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Metastasis is the leading cause of cancer mortality and cancer cell migration is an essential stage of metastasis. We identified benproperine (Benp, a clinically used antitussive drug) as an inhibitor of cancer cell migration and an anti-metastatic agent. Benp selectively inhibited cancer cell migration and invasion, which also suppressed metastasis of cancer cells in animal models. Actin-related protein 2/3 complex subunit 2 (ARPC2) was identified as a molecular target of Benp by affinity column chromatography with Benp-tagged Sepharose beads. Benp bound directly to ARPC2 in cells, which was validated by pull-down assay using Benp-biotin and label-free biochemical methods such as the drug affinity responsive target stability (DARTS) and cellular thermal shift assay (CETSA). Benp inhibited Arp2/3 function, showing disruption of lamellipodial structure and inhibition of actin polymerization. Unlike Arp2/3 inhibitors, Benp selectively inhibited the migration of cancer cells but not normal cells. ARPC2-knockdown cancer cells showed defective cell migration and suppressed metastasis in an animal model. Therefore, ARPC2 is a potential target for anti-metastatic therapy, and Benp has the clinical potential to block metastasis. Furthermore, Benp is a useful agent for studying the functions of the Arp2/3 complex in cancer cell migration and metastasis.

2′-Hydroxycinnamaldehyde (HCA), the active component isolated from the stem bark of Cinnamomum cassia, exerts anticancer effects through multiple mechanisms. We recently determined that HCA inhibits signal transducer and activator of transcription 3 (STAT3) signaling in prostate cancer cells. Because STAT3 overactivation has been closely associated with the development of psoriasis, a chronic autoimmune skin disease, we examined whether HCA ameliorates skin lesions in an imiquimod-induced psoriasis-like mouse model. The results showed that intraperitoneal administration of HCA alleviated imiquimod-induced psoriasis-like dermatitis, epidermal thickening, dermal infiltration of inflammatory cells, and proinflammatory cytokine production. Mechanistically, HCA inhibited pyruvate kinase isozyme M2 and STAT3 signaling, leading to the suppression of T cell activation, Th17 cell differentiation, and keratinocyte hyperproliferation. These results suggest that HCA may be a new treatment for psoriasis and other STAT3-mediated skin disorders, such as infection, inflammation and carcinogenesis., Psoriasis: Cinnamon bark derivative reduces skin inflammation in mice An active compound found in cinnamon bark could help alleviate the symptoms of psoriasis according to a study in mice by researchers in South Korea. A team led by Eun Ju Bae and Byung-Hyun Park from Chonbuk National University in Jeonju administered 2′-hydroxycinnamaldehyde, a molecule derived from cinnamon, to mice with drug-induced psoriatic skin lesions. After the treatment, the mice showed fewer signs of skin irritation, with less inflammation and no detectable ill effects. The researchers showed that the drug blocked an enzyme that normally activates a central regulator of immune responses in the skin. Consequently, fewer proinflammatory T cells infiltrated the skin and epidermal cells did not grow out of control. The findings highlight the potential of a natural product to safely treat psoriasis and related inflammatory disorders.

trans-Cinnamaldehyde (CA) is the main bioactive component of cinnamon extract obtained from the stem bark of Cinnamomum cassia Blume. CA shows diverse biological activities in diabetes, neuropathy, cardiovascular diseases, and cancer in particular. To develop CA as a promising anticancer agent for clinical application, numerous research groups have investigated the effects of natural and synthetic derivatives of CA in cancer. CA and its derivatives increase the intracellular reactive oxygen species (ROS) levels of cancer cells, which trigger apoptotic cell death through activation of the mitochondrial and ER pathways. In addition to ROS generation, CA and its derivatives induce oxidative stress by inhibiting antioxidant enzymes and thereby disrupting redox homeostasis. Recently, multiple binding targets of CA and HCA involved in the regulation of oxidative stress-induced apoptosis have been identified. In this review, we focus on the mode of action of CA and its derivative in cancer cells and their effect on ROS generation and oxidative stress.

Piperlongumine (PL), isolated from Piper longum L., is receiving intense interest due to its selectively ability to kill cancer cells but not normal cells. We synthesized a number of analogues by replacing the cyclic amide of PL with aliphatic amides to explore structural diversity. Compound CG-06 had the strongest cytotoxic profile of this series, showing potent effects in human prostate cancer DU-145 cells, in which signal transducer and activator of transcription 3 (STAT3) is constitutively active. CG-06 inhibited STAT3 phosphorylation at tyrosine 705 in a dose- and time dependent manner in DU-145 cells and suppressed IL-6-induced STAT3 phosphorylation at Tyr-705 in DU-145 and LNCaP cell lines. CG-06 decreased the expression levels of STAT3 target genes, such as cyclin A, Bcl-2, and survivin. Notably, we used drug affinity responsive target stability (DARTS) to show that CG-06 binds directly to STAT3, and the reactive oxygen species (ROS) scavenger N-acetyl cysteine (NAC) rescued the CG-06-induced suppression p-STAT3. Our results suggest that CG-06 is a novel inhibitor of STAT3 and may be a useful lead molecule for the development of a therapeutic STAT3 inhibitor.

The roles and significance of signal transducer and activator of transcription 3 (STAT3) in human cancers have been extensively studied and STAT3 is a promising therapeutic target for cancer drug discovery. During the screening of natural products to identify STAT3 inhibitors, we identified geranylnaringenin (CG902), which decreased luciferase activity in a dose-dependent manner. CG902 specifically inhibited STAT3 phosphorylation at Tyr-705 in DU145 prostate cancer cells and decreased the expression levels of STAT3 target genes, such as cyclin D1, cyclin A, and survivin. Notably, the knockdown of the SHP-2 gene by small interfering RNA suppressed the ability of CG902 to inhibit STAT3 activation and CG902 activated the phosphatase activity of SHP-2 through direct interaction with SHP-2 and induced the phosphorylation of SHP-2. The interactions between CG902 and SHP-2 were confirmed by pull-down assay using biotinylated CG902. The interactions were also further validated by the drug affinity responsive target stability (DARTS) and cellular thermal shift assay (CETSA). The inhibitory effect of CG902 on cell growth was confirmed using the DU145 mouse xenograft model. We propose that CG902 inhibits STAT3 activity through a mechanism that involves the interactions between CG902 and SHP-2, and the phosphorylation of SHP-2, which leads to SHP-2 activation in DU145 cells. CG902 is the first compound to regulate STAT3 activity via the modulation of SHP-2 activity, and our results suggest that CG902 is a novel inhibitor of the STAT3 pathway and an activator of SHP-2, and may be a useful lead molecule for the development of a therapeutic STAT3 inhibitor.

PPARγ agonists induced obesity in animal models as a side effect. Microarray experiments reveal that PPARγ agonist upregulates the expression of lipin-1 and this upregulation is correlated with the activity of the agonists. Lipin-1 induced by PPARγ agonists decreased the levels of PPARγ and ERK1/2 phosphorylation through direct interaction with these proteins in 3T3-L1 cells. In PPARγ agonist-treated 3T3-L1 preadipocytes, the knockdown of lipin-1 expression by small interfering RNA inhibited the adipogenesis that was induced by PPARγ agonists. In contrast, PPARγ2 expression was increased, and lipid droplets were accumulated in lipin-1-overexpressing 3T3-L1 adipocytes. Rosiglitazone (RGZ), a strong PPARγ agonist, synergistically promoted PPARγ dephosphorylation and adipogenesis in lipin-1-overexpressing 3T3-L1 preadipocytes. Therefore, lipin-1 has dual functions as a transcriptional cofactor and phosphatidate phosphatase (PAP) in the differentiation of preadipocyte cells induced by strong PPARγ agonists. In addition, the adipogenesis of 3T3-L1 cells was markedly upregulated by diacylglycerol (DAG), which was produced by lipin-1. Therefore, lipin-1 induction by PPARγ agonists might be an important factor in understanding the biological mechanism of the agonists’ adverse effects, and this information may be valuable in the development of type-2 diabetes mellitus (T2DM) therapeutics with reduced adverse effects and greater tolerability.

Cancer cells, compared to normal cells, are under oxidative stress associated with an elevated level of reactive oxygen species (ROS) and are more vulnerable to oxidative stress induced by ROS generating agents. Thus, manipulation of the ROS level provides a logical approach to kill cancer cells preferentially, without significant toxicity to normal cells, and great efforts have been dedicated to the development of strategies to induce cytotoxic oxidative stress for cancer treatment. Fenton reaction is an important biological reaction in which irons convert hydrogen peroxide (H2O2) to highly toxic hydroxyl radicals that escalate ROS stress. Here, we report Fenton reaction-performing polymer (PolyCAFe) micelles as a new class of ROS-manipulating anticancer therapeutic agents. Amphiphilic PolyCAFe incorporates H2O2-generating benzoyloxycinnamaldehyde and iron-containing compounds in its backbone and self-assembles to form micelles that serve as Nano-Fenton reactors to generate cytotoxic hydroxyl radicals, killing cancer cells preferentially. When intravenously injected, PolyCAFe micelles could accumulate in tumors preferentially to remarkably suppress tumor growth, without toxicity to normal tissues. This study demonstrates the tremendous translatable potential of Nano-Fenton reactors as a new class of anticancer drugs.

The positively charged surface with a 50KRSIK54 motif is the main interaction site of hAng for both heparin and DNA binding, providing an insight into the potential role of the 50KRSIK54 motif for the internalization and promoter binding of hAng, which is essential for the regulation of angiogenesis.

To identify signal transducer and activator of transcription factor 3 (STAT3) inhibitors, we generated STAT3-dependent gene expression signature by analyzing gene expression profiles of DU145 cancer cells treated with STAT3 inhibitor, piperlongumine and 2-hydroxycinnamaldehyde. Then we explored gene expression signature-based strategies using a connectivity map database and identified several STAT3 inhibitors, including ethacrynic acid (EA). EA is currently used as a diuretic drug. EA inhibited STAT3 activation in DU145 prostate cancer cells and consequently decreased the levels of STAT3 target genes such as cyclin A and MCL-1. Furthermore, EA treatment inhibited tumor growth in mice xenografted with DU145 cells and decreased p-STAT3 expression in tumor tissues. Knockdown of Src homology region 2 domain-containing phosphatase-2 (SHP2) or Protein tyrosine phosphatase 1B (PTP1B) gene expression by siRNA suppressed the ability of EA to inhibit STAT3 activation. When EA was combined with an activator of SHP2 or PTP1B, p-STAT3 expression was synergistically decreased; when EA was combined with an inhibitor of SHP2 or PTP1B, p-STAT3 expression was rescued. By using an affinity pulldown assay with biotinyl-EA, EA was shown to associate with SHP2 and PTP1B in vitro. Additionally, the drug affinity responsive target stability (DARTS) assay confirmed the direct binding of EA to SHP2 and PTP1B. SHP2 is activated by EA through active phosphorylation at Y580 and direct binding to SHP2. Collectively, our results suggest that EA inhibits STAT3 activity through the modulation of phosphatases such as SHP2 and PTP1B and may be a potential anticancer drug to target STAT3 in cancer progression.

The aim of the present study was to evaluate the effect of the herbal medicine, tanshinone IIA (Tan IIA), on vestibular schwannoma (VS) cells and assess the functional targets of Tan IIA. HEI‑193 cells and Nf2‑/‑mouse Schwann (SC4) cells were used to investigate the inhibitory effects of Tan IIA on VS. Cell viability was measured using an MTT assay and apoptosis was assessed by flow cytometry. Western blot analysis and reverse transcription quantitative polymerase chain reaction (RT‑qPCR) were performed to assess the expression of hypoxia‑inducible factor‑1α (HIF‑1α) and its signaling pathways. In addition, the effect of Tan IIA on HIF‑1α transcription was determined using a luciferase reporter assay. Schwannoma cell proliferation was observed to be inhibited as the Tan IIA concentration increased under normoxic and hypoxic conditions. Furthermore, Tan IIA induced apoptosis in the HEI‑193 cells and inhibited the protein expression of HIF‑1α in the HEI‑193 cells under hypoxia, thus repressing the transcriptional activity of HIF‑1α. The present study demonstrated that HIF‑1α is expressed in hypoxic VS cells and Tan IIA inhibits VS cells by suppressing the activity of HIF‑1α. In conclusion, these results indicate that Tan IIA is a potential chemotherapeutic agent for the treatment of VS.

During the search for signal transducer and activator of transcription 3 (STAT3) inhibitors from natural products, methyllucidone, isolated from Lindera species (Lauraceae), was identified as a STAT3 inhibitor. Methyllucidone inhibited STAT3 phosphorylation at tyrosine 705 in a dose- and time dependent manner in DU145 prostate cancer cells and suppressed IL-6-induced STAT3 phosphorylation at Tyr-705 in LNCaP cells. Methyllucidone decreased the expression levels of STAT3 target genes, such as cyclin D1, cyclin A, Bcl-2, Mcl-1, and survivin. Methyllucidone inhibited DU145 cell growth and induced apoptosis by arresting the cell cycle at G1 phase. Notably, knockdown of the MEG2 gene by small interfering RNA suppressed the ability of methyllucidone to inhibit STAT3 activation. Methyllucidone regulates STAT3 activity by modulating MEG2 expression, and our results suggest that this compound is a novel inhibitor of the STAT3 pathway and may be a useful lead molecule for the development of a therapeutic STAT3 inhibitor.

Cancer cells are under oxidative stress due to a large production of reactive oxygen species (ROS), which involve in cell proliferation and cancer promotion and progression. On the other hand, ROS promotes cell death, depending on the rate of ROS production and the activity of antioxidant systems. Recently, "oxidation therapy" has arisen as a promising anticancer strategy, which can be achieved by inducing the generation of cytotoxic level of ROS or inhibiting the antioxidant systems in tumor cells. Here, we report oxidative stress amplifying nanoplatforms as novel anticancer therapeutics, which are able not only to suppress antioxidant but also to generate ROS simultaneously in acidic tumor microenvironments. The oxidative stress amplifying nanoplatforms are composed of dual pH-sensitive PBCAE copolymer, polymeric prodrug of BCA (benzoyloxycinnamaldehyde) and heme oxygenase-1 (HO-1) inhibiting zinc protoporphyrin (ZnPP). PBCAE was designed to incorporate ROS-generating BCA in its backbone via acid-cleavable acetal linkages and self-assemble to form micelles that encapsulate ZnPP. In vitro proof-of-concept studies revealed that ZnPP encapsulated in PBCAE micelles suppressed HO-1 to make cancer cells more vulnerable to BCA-induced ROS, leading to enhanced apoptotic cell death. In addition, ZnPP-loaded PBCAE micelles significantly suppressed the tumor growth in human cancer xenograft mouse models. We believe that oxidative stress amplifying micellar nanoparticles have a great potential as novel redox anticancer therapeutics.

The constitutive activation of STAT3 in human cancers causes the abnormal proliferation and survival of cancer cells, and thus, STAT3 is a therapeutic target of antitumor drugs. We screened a small-molecule library of 8600 synthetic compounds from the “Korea Chemical Bank” to identify inhibit STAT3 activity using a cell-based luciferase assay system. KT-18618 (( Z )- N -(4-chlorophenyl)- N -methyl-2-[1,3,3,3,-tetrafluoro-2-(thiophen-2-yl)prop-1-enyloxy]-acetamide) was selected as a novel inhibitor of the JAK/STAT3 pathway. KT-18618 inhibited STAT3 phosphorylation and the expression of STAT3-regulated genes. The inhibition of STAT3 phosphorylation led to the apoptosis of MDA-MB-468 cells. We postulated that the inhibition of the JAK family of proteins or c-Src inhibited STAT3 phosphorylation. Interestingly, the phosphorylation of these kinases was only mildly inhibited, but the phosphorylation of STAT3 was completely inhibited. This result implies that the inhibition of STAT3 phosphorylation by KT-18618 is an independent event that occurs through the phosphorylation of upstream kinases. Co-immunoprecipitation experiments revealed that KT-18618 inhibited the JAK3-STAT3 interaction. Moreover, JAK3 molecules were captured by biotinylated KT-18618, implying that KT-18618 bound to JAK3 molecules. Additionally, 1 μM KT-18618 inhibited JAK3 kinase activity by approximately 28% in an in vitro kinase assay. From these results, we suggest that KT-18618 binds to JAK3 molecules and disrupts the JAK3-STAT3 interaction, which leads to the inhibition of STAT3 phosphorylation. KT-18618 is the first inhibitor of the JAK3-STAT3 interaction.

Signal Transducer and Activator of Transcription 3 (STAT3) is a cytoplasmic factor that mediates intracellular signaling commonly generated at cell surface receptors and transmits it to the nucleus. In previous research studies we synthesized several molecules inhibiting STAT3 and among them oxadiazole MD77 and pyridazinone I showed an interesting activity. For the first one, a direct inhibition mechanism was determined, while I interfered within the STAT3 pathway at a different level. In order to discover novel compounds possibly endowed with an improved activity, we decided to merge their scaffolds on the basis of their calculated conformational properties. Therefore we designed and synthesized the chimera diastereomers 3-oxo-N-(4-(trifluoromethyl)phenyl)-2,3,4,4a,5,6-hexahydrobenzo[h]cinnoline-6-carboxamide (1, 2) and 3-oxo-N-(4-(trifluoromethyl)phenyl)-2,3,5,6-tetrahydrobenzo[h]cinnoline-6-carboxamide (3) as racemate and their enantiomeric separation was performed. Modeling studies and theoretical prediction of [α]D values were carried out beside NMR studies which allowed us to assign 1 and 2 relative configurations.

Heat shock factor 1 (HSF1) enhances the survival of cancer cells under various stresses. The knock-out of HSF1 impairs cancer formation and progression, suggesting that HSF1 is a promising therapeutic target. To identify inhibitors of HSF1 activity, we performed cell-based screening with a library of marketed and experimental drugs and identified cantharidin as an HSF1 inhibitor. Cantharidin is a potent antitumor agent from traditional Chinese medicine. Cantharidin inhibited heat shock-induced luciferase activity with an IC50 of 4.2 μm. In contrast, cantharidin did not inhibit NF-κB luciferase reporter activity, demonstrating that cantharidin is not a general transcription inhibitor. When the HCT-116 colorectal cancer cells were exposed to heat shock in the presence of cantharidin, the induction of HSF1 downstream target proteins, such as HSP70 and BAG3 (Bcl-2-associated athanogene domain 3), was suppressed. HSP70 and its co-chaperone BAG3 have been reported to protect cells from apoptosis by stabilizing anti-apoptotic Bcl-2 family proteins. As expected, treating HCT-116 cancer cells with cantharidin significantly decreased the amounts of BCL-2, BCL-xL, and MCL-1 protein and induced apoptotic cell death. Chromatin immunoprecipitation analysis showed that cantharidin inhibited the binding of HSF1 to the HSP70 promoter and subsequently blocked HSF1-dependent p-TEFb recruitment. Therefore, the p-TEFb-dependent phosphorylation of the C-terminal domain of RNA polymerase II was blocked, arresting transcription at the elongation step. Protein phosphatase 2A inhibition with PP2CA siRNA or okadaic acid did not block HSF1 activity, suggesting that cantharidin inhibits HSF1 in a protein phosphatase 2A-independent manner. We show for the first time that cantharidin inhibits HSF1 transcriptional activity.

PRL-3, phosphatase of regenerating liver-3, plays a role in cancer progression through its involvement in invasion, migration, metastasis, and angiogenesis. We synthesized rhodanine derivatives, CG-707 and BR-1, which inhibited PRL-3 enzymatic activity with IC50 values of 0.8 μM and 1.1 μM, respectively. CG-707 and BR-1 strongly inhibited the migration and invasion of PRL-3 overexpressing colon cancer cells without exhibiting cytotoxicity. The specificity of the inhibitors on PRL-3 phosphatase activity was confirmed by the phosphorylation recovery of known PRL-3 substrates such as ezrin and cytokeratin 8. The compounds selectively inhibited PRL-3 in comparison with other phosphatases, and CG-707 regulated epithelial-to-mesenchymal transition (EMT) marker proteins. The results of the present study reveal that rhodanine is a specific PRL-3 inhibitor and a good lead molecule for obtaining a selective PRL-3 inhibitor.

Here, antitumor mechanism of cinnamaldehyde derivative CB-PIC was elucidated in human SW620 colon cancer cells. CB-PIC significantly exerted cytotoxicity, increased sub-G1 accumulation, and cleaved PARP with apoptotic features, while it enhanced the phosphorylation of AMPK alpha and ACC as well as activated the ERK in hypoxic SW620 cells. Furthermore, CB-PIC suppressed the expression of HIF1 alpha, Akt, and mTOR and activated the AMPK phosphorylation in hypoxic SW620 cells. Conversely, silencing of AMPKα blocked PARP cleavage and ERK activation induced by CB-PIC, while ERK inhibitor PD 98059 attenuated the phosphorylation of AMPKα in hypoxic SW620 cells, implying cross-talk between ERK and AMPKα. Furthermore, cotreatment of CB-PIC and metformin enhanced the inhibition of HIF1α and Akt/mTOR and the activation of AMPKα and pACC in hypoxic SW620 cells. In addition, CB-PIC suppressed the growth of SW620 cells inoculated in BALB/c athymic nude mice, and immunohistochemistry revealed that CB-PIC treatment attenuated the expression of Ki-67, CD34, and CAIX and increased the expression of pAMPKα in CB-PIC-treated group. Interestingly, CP-PIC showed better antitumor activity in SW620 colon cancer cells under hypoxia than under normoxia, since it may be applied to chemoresistance. Overall, our findings suggest that activation of AMPKα and ERK mediates CB-PIC-induced apoptosis in hypoxic SW620 colon cancer cells.

Obovatol, a major constituent of the leaves of Magnolia obovata Thunb, is known to inhibit nuclear factor-κB activity and arachidonic acid-induced platelet aggregation. This study was performed to identify the metabolites of obovatol in human liver microsomes. Human liver microsomes incubated with obovatol in the presence of NADPH and/or UDPGA resulted in the formation of six metabolites, M1-M6. M1 and M2 were identified as hydroxyobovatol, on the basis of liquid chromatography/tandem mass spectrometric (LC-MS/MS) analysis. M1, M2 and obovatol were further metabolized to their glucuronide conjugates, obovatol-glucuronide (M3), obovatol-diglucuronide (M4) and hydroxyobovatol-glucuronide (M5 and M6). The inhibitory potency of obovatol on eight major human P450s was also investigated in human liver microsomes. In these experiments, obovatol strongly inhibited CYP2C19-mediated S-mephenytoin hydroxylase activity with an IC(50) value of 0.8 µM, which could have implications for drug-drug interactions.

The signal transducer and activator of transcription 3 (STAT3) is constitutively activated in cancer cells. Therefore, blocking the aberrant activity of STAT3 in tumor cells is a validated therapeutic strategy. To discover novel inhibitors of STAT3 activity, we screened against microbial natural products using a dual-luciferase assay. Using the microbial metabolome library, we identified cosmomycin C (CosC), which was isolated from the mycelium extract of Streptomyces sp. KCTC19769, as a STAT3 pathway inhibitor. CosC inhibited STAT3 (Tyr705) phosphorylation and subsequent nuclear translocation in MDA-MB-468 breast cancer cells. CosC-mediated inhibition of STAT3 signaling pathway was confirmed by suppressed expression of STAT3 downstream target proteins including cyclin D1, Bcl-xL, survivin, Mcl-1, and VEGF in CosC-treated MDA-MB-468 cells. Flow cytometry showed that CosC caused accumulation in the G0–G1 phase of the cell cycle and induced apoptosis via PARP cleavage and caspase-3 activation. Based on these findings, CosC may be a potential candidate for modulation of STAT3 pathway.

2-Hydroxycinnamaldehyde (HCA) and curcumin have been reported to have antitumor effects against various human tumor cells in vitro and in vivo by generation of ROS. Aldehyde-free HCA analogs were synthesized based on the structure of curcumin, which we have called 2-hydroxycurcuminoids. The hydroxyl group of curcuminoids enhances the ability to generate ROS. 2-Hydroxycurcuminoid (HCC-7) strongly inhibited the growth of SW620 colon tumor cells with a GI(50) value of 7μM, while the parent compounds, HCA and curcumin, displayed GI(50) values of 12 and 30μM, respectively. HCC-7 was found to induce apoptosis through the reactive oxygen species-mitochondria pathway and cell cycle arrest at G2/M phase.

The methanolic extract of the roots of Rubia akane (Rubiaceae) was found to show inhibitory activity on phosphatase of regenerating liver-3 (PRL-3). Bioassay-guided fractionation of the methanolic extract resulted in the isolation of two anthraquinone compounds, 2-methyl-1,3,6-trihydroxy-9,10-anthraquinone-3-O-(6'-O-acetyl)-α-rhamnosyl(1→2)-β-glucoside and 2-methyl-1,3,6-trihydroxy-9,10-anthraquinone, as inhibitors on PRL-3. These compounds inhibited PRL-3 in a dose-dependent manner with IC₅₀ values of 5.2 and 1.3 μg/mL, respectively.

Hypoxia-inducible factor (HIF)-1 is a therapeutic target in solid tumors. We report the novel benzimidazole analogue AC1-004, obtained from a chemical library using an HRE-dependent cell-based assay in colorectal carcinoma HCT-116 cells. The accumulation of hypoxia-induced HIF-1alpha was inhibited by compound AC1-004 in various cancer cells, including HCT-116, MDA-MB435, SK-HEP1, and Caki-1. Further, AC1-004 down-regulated VEGF and EPO, target genes of HIF-1, and inhibited in vitro tube formation of HUVEC, suggesting its potential inhibitory activity on angiogenesis. Importantly, AC1-004 was found to regulate the stability of HIF-1alpha through the Hsp90-Akt pathway, leading to the degradation of HIF-1alpha. An in vivo antitumor study demonstrated that AC1-004 reduced tumor size significantly (i.e., by 58.6%), without severe side effects. These results suggest the benzimidazole analogue AC1-004 is a novel HIF inhibitor that targets HIF-1alpha via the Hsp90-Akt pathway, and that it can be used as a new lead in developing anticancer drugs.

The pharmacokinetics and metabolism of 2'-benzoyloxycinnamaldehyde (BCA) was characterized in male Sprague-Dawley rats as part of the preclinical evaluations for developing this compound as an antitumour agent. BCA was not detected in the plasma following either intravenous or oral dose, whereas its putative metabolites 2'-hydroxycinnamaldehyde (HCA) and o-coumaric acid were present at considerable levels. In separate pharmacokinetics studies, HCA exhibited a high systemic clearance and a large volume of distribution, whereas both pharmacokinetic parameters were much lower for o-coumaric acid. The terminal half-life of both metabolites was approximately 2 h. BCA was converted rapidly to HCA in rat serum, liver microsomes and cytosol in vitro; HCA was subsequently converted to o-coumaric acid in a quantitative manner only in the liver cytosol. In addition, the formation of o-coumaric acid was inhibited significantly by menadione, a specific inhibitor for aldehyde oxidase. Taken collectively, the results suggest that the rapid systemic clearance of HCA is likely due mainly to hepatic clearance occurring from aldehyde oxidase-catalysed biotransformation to o- coumaric acid. In conclusion, the present work demonstrates that the anticancer drug candidate BCA is highly likely to work as its active metabolite HCA in the body.

Neolignans such as obovatol, honokiol, and magnolol have been previously reported to show various biological activities including anti-inflammation and antitumor effects. This is the first demonstration on the in vivo antitumor effect of obovatol on human colorectal carcinoma SW620 cells. Nude mice were implanted with SW620 cells and fed with vehicle or 5 mg/kg/d dose of obovatol for 20 days. Obovatol inhibited tumor growth that accounted for 50% decrease in tumor volume and 44.6% decrease in tumor weight at the end of the experiment without any adverse health effect. In nude mice bearing SW620-incubated tumor, obovatol exhibited more potent antitumor activity than honolkiol. In addition, DNA flow cytometric analysis shows that obovatol progresses to apoptosis as detected by flow cytometry after double staining with annexin V and propidium iodide. Thus, we suggest that obovatol is a potent inducer of cell apoptosis in SW620 cells, and a potent antitumor agent.

The methanol extract from the aerial parts of Aceriphyllum rossii was fractionated into ethyl acetate, n-BuOH and layers through solvent fractionation. Repeated silica gel column chromatography of EtOAc and n-BuOH layers afforded five flavonol glycosides. They were identified as astragalin (1), kaempferol 3-O--L-rhamnopyranosyl (16)--D-glucopyranoside (2), rutin (3), kaempferol 3-O--L-rhamnopyranosyl (14)--L-rhamnopyranosyl 16)--D-glucopyranoside (4), and quercetin 3-O--L-rhamnopyranosyl (14)--L-rhamnopyranosyl (16)--D-glucopyranoside (5) on the basis of spectroscopic data. All of them showed an inhibition in farnesyl protein tranferase (FPTase) activity, and rutin (3) inhibited the growth of rat H-ras cell and the cell migration of human umbilical vein endothelial cells (HUVECs).

A series of cinnamyl compounds related to 2′-hydroxycinnamaldehyde were synthesized and their antitumor effects against human cancer cells evaluated. Hydroxylamine derivative 6 inhibited the growth of human cancer cells and human colon tumor xenograft in nude mice. Its antitumor effects belong to the induction of apoptosis and arresting cell cycle at G2/M phase, which is confirmed by detection of apoptosis markers and cell cycle analysis.

Because the activation of matrix metalloproteinases (MMP) is a key factor in the metastatic process, compounds with the ability to inhibit MMP activity have a potential in the treatment of tumor. From the examination of 2000 plant extracts, obovatal isolated from the extract of the leaves of Magnolia obovata THUNB was a potent inhibitor of MMP-2 enzyme in vitro. In human fibrosarcoma cells (HT1080) activated with MMP-2, obovatal inhibited MMP-2 enzyme activity and expression. In addition, the compound blocked migration and invasion of the cells. This study demonstrates that obovatal exerts its anticancer effects through blocking migration and invasion by inhibition of MMP-2 expression and activity, and also will be a good lead molecule for the development of anti-tumor drug.

The methanolic extract of the leaves of Liriodendron tulipifera was found to show inhibitory activity towards farnesyl protein transferase (FPTase). Bioassay-guided fractionation of the methanolic extract resulted in the isolation of lipiferolide, an inhibitor of FPTase. This compound inhibited the FPTase activity in a dose-dependent manner, and showed cell growth inhibitory activity against several tumor cells.

In the course of search for potent chitin synthase inhibitors from natural resources, a novel chitin synthases inhibitor, 2'-benzoyloxycinnamaldehyde (2'-BCA) (I), was isolated from the aerial parts of Pleuropterus ciliinervis NAKAI. 2'-BCA inhibited chitin synthase 1 and 2 of Saccharomyces cerevisiae with the IC50s of 54.9 and 70.8 microg/ml, respectively, whereas it exhibited no inhibitory activity for chitin synthase 3 up to 280 microg/ml. Its derivatives, 2'-chloro- (V) and 2(-bromo-cinnamaldehyde (VI), each showed 1.9 and 2.7-fold stronger inhibitory activities than 2'-BCA, with the IC50s of 37.2 and 26.6 microg/ml, respectively. Especially, the IC50 of compound VI against chitin synthase 2 represented 1.7-fold more potent inhibitory activity than polyoxin D, a well-known chitin synthase inhibitor. Furthermore, compounds V and VI showed potent antifungal activities against various fungi including human pathogenic fungi, with a particularly strong inhibitory activity against Cryptococcus neoformans (MIC = 16 microg/ml). Although the chemical synthesis of this compound has been reported, the present study is the first report to describe the isolation of 2'-BCA from natural resources and chitin synthases inhibitory activities of its derivatives. These results suggested that 2'-BCA and its derivatives can potentially serve as useful lead compounds for development of antifungal agents.

As a key step in achieving low-cost, easily accessible anti-cancer therapy for low- and middle-income countries, we recently established the scientific basis for the folkloric use of Artocarpus altilis for the treatment of cancer by investigating the geranyl dihydrochalcone (CG-901) content and its interference with signal transducer and activator of transcription 3 (STAT3) phosphorylation and blockage of further downstream signaling. In the current study, the CG-901 upstream target was queried by chemical fingerprinting similarity assessment, semi-empirical (PM6ESCF) QMMM and molecular dynamics (MD) simulation. Moderate (∼0.4) to high (∼0.7) Tanimoto scores were found when the CG-901 scaffold was compared to ligands co-crystallized with Janus kinases (JAK) 1-3. High negative energy values were obtained when the CG-901 was treated semi-empirically (PM6ESCF) within the classical field of JAK (1-3). Multiple nanosecond MD simulations showed that CG-901 did not cause any large structural perturbations in the nucleotide-binding, activation and catalytic loops within the kinase (JH1) domain of JAK (1-3); however, it reduced the energy required to attain metastability along the path to energy minima conformation. In comparison to JAK1 and Apo-state JAK2, JAK2-bound CG-901 exhibited a highly re-organized key intra-domain protein network; indicating atomic level interference with inter-residue communication. In conclusion, CG-901 isolated from A. altilis represents a broad-spectrum JAK inhibitor, which may underlie the mechanism of STAT3 phosphorylation blockage. Graphical abstract Upper panel Janus kinase 2 upstream signaling pathway. Lower panel Apo-JAK2 (left) and CG-901-bound JAK2 (right).

Signal transducer and activator of transcription 3 (STAT3) is constitutively activated in various human cancers and has been used as a therapeutic target for tumors. This study screened natural products to identify compounds that inhibit STAT3 activity using a STAT3-dependent luciferase reporter system. Sugiol was identified as a compound that decreased luciferase activity in a dose-dependent manner. Sugiol specifically inhibited STAT3 phosphorylation at Tyr-705 in DU145 prostate cells, and this inhibition was independent of the STAT3 upstream kinase. Sugiol induced cell cycle arrest and decreased the expression levels of STAT3 target genes, such as cyclin D1, cyclin A, and survivin. Notably, we observed that sugiol interacted with transketolase, an enzyme in central metabolism, and increased ROS levels leading to the activation of ERK, which inhibits STAT3 activity. The protein phosphatase MEG2 was also responsible for sugiol-induced STAT3 dephosphorylation. The inhibitory effect of sugiol on cell growth was confirmed using the DU145 mouse xenograft model. We propose that sugiol inhibits STAT3 activity through a mechanism that involves the inhibition of transketolase, which leads to increased ROS levels and MEG2 activation in DU145 cells. Therefore, sugiol is the first compound regulating STAT3 activity via modulation of cancer metabolic pathway and we suggest the use of sugiol as an inhibitor of the STAT3 pathway for the treatment of human solid tumors with activated STAT3.

Two biflavonoids, ginkgetin (1) and sciadopitysin (2), were isolated from the MeOH extract of the young branches of Taxus cuspidata, which inhibited phosphatase of regenerating liver-3 (PRL-3) with IC50 values of 25.8 and 46.2 microM, respectively. This is the first report on PRL-3 inhibitors, isolated from natural sources.

Twigs from Ilex macropoda were extracted with MeOH, and the concentrated extracts were partitioned with CH2Cl2, EtOAc, n-BuOH, and H2O. Repeated column chromatography of the CH2Cl2 fraction ultimately resulted in the isolation of two compounds, via activity-guided fractionation, using ACAT inhibitory activity measurements. According to the physico-chemical data, the chemical structures of these isolated compounds were identified as lupeol (1) and betulin (2). Compounds 1 and 2 were shown to inhibit the activity of hACAT-1 and hACAT-2 in a dose-dependent manner, and compounds 1 and 2 inhibited hACAT-1 with IC50 values of 48 and 83 microM, respectively.

Two cyclic diarylheptanoids, garugamblin-3 (1) and acerogenin L (2), isolated from the MeOH extract of the fruits of Alnus japonica Steud., inhibited human low-density lipoprotein (LDL) oxidation in the thiobarbituric acid-reactive substance assay with IC50 values of 2.9 and 1.7 microM, respectively, and they also inhibited cell-mediated LDL oxidation more than five times more strongly than that of a well-known antioxidant, probucol, at a concentration of 10 microM. 1 had no effect on the anti-atherogenesis in low-density lipoprotein receptor- deficient mice.

Two cyclic diarylheptanoids, garugamblin-3 (1) and acerogenin L (2), isolated from the MeOH extract of the fruits of Alnus japonica Steud., inhibited human low-density lipoprotein (LDL) oxidation in the thiobarbituric acid-reactive substance assay with IC(50) values of 2.9 and 1.7 microM, respectively, and they also inhibited cell-mediated LDL oxidation more than 5 times more strongly than that of a well-known antioxidant, probucol, at a concentration of 10 microM. Compound (1) had no effect on the anti-atherogenesis in low-density lipoprotein receptor-deficient mice.

Cell migration is a prerequisite for cancer invasion and metastasis, suggesting cell motility as a potential therapeutic target for cancer treatment. A synthetic library was screened to identify inhibitors of tumor cell migration. From this, we discovered that CAC-1098 (aurintricarboxylic acid) and CBI-0997 (5-(2,4-dimethoxy-5-ethylphenyl)-4-(4-bromophenyl) isoxazole) inhibited migration of MDA-MB-231 cells with IC50 = 5 and 50 nM, respectively. We synthesized KRIBB3 (5-(5-ethyl-2-hydroxy-4-methoxyphenyl)-4-(4-methoxyphenyl) isoxazole) by replacing the bromide group of CBI-0997 with a methoxyl group. Like CBI-0997, KRIBB3 has anti-migratory and anti-invasive activities in MDA-MB-231 cells. Because KRIBB3 has a better drug-like structure, we focused our effort on further understanding its anti-migratory mechanism. Biotinyl-KRIBB3 was synthesized as an affinity probe for identification of KRIBB3-binding proteins. Using affinity chromatography, we identified Hsp27 as a target protein of KRIBB3 in vitro. Treatment of MDA-MB-231 cells with phorbol 12-myristate 13-acetate induced protein kinase C-dependent phosphorylation of Hsp27 and tumor cell migration. In contrast, treatment of MDA-MB-231 cells with KRIBB3 blocked phorbol 12-myristate 13-acetate-induced phosphorylation of Hsp27 and tumor cell migration. Furthermore, overexpression of Hsp27 antagonized the inhibitory effect of KRIBB3 on tumor cell invasion, and knockdown of Hsp27 using small interfering RNA inhibited tumor cell migration. Overall, our results demonstrate that KRIBB3 inhibits tumor cell migration and invasion by blocking protein kinase C-dependent phosphorylation of Hsp27 through its direct binding to Hsp27.

Four cyclopentenediones, farnesyl protein transferase inhibitors, and anti-tumor compounds were isolated from the methanolic extract of the fruits of Lindera erythrocarpa Makino (Lauraceae). The structure of the compounds was determined by spectral data including NMR and mass spectrometry, and cyclopentenediones such as methyllinderone (1), methyllucidone (2), lucidone (3), and linderone (4) were identified by comparing their reported spectral data with that of the literature values. Compounds 1-4 inhibited farnesyl protein transferase with IC50 value of 55.3+/-4.1, 42+/-1.9, 103+/-5.1, and 40+/-3.5 microM, respectively. Isolated compounds also inhibited the growth of various human cancer cell lines in a dose-dependent manner. Especially, Compounds 1 and 2 selectively inhibited the growth of H-ras-transformed rat-2 cell lines in comparison with normal rat-2 cells with a GI50 value of 0.3 and 0.85 microM, respectively. Methyllucidone strongly inhibited the growth of human cancer cells and colon tumor xenografted in nude mice. The anti-tumor effects of the compound were further confirmed with caspase-3 activation and degradation of PARP. The results suggest that methyllucidone can be a potential anti-cancer agent against H-ras-transformed tumor and will also be a good lead molecule for the development of anti-tumor drug.

2'-Hydroxycinnamaldehyde (HCA) inhibits cell growth of several human cancer cells with unknown mechanisms. We investigated the inhibitory effect of HCA on TNF-alpha-induced cell growth and possible signal pathway in SW620 colon cancer cells. HCA inhibited TNF-alpha-induced SW620 colon cell growth in time- and dose-dependent manner through induction of apoptotic cell death. Parallel with inhibitory effect on cell growth, HCA dose dependency inhibited TNF-alpha-induced activation of NF-kappaB accompanied with inhibition of the translocation of p50. HCA also induced expression of caspase-3 and Bax, but decreased Bcl-2. HCA furthermore activated ERK pathway, and ERK inhibitor reversed inhibitory effect of HCA on cell growth and transcriptional activation of NF-kappaB. These results demonstrate that HCA inhibits cell growth through induction of apoptotic cell death by ERK pathway-dependent NF-kappaB inactivation.

Diarylheptanoids [curcumin (1), demethoxycurcumin (2), bisdemethoxycurcumin (3), bisdimethoxymethylcurcumin (4), and 1,2-dihydrobis(de-O-methyl)curcumin (5)] were isolated from the methanolic extract of Curcuma longa L. and a new cyclic diarylheptanoid (6) and a known Compound 7 were isolated from fruits of Alnus japonica Steud. Diarylheptanoids (1-3) inhibited farnesyl protein transferase (FPTase) with an IC50 of 29-50 microM. The other compounds very mildly inhibited FPTase, therefore, the inhibitory activity on FPTase very much depends on the structure of diarylheptanoids.

Flavonoids are well-known phytochemicals that are produced by various plants in high quantities. The chemopreventive activity of flavonoids is dependent on their structural features. The studies of structure-FPTase inhibitory activity indicated that the number, position and substitution of hydroxyl groups of the A and B rings of flavonoid, and unsaturation of the C2-C3 bond are important factors affecting inhibition on FPTase by flavonoids. A couple of flavonoids inhibited FPTase and also the growth of human tumor cell lines, especially butein, which strongly inhibited the growth of colon cancer cell line (HCT116). However, flavanones and flavanols did not inhibit FPTase nor the growth of tumor cells.

Members of the Artemisia genus are important medicinal plants found throughout the world. Arteminolides A–D ( 1 – 4 ), isolated from the aerial parts of Artemisia, have an inhibitory activity on farnesyl-protein transferase (FPTase; EC 2.5.1.29) in in vitro assay. This study was carried out with the purpose of validating anti-tumor effects of the compounds in human tumor cells and mouse xenograft model. The arteminolides inhibited tumor cell growth in a dose-dependent manner. Furthermore, arteminolide C ( 3 ) blocked in vivo growth of human colon and lung tumor xenograft without the loss of body weight in nude mice.

Cinnamaldehydes have been shown to have inhibitory effects on farnesyl protein transferase (FPTase; EC 2.5.1.29) in vitro, angiogenesis, cell-cell adhesion, and tumor cell growth and to be immunomodulators. However, the mechanisms responsible for these effects remain unknown. To elucidate the molecular mechanism of the cinnamaldehyde derivative CB403 for growth inhibition, CB403 was synthesized from 2'-hydroxycinnamaldehyde. CB403-treated cells were weakly adherent to the culture dishes. In addition, CB403 inhibited tumor growth in these cells in a concentration-dependent manner. FACS analysis using human cancer cells treated with this compound showed cell cycle arrest in mitosis, which was correlated with a marked increase in the amount of cyclin B1. Furthermore, CB403 blocked in vivo growth of human colon and breast tumor xenografts without loss of body weight in nude mice. These results support the hypothesis that the cinnamaldehyde derivative CB403 exerts cytostatic properties by inducing mitotic arrest in cancer cells.

The flavones 5,6-dihydroxy-7,3',4'-trimethoxyflavone ( 1), 5,6,4'-trihydroxy-7,3'-dimethoxyflavone ( 2), 5-hydroxy-3',4',6,7-tetramethoxyflavone, 5,7,3'-trihydroxy-6,4',5'-trimethoxyflavone, ladanein, and hispidulin were isolated from the methanolic extracts of the aerial parts of Artemisia argyi and structures of the compounds were elucidated on the basis of their spectral data. These flavones inhibited farnesyl protein transferase with IC 50 values of 25 - 200 microg/mL. Compound 2 inhibited proliferation of a couple of tumor cell lines and also inhibited neovascularization in a chick chorioallantoic membrane assay. Without loss of body weight of nude mice, compounds 1 and 2 inhibited growth of a colon tumor (SW620) by 44.6 % and 14.6 %, respectively.

Sesquicillin, isolated from fungal fermentation broth, strongly induced G1 phase arrest in human breast cancer cells. During G1 phase arrest, the expression level of cyclin D1, cyclin A, and cyclin E was decreased, and the expression of CDK (cyclin-dependent-kinase) inhibitor, protein p21Waf1/Cip1, was increased in a time-dependent manner in a breast cancer cell MCF-7. Interestingly, the G1 phase arrest induced by sesquicillin also occurred independently of the tumor suppressor protein, p53. Sesquicillin inhibits the proliferation of MCF-7 via G1 phase arrest in association with the induction of CDK inhibitor protein, p21Waf1/Cip1, and the reduction of G1 phase related-cyclin proteins.

A new sesquiterpene–monoterpene lactone, artemisolide, was isolated from the aerial parts of Artemisia argyi. The structure of artemisolide was elucidated by spectroscopic data including HMBC and NOESY. The structure was confirmed by X-ray crystallographic analysis. Artemisolide exhibited in vitro cytotoxic activity with GI50 values of 2–8 μM against cancer cell lines.

The first example of tripeptide inhibitors of farnesyltransferase with sub-micromolar inhibition activity was developed based on the fact that CVFM is not a substrate for farnesyltransferase.

Dynamin I is a key molecule required for the recycling of synaptic vesicles in neurons, and it has been known that dynamin I gene expression is induced during neuronal differentiation. Our previous studies established that neuronal restriction of dynamin I gene expression is controlled by Sp1 and nuclear factor-kappaB-like element-1. Here, using a series of deletion constructs and site-directed mutation, we found that transcription of dynamin I gene during neuronal differentiation of N1E-115 cells is controlled primarily by the Sp1 element located between -13 to -4 bp of the dynamin I promoter. Gel shift analysis demonstrated that in addition to Sp1, Sp3 could interact with this Sp1 element. The requirement for Sp family transcription factors in dynamin I gene expression was confirmed by using mithramycin, an inhibitor of Sp1/Sp3 binding. Mithramycin repressed dynamin I gene expression and resulted in blocking of neuronal differentiation of N1E-115 cells. The localization of the dynamin I protein was also restricted in the peripheral region of the nucleus by the mithramycin treatment. Thus, all of our results suggest that induction of dynamin I gene expression during N1E-115 cell differentiation is modulated by Sp1/Sp3 interactions with the dynamin I promoter, and its expression is important for neuronal differentiation of the N1E-115 cells.

In the course of the search for inhibitors of ScCHS2 from natural sources, we have isolated a new type of chitin synthase 2 inhibitor, obovatol, which has a biphenol skeleton, from Magnolia obovata. Obovatol inhibited chitin synthase 2 activity of Saccharomyces cerevisiae with an IC(50) of 38 microM. Its derivative, tetrahydroobovatol, inhibited chitin synthase 2 activity under the same conditions with an IC(50) of 59 microM. These compounds exhibited no inhibitory activity for ScCHS3, and showed less inhibitory activity for chitin synthase 1 than for chitin synthase 2 (IC(50) > 1 mM). These results indicated that obovatol and tetrahydroobovatol are specific inhibitors of ScCHS2. They also inhibited CaCHS1, which is structurally and functionally analogous to ScCHS2, with similar IC(50)s to ScCHS2 (IC(50) 28 and 51 microM, respectively). The compounds exhibited mixed competitive inhibition with respect to UDP-N-acetyl-D-glucosamine as substrate [inhibition constant (K(i)) 21.8 microM for obovatol and 23.1 microM for tetrahydroobovatol]. Furthermore, they showed antifungal activities against various pathogenic fungi, with a particularly strong inhibitory activity against Cryptococcus neoformans (MIC 7.8 mg/L). The results indicate that obovatol and tetrahydroobovatol can potentially serve as antifungal agents.

2′-Hydroxycinnamaldehyde inhibits breast cancer cell invasion. This study examined whether 2′-hydroxycinnamaldehyde, acting as a Michael acceptor, interferes with the ligand binding of low-density lipoprotein receptor-related protein 1 to mediate breast cancer cell invasion. Low-density lipoprotein receptor-related protein 1, one of the direct molecular targets of 2′-hydroxycinnamaldehyde, is a multifunctional endocytic receptor. Changes in the thiol oxidation status of cell surface receptor proteins may function as a molecular switch, influencing ligand(s) binding. The oxidation status of extracellular cysteine thiol groups in MCF-7 and MDA-MB-231 cells was examined using a fluorescence-activated cell sorter with thiol-specific fluorescent probes; Matrigel invasion and wound-healing assays were performed to determine the effects of 2′-hydroxycinnamaldehyde on in vitro cell migration. The molecular mechanisms by which 2′-hydroxycinnamaldehyde acts were evaluated by transient knockdown using siRNA or inhibition of low-density lipoprotein receptor-related protein 1 by receptor-associated protein treatment. 2′-Hydroxycinnamaldehyde increased α-2-macroglobulin binding to low-density lipoprotein receptor-related protein 1, which was alleviated by pretreatment of cells with N-acetylcystein. 2′-Hydroxycinnamaldehyde decreased the extracellular pepsin concentration significantly in a low-density lipoprotein receptor-related protein 1- and α-2-macroglobulin-dependent manner. The anti-invasive effect of 2′-hydroxycinnamaldehyde was mitigated with receptor-associated protein pretreatment, suggesting that low-density lipoprotein receptor-related protein 1 is essential for the effects of 2′-hydroxycinnamaldehyde. From these data, we suggest that 2′-hydroxycinnamaldehyde increases the cysteine thiol oxidation status of low-density lipoprotein receptor-related protein 1 extracellular domains, which results in α-2-macroglobulin ligand binding stimulation. Therefore, pepsin clearance in a low-density lipoprotein receptor-related protein 1-α-2-macroglobulin-dependent manner might be an important molecular mechanism in 2′-hydroxycinnamaldehyde exerting its anti-invasive action on breast cancer cells. Furthermore, our data may provide an opportunity to promote the importance of the thiol oxidation status of cell surface receptor proteins for regulating cellular signaling pathways that are important in cancer progression.