Development of 3-methyl/3-(morpholinomethyl)benzofuran derivatives as novel antitumor agents towards non-small cell lung cancer cells

As one of the most lethal malignancies, lung cancer is considered to account for approximately one-fifth of all malignant tumours-related deaths worldwide. This study reports the synthesis and in vitro biological assessment of two sets of 3-methylbenzofurans (4a–d, 6a–c, 8a–c and 11) and 3-(morpholinomethyl)benzofurans (15a–c, 16a–b, 17a–b and 18) as potential anticancer agents towards non-small cell lung carcinoma A549 and NCI-H23 cell lines, with VEGFR-2 inhibitory activity. The target benzofuran-based derivatives efficiently inhibited the growth of both A549 and NCI-H23 cell lines with IC₅₀ spanning in ranges 1.48–47.02 and 0.49–68.9 µM, respectively. The three most active benzofurans (4b, 15a and 16a) were further investigated for their effects on the cell cycle progression and apoptosis in A549 (for 4b) and NCI-H23 (for 15a and 16a) cell lines. Furthermore, benzofurans 4b, 15a and 16a displayed good VEGFR-2 inhibitory activity with IC₅₀ equal 77.97, 132.5 and 45.4 nM, respectively.     

Induction of a Mitosis Delay and Cell Lysis by High-Level Secretion of Mouse α-Amylase from Saccharomyces cerevisiae

Some foreign proteins are produced in yeast in a cell cycle-dependent manner, but the cause of the cell cycle dependency is unknown. In this study, we found that Saccharomyces cerevisiae cells secreting high levels of mouse α-amylase have elongated buds and are delayed in cell cycle completion in mitosis. The delayed cell mitosis suggests that critical events during exit from mitosis might be disturbed. We found that the activities of PP2A (protein phosphatase 2A) and MPF (maturation-promoting factor) were reduced in α-amylase-oversecreting cells and that these cells showed a reduced level of assembly checkpoint protein Cdc55, compared to the accumulation in wild-type cells. MPF inactivation is due to inhibitory phosphorylation on Cdc28, as a cdc28 mutant which lacks an inhibitory phosphorylation site on Cdc28 prevents MPF inactivation and prevents the defective bud morphology induced by overproduction of α-amylase. Our data also suggest that high levels of α-amylase may downregulate PPH22, leading to cell lysis. In conclusion, overproduction of heterologous α-amylase in S. cerevisiae results in a negative regulation of PP2A, which causes mitotic delay and leads to cell lysis.     

Curcumin Inhibits Non-Small Cell Lung Cancer Cells Metastasis through the Adiponectin/NF-κb/MMPs Signaling Pathway

Adipose tissue is now considered as an endocrine organ involved in metabolic and inflammatory reactions. Adiponectin, a 244–amino acid peptide hormone, is associated with insulin resistance and carcinogenesis. Curcumin (diferuloylmethane) is the principal curcuminoid of the popular Indian spice, turmeric. Curcumin possesses antitumor effects, including the inhibition of neovascularization and regulation of cell cycle and apoptosis. However, the effects of adiponectin and curcumin on non-small cell lung cancer (NSCLC) remain unclear. In this study, we evaluated the expression of adiponectin in paired tumors and normal lung tissues from 77 patients with NSCLC using real-time polymerase chain reaction, western blotting, and immunohistochemistry. Kaplan–Meier survival analysis showed that patients with low adiponectin expression ratio (<1) had significantly longer survival time than those with high expression ratio (>1) (p = 0.015). Curcumin inhibited the migratory and invasive ability of A549 cells via the inhibition of adiponectin expression by blocking the adiponectin receptor 1. Curcumin treatment also inhibited the in vivo tumor growth of A549 cells and adiponectin expression. These results suggest that adiponectin can be a prognostic indicator of NSCLC. The effect of curcumin in decreasing the migratory and invasive ability of A549 cells by inhibiting adiponectin expression is probably mediated through NF-κB/MMP pathways. Curcumin could be an important potential adjuvant therapeutic agent for lung cancer in the future.     

Protective roles of cytoplasmic p21Cip1 /Waf1 in senolysis and ferroptosis of lung cancer cells

ObjectiveTherapy‐induced senescent cancer cells increase the expression of the cyclin‐dependent kinase inhibitors p16^Ink4a and p21^Cip1/Waf1. Given that p21 regulates not only the cell cycle but also cell death, we investigated the roles of p21 in cell death using a p16‐negative A549 human lung adenocarcinoma cell line.MethodsSenescence was induced by doxorubicin (DXR) or pemetrexed (PEM). The protein expression of p21 was examined by immunoblot. Cell death, reactive oxygen species (ROS) and lipid peroxidation were determined by flow cytometry. ABT‐263 and ABT‐737 were used as senolytic drugs. In vivo growth of A549 cells with different levels of p21 and their sensitivity to PEM were examined in xenograft models.ResultsDXR‐induced senescent A549 cells increased the expression of cytoplasmic p21, and the sensitivity to ABT‐263 was augmented in p21‐knockout A549 (A549‐KOp21) cells. A similar senolytic effect was observed when PEM was combined with ABT‐737. PEM alone induced a higher level of non‐apoptotic cell death, ferroptosis, in A549‐KOp21 cells than in A549 cells. Although there was no difference in the level of lipid peroxidation, ROS levels were higher in PEM‐treated A549‐KOp21 cells than in PEM‐treated A549 cells. A loss of p21 increased the sensitivity of A549 cells to PEM both in vitro and in vivo. A clinical database analysis showed that CDKN1A ^high lung adenocarcinoma patients had a poorer prognosis compared to CDKN1A ^low patients.ConclusionCytoplasmic p21, which was increased in therapy‐induced senescent lung cancer cells, plays protective roles in senolysis and ferroptosis.

Doxorubicin or pemetrexed could induce senescence in p21‐expressing parental A549 cells and increase the expression of cytoplasmic p21. However, such drug‐induced senescent A549 cells were relatively resistant to apoptosis by senolytic drugs. By contrast, p21‐knockout A549 (A549‐KOp21) cells increased their sensitivity to senolytic drugs. On the other hand, pemetrexed induced a higher level of non‐apoptotic cell death, ferroptosis, in A549‐KOp21 cells than in A549 cells. These findings highlight the protective roles of cytoplasmic p21 against senolysis and ferroptosis in therapy‐induced senescent lung cancer cells.     

The Use of Knockout Mice Reveals a Synergistic Role of the Vav1 and Rasgrf2 Gene Deficiencies in Lymphomagenesis and Metastasis

Background

Vav1 and RasGRF2 are GDP/GTP exchange factors for Ras superfamily GTPases with roles in the development and/or effector functions of T–lymphocytes.

Methodology/Principal Findings

Given that the phenotype of Vav1 ^–/–, Rasgrf2 ^–/– and Vav1 ^–/–;Rasgrf2 ^–/– mice has been studied so far in young animals, we decided to explore the long–term consequences of the inactivation of those loci in the immune system. Unexpectedly, our studies revealed that the inactivation of the Vav1 proto–oncogene favors the formation of lymphoblastic lymphoma–like tumors in aging mice. Those tumors, that can be found either localized exclusively inside the thymus or widely disseminated in hematopoietic and non–hematopoietic tissues, are composed of CD3⁺ lymphoblasts that display heterogeneous combinations of CD4 and CD8 surface markers. Interestingly, the additional deletion of the Rasgrf2 gene induces a shortening in the latency period for the development of those tumors, an increase in the percentage of disseminated tumors outside the thymus and, as a result, higher mortality rates.

Conclusions/Significance

These data reveal unexpected negative roles for Vav1 and RasGRF2 in different stages of T–cell lymphoma progression. They also suggest that the inactivation of Vav1 function may represent an inadequate strategy to treat T–cell lymphomas, especially those associated with low levels of Rasgrf2 gene expression.     

Involvement of general control nonderepressible kinase 2 in cancer cell apoptosis by posttranslational mechanisms

General control nonderepressible kinase 2 (GCN2) is a promising target for cancer therapy. However, the role of GCN2 in cancer cell survival or death is elusive; further, small molecules targeting GCN2 signaling are not available. By using a GCN2 level-based drug screening assay, we found that GCN2 protein level critically determined the sensitivity of the cancer cells toward Na⁺,K⁺-ATPase ligand–induced apoptosis both in vitro and in vivo, and this effect was largely dependent on C/EBP homologous protein (CHOP) induction. Further analysis revealed that GCN2 is a short-lived protein. In A549 lung carcinoma cells, cellular β-arrestin1/2 associated with GCN2 and maintained the GCN2 protein level at a low level by recruiting the E3 ligase NEDD4L and facilitating consequent proteasomal degradation. However, Na⁺,K⁺-ATPase ligand treatment triggered the phosphorylation of GCN2 at threonine 899, which increased the GCN2 protein level by disrupting the formation of GCN2–β-arrestin–NEDD4L ternary complex. The enhanced GCN2 level, in turn, aggravated Na⁺,K⁺-ATPase ligand–induced cancer cell apoptosis. Our findings reveal that GCN2 can exert its proapoptotic function in cancer cell death by posttranslational mechanisms. Moreover, Na⁺,K⁺-ATPase ligands emerge as the first identified small-molecule drugs that can trigger cancer cell death by modulating GCN2 signaling.     

Maternal smoking and the retinoid pathway in the developing lung

Background

Maternal smoking is a risk factor for pediatric lung disease, including asthma. Animal models suggest that maternal smoking causes defective alveolarization in the offspring. Retinoic acid signaling modulates both lung development and postnatal immune function. Thus, abnormalities in this pathway could mediate maternal smoking effects. We tested whether maternal smoking disrupts retinoic acid pathway expression and functioning in a murine model.

Methods

Female C57Bl/6 mice with/without mainstream cigarette smoke exposure (3 research cigarettes a day, 5 days a week) were mated to nonsmoking males. Cigarette smoke exposure continued throughout the pregnancy and after parturition. Lung tissue from the offspring was examined by mean linear intercept analysis and by quantitative PCR. Cell culture experiments using the type II cell-like cell line, A549, tested whether lipid-soluble cigarette smoke components affected binding and activation of retinoic acid response elements in vitro.

Results

Compared to tobacco-naïve mice, juvenile mice with tobacco toxin exposure had significantly (P < 0.05) increased mean linear intercepts, consistent with an alveolarization defect. Tobacco toxin exposure significantly (P < 0.05) decreased mRNA and protein expression of retinoic acid signaling pathway elements, including retinoic acid receptor alpha and retinoic acid receptor beta, with the greatest number of changes observed between postnatal days 3–5. Lipid-soluble cigarette smoke components significantly (P < 0.05) decreased retinoic acid-induced binding and activation of the retinoic acid receptor response element in A549 cells.

Conclusions

A murine model of maternal cigarette smoking causes abnormal alveolarization in association with altered retinoic acid pathway element expression in the offspring. An in vitro cell culture model shows that lipid-soluble components of cigarette smoke decrease retinoic acid response element activation. It is feasible that disruption of retinoic acid signaling contributes to the pediatric lung dysfunction caused by maternal smoking.     

Development of a novel method to evaluate sialylation of glycoproteins and analysis of gp96 sialylation in Hela,SW1990 and A549 cell lines

Background

Glycoproteins play a critical role in the cellular activities of eukaryotes. Sialic acid is typically the outermost monosaccharide of glycolipids and glycoproteins, and is necessary for normal development.

Results

A strategy based on avidin–biotin affinity was established to enrich sialylated glycoproteins from HeLa cervical carcinoma, SW1990 pancreatic adenocarcinoma, and A549 lung adenocarcinoma cells. Using HPLC–MS/MS, western blot, real-time PCR, and enzyme-linked immunosorbent assay, gp96 was identified in all three cell lines. No significant difference in the protein expression of gp96 was detected at the whole cell level, but the amount of biotinylated gp96 in SW1990 cells was 30–40 % lower than that in A549 and HeLa cells, and the amount of sialylated gp96 in SW1990 cells was 30 % lower than that in A549 and HeLa cells. Immunoblotting results showed that the expression of sialyltransferase proteins in the total cell lysates from HeLa and A549 cells were higher than that in SW1990 cells.

Conclusions

We established a new method for investigating the expression and sialylation of glycoproteins using metabolic labeling, click chemistry, and avidin–biotin affinity. We successfully used this method to purify sialylated glycoproteins from cancer cell lines. Our results showed that the levels of gp96 sialylation varied across different cancer cell lines, and this may be because of differences in sialyltransferase expression.     

Pre-clinical evaluation of cinobufotalin as a potential anti-lung cancer agent

Lung cancer is a major cause of cancer-related mortality in the United States and around the world. Due to the pre-existing or acquired chemo-resistance, the current standard chemotherapy regimens only show moderate activity against lung cancer. In the current study, we explored the potential anti-lung cancer activity of cinobufotalin in vivo and in vitro, and studied the underlying mechanisms. We demonstrated that cinobufotalin displayed considerable cytotoxicity against lung cancer cells (A549, H460 and HTB-58 lines) without inducing significant cell apoptosis. Our data suggest that mitochondrial protein cyclophilin D (Cyp-D)-dependent mitochondrial permeability transition pore (mPTP) opening mediates cinobufotalin-induced non-apoptotic death of lung cancer cells. The Cyp-D inhibitor cyclosporine A (CsA), the mPTP blocker sanglifehrin A (SfA), and Cyp-D shRNA-silencing significantly inhibited cinobufotalin-induced mitochondrial membrane potential (MMP) reduction and A549 cell death (but not apoptosis). Using a mice xenograft model, we found that cinobufotalin inhibited A549 lung cancer cell growth in vivo. Thus, cinobufotalin mainly induces Cyp-D-dependent non-apoptotic death in cultured lung cancer cells. The results of this study suggest that cinobufotalin might be further investigated as a novel anti-lung cancer agent.     

Frondoside A Suppressive Effects on Lung Cancer Survival,Tumor Growth,Angiogenesis, Invasion,and Metastasis

A major challenge for oncologists and pharmacologists is to develop less toxic drugs that will improve the survival of lung cancer patients. Frondoside A is a triterpenoid glycoside isolated from the sea cucumber, Cucumaria frondosa and was shown to be a highly safe compound. We investigated the impact of Frondoside A on survival, migration and invasion in vitro, and on tumor growth, metastasis and angiogenesis in vivo alone and in combination with cisplatin. Frondoside A caused concentration-dependent reduction in viability of LNM35, A549, NCI-H460-Luc2, MDA-MB-435, MCF-7, and HepG2 over 24 hours through a caspase 3/7-dependent cell death pathway. The IC50 concentrations (producing half-maximal inhibition) at 24 h were between 1.7 and 2.5 µM of Frondoside A. In addition, Frondoside A induced a time- and concentration-dependent inhibition of cell migration, invasion and angiogenesis in vitro. Frondoside A (0.01 and 1 mg/kg/day i.p. for 25 days) significantly decreased the growth, the angiogenesis and lymph node metastasis of LNM35 tumor xenografts in athymic mice, without obvious toxic side-effects. Frondoside A (0.1–0.5 µM) also significantly prevented basal and bFGF induced angiogenesis in the CAM angiogenesis assay. Moreover, Frondoside A enhanced the inhibition of lung tumor growth induced by the chemotherapeutic agent cisplatin. These findings identify Frondoside A as a promising novel therapeutic agent for lung cancer.     

Myeloid-Derived Suppressor Cells Modulate Immune Responses Independently of NADPH Oxidase in the Ovarian Tumor Microenvironment in Mice

The phagocyte NADPH oxidase generates superoxide anion and downstream reactive oxidant intermediates in response to infectious threat, and is a critical mediator of antimicrobial host defense and inflammatory responses. Myeloid-derived suppressor cells (MDSCs) are a heterogeneous population of immature myeloid cells that are recruited by cancer cells, accumulate locally and systemically in advanced cancer, and can abrogate anti-tumor immunity. Prior studies have implicated the phagocyte NADPH oxidase as being an important component promoting MDSC accumulation and immunosuppression in cancer. We therefore used engineered NADPH oxidase-deficient (p47^phox−/−) mice to delineate the role of this enzyme complex in MDSC accumulation and function in a syngeneic mouse model of epithelial ovarian cancer. We found that the presence of NADPH oxidase did not affect tumor progression. The accumulation of MDSCs locally and systemically was similar in tumor-bearing wild-type (WT) and p47^phox−/− mice. Although MDSCs from tumor-bearing WT mice had functional NADPH oxidase, the suppressive effect of MDSCs on ex vivo stimulated T cell proliferation was NADPH oxidase-independent. In contrast to other tumor-bearing mouse models, our results show that MDSC accumulation and immunosuppression in syngeneic epithelial ovarian cancer is NADPH oxidase-independent. We speculate that factors inherent to the tumor, tumor microenvironment, or both determine the specific requirement for NADPH oxidase in MDSC accumulation and function.     

内吞适配蛋白Epsin在非小细胞肺癌发生中的作用研究

目的:探讨内吞适配蛋白Epsin在非小细胞肺癌发生中的潜在作用。方法:选择体外培养的人非小细胞肺癌细胞(A549),筛选Epsin 1和Epsin 2 shRNA干扰效率达标的细胞。将裸鼠随机分为3组,每组10只,第1、2组裸鼠分别经胸腔植入人非小细胞肺癌细胞(A549)及epsin表达敲减的A549细胞,第3组注射等量的生理盐水,比较1、2组小鼠肿瘤体积的变化。8周后,处死所有裸鼠,留取肺组织及肿瘤组织,通过免疫荧光染色检测非肿瘤(正常)肺和致瘤性肺组织中的epsin 1和2的蛋白质水平。用实时定量PCR(qRT-PCR)来研究epsin 1和2的基因表达水平。结果:肺肿瘤组织epsin1和2的m RNA和蛋白表达均显著高于正常肺组织中(P0.05)。种植epsin表达敲减的A549细胞裸鼠肿瘤生长速度及体积均大于种植正常A549细胞的裸鼠肿瘤。结论:Epsins表达上调可能促进非小细胞肺癌肿瘤的发生发展,而敲减epsins的表达可能为未来的非小细胞肺癌的治疗提供新的治疗靶点。     

Eurycomanone suppresses expression of lung cancer cell tumor markers, prohibitin, annexin 1 and endoplasmic reticulum protein 28

Bioactive compounds from the medicinal plant, Eurycoma longifolia Jack have been shown to promote anti-proliferative effects on various cancer cell lines. Here we examined the effects of purified eurycomanone, a quassinoid found in Eurycoma longifolia Jack extract, on the expression of selected genes of the A549 lung cancer cells. Eurycomanone inhibited A549 lung cancer cell proliferation in a dose-dependent manner at concentrations ranging from 5 to 20 μg/ml. The concentration that inhibited 50% of cell growth (GI₅₀) was 5.1 μg/ml. The anti-proliferative effects were not fully reversible following the removal of eurycomanone, in which 30% of cell inhibition still remained (p < 0.0001, T-test). At 8 μg/ml (GI₇₀), eurycomanone suppressed anchorage-independent growth of A549 cells by >25% (p < 0.05, T-test, n = 8) as determined using soft agar colony formation assay. Cisplatin, a chemotherapy drug used for the treatment of non small cell lung cancer on the other hand, inhibited A549 cells proliferation at concentrations ranging from 0.2 μg/ml to 15 μg/ml with a GI₅₀ of 0.58 μg/ml. The treatment with eurycomanone reduced the abundance expression of the lung cancer markers, heterogeneous nuclear ribonucleoprotein (hnRNP) A2/B1, p53 tumor suppressor protein and other cancer-associated genes including prohibitin (PHB), annexin 1 (ANX1) and endoplasmic reticulum protein 28 (ERp28) but not the house keeping genes. The mRNA expressions of all genes with the exception of PHB were significantly downregulated, 72 h after treatment (p < 0.05, T-test, n = 9). These findings suggest that eurycomanone at viable therapeutic concentrations of 5-20 μg/ml exhibited significant anti-proliferative and anti-clonogenic cell growth effects on A549 lung cancer cells. The treatment also resulted in suppression of the lung cancer cell tumor markers and several known cancer cell growth-associated genes.     

Small Molecule Antagonists of the Wnt/Beta-Catenin Signaling Pathway Target Breast Tumor-Initiating Cells in a Her2/Neu Mouse Model of Breast Cancer

Background

Recent evidence suggests that human breast cancer is sustained by a minor subpopulation of breast tumor-initiating cells (BTIC), which confer resistance to anticancer therapies and consequently must be eradicated to achieve durable breast cancer cure.

Methods/Findings

To identify signaling pathways that might be targeted to eliminate BTIC, while sparing their normal stem and progenitor cell counterparts, we performed global gene expression profiling of BTIC- and mammary epithelial stem/progenitor cell- enriched cultures derived from mouse mammary tumors and mammary glands, respectively. Such analyses suggested a role for the Wnt/Beta-catenin signaling pathway in maintaining the viability and or sustaining the self-renewal of BTICs in vitro. To determine whether the Wnt/Beta-catenin pathway played a role in BTIC processes we employed a chemical genomics approach. We found that pharmacological inhibitors of Wnt/β-catenin signaling inhibited sphere- and colony-formation by primary breast tumor cells and primary mammary epithelial cells, as well as by tumorsphere- and mammosphere-derived cells. Serial assays of self-renewal in vitro revealed that the Wnt/Beta-catenin signaling inhibitor PKF118–310 irreversibly affected BTIC, whereas it functioned reversibly to suspend the self-renewal of mammary epithelial stem/progenitor cells. Incubation of primary tumor cells in vitro with PKF118–310 eliminated their capacity to subsequently seed tumor growth after transplant into syngeneic mice. Administration of PKF118–310 to tumor-bearing mice halted tumor growth in vivo. Moreover, viable tumor cells harvested from PKF118–310 treated mice were unable to seed the growth of secondary tumors after transplant.

Conclusions

These studies demonstrate that inhibitors of Wnt/β-catenin signaling eradicated BTIC in vitro and in vivo and provide a compelling rationale for developing such antagonists for breast cancer therapy.     

TGF-β1 exposure induces epithelial to mesenchymal transition both in CSCs and non-CSCs of the A549 cell line,leading to an increase of migration ability in the CD133+ A549 cell fraction

Metastasis is the leading cause of death by cancer. Non-small-cell lung cancer (NSCLC) represents nearly 85% of primary malignant lung tumours. Recent researches have demonstrated that epithelial-to-mesenchymal transition (EMT) plays a key role in the early process of metastasis of cancer cells. Transforming growth factor-β1 (TGF-β1) is the major inductor of EMT. The aim of this study is to investigate TGF-β1''s effect on cancer stem cells (CSCs) identified as cells positive for CD133, side population (SP) and non-cancer stem cells (non-CSCs) identified as cells negative for CD133, and SP in the A549 cell line. We demonstrate that TGF-β1 induces EMT in both CSC and non-CSC A549 sublines, upregulating the expression of mesenchymal markers such as vimentin and Slug, and downregulating levels of epithelial markers such as e-cadherin and cytokeratins. CSC and non-CSC A549 sublines undergoing EMT show a strong migration and strong levels of MMP9 except for the CD133⁻ cell fraction. OCT4 levels are strongly upregulated in all cell fractions except CD133⁻ cells. On the contrary, wound size reveals that TGF-β1 enhances motility in wild-type A549 as well as CD133⁺ and SP⁺ cells. For CD133⁻ and SP⁻ cells, TGF-β1 exposure does not change the motility. Finally, assessment of growth kinetics reveals major colony-forming efficiency in CD133⁺ A549 cells. In particular, SP⁺ and SP⁻ A549 cells show more efficiency to form colonies than untreated corresponding cells, while for CD133⁻ cells no change in colony number was observable after TGF-β1 exposure. We conclude that it is possible to highlight different cell subpopulations with different grades of stemness. Each population seems to be involved in different biological mechanisms such as stemness maintenance, tumorigenicity, invasion and migration.     

Honokiol Inhibits Non-Small Cell Lung Cancer Cell Migration by Targeting PGE2-Mediated Activation of β-Catenin Signaling

Lung cancer remains a leading cause of death due to its metastasis to distant organs. We have examined the effect of honokiol, a bioactive constituent from the Magnolia plant, on human non-small cell lung cancer (NSCLC) cell migration and the molecular mechanisms underlying this effect. Using an in vitro cell migration assay, we found that treatment of A549, H1299, H460 and H226 NSCLC cells with honokiol resulted in inhibition of migration of these cells in a dose-dependent manner, which was associated with a reduction in the levels of cyclooxygenase-2 (COX-2) and prostaglandin E₂ (PGE₂). Celecoxib, a COX-2 inhibitor, also inhibited cell migration. Honokiol inhibited PGE₂-enhanced migration of NSCLC cells, inhibited the activation of NF-κB/p65, an upstream regulator of COX-2, in A549 and H1299 cells, and treatment of cells with caffeic acid phenethyl ester, an inhibitor of NF-κB, also inhibited migration of NSCLC cells. PGE₂ has been shown to activate β-catenin signaling, which contributes to cancer cell migration. Therefore, we checked the effect of honokiol on β-catenin signaling. It was observed that treatment of NSCLC cells with honokiol degraded cytosolic β-catenin, reduced nuclear accumulation of β-catenin and down-regulated matrix metalloproteinase (MMP)-2 and MMP-9, which are the down-stream targets of β-catenin and play a crucial role in cancer cell metastasis. Honokiol enhanced: (i) the levels of casein kinase-1α, glycogen synthase kinase-3β, and (ii) phosphorylation of β-catenin on critical residues Ser⁴⁵, Ser^33/37 and Thr⁴¹. These events play important roles in degradation or inactivation of β-catenin. Treatment of celecoxib also reduced nuclear accumulation of β-catenin in NSCLC cells. FH535, an inhibitor of Wnt/β-catenin pathway, inhibited PGE₂-enhanced cell migration of A549 and H1299 cells. These results indicate that honokiol inhibits non-small cell lung cancer cells migration by targeting PGE₂-mediated activation of β-catenin signaling.