drugs. 54(2023) 1487-1497. 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Also discoverable on Platform About Our Team In Review Editorial Policies Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-3366517","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":289829799,"identity":"fb2d6d6e-52fc-4879-a41a-2b31b819d064","order_by":0,"name":"哲 张","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAyElEQVRIiWNgGAWjYBAC+/7GhgMSP2rkGNsbiNRiIHG48YFlzzFj5p4DxGphSG82qGBjTmyfkUCkFnOGg20SN3jYEntnPt54g6HGJpqgFsvmxjbJGRYyxjNnpxVbMBxLy20gqOfAwTZpCR422Y2zc8wkGBsOE6MlsU36Dxsz4/6bZ4jUYnAgsdlAgo1ZsXEGD5FaJGccbHwgCQxkxh6gXxKI8Qs/f/sDaFQe3njjQ40NEX5BdqREAinKIVpI1TEKRsEoGAUjAwAAc1pEShaO8VcAAAAASUVORK5CYII=","orcid":"https://orcid.org/0000-0001-9232-101X","institution":"The First Affiliated Hospital of Xi'an Jiaotong University","correspondingAuthor":true,"submittingAuthor":false,"prefix":"","firstName":"哲","middleName":"","lastName":"张","suffix":""},{"id":289829800,"identity":"6c1265ae-94a6-48a9-9302-3528932368be","order_by":1,"name":"xin zhao","email":"","orcid":"","institution":"The First Affiliated Hospital of Xi'an Jiaotong University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"xin","middleName":"","lastName":"zhao","suffix":""},{"id":289829801,"identity":"e5b4a8f8-8fa5-4b71-955e-a1611a1a21bb","order_by":2,"name":"lian sun","email":"","orcid":"","institution":"The First Affiliated Hospital of Xi'an Jiaotong University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"lian","middleName":"","lastName":"sun","suffix":""},{"id":289829802,"identity":"97e37f11-56a0-48c7-a50d-0b54b77f1750","order_by":3,"name":"jun wang","email":"","orcid":"","institution":"The First Affiliated Hospital of Xi'an Jiaotong University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"jun","middleName":"","lastName":"wang","suffix":""},{"id":289829803,"identity":"abe04e28-050f-4fd1-a0c4-ca479f5ddf46","order_by":4,"name":"jing du","email":"","orcid":"","institution":"The First Affiliated Hospital of Xi'an Jiaotong University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"jing","middleName":"","lastName":"du","suffix":""},{"id":289829804,"identity":"5f9b440a-bd4b-4238-afc9-957b6caa5a05","order_by":5,"name":"ying zhang","email":"","orcid":"","institution":"The First Affiliated Hospital of Xi'an Jiaotong University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"ying","middleName":"","lastName":"zhang","suffix":""},{"id":289829805,"identity":"a99ca256-ac57-4a49-b2e1-6f08b3df67f3","order_by":6,"name":"ying Yuan","email":"","orcid":"","institution":"The First Affiliated Hospital of Xi'an Jiaotong University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"ying","middleName":"","lastName":"Yuan","suffix":""}],"badges":[],"createdAt":"2023-09-18 14:46:01","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-3366517/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-3366517/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":54672708,"identity":"77a8cbb8-f07d-4c8d-aad4-ac8ae620b642","added_by":"auto","created_at":"2024-04-15 05:32:50","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":668091,"visible":true,"origin":"","legend":"\u003cp\u003eHPLC analysis of chemical components in aqueous QRYLD extracts.\u003c/p\u003e\n\u003cp\u003e(A) Drug standards. (B) Aqueous QRYLD extracts. 1. chlorogenic acid; 2. caffeic acid; 3. quercetin; 4. rutin; 5. ferulic acid; 6. luteolin.\u003c/p\u003e","description":"","filename":"1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-3366517/v1/6be27222c333d6dc354095b9.jpg"},{"id":54672731,"identity":"88026787-768f-4db2-9c53-1bd4775991f5","added_by":"auto","created_at":"2024-04-15 05:32:51","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":1359110,"visible":true,"origin":"","legend":"\u003cp\u003eBioinformatic analysis of the transcriptomes in QRYLD-treated MCF_7 cells. (A) A Heatmap analysis of differentially expressed genes (DEGs). 1-3: MCF-7 controls. 4-6: the QRYLD-treated MCF-7 cells. (B) The PPI analysis of the potential interactions among the targets of QRYLD. (C) Enrichment analysis of DEGs.\u003c/p\u003e","description":"","filename":"2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-3366517/v1/cc643590187a61c81e795ae7.jpg"},{"id":54672729,"identity":"c5f5da5f-e1ff-4ad8-aa6a-81fca90fc056","added_by":"auto","created_at":"2024-04-15 05:32:51","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":2776628,"visible":true,"origin":"","legend":"\u003cp\u003eQRYLD treatment, like MnSOD silencing, attenuates the malignant behaviors of MCF-7 cells in vitro. (A) CCK-8 assays determined the effect of QRYLD on cell viability. MCF-7 cells were treated with vehicle or the indicated doses of QRYLD-medicated serum, together with Ad-sh MnSOD and lent MnSOD MCF7 cells were tested in triplicate for their viability by CCK-8 assays at the indicated time points. (B) Transwell invasion assays evaluated the effects of QRYLD on cell invasion. (C) The effects of QRYLD on the wound healing of the indicated MCF-7 cells. (D) Flow cytometry analysis of the effects of QRYLD on cell apoptosis.\u003c/p\u003e","description":"","filename":"3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-3366517/v1/08870103a25d4375065916e3.jpg"},{"id":54672733,"identity":"f38f540f-a118-480c-853d-7884d5c231f9","added_by":"auto","created_at":"2024-04-15 05:32:51","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":2537601,"visible":true,"origin":"","legend":"\u003cp\u003eQRYLD treatment, like MnSOD silencing modulates the Warburg effect in MCF-7 cells. (A,B) The effect of QRYLD on the levels of glucose uptake and lactic acid production. (C) The expression of key regulatory factors in the Warburg effect process. (D) The expression of metabolic enzymes in the Warburg effect process. (E) The expression of MnSOD/CaMKII/AMPK pathway events in the Warburg effect process.\u003c/p\u003e","description":"","filename":"4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-3366517/v1/7d3780bb32127ee10b61c351.jpg"},{"id":54672728,"identity":"19bdcb61-2730-42ae-b243-da5663d9e22a","added_by":"auto","created_at":"2024-04-15 05:32:50","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":3285075,"visible":true,"origin":"","legend":"\u003cp\u003eQRYLD treatment suppresses the tumor growth in mice. (A) The tumor volumes in mice were measured every other day for 4 consecutive weeks. (B) The body weights of mice were measured every other day for 4 consecutive weeks. (C) The photoimage of all dissected tumors and their weights. (D) Immunohistochemistry analysis of MnSOD, CaMKII and AMPK expression in tumor tissues of each group of mice.\u003c/p\u003e","description":"","filename":"5.jpg","url":"https://assets-eu.researchsquare.com/files/rs-3366517/v1/fa65b62ae6ae13ccf2ede4d8.jpg"},{"id":54672732,"identity":"0ee97d6a-fab8-481b-9e4a-a559f1a3e3fa","added_by":"auto","created_at":"2024-04-15 05:32:51","extension":"jpg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":441589,"visible":true,"origin":"","legend":"\u003cp\u003eThe mechanism of QRYLD attenuates the MnSOD/CaMKII/AMPK signaling and Warburg effect\u003c/p\u003e","description":"","filename":"6.jpg","url":"https://assets-eu.researchsquare.com/files/rs-3366517/v1/a0972f28c70a0975542157e1.jpg"},{"id":54673131,"identity":"e6161ec8-1de7-4295-82a4-fb27b5e18254","added_by":"auto","created_at":"2024-04-15 05:40:51","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":565615,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-3366517/v1/8d09c5f9-9a41-4bbb-9a49-bbc7bd362c34.pdf"}],"financialInterests":"","formattedTitle":"Qing-Re-Yi-Liu decoction suppresses the malignant behaviors of breast cancer cells by attenuating the MnSOD/CaMKII/AMPK signaling and Warburg effect","fulltext":[{"header":"Introduction","content":"\u003cp\u003eBreast cancer is the most common malignant tumor in women worldwide. Currently, therapeutic strategies for the intervention of breast cancer include surgical resection of tumors, neoadjuvant and post-operative therapies, such as chemotherapy, target therapy, radiotherapy, immunotherapy and others. Traditional Chinese medicine (TCM) has been used for the treatment of breast cancer. There are many studies on the prevention and intervention of TCM for breast cancer.\u003c/p\u003e \u003cp\u003eIn the theory of TCM, the tumor development and progression are attributed to the accumulation of heat and toxin, and therapeutic strategies to clear heat and detoxify are important for the control of tumor growth. However, the pharmacological mechanisms underlying the actions of these TCMs in antitumor activity have not been clarified. Previous studies have shown that \u003cem\u003eScutellaria baicalensis\u003c/em\u003e and its bioactive ingredients have potent activities against the malignancy of breast cancer cells[\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]and treatment with ethanol extracts of \u003cem\u003eAndrographis paniculata\u003c/em\u003e inhibits the proliferation of breast cancer MCF-7 cells in a dose-dependent manner[\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e].Similarly, treatment with \u003cem\u003eCoptidis rhizoma\u003c/em\u003e limits the growth of 4T1 breast cancer in mice by inducing their cell cycle arrest in G1 phase and suppressing DNA synthesis[\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]while treatment with \u003cem\u003eRadix isatidis\u003c/em\u003e attenuates the malignant behaviors of SK-BR-1 and MDA-MB-231 cells in a time-dependent manner by inducing cell cycle arrest in G2/M phases and apoptosis[\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. Furthermore, treatment with different doses of \u003cem\u003ePulsatilla saponin\u003c/em\u003e can inhibit the proliferation of MCF-7 cells in a dose-dependent manner[\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. Although many TCMs have been used in clinical practices for the intervention of breast cancer, the therapeutic efficacy of these TCMs is limited. Hence, discovery of new and safe TCMs, particularly for those with potent activities of heat clearing and detoxification is urgently needed.\u003c/p\u003e \u003cp\u003eThe Qing-Re-Yi-Liu decoction (QRYLD) is a clinical effective prescription for the treatment of breast cancer given its heat clearing and detoxification activities. Our preliminary studies have found that treatment with QRYLD inhibits the proliferation of breast cancer MCF-7 cells, which is associated with inhibition of the Warburg effect (aerobic glycolysis). The current studies aimed at investigating the pharmacological effect of QRYLD on the malignancy of MCF-7 breast tumors and their Warburg effect as well as the molecular mechanisms underlying the pharmacological action of QRYLD in breast cancer.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e1. QRYLD\u003c/h2\u003e \u003cp\u003eQRYLD is composed of \u003cem\u003eTaraxacum officinala, Iphigenia indica, Forsythia suspensa, Cortex moutan, Hedyotis diffusa, Lonicera japonica, Prunella vulgaris and Lycii cortex\u003c/em\u003e root of T\u003cem\u003earaxacum officinala L., Iphigenia indica L, Forsythia suspense L, Cortex moutan L, Hedyotis diffusa L, Lonicera japonica L, Prunella vulgaris L. and Lycii cortex L.\u003c/em\u003e, and the detailed components of ORYLD are shown in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e(The plant name has been checked with \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ewww.worldfloraonline.org\u003c/span\u003e\u003cspan address=\"http://www.worldfloraonline.org\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e). These medicinal herbs were obtained from the TCM Pharmacy of the First Affiliated Hospital of Xi'an Jiaotong University (Xi'an, Shaanxi, China) and were identified by experts in the Pharmacy Department. The QRYLD was soaked in 10 volumes of water and cooked at 100 \u0026deg; C for 1 hour. The decoction was collected and the herbs were cooked with another 10 volumes of water for 40 minutes for collection of the decoction. The collected two decoctions were mixed, filtered through a filter, concentrated and dried using a spray dryer (Lemal, Changzhou, China). The dried ORYLD powder was dissolved in water and the chemical components in the aqueous ORYLD extracts were identified by HPLC using Agilent 1260 InfinityII liquid chromatography (Agilent Technologies, California, USA).\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eThe composition of QRYLD and List of selected plant material traditionally use in diseases\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"8\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c8\" colnum=\"8\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eFamily\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eSpecies\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eChinese name\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eDosage(g)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003ePart\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003eLot No.\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c7\"\u003e \u003cp\u003eTraditional use\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c8\"\u003e \u003cp\u003eReferences\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAsteraceae\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eTaraxacum L.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003ePugongying\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e30\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eherba\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e210601\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eanti-bacterial, anti-oxidant, anti-cancer, and anti-rheumatic activities\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e[\u003cspan additionalcitationids=\"CR32 CR33\" citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eColchicaceae\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eIphigenia indica L.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eShancigu\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e30\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003ecaulis\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e210501\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eAnti-cancer, anti-inflammatory, pain relieving, blood pressure lowering, cough relieving, asthma relieving\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e[\u003cspan additionalcitationids=\"CR36\" citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e]\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eOleaceae\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eForsythia suspensa L.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eLianqiao\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e15\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003efructus\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e210801\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eAnti-bacterial, anti-pyretic, anti-emetic, anti-inflammatory, diuretic, anti-liver injury, cardiotonic, diuretic, anti-hypertensive\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e[\u003cspan additionalcitationids=\"CR39 CR40\" citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e]\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePaeoniaceae\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eCortex Moutan L.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eMudanpi\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e15\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eRadix bark\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e210701\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eAnti-bacterial, anti-inflammatory, anti- allergic, liver protective, hypoglycemic, anti-cancer\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e[\u003cspan additionalcitationids=\"CR43 CR44\" citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e]\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eRubiaceae\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eHedyotis diffusa L.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eBaihuasheshecao\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e30\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eherba\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e210601\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eAnti- inflammatory, anti-cancer, analgesic, liver and gallbladder protection\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e[\u003cspan additionalcitationids=\"CR47 CR48\" citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e]\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCaprifoliaceae\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eLonicera japonica L.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eJinyinhua\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e15\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eFlos\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e210601\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eAnti-inflammatory, anti-pyretic, anti- endotoxin, hypolipidemic\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e[\u003cspan additionalcitationids=\"CR51 CR52\" citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e]\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eLamiaceae\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003ePrunella vulgaris L.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eXiakucao\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e15\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003efructus\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e210801\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eHypotension, hypoglycemic, anti- bacterial, anti-inflammatory, anti- allergic, and anti-viral\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e[\u003cspan additionalcitationids=\"CR55 CR56\" citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e57\u003c/span\u003e]\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCanellaceae\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eLycii Cortex L.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eDigupi\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e15\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eRadix bark\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e210501\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eAnti-pyretic, anti-hypertensive, hypoglycemic, anti-cancer, and lipid-lowering\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e[\u003cspan additionalcitationids=\"CR59\" citationid=\"CR58\" class=\"CitationRef\"\u003e58\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e60\u003c/span\u003e]\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003ctfoot\u003e \u003ctr\u003e\u003ctd colspan=\"8\"\u003eThe plant name has been checked with \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://www.theplantlist.org\u003c/span\u003e\u003cspan address=\"http://www.theplantlist.org\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e and \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e\u003ca href=\"http://www.worldfloraonline.org\" target=\"_blank\"\u003ewww.worldfloraonline.org\u003c/a\u003e\u003c/span\u003e\u003cspan address=\"http://www.worldfloraonline.org\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/td\u003e\u003c/tr\u003e \u003c/tfoot\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003e2. Preparation of QRYLD-medicated serum\u003c/p\u003e \u003cp\u003eBalb/c mice at 3\u0026ndash;5 weeks of age were from the Laboratory Animal Center, Xi`an Jiaotong University Health Science Center and treated with 0.3 ml of QRYLD in saline at low-dose (LD, 5.6875g\u0026middot;kg-1\u0026middot;d-1), medium-dose (MD, 11.375g\u0026middot;kg-1\u0026middot;d-1) or high-dose (HD, 22.75g\u0026middot;kg-1\u0026middot;d-1), based on the ratio of body surface area of humans to mice by gavage daily for consecutive 7 days. Two hours after the last gavage, their blood samples were collected for preparation of serum samples. After being heated at 56\u0026deg;C for 30 minutes, the serum samples were filtered through a 0.45 \u0026micro;M filter and used as the ORYLD-medicated serum for cell culture. A 10% of medicated serum in RPMI-1640 medium was prepared (Solarbio, Beijing, China).\u003c/p\u003e \u003cp\u003e3. Cell culture\u003c/p\u003e \u003cp\u003eHuman breast cancer MCF-7 cells were obtained from Shanghai Institute of Cell Biology, China and cultured in RPMI-1640 medium (Solarbio) containing 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin at 37℃ in a 5% CO\u003csub\u003e2\u003c/sub\u003e incubator.\u003c/p\u003e \u003cp\u003e4. Transfection and transduction\u003c/p\u003e \u003cp\u003e293T cells were transfected with plasmids for lentivirus packaging and MnSOD expression or control GFP expression using lipofectamine 3000 to produce lent-MnSOD or control GFP virions by Jinsirui Biotechnology, Nanjing, China. Similarly, HEK293 cells were transfected with the adenovirus packaging plasmids and the plasmid for the expression of MnSOD-specific shRNA or control shRNA to generate Ad-sh-MnSOD or control Ad-sh virions by Jinsirui Biotechnology. MCF-7 cells were transduced with lentivirus for stable MnSOD over-expression at a MOI of 10 to generate Lent-MnSOD cells. The transduced cells were treated with 4 \u0026micro;g/ml of puromycin to induce stable expression. Furthermore, MCF-7 cells were infected with the Ad-sh-MnSOD or control Ad-sh at a MOI of 10 and treated with 2 \u0026micro;g/ml of puromycin to generate stable MnSOD silencing MCF-7 cells. The efficiency of MnSOD over-expression or silencing was examined by Western blot.\u003c/p\u003e \u003cp\u003e5. CCK-8 assay\u003c/p\u003e \u003cp\u003eWild-type, Ad-sh MnSOD, lent MnSOD MCF-7 cells (1\u0026times;10\u003csup\u003e4\u003c/sup\u003e cells/well) were cultured in 96-well plates and treated with vehicle PBS, while the wild-type MCF-7 cells were treated with 10% of LD, MD, or HD of QRYLD-medicated serum, respectively. The proliferation of each group of cells was tested in triplicate for 24, 48 and 72 hours, respectively using CCK-8 assays. During the last 4-hour culture, individual wells were added with 10 \u0026micro;L of CCK-8 solution (Beyotime, Shanghai, China) and the viability of each group of cells was measured for the absorbance of each well at 450 nm using a microplate reader.\u003c/p\u003e \u003cp\u003e6. Transcriptome analysis\u003c/p\u003e \u003cp\u003eMCF-7 cells were treated with vehicle as the control or MD of QRYLD-medicated serum for 48 hours. Their total RNA was extracted using Trizol (Life Technologies, California, USA) and the contained mRNAs were sorted using Oligotex mRNA sorting kit (Qiagen, Germany), per the manufacturer\u0026rsquo;s instructions. After qualification and quantification of mRNA in Illumina HiSeq\u003csup\u003eTM\u003c/sup\u003e2000, mRNA samples from each group were reverse-transcribed into cDNA to generate the cDNA libraries using SMART-Seq V4 kit (Takara Bio, USA). The cDNA libraries were sequenced in NovaSeq 6000 machine (Illumina, USA). The differentially expressed genes (DEGs) were defined when a p-value of \u0026lt;\u0026thinsp;0.05 or a fold change\u0026thinsp;\u0026gt;\u0026thinsp;2.0 or \u0026lt;\u0026thinsp;0.5. The biological nature of DEGs was analyzed using DAVID Bioinformatics Resources 6.7 and their biological processes or signaling pathways were analyzed by gene ontology (GO) and KEGG. In addition, the potential connections among these biological processes and signaling pathways of the DEGs were analyzed by the protein-protein interaction (PPI) using Cytoscape software.\u003c/p\u003e \u003cp\u003e7. Transwell invasion assay\u003c/p\u003e \u003cp\u003eMCF-7 (4\u0026times;10\u003csup\u003e4\u003c/sup\u003ecells/well) were treated with vehicle or MD of QRYLD-medicated serum for 48 hours. The cells, together with the same number of Ad-sh-MnSOD MCF-7, lent-MnSOD MCF-7 cells, were cultured into the upper chamber that had been coated with Matrigel (Thermo Fisher Scientific, Massachusetts, USA) and the bottom chamber was filled with 10% FBS medium. After being cultured for 24 hours, the cells on the upper chamber surface membrane were removed and the invaded cells on the upper chamber bottom surface were fixed in 4.0% paraformaldehyde and stained with 0.1% crystal violet solution for 10 minutes, followed by photoimaging under a Leica DMil inverted microscope (Leica, Wetzlar, Germany). The number of invaded cells was counted in a blinded manner.\u003c/p\u003e \u003cp\u003e8. Wound healing assay\u003c/p\u003e \u003cp\u003eMCF-7 were treated with vehicle or MD of QRYLD-medicated serum for 48 hours. The cells, together with the same number of Ad-sh-MnSOD MCF-7, lent-MnSOD MCF-7 cells (1\u0026times;10\u003csup\u003e6\u003c/sup\u003e cells/well) were cultured in 6-well plates. When the cells reached 95% confluency, the monolayer cells were wounded using a 10-\u0026micro;L sterile pipette tip. The wounded areas were photoimaged immediately after scratch and 48-hour culture. The wound healing ability of each group of cells was analyzed using ImageJ software.\u003c/p\u003e \u003cp\u003e9. Apoptosis Detection\u003c/p\u003e \u003cp\u003eAd-sh-MnSOD MCF-7, lent-MnSOD MCF-7, and MCF-7 cells that had been treated with vehicle saline or MD of QRYLD-medicated serum for 48 hours were harvested and stained with Annexin V-isothiocyanofluorescein (FITC) (Solarbio) and propidium iodide (PI) (Solarbio) for 10 minutes in the dark. The percentages of apoptotic cells were analyzed by flow cytometry and the data were analyzed by FlowJo software.\u003c/p\u003e \u003cp\u003e10. Western blotting\u003c/p\u003e \u003cp\u003eAd-sh-MnSOD MCF-7, lent-MnSOD MCF-7, and MCF-7 cells that had been treated with vehicle saline or MD of QRYLD-medicated serum for 48 hours were harvested and lyzed in lysis buffer, followed by centrifugation. After measurement of protein concentrations using a BCA kit, the cell lysates (30 \u0026micro;g/lane) were resolved in sodium dodecyl-sulfate polyacrylamide gel electrophoresis (SDS-PAGE) on 10% gels and transferred onto polyvinylidene difluoride (PVDF) membranes. After being blocked with 5% skimmed dry milk powder in TBST for 2 hours, the membranes were probed with primary antibodies against MnSOD, CaMKII, AMPK, HIF-1α, Glut-1, c-myc, HK-2, PFK-1, LDH-A, PKM-2 and β-actin (1:1000, Abiowell Biotechnology, Changsha, China) at 4℃ overnight, and reacted with horseradish peroxidase (HRP)-conjugated secondary antibodies (Abiowell Biotechnology), followed by developing with the enhanced chemiluminescent reagents. The data were analyzed by densitometric scanning using ImageJ software.\u003c/p\u003e \u003cp\u003e11. Glucose uptake and lactate levels\u003c/p\u003e \u003cp\u003eMCF-7 cells were treated with vehicle saline or MD of QRYLD-medicated serum for 48 hours. The impact of QRYLD treatment on glucose uptake in those cells, together with the same number of Ad-sh-MnSOD MCF-7, lent-MnSOD MCF-7 was tested using the 2-deoxyglucose (2-DG)-based colorimetric glucose uptake assay kit (abcam), per the manufacturers\u0026rsquo; protocol. Briefly, the cells were glucose-starved for 40 minutes, stimulated with insulin for 20 minutes and exposed to 1 mM 2-DG for 20 minutes, followed by washing. The cells were lyzed, frozen/thawed and heated at 80\u0026deg;C for 40 minutes. After neutralization, the samples were reacted with the reaction mixture, neutralized and reacted with the reaction B. The levels of 2-DG6P-related NADPH in each group of samples were measured for the absorbance at 412 nm in a microplate reader. The concentrations of glucose uptake were calculated, based on the standard curve established using the standard 2-DG6P provided by the manufacturer. In addition, the levels of lactic acid in the supernatants of cultured cells were measured by a fully automated biochemical analyzer (Solarbio).\u003c/p\u003e \u003cp\u003e12. Animal studies\u003c/p\u003e \u003cp\u003eFemale Balb/c nude mice were obtained from the Laboratory Animal Center, Xi`an Jiaotong University Health Science Center and housed in a specific pathogen-free (SPF) facility in our university. The mice were randomized and implanted with 3x10\u003csup\u003e6\u003c/sup\u003e MCF-7, Ad-sh-MnSOD MCF-7 or lent MnSOD MCF-7 cells in 100 \u0026micro;l of saline into their breast fat pads. Some mice with MCF-7 cells were treated intravenously with 11.375g kg\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e d\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e QRYLD in 0.3 ml of saline daily beginning on day 5 post inoculation for 10 consecutive days. Other groups of mice received the same volume of saline injection (n\u0026thinsp;=\u0026thinsp;6 per group). The volumes of implanted tumors and body weights of mice were measured every other day for 4 weeks. The mice were euthanized and their tumor tissues were dissected, photoimaged and weighed. The tumor tissues were fixed in 10% formalin and paraffin-embedded. The tumor tissue sections (4 \u0026micro;m) were subjected to immunohistochemistry using primary antibodies against MnSOD (1:100), CaMKII (1:200), and AMPK (1:2000) (Abiowell Biotechnology), counterstained with hematoxylin, and analyzed using ImageJ software. All animal experiments were performed strictly following applicable national and institutional guidelines and were approved by the Ethics Committee of the First Affiliated Hospital of Xi'an Jiaotong University (No. 2017 Lunshenkezi No. 44, dated March 2, 2017).\u003c/p\u003e \u003cp\u003e13. Statistical analysis\u003c/p\u003e \u003cp\u003eData are shown as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SEM and the difference among groups was analyzed by one-way ANOVA and post hoc Tukey\u0026rsquo;s test. The comparison between groups was performed using Student \u003cem\u003et\u003c/em\u003e-test. All statistical analyses were conducted using GraphPad Prism 8 software (GraphPad Software Company, California, USA). The difference was thought statistically significant when a \u003cem\u003eP\u003c/em\u003e-value of \u0026lt;\u0026thinsp;0.05.\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cp\u003e1. QRYLD aqueous extracts contain many bioactive components\u003c/p\u003e \u003cp\u003eTo understand the biological functions of QRYLD aqueous extracts, we prepared aqueous extracts of 3300 g QRYLD and achieved 410 g of QRYLD aqueous extracts in powder, leading to a powder recovery rate of approximately 55%. HPLC analyses revealed that there were six main chemical compounds at high contents (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e) and they were chlorogenic acid (133.21\u0026thinsp;\u0026plusmn;\u0026thinsp;19.05 mg/g), caffeic acid (91.22\u0026thinsp;\u0026plusmn;\u0026thinsp;11.38 mg/g), quercetin (39.88\u0026thinsp;\u0026plusmn;\u0026thinsp;2.96mg/g), rutin (125.11\u0026thinsp;\u0026plusmn;\u0026thinsp;16.29 mg/g), ferulic acid (189.53\u0026thinsp;\u0026plusmn;\u0026thinsp;23.58 mg/g), and luteolin (62.39\u0026thinsp;\u0026plusmn;\u0026thinsp;8.23 mg/g). Hence, the QRYLD aqueous extracts contained several chemical compounds with high bioactivity and their interaction may contribute to the functions of QRYLD.\u003c/p\u003e \u003cp\u003e2. QRYLD targets several biological processes and signaling pathways in breast cancer cells.\u003c/p\u003e\u003cp\u003eNext, we explored whether QRYLD treatment could modulate the transcriptome profiles in MCF-7 cells. Following treatment with, or without, QRYLD-medicated serum for 48 hours, the transcriptomes in the untreated and QRYLD-treated MCF-7 cells were analyzed by RNA sequencing. The results indicated that there were 2918 DEGs, of them, 1693 genes were up-regulated while 1225 DEGs were down-regulated in the QRYLD-treated cells. It was notable that there were 293 DEGs belonging to oncogenes and tumor suppressor genes, and among them, 133 DEGs were down-regulated and 160 up-regulated in the QRYLD-treated cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA). Bioinformatic analyses predicted that these 293 DEGs were mainly involved in the metabolism-related signaling pathways or biological processes. The GO and KEGG analyses unveiled that the top 50 DEGs were directly related to the Warburg effect (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB). The connections among these targets and signal pathways predicted that these 293 DEGs were mainly involved in response to oxidative stress, cancer-related signaling pathways, glycogen synthesis and metabolism, pentose phosphate pathway (PPP), TCA cycle, glucose metabolism regulation, gluconeogenesis, glycolysis, and the MnSOD MAPK signaling (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC). Apparently, MnSOD may be a target of QRYLD in breast cancer.\u003c/p\u003e \u003cp\u003e3. QRYLD treatment limits the malignant behaviors of MCF-7 cells\u003c/p\u003e \u003cp\u003eTo understand the importance of MnSOD in the malignant behaviors of breast cancer cells, we generated stable MnSOD-silencing Ad-sh-MnSOD and MnSOD over-expressing Lent-MnSOD MCF-7 cells. Subsequently, wild-type MCF-7 cells were treated with vehicle or the LD, MD, or HD of QRYLD-medicated serum and their proliferation was tested by CCK-8 assays. The data indicated that compared with the control MCF-7 group, the proliferation activity of Ad-sh MnSOD MCF-7 cells was significantly reduced (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01), while the proliferation activity of lent MnSOD MCF-7 cells was significantly enhanced (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01). The proliferation activity of the QRYLD-treated MCF-7 cells (LD, MD, or HD of QRYLD) was significantly reduced (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05, Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA). Notably, treatment with MD of QRYLD-mediated serum achieved 50% inhibition of cell proliferation at 48 hours post treatment in MCF-7 cells. Accordingly, we used the MD of QRYLD-medicated serum for the subsequent experiments in vitro. Transwell invasion assays exhibited that the number of invaded cells in the QRYLD group was significantly reduced, relative to those in the control and MnSOD over-expression groups, but still slightly greater than those in the MnSOD silencing Ad-sh-MnSOD MCF-7 cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB). A similar pattern of wound healing ability was observed among the different groups of cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC). Thus, treatment with QRYLD, like MnSOD silencing, limited the malignant behaviors of MCF-7 cells in vitro.\u003c/p\u003e \u003cp\u003eFlow cytometric analyses revealed that the percentages of apoptotic MCF-7 cells in the QRYLD group were significantly higher than those in the control and the MnSOD over-expressing lent-MnSOD MCF-7 cells, but lower than that in the MnSOD-silencing Ad-sh-MnSOD MCF-7 cells (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01, Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eD). These indicated that QRYLD treatment, like MnSOD silencing, promoted MCF-7 cell apoptosis, which might also contribute to its ability to limit the malignant behaviors of MCF-7 cells.\u003c/p\u003e \u003cp\u003e4. QRYLD treatment inhibits glucose uptake, lactic acid production and reduces the Warburg effect in MCF-7 cells by attenuating the MnSOD/CaMKII/AMPK signaling\u003c/p\u003e\u003cp\u003eTo further understand the pharmacological action of QRYLD, we tested the impact of QRYLD treatment on glucose uptake and lactic acid production in MCF-7 cells. Following treatment with MD of QRYLD-medicated serum for 48 hours, the levels of glucose uptake in the different groups of MCF-7 cells were measured by 2-DG based colorimetric assays. The results exhibited that the levels of glucose uptake in the QRYLD-treated MCF-7 cells were significantly less than that in the control and MnSOD over-expressing MCF-7 cells, but higher than that in the MnSOD silencing MCF-7 cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA) and a similar pattern of lactic acid levels in the supernatants of cultured cells was detected in the different groups of cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB). The decreased glucose uptake and lactic acid production evidenced that QRYLD treatment reduced the Warburg effect in MCF-7 cells.\u003c/p\u003e \u003cp\u003eIt is well known that several regulators are crucial for the Warburg effect in cancers. To explore how QRYLD treatment reduced the Warburg effect in MCF-7 cells, we tested the relative levels of HIF-1α, Glut-1, and C-Myc expression in the different groups of cells by Western blot. Similarly, the relative levels of HIF-1α, Glut-1, and C-Myc expression in the QRYLD-treated MCF-7 cells were significantly lower than that in the control and MnSOD over-expressing MCF-7 cells, but higher than that in the MnSOD-silencing MCF-7 cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC). A similar pattern of HK-2, PFK-1, LDH-A and PKM-2 expression was observed in the different groups of cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eD). These data indicated that QRYLD treatment, like MnSOD silencing, inhibited the expression of these regulators and metabolic enzymes, contributing to its inhibition on the Warburg effect in MCF-7 cells.\u003c/p\u003e \u003cp\u003eGiven the importance of MnSOD in the pharmacological functions of QRYLD, we tested whether QRYLD treatment could modulate the MnSOD/CaMKII/AMPK signaling in MCF-7 cells by Western blot. As expected, the relative levels of MnSOD expression in the QRYLD-treated MCF-7 cells were significantly lower than that in the control and MnSOD over-expressing MCF-7 cells, but higher than that in the MnSOD silencing MCF-7 cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eE). These demonstrated high efficiency of MnSOD over-expression and silencing and also suggest that QRYLD may target MnSOD expression in MCF-7 cells. A similar pattern of CaMKII and AMPK expression was observed in the different groups of cells. Collectively, these data provided novel evidence to demonstrate that QRYLD treatment, like MnSOD silencing, inhibited glucose uptake and lactic acid production to reduce the Warburg effect in MCF-7 cells by reducing the expression of several regulators and metabolic enzymes and attenuating the MnSOD/CaMKII/AMPK signaling\u003c/p\u003e \u003cp\u003e5. QRYLD treatment inhibits the growth of xenograft breast tumors in mice\u003c/p\u003e \u003cp\u003eFinally, we tested the therapeutic effect of QRYLD treatment on the growth of xenograft breast cancer in mice. Following implantation with wild-type, MnSOD silencing or over-expressing MCF-7 cells, the mice with wild-type MCF-7 cells were randomized and treated intravenously with vehicle or QRYLD daily for 10 consecutive days. The dynamic growth of implanted tumors in the different groups of mice was monitored and the results revealed that compared with the control group, QRYLD treatment, like MnSOD silencing, significantly decreased the tumor volumes and weights in mice while MnSOD over-expression significantly increased the tumor volumes and weights in mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA and B). In contrast, there was no significant difference in body weights among the different groups of mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eC). Immunohistochemistry displayed that compared with the control tumors, the tumors from the QRYLD-treated mice, like the MnSOD silencing tumors, exhibited a reduced level of MnSOD, CaMKII and AMPK expression in mice while the levels of MnSOD, CaMKII and AMPK expression in the MnSOD over-expressing tumors significantly increased (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eD). Together, these data indicated that QRYLD treatment significantly inhibited the growth of implanted xenograft breast tumors in mice by attenuating the MnSOD/CaMKII/AMPK signaling.\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eThe SOD family is an important antioxidant system. Among them, MnSOD is a manganese-contained homotetramer in the mitochondrial matrix[\u003cspan additionalcitationids=\"CR7 CR8 CR9\" citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. MnSOD expression is usually up-regulated in cancer cells, and can promote the release of hydrogen peroxide in mitochondria, and up-regulate the expression of CaMKII, which can phosphorylate and activate AMPK. AMPK is an important energy receptor in cells and can enhance glucose glycolysis and the Warburg effect[\u003cspan additionalcitationids=\"CR12 CR13 CR14\" citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. The AMPK activation is also crucial for the cellular responses to stress, autophagy, survival and the growth of cancer cells[\u003cspan additionalcitationids=\"CR17 CR18 CR19\" citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. Previous studies have shown that MnSOD over-expression up-regulates HIF-1α expression to regulate the cellular redox reactions and malignant behaviors of MDA-MB-231 cells in a hypoxic condition by eliminating superoxide anion[\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e, \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e].Accordingly, the MnSOD is a potent antioxidant to support the survival of cells under a hypoxic condition. MnSOD can shift oxidative phosphorylation into glycolysis in human triple negative breast cancer cells by transmitting the glucose signal and changing the oxygen transmission pathway[\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e, \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. In this study, we found that MnSOD over-expression enhanced glycolysis in MCF-7 cells by enhancing CaMKII and AMPK expression, and promoted the malignant behaviors, such as proliferation, migration, wound healing and invasion of MCF-7 cells. Conversely, MnSOD silencing dramatically decreased glucose uptake, lactic acid production, Glut-1 expression, the hallmarks of reduced levels of glycolysis and MnSOD silencing MCF-7 cells displayed less malignant aggressiveness in vitro, consistent with previous findings[\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. Our data were in disagreement with a previous observation that MnSOD deficiency does not significantly alter the levels of glycolysis in breast cancer cells[\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. The difference may stem from different breast cancer cell lines with varying metabolic state. Actually, MnSOD silencing promoted the occurrence of oxidative phosphorylation[\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e] In breast cancer cells, MnSOD may maintain a stable increase in hydrogen peroxide contents, thus maintaining the activation of AMPK and the transformation of intracellular metabolic mode into glycolysis while MnSOD silencing or inhibiting the activation of AMPK can reduce the growth vitality of cells[\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. Hence, the MnSOD/CaMKII/AMPK axis may be crucial for supporting the bioenergetics of cancer cells, and can promote the progression of breast cancer[\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eIn our experiment, we found that MnSOD silencing also decreased the levels of CaMKII and AMPK expression in MCF-7 cells. Furthermore, MnSOD silencing also significantly reduced the expression levels of HK-2, PFK-1, LDH-A and PKM-2, the key metabolic enzymes in the Warburg effect process in MCF-7 cells. As a result, the MnSOD silenced MCF-7 cells exhibited less malignant aggressiveness in vitro and in vivo, which were associated with decreased levels of CaMKII and AMPK expression. In contrast, MnSOD over-expression also enhanced the expression of CaMKII, AMPK, key metabolic enzymes and critical regulators, such as HIF-1α, Glut-1and c-myc, in MCF7 cells, accompanied by enhanced malignant aggressiveness in vitro and in vivo. These paralleling data demonstrated that the MnSOD/CaMKII/AMPK axis was the key factor to regulate the progression of breast cancer and suggested that this axis may be served as a therapeutic target for developing therapies for the intervention of breast cancer.\u003c/p\u003e \u003cp\u003eHow did the QRYLD modulate the malignant behaviors of MCF-7 cells? We speculate that QRYLD treatment may attenuate the MnSOD/CaMKII/AMPK signaling to inhibit the Warburg effect and malignant behaviors of MCF-7 cells. Evidently, RNAseq analysis indicated that QRYLD treatment modulated the expression of many genes, including down-regulated MnSOD in MCF-7 cells. Bioinformatic analysis unveiled that the DEGs mainly participated in the metabolism-related signaling pathways, such as the MnSOD/MAPK signaling pathway, and biological processes, including the Warburg effect. Furthermore, QRYLD treatment significantly inhibited the malignant behaviors, such as the proliferation, migration and invasion of MCF-7 cells as well as the tumor growth in vivo. In addition, QRYLD treatment also decreased the expression levels of MnSOD, CaMKII and AMPK, the key metabolic enzymes in the Warburg effect process and critical regulators of HIF-1α, Glut-1and c-myc in MCF-7 cells as well as in the grown tumors in vivo. Interestingly, the therapeutic effects of QRYLD treatment were similar to that of MnSOD silencing in MCF-7 cells although they were less effective than the MnSOD silencing did. These data support the notion that QRYLD may suppress the expression of MnSOD and attenuate the MnSOD/CaMKII/AMPK signaling to inhibit the Warburg effect and malignant behaviors of MCF-7 cells. Our novel findings may provide new pharmacological mechanisms by which QRYLD treatment inhibits the malignancy of breast cancer(Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eIn conclusion, our data indicated that the QRYLD contained several bioactive components and treatment with QRYLD significantly modulated the expression of many genes, including metabolism-related MnSOD. QRYLD treatment, like MnSOD silencing, significantly suppressed the malignant behaviors and the Warburg effect in MCF-7 cells as well as tumor growth in vivo, associated with inhibition of the MnSOD/CaMKII/AMPK signaling. Our findings may provide new insights into the pharmacological actions of QRYLD in inhibiting breast cancer growth and unveil new therapeutic targets for designing therapies for the intervention of breast cancer.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eQRYLD\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eQing-Re-Yi-Liu decoction\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eMnSOD\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eManganese superoxide dismutase\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eCaMKII\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eCalmodulin dependent kinase II\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eAMPK\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eAMP-activated protein kinase\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eGlut-1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eGlucose transporter-1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eHIF-1\u0026alpha;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eHypoxia-inducible factor 1\u0026alpha;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eHK-2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eHexokinase-2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003ePFK-1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003ePhosphofructokinase-1\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eLDH-A\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eLactate Dehydrogenase-A\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003ePKM-2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003ePyruvate kinase isozyme-2\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThis work was funded by grants from the National Natural Science Foundation of China (no.\u0026nbsp;81703899),\u0026nbsp;Shaanxi Provincial Natural Science Basic Research Program (no.\u0026nbsp;2017JM8068),\u0026nbsp;Shaanxi Provincial Administration of Traditional Chinese Medicine Project(no.15-JC021),\u0026nbsp;Xi'an Jiaotong University First Affiliated Hospital General Project(no.\u0026nbsp;2021ZXY-06),\u0026nbsp;The second batch of provincial-level Chinese medicine young and middle-aged scientific and technological backbone projects (no.\u0026nbsp;2023-ZQNY-008).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflicts of interest\u003c/strong\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe authors declare no conflicts of interest regarding the publication of this paper.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eLianqing Sun\u0026nbsp;provided the plant materials.\u0026nbsp;Xinhan Zhao\u0026nbsp;performed screening of extracts.\u0026nbsp;Junxia Wang\u0026nbsp;performed phytochemical analysis.Jing Du\u0026nbsp;performed the vivo and vitro experiments.\u0026nbsp;Ying Zhang\u0026nbsp;and\u0026nbsp;Ying Yuan\u0026nbsp;conceived and supervised the study.\u0026nbsp;Zhe Zhang\u0026nbsp;drafted the manuscript.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eAll authors read and approved the final manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of Data and Materials\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe data included in this investigation are available from the corresponding author.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics Approval and Consent to Participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll experimental protocols were implemented following the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health (NIH Publication No. 85-23, revised 1996). The materials and data are carried out in accordance with the Declaration of Helsinki. The study was approved by the Ethics Committee of the First Affiliated Hospital of Xi'an Jiaotong University (No. 2017 Lunshenkezi No. 44, dated March 2, 2017).\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eYu P., Li JY., Luo YQ., Sun JY., Hu YF.,Lin B.,Meng XL., Xiang L., Mechanistic role of \u003cem\u003eScutellaria baicalensis georgi\u003c/em\u003e in breast cancer therapy, Am.J.Chin.Med. 51 (2023) 279-308. https://doi.org/10.1142/S0192415X23500155.\u003c/li\u003e\n\u003cli\u003eSholihah MM.,Indarto D.,Pramana TY., The inhibitory effect of \u003cem\u003eAndrographis paniculata\u003c/em\u003e extract on proliferation of breast cancer cell line, Iop. Con. 546(2019) 20-29.\u003c/li\u003e\n\u003cli\u003eWang B., Xu SY., Liu JX., Li YF.,Xu HS., Han B.,Zhang J., Li XG., Ye XL., Total alkaloids of \u003cem\u003eCoptidis rhizoma\u003c/em\u003e combined with exercise inhibits tumor growth of orthotopically transplanted 4T1 breast cancer mice by blocking cell cycle G_1/S transformation, Chin.J.Chin.Med. 44(2019) 1635-1641.\u003c/li\u003e\n\u003cli\u003eLiu ZY.,Tang Y., Zhou RR., Shi XS., Zhang HM., Liu TF., Lian ZL., Shi XY., Bi-directional solid fermentation products of trametes robiniophila murr with \u003cem\u003eRadix isatidis\u003c/em\u003e inhibit proliferation and metastasis of breast cancer cells, J.Chin.Med.Assoc. 81(2018) 520-530.\u003c/li\u003e\n\u003cli\u003eZou XJ., Hao YY., Hu YD., Xie SX., Bai W., \u003cem\u003ePulsatilla saponinA\u003c/em\u003e affects proliferation and radiosensitivity of breast cancer cells by regulating miR-24-3p/RNF2 expression, Chin.J.Path. 36(2020) 865-870.\u003c/li\u003e\n\u003cli\u003eKim NY., Ha IJ., Um JY., Kumar AP., Sethi G., Ahn KS., Loganic acid regulates the transition between epithelial and mesenchymal-like phenotypes by alleviating MnSOD expression in hepatocellular carcinoma cells, Life.Sci. 317(2023)1-12. https://doi.org/10.1016/j.lfs.2023.121458.\u003c/li\u003e\n\u003cli\u003eValentino RB., The structure-function relationships and physiological roles of MnSOD mutants, Bios.Rep. 42(2022) 1-12. https://doi.org/10.1042/BSR20220202.\u003c/li\u003e\n\u003cli\u003eAl Haq AT., Tseng HY., Chen LM., Wang CC., Hsu HL., Targeting prooxidant MnSOD effect inhibits triple-negative breast cancer (TNBC) progression and M2 macrophage functions under the oncogenic stress, Cell.Death Dis.13(2022) 1-13.\u003c/li\u003e\n\u003cli\u003eLiu SS., Hu JQ., Gu JF., Ni AM., Tang WH., Liu XY., Combined oncolytic adenovirus carrying MnSOD and mK5 genes both regulated by survivin promoter has a synergistic inhibitory effect on gastric cancer,Neoplasma. 69(2022) 36-48.\u003c/li\u003e\n\u003cli\u003eGius D.,.Lysine 68 acetylation directs MnSOD as a tetrameric detoxification complex versus a monomeric tumor promoter, Clin.Cancer.Res. 27(2021) 1-9.\u003c/li\u003e\n\u003cli\u003eQuiros-Gonzalez I., Gonzalez-Menendez P., Alcon-Rodriguez S., Hevia, D. Sainz, RM.,Mayo JC., Androgen-dependent prostate cancer cells reprogram their metabolic signature upon Glut-1 upregulation by manganese superoxide dismutase,Free.Radi.Bio.Med. 165(2021) 1-12. https://doi.org/10.1016/j.freeradbiomed.2020.12.419.\u003c/li\u003e\n\u003cli\u003eGao YC., Zhu YM., Tran EL., Tokars V., Dean AE., Quan SH., Gius D., MnSOD Lysine 68 acetylation leads to cisplatin and doxorubicin resistance due to aberrant mitochondrial metabolism, Int.J.Biol.Sci. 17(2021) 1203-1216.\u003c/li\u003e\n\u003cli\u003eFu ZM., Cao XC., Liu LH., Cao XZ., Cui YH., Li X., Quan MF., Ren KQ., Chen A.,Xu C., Qiu YB., Chen XD., Wang Z., Cao JG., Genistein inhibits lung cancer cell stem-like characteristics by modulating MnSOD and FoxM1 expression, Oncol.Lett. 20(2020) 2506-2515. https://doi.org/10.3892/ol.2020.11802\u003c/li\u003e\n\u003cli\u003eTorrens-Mas M., Cordani M., Mullappilly N., Pacchiana R., Riganti C.,Palmieri M., Pons DG., Roca P., Oliver J., Donadelli M., Mutant p53 induces SIRT3/MnSOD axis to moderate ROS production in melanoma cells, Arch.Bio.Bio. 679(2020) 1-10. https://doi.org/10.1016/j.abb.2019.108219.\u003c/li\u003e\n\u003cli\u003eLi QY., Sun Y., Liu BW., Li JB., Hao X., Ge WZ., Zhang XM., Bao SQ., Gong JM., Jiang ZH ., Qiu CJ., Zhao LQ., Zhao YP., Chen Y., Yang XJ., Ding YH., Wu ZZ., ACT001 modulates the NF-kappa B/MnSOD/ROS axis by targeting IKK beta to inhibit glioblastoma cell growth, J.Mol.Med. 98(2020) 263-277.\u003c/li\u003e\n\u003cli\u003eCao XC., Liu LH., Yuan Q., Li X., Cui YH., Ren KQ., Zou C., Chen A., Xu C., Qiu YB., Quan MF., Zhang JS., Cao JG., Chen XD., Isovitexin reduces carcinogenicity and stemness in hepatic carcinoma stem-like cells by modulating MnSOD and FoxM1, J.Exp.Clin.Cancer.Res. 38(2019) 1-18. https://doi.org/10.1186/s13046-019-1244-6.\u003c/li\u003e\n\u003cli\u003eZhu YM., Zou XH., Dean AE., O\u0026apos; Brien J., Gao YC., Tran EL., Park SH., Liu GX., Kieffer MB ., Jiang HY., Stauffer ME., Hart R., Quan SH., Satchell KJF., Horikoshi N., Bonini M., Gius D., Lysine 68 acetylation directs MnSOD as a tetrameric detoxification complex versus a monomeric tumor promoter, Nat.Commun. 10(2019) 1-15. https://doi.org/10.1038/s41467-019-10352-4.\u003c/li\u003e\n\u003cli\u003eFan JJ., Hsu WH., Hung HH., Zhang WJ., Lee YLA., Chen KC., Chu CY., Ko TP., Lee MT., Lin CW., Cheng CH., Reduction in MnSOD promotes the migration and invasion of squamous carcinoma cells, Int.J.Oncol. 54(2019) 1639-1650. https://doi.org/10.3892/ijo.2019.4750.\u003c/li\u003e\n\u003cli\u003eMa SJ., Fu A., Lim S., Chiew GGY., Luo KQ., MnSOD mediates shear stress-promoted tumor cell migration and adhesion, Free.Radi.Bio.Med. 129(2018) 46-58. https://doi.org/10.1016/j.freeradbiomed.2018.09.004.\u003c/li\u003e\n\u003cli\u003eLiu F., Yuan Q., Cao XC., Zhang JL., Cao JG., Zhang JS., Xia LQ., Isovitexin suppresses stemness of lung cancer stem-Like cells through blockage of MnSOD/CaMKII/AMPK Signaling and glycolysis inhibition, Biomed.Res.Int. 21(2021) 1-15. https://doi.org/10.1155/2021/9972057.\u003c/li\u003e\n\u003cli\u003eJetawattana S., Venkataraman S., Domann F., Oberley L., A role for MnSOD and p53 in regulating HIF-1 alpha in breast cancer cell lines, Free.Radi.Bio.Med. 39(2005) 166-176.\u003c/li\u003e\n\u003cli\u003eJetawattana S., Venkataraman, S., Teoh MLT., Oberle, LW., Redox regulation of HIF-1 alpha by MnSOD via ROS, Free.Radi.Bio.Med. 41(2006) 110-119.\u003c/li\u003e\n\u003cli\u003eHe CX., Hart P., Fricano K., Vargas M., Thieraud K., de Abreu AL., Bonini M., Mangenese superoxide dismutase (MnSOD) promotes stem-like cell phenotypes in breast cancer, Faseb.J. 31(2017) 809-815.\u003c/li\u003e\n\u003cli\u003eAl Haq AT., Tseng HY., Chen LM., Wang CC., Hsu HL., Targeting prooxidant MnSOD effect inhibits triple-negative breast cancer (TNBC) progression and M2 macrophage functions under the oncogenic stress, Cell.Death.Dis. 13(2022) 1-15. \u003c/li\u003e\n\u003cli\u003eHart PC., Fricano KA., Minshall R., Bonini M., Loss of caveolin-1 results in a MnSOD-dependent glycolytic shift in breast cancer, Free.Radi.Bio.Med. 53(2012) 43-53. https://doi.org/10.1016/j.freeradbiomed.2012.10.112.\u003c/li\u003e\n\u003cli\u003eAmbrosone CB., Choi JY., Barlow W., Yeh IT., Hayes DF., Davis W., Rae JM., Nowak N., Conroy J., Li Y., Den Haese J., Lehman T., Hutchins LF., Livingston R., Ravdin P., Albain K., Pharmacogenetics and breast cancer treatment outcomes: results on oxidative stress related genotypes (MPO, MnSOD) from a southwest oncology group trial (S8897), Int.J.Biol.Marker. 22(2007) 61-69.\u003c/li\u003e\n\u003cli\u003eWeydert CJ., Zhang Y., Sun W., Waugh TA., Teoh MLT., Andriinga KK., Aykin-Burns N., Spitz DR., Smith BJ., Oberley LW., Increased oxidative stress created by adenoviral MnSOD or CuZnSOD plus BCNU (1,3-bis(2-chloroethyl)-1-nitrosourea) inhibits breast cancer cell growth, Free.Radi.Bio.Med. 44(2008) 856-867. https://doi.org/10.1016/j.freeradbiomed.2007.11.009\u003c/li\u003e\n\u003cli\u003eQuiros-Gonzalez I., Gonzalez-Menendez P., Alcon-Rodriguez S., Hevia D., Sainz RM., Mayo JC., Androgen-dependent prostate cancer cells reprogram their metabolic signature upon Glut-1 upregulation by manganese superoxide dismutase (Mnsod/SOD2), Free.Radi.Bio.Med. 165(2021) 97-107. https://doi.org/10.1016/j.freeradbiomed.2020.12.419\u003c/li\u003e\n\u003cli\u003eHart PC., Mao M., de Abreu ALP., Ansenberger-Fricano K., Ekoue DN., Ganini D., Kajdacsy-Balla A., Diamond AM., Minshall RD., Consolaro MEL., Santos JH., Bonini MG., MnSOD upregulation sustains the Warburg effect via mitochondrial ROS and AMPK-dependent signalling in cancer, Nat.Commun. 6(2015) 1-14.\u003c/li\u003e\n\u003cli\u003eAnsenberger-Fricano K., Mao M., Green PD., Hall A., Kajdacsy-Balla A., Santos JA., Bonini MG., MnSOD regulation of AMPK-Mediated transition to glycolysis in breast cancer, Free.Radi.Bio.Med. 53(2012) 39-49. https://doi.org/10.1016/j.freeradbiomed.2012.10.100.\u003c/li\u003e\n\u003cli\u003eYe DY., Sun J., Li YQ., Evaluation of toxicity, bacteriostatic, analgesic, anti-inflammatory, and antipyretic activities of \u003cem\u003eHuangqin-Honghua-Pugongying-Jinyinhua\u003c/em\u003e extract, VET,SCI.8(2021) 1-12.\u003c/li\u003e\n\u003cli\u003eMin F., Xiao Z., Huaping S.,Yakong Z., Dandelion (\u003cem\u003eTaraxacum genus\u003c/em\u003e): A review of chemical constituents and pharmacological effects, Mol. 28(2023) 1-31.https://doi.org/10.3390/molecules28135022.\u003c/li\u003e\n\u003cli\u003eZhang Y., Hu YF., Li W., Xu GY., Wang KR., Li L., Luo H., Zou L., Wu JS., Updates and advances on pharmacological properties of \u003cem\u003eTaraxacum mongolicum\u003c/em\u003e Hand.-Mazz and its potential applications, Food Chem. 373(2022) 131-143. https://doi.org/10.1016/j.foodchem.2021.131380.\u003c/li\u003e\n\u003cli\u003eMehdi Sharifi-Rad,Thomas H. Roberts, Karl R. Matthews, Camila F. Bezerra, Maria Flaviana B. Morais-Braga, Henrique D.M. Coutinho, Farukh Sharopov, Bahare Salehi, Zubaida Yousaf, Majid Sharifi-Rad, Mar\u0026iacute;a del Mar Contreras, Elena Maria Varoni, Deepa R. Verma, Marcello Iriti, Javad Sharifi-Rad, Ethnobotany of the genus \u003cem\u003eTaraxacum\u003c/em\u003e\u0026mdash;phytochemicals and antimicrobial activity, Phy.Res. 32(2018) 2131-2145. https://doi.org/10.1002/ptr.6157.\u003c/li\u003e\n\u003cli\u003eWang Y., Tang J., Sun P., Lin S., Zhang X.,.Effects of \u003cem\u003eIphigenia indica\u003c/em\u003e extract on proliferation and apoptosis of human colorectal cancer cell line SW480, Chin.J.Basic.Med. 27(2021) 1754-1758.\u003c/li\u003e\n\u003cli\u003eTang RP., Yang YH., Su HL., Huang GB.,Study on pollen viability of \u003cem\u003eIphigenia indica\u003c/em\u003e. Southwest.Chin.J.Agr.Sci. 22(2009) 153-155.\u003c/li\u003e\n\u003cli\u003eMukhopadhyay MJ, Mukhopadhyay S.,High frequency in vitro propagation through somatic embryogenesis of \u003cem\u003eIphigenia indica\u003c/em\u003e \u003cem\u003ekunth et benth\u003c/em\u003e, an endangered medicinal and colchicines yielding herb of commercial interest, Cytologia. 73(2008) 97-103.\u003c/li\u003e\n\u003cli\u003eJun L., Liyao L., Zhen J., Jing C., Zuguo Z., Yi Z., Zhujin X., Zhen G., Antibacterial potential of \u003cem\u003eForsythia suspensa\u003c/em\u003e polysaccharide against resistant enterobacter cloacae with SHV-12 extended-spectrum \u0026beta;-lactamase, J.Cell.Mol.Med. 24(2020) 8763-8771. https://doi.org/10.1111/jcmm.15510.\u003c/li\u003e\n\u003cli\u003eZhao L., Li W., Dai SJ., Liu RX., Xie ZP., Zhang SM., Yue XD., Alkaloids bearing rare skeletons from \u003cem\u003eForsythia suspensa\u003c/em\u003e with anti-inflammatory and anti-viral activities in vitro, Phytochem. 186(2021) 1-12. https://doi.org/10.1016/j.phytochem.2021.112739.\u003c/li\u003e\n\u003cli\u003eShao SY., Zhang F., Yang YN., Feng ZM., Jiang JS., Zhang PC., Neuroprotective and anti-inflammatory phenylethanoid glycosides from the fruits of \u003cem\u003eForsythia suspense\u003c/em\u003e, Bioorg,Chem. 113(2021) 1-15. https://doi.org/10.1016/j.bioorg.2021.105025.\u003c/li\u003e\n\u003cli\u003eLi W., Sun LT., Zhao L., Yue XD., Dai SJ., New C-9-monoterpenoid alkaloids featuring a rare skeleton with anti-inflammatory and antiviral activities from \u003cem\u003eForsythia suspense\u003c/em\u003e, Chem. Biodivers. 19(2022) 1-7.\u003c/li\u003e\n\u003cli\u003eWang R., Wang XC., Xia MQ., Yang LX., Cheng WK., Song QQ.,Combining network pharmacology with chromatographic fingerprinting and multicomponent quantitative analysis for the quality evaluation of \u003cem\u003eMoutan cortex\u003c/em\u003e, Bio.Chr. 36(2022) 1-12.https://doi.org/10.1002/bmc.5434\u003c/li\u003e\n\u003cli\u003eYang SC., Liu XY., He JY., Liu MH., Insight into seasonal change of phytochemicals, antioxidant, and anti-aging activities of root bark of \u003cem\u003epaeonia suffruticosa (Cortex moutan)\u003c/em\u003e combined with multivariate statistical analysis, Mol. 26(2021) 1-19.https://doi.org/10.3390/molecules26206102.\u003c/li\u003e\n\u003cli\u003eZhang YC., Wu XY., Wang XH., Zeng Y., Liao YX., Zhang RZ., Zhai FQ., Zeng ZL., Grey relational analysis combined with network pharmacology to identify antioxidant components and uncover its mechanism from \u003cem\u003eMoutan cortex\u003c/em\u003e, Front Pharmacol. 12(2021) 1-14. https://doi.org/10.3389/fphar.2021.748501\u003c/li\u003e\n\u003cli\u003eZhou SJ., Lin HJ., Meng J., Discrimination and chemical composition quantitative model of Raw \u003cem\u003eMoutan cortex\u003c/em\u003e and \u003cem\u003eMoutan cortex\u003c/em\u003e carbon based on electronic nose, Math.Biosci.Eng. 19(2022) 9079-9097.\u003c/li\u003e\n\u003cli\u003eWu ZH., Yin B., You FM., Molecular mechanism of anti-colorectal cancer effect of \u003cem\u003eHedyotis diffusa\u003c/em\u003e willd and its extracts, Front Pharmacol. 13(2022) 1-10.https://doi.org/10.3389/fphar.2022.820474\u003c/li\u003e\n\u003cli\u003eLu L.Effect of \u003cem\u003eHedyotis diffusa\u003c/em\u003e Polysaccharide on Apoptosis of Thyroid Cancer Cells, Pak.J.Zool. 54(2022) 1803-1810. https://dx.doi.org/10.17582/journal.pjz/20201101141148.\u003c/li\u003e\n\u003cli\u003eDong HP., Zhang Q., \u003cem\u003eHedyotis diffusa\u003c/em\u003e willd extract protects rat renal tubular epithelial cells from high glucose-induced injury by inhibiting PI3K/AKT signaling pathway, Scie. 48(2022) 532-537.\u003c/li\u003e\n\u003cli\u003eXu L, Li Y., Ji JJ., Lai YH., Chen J., Ding T., Li HC., Ding B., Ge WH., The anti-inflammatory effects of \u003cem\u003eHedyotis diffusa\u003c/em\u003e willd on SLE with STAT3 as a key target, J. Ethnopharmacol. 298(2022) 1-14. https://doi.org/10.1016/j.jep.2022.115597.\u003c/li\u003e\n\u003cli\u003eYang R., Lu Y., Hao H., Zhang M., Xuan J., Zhang YQ., Research progress on chemical constituents and pharmacological activities of iridoid glycosides in \u003cem\u003eLonicera japonica\u003c/em\u003e, Chin.J.Chin. 46(2021) 2746-2752.\u003c/li\u003e\n\u003cli\u003eJiang HF., Chen C., Jiang XY., Shen JL., Ling F., Li PF., Wang GX., Wang GX., \u003cem\u003eLuteolin\u003c/em\u003e in \u003cem\u003eLonicera japonica\u003c/em\u003e inhibits the proliferation of white spot syndrome virus in the crayfish procambarus clarkia, Aquaculture. 550(2022) 1-10. https://doi.org/10.1016/j.aquaculture.2021.737852.\u003c/li\u003e\n\u003cli\u003eXu N., Du LH., Chen YC., Zhang JH., Zhu QF., Chen R., Peng GP., Wang QM., Yu HZ., Rao LQ., \u003cem\u003eLonicera japonica\u003c/em\u003e thunb as a promising antibacterial agent for bacillus cereus ATCC14579 based on network pharmacology, metabolomics, and in vitro experiments, Rsc.Adv. 13(2023) 15379-15390. https://doi.org/10.1039/D3RA00802A.\u003c/li\u003e\n\u003cli\u003eYang XP., Yu AQ., Hu WJ., Zhang ZJ., Ruan Y., Kuang HX., Wang M., Extraction, purification, structural characteristics, health benefits, and application of the polysaccharides from \u003cem\u003eLonicera japonica\u003c/em\u003e thumb, A.Rev.Mol. 28(2023) 1-11. https://doi.org/10.3390/molecules28124828.\u003c/li\u003e\n\u003cli\u003eDar Khursheed Ahmad, Senthilmurugan S, Venkatesan A, Antibacterial, antioxidant potential and functional group analysis of kashmir grown \u003cem\u003ePrunella vulgaris L.\u003c/em\u003e root extract, Int.J.Bot.Stu. 7(2022) 19-25.\u003c/li\u003e\n\u003cli\u003eBai HT., Wang R., Li YL., Liang X., Zhang JH., Sun N., Yang J., Network pharmacology analysis, molecular docking, and in vitro verification reveal the action mechanism of \u003cem\u003ePrunella vulgaris L\u003c/em\u003e. in treating breast vancer, Evid-Based.Compl.Alt. 12(2022) 1-19.https://doi.org/10.1155/2022/5481563\u003c/li\u003e\n\u003cli\u003eDar KA., Senthilmurugan S., Ali S., Al-Sadoon MK., Paray BA., Studies on the isolation and identification of the antibacterial compound from \u003cem\u003ePrunella vulgaris L\u003c/em\u003e. flower extract, J.King.Saud.Univ.Sci. 34(2022) 1-6. https://doi.org/10.1016/j.jksus.2022.102324.\u003c/li\u003e\n\u003cli\u003eKim J., Lee CG., Hwang S., Yun SH., Uprety LP., Oh KI., Singh S., Yoo J., Jeong H., Yong Y ., Anti-osteoarthritic effects of \u003cem\u003ePrunella Vulgaris\u003c/em\u003e and \u003cem\u003eGentiana lutea\u003c/em\u003e in vitro and in vivo, Antioxidants. 12(2023) 47-57. https://doi.org/10.3390/antiox12010047.\u003c/li\u003e\n\u003cli\u003eChen HX., Olatunji OJ., Zhou YF., Anti-oxidative, anti-secretory and anti-inflammatory activities of the extract from the root bark of \u003cem\u003eLycium chinense\u003c/em\u003e (\u003cem\u003eCortex Lycii\u003c/em\u003e) against gastric ulcer in mice, J.Nat.Med-Tokyo. 70(2016) 610-619.\u003c/li\u003e\n\u003cli\u003eGuo ZH., Yin H., Wu T., Wu SF., Liu LY., Zhang L., He YL., Zhang R., Liu N., Study on the mechanism of \u003cem\u003eCortex Lycii\u003c/em\u003e on lung cancer based on network pharmacology combined with experimental validation, J.Ethno. 293(2022) 1-10. https://doi.org/10.1016/j.jep.2022.115280.\u003c/li\u003e\n\u003cli\u003eZhao Sl., Yuan Z., Zhu YR., Ding HQ., Zheng XJ., Zhang JZ., Liu DL., Mechanism of hypolipidemic effect of \u003cem\u003eLycii cortex\u003c/em\u003e based on network pharmacology and molecular docking, Chin. Trad.Drugs. 54(2023) 1487-1497.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"breast-cancer-research-and-treatment","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"brea","sideBox":"Learn more about [Breast Cancer Research and Treatment](https://www.springer.com/journal/10549)","snPcode":"10549","submissionUrl":"https://submission.nature.com/new-submission/10549/3","title":"Breast Cancer Research and Treatment","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"QRYLD, MnSOD, malignant behaviors, Warburg effect, CaMKII/AMPK signaling","lastPublishedDoi":"10.21203/rs.3.rs-3366517/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-3366517/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eBackground\u003c/h2\u003e \u003cp\u003eThe Qing-Re-Yi-Liu decoction (QRYLD) is a clinical effective prescription for the treatment of breast cancer due to its activity of heat clearing and detoxification. Our preliminary studies have found that QRYLD can interfere with the Warburg effect of breast cancer cells, inhibiting the proliferation of breast cancer MCF-7 cells.The chemical components and molecular mechanisms underlying the actions of QRYLD in regulating the Warburg effect in breast cancer cells are still unclear.\u003c/p\u003e\u003ch2\u003eMethods\u003c/h2\u003e \u003cp\u003eThe bioactive components of QRYLD aqueous extracts were detected by HPLC. The biological processes and signaling pathways in MCF-7 cells of QRYLD targets were measured with transcriptome analysis. The effect of QRYLD on the malignant behaviors of MCF-7 cells were analyzed by CCK-8 assay,transwell invasion assay, wound healing assay, apoptosis detection. The effect of QRYLD on glucose uptake, lactic acid production and Warburg effect in MCF-7 cells assessed by colorimetry and western blotting. The volumes of xenograft breast tumors and body weights of mice were measured, and the effect of QRYLD on the tumor tissues was assessed with immunohistochemistry.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e \u003cp\u003eHere, we show that the QRYLD aqueous extracts contain several bioactive components. Analysis of transcriptomes indicated that QRYLD treatment altered the expression of many genes, such as manganese superoxide dismutase (MnSOD), that were involved in biological processes and signaling pathways, particularly for glucose metabolism in MCF-7 cells. Functionally, QRYLD treatment, like MnSOD silencing, inhibited the malignant behaviors of MCF-7 and enhanced their apoptosis while MnSOD over-expression had opposite effects. Furthermore, QRYLD treatment, like MnSOD silencing, limited glucose uptake and lactic acid production in MCF-7 cells, which were associated with a decrease in the relative levels of Glut-1, HIF-1α, c-Myc, HK-2, PFK-1, LDH-A, PKM-2, MnSOD, calmodulin dependent kinase II (CaMKII) and AMPK expression. Finally, treatment with QRYLD, like MnSOD silencing, significantly mitigated the growth of xenograft MCF-7 tumors in mice and reduced the expression of MnSOD, CaMkII and AMPK expression in the tumors.\u003c/p\u003e\u003ch2\u003eConclusion\u003c/h2\u003e \u003cp\u003eThese data suggest that QRYLD may target MnSOD to attenuate the MnSOD/CaMKII/AMPK signaling, leading to inhibition of the Warburg effect and malignant behaviors in MCF-7 cells. These findings may provide new insights into the pharmacological mechanisms underlying the actions of QRYLD in inhibiting the Warburg effect and malignant behaviors of breast cancer cells.\u003c/p\u003e","manuscriptTitle":"Qing-Re-Yi-Liu decoction suppresses the malignant behaviors of breast cancer cells by attenuating the MnSOD/CaMKII/AMPK signaling and Warburg effect","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-04-15 05:32:45","doi":"10.21203/rs.3.rs-3366517/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Reject Low Priority","date":"2024-05-22T12:21:15+00:00","index":"","fulltext":""},{"type":"reviewersInvited","content":"","date":"2024-04-10T18:07:21+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"Breast Cancer Research and Treatment","date":"2023-09-21T11:41:02+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2023-09-20T14:25:03+00:00","index":"","fulltext":""},{"type":"submitted","content":"Breast Cancer Research and Treatment","date":"2023-09-20T09:54:28+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"breast-cancer-research-and-treatment","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"brea","sideBox":"Learn more about [Breast Cancer Research and Treatment](https://www.springer.com/journal/10549)","snPcode":"10549","submissionUrl":"https://submission.nature.com/new-submission/10549/3","title":"Breast Cancer Research and Treatment","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"aacf3ffd-9e96-4976-b7a8-5834a8bb6542","owner":[],"postedDate":"April 15th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[],"tags":[],"updatedAt":"2024-07-17T05:01:48+00:00","versionOfRecord":[],"versionCreatedAt":"2024-04-15 05:32:45","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-3366517","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-3366517","identity":"rs-3366517","version":["v1"]},"buildId":"zQwnuV7TCBrMSSSToR1PI","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}