Diverse modulatory effects of bibenzyls from Dendrobium species on human immune cell responses under inflammatory conditions.

Dendrobium plants are widely used in traditional Chinese medicine. Their secondary metabolites such as bibenzyls and phenanthrenes show various pharmacological benefits such as immunomodulation and inhibitory effects on cancer cell growth. However, our previous study also showed that some of these promising compounds (i.e., gigantol and cypripedin) also induced the expression of inflammatory cytokines including TNF in human monocytes, and thus raising concerns about the use of these compounds in clinical application. Furthermore, the effects of these compounds on other immune cell populations, apart from monocytes, remain to be investigated. In this study, we evaluated immunomodulatory effects of seven known bibenzyl compounds purified from Dendrobium species in human peripheral blood mononuclear cells (PBMCs) that were stimulated with lipopolysaccharide (LPS). Firstly, using flow cytometry, moscatilin (3) and crepidatin (4) showed the most promising dose-dependent immunomodulatory effects among all seven bibenzyls, determined by significant reduction of TNF expression in LPS-stimulated CD14+ monocytes. Only crepidatin at the concentration of 20 µM showed a significant cytotoxicity, i.e., an increased cell death in late apoptotic state. In addition, deep immune profiling using high-dimensional single-cell mass cytometry (CyTOF) revealed broad effects of Dendrobium compounds on diverse immune cell types. Our findings suggest that to precisely evaluate therapeutic as well as adverse effects of active natural compounds, a multi-parameter immune profiling targeting diverse immune cell population is required.

Secondary Metabolites in the Dendrobium heterocarpum Methanolic Extract and Their Impacts on Viability and Lipid Storage of 3T3-L1 Pre-Adipocytes.

Although many natural products have proven their potential to regulate obesity through the modulation of adipocyte biology, none of them has yet been approved for clinical use in obesity therapy. This work aims to isolate valuable secondary metabolites from an orchid species (Dendrobium heterocarpum) and evaluate their possible roles in the growth and differentiation of 3T3-L1 pre-adipocytes. Six compounds were isolated from the orchid's methanolic extracts and identified as amoenylin (1), methyl 3-(4-hydroxyphenyl) propionate (2), 3,4-dihydroxy-5,4'-dimethoxybibenzyl (3), dendrocandin B (4), dendrofalconerol A (5), and syringaresinol (6). Among these phytochemicals, compounds 2, 3, and 6 exhibited lower effects on the viability of 3T3-L1 cells, offering non-cytotoxic concentrations of ≲10 µM. Compared to others tested, compound 3 was responsible for the maximum reduction of lipid storage in 3T3-L1 adipocytes (IC50 = 6.30 ± 0.10 µM). A set of protein expression studies unveiled that compound 3 at non-cytotoxic doses could suppress the expression of some key transcription factors in adipocyte differentiation (i.e., PPARγ and C/EBPα). Furthermore, this compound could deactivate some proteins involved in the MAPK pathways (i.e., JNK, ERK, and p38). Our findings prove that D. heterocarpum is a promising source to explore bioactive molecules capable of modulating adipocytic growth and development, which can potentially be assessed and innovated further as pharmaceutical products to defeat obesity.

Anti-Inflammatory Activity of Oxyresveratrol Tetraacetate, an Ester Prodrug of Oxyresveratrol, on Lipopolysaccharide-Stimulated RAW264.7 Macrophage Cells.

Oxyresveratrol (OXY) has been reported for its anti-inflammatory activity; however, the pharmaceutical applications of this compound are limited by its physicochemical properties and poor pharmacokinetic profiles. The use of an ester prodrug is a promising strategy to overcome these obstacles. In previous researches, several carboxylate esters of OXY were synthesized and oxyresveratrol tetraacetate (OXY-TAc) was reported to possess anti-melanogenic and anti-skin-aging properties. In this study, in addition to OXY-TAc, two novel ester prodrugs of OXY, oxyresveratrol tetrapropionate (OXY-TPr), and oxyresveratrol tetrabutyrate (OXY-TBu), were synthesized. Results from the Caco-2-permeation assay suggested that synthesized ester prodrugs can improve the membrane-permeation ability of OXY. The OXY-TAc exhibited the most significant profile, then this prodrug was chosen to observe anti-inflammatory activities with lipopolysaccharide (LPS)-stimulated RAW264.7 macrophages. Our results showed that OXY-Tac significantly alleviated secretion of several pro-inflammatory mediators (nitric oxide (NO), interleukin-6 (IL-6), and tumor necrosis factor-alpha (TNF-α)), mitigated expression of enzyme-regulated inflammation (inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2)), and suppressed the MAPK cascades. Interestingly, the observed anti-inflammatory activities of OXY-TAc were more remarkable than those of its parent compound OXY. Taken together, we demonstrated that OXY-TAc improved physicochemical and pharmacokinetic profiles and enhanced the pharmacological effects of OXY. Hence, the results in the present study would strongly support the clinical utilities of OXY-TAc for the treatment of inflammation-related disorders.

Stemness-Suppressive Effect of Bibenzyl from Dendrobium ellipsophyllum in Human Lung Cancer Stem-Like Cells.

Cancer stem-like cells (CSCs) are key mediators driving tumor initiation, metastasis, therapeutic failure, and subsequent cancer relapse. Thus, targeting CSCs has recently emerged as a potential strategy to improve chemotherapy. In this study, the anticancer activity and stemness-regulating capacity of 4,5,4'-trihydroxy-3,3'-dimethoxybibenzyl (TDB), a bibenzyl extracted from Dendrobium ellipsophyllum, are revealed in CSCs of various human lung cancer cells. Culture with TDB (5-10 µM) strongly abolished tumor-initiating cells in lung cancer H460, H23, and A549 cells in both anchorage-dependent and anchorage-independent colony formation assays. Through the 3D single-spheroid formation model, attenuation of self-renewal capacity was observed in CSC-enriched populations treated with 1-10 µM TDB for 7 days. Flow cytometry analysis confirmed the attenuation of %cell overexpressing CD133, a CSC biomarker, in TDB-treated lung cancer spheroids. TDB at 5-10 µM remarkably suppressed regulatory signals of p-Akt/Akt, p-GSK3ß/GSK3ß, and ß-catenin corresponding to the downregulated mRNA level of stemness transcription factors including Nanog, Oct4, and Sox2. Moreover, the antiapoptosis Bcl-2 and Mcl-1 proteins, which are downstream molecules of Akt signaling, were evidently decreased in CSC-enriched spheroids after culture with TDB (1-10 µM) for 24 h. Interestingly, the diminution of Akt expression by specific siAkt effectively reversed suppressive activity of TDB targeting on the CSC phenotype in human lung cancer cells. These findings provide promising evidence of the inhibitory effect of TDB against lung CSCs via suppression of Akt/GSK3ß/ß-catenin cascade and related proteins, which would facilitate the development of this bibenzyl natural compound as a novel CSC-targeted therapeutic approach for lung cancer treatment.

Bibenzyl analogue DS-1 inhibits MDM2-mediated p53 degradation and sensitizes apoptosis in lung cancer cells.

BACKGROUND: Lung cancer is a leading fatal malignancy due to the high incidence of treatment failure. Dysfunction of the tumor suppressor p53 contributes to cancer initiation, progression, and therapeutic resistance. Targeting MDM2, a negative regulator of p53, has recently attracted interest in cancer drug research as it may restore tumor suppressive function. PURPOSE: The present study aimed to investigate the effect of 3,4-dihydroxy-5,4'-dimethoxybibenzyl (DS-1) on targeting MDM2 and restoring p53 function in lung cancer cells. METHODS: The efficacy of DS-1 alone or in combination with cisplatin in lung cancer cells was determined by MTT, nuclear staining, and annexin V/PI assay. The expression of apoptosis-related proteins was determined by western blot analysis. To evaluate the role of DS-1 on the stabilization and degradation of p53, cycloheximide chasing assay and immunoprecipitation were conducted, and the active form of p53 was investigated by immunofluorescent staining assay. To confirm and demonstrate the site interaction between DS-1 and the MDM2 protein, in silico computational analysis was performed. RESULTS: DS-1 exhibited a cytotoxic effect and sensitized lung cancer cells to cisplatin-induced apoptosis. DS-1 caused a significant increase in the cellular level of p53 protein, while the active form of p53 (phosphorylation at Ser15) was unaltered. DS-1 treatment in combination with cisplatin could enhance activated p-p53 (Ser15) and p53 downstream signaling (Bax, Bcl-2, and Akt), leading to a higher level of apoptosis. Immunoprecipitation analysis revealed that DS-1 decreased the p53-ubiquitin complex, a prerequisite step in p53 proteasomal degradation. Molecular docking simulation further evidenced that DS-1 interacts with MDM2 within the p53-binding domain by carbon-hydrogen bond interaction at Lys27, π-alkyl interactions at Ile37 and Leu30, and van der Waals interactions at Ile75, Val51, Val69, Phe67, Met38, Tyr43, Gly34, and Phe31. Treatment by DS-1 and cisplatin in patient-derivated primary lung cancer cells showed consistent effects by increasing cisplatin sensitivity. CONCLUSIONS: Our findings provide evidence that DS-1 is an MDM2 inhibitor and its underlying mechanism involves MDM2 binding and p53 induction, which may benefit the development of this compound for lung cancer treatment.

Four Novel Phenanthrene Derivatives with α-Glucosidase Inhibitory Activity from Gastrochilus bellinus.

Four new phenanthrene derivatives, gastrobellinols A-D (1-4), were isolated from the methanolic extract of Gastrochilus bellinus (Rchb.f.) Kuntze, along with eleven known phenolic compounds including agrostophyllin (5), agrostophyllidin (6), coniferyl aldehyde (7), 4-hydroxybenzaldehyde (8), agrostophyllone (9), gigantol (10), 4-(methoxylmethyl)phenol (11), syringaldehyde (12), 1-(4'-hydroxybenzyl)-imbricartin (13), 6-methoxycoelonin (14), and imbricatin (15). Their structures were determined by spectroscopic methods. Each isolate was evaluated for α-glucosidase inhibitory activity. Compounds 1, 2, 3, 7, 9, 13, and 15 showed higher activity than the drug acarbose. Gastrobellinol C (3) exhibited the strongest α-glucosidase inhibition with an IC50 value of 45.92 µM. A kinetic study of 3 showed competitive inhibition on the α-glucosidase enzyme. This is the first report on the phytochemical constituents and α-glucosidase inhibitory activity of G. bellinus.

(+)-7-O-Methylisomiroestrol, a new chromene phytoestrogen from the Pueraria candollei var. mirifica root.

(+)-7-O-Methylisomiroestrol (MeI), a novel chromene, was discovered as a phytoestrogen in the Pueraria candollei var. mirifica (Airy Shaw & Suvat.) Niyomdham (PM) root having been used as an active agent against oestrogen depletion disorders. The identification of PM phytochemicals is crucial for the development of standardised botanical drugs of PM. MeI was purified from the root cortex of PM, and its structure was elucidated using NMR and mass spectrometry. The content of MeI in the root bark of the PM root was 2.1-6.5 × 10-3% (w/w). The oestrogenic potency of MeI was stronger than that of isomiroestrol but less than that of deoxymiroestrol and miroestrol. Therefore, MeI is a new oestrogenic biomarker for the effective chemical standardisation of the PM extract for health product development.

Erianthridin Induces Non-small Cell Lung Cancer Cell Apoptosis through the Suppression of Extracellular Signal-regulated Kinase Activity.

Due to the high mortality of lung cancer, natural derivative compounds have been promoted as versatile sources for anticancer drug discovery. Erianthridin, a phenanthrene compound isolated from Dendrobium formosum, exhibits intriguing apoptosis-inducing effects in non-small cell lung cancer cells. Apoptotic nuclei staining assays showed that apoptotic cells with DNA fragmentation and apoptotic bodies were apparent, and an increase in annexin V-FITC-positive cells were found in cells treated with erianthridin. The apoptosis protein markers for cleaved caspase-3 and cleaved poly-ADP-ribose polymerase were significantly upregulated in response to erianthridin. A mechanistic investigation revealed that erianthridin was able to attenuate extracellular signal-regulated kinase activity and thereby mediate apoptosis through the modulation of Bcl-2 family protein levels. U0126, an extracellular signal-regulated kinase inhibitor, augmented the apoptosis-inducing effect of erianthridin; in contrast, overexpression of exogenous extracellular signal-regulated kinase substantially abrogated erianthridin activity. Furthermore, an in vitro 3D tumorigenesis assay showed that erianthridin was able to potentially suppress lung cancer cell proliferation. This study is the first to report a promising cytotoxic effect of erianthridin, which provides preclinical evidence for further research and development of this compound.

New Fluorene Derivatives from Dendrobium gibsonii and Their α-Glucosidase Inhibitory Activity.

Two new compounds, dihydrodengibsinin (1) and dendrogibsol (2), were isolated from the whole plant of Dendrobium gibsonii, together with seven known compounds (3-9). The structures of the new compounds were elucidated by their spectroscopic data. All these isolates were evaluated for their α-glucosidase inhibitory activities. Dendrogibsol (2) and lusianthridin (7) showed strong α-glucosidase inhibitory activity when compared with acarbose. An enzyme kinetic study revealed that dendrogibsol (2) is a noncompetitive inhibitor of α-glucosidase.

Thermal Degradation Kinetics and pH-Rate Profiles of Iriflophenone 3,5-C-ß-d-diglucoside, Iriflophenone 3-C-ß-d-Glucoside and Mangiferin in Aquilaria crassna Leaf Extract.

The health benefits of the Aquilaria crassna Pierre ex Lecomte leaf extract (AE) make it very useful as an ingredient in food and pharmaceutical products. Iriflophenone 3,5-C-ß-d-diglucoside (1), iriflophenone 3-C-ß-d-glucoside (2) and mangiferin (3) are bioactive compounds of AE. We assessed the stability of AE by investigating the thermal degradation kinetics and shelf-life (t90%) of compounds 1, 2 and 3 using Arrhenius plot models and studied their pH-rate profiles. The results demonstrate that 1 and 2 were degraded, following a first-order kinetic reaction. The degradation of 3 followed first-order reaction kinetics when present in a solution and second-order reaction kinetics in the dried powder form of the extract. According to the first-order kinetic model, the predicted shelf-life (t90%) of the extract at 25 °C in dried form for compound 1 was 989 days with activation energy 129.86 kJ·mol-1, and for 2 it was 248 days with activation energy 110.57 kJ·mol-1, while in the extract solution, the predicted shelf-life of compounds 1-3 was 189, 13 and 75 days with activation energies 86.83, 51.49 and 65.28 kJ·mol-1, respectively. In addition, the pH-rate profiles of 1-3 indicated that they were stable in neutral to acidic environments.

Immune modulatory effect of a novel 4,5-dihydroxy-3,3´,4´-trimethoxybibenzyl from Dendrobium lindleyi.

Dendrobium bibenzyls and phenanthrenes such as chrysotoxine, cypripedin, gigantol and moscatilin have been reported to show promising inhibitory effects on lung cancer growth and metastasis in ex vivo human cell line models, suggesting their potential for clinical application in patients with lung cancer. However, it remains to be determined whether these therapeutic effects can be also seen in primary human cells and/or in vivo. In this study, we comparatively investigated the immune modulatory effects of bibenzyls and phenanthrenes, including a novel Dendrobium bibenzyl derivative, in primary human monocytes. All compounds were isolated and purified from a Thai orchid Dendrobium lindleyi Steud, a new source of therapeutic compounds with promising potential of tissue culture production. We detected increased frequencies of TNF- and IL-6-expressing monocytes after treatment with gigantol and cypripedin, whereas chrysotoxine and moscatilin did not alter the expression of these cytokines in monocytes. Interestingly, the new 4,5-dihydroxy-3,3',4'-trimethoxybibenzyl derivative showed dose-dependent immune modulatory effects in lipopolysaccharide (LPS)-treated CD14lo and CD14hi monocytes. Together, our findings show immune modulatory effects of the new bibenzyl derivative from Dendrobium lindleyi on different monocyte sub-populations. However, therapeutic consequences of these different monocyte populations on human diseases including cancer remain to be investigated.

Constituents of Huberantha jenkinsii and Their Biological Activities.

The phytochemical investigation of Huberantha jenkinsii resulted in the isolation of two new and five known compounds. The new compounds were characterized as undescribed 8-oxoprotoberberine alkaloids and named huberanthines A and B, whereas the known compounds were identified as allantoin, oxylopinine, N-trans-feruloyl tyramine, N-trans-p-coumaroyl tyramine, and mangiferin. The structure determination was accomplished by spectroscopic methods. To evaluate therapeutic potential in diabetes and Parkinson's disease, the isolates were subjected to assays for their α-glucosidase inhibitory activity, cellular glucose uptake stimulatory activity, and protective activity against neurotoxicity induced by 6-hydroxydopamine (6-OHDA). The results suggested that mangiferin was the most promising lead compound, demonstrating significant activity in all the test systems.

New bisbibenzyl and phenanthrene derivatives from Dendrobium scabrilingue and their α-glucosidase inhibitory activity.

Phytochemical investigation of the whole plant of Dendrobium scabrilingue resulted in the isolation of two new compounds namely dendroscabrols A (1) and B (2), along with eight known compounds (3-10). The structures of these compounds were determined by NMR and HR-ESI-MS experiments. All of the isolates were evaluated for their α-glucosidase inhibitory effect. Dendroscabrol B (2) and RF-3192C (10) showed the most potent α-glucosidase inhibitory activity. Dendroscabrol A (1), gigantol (5), coelonin (7) and lusianthridin (9) also exhibited strong activity as compared with the positive control acarbose.

Comparative pharmacokinetics of oxyresveratrol alone and in combination with piperine as a bioenhancer in rats.

BACKGROUND: Oxyresveratrol is a major bioactive component derived from the heartwood of Artocarpus lacucha. This compound exerts several biological activities, including neuroprotective effects in vitro and in vivo. However, there is limited pharmacokinetic information on this compound, especially its distribution in neuronal tissue and its route of excretion. The aim of this study was to investigate the pharmacokinetic profiles of oxyresveratrol alone and in combination with piperine as a bioenhancer in rats. METHODS: Male Wistar rats were administered with oxyresveratrol 10 mg/kg, oxyresveratrol 10 mg/kg plus piperine 1 mg/kg via intravenous or oxyresveratrol 100 mg/kg, oxyresveratrol 100 mg/kg plus piperine 10 mg/kg via oral gavage. Plasma, internal organs, urine, and feces were collected. Determination of the oxyresveratrol concentration in biological samples was performed by liquid chromatography tandem mass spectrometry. RESULTS: The combination with piperine had shown a significantly higher maximum concentration in plasma approximately 1500 µg/L within 1-2 h after oral dosing, and could increase oral bioavailability of oxyresveratrol approximately 2-fold. Oxyresveratrol could widely distributed most of the internal organs with a tissue to plasma ratio of 10-100 fold within 5 min after dosing. Urinary excretion of oxyresveratrol glucuronide was the major route of excretion after administration of oxyresveratrol alone and in combination with piperine. CONCLUSION: The addition of piperine could enhance some of the pharmacokinetic properties of oxyresveratrol via both intravenous and oral administration. This pharmacokinetic information will be useful for appropriate strategies to develop oxyresveratrol as a phytopharmaceutical product.

Exploring Novel Cocrystalline Forms of Oxyresveratrol to Enhance Aqueous Solubility and Permeability across a Cell Monolayer.

Oxyresveratrol (ORV) is a naturally extracted compound with many pharmacological activities. However, information about the crystalline form is not known when considering the development of a form for oral dosage. Cocrystal engineering offers drug molecular understanding and drug solubility improvements. Thus, we attempted cocrystallization of ORV using 10 carboxylic acids as a coformer at a 1:1 M ratio. Each combination was processed with liquid-assisted grinding, solvent evaporation and a slurry method, then characterized by powder X-ray powder diffraction (PXRD), conventional and low-frequency Raman spectroscopy and thermal analysis. The solubility, dissolution and permeation studies across Caco-2 cell monolayers were conducted to evaluate the ORV samples. A screening study revealed that an ORV and citric acid (CTA) cocrystal formed by ethyl acetate-assisted grinding had characteristic PXRD peaks (14.0 and 16.5°) compared to those of ORV dihydrate used as a starting material. Low-frequency Raman measurements, with peaks at 100 cm-1, distinguished potential cocrystals among three processing methods while conventional Raman could not. An endothermic melt (142.2°C) confirmed the formation of the novel crystalline complex. The solubility of the cocrystal in the dissolution media of pH 1.2 and 6.8 was approximately 1000 µg/mL, a 1.3-fold increase compared to ORV alone. In vitro cytotoxicity studies showed that the cocrystal and physical blend were not toxic at concentrations of 25 and 12.5 µM ORV, respectively. The ORV-CTA cocrystal enhanced the cellular transport of ORV across Caco-2 monolayers. Therefore, cocrystallization could be used to improve aqueous solubility and permeability, leading to better oral bioavailability of ORV.

Lusianthridin targeting of lung cancer stem cells via Src-STAT3 suppression.

BACKGROUND: Cancer stem cells (CSCs) are well-recognized as a majority cause of treatment failure and can give rise to relapse. The discovery of compounds attenuating CSCs' properties is crucial for enabling advances in novel therapeutics to limit recurrence. CSCs' features in lung cancer are regulated through a reduction in Src-STAT3-c-Myc, which drives cancer progression, drug resistance, and metastasis. METHODS: The effect of lusianthridin suppresses CSC-like phenotypes was determined by 3D culture and anchorage independent growth. The expression of CSC markers and associated proteins were determined by Western blot analyses. Protein ubiquitination and degradation were assessed using immunoprecipitation. RESULTS: Herein, we report that lusianthridin, a pure compound from Dendrobium venustum, dramatically suppressed CSCs in lung cancer cells as verified by several CSC phenotype assessments and CSC markers. The CSC phenotypes in lusianthridin-treated cells were suppressed through downregulation of Src-STAT3-c-Myc pathways. Ectopic Src introduced by the transfection augmented CSC phenotypes in lung cancer cells through STAT3 (increased active p-STAT3Tyr705) and c-Myc signals, while the ShRNA-Src transfection or Src inhibitor dasatinib exhibited opposite results. Treatment of the Src-overexpressing cells with lusianthridin resulted in the reversal of active STAT3 (p-STAT3Tyr705) and c-Myc as well as the CSC marker CD133. Importantly, we confirmed the CSC-targeted activity of lusianthridin in CSC-rich primary lung cancer cells. The compound dramatically inhibited the formation of tumor spheres of primary lung cancer cells. Finally, we demonstrated that after CSC-attenuation by lusianthridin, the lung cancer cells exhibited significantly higher susceptibility to chemotherapeutic drugs. Such a sensitizing effect caused by pro-survival suppression and pro-apoptotic induction together with the abolishment of stemness indicated by the decrease in CSC markers CD133, ABCG2, and ALDH1A1. CONCLUSION: These findings revealed a novel pharmacological action and the underlying mechanism of lusianthridin in negatively regulating CSC-like phenotypes and sensitizing resistant cancer cells to cemetery.

Development of monoclonal antibody-based enzyme-linked immunosorbent assay for quantitative quality control of Derris scandens (Roxb.) Benth.

Derris scandens (Roxb.) Benth. is a medicinal plant used for treatment of musculoskeletal pain in Thai traditional medicines. Its stem contains active compound genistein-7-O-[α-rhamnopyranosyl-(1 to 6)-ß-glucopyranoside] (GTG) which is used as a biomarker for standardization of D. scandens extracts. As an alternative for rapid quantitation of GTG, a monoclonal antibody against GTG was prepared and applied for an indirect competitive enzyme-linked immunosorbent assay (ELISA) to determine GTG in plants and herbal products. The established method provided a quantification range of 0.31-10 µg/mL with a limit of detection of 0.29 µg/mL. The assay was validated for precision and accuracy by intra- and interassay variation analyses, recovery test, and comparison analysis between the amounts of GTG determined by ELISA and HPLC. The results exhibited that the developed ELISA is sensitive and effective for determination of GTG in D. scandens plant materials and herbal products.

Chrysotobibenzyl inhibition of lung cancer cell migration through Caveolin-1-dependent mediation of the integrin switch and the sensitization of lung cancer cells to cisplatin-mediated apoptosis.

BACKGROUND: A Lung cancer death account for approximately 1 in 5 of all cancer-related deaths and is particularly virulent due to its enhanced metastasis and resistance to chemotherapy. Chrysotobibenzyl has been reported to decrease cell metastasis, according to the results of an anchorage-independent growth assay; however, its underlying mechanism has not been investigated yet. PURPOSE: The aim of this study was to investigate the effect of chrysotobibenzyl on lung cancer cell migration and drug sensitization and its mechanism. METHODS: Cell viability, cell proliferation and drug sensitization were determined by MTT assay. Cell migration was analyzed using a wound-healing assay. Transwell migration and invasion were analyzed using Boyden chamber assay. Mechanisms of chrysotobibenzyl against metastasis including cell migration, invasion, and epithelial to mesenchymal transition (EMT) were evaluated by Western blot analysis and immunofluorescence. RESULTS: Treatment with chrysotobibenzyl was applied at concentrations of 0-50 µM and the results showed non-cytotoxicity in human lung cancer cells (H460, H292, A549, and H23) and other non-cancerous human cells (HCT116, primary DP1 and primary DP2). However, 50 µM of chrysotobibenzyl significantly altered cell proliferation in H292 cells at 48 h. In addition, 1-50 µM of chrysotobibenzyl significantly inhibited H460 and H292 cell migration, invasion, filopodia formation, and decreased EMT in a dose-dependent manner at 48 h, which were correlated with reduced protein levels of integrins ß1, ß3, and αν, p-FAK, p-AKT, Cdc42, and Cav-1. We also established shRNA-Cav-1-transfected (shCav-1) H460 and H292 cells. shCav-1 transfected cells can decrease cell migration and downregulate the expression of integrins ß1, ß3, and αν when compared with the control. Moreover, chrysotobibenzyl was shown to suppress EMT indicated by the reduction of EMT markers (Vimentin, Snail, and Slug), and sensitize lung cancer cells to cisplatin-mediated apoptosis. CONCLUSION: Treatment with chrysotobibenzyl inhibited lung cancer cell migration via Cav-1, integrins ß1, ß3, and αν, and EMT suppressions. The downregulation of integrins in response to the compound not only inhibited cell metastasis, but also sensitized lung cancer cells to cisplatin-mediated apoptosis.