Platinum-derived chemotherapy medications are often combined with other conventional therapies for treating different tumors, including colorectal cancer. However, the development of drug resistance and multiple adverse effects remain common in clinical settings. Thus, there is a necessity to find novel treatments and drug combinations that could effectively target colorectal cancer cells and lower the probability of disease relapse. To find potential synergistic interaction, we designed multiple different combinations between cisplatin, cannabidiol, and intermittent serum starvation on colorectal cancer cell lines. Based on the cell viability assay, we found that combinations between cannabidiol and intermittent serum starvation, cisplatin and intermittent serum starvation, as well as cisplatin, cannabidiol, and intermittent serum starvation can work in a synergistic fashion on different colorectal cancer cell lines. Furthermore, we analyzed differentially expressed genes and affected pathways in colorectal cancer cell lines to understand further the potential molecular mechanisms behind the treatments and their interactions. We found that synergistic interaction between cannabidiol and intermittent serum starvation can be related to changes in the transcription of genes responsible for cell metabolism and cancer’s stress pathways. Moreover, when we added cisplatin to the treatments, there was a strong enrichment of genes taking part in G2/M cell cycle arrest and apoptosis. According to the World Health Organization, colorectal cancer (CRC) is the third most common cancer and the second leading cause of cancer-related deaths worldwide. It is often diagnosed at later stages when the treatment options are limited .CRC is not a single disease but rather a genetically heterogeneous group of oncological pathologies . In 2014Despite the implementation of various preventive strategies, screening protocols, and a diverse range of treatment choices, CRC remains a prominent contributor to global disease prevalence and mortality. There is a pressing demand for the development of new, more efficient prevention methods and therapeutic strategies that involve drug combinations. According to recent experimental findings, cannabinoids might emerge as promising contenders in this regard ,13,14,15Over recent years, multiple experimental data have provided evidence of the antioncogenic impact of cannabinoids on CRC ,18,19,20CBD is a partial agonist of cannabinoid receptors 1 and 2 (CB1 and CB2) ,23. AddiOne of the best-described anticancer effects of CBD is the activation of NOXA, suppressing mTOR/AKT signaling and MAPK pathways . In CRCsIn a study involving the combination of CBD with the platinum drug oxaliplatin, it was shown that oxaliplatin resistance can be overcome by adding CBD to the treatment, which results in autophagy-mediated cell death and the formation of free radicals by dysfunctional mitochondria in resistant cells . FurtherA DNA crosslinking agent, cisplatin, is used in multiple combination therapies with paclitaxel, tegafur-uracil, doxorubicin, gemcitabine, and vitamin D . UnfortuOne of the crucial pathways of cisplatin-induced apoptotic death is the activation of p38 MAPK, which causes transcription of PUMA and NOXA through p53 activation ,51. HayaHumanity has been practicing fasting for centuries. However, only recent years’ discoveries have shown the health benefits of simple time-adapted food consumption and calorie deprivation. Fasting helps reprogram cellular stress-related adaptation mechanisms and energy metabolism and boosts cellular defense mechanisms. It was shown in animal models that intermittent fasting (IF) could protect against diabetes mellitus, cardiovascular pathologies, neurodegeneration, and cancer. Recent human trials showed that IF helps fight obesity, hypertension, asthma, and rheumatoid arthritis. Fasting may lengthen the lifespan of bacteria, yeast, worms, and mice ,55,56,57Research showed that serum starvation in vitro and short-term food starvation in vivo reduced levels of growth factor stimulation ,59,60. IAnother animal study including fasting showed that alternate-day fasting in mice reduced the incidence of lymphomas . The samIn metastatic animal models, the combination of fasting and chemotherapy resulted in 20–60% cancer-free survival compared to chemotherapy or fasting alone ,67. AddiThe inhibition of the glycolytic pathway in cancer cells could lead to stress overload due to constant proliferative signaling from oncogenes . Cancer The mentioned metabolic changes in cancer cells might become one of the targets for anticancer therapy. As we discussed before, fasting affects metabolism in cancer cells, leading to differential stress sensitization. Additionally, cannabinoids are also known to regulate stress survival pathways in cancer cells. Thus, it makes sense to test these treatments individually and in combination, to assess if there is a cytotoxic effect on CRC cells and whether these treatments can act in synergy.First, to evaluate the treatment effectiveness, we established the time and dose-dependent effects  and calcNext, we combined various treatments in CRC cell lines to test if any combination had a synergistic interaction . Actual SNF, and apoptosis, such as BAX and FAS. These results were somewhat expected based on the drug mechanisms of action , which binds to cyclin/cyclin-dependent kinase 2 or 4 complexes and functions as a regulator of the G1 cell cycle, could support the notion of G1/S cell cycle arrest. Increased RGCC gene expression is also responsible for cell cycle regulation and is induced by p53 under DNA damage. Additionally, higher levels of BTG anti-proliferation factor 2 could have helped with G1/S cell cycle arrest. The increased levels of stratifin (SFN) transcript, whose product binds to translation initiation factors and functions as a regulator of translation during mitosis, help prevent DNA errors during mitosis and could indicate cancer cell survival. The increased transcription of the SESN1 gene, coding for sestrin 1, as induced by p53 and serving as a potent inhibitor of mTOR, would help in cisplatin’s cytotoxic effects. Although the increased levels of polo-like kinase 2 might also indicate a strong stimulus for cell proliferation, the weight of anti-proliferative signaling was stronger, which supports our MTT results. The increased levels of fibroblast growth factor 2 (FGF2) and syndecan 1 (SDC1), responsible for cell binding, cytoskeletal organization, interactions with the extracellular matrix (ECM), and cell migration, could stimulate epithelial-to-mesenchymal transition (EMT) and invasiveness of the HCT-116 cell line. These are the signs of cancer cell progression.One of the most enriched terms under cisplatin action on the HCT-116 CRC cell line was TP53-regulated transcription of the cell death gene . The incBased on the dot plot data, CBD significantly affected carbohydrate metabolism, PPAR, receptor-type tyrosine-protein phosphatase, TGF-β, and MAPK signaling C.PFKFB4, which regulates fructose-2,6-bisphosphate and responds to hypoxia to help cancer cells produce more ATP (HK2) expression involved in the rapid activation of glycolysis in cancer cells. The transcript for glucose-6-phosphate isomerase (GPI) was decreased, too. The function of GPI, along with the regulation of glucose metabolism, is acting as an autocrine motility factor, a tumor-secreting cytokine, and an inducer of angiogenesis. This shows that the addition of CBD inhibited transcription of factors that help cancer cells in energy and oxygen scarcity and might prevent cancer progression.Furthermore, CBD decreased the expression of genes responsible for glucose metabolism, glycolysis , and glumore ATP D. There FOSB, EGR3, and EGR1. The FOSB gene codes for the FosB proto-oncogene, AP-1 transcription factor, which can activate cell proliferation, differentiation, and transformation in cancer cells. EGR3 and EGR1, transcripts for the early growth response 3 and 1, are regulators of cell mitogenesis and differentiation.Addition of CBD changed the expression of NGF-stimulated transcription kinase and downregulated genes such as SMAD7 and SMAD3. SMAD7 was shown to antagonize signaling by the TGF-β type 1 receptor and inhibit TGF-β by associating with their receptors and preventing SMAD2 access. The upregulation of SMAD3, which is part of the transcription factor complex and can serve as a tumor suppressor, has a similar effect. There was also downregulation of gene expression of IL1RAPL1, which is a part of the IL-1 receptor family.CBD showed modulation of TGF-β signaling pathways, including genes such as ABCA1, the ATP binding subfamily A member 1, which functions as a cholesterol efflux pump and is regulated by the PPAR signaling system. The ABC family transporters are commonly responsible for chemotherapy drug resistance. Thus, CBD might reduce the likelihood of drug resistance development. Furthermore, there was upregulation of CYP1A1, a gene coding for monooxygenase enzymes that take part in drug metabolism.Interestingly, CBD decreased the expression of MAP3K8 transcript, an oncogene coding for mitogen-activated protein kinase 8, which can activate both MAP and JNK pathways as well as pro-inflammatory pathways involving activation of NFκB.The decreased transcription of factor JUN could inhibit T-cell activation-induced death and reduce transcription in response to Toll-like receptor signaling. JUN transcript is also responsible for KRAS-mediated transcriptional activation of USP28 in CRC. Furthermore, it takes part in the MyD88-dependent cascade initiated by endosome signaling. Additionally, CBD increased the expression of CD22, which helps in B-cell activation. Finally, there was a decreased level of Based on Reactome data for differential expression analysis in HCT-116 CRC cells, CBD had various effects on gene expression. It decreased the expression of genes responsible for glucose metabolism, glycolysis, and gluconeogenesis, which may reduce tumor growth. It could also downregulate cell mitogenesis and differentiation genes. Additionally, CBD modulated pathways involving TGF-β signaling. This could decrease the likelihood of the development of drug resistance. In summary, these findings suggest that CBD may have potential therapeutic benefits for treating CRC. However, more experimental data would be needed to support this notion.Based on dot plot analysis, ISS changed multiple pathways involved in CRC cell survival, lipid metabolism, extracellular matrix organization, ERBB2 receptor signaling, as well as the PI3K/AKT pathway E.ARC, EGR1, EGR2, EGR3, EGR4, FOS, FOSB, FOSL1, ID2, ID3, ID4, JUNB, JUND, NAB2, SRF, and TRIB1 , which is a transcription factor that regulates gene expression in response to extracellular signals, could indicate the activation of pro-survival mechanisms in tested cancer cells.There was a strong upregulation of multiple genes that participate in NTRK1 signaling, including nd TRIB1 F. Most lTF (coding for iron-binding factor) and VGF, a neuropeptide precursor implicated in cancer progression and metastasis. Downregulation of TF and VGF can have different effects on CRC, with TF downregulation potentially leading to reduced proliferation and increased apoptosis, while VGF downregulation may decrease migration and invasion of CRC cells.On the other hand, there was a downregulation of DUSP1, DUSP10, DUSP2, DUSP4, DUSP5, DUSP6, and DUSP8. The upregulation of the listed DUSP genes in CRC can have different effects on MAPK signaling and may contribute to the development and progression of the disease. For instance, the upregulation of DUSP6 may inhibit ERK1/2 activity and prevent apoptosis. DUSP8 is a phosphatase that dephosphorylates and deactivates JNK and p38 MAPKs, and it was shown to inhibit cell proliferation and migration of cancer cells. However, the upregulation of DUSP2 has been shown to have both pro- and anti-tumorigenic effects in different cancer types. Overall, the effects of upregulating DUSP in CRC can be complex and may depend on the specific DUSP isoform.There were multiple genes that were upregulated under the RAF-independent MAPK1/3 activation term , which iEGR1, GBP2, GBP5, IFI35, IFI6, IFIT1, IFIT3, IFIT5, MX1, OAS1, OAS3, OASL, SMAD7, STAT1, and UBE2L6, were also upregulated. Interferon signaling is thought to have anti-tumor effects by promoting apoptosis and inhibiting tumor cell proliferation and migration. On the other hand, some upregulated genes, such as STAT1 and SMAD7, could have pro-tumor effects. STAT1 is a transcription factor involved in immune system activation in response to viral infection, and it has been shown to promote tumor growth and survival. Additionally, SMAD7 is involved in the TGF-β signaling pathway and can promote tumor growth and metastasis.Multiple genes responsible for interferon signaling, including HMGCS1 gene, which is also involved in the synthesis of ketone bodies) was increased. There were a few genes that were downregulated, too. Decreased levels of ANGPTL4—coding for angiopoietin-like protein 4, which regulates lipid and glucose metabolism and the growth of new blood vessels—could lead to inhibition of angiogenesis in cancer cells. Interestingly, there was a decreased expression of NR1D1 (nuclear receptor subfamily 1 group D member 1), which is involved in the regulation of circadian rhythms by repressing the expression of core clock components. NR1D1 also regulates lipid metabolism, adipogenesis, gluconeogenesis, and the macrophage inflammatory response.Another interesting effect of ISS was changes in the regulation of lipid metabolism via PPARα signaling. The expression of genes responsible for making medium-chain acyl-CoA dehydrogenase (ACADM) and supporting long-chain fatty acid synthesis and cholesterol metabolism , which takes part in the regulation of cell growth, proliferation, differentiation, and survival, could be one of the important targets in cancer therapy. Additionally, decreased levels of NRG1 transcript could decrease proliferation and survival. Moreover, lowering PRKCA  and PTK6 (protein tyrosine kinase 6) could also inhibit cell growth, proliferation, and cancer progression.The genes AREG (amphiregulin), BTC , EREG (epiregulin), FGF9 (fibroblast growth factor 9), HBEGF (heparin-binding EGF-like growth factor), NRG2 (neuregulin 2), PIK3AP1 (phosphoinositide-3-kinase adaptor protein 1), and PIK3R3 (phosphoinositide-3-kinase regulatory subunit 3). All the mentioned genes take part in the activation of cell growth, proliferation, insulin signaling, and cancer progression, which could result in unwanted effects of ISS in cancer. On the other hand, EGFR, NRG1, and PDGFA were downregulated under ISS. The epidermal growth factor receptor, coded by the EGFR gene, triggers a signaling pathway that promotes cell growth and proliferation of cancer cells. Thus, inhibition of EGFR by ISS could be beneficial in CRC therapy.The following genes were overexpressed in PI3K signaling under ISS treatment: BMP4, CAPN8, COL3A1, COL5A2, FBLN5, ITGA1, SERPINE1, SPOCK3, THBS1, and TLL1. The bone morphogenic protein encoded by the BMP4 gene, which plays a role in cell differentiation and proliferation, was overexpressed under ISS. CAPN8, coding for calpain 8 protein, a calcium-dependent cysteine protease, plays a role in cytoskeletal organization. The increased expression of genes encoding collagen type 3 and type 5 could indicate stimulation of connective tissue formation by cancer cells. Increased expression of the FBLN5 gene encoding fibulin 5 could help promote the adhesion of endothelial cells and help develop new arteries. Moreover, ITGA1 codes for an alpha-1 subunit of integrin receptors. SERPINE1 (plasminogen activator inhibitor 1) is responsible for inhibiting fibrinolysis. Thus, an increased expression of the listed genes could help with the reorganization of extracellular tissue to the cancer cells’ advantage.Under ISS treatment, many genes that code for the extracellular matrix organization were upregulated, including At the same time, multiple gene transcripts taking part in extracellular matrix organization were decreased. These included ADAMTS16, COL13A1, COL4A3, CTSK, DDR2, EFEMP2, FBLN2, FBN1, ITGA10, ITGA5, LAMA4, LAMB3, LAMC1, LAMC2, LOXL2, LTBP3, MMP16, P4HA1, P4HA2, PDGFA, PLOD1, and PRKCA. The ADAMTS16 gene encodes ADAM metallopeptidase with thrombospondin type 1. COL13A1 and COL4A3 encode collagen types 13 and 4, respectively. Collagen type 4 is a main structural component of basement membranes and helps with the formation of new vessels. CTSK encoding cathepsin K, a lysosomal cysteine proteinase that could contribute to tumor invasiveness, was also decreased. DDR2 is a gene encoding the discoidin domain receptor subclass of the receptor tyrosine kinase protein family. This protein is a collagen-induced receptor that activates signal transduction pathways involved in cell adhesion, proliferation, and extracellular matrix remodeling. EFEMP2, a gene encoding EGF-containing fibulin extracellular matrix protein 2, is part of many ECM proteins involved in elastic fiber formation and connective tissue development; specifically, it participates in terminal differentiation and maturation of smooth muscle cells. FBLN2 codes for fibulin 2, an ECM protein that plays a role in organ development and can bind to fibrillin. The decreased levels of FBN1, encoding fibrillin 1, an ECM glycoprotein that is a structural component of calcium-binding microfibrils, could lead to the inhibition of cancer cell invasion. ITGA10 and ITGA5 code for integrin subunit alpha 10 and 5, which are collagen-binding proteins involved in cell-matrix adhesion. LAMA4 , LAMB3 (laminin B1k chain), LAMC1 (laminin subunit gamma 1), and LAMC2 (laminin subunit gamma 2) were decreased, too. Laminins, a family of ECM glycoproteins, are one of the main noncollagenous constituents of basement membranes, which are implicated in cell adhesion, differentiation, migration, and metastasis. Decreased LOXL2, encoding lysyl oxidase-like 2 delta E13 , could contribute to the inhibition of cancer invasiveness. LTBP3 (latent TGF-β binding protein 3), which encodes a protein that forms a complex with TGF-β and plays a structural role in the extracellular matrix, was also reduced. MMP16  takes part in the breakdown of ECM, tissue remodeling, and metastasis. P4HA1 and 2  encode a key component of prolyl hydroxylase, a major enzyme in collagen synthesis. PDGFA (platelet-derived growth factor subunit A) encodes the protein that is a part of PDGF, which is essential for cell proliferation, cell migration, survival, angiogenesis, and chemotaxis. PLOD1  catalyzes the hydroxylation of lysyl residues in collagen-like peptides. PRKCA  belongs to a family of serine–threonine protein kinases that can be activated by calcium. Its protein product takes part in cell adhesion, cell transformation, cell cycle checkpoint, cell volume control, tumorigenesis, and angiogenesis.Overall, the reduction of the vast majority of factors that take part in CRC’s invasion and metastatic mechanisms could represent a beneficial effect of ISS. Additionally, activation of lipid metabolism could be beneficial when ISS is combined with CBD, which has been shown to strongly suppress the metabolism of carbohydrates.Based on cell viability data, we established that combining cisplatin with CBD had an antagonistic interaction A. DifferIn addition to the results for p53 regulation of apoptosis, the results based on the Reactome dot plot showed the differential gene expression in the TGF-β signaling, extracellular matrix organization, circadian clock regulation, and checkpoints defects .BHLHE40 (basic helix–loop–helix family member E40) gene that controls the body’s circadian clock was downregulated. Also, the expression of NFIL3 , a transcriptional regulator of genes responsible for the circadian clock, was increased. This might indicate changes in the regulation of circadian rhythms under the combination of CBD and cisplatin.The Moreover, there was an increased expression of genes responsible for the extracellular matrix organization and cancer cell invasiveness, including collagen, laminins, and FGF2 activation, in response to CBD and cisplatin. On the other hand, the increased expression of SMAD7 could prevent the activation of TGF-β and inhibit EMT in cancer cells.CCNE2) and CDC25A (cell division cycle 25A) and decreased transcripts for CDK inhibitor 1C (CDKN1C) could stimulate cell cycle progression even with damaged DNA. This could also be one of the reasons why CBD with cisplatin had antagonistic interaction.Cisplatin is known to activate apoptosis due to its damaging effect on the DNA. However, our results showed that the increased levels of cyclin E2  expression, which is involved in the rapid activation of glycolysis in cancer cells. The transcript for glucose-6-phosphate isomerase (GPI) was decreased, too. Along with the regulation of glucose metabolism, the function of GPI is to act as an autocrine motility factor, a tumor-secreting cytokine, and an inducer of angiogenesis. This shows that the addition of CBD inhibited transcription of factors that help in the survival of cancer cells under energy and oxygen scarcity and might prevent cancer progression.Based on the Reactome data for differential expression analysis in HCT-116 CRC cells, CBD in combination with cisplatin decreased the expression of genes responsible for glucose metabolism, glycolysis, and gluconeogenesis, similar to CBD treatment alone. Increased expression of Overall, based on our mRNA expression results, we suggest that the antagonistic effect of combining CBD and cisplatin was the result of lower fold enrichment in genes responsible for apoptosis when compared to cisplatin alone in the HCT-116 CRC cell line. However, our data would need to be confirmed with additional experiments involving other cell viability and apoptosis assays, as well as protein expression analysis for selected genes.The combination of cisplatin and ISS showed moderate antagonistic effects in the HCT-116 CRC cell line B. HoweveBDNF), early growth response proteins 1-4 (EGR1-4), and parts of AP-1 transcription factors (FOS and JUNB) indicated activation of cell survival mechanisms and cell proliferation. Increased levels of BCL2 indicate inhibition of apoptosis. A higher SRF could also stimulate cell proliferation. SRF is a part of the immediate-early genes that regulate the cell cycle, and apoptosis that couples cellular gene expression to cytoskeletal dynamics; upregulation of SRF could indicate strong pro-survival signals in cancer cells. Moreover, increased levels of TP53I3 (encoding tumor protein P53 inducible protein 3) and its effector GADD45A indicated possible activation of p53-mediated DNA repair.Furthermore, the elevated levels of brain-derived neurotrophic factor , a mitochondrial enzyme that catalyzes the oxidative decarboxylation of pyruvate and regulates the homeostasis of carbohydrate fuels in mammals, would suggest inhibition of the Krebs cycle and oxidative phosphorylation in HCT-116 cells.The elevated BMP4 (bone morphogenic protein), CEACAM1 (CEA cell adhesion molecule), COL5A2 , LAMA2 , NTN4 (netrin 4), PDGFB (platelet-derived GF subunit B), SERPINE1 (serpin family E member 1), SPOCK3 (osteonectin), and THBS1 (thrombospondin 1) indicated a wide variety of responses related to interactions with the extracellular matrix, which can represent an attempt of cancer cells to activate its invasive potential. Furthermore, the elevated levels of ARC, a gene coding for an activity-regulated cytoskeleton-associated protein involved in cell migration, could lead to cancer cell progression. Another protein regulating microtubular and vesicular transport that could cause cancer cell progression was dynamin 3 (DNM3); the expression of DNM3 was also increased.The increased levels of CRKL) and Ras-like without CAAX 1 (RIT1), the GTPase regulating the p38 MAPK-dependent signaling cascade that responds to cellular stress, as well as MAPK7, could lead to the reduction of cancer cell proliferation and inhibition of its survival. Moreover, decreased levels of transcripts responsible for the formation of some collagens, laminins, and metalloproteases could lead to the suppression of cancer cell invasiveness.However, with the lowering the expression of genes coding for proteins that take part in Ras signaling, including CRK-like proto-oncogene adaptor protein , and MHC class II (HLA-DRA) indicated strong stimulation of IFN signaling, which could result in activation of macrophages in the tumor microenvironment. Additionally, the listed transcripts participate in the negative regulation of apoptosis and inhibition of cell proliferation by activating p21 and p27.The increased transcripts for Based on the mRNA expression results, we observed an activation of multiple pro-survival and pro-invasive mechanisms under the combination of ISS and cisplatin in HCT-116. Thus, we would not recommend this treatment combination in MMR-deficient CRC. However, we would suggest conducting further experiments to support this notion.Based on the Reactome dot plot, the genes that take part in MAPK and PI3K/AKT signaling, carbohydrate metabolism, NGF signaling, and apoptosis were differentially expressed when we compared the combination of CBD and ISS to the DMSO control in the HCT-116 CRC cell line K.FAS, PERP, PMAIP1, and TP53I3. All the mentioned genes might help with stimulating apoptosis. For instance, FAS codes for the cell surface death receptor, a TNF superfamily of receptors taking part in the extrinsic apoptotic pathway. PERP is a p53 apoptosis effector related to PMP22. Also, PMAIP1 (phorbol-12-myristate-13-acetate-induced protein 1) is a pro-apoptotic subfamily within the BCL-2 protein family that determines whether a cell commits to apoptosis. Lastly, TP53I3 is involved in p53-mediated cell death and could further assist in the suppression of CRC cell growth.When we analyzed each major Reactome term for differentially expressed genes, multiple pathways of interest were changed L. The foSimilar to CBD alone, the combination of CBD and ISS inhibited multiple genes responsible for carbohydrate metabolism. The inhibition of hexokinases 1 and 2 and other enzymes that are part of glycolysis and gluconeogenesis was downregulated. It was shown that many tumors rely on glycolysis as a major source of energy production. Thus, inhibition of carbohydrate metabolism could drastically affect cancer cells’ survival .AREG, BTC, DUSP1, DUSP2, DUSP4, DUSP5, EGF, FGF19, FGF2, FGF9, HBEGF, ICMT, PDGFB, PSMB10, and PSMB9. The increased levels of amphiregulin (AREG), epidermal growth factors, fibroblast growth factors 19, 2, and 9, and platelet-derived growth factor subunit B transcripts, which play essential roles in the stimulation of cancer cell proliferation, survival, EMT, and invasion, could be responsible for cell survival under CBD and ISS treatments. Increased transcripts of heparin-binding EGF-like growth factor (HBEGF), a positive regulator of the epidermal growth factor receptor, and protein kinase B gene might overstimulate stress response pathways and cell survival in a way that could be damaging to cancer cells. Additionally, there was an increased expression of dual-specificity phosphatases 1, 2, 4, and 5 , negative regulators of MAPK1/ERK2 SAPK/JUN, p38, which act result in response to environmental stress and negative regulation of cellular proliferation. Also, the downregulation of fibroblast growth factor receptors 3 and 4  would result in inhibition of cell proliferation. Furthermore, the decreased expression of kinase suppressor Ras 2 (KSR2) and RAS guanyl-releasing protein 1 (RASGRP1), which promotes BRAF-mediated phosphorylation of MAP2K1/MEK1 and helps in the activation of Ras, would also contribute to the inhibition of cell proliferation. Additionally, inhibition of fibronectin 1 (FN1), involved in cell adhesion, migration, and invasion of cancer cells, could suppress tumor progression.There was also an increase in the expression of RAF/MAP kinase cascade genes, including CHUK) and NFκB inhibitor alpha (NFKBIA) could suggest inhibition of inflammatory cytokine formation. Phosphoinositide-3-kinase regulatory subunit 3 (PIK3R3), a regulatory subunit of PI3K, was also increased, which could point to the overactivation of the PI3K/AKT pathway. Moreover, the higher levels of the nuclear receptor subfamily 4 group A member 1 (NR4A1) gene transcript, encoding the steroid–thyroid hormone–retinoid receptor protein, a transcription factor induced by the serum stimulation, could further indicate an attempt of cancer cells to stimulate cell proliferation. On the contrary, decreased levels of neuregulin 1 (NRG1), a direct ligand of ERBB3 and ERBB4 tyrosine kinase receptors, which activate ERBB receptors, could be responsible for the inhibition of cancer cell growth. Additionally, the downregulated fibroblast growth factor receptors 3 and 4, PDGFA, and GAB1 (GRB2-associated binding protein 1), which take part in FGF signaling, indicate downregulation of this pathway and possible suppression of tumor cell invasiveness and EMT.Next, we analyzed changes in the “PI3K/AKT signaling in cancer” term. The increased levels of transcripts for a component of inhibitor of NFκB (BDNF (brain-derived neurotrophic factor), EGR 1-4 (early growth response proteins 1-4), JUNB (transcription factor jun-B), which enables sequence-specific dsDNA binding activity and is a part of AP-1 transcription factors, and SRF, which stimulates cell proliferation and takes part in immediate-early genes, might indicate stimulation of cell proliferation. Moreover, increased TIAM (T-lymphoma invasion and metastasis-inducing protein 1), a guanine nucleotide exchange factor (GEF) that regulates RAC1 signaling pathways affecting cell shape, migration, adhesion, growth, survival, actin cytoskeleton formation, endocytosis, and membrane trafficking, could contribute to activation of cancer cell migration and metastasis.The increased signaling by the “NTRK1 (neurotrophic tyrosine receptor kinase gene fusion)” term could promote cancer cell growth. The elevated levels of CRKL (CRK-like proto-oncogene adaptor protein), which has been shown to activate the RAS and JUN kinase signaling pathways and transform fibroblasts in a RAS-dependent fashion; MAPK7 (mitogen protein kinase 7), involved in cell proliferation and transcription regulation; and RIT1 (Ras-like without CAAX 1—Ras-related GTPase), involved in regulating p38 MAPK-dependent signaling cascades related to cellular stress, could support the hypothesis that a combination of CBD and ISS can have an anti-proliferative effect.On the other hand, lower levels of ARC (activity-regulated cytoskeleton-associated protein) involved in cell migration and cytoskeleton organization may indicate decreased cancer cell invasion. Additionally, decreased CHUK) inhibitor component and NFKBIA suggests the downregulation of NFkB signaling. Higher levels of NLR family CARD domain containing 5 (a caspase-recruitment-domain-containing NLR family playing an important role in the cytokine response through inhibition of NFkB) and negative regulation of IFN signaling could also contribute to inhibiting pro-inflammatory cascades. Additionally, the downregulation of IL1RAP (IL1 receptor accessory protein), which helps in the activation of signaling events of IL-1-responsive genes NFKB2, NOD1, NOD2, members of Nod1/Apaf-1 family proteins that take part in the immune response to LPS and activation of NFkB, and TRAF6  suggests that NFkB signaling was substantially downregulated under our treatment.Based on the “IL-1 signaling” term, there was an overall inhibition of NFkB signaling. As we already mentioned, increased expression of an NFkB kinase complex , could cause delays in the onset of anaphase. The decreased levels of centromere proteins A, F, E, I, and L would disturb the regular mitotic behavior of chromosomes, leading to improper centromere structures. A decrease in CLASP1, a nonmotor microtubule-associated protein involved in regulating microtubule dynamics at the kinetochores, also indicated a disturbance of proper mitosis. Lower levels of NUF2, the component of the NDC80 kinetochore complex, would suggest improper chromosome segregation and spindle checkpoint activity. Additionally, MAD2L1  was decreased, suggesting arrest in the M phase. PLK1  was also decreased. Furthermore, decreased expression of RAD21, a gene involved in the repair of DNA double-strand breaks and chromatin cohesion during mitosis, would suggest disturbance of the homologous recombination repair system.The increased levels of CDK1), cell division cycle 20 (CDC20), and cyclin B1 and B2 (CCNB1 and CCNB2) would suggest strong inhibition of cell cycle progression in the G2/M phase.Moreover, the lowering of cyclin-dependent kinase 1  would further suggest inhibition of pro-survival mechanisms and activation of apoptosis.Similar to cisplatin alone, p53-mediated pro-apoptotic factors such as FAS and BAX transcripts were elevated. Additionally, decreased levels of JUNB protooncogene, an AP-1 transcription factor subunit that regulates cell proliferation. Additionally, the increased levels of HIF1α and FGF2 indicated activation of survival pathways in cancer cells and possible stimulation of EMT, which would constitute unwanted effects of combinational treatment. Additionally, increased Ras homolog family member U, a Rho GTPase activating PAK1 and JNK1, can induce the formation of filopodium and dissolving stress fibers. It also mediates the effects of Wnt signaling in regulating cell morphology, cytoskeletal organization, and cell proliferation. Moreover, there was an increase in TNFRSF1B, a gene encoding a member of the TNF-receptor superfamily. Together with TNFR1, this forms a heterocomplex that mediates the recruitment of c-IAP1 and c-IAP2, which could help in cancer cell survival.At the same time, multiple transcripts responsible for the tumor’s cell survival mechanisms were also kicking in. There were increased levels of BTC) levels could indicate the increased production of EGF-like proteins to stimulate cancer cell growth. Additionally, higher levels of FGF1, FGF2, FGF9, and FGF19 indicate a strong stimulation of FGF signaling, which could stimulate broad mitogenic and cell survival mechanisms, including angiogenesis, invasion, and metastasis. When elevated, heparin-binding EGF-like growth factor (HBEGF) could stimulate PI3K/AKT pro-survival signaling. Moreover, elevated levels of PDGF, TGF-α, and PI3K regulatory subunit 3 also indicated activation of PI3K/AKT signaling.The increased betacellulin (FGFR4) and EGFR could decrease cell proliferation, migration, and lipid and glucose metabolism. Lower levels of IL-1 receptor accessory protein could reduce IL-1-dependent activation of NFkB and result in an anti-inflammatory effect. Additionally, SNAIL1, a strong inducer of EMT, was also decreased, which would indicate inhibition of tumor cell progression. Decreased expression of RPAGD  could inhibit CRC cell growth.On the other hand, reduced expression of fibroblast growth factor receptor 4 , and CBD was also sourced from Sigma-Aldrich . DMSO (Dimethyl sulfoxide anhydrous) was purchased from Thermo Fisher Scientific . CBD (10 mg/mL) was dissolved in methanol and stored at −20℃. The stock solution of cisplatin (100 μM) was dissolved in DMSO and kept at −20℃ for no longer than three weeks.The experiments were performed on three human CRC cell lines, HT-29 (HTB-38™), HCT-116 (CCL-247™), and LS-174T (CL188™), and immortalized human colonic epithelial cell line HCEC-1CT . The CRC cell lines HCT-116 (CCL-247™) and LS-174T (CL188™) were purchased from ATCC . HT-29 (HTB-38™) was a kind gift from Dr. Roy Golsteyn’s Laboratory at the University of Lethbridge. All cell lines were cultured according to the manufacturer’s instructions. To establish IC50s, a range of concentrations (1–15 μM) of cisplatin was obtained by diluting cisplatin in the fresh complete media, and those were tested on CRC and a normal colon epithelial cell line.To establish IC50s, a range of concentrations (2–12 μM) of pure CBD were obtained by diluting the cannabinoids in the fresh complete media, and those were tested on CRC and normal cell lines.TM). The intermittent serum starvation (ISS) was recreated by depriving cells of FBS for 16 h and reintroducing media with FBS for 8 h. The experiments lasted for five consecutive days in all cell lines. The first combination group included complete media for 24 h with the addition of cisplatin (8 h) and/or CBD (16 h). The second combination group included ISS for 16 h with the introduction of CBD (16 h), cisplatin (8 h), and their combination.To maintain the same nutritional supply for each CRC cell line, the cells were cultivated in RPMI-1640 medium -2, 5-diphenyltetrazolium bromide] assay. An MTT assay  was used to evaluate the effects of cisplatin, CBD, and ISS on CRC and normal epithelial colon cell viability. 2 for 24 h. Treatments were changed daily with the normalized concentrations of methanol and DMSO. Cells were incubated to 80–90% confluency in 10 cm Petri dishes. Next, cells were trypsinized with trypsin/EDTA . After trypsinization and centrifuging, fresh medium was added to the cells, and one (for experiments with complete media) or three (for ISS experiments) thousand cells per well were plated in triplicate into flat-bottomed 96-well plates in 100 μL of the appropriate medium. The cells were allowed to adhere to the plate surface overnight and were exposed to treatment for five days. Cells were then maintained at 37 °C in a humidified atmosphere containing 5% CO2 incubator for 4 h. After the incubation, 100 μL of MTT solution was added to each well, and the plates continued to incubate at 37 °C overnight. Absorbance was measured at 595 nm using a FLUOstar Omega microplate reader . Results were calculated by comparing the treatments to the appropriate controls. All treatments were in triplicate, and each test was performed in at least three independent experiments. Every 24 h of treatment, 10 μL of MTT kit I  was added, and plates were incubated at 37 °C in the CO5 cells/well. After 24 h of incubation, cells were exposed to treatments for 72 h. The culture medium was replaced with a fresh treatment medium every 24 h and at specific time points (at the 8th and 16th hours during a 24-h period) for ISS experiments in the HCT-116 cell line. At the endpoint, cells were washed twice with ice-cold PBS and harvested using the TRIzol™ Reagent (Invitrogen). Total RNA extraction followed the TRIzol Reagent protocol . Immediately after the isolation, the quality and quantity of the extracted RNA were assessed using a NanoDrop 2000/2000c Spectrophotometer . Additionally, the integrity of RNA samples was assessed using agarose gel electrophoresis. Once grown to 85–90% confluency in a Petri dish, cells were detached using trypsin/EDTA  and re-plated in 6-well plates at a density of 6 × 10® Ultra™ RNA Library Prep Kit for Illumina® (New England BioLabs). Massive parallel sequencing was performed using the Illumina NovaSeq6000 S4 . Base calling and demultiplexing were performed by the sequencing provider (Genome Quebec). Libraries were generated from 250 ng of total RNA using the kit Illumina® Stranded mRNA Prep, Ligation (Illumina), as per the manufacturer’s recommendations. Adapters and PCR primers were purchased from Illumina. Libraries were quantified using the KAPA Library Quantification Kits—Complete kit  (Kapa Biosystems). The average size fragment was determined using a LabChip GXII  instrument. The libraries were normalized and pooled and then denatured in 0.05 N NaOH and neutralized using HT1 buffer. The pool was loaded at 175 pM and 200 pM on an Illumina NovaSeq S4 lane using the Xp protocol as per the manufacturer’s recommendations. The run was performed for 2 × 100 cycles (paired-end mode). A phiX library was used as a control and mixed with libraries at the 1% level. Base calling was performed with RTA v3.4.4. The program bcl2fastq2 v2.20 was then used to demultiplex samples and generate fastq reads.The RNA samples from the HCT-116 CRC cell line were shipped to Génome Québec  for mRNA library preparation and next-generation sequencing. The stranded mRNA libraries were prepared using the NEBNexthttps://www.bioinformatics.babraham.ac.uk/projects/fastqc/, accessed in May–June 2023. Sequencing reads were trimmed of adapter sequences and low-quality bases using Trimmomatic. Trimmed sequence files were examined with FastQC to verify the trimming results.Initial quality control was conducted using FastQC v0.11.9 https://support.illumina.com/sequencing/sequencing_software/igenome.html, accessed in May–June 2023). Mapping was performed using splice-aware aligner HISAT2 2.1.0 [http://qualimap.conesalab.org/, accessed in May–June 2023 [Trimmed sequencing reads were mapped to the human genome  downloaded from the Illumina iGenome website  and principal components analysis (PCA) to investigate the relationship between samples and detect potential outliers. Prior to HC and PCA analysis, DESeq2-normalized values underwent variance stabilizing transformation using the vst function from DESeq2 . HC was p-value) kept at 0.1. Multiple-comparison adjustment was performed using the Bejamini–Hochberg procedure. Significantly changed genes were visualized as MA plots, volcano plots and heatmaps built using pheatmap v.1.0.12 and ggplot2 v.3.4.0. The genes involved in the analysis were annotated with Entrez IDs and descriptions using BiomaRt 2.54.0 [p values below 0.05 and absolute log2 fold change over 0.59 (~1.5 fold change) were considered significant.Differentially expressed genes (DEGs) were detected with the DESeq2 function results using the default options. DESeq2 uses the Wald test to determine significantly changed genes between groups. The independent filtering option was set to TRUE with the alpha threshold (adjusted t 2.54.0 . Genes wGene ontology (GO) and Reactome pathway enrichment analysis was conducted using pathfindR v.1.64.0, an R package that identifies significantly enriched biological terms utilizing active subnetworks. The main pathfindR function was used with the following options: gene_sets = “GO-All” , adj_method = “fdr”, enrichment_threshold = 0.05, pin_name_path = “STRING”, search_method = “GR”, max_gset_size = 500. The same options were applied to pathfindR enrichment of Reactome analysis. PathfindR output includes a table with enriched terms, a table of enriched term clusters, dot plots, gene-term maps, and upset plots. Clustering analysis and corresponding plots were prepared using corresponding pathfindR functions with the default settings.p-values below 0.05 were considered statistically significant.Additionally, pathway topology analysis was performed with the Signaling Pathway Impact Analysis (SPIA) tool (version 2.50.0) . Reactomwww.genecards.org, accessed in May–June 2023 [The analysis of the pathway results, including gene functions and interactions, was performed with the help of GeneCards—the human gene database une 2023 ,84.t-test was employed to determine the statistical significance between treatment groups for cell viability experiments. Results were presented as the mean ± SD of data. p < 0.05 was considered statistically significant.GraphPad Prism 9.0 software was used to calculate two- and one-way analyses of variance (ANOVA), and the two-tailed Student’s To determine synergism, additivity, or antagonism, we performed drug combination analysis based on the Chou–Talalay method using CompuSyn 1.0 software ,86,87. ICisplatin is an effective chemotherapy agent that is used for the treatment of multiple cancers . It is oCisplatin has a strong pro-apoptotic effect ,51, whicAccording to the mRNA expression data obtained from the HCT-116 CRC cell line, cisplatin had multiple effects. It reduced multiple metabolic pathways that cancer cells depend on for growth and proliferation. Cisplatin induced cytotoxic effects by increasing the expression of several genes involved in both the intrinsic and extrinsic pathways of apoptosis. The fold enrichment was one of the highest for TP53 regulation of transcription of cell death genes under cisplatin treatment . Our datAs we previously mentioned, the mechanisms of anticancer effects of cannabinoids include the activation of apoptosis, endoplasmic reticulum (ER) stress response, downregulation of survivin (inhibitor of apoptosis), and a decrease in RAS/MAPK and PI3K/AKT signaling ,16,20,21SMAD7 and SMAD3 transcripts’ increase, which resulted in the inhibition of TGF-β, a common pathway  that is affected in CRCs [ABCA1 transcript, which belongs to the ABC family of transporters that often contribute to chemotherapy drug resistance. Thus, CBD could possibly reduce the development of drug resistance by decreasing drug efflux pumps.Adding CBD modulated TGF-β signaling via  in CRCs ,92. Inteγ, and IL-6 due to the activation of NFkB and STAT3 can lead to the formation of aberrant cryptic foci (ACFs) and adenomas that eventually can develop into adenocarcinomas. Moreover, continuous activation of COX-2 increases KRAS signaling and promotes tumor survival, progression, and metastatic potential [MAP3K8 transcript, an oncogene coding for mitogen-activated protein kinase 8, which can activate both MAP and JNK pathways as well as pro-inflammatory pathways involving activation of NFκB. This could be one of the mechanisms of CBD preventive action on CRCs’ development.A persistent chronic inflammatory response in some CRCs with increased levels of TNF, IL-17, IL-23, IFN-otential ,94,95. APFKFB4, which regulates fructose-2,6-bisphosphate and responds to hypoxia to help cancer cells produce more ATP. There was also a decrease in hexokinase 2, which is involved in the rapid activation of glycolysis in cancer cells. Furthermore, our data showed that the addition of CBD inhibited the transcription of factors that help cancer cells with energy and oxygen scarcity. Thus, it could prevent cancer cell survival and the development of cancer progression. The observed mechanisms of CBD regarding glucose metabolism were of high interest to us because they could assist with the mechanisms of serum deprivation when two treatments were combined.Additionally, we observed the decreased expression of genes responsible for glucose metabolism, glycolysis, and gluconeogenesis, such as FOSB, EGR3, and EGR1. CBD had an impact on TGF-β signaling pathways and decreased the expression of ABCA1 and ANGPTL4, potentially reducing the development of drug resistance. Conversely, it increased the expression of oncogenes like JUN and MAPK3K8 transcripts. In summary, these results suggest that CBD may have therapeutic potential for the MSI subtype of CRC. However, further experimental data would be required to support this idea, such as apoptosis assay, protein expression analysis of selected pathways, and animal models.Additionally, CBD downregulated cell mitogenesis and differentiation genes, specifically Studies have demonstrated that both serum starvation in vitro and short-term food deprivation in vivo can decrease growth factor stimulation levels ,59,60. WThe mRNA expression analysis of ISSs’ effects in HCT-116 showed that serum deprivation activated multiple survival pathways, including PI3K/AKT signaling. ISS downregulated the expression of EGFR, which could inhibit cell growth, proliferation, and survival of cancer cells. On the other hand, increased levels of serum response factor and PI3K signaling may indicate the activation of survival pathways in CRC cells. Multiple genes that take part in the downregulation of MAPK/ERK1/2 signaling were upregulated, which could lead to the prevention of apoptosis. The numerous genes taking part in extracellular matrix reorganization were changed, too. The decreased levels of some integrins, laminins, MMPs, and PDGFs could point to reduced invasiveness of the HCT-116 cells under ISS treatment. ISS also inhibited carbohydrate metabolism and modulated lipid metabolism similarly to the CBD treatment alone.Based on the literature review, MTT, and mRNA expression data, we decided to test CBD, cisplatin, and ISS in different combinations to see if we could achieve a synergistic effect. Thus, our next step was to look at different treatment interactions and their molecular mechanisms.As mentioned previously, the activation of the p38 MAPK pathway plays a crucial role in cisplatin-induced apoptotic death as it triggers the transcription of PUMA and NOXA via p53 activation ,51. Our Unfortunately, the combination of CBD and cisplatin had an antagonistic effect. Although we observed an activation of pro-apoptotic genes, the fold enrichment of cell death transcripts was much higher in cisplatin alone compared to cisplatin with CBD. We suggest that one of the reasons might be an increased basal activity of pro-survival pathways under CBD treatment due to its modulative role in carbohydrate and lipid metabolism.It was previously shown that serum starvation sensitized cancer cells to cisplatin while protecting normal cells . In normThe inhibition of the glycolytic pathway in cancer cells could lead to stress overload due to constant proliferative signaling from oncogenes . Cancer Glycolysis is activated by the PI3K pathway and its downstream target, AKT, which can trigger cell hypertrophy, enhance glycolysis, and activate cell survival . AKT mayPrevious studies showed that many cancer cells have higher glucose uptake rates and rely on glycolysis and lactic acid fermentation even when oxygen is present, known as the Warburg effect . The PI3The potential of modulating tumor metabolism for therapeutic purposes has not been extensively studied. However, several methods for influencing tumor metabolism could have therapeutic implications. These methods include inhibiting HIF-1α from decreasing angiogenesis, re-establishing p53 to activate apoptosis, and suppressing the PI3K/AKT/mTOR signaling pathway to inhibit cell growth and proliferation ,104. AddBased on our results, we suggest that the strong synergistic effect between ISS and CBD was due to the activation of AKT signaling and simultaneous inhibition of glycolysis and oxidative phosphorylation , which pThe capacity of cisplatin to create DNA crosslinks results in the activation of cell cycle checkpoints. Cisplatin leads to a temporary pause in the S phase of the cell cycle, aided by p16. Cdc2-cyclin A or B kinase is strongly inhibited, resulting in more noticeable G2/M cell cycle arrest. In addition, cisplatin triggers the activation of ATM and ATR, which includes the phosphorylation of the p53 protein . Our resIn summary, our work used cell viability outcomes and mRNA expression evaluations to discern treatment synergies within CRC cell lines, shedding light on the underlying molecular mechanisms of these drug interactions. Nevertheless, additional studies are imperative to support our claims. We acknowledge that the drawback of our study is that it was based on the analyses of cell viability and mRNA expression alone. To solidify our hypotheses, additional research is needed. Future studies will have to analyze gene expression at the protein level in specific pathways, conduct apoptosis assays for various treatments, and employ animal models featuring intermittent fasting and drug combinations. Such an approach could yield more dependable data with potential clinical implications.


Ion channels play a crucial role in a wide range of biological processes, including cell cycle regulation and cancer progression. In particular, the transient receptor potential (TRP) family of channels has emerged as a promising therapeutic target due to its involvement in several stages of cancer development and dissemination. TRP channels are expressed in a large variety of cells and tissues, and by increasing cation intracellular concentration, they monitor mechanical, thermal, and chemical stimuli under physiological and pathological conditions. Some members of the TRP superfamily, namely vanilloid (TRPV), canonical (TRPC), melastatin (TRPM), and ankyrin (TRPA), have been investigated in different types of cancer, including breast, prostate, lung, and colorectal cancer. TRP channels are involved in processes such as cell proliferation, migration, invasion, angiogenesis, and drug resistance, all related to cancer progression. Some TRP channels have been mechanistically associated with the signaling of cancer pain. Understanding the cellular and molecular mechanisms by which TRP channels influence cancer provides new opportunities for the development of targeted therapeutic strategies. Selective inhibitors of TRP channels are under initial scrutiny in experimental animals as potential anti-cancer agents. In-depth knowledge of these channels and their regulatory mechanisms may lead to new therapeutic strategies for cancer treatment, providing new perspectives for the development of effective targeted therapies. According to the World Health Organization (WHO), cancer comprises a large group of diseases characterized by the rapid growth of abnormal cells that invade neighboring parts of the body, with the capacity to spread to other organs. More than 100 types of cancer have been identified so far . The leaIon channels are integral membrane proteins containing an aqueous pore that facilitates the mobilization of certain ions between cell compartments playing an essential role in cell functioning . They reIncreasing awareness of the ion channel’s role in tumor progression has led to the consideration of cancer as a channelopathy, or as a disease characterized by a profound alteration in ion channel function . A speci2+), potassium (K+), and sodium (Na+) channels are examples of channels involved in tumor growth and metastasis [2+ is an important second messenger whose intracellular levels control several downstream signaling pathways, such as apoptosis and cell migration, functions that typically affect cancer growth [Calcium , canonical (TRPC1–7), melastatin (TRPM1–8), mucolipins TRPML1–3), non-mechanoreceptor potential C , polycystins (TRPP1–5), and vanilloid TRPV1–6)  Figure . Except , non-mec  Figure TRP channels, almost ubiquitously expressed in cells and tissues, play important roles in health and disease ,58, inclTRPA1, the only member of the ankyrin subfamily, is a polymodal channel that can be activated by a wide variety of noxious external stimuli, such as irritants, often associated with pain and inflammation, and intense cold ,61,62,632+-dependent antiapoptotic pathways by promoting ROS resistance [In cancer, TRPA1 activation in prostate tumor endothelial cells acts as a modulator of angiogenesis, since its activation promotes neovascularization, endothelial cell migration, and tubulogenesis in vitro in models of human prostate cancer . TRPA1 asistance . Specifisistance . Furthersistance .2O2, an observation in line with previous findings that have indicated the channel as an oxidative stress sensor and amplifier, a function that might affect tumor cells and proliferation [The TRPA1 protein has been identified in benign human skin lesions , in cutaneous thin (pT1) and thick (pT4) melanomas, and in two different melanoma cell lines (SK-MEL-28 and WM266-4) . In sampferation .2+-dependent NADPH oxidase-1 (NOX1), amplified the oxidative stress to sustain pain signals [Recent studies have underlined the role of TRPA1 in cancer pain. In a mouse model of cancer induced by the subcutaneous inoculation of melanoma B16-F10 cells, neuronal TRPA1 has been proposed to mediate mechanical and cold hypersensitivity and thigmotaxis behavior . However signals ,92. Schw signals .Pain is also a recurrent symptom of cancer that becomes more frequent and debilitating in the presence of bone metastases, which are a common consequence of many primary tumors, including breast cancer . A promi3) [2+ ions into the cytoplasm, leading to an increase in the intracellular Ca2+ concentration. This Ca2+ influx triggers downstream signaling pathways and modulates the activity of various enzymes and transcription factors, ultimately influencing cell function. Aberrant TRPC channel activity has been associated with several diseases and pathological conditions, including cardiovascular disorders, neurodegenerative diseases, and cancer. Therefore, TRPC channels have emerged as potential therapeutic targets, and efforts are underway to develop drugs that modulate their activity for the treatment of these conditions.There are seven known members of the TRPC family, from TRPC1 to TRPC7. TRPC channels are widely expressed in many tissues and cells, including neurons, muscle cells, and epithelial cells. TRPC channels are involved in various physiological processes, such as sensory perception, smooth muscle contraction, hormone secretion, and cell migration . TRPC ch3) ,99. UponTRPC1 is expressed in various types of cancer, including breast, pancreatic, lung, and glioblastoma multiforme. Its dysregulation has been proposed as a prognostic marker for some types of cancer , includi2+ signaling has been implicated in glioma pathogenesis and cell biology influencing cell proliferation, migration, invasion, and angiogenesis [2+-permeable channels, have been shown to regulate Ca2+ influx in glioma cells [2+ influx through TRPC1 channels activates downstream signaling pathways, leading to cell proliferation and survival [2+ entry (SOCE) [Aberrant Caogenesis . TRPC1 cma cells . Increassurvival . A loss y (SOCE) .2+ signaling can activate proteases and matrix metalloproteinases, facilitating the breakdown of the extracellular matrix and promoting glioma cell invasion [TRPC1 channels have been found to promote glioma cell migration through their association with focal adhesion proteins and cytoskeletal rearrangement. Furthermore, TRPC1-mediated Cainvasion . TRPC1 cinvasion .2+ influx has been suggested as an endothelial cell attraction factor in prostate cancer, thus promoting angiogenesis [The TRPC3 protein might contribute to the development of tumor senescent phenotypes. Its downregulation in stromal cells promotes cellular senescence, sustaining inflammation and tumor growth in vivo . TRPC3-mogenesis ,113. TRPogenesis . The TRPogenesis .2+/calmodulin-dependent protein kinase beta/adenosine monophosphate-activated protein kinase alpha/mechanistic target of rapamycin (CaMKKβ/AMPKα/mTOR) pathway [2+ concentration and mesenchymal biomarker expression, promotes cell migration, invasion, and proliferation [An aberrant Wnt/β-catenin signaling cascade facilitates cell renewal, proliferation, and differentiation in several cancer types . The act pathway . It has  pathway . The TRP pathway . TRPC5 oferation .+/Ca2+ exchanger 1 (NCX1), and TRPC6 activation regulates TGFβ, thus inducing the formation of a TRPC6/NCX1 molecular complex [Growing evidence has reported that the pattern of the expression of TRPC6 proteins is upregulated in several pathophysiological conditions, including cancer. TRPC6 has been found to be overexpressed in breast cancer biopsy tissues compared to normal breast tissues . Human b complex . The exp complex . An incr complex .TRPM channels are a family of ion channels that regulate sensory perception, cellular homeostasis, and signal transduction. TRPM channels respond to a broad array of stimuli, including temperature, touch, pain, osmolarity, and chemical signals. They are expressed in various tissues and cell types throughout the body, highlighting their importance in numerous physiological functions. TRPM channels have gained significant attention in the field of cancer research due to their potential involvement in tumor progression and metastasis. Several members of the TRPM channel family have been implicated in cancer development and are investigated as potential therapeutic targets ,127.2+/calmodulin-dependent protein kinase II (CaMKII), which facilitates the binding of CaMKII with protein kinase B (AKT) and activates AKT, promoting melanoma cell colony formation, mobility, and an increase in tumor growth [TRPM1 gene expression has been identified in benign nevi, dysplastic nevi, and cutaneous melanomas, with a negative association between its presence and melanoma aggressiveness ,129. In r growth .TRPM2 is highly expressed in many human cancers , where its activation increases malignant cell survival . The mod2+ influx and decreases activity, autophagy, antioxidant response, and mitochondrial function, thus impairing tumor cell survival [2+ influx influences the release of inflammatory mediators that promote tumor growth and angiogenesis.Channel activation also modulates oxidative stress production. TRPM2 opening modulates the hypoxia-inducible transcription factor 1/2α (HIF-1/2α) signaling cascade, including proteins involved in oxidant stress, glycolysis, and mitochondrial function in neuroblastoma xenograft models . TRPM2 isurvival ,140,141.survival . Additio2+ signaling and homeostasis [2+ influx, which elicits the activation of CaMKII, 5′-AMP-activated protein kinase (AMPK), and Unc-51-like autophagy-activating kinase 1 (ULK1), as well as the formation of phagophore [TRPM3 plays pleiotropic roles in cellular Caeostasis . TRPM3 heostasis ,145,146.eostasis . An incragophore .MiR-204 is an intron micro-RNA (miRNA) located between exons 7 and 8 of the TRPM3 gene. A reduction in miR-204 induced by the higher methylation of host gene TRPM3 in gliomas can promote cell migration and enhance cell stemness . It has In physiological conditions, TRPM4 controls cell migration . It reguTRPM4 channel expression has been described in several cancers, including prostate ,156,157,2+), thus assuming a relevant role in epithelial Mg2+ transport and active Mg2+ absorption, especially in the gut and kidneys [TRPM6 is mostly expressed in the kidneys, distal small intestine, and colon . The TRP kidneys . Hypomag kidneys , and it  kidneys . The dow kidneys .TRPM8, also defined as a “cold receptor”, as it is activated by chemical cooling agents (such as menthol) , exhibitTRPM8 involvement in the death and apoptosis of bladder cancer cells is due to the modulation of mitochondrial activity ,177. It The endolysosomal TRPML subfamily consists of the TRPML1, TRPML2, and TRPML3 proteins. TRPMLs share roughly 40% of amino acid sequence similarity and have important roles in ion homeostasis, membrane trafficking, exocytosis, and autophagy ,184. TwoTRPML1, mainly located in lysosomes, promotes cation efflux into the cytosol , thus coRecently, an increased TRPML1 expression level has been correlated with poor clinical outcomes of pancreatic ductal adenocarcinoma patients , signifiWhile TRPML1 is mainly localized in late endosomes and lysosomes in all tissues, TRPML2 is primarily expressed in myeloid and lymphoid cell lineages as recycling endosomes . Due to A link between TRPML2 expression and cancer has been investigated in different tumor types. Overall, a pro-tumorigenic role of TRPML2 has recently been proposed . TRPML2 Differing from TRPML1, which is ubiquitously expressed in all tissues, TRPML3 is mainly expressed in specific organs, such as the kidneys, lungs, back skin, and thymus ,202. TRP2+ release, wound healing, and cell invasion, which collaboratively diminishes the SMAD family member 4 (Smad4), STAT3, SNAIL, SLUG, and TWIST expression.The TRPP family consists of three members: TRPP2, TRPP3, and TRPP5. TRPP2 has been demonstrated to be an active coordinator in TRP channel heteromerization. The physical interaction of TRPP2, also known as polycystin-2 (PKD2), with polycystin-1 (PKD1) was identified 26 years ago . A study2+, Na+, and K+ across the cell membrane. TRPV channels are characterized by their sensitivity to multiple physical and chemical stimuli, including temperature, pH, mechanical stress, and various endogenous and exogenous compounds.TRPV channels are a group of ion channels that play crucial roles in sensory signaling and are involved in a wide range of physiological processes. These channels are named after their founding member, vanilloid 1 (TRPV1), which deorphanized the so-called receptor for capsaicin, the spicy ingredient of chili peppers ,210. TRPTRPV channels have been increasingly involved in cancer development and progression. Emerging evidence suggests that these channels play significant roles in various aspects of cancer biology, including tumor cell proliferation, migration, invasion, angiogenesis, and resistance to therapy. The dysregulation of TRPV channels in cancer suggests their potential as therapeutic targets. Modulating the activity of TRPV channels could help to inhibit tumor growth and metastasis and enhance the efficacy of existing therapies.2+ from these intracellular stores. The capsaicin-induced stimulation of TRPV1 triggers the apoptosis of human urothelial cancer cells via the activation of the ataxia telangiectasia mutated/CHK2/p53DNA damage response and Fas/CD95-mediated apoptotic pathways [2+ signaling. TRPV1 is functionally expressed in human esophageal squamous cells, and thermo-TRPVs might play an important role in the development of esophageal squamous cells [The TRPV1 channel is the selective molecular target of the vanilloid capsaicin, but it can also be gated by acid (pH < 6.5), ethanol, and heat ,211,212.pathways . In tumous cells . It has 2+/CaMKKβ/AMPK pathway, and its downregulation has been associated with poor survival in human gastric cancer patients. Thus, TRPV1 upregulation and its downstream signaling may represent promising targets for gastric cancer prevention and therapy [An elevated TRPV1 expression has been proven in squamous cell carcinoma of the human tongue, lung cancer, and breast cancer ,218,219. therapy . Little While TRPV1 expression is primarily localized to the plasma membrane , TRPV2 i2+ level. In prostate cancer, TRPV2 overexpression is also correlated with castration-resistant phenotype and metastasis [In multiple myeloma patients, TRPV2 overexpression is associated with bone tissue damage and a poor prognosis. A loss or inactivation of TRPV2 also increases glioblastoma cell proliferation and provokes resistance to CD95-induced apoptotic cell death . TRPV2 mtastasis . Recent tastasis . TRPV2 otastasis .2+-permeable nonselective cation channel broadly expressed in skin keratinocytes, along with oral and nasal epithelia [TRPV3 is a Capithelia . Althougpithelia ,231. TheThe TRPV4 channel is a non-selective cation channel that is extensively expressed in several tissues, where it acts as a molecular sensor and a transducer that regulates a variety of functional activities ,235,236.An abnormal TRPV4 expression is linked to gastric, liver, pancreatic, colorectal, lung, and breast cancers ,240. The2+-selective ion channel widely expressed in many tissues, including urinary bladder and kidney, where it acts as a gatekeeper of active Ca2+ reabsorption [TRPV5 is a Casorption ,249. Altsorption .2+ deficiency triggers abnormal colonic growth and increases colon cancer risk with well-known mechanisms [2+ homeostasis. There was a presence of TRPV5 and TRPV6 in sporadic parathyroid adenomas and normal parathyroid glands co-localized with Ca2+-sensing receptors on the membrane surface, although immunoreactivity was present in the cytosol and around the nuclei. An increased expression of both channels in adenoma compared to normal glands implies a relationship between cell Ca2+ signaling and pathological processes [2+-induced IGF signaling is mediated by TRPV5-associated membrane depolarization. These results disclose a novel signaling mechanism that results in abnormal epithelial proliferation associated with Ca2+ deficiency [Cachanisms . The parrocesses . A recenficiency . A reducficiency .2+ channel widely expressed by the epithelial tissues of many tissues, such as the intestines, kidneys, placenta, epididymis, and exocrine glands [TRPV6 is a membrane Cae glands . The expe glands ,256. TRPe glands , and in e glands .2+ influx maintains the inactive state by targeting IGF-mediated AKT-TOR and ERK signaling. A recent study showed that, in zebrafish epithelia and human colon carcinoma cells, TRPV6 diminishes intracellular Ca2+ levels and activates protein phosphatase 2A (PP2A), which downregulates IGF signaling and endorses the inactive state. This suggests that TRPV6 mediates a constitutive Ca2+ influx into epithelial cells to continuously suppress growth factor signaling and maintain the quiescent state [2+ signaling [Mechanistically, TRPV6-mediated Cant state . In addint state . One stuignaling   aims to investigate the role of the TRPA1 channel in oral cancer pain. In particular, the study evaluates the mechanical (von Frey testing) and chemical (capsaicin and AITC) response in oral cancer patients and compares their sensitivities to healthy subjects. Another interventional study (NCT04923412) aims to investigate TRPA1 and TRPV1 expression in non-small cell lung cancer (NSCLC) patients before and after surgery to quantitatively measure injuries of the vagus nerve during mediastinal lymph node dissection. A lobectomy with lymph node dissection represents the standard surgical method. However, about 60% of patients experience postoperative chronic cough during the first year after surgery. In this trial, the investigators provide a new basis for safe and effective new surgical techniques, as well as possible new biomarkers (TRPA1 and TRPV1) related to postoperative cough and pulmonary complications.Regarding TRPV1, two cancer clinical trials have been reported so far. A phase III study (NCT04572776) is currently investigating the role of TRPV1 in intractable cancer pain through a single epidural administration of resiniferatoxin vs. the standard of care. A small prospective study (NCT02666976) evaluates the modulation of inflammatory biomarkers, including TRPV1, in patients affected by gastrointestinal subepithelial tumors. One observational study (NCT05507879) evaluates the role of TRPC6 as a predictive biomarker of chemotherapy-related cardiac toxicity in patients with breast cancer. A small interventional phase I clinical trial (NCT01578564) tests the safety and tolerability of a TRPV6 inhibitor in subjects with advanced ovarian cancer or other cancers known to overexpress the TRPV6 channel.Over the years, researchers have revealed that TRP channels play crucial roles in cellular homeostasis and represent an efficient and timely interface between the environment and the body. The dysregulation of TRP channels has been associated with numerous cancer types, highlighting their potential as therapeutic targets ,23.. The


GPER knockout mice, significant progress has been made in understanding the function of GPER. In this review, the tissue and cellular localizations, endogenous and exogenous ligands, and signaling pathways of GPER are systematically summarized in diverse physiological and diseased conditions. This article further emphasizes the role of GPER in vascular pathology and physiology, focusing on the latest research progress and evidence of GPER as a promising therapeutic target in hypertension, pulmonary hypertension, and atherosclerosis. Thus, selective regulation of GPER by its agonists and antagonists have the potential to be used in clinical practice for treating such diseases.Objective: Estrogen is indispensable in health and disease and mainly functions through its receptors. The protection of the cardiovascular system by estrogen and its receptors has been recognized for decades. Numerous studies with a focus on estrogen and its receptor system have been conducted to elucidate the underlying mechanism. Although nuclear estrogen receptors, including estrogen receptor-α and estrogen receptor-β, have been shown to be classical receptors that mediate genomic effects, studies now show that GPER mainly mediates rapid signaling events as well as transcriptional regulation via binding to estrogen as a membrane receptor. With the discovery of selective synthetic ligands for GPER and the utilization of  However, the inhibition of these receptors hardly abolishes the protective effect of estrogen, indicating that a novel, unidentified receptor that was yet to be discovered was the actual mediator. This was later characterized as the G protein-coupled estrogen membrane receptor (GPER) [Cardiovascular disease (CVD) is currently the primary cause of death worldwide. In 2020, an estimated 19.05 million people died from CVD globally and 207.1 of 100,000 people died from hypertension and stroke, the prevalence of which was higher than that of cancer and chronic diseases . The incGPER is present throughout the cardiovascular system, implying a critical role in maintaining cardiovascular physiological function. GPER activation by G1 restored glucose homeostasis and reduced body weight in multiple preclinical male and female models associated with obesity, diabetes, and metabolic abnormalities . In blooGPER was initially identified as an orphan receptor with no known ligand, and belongs to the superfamily of 7-transmembrane G protein-coupled receptors (GPCRs). It was later named GPR30 based on the sequential numbering scheme for orphan receptors . GPR30 wGPER is widely distributed in various tissues of the human body. GPER first attracted considerable attention in reproduction as an estrogen receptor that functioned in the breast ,20, ovarThe cellular localization of GPER has been a subject of much debate since its discovery. Most GPCRs are lipophilic, localized to the plasma membrane , and theGPER knockout mice showed that the loss of GPER increases blood pressure, eliminates E2-stimulated insulin release, and affects glucose metabolism, thereby leading to visceral obesity [GPER knockout mice showed an early increase in atherosclerotic progression [GPER-deficient mice exhibited impaired left ventricular cardiac function, left ventricular enlargement, and impaired systolic and diastolic function [GPER deletion caused left ventricular dysfunction and adverse remodeling in mice. Further specific spectral analysis showed that mitochondria-related genes were enriched in females of GPER KO mice, while genes related to the inflammatory response were enriched in male GPER KO mice [Estrogen is a steroid hormone which is secreted mainly from the ovaries, and four natural estrogens in females are biosynthesized: estrone (E1), 17β-estradiol (E2), 17α-estradiol (E2), and estriol (E3) ,54. Amon obesity ,58. Hype obesity . In addigression . In the function , or redufunction . Wang et KO mice . This in KO mice ,64. GPERα and ERβ [s protein and a pertussis toxin (PTX)-sensitive Gαi/o protein, which activate intracellular signaling cascades using cAMP and Ca2+ as second messengers [α also activates PI3K, which does not involve EGFR transactivation [GPER binds to estradiol with high affinity and was thought to mediate rapid non-genomic effects distinct from those of ER and ERβ ,18,65,66ssengers ,17,65,66ssengers . Throughssengers , inducesssengers , and prossengers , and prossengers ,72. In assengers ,74,75,76ssengers , apoptosssengers ,79, and ssengers ,81. It stivation ,83. Notativation . In additivation ,86,87, ttivation ,89,90,91As a vasoactive steroid, aldosterone has recently attracted much interest, not only due to its great importance in the regulation of salt and water balance, but also because of its key role in cardiovascular metabolism and function. The classical effects of aldosterone are mediated by the activation of mineralocorticoid receptors (MRs) and subsequent transcriptional regulatory mechanisms . HoweverDespite accumulating evidence for aldosterone’s actions through GPER, there remains significant speculation that aldosterone directly binds to GPER ,98,99. HThe mechanism of the downstream signal pathways via the interaction between aldosterone and GPER are not well understood. Recent studies have revealed that aldosterone trans-activates EGFR via GPER in adult rat ventricular myocytes, followed by reactive oxygen species (ROS) production and PI3K/AKT-dependent pathway activation, ultimately resulting in the stimulation of cardiac sodium/bicarbonate cotransporter (NBC) ,103. In cyp11b2 mRNA through the activation of GPER, which is the last rate-limiting enzyme for aldosterone synthesis and up-regulates aldosterone secretion. This aldosterone-induced aldosterone production may, under certain circumstances, such as volumetric hypotension, physiologically increase aldosterone production to maintain blood pressure [β was stated to inhibit aldosterone synthase expression and aldosterone synthesis through the finding that the selective blockade of ERβ markedly increased aldosterone expression, which was mimicked by the GPER-selective agonist G-1 and abolished by co-treatment with either the GPER antagonist G-15 or a selective protein kinase A inhibitor [β [β, and after menopause, the loss of estrogen can lead to an increase in blood pressure through GPER-mediated aldosterone release [GPER has also been shown to regulate aldosterone synthesis. Aldosterone increases the expression of pressure . In contnhibitor . Thus, ebitor [β . In prem release , which mα/β at concentrations up to 10 μM [In addition to endogenous steroid hormones, many synthetic estrogenic compounds, as well as phytoestrogenic compounds of natural origin, have also been shown to bind GPER to exert their effects. Ligand-binding studies revealed that GPER binds E2 with much weaker binding affinities at concentrations from Kd = 3 to 6 nM  than clato 10 μM , which pto 10 μM . Notablyto 10 μM . Similarto 10 μM ,117. Thito 10 μM . Therefoα/β similar to G-1 [After the discovery of G-1, researchers subsequently synthesized the first GPER antagonist G-15  and its r to G-1  in the ar to G-1 . Two addr to G-1 . The disr to G-1 ,123,124,r to G-1 ,126, atrr to G-1 ,128, kepr to G-1 ,124. Lasr to G-1 ,130,131,r to G-1 , Equol [r to G-1 , Oleuropr to G-1 , Hydroxyr to G-1 , Resverar to G-1 ,136,137,r to G-1 , Tectorir to G-1 , Zearaler to G-1 ,141, andr to G-1 . The strHypertension (HTN) is a multifactorial disease, defined as a systolic blood pressure (SBP) greater than 130 mmHg or diastolic blood pressure (DBP) higher than 80 mmHg ,147, andα [Animal experiments proved that GPER could mediate vasodilation in rats and reduce blood pressure in normotensive rats . In the α , but GPEα . 2+ levels through transient receptor potential channel (TRPC) isoforms, leading to the phosphorylation of calmodulin-dependent protein kinase 2 (CaMKKβ), AMPK, and calmodulin-dependent protein kinase II (CaMKIIα), which in turn increase the expression of eNOS through the activation of histone deacetylase 5 (HDAC5) and the Kruppel-like factor 2 (KLF2) signaling pathway [2+ influx channel opening and myosin light chain phosphatase (MLCP) activation [2+ channel opening via the endothelium-dependent PKG pathway and the endothelium-independent PKA and Epac/Rap1 pathways , defined as elevated mean pulmonary arterial pressure , is characterized by the pathological elevation of pulmonary artery pressure leading to remodeling of the right ventricle and ultimately to severe right heart failure . The preroblasts . In femaroblasts .GPER-deficient female mice, and aged male GPER-deficient mice exhibited dyslipidemia and inflammation [α and ERβ had no effect on the estrogen-mediated attenuation of LDL trans-endocytosis. Instead, estrogen’s effect on LDL transcytosis was blocked by the depletion of GPER [α or ERβ.Atherosclerosis is characterized by plaque formation leading to the stenosis of the arterial wall, and is associated with a long-term state of dyslipidemia and vascular inflammation . Correlaammation . In a paammation , and treammation . The effammation . Meanwhiammation , suggestammation . Estroge of GPER . Apparenα both reduced medial vascular hypertrophy and inflammatory processes after carotid artery ligation [αER knockout mice but not in GPER knockout mice [Further evidence for the anti-inflammatory effects of GPER in blood vessels and inflammatory cells was obtained in in vitro studies. In human umbilical vein vessels, GPER activation attenuated the TNF-α-induced upregulation of proinflammatory proteins, which was reversed by G-15 . Howeverligation . The selligation . Remarkaligation . The selligation . In addiligation . In autoout mice . In a heout mice . Indeed,out mice  and macrout mice . ApoE-deficient mice, GPER attenuated endothelial cell apoptosis, which reduced plasma lipid levels and prevented atherosclerotic plaque formation [GPER deletion [It is well accepted that the maintenance of the integrity and physiological function of endothelial cells is vital for vascular function, the dysregulation of which is the initial step for the development of atherosclerosis . In a raormation . The kaeormation . The inhormation ,196. G-1deletion . In cultdeletion . Estrogedeletion . In addideletion . ThroughAs an estrogen receptor, GPER is widely distributed throughout the body, and its beneficial effects have been demonstrated in cardiovascular diseases. The binding of GPER to aldosterone has been demonstrated, but the exact mechanism remains to be elucidated. In addition, many natural and synthetic molecules are also able to activate or inhibit GPER, but further studies are needed to determine how they affect disease and health through GPER. In blood vessels, GPER is indispensable in the physiological functions of endothelial cells and smooth muscle cells. Given that the incidence of CVD is lower in premenopausal women than in age-matched men, it is likely that estrogen plays a crucial role in this disparity . However


We designed this study to determine whether there is a link between vitamin D levels and sensitivity to thyroid hormone and to provide a new perspective for studying the relationship between vitamin D and thyroid disease.Our study included 8,126 participators from the National Health and Nutrition Examination Survey (NHANES) database between 2007 and 2012. We used weighted multiple linear regression models to enquire the connection between serum vitamin D levels and thyroid hormone sensitivity indicators, including the following: Thyroid-stimulating hormone index (TSHI), Free Triiodothyronine/Free thyroxine (FT3/FT4), Thyroid Feedback Quantile-based Index (TFQI), and Thyrotroph Thyroxine Resistance Index (TT4RI). Finally, we used constrained cubic splines to explore possible nonlinear relationships. All data cleaning and statistical analyses were performed using R software.The final Results were reached after adjusting for various confounding factors. We found a U-shaped relationship between TFQI and serum vitamin D, and the lowest TFQI appeared when the serum vitamin D concentration was 25.77ng/ml. However, an inverse U-shaped relationship was found between FT3/FT4 and vitamin D levels. When the serum vitamin D concentration was 25.43ng/ml, the ratio of FT3/FT4 was the highest.In the US population, our study concluded that FTQI and FT3/FT4 were U-shaped or inverse-U-shaped with serum vitamin D levels respectively after several adjustments. Therefore, FTQI and FT3/FT4 are considered indicators of the complex relationship between thyroid hormone resistance and vitamin D metabolism. In the future, more complex prospective investigations are needed to confirm these findings and find a causal link between them. In humans, Vitamin D2 is obtained by ingesting food, especially dietary vegetables, and taking oral supplements. Vitamin D3 can be obtained in several ways: synthesized by skin exposure to UV-B radiation, food intake such as various Marine fish, and oral supplements . After oIn clinical practice, we evaluate the thyroid function status of patients by measuring serum TSH, FT3 and FT4 levels. However, we cannot fully understand thyroid hormone homeostasis with these three indicators alone –9. IncreIn addition to bone health, vitamin D has many other functions, of which the two most important non-skeletal functions include: immunomodulatory and anti-inflammatory functions. Therefore, it may help prevent or improve inflammation and participate in the regulatory pathways of immune mediated tissue injury. The correlation between autoimmune thyroid disease and vitamin D was controversial. A systematic review exploring the relationship between autoimmune thyroid disease and vitamin D levels demonstrated that Grave’s disease and Hashimoto thyroiditis were both relevant to lower levels of vitamin D . However22.1This cross-sectional study used database from the NHANES, an ongoing study conducted by the Centers for Disease Control (CDC) since the 1980s, with the aim of assessing the nutritional and health status of American residents and developing health guidelines. We collected demographic, questionnaire, examination and laboratory information from three consecutive NHANES cycles from 2007 to 2012. As shown in 2.2Concentrations of Serum 25(OH)D were detected at the Division of Laboratory Sciences, National Center for Environmental Health, Centers for Disease Control and Prevention, Atlanta, GA, using a method called ultra-high performance liquid chromatography-tandem mass spectrometry. In our study, we first determined the classification nodes based on the guidelines of the Endocrine Society, and then divided the subjects into three categories according to the serum 25 (OH) D levels. Serum 25(OH)D concentrations <20 ng/mL was regarded as VD deficiency, serum 25(OH)D concentrations between 20 and 29.9 ng/ml was regarded as VD insuficiency and serum 25(OH)D concentrations≥ 30.0ng/mL was regarded as VD suficiency .2.3The TT3, FT3 and TT4 were detected using a competitive binding immunoenzymatic assay. The assay of Free T4 was a two-step enzyme immunoassay. The Free T4 assay was a two-step enzyme immunoassay. The detection of sensitive human thyroid stimulating hormone was performed by a two-site “ sandwich “ immunoenzyme test. TgAb and TPOAb (IU/mL) were assessed by a sequential two-step immunoenzymatic “sandwich” assay, while Tg levels (ng/ml) were detected by a simultaneous one-step “sandwich” assay.2.4FT3/FT4, TT4RI, TFQI and TSHI constitute thyroid hormone sensitivity indicators, and their calculation formulas are as follows:(1) FT3/FT4 =FT3/FT4 (2) TT4RI =FT4 (pmol/L) ∗ TSH (mIU/L) (3) TFQI = cdfFT4 − (1−cdfTSH) (4) TSHI = ln TSH (mIU/L) + 0.1345∗ FT4 (pmol/L) Sensitivity of peripheral tissue to thyroid hormone raised with the augment of FT3/FT4 ratio. The central thyroid hormone sensitivity decreased with the augment of TSHI and TT4RI index. TFQI is an indicator used to assess the central thyroid hormone sensitivity, and it was computed based on the empirical cumulative distribution function, with values ranging from -1 to 1. Referring to conclusions drawn from the former studies, we defined TFQI=0 as the reference standard, indicating that the HPT axis had a normal sensitivity to FT4 changes. A value less than zero indicated that the HPT axis had a higher sensitivity to FT4 variations, while a value greater than zero represented that human HPT axis responded slowly to FT4 variations .2.5In accordance with previous similar studies, we believed that the below variables may affect our statistical results, so we identified them as confounders in this study: gender, age, race, educational background, marriages, Family income to poverty ratio, drinking behavior, smoking behavior, physical activities, BMI, diabetes, hypertension, glycohemoglobin, albumin (g/dL), serum calcium(mg/dL), blood urea nitrogen (mg/dL), cholesterol(mg/dL), triglycerides(mg/dL), phosphorus (mg/dL), serum uric acid (mg/dL)and creatinine (mg/dL) . The rac2.6Following NHANES’s suggestion, sample weights based on stratified, multi-stage probabilistic sampling design were used for all analyses. Continuous variables were displayed as mean standard deviation, and categorical variables were shown as percentage of subjects. According to different levels of serum vitamin D, the study subjects were divided into three groups. P values were calculated using the weighted chi-square test and the linear regression model. We used a weighted multiple linear regression model to investigate the relationship between serum vitamin D levels and sensitivity to thyroid hormone indices in three different models. Model 1 adjusted no variable. Model2 adjusted for the following variables: age, race, gender, family income to poverty ratio, educational background, marriages. Model 3 further adjusted for age, race, gender, family income to poverty ratio, educational background, marriages, physical activities, BMI, smoking behavior, drinking behavior, diabetes, hypertension, glycohemoglobin, serum urea nitrogen, cholesterol, triglycerides, serum calcium, phosphorus, creatinine, serum uric acid, and albumin.Restricted cubic spline with three knots at the 10th, 50th, 90th were conducted to explore the nonlinear relationship between serum vitamin D levels and sensitivity to thyroid hormone indices after adjusting for cofounding factors, including age, race, gender, educational background, marriages, family income to poverty ratio, physical activities, BMI, smoking behavior, drinking behavior, diabetes, hypertension, glycohemoglobin, serum urea nitrogen, albumin, phosphorus, serum calcium, cholesterol, triglycerides, serum uric acid, and creatinine.All analyses in the study were weighted to represent the US population. When the P-value less than 0.05, we considered the difference to be statistically significant. We Performed all statistical analyses using R (version 4.2.2).33.1The weighted demographic and socioeconomic characteristics grouped into three categories based on serum vitamin D levels were presented in Several demographic factors were found to differ significantly among the three groups. Participants in the insufficient and adequate groups had a higher probability to be seniors, people with higher education, non-Hispanic white, married, and have a higher ratio of family income to poverty than those in the deficient group. At the same time, referring to smoking behavior, physical activities, and drinking behavior, the three groups differed significantly.Comparison of clinical characteristics among the three groups also differ significantly in most of the factors. Normal-weight participants were more likely to be in the Sufficiency group. Nearly 12.5% of the deficiency group had diabetes, compared to 8.0% and 9.8 in the Sufficiency and insufficiency groups. Meanwhile, there were significant differences in glycohemoglobin, albumin, serum calcium, blood urea nitrogen, cholesterol, creatinine, triglycerides, serum uric acid, and phosphorus among the three groups. In addition, we also found significant differences in TSHI, FT4, FT3, and FT3/FT4 levels among the three groups.3.2We found that there was no linear correlation between FT3/FT4, TSHI, TFQI, TT4RI and serum vitamin D levels after adjusting confounding factors. The outcomes of weighted multiple linear regression analyses are shown in The restricted cubic splines were further adopted to explore the nonlinear association between FT3/FT4, TSHI, TFQI, TT4RI and vitamin D levels. Outcomes are shown in 4Our study evaluated the connection between thyroid hormone sensitivity and vitamin D levels using The NHANES database. There are many studies on the role of vitamin D in the occurrence of Thyroid diseases, but we still cannot reach a completely unified conclusion. In our view, we are the first team to study the associations between vitamin D and thyroid hormone sensitivity. TFQI and FT3/FT4 were found to be nonlinearly correlated with vitamin D levels, meaning that both the central and peripheral thyroid hormone sensitivity were nonlinearly correlated with vitamin D. We found a U-shaped relationship between TFQI and vitamin D, while an inverse U-shaped relationship between FT3/FT4 and vitamin D. There was no consistent association between vitamin D levels and TSHI, TT4RI.Serum vitamin D status is closely related to many thyroid diseases. The research of Chao et al. shows that the level of 25(OH)D in the non-HT group was higher than that in the HT group . SimilarOur study showed statistically significant differences in FT3 and FT4 among groups with different vitamin D levels. But TSH levels between the three groups were not statistically significant. The conclusions of previous relevant studies were controversial. Sandeep et al. discovered a positive link between 25 (OH) D and FT3, FT4 levels, while TSH showed the opposite trend . HoweverTFQI was originally recommended by Laclaustra et al. to assess the extent of thyroid hormone resistance and its relationship with other diseases. Actually, for the reason that our team is the earliest to investigate the connection between thyroid hormone sensitivity and serum vitamin D levels, there is little comparative data available. In our study, we found a U-shaped relationship between TFQI and serum vitamin D levels, while an inverse U-shaped relationship between FT3/FT4 and serum vitamin D levels. When serum vitamin D levels were more than 25.77ng/ml, the TFQI increased with the increase of vitamin D levels. The inflection point value of the curve between vit D and FT3/FT4 was 25.43 ng/ml. Vit D levels were negatively correlated with FT3/FT4when above this value. More recently, Experts have confirmed that a vitamin D concentration of 30-40 ng/mL is sufficient to maintain bone health, and higher levels of vitamin D do not provide additional benefits. Vitamin D levels above 40ng/mL were not found to have any further benefits for bone health. Similarly, in order to achieve good central and peripheral sensitivity at the same time, vitamin D levels must be maintained within the appropriate range, neither too high nor too low.The pituitary secreted thyrotropin, which stimulated the thyroid to produce thyroxine (T4) and triiodothyronine (T3), of which T4 was the main hormone produced and released by the thyroid, which was converted to T3 by the deiodination of deiodinase in the peripheral. Vitamin D was involved in this complex process to regulate TH levels in the body . ThyroidAlrefaie et al. proved that vitamin D3 was associated with increased levels of type 2 deiodinase (DIO2) and peripheral conversion of TH  in multiple tissues of diabetic rats, and vitamin D supplementation could reduce TSH values to normal levels in diabetic rats . A negatHowever, the relationship between vitamin D and Thyroid hormones may be mutual. At present, little is known about the effect of Thyroid hormones on vitamin D metabolism. Bouillon et al. found that 1,25-dihydroxyvitamin D3 (the active form of vitamin D) decreased in patients with hyperthyroidism, increased in patients with hypothyroidism, and maintained a relatively normal concentration of vitamin D after thyroid function returned to normal . This maThe major highlight of this survey is that we use the NHANES database, which includes data of participants from the whole nation. Due to the large enough sample capacity, we can get more trustworthy conclusion of the U shape relationship between vitamin D and thyroid hormone sensitivity. In addition, confounding factors were taken into account in our analysis. The primary shortcomings of this survey are listed below (1):We cannot speculate on the causal relationship between Thyroid hormone sensitivity and vitamin D levels because this is a retrospective cross-sectional study; (2) On account of the restrictions of NHANES data, some significant variables associated with vitamin D such as seasonal changes, geographical locations and light duration are not available; (3) Some variables such as physical activities, drinking behavior, and smoking behavior are self-judged and therefore the accuracy cannot be fully guaranteed.5In the US population, our study concluded that FTQI and FT3/FT4 were U-shaped or inverse-U-shaped with serum vitamin D levels respectively after several adjustments. Therefore, FTQI and FT3/FT4 are considered indicators of the complex relationship between thyroid hormone resistance and vitamin D metabolism. In the future, more complex prospective investigations are needed to confirm these findings and find a causal link between them.The original contributions presented in the study are included in the article/The studies involving humans were approved by The NCHS Research Ethics Review Board. The studies were conducted in accordance with the local legislation and institutional requirements. The participants provided their written informed consent to participate in this study. Written informed consent was obtained from the individual(s) for the publication of any potentially identifiable images or data included in this article.Concept and design: SC, XL, YZ. Data acquisition: WY, ZG. Retrieval and analysis of data: SC, WY. Drafting and interpretation of data: SC. Critical revision: XL, YZ. All authors read and approved the final manuscript.