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| ORIGINAL ARTICLE |
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| Year : 2023 | Volume
: 9
| Issue : 3 | Page : 284-296 |
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The mechanism of total ginseng extracts in the treatment of lung cancer progression based on network pharmacology and experimental validation
Hong-Kuan Hana1, Cheng Qian1, Meng-Yao Song1, Teng Zhang1, Chun-Mei Yang2, Ren-Jun Gu3, Xian Zhou4, Zhong-Hong Wei1, Yang Zhao2, Yin Lu1
1 Jiangsu Key Laboratory for Pharmacology and Safety Evaluation of Chinese Materia Medica, School of Pharmacy; Jiangsu Joint International Research Laboratory of Chinese Medicine and Regenerative Medicine, Nanjing University of Chinese Medicine, Nanjing, China
2 Jiangsu Key Laboratory for Pharmacology and Safety Evaluation of Chinese Materia Medica, School of Pharmacy; Department of Biochemistry and Molecular Biology, School of Medicine and Holistic Integrative Medicine, Nanjing University of Chinese Medicine, Nanjing, China
3 School of Chinese Medicine, School of Integrated Chinese and Western Medicine, Nanjing University of Chinese Medicine, Nanjing, China
4 NICM Health Research Institute, Western Sydney University, Westmead, NSW, Australia
| Date of Submission | 27-Feb-2023 |
| Date of Acceptance | 17-May-2023 |
| Date of Web Publication | 11-Sep-2023 |
Correspondence Address:
Prof. Yang Zhao
School of Medicine and Holistic Integrative Medicine, Nanjing University of Chinese Medicine, 138 Xianlin Avenue, Nanjing 210023
China
Prof. Yin Lu
School of Pharmacy, Nanjing University of Chinese Medicine, 138 Xianlin Avenue, Nanjing 210023
China
Dr. Zhong-Hong Wei
School of Pharmacy, Nanjing University of Chinese Medicine, 138 Xianlin Avenue, Nanjing 210023
China
 Source of Support: None, Conflict of Interest: None
DOI: 10.4103/2311-8571.385513
Objective: The objective of this study was to investigate the mechanism by which total ginseng extract (TGE) inhibits the progression of lung cancer through network pharmacology and experimentation. Materials and Methods: A Lewis lung carcinomas (LLC) model was established by injecting cancer cells through the tail vein and through administering different doses of TGE. The infiltrated immune cells into the microenvironment of lung cancer metastasis through flow cytometry were evaluated, and the messenger RNA (mRNA) expression levels of various immune cell-related chemokines were determined using quantitative reverse transcription–polymerase chain reaction. Therapeutic targets and signaling pathways of TGE in nonsmall cell lung cancer were investigated using systematic pharmacology and virtual docking. Immunoblotting was performed to determine the impacts of TGE on migration-related proteins. Results: Flow cytometry showed that 1.82 g/kg TGE increased the infiltrated T cells and inhibited the recruitment of myeloid cells, which was caused by decreased mRNA expression of chemokines after TGE treatment. Gene ontology and Kyoto Encyclopedia of Genes and Genomes analyses showed that the delayed progression of lung cancer by TGE might be related to the promotion of lung cancer cell apoptosis-associated signaling pathways. The virtual docking results indicated that the active components of ginseng are directly bound to apoptosis-related proteins. Immunoblotting showed that TGE inhibited tumor metastasis by regulating the expression of migration-related proteins. Conclusions: The study reveals the potential mechanism of ginseng extract in the treatment of lung cancer progression and provides a reliable basis for its clinical application.
Keywords: Ginseng, lung cancer, mechanism, metastasis, network pharmacology
How to cite this article:
Hana HK, Qian C, Song MY, Zhang T, Yang CM, Gu RJ, Zhou X, Wei ZH, Zhao Y, Lu Y. The mechanism of total ginseng extracts in the treatment of lung cancer progression based on network pharmacology and experimental validation. World J Tradit Chin Med 2023;9:284-96 |
How to cite this URL:
Hana HK, Qian C, Song MY, Zhang T, Yang CM, Gu RJ, Zhou X, Wei ZH, Zhao Y, Lu Y. The mechanism of total ginseng extracts in the treatment of lung cancer progression based on network pharmacology and experimental validation. World J Tradit Chin Med [serial online] 2023 [cited 2023 Sep 25];9:284-96. Available from: https://www.wjtcm.net/text.asp?2023/9/3/284/385513 |
| Introduction | |  |
Currently, lung cancer remains the disease that causes the greatest number of patient deaths worldwide.[1],[2] Nonsmall cell lung cancer (NSCLC) is one of the major subtypes of lung cancer, accounting for 85% of all cancer cases.[3],[4] Clinically, chemotherapeutic agents (e.g. paclitaxel and cisplatin) and molecular-targeted agents (e.g. the estimated glomerular filtration rate inhibitor gefitinib) are commonly used to treat patients with NSCLC.[5],[6] Although these drugs improve the prognosis of patients, the obvious cytotoxicity of chemotherapy drugs and limitations of resistance to molecular-targeted drugs have caused many problems.[7],[8] In addition, NSCLC metastases have not been effectively treated.[9],[10] Therefore, innovative anti-metastatic drugs are urgently needed for the treatment of NSCLC.
Traditional Chinese medicine (TCM) is considered for treating diseases for nearly 5000 years across China.[11],[12] The molecular mechanism of TCM is continuously being revealed.[13],[14],[15] At present, most studies have shown that a single active component or herbal extract has good anticancer activity[16],[17],[18] and that relevant prescriptions are used in the clinical supplementary treatment of cancer patients.[19],[20]
Ginseng is the most commonly used medicinal for the adjuvant treatment of cancer, as it has a tremendous effect on the regulation of immunity.[21],[22] Its active components, such as ginsenosides, polysaccharides, and sterol, have been reported to have therapeutic effects in NSCLC.[23],[24],[25] However, TCM has multitarget and multilink mechanisms for treating diseases. Thus, the potential mechanism of ginseng in the treatment of NSCLC needs to be clarified.
In the study, we used Lewis lung carcinomas (LLC) cells to construct a mouse lung cancer model; after treatment of this model with total ginseng extract (TGE), we found that a high-dose TGE significantly inhibited the growth of lung cancer, while TGE improved the immune microenvironment of lung cancer. Network pharmacology and virtual docking results showed that the active components of TGE might constrict lung cancer growth by inducing apoptosis. Subsequently, we performed molecular biology experiments to verify these results. In conclusion, our findings confirmed that TGE inhibits NSCLC metastasis through modifying the tumor microenvironment and inducing apoptosis.
| Materials and Methods | | |
Antibodies
Primary Ras homolog gene family member A (RhoA) antibodies were from ZENBIO (China); the phosphorylated myosin light chain (MLC-2) (3674S) primary antibody was attained from Cell Signaling Technology, Inc.(Beverly, MA, USA); β-actin (81115-1-RR) antibody was obtained from Proteintech (Chicago, IL, USA); primary MLC-2 (DF7911) and Rho-associated protein kinase (ROCK) (DF7466) antibodies were obtained from Affinity (Melbourne, Australia); and PE anti-mouse CD8a (162303), FITC anti-mouse CD3 (100203), PE anti-mouse Ly6C (128007), APC anti-mouse CD4 (100411), FITC anti-mouse CD45 (103107), APC anti-mouse CD11b (101211), Per/Cy5.5 anti-mouse Ly6G (127616), and PE/Cy7 anti-mouse F4/80 (123113) antibodies were bought from BioLegend (San Diego, CA, USA).
Preparation of total ginseng extract
As described in previous studies,[26] Chinese herbal slices of ginseng were provided by Jiangsu Province Hospital of Chinese Medicine (Nanjing, China). Ginseng slices were added to water and 95% ethanol (solid-to-liquid ratio 1:8), with reflux extraction for 1 h. The ethanol and aqueous extracts were evaporated, concentrated, and mixed, which were then freeze-dried.
Cell culture
Mouse LLC cell line was obtained from the Chinese Academy of Sciences (Shanghai, China). The cell line was cultured at 37°C with 5% CO2 in Dulbecco's Modified Eagle Medium with 10% fetal bovine serum (CellMax, China).
Lentivirus transfection
LLC cells were then transfected with the lentivirus from GeneChem Co., Ltd (Shanghai, China) and prepared for next experiments.
Animals model and experimental design
C57BL/6 mice were purchased from Shanghai SLAC Laboratory Animal Co., Ltd (Shanghai, China). As for the breeding environment, the mice were kept at the Experimental Animal Center of Nanjing University of Chinese Medicine. The experimental protocols were approved by the animal care and Use Committee of Nanjing University of Chinese Medicine (approval number: No. 202206A075). To establish a lung cancer metastasis model, LLC cells were injected by the tail vein. Specifically, 1 × 106 cells/mL was injected intravenously into mice, and the injection volume of each mouse was 100 μL. The model group mice were gavaged with equivalent normal saline. The low-dose, medium-dose, and high-dose treatment groups were gavaged with 0.45 g/kg, 0.91 g/kg, and 1.82 g/kg TGE, respectively. The body weight of mice was constructed weekly.
In vivo imaging system
Before imaging, each mouse was injected with D-luciferin (Yeasen, China). Using IVIS Spectrum (PerkinElmer, Waltham, MA, USA), in vivo bioluminescence was used to track tumor metastasis.
Flow cytometry
As mentioned in the previous method,[26] a solution of collagenase type I and DNase I (Sigma, USA) in phosphate-buffered saline (PBS) was used to mince fresh lung tissue on ice before lysing it at 37°C. After being lysed in a bath of water for 1 h, the tissue-containing lysate was filtered through a 100 μm cell mesh, and the part of the tissue that could not be filtered was removed by grinding. The filtered cell suspension was then centrifuged at 1500 rpm for 5 min to produce the cell pellets. After washing with PBS, samples were stained with corresponding antibodies at 4°C. Samples were examined by CytoFlex (Beckman Cytoflex, Brea, USA), and the data were analyzed by FlowJo software (Version 10.4, Franklin Lake, USA). CytoFlex (Beckman Cytoflex, Brea, USA) was used to investigate single-cell suspensions, and FlowJo software (Version 10.4, Franklin Lake, USA) was used to analyze the data.
Hematoxylin and eosin staining
Hematoxylin and eosin (H and E) staining was conducted through the manufacturer's protocol (LEAGENE, China). Sections were observed and imaged by Mantra Pathology (PerkinElmer, MA, USA).
Network pharmacology analysis
As demonstrated in previous studies,[26],[27] the active compounds in ginseng were screened for using the traditional Chinese medicine systems pharmacology database and analysis platform (TCMSP) database. The screening conditions were as follows: oral bioavailability value ≥30%, drug-likeness value ≥0.18, Caco-2 permeability value ≥−0.4, blood–brain barrier value ≥−0.3, and half-life value ≥4 [Table 1]. Effective compounds target prediction was made using the SEA search tool, SwissTarget, and TCMSP, using Cytoscape software (Version 3.9.0) to construct an ingredient-target network. For the screening of disease targets, the OMIM and GeneCards databases were used to search the keyword “NSCLC.” Target intersection analysis was performed by HiPlot (https://hiplot.com.cn/). For protein–protein interactions, the STRING database was used for analysis, and further visualization was carried out using Cytoscape software. Gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses were conducted through DAVID and visualized by HiPlot.
Molecular docking simulation
Molecular docking was conducted using AutoDock software. The structures of the bioactive compounds were obtained from the PubChem database (https://pubchem.ncbi.nlm.nih.gov/), and three-dimensional protein structures were obtained from the Protein Data Bank (http://www.rcsb.org/pdb/home/home.do). The specific numbers were caspase-3 (1 nme) and caspase-9 (3d9t). The docking images were visualized using PyMoL.
Western blotting
By homogenizing lung tissues in a mixed lysis solution comprising protease inhibitors, phosphatase inhibitors, and RIPA (Beyotime, China), lung tissues were extracted for entire protein extraction. Following the manufacturer's directions, protein concentrations were determined using a Thermo Fisher Scientific protein assay. For the western blot examination, aliquots were employed. With the help of a wet transfer system (Bio-Rad, USA), the samples' total proteins were separated with sodium dodecyl sulfate-polyacrylamide gel electrophoresis and then transferred to polyvinylidene fluoride membranes (PVDF). PVDF membranes were blocked overnight at 4°C with 5% nonfat milk in Tris-buffered saline and Tween 20, then, prime antibodies were added. The samples were then run through a Bio-Rad imaging system, treated with the appropriate secondary antibody, and then, detected with western blots using enhanced chemiluminescence reagents (Biosharp, China). The National Institutes of Health in the United States carried out all western blot studies in triplicate and quantified the results using ImageJ software.
Quantitative reverse transcription–polymerase chain reaction analysis
Total RNA was isolated from lung tissue using the TRIzol reagent, and aliquots of 5 g RNA were reverse-transcribed into cDNA using the HiScript II QRT SuperMix for quantitative polymerase chain reaction (PCR) reagents and the SYBR Mix following the manufacturer's protocol (Yeasen, China). PCR amplification and detection were carried out with the aid of Applied Biosystems' ABI 7500 equipment. [Table 2] provides a list of the primer sequences.
Statistical analysis
All data were shown as mean ± standard deviation. Statistical analysis was performed using GraphPad Prism software (Version 8.0, San Diego, CA, USA), and one-way analysis of variance (ANOVA) was used for two more groups. Analysis was performed with two-way ANOVA for body weight curves with multiple variables, followed by the Bonferroni post hoc test for comparison of body weight at multiple individual time points. P < 0.05 represents a significant difference.
| Results | | |
Total ginseng extract inhibits lung cancer cell metastasis in vivo
To determine the therapeutic effects of TGE in vivo, we established lung cancer metastasis model by injecting LLC cells into the tail vein and administering different doses of TGE [Figure 1]a. Although we found that, compared to the model group, the intervention with TGE did not alter body weight through weekly weight monitoring [Figure 1]b, the high dose of TGE obviously reduced the lung organ index [Figure 1]c. Consistent with this result, live imaging showed that, compared to the model group, high-dose TGE treatment reduced metastasis [Figure 1]d and [Figure 1]f. Next, we used H and E staining to observe lung pathology. Similarly, a high dose of TGE markedly reduced the number of pulmonary surface nodules [Figure 1]e and [Figure 1]g. These results showed that a high dose of TGE significantly inhibited lung cancer metastasis in vivo. | Figure 1: Total ginseng extract inhibits lung cancer cell metastasis in vivo. (a) Animal experimental design. (b) Weekly body weight of each group (n = 5). (c) Organ index of lung in each group (n = 5). (d) Representative live imaging of each group on the 28th day of the experiment (n = 5). (e) Representative surface nodules of the lung by hematoxylin and eosin staining on the 28th day in each group. Scale bars, 200 μm. (f). In vivo imaging system for lung fluorescence quantification on the 28th day of each group (n = 5). (g) Quantitative statistics of the number of lung surface nodules (n = 5). All Data are shown as mean ± standard deviation. * P < 0.05 versus the model group
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Total ginseng extract ameliorates the lung cancer immune microenvironment
Previous studies have shown that ginseng regulates immunity against diseases. Therefore, we investigated whether TGE alters the immune microenvironment in lung cancer. Using flow cytometry, we found that TGE treatment significantly increased the proportion of CD4+ and CD8+ T cells [Figure 2]a and [Figure 2]b. Recent research has reported that immunosuppression is a characteristic of the metastatic microenvironment, in which monocytes, neutrophils, and macrophages play important roles.[28],[29],[30],[31] These cells weaken the antitumor-killing effect of T cells by inhibiting their recruitment and function. Therefore, we investigated whether TGE affected the recruitment of these cells in the microenvironment. Flow cytometric analysis further showed that 1.82 g/kg TGE intervention prominently inhibited the aggregation of neutrophils (CD45+ CD11b+ Ly6G+), monocytes (CD45+ CD11b+ Ly6C+), and macrophages (CD45+ CD11b+ F4/80+) in the lung [Figure 2]c and [Figure 2]d. It has been proven that the chemokine CXCL9 is related to the recruitment of CXCL10 and T cells,[32],[33],[34] while CXCL2 and CCL2 could recruit neutrophils, monocytes, and macrophages.[35],[36],[37],[38] We further verified whether TGE regulated the expression of chemokines in the lungs and, thus, altered the immune microenvironment. The results of the quantitative reverse transcription–polymerase chain reaction were consistent with the flow cytometry results. TGE treatment increased CXCL9 and CXCL10 expression and decreased CXCL2 and CCL2 messenger RNA expression [Figure 2]e. In general, the above data showed that the intervention improved the recruitment of immune cells in the metastatic niche of lung cancer through the regulation of chemokines. | Figure 2: Total ginseng extract ameliorates the lung cancer immune microenvironment. (a) Representative images of flow cytometry analysis of CD4+ and CD8+ T cells in lungs. (b) Quantification of the percentage of CD4+ and CD8+ T cells in each group (n = 5). (c) Representative flow cytometry analysis of the distribution of monocytes, neutrophils, and macrophages in each group. (d) Quantification of the percentage of monocytes, neutrophils, and macrophages in each group (n = 5). (e) The level of Cxcl9, Cxcl10, Cxcl2, and Ccl2 messenger RNA expression in the lungs in each group. (n = 5). All data are shown as mean ± standard deviation. * P < 0.05, ** P < 0.01, *** P < 0.001. Versus the model group
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Network pharmacology reveals the potential target of total ginseng extract in the treatment of lung cancer
Network pharmacology has become a prominent means of studying the molecular mechanisms of TCM.[39],[40],[41],[42] To this end, we identified the potential active ingredients of ginseng and obtained the targets of the active compound through the TCMSP database, thus constructing a component-target network diagram [Figure 3]a. Next, we obtained the treatment targets for NSCLC from the OMIM and GeneCards databases. The OMIM database contained 468 targets and the GeneCards database contained 2976 targets, including 93 duplicate targets between the two databases [Figure 3]b. We further intersected the disease targets with active ingredient targets to obtain 56 duplicate targets [Figure 3]c. We then constructed a network diagram of 56 repetitive targets and their corresponding active components and diseases to obtain an active component-target-disease network diagram [Figure 3]d. We used the STRING database to determine protein–protein interactions and obtained core targets, including cysteinyl aspartate-specific proteinase-3 (caspase-3), caspase-9, peroxisome proliferator-activated receptor gamma (PPARG), and other core targets [Figure 3]e. In summary, we used network pharmacology to identify multiple components of TGE and its potential targets for the treatment of NSCLC. | Figure 3: Network pharmacology reveals the potential target of total ginseng extract in the treatment of lung cancer. (a) Network diagram of the active components of ginseng and their corresponding targets. (b) OMIM and GeneCards database concerning targets of nonsmall cell lung cancer (NSCLC) displayed through Venn map. (c) Intersection of the targets of Ginseng active ingredients and the disease targets obtained through OMIM and GeneCards, displayed as a Venn map. (d) Network diagram of common targets of Ginseng active ingredients and NSCLC. (e) The protein–protein interaction network of targets
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Total ginseng extract inhibits the progression of lung cancer through multiple pathways
We identified 56 potential targets for ginseng treatment of NSCLC. Thus, we conducted a GO enrichment analysis of the 56 targets. Through the enrichment analysis results, we found that these core target genes were related to biological processes (BP), such as positive regulation of the apoptotic process and positive regulation of the ERK1 and ERK2 cascades [Figure 4]a. Molecular function analysis showed that 56 core targets were related to protein homodimerization and enzyme binding [Figure 4]b. In addition, cellular components indicated that core targets were involved in membrane rafts and cell surfaces [Figure 4]c. We conducted a KEGG analysis on these core targets to investigate the signaling pathways. The enrichment analysis showed that the enriched pathways included apoptosis, the PI3K-AKT pathway, and other diverse signaling pathways [Figure 4]d. Therefore, TGE may affect NSCLC progression by influencing diverse BP and pathways. | Figure 4: Total ginseng extract inhibits the progression of lung cancer through multiple pathways. (a-c) Histogram of top five of core protein gene ontology analysis, (a) biological process, (b) cell component, and (c) molecular function. (d) The top 30 KEGG enrichment analysis of the 56 core targets
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Total ginseng extract active ingredient has direct binding with multiple core targets
To further verify the binding activity of the active components with the crucial target, we selected five components with the highest degree values along with five proteins for the molecular docking assay. We found that the two core target proteins, caspase-3 and caspase-9, related to apoptosis, and the active component with the highest degree value in ginseng, beta-sitosterol, had good binding activity. The binding energy is −7.7 kcal/mol and −10.68 kcal/mol, respectively [Figure 5]a and [Figure 5]b. In addition, we investigated the docking activity of other active components and apoptosis-related proteins, such as Bcl2-associated X (Bax) and B-cell CLL/lymphoma 2 (Bcl-2). We found that most of the active compounds bound directly to these proteins [Figure 5]c, suggesting that the active components of ginseng had a regulatory effect on core target proteins. In conclusion, we verified that the active ingredients in ginseng are directly bound to the core target through molecular docking simulations, indicating that these active ingredients might inhibit the progression of NSCLC by acting on apoptosis-related proteins. | Figure 5: Total ginseng extract active ingredient has direct binding with multiple core targets. (a and b) Molecular docking diagram of beta-sitosterol binding with Caspase-3 protein, with binding energy of −7.7 kcal/mol, and beta-sitosterol binding with Caspase-9 protein, with binding energy of −10.68 kcal/mol. (c) The heat map of the binding energy of the top five ginseng components and the core targets obtained by degree value analysis. (Unit: kcal/mol)
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Total ginseng extract regulates migration-related protein to inhibit the lung cancer metastasis
The enhanced movement of tumor cells drives cancer metastasis, and the inhibition of tumor cell migration could be an effective treatment strategy. Therefore, we investigated whether TGE regulates the expression of migration-related proteins. We measured the levels of ROCK, RhoA, MLC2, and p-MLC2 protein expression through immunoblotting. Previous studies report that both ROCK and RhoA are involved in migration and tumor cell invasion.[43] Activation of RhoA-ROCK signaling can cause the phosphorylation of downstream MLC2.[44],[45] Western blotting showed that 1.82 g/kg TGE remarkedly reduced the expression of ROCK and RhoA and decreased MLC-2 phosphorylation [Figure 6]. These data suggested that TGE inhibited lung cancer metastasis by modulating the RhoA/ROCK/MLC2 signaling pathway. | Figure 6: Total ginseng extract regulates migration-related protein to inhibit lung cancer metastasis. (a) Western blot image of β-actin, Rho-associated protein kinase (ROCK), Ras homolog gene family member A (RhoA), Myosin light chain-2 (MLC2), p-MLC2 (n = 3). (b-d) The quantification of (b) ROCK, (c) RhoA, and (d) p-MLC2 protein expressions (n = 3). All data are shown as means ± standard deviation. * P < 0.05, ** P < 0.01, *** P < 0.001 versus the model group
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| Discussion | | |
Lung cancer is one of the deadliest cancers worldwide.[46],[47] As one of the most important subtypes, NSCLC has a 5-year overall survival rate of only 15%.[48],[49] More than 70% of NSCLC patients die from metastasis.[9] Metastasis (from initial primary tumor growth to angiogenesis, endocytosis, blood flow survival, exosmosis, and metastatic growth) is a complex process.[50],[51] Therefore, medical research and development for the treatment of metastasis are major tasks.
TCM has been used in many countries to treat cancer, cardiovascular conditions, and other diseases.[52],[53] Ginseng is a commonly used herb for the clinical treatment of lung cancer.[54],[55] However, the underlying mechanism remains unclear because of its complex components. Recently, the development and application of multi-omics, network pharmacology, and molecular biology in TCM have provided powerful means to confirm the potential mechanisms of TCM in treating diseases.[56],[57]
In the study, we established a lung cancer metastasis model by injecting LLC cells into the tail vein of a mouse model and by administering three doses of TGE. We found that the treatment had no significant impact on the body weight of the mice, but that a high-dose TGE significantly reduced the lung organ index. H and E staining and live imaging analyses suggested that high-dose TGE prominently inhibited the tumor burden. We concluded that TGE might have an inhibitory effect on lung cancer metastasis.
The metastatic microenvironment has been the focus of many studies on tumor metastasis. Immunosuppression is one of the most important characteristics, which is manifested by the weakening of the proliferation and activation of T and NK cells with a tumor cell-killing effect, while other immune cells, such as neutrophils, monocytes, and macrophages, are largely recruited and polarized into phenotypes that promote tumor progression; for example, neutrophils are polarized into the N2 type and macrophages are polarized into the M2 type.[58],[59] Therefore, a reversal of this immunosuppressive phenotype is a potential therapeutic strategy against cancer. Previous studies and clinical data have shown that ginseng can regulate and enhance immunity. Therefore, we explored whether TGE affects the metastatic niche in lung cancer. Flow cytometry analysis indicated that TGE expanded the numbers of T cells and decreased the recruitment of neutrophils, monocytes, and macrophages. The recruitment of immune cells depends on chemokine signal transduction. We found that chemokines CXCL9, CXCL10, CXCL2, and CCL2, which are related to T cells and bone marrow-derived cells, showed similar results.
The multicomponent and multitarget mechanisms of TCM have brought many challenges to modern research. Network pharmacology has provided ideas for research on the mechanisms behind TCM. Many studies have applied network pharmacology to explore the potential molecular mechanisms of TCM in the treatment of diseases.[60],[61] We identified 16 active ingredients of ginseng and potential core targets thereof for NSCLC treatment. Through GO and KEGG analyses, we found that these potential therapeutic targets were related to apoptosis, PI3K/Akt, and other BP and signaling pathways, indicating that ginseng treatment of NSCLC involves multiple pathways.
To further determine the molecular interaction between the relevant active components in ginseng and the core target protein, we conducted a molecular docking analysis between the five active components with the highest scores and the apoptosis-related protein. The docking results showed that the five components of ginseng (beta-sitosterol, stigmasterol, Inermin, Frutinone A, and fumarine) had good docking activity with five proteins related to apoptosis: caspase-3, caspase-9, PPARG, Bcl-2, and BAX. We verified the binding ability of the active ingredients in ginseng as core targets of NSCLC.
Finally, we discussed the expression of RhoA, ROCK, and the downstream molecules MLC2 and p-MLC2, which have been reported to be related to the migration of tumor cells.[62],[63] TGE significantly inhibited the expression of RhoA and ROCK proteins and the phosphorylation of MLC-2. These results suggested that TGE inhibited metastasis by regulating the RhoA/ROCK/MLC2 axis. Previous studies have focused on the role of the active ingredients of ginseng in lung cancer metastasis. Ginsenoside Rg3 inhibits lung cancer metastasis through modifying extracellular signal-regulated kinases, while ginsenoside Rk1 and Rg5 inhibit lung metastasis by regulating transforming growth factors-β-mediated epithelial–mesenchymal transformation.[64],[65] Total ginsenoside extracts activate endoplasmic reticulum stress and induce autophagy in NSCLC cells.[25] However, there had been little research about the effects of TGE on lung cancer metastasis. In this study, we used TGE to simulate clinical ginseng treatment methods and explored its effects on lung cancer metastasis. We found that TGE also had an inhibitory impact on lung cancer metastasis, which might be related to its multicomponent and multitarget mechanisms. In summary, our study provides a basis for the use of the herb ginseng for the treatment of lung cancer metastasis.
This study has some limitations. First, the anticancer efficacy of TGE needs to be verified in additional cancer models. Furthermore, we only carried out molecular docking to determine binding. In future research, we will investigate the inhibitory effects of active compounds from TGE on these core targets.
| Conclusions | | |
Our study verifies that TGE inhibits lung cancer metastasis by improving the immune microenvironment, inducing apoptosis, and inhibiting tumor cell migration, thus providing a basis for the clinical application of ginseng.
Acknowledgment
We thank the Experimental Center for Science and Technology at the Nanjing University of Chinese Medicine for providing resources.
Financial support and sponsorship
This study was supported by projects of the National Nature Science Foundation of China (81961128020 and 81973734), Jiangsu Province Traditional Chinese Medicine Leading Talents Program (SLJ0229), and Jiangsu Specially Appointed Professorship Foundation (013038021001).
Conflicts of interest
There are no conflicts of interest.
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[Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5], [Figure 6]
[Table 1], [Table 2]
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