Cancer presents a substantial global health challenge and ranks among the leading causes of mortality worldwide. In 2020, GLOBOCAN reported approximately 19.3 million new cancer cases, resulting in nearly 10.0 million deaths26. The most frequently diagnosed cancers included breast cancer (11.7%), lung cancer (11.4%), colorectal cancer (10.0%), prostate cancer (7.3%), and stomach cancer (5.6%). Projections suggest a 47% increase in the global cancer burden, with an estimated 28.4 million cases expected by the year 204026. GPCRs, which constitute an extensive and diverse family of cell surface signaling receptors, have been implicated in numerous cancer types. Dysregulation of GPCR activity within cancer cells can manifest through various mechanisms, such as aberrant overexpression, activating mutations resulting in gain-of-function effects, and increased production and secretion of agonists. GPCRs are often overexpressed in several cancer types, including human chronic lymphocytic leukemia, breast cancer, colon cancer, pancreatic ductal adenocarcinoma, and cancer-associated fibroblasts (CAFs) cell lines27. For instance, protease-activated receptors (PARs), which constitute a distinct class of GPCRs associated with cancer, display notable expression and overexpression in solid tumors and cancer cells, including those of breast cancer, colon cancer, and melanoma28. The high expression of GPCRs in cancer cells suggest their potential involvement on malignant progression. Understanding the activation of GPCRs is paramount as it offers insights into the underlying mechanisms that can be targeted for the development of anticancer drugs. GPCRs play a pivotal role in regulating various pathways, such as Wnt, MAPK, and PI3K signaling, which are often disrupted in cancer due to mutations29. Formylpeptide receptor-2 (FPR2), a GPCR identified in colon cancer cells, activates the protein kinase B pathway (AKT pathway), thereby contributing to drug resistance30. However, the expression levels and molecular mechanisms of GPCRs may vary among different cancer types, owing to the distinct genetic and biochemical characteristics specific to each type of cancer. Currently, there are FDA-approved drugs available for cancer treatment that specifically target GPCRs. These include cabergoline, which targets the dopamine receptor D1 (DRD1) for neuroendocrine and pituitary cancer; lanreotide, targeting the somatostatin receptor (SSTR) for pancreatic cancer; Degarelix, aimed at the gonadotropin-releasing hormone receptor (GnRH) for prostate cancer; and Raloxifene, directed at the estrogen receptor (ER) for breast cancer31. Moreover, various other GPCRs, such as FPR2, Galphas-coupled beta-adrenergic receptor, Angiotensin II type 1 receptor (AT1R), GPR160, and ACKR3, have been proposed as potential targets for cancer treatment31. The increased expression of GPCRs and their associated signaling proteins in cancer cells presents promising opportunities for novel therapeutic targets, offering distinctive therapeutic possibilities.Network pharmacology has emerged as a valuable approach for predictive analyses, particularly in complex diseases such as cancer32. In this study, we employed a comprehensive network pharmacology approach to elucidate the potential molecular mechanisms of GG compounds against cancer. Our approach encompassed drug-likeness evaluation, target identification, GO and KEGG pathway analysis, as well as PPI analysis. Through the integration of drug and disease databases, we identified a total of 265 common targets for constructing a PPI network. Among these targets, 260 nodes demonstrated protein-protein interactions (Fig. 2A and B). Subsequent KEGG analysis unveiled the specific pathways influenced by GG in GPCRs-related cancer. Notable pathways encompassed pathways in cancer, insulin resistance, chemical carcinogenesis, PI3K-AKT signaling pathway, microRNAs in cancer, endocrine resistance, proteoglycans in cancer, neurotrophin signaling pathway, MAPK signaling pathway, and EGFR tyrosine kinase inhibitor resistance. These pathways play pivotal roles in various facets of tumor development and progression, including tumor cell growth, apoptosis evasion, angiogenesis, invasion, and metastasis. This underscores their significant involvement in the pathological processes underpinning cancer33,34,35. After employing cytoHubba plug-ins in Cytoscape, we identified a set of 10 hub genes that demonstrate significant importance. These hub genes, namely MAPK3, SRC, EGFR, STAT3, ESR1, MTOR, CCND1, PPARG, BCL2L1, and PTGS2, play crucial roles in cellular signaling pathways. In particular, MAPK3, also recognized as ERK1, holds paramount significance as a key molecule in the ERK/MAPK pathway, a pivotal cell signaling pathway. The upregulation and heightened activity of MAPK3 are consistently linked to the initiation, progression, cancer cell migration, drug resistance in various carcinoma types, including liver, thyroid, lung, and gastric cancers36. It phosphorylates cytoplasmic proteins downstream, activating numerous nuclear transcription factors associated with apoptosis and cell proliferation29. Conversely, SRC shows abnormal overexpression and activation in various cancer types, including glioblastoma, liver, lung, colon, breast, bladder, and pancreatic cancers30,31. This heightened SRC expression significantly contributes to the progression of these malignancies. SRC can be activated by a diverse array of extracellular signals originating from integrins, G-protein-linked receptors, steroid receptors, and receptor tyrosine kinases (RTKs)37. Upon activation, SRC triggers the Ras/Raf/ERK signaling cascade and activates other kinases such as PI3K, MAPK, and AKT31. Functioning as an oncoprotein, SRC regulates transformed cells and plays a pivotal role in tumor progression and metastasis38. EGFR, a key hub gene in the growth factor receptor family with intrinsic tyrosine kinase activity, plays a central role in tumorigenesis, particularly in lung, breast, and glioblastoma cancers39. Elevated EGFR expression is associated with poor cancer patient prognoses. Ligand-induced EGFR activation triggers multiple signaling pathways, including Ras/MAPK, PI3K/AKT, and PLC/PKC cascades, making EGFR a target in current cancer therapies39. STAT3, an oncogene within the STAT family, undergoes activation via JAK, EGFR, and GPCR, resulting in its phosphorylation and subsequent nuclear translocation. Inside the nucleus, STAT3 upregulates target genes such as Bcl-xL, Cyclin D1, and VEGF40. STAT3 plays a crucial role in tumor cell proliferation, invasion, migration, resistance to therapy, and the prediction of poor prognoses41. Persistent STAT3 activation is consistently observed in esophageal squamous cell carcinoma, colorectal cancer, lung cancer, and gastric cancer, emphasizing its potential as a therapeutic target in cancer treatment42. Estrogen receptor (ESR1), a transcription factor, exerts significant influence on cell proliferation and differentiation in target tissues. ESR1, implicated in breast cancer, endometrial cancer, and osteoporosis, plays a central role in regulating gene expression associated with the cell cycle, proliferation, and apoptosis, involving key factors such as IGF1, Cyclin D1, c-Myc, FOXM1, GREB1, BCL2, and CXCL1243. MTOR, a serine/threonine kinase, plays a role in regulating a wide range of cellular processes, including cell survival, growth, metabolism, protein synthesis, autophagy, and homeostasis. Its importance spans across various cancer types, with regulatory interconnections with pathways such as PI3K/AKT, MAPK, VEGF, NF-κB, and p5344. Cyclin D1 (CCND1), a crucial regulator facilitating the transition from G1 to S phase in the cell cycle, has been found to be upregulated in a variety of solid and hematologic tumors45. Peroxisome proliferator-activated receptor gamma (PPARG), a ligand-inducible transcription factor and member of the nuclear receptor superfamily, regulates various physiological processes, including immunity, inflammation, vascular functions, cellular proliferation, differentiation, development, and apoptosis. Elevated PPARG expression has been noted in multiple cancer types, suggesting the potential for PPARG antagonists as therapeutic options for cancer treatment46. BCL2L1, also known as BCL-X, plays a pivotal role in enhancing cell survival and exerts anti-apoptotic effects, especially in cancer cells. The overexpression of BCL2L1 imparts resistance to chemotherapeutic agents, underscoring the potential for therapeutic interventions that target BCL2L1 in effective cancer treatment47. Prostaglandin-endoperoxide synthase 2 (PTGS2), an enzyme involved in prostaglandin synthesis, is induced by inflammation and expressed in tumor epithelial cells, especially in colorectal cancer and non-small cell lung cancer. Excessive prostaglandin production is linked to various facets of lung cancer progression, including angiogenesis, metastasis, and immunosuppression48. Significantly, BCL2L1 and PTGS2 were omitted from further investigation as they do not directly engage in or act via a GPCR mechanism.Through molecular docking analyses, it was determined that (-)-viniferin, gnetin A, and the resveratrol dimer exhibited strong binding affinity toward SRC, EGFR, and MTOR, respectively. Particularly, gnetin C demonstrated a robust binding force with MAPK3 and PTGS2. A close examination of the protein-ligand interactions suggests that the GG extracts possess effective binding capabilities with these specific targets, achieving lower binding energies primarily through the formation of multiple hydrogen bonds. (-)-Viniferin, a stilbenoid compound, corresponds to cis-epsilon, whereas the resveratrol dimer corresponds to trans-epsilon-viniferin. (-)-Viniferin exhibited varying IC50 values in different cancer cell lines: 20.1 µM in C6 cells (Glioma), 76.2 µM in HepG2 cells (Hepatoblastoma), 21.5 µM in HeLa cells (Breast cancer), 47.2 µM in MCF-7 cells (Breast cancer), and 90.2 µM in HT-29 cells (Colorectal adenocarcinoma)49. Notably, this compound demonstrated activity in P-388 cells (Leukemia) with an IC50 value of 18.1 ± 0.7 µM50. Additionally, trans-epsilon-viniferin displayed variability in different cancer cell lines, with IC50 values as follows: 85.5 ± 8.1 µM in COLO205 cells (Colon carcinoma), 13.9 ± 0.1 µM in HT-29 cells (Colorectal adenocarcinoma), 7.7 ± 0.2 µM in HepG2 cells (Hepatoblastoma), 9.3 ± 0.3 µM in AGS cells (Gastric adenocarcinoma), and 5.6 ± 1.4 µM in HL-60 cells (Human leukemia)51. Previous studies have highlighted the antiproliferative and apoptosis-inducing effects of epsilon-viniferin on various cancer cell lines, including those of osteosarcoma, non-small cell lung cancer, human hepatoma, colon cancer, glioblastoma, and human melanoma52,53,54. Epsilon-viniferin has shown significant anticancer activity by modulating various pathways, including the induction of apoptosis, inhibition of cell cycle progression, and suppression of invasion and migration. Notably, in non-small cell lung cancer cells (A549 cells), epsilon-viniferin induces apoptosis by downregulating phospho-AKT expression and upregulating cleaved PARP and cleaved caspase-3 expression52. It also inhibited epithelial-mesenchymal transition (EMT), invasion, and migration in non-small cell lung cancer cells induced by TGF-β1 or IL-1β. These effects were achieved through the reduction of TGF-β1-induced reactive oxygen species (ROS) production and the downregulation of key factors associated with EMT and metastasis, including MMP2, vimentin, Zeb1, Snail, p-SMAD2, p-SMAD3, and ABCG2 in A549 cells. Furthermore, in xenograft metastatic mouse models of A549 cells, epsilon-viniferin significantly inhibited lung metastasis55. By modulating key regulators of the cell cycle, such as cyclins A, E, and D1, along with their associated CDK-1 and − 2, epsilon-viniferin compounds effectively disrupt melanoma cell cycle progression, particularly during the S phase54. Additionally, treatment with epsilon-viniferin remarkably suppressed lung cancer progression in nude mice bearing A549-cell xenografts52. In this study, we explored cis- and trans-epsilon-viniferin as inhibitors of SRC and MTOR. SRC, a protein involved in interactions with transmembrane receptors like integrin/FAK, RTKs, and GPCRs, activates critical downstream signaling pathways, including MAPK, ERK, PI3K/AKT/mTOR, IL-6/JAK/STAT3, and Rho/ROCK pathways. These pathways drive essential cancer processes, including survival, proliferation, angiogenesis, migration, invasion, and metastasis56. Therefore, the targeted inhibition of SRC and MTOR by cis- and trans-epsilon-viniferin highlights their potential to modulate and inhibit crucial signaling molecules in cancer cells, as discussed earlier. However, in vitro anti-cancer experiments should confirm the mechanism of action of cis- and trans-epsilon-viniferin, and variations in the mechanism might be observed depending on the type of cancer. Furthermore, epsilon-viniferin exhibits favorable drug-like properties and demonstrates promising ADME pharmacokinetic characteristics. These attributes position epsilon-viniferin as a promising and effective therapeutic agent with the potential to revolutionize current cancer treatment strategies.Gnetin A and gnetin C are resveratrol derivatives that fall under the stilbene class, and they have been extracted from various species within the Gnetaceae family11. Gnetin A shares a similar chemical structure with gnetin C. In this study, gnetin A displayed a robust binding affinity to the tyrosine kinase of EGFR. However, there is currently limited information available regarding the anticancer properties of gnetin A, highlighting the need for further in vitro investigations in our future studies. Molecular docking of active compounds extracted from GG revealed the presence of chemical structures based on resveratrol. Resveratrol, a natural stilbenoid compound, has the ability to undergo oxidative coupling of two to eight resveratrol units, leading to the formation of oligostilbenoids in various plant families57. This natural phytoalexin compound, stilbenoid, is abundant in plants and notably prominent in red wine58. Researchers have shown considerable interest in its multiple health benefits, including cardiovascular protection, anti-inflammatory properties, anti-metastatic effects, and anti-cancer activities59. Gnetin C has demonstrated significant anticancer activity by inhibiting proliferation, migration, and angiogenesis in various cancer cell types and in in vivo models. Clinically achievable concentrations of gnetin C have been shown to significantly inhibit proliferation and induce apoptosis in pancreatic, prostate, breast, and colon cancer cells60. Gnetin C exhibited varying IC50 values in cancer cell proliferation, with the following results: 16.29 ± 1.11 µM in PANC-1 cells (pancreatic cancer), 13.83 ± 0.92 µM in AsPC-1 cells (pancreatic cancer), 12.22 ± 1.45 µM in Pan-02 cells (mouse pancreatic cancer), 10.28 ± 0.79 µM in PC-3 cells (prostate cancer), 9.85 ± 2.60 µM in DU-145 cells (prostate cancer), 8.95 ± 0.92 µM in LNCaP cells (prostate cancer), 9.01 ± 0.15 µM in PTEN-CaP8 cells (mouse prostate cancer), 13.13 ± 0.61 µM in MCF-7 cells (breast cancer), 11.78 ± 1.45 µM in HT-29 cells (breast cancer), and 11.3 ± 0.60 µM in Colon-26 (mouse colon cancer)60. MSE, which contains the active ingredient gnetin C, has been found to inhibit tumor growth, intratumoral angiogenesis, and the development of liver metastasis in mice with Colon-26 tumors52. The antitumor properties of gnetin C have been attributed to its ability to suppress the ERK1/2 pathway, leading to the inhibition of proliferation, migration, and tube formation in human umbilical vein endothelial cells61. Gnetin C has also demonstrated potent antitumor effects against patient-derived acute myeloid leukemia (AML) cells by targeting both the ERK1/2 and AKT/mTOR signaling pathways, which play critical roles in the survival and growth of AML cells61,62. The inhibition of the MAPK/ERK1/2 and AKT pathways by gnetin C in leukemia cells results in the inactivation of downstream members, including activated p90 ribosomal S6 kinase (RSK1) and mitogen- and stress-activated protein kinase (MSK1/2), leading to cell cycle arrest, apoptosis, and the inhibition of cell growth63. In our study, we propose that gnetin C exhibits a high binding affinity towards MAPK3 or ERK1, supporting previous findings and providing further evidence of its potential therapeutic efficacy and its ability to target crucial signaling pathways in cancer cells. Gnetin C has demonstrated inhibitory effects on PTGS2 (COX-2). Cyclooxygenase-2 (COX-2) is highly expressed in various human cancers, including colorectal, breast, ovarian, uterine cervix, lung, head, and neck cancers. The NF-κB/IκB pathway, which is regulated by the PI3K/AKT and ERK signaling pathways, plays a crucial role in inducing COX-2 expression. Elevated COX-2 expression can inhibit apoptosis, promote angiogenesis and invasiveness, contribute to inflammation and immunosuppression, and facilitate the conversion of procarcinogens into carcinogens, thereby promoting tumorigenesis64. Gnetin C, found in MSE, has demonstrated anti-inflammatory effects by downregulating IL-1β, a proinflammatory cytokine, in a mouse model. This effect may be associated with its inhibitory effects on COX-265. These findings emphasize the potential of gnetin C as a promising therapeutic agent for cancer treatment. It can inhibit not only COX-2 and its associated pro-tumorigenic effects but also exert anti-inflammatory actions by regulating IL-1β. Furthermore, gnetin C has been shown to downregulate the expression of MTA1 and exhibit significant inhibitory effects on cell viability, colony formation, cell death induction, and migration in DU145 and PC3M prostate cancer cells66. MTA1, a chromatin modifier protein, plays a significant role in promoting aggressiveness and metastasis in prostate cancer through its overexpression67. It activates downstream targets involved in inflammation, cell survival, and invasion, contributing to prostate cancer progression and metastasis. Alterations in MTA1 levels impact the PTEN/AKT pathway among other downstream pathways68. Treatment with gnetin C at a dose of 50 mg/kg results in the most potent suppression of tumor progression, as evidenced by reduced mitotic activity, angiogenesis, a significant increase in apoptosis, and confirmed downregulation of MTA1, Cyclin D1, and Notch 2 in xenograft prostate tumor tissues69. Recent studies have shown that gnetin C supplementation effectively reduces the progression of prostate cancer in a mouse model by inhibiting MTA1, which leads to decreased cell proliferation, inflammation, and angiogenesis. Notably, the administration of gnetin C did not result in any observable toxicity in mice13. In silico investigation of gnetin C through ADME analysis revealed favorable gastrointestinal absorption, the absence of blood-brain barrier permeation, and no inhibition of CYP enzymes, except for being a CYP2C9 inhibitor (Table 2). However, ADME properties are solely based on computational predictions in this study, and experimental studies will be necessary in subsequent work. In line with preclinical and clinical investigations, a 2-week daily consumption of pure gnetin C in healthy volunteers did not lead to any adverse events. Furthermore, gnetin C exhibited improved pharmacokinetic properties, including higher bioavailability, when compared to resveratrol70,71,72. Molecular docking models have offered valuable insights into the mechanism of action of these compounds on GPCRs-related targets, shedding light on their inhibitory effects on cancer. These insights find support in in vitro and in vivo experiments, providing preliminary confirmation of the therapeutic potential of various active components in GG. These components target key pathways that are implicated in cancer. Moreover, there are several previous studies reported about in silico analysis of active compounds of GG. The natural compound epsilon-viniferin, an oligostilbene (a resveratrol dimer), binds to the hinge region between the α- and β-subunits of AMPK, interacting with its active site and contributing to improved hyperglycemia and hyperlipidemia. epsilon-viniferin’s hydroxyl groups form crucial hydrogen bonds with AMPK, involving residues ARG10 and LYS31 (α-subunit), as well as THR106, ARG107, and ASP108 (β-subunit)73. Gnetin C and trans-epsilon-viniferin bind to the active site of ACE (angiotensin-converting enzyme) through hydrogen bonds or hydrophobic interactions, with free energy binding values of -8.51 and − 8.13 kcal/mol, respectively74. HER2 proteins are pivotal in breast cancer cell growth and differentiation. epsilon-viniferin binds to HER2 receptors with a binding energy of -10.45 kcal/mol, forming six hydrogen bonds at MET766, CYS775, ASP855, LEU788, GLN791, and ASN84275. In an in silico study, Gnetin C and trans-resveratrol, active compounds from melinjo seeds, were assessed for binding affinity against ERα in breast cancer cells (MCF-7). The results revealed that both Gnetin C and trans-resveratrol can bind to the same amino acids (VAL54B, TYR55B, TYR216B, TRP227B, and LEU306B) with docking scores of -6.0 and − 7.9 kcal/mol, respectively76. In an in silico molecular docking study, gnetin C was evaluated for its interaction with the target protein VHR (Vaccinia H-1 related phosphatase), a receptor involved in multiple signaling pathways (MAPK, JNK, ERK1, p38, EGFR, and ErbB2/HER2) and crucial for the proliferation of HeLa cervical cancer cells. The docking results indicated that gnetin C binds to VHR with a binding energy of -8.3 kcal/mol, inhibiting receptor activity by interacting with specific amino acid residues: SER129, PRO162, ASN163, and GLU12677. VHR plays significant roles in cellular signaling, including cell-cycle regulation, DNA damage response, MAPK signaling, platelet activation, and angiogenesis77. Consequently, gnetin C and (-)-viniferin demonstrated specific binding to molecular targets in cancer, such as HER2, EGFR, ERα, and VHR, as revealed by in silico molecular docking studies. This study found that gnetin C and (-)-viniferin exhibit notable binding affinity to MAPK3 and SRC, with strong binding energy and hydrogen bond formation at the active sites of these targets. Therefore, gnetin C and (-)-viniferin inhibited MAPK3 and SRC, suggesting potential interactions with HER2, EGFR, ERα, and VHR. This could disrupt cancer cell signaling and growth. MAPK3 (ERK1) and SRC are signaling molecules associated with HER2, EGFR, and ERα in lung and breast cancer78,79. MAPK3 is also promoted by GPCRs via the Gαq/PLCβ/PKC pathway80, while SRC kinases form direct associations with GPCRs, resulting in complex interplay81. These kinases are also linked to cellular growth, cancer, and growth factor receptor tyrosine kinases. The SRC and STAT3 pathways serve as potential effectors for G proteins82. GPCRs activate STAT3 through JAKs, leading to cancer progression83. Additionally, our study reported that the resveratrol dimer and gnetin C exhibit substantial binding affinity to mTOR with strong binding energies. mTOR (mTORC2) activation is enhanced by β- and α-adrenergic signaling through GPCRs and the presence of growth factors via RTK receptors, and it also occurs in membrane subcellular compartments84. Previously, gnetin C has shown significant antitumor effects against patient-derived acute myeloid leukemia (AML) cells by targeting the ERK1/2 and AKT/mTOR signaling pathways, which are crucial for the survival and proliferation61,62. Therefore, mTOR, MAPK3, and SRC are key targets directly associated with the GPCR family and are targeted by gnetin C and (-)-viniferin, suggesting that the active compounds from GG may act as potent inhibitors of these targets, directly impacting GPCR-related pathways.In conclusion, the consistent demonstration of GPCRs involvement in tumor progression and metastasis across various cancer types highlights their potential as promising targets for cancer treatment. Among the compounds found in the GG collection, a total of 13 exhibited drug-like properties, meeting the criteria of the Rule of Five (RO5). Through network pharmacology analysis, we identified 8 potential central targets related to GPCR for these GG compounds, including MAPK3, SRC, EGFR, STAT3, ESR1, MTOR, CCND1, and PPARG. By employing network pharmacology and molecular docking analysis, we predict that (-)-viniferin, gnetin A, gnetin C, and resveratrol dimer have the potential to exert an anti-cancer effect through interactions with multiple proteins, such as MAPK3, SRC and mTOR. These targets are closely associated with essential cellular processes in cancer, including proliferation, migration, apoptosis, and angiogenesis. However, it is imperative to conduct further in vitro and in vivo experimental studies to validate the effectiveness of these compounds and elucidate their mechanisms of action against specific types of cancer.
