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Because signal transducer and activator of transcription 3 (STAT3) is constitutively activated in most human solid tumors and is involved in the proliferation, angiogenesis, immune evasion, and antiapoptosis of cancer cells, researchers have focused on STAT3 as a target for cancer therapy. We screened for natural compounds that inhibit the activity of STAT3 using a dual-luciferase assay. Cryptotanshinone was identified as a potent STAT3 inhibitor. Cryptotanshinone rapidly inhibited STAT3 Tyr705 phosphorylation in DU145 prostate cancer cells and the growth of the cells through 96 hours of the treatment. Inhibition of STAT3 Tyr705 phosphorylation in DU145 cells decreased the expression of STAT3 downstream target proteins such as cyclin D1, survivin, and Bcl-xL. To investigate the cryptotanshinone inhibitory mechanism in DU145 cells, we analyzed proteins upstream of STAT3. Although phosphorylation of Janus-activated kinase (JAK) 2 was inhibited by 7 μmol/L cryptotanshinone at 24 hours, inhibition of STAT3 Tyr705 phosphorylation occurred within 30 minutes and the activity of the other proteins was not affected. These results suggest that inhibition of STAT3 phosphorylation is caused by a JAK2-independent mechanism, with suppression of JAK2 phosphorylation as a secondary effect of cryptotanshinone treatment. Continuing experiments revealed the possibility that cryptotanshinone might directly bind to STAT3 molecules. Cryptotanshinone was colocalized with STAT3 molecules in the cytoplasm and inhibited the formation of STAT3 dimers. Computational modeling showed that cryptotanshinone could bind to the SH2 domain of STAT3. These results suggest that cryptotanshinone is a potent anticancer agent targeting the activation STAT3 protein. It is the first report that cryptotanshinone has antitumor activity through the inhibition of STAT3. [Cancer Res 2009;69(1):193–202]

Supplementary Figures 1-3 from Cryptotanshinone Inhibits Constitutive Signal Transducer and Activator of Transcription 3 Function through Blocking the Dimerization in DU145 Prostate Cancer Cells

Signal transducer and activator of transcription 3 (STAT3) plays a critical role in the formation and growth of human cancer. Therefore, STAT3 is a therapeutic target for cancer drug discovery. Acacetin, a flavone present in various plants, inhibits constitutive and inducible STAT3 activation in STAT3-activated DU145 prostate cancer cells. Acacetin inhibits STAT3 activity by directly binding to STAT3, which we confirmed by a pull-down assay with a biotinylated compound and two level-free methods, namely, a drug affinity responsive target stability (DARTS) experiment and a cellular thermal shift assay (CETSA). Acacetin inhibits STAT3 phosphorylation at the tyrosine 705 residue and nuclear translocation in DU145 cells, which leads to the downregulation of STAT3 target genes. Acacetin then induces apoptosis in a time-dependent manner. Interestingly, acacetin induces the production of reactive oxygen species (ROS) that are not involved in the acacetin-induced inhibition of STAT3 activation because the suppressed p-STAT3 level is not rescued by treatment with GSH or NAC, which are general ROS inhibitors. We also found that acacetin inhibits tumor growth in xenografted nude mice. These results suggest that acacetin, as a STAT3 inhibitor, could be a possible drug candidate for targeting STAT3 for the treatment of cancer in humans.

ARPC2 is a subunit of the Arp2/3 complex, which is essential for lamellipodia, invadopodia and filopodia, and ARPC2 has been identified as a migrastatic target molecule. To identify ARPC2 inhibitors, we generated an ARPC2 knockout DLD‐1 human colon cancer cell line using the clustered regularly interspaced short palindromic repeats/CRISPR‐associated protein 9 (CRISPR/Cas9) system and explored gene signature‐based strategies, such as a connectivity map (CMap) using the gene expression profiling data of ARPC2 knockout and knockdown cells. From the CMap‐based drug discovery strategy, we identified pimozide (a clinically used antipsychotic drug) as a migrastatic drug and ARPC2 inhibitor. Pimozide inhibited the migration and invasion of various cancer cells. Through drug affinity responsive target stability (DARTS) analysis and cellular thermal shift assay (CETSA), it was confirmed that pimozide directly binds to ARPC2. Pimozide increased the lag phase of Arp2/3 complex‐dependent actin polymerization and inhibited the vinculin‐mediated recruitment of ARPC2 to focal adhesions in cancer cells. To validate the likely binding of pimozide to ARPC2, mutant cells, including ARPC2F225A, ARPC2F247A and ARPC2Y250F cells, were prepared using ARPC2 knockout cells prepared by gene‐editing technology. Pimozide strongly inhibited the migration of mutant cells because the mutated ARPC2 likely has a larger binding pocket than the wild‐type ARPC2. Therefore, pimozide is a potential ARPC2 inhibitor, and ARPC2 is a new molecular target. Taken together, the results of the present study provide new insights into the molecular mechanism and target that are responsible for the antitumor and antimetastatic activity of pimozide., Pimozide is identified as a migrastatic drug and ARPC2 inhibitor from connectivity map‐based drug discovery strategy. Pimozide inhibits migration and invasion in various cancer cell lines, and suppresses metastasis in an in vivo antimetastatic assay. Through drug affinity responsive target stability (DARTS) analysis and cellular thermal shift assay (CETSA), it was confirmed that pimozide directly binds to ARPC2.

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.

Simple Summary We attempted to identify target proteins and compounds that can be used to overcome EGFR-TKI resistance in NSCLC. To accomplish this, we generated EGFR inhibitor erlotinib-resistant HCC827-ErlR cells and obtained a list of differentially expressed genes. Then, we performed connectivity map analysis and identified heat shock factor 1 (HSF1) as a potential target protein to overcome erlotinib resistance. Using specific HSF1 shRNAs and KRIBB11 (N2-(1H-Indazol-5-yl)-N6-methyl-3-nitropyridine-2,6-diamine), we proved the effectiveness of HSF1 inhibition for overcoming erlotinib resistance in vitro. In addition, we proved the efficacy of emetine in inhibiting HSF1 activity and the tumor growth of erlotinib-resistant PC9-ErlR cells in a mouse model. Abstract Although EGFR-TKI treatment of NSCLC (non-small-cell lung cancer) patients often achieves profound initial responses, the efficacy is transient due to acquired resistance. Multiple receptor tyrosine kinase (RTK) pathways contribute to the resistance of NSCLC to first- and third-generation EGFR-TKIs, such as erlotinib and osimertinib. To identify potential targets for overcoming EGFR-TKI resistance, we performed a gene expression signature-based strategy using connectivity map (CMap) analysis. We generated erlotinib-resistant HCC827-ErlR cells, which showed resistance to erlotinib, gefitinib, osimertinib, and doxorubicin. A list of differentially expressed genes (DEGs) in HCC827-ErlR cells was generated and queried using CMap analysis. Analysis of the top 4 compounds from the CMap list suggested HSF1 as a potential target to overcome EGFR-TKI resistance. HSF1 inhibition by using HSF1 shRNAs or KRIBB11 decreased the expression of HSF1 downstream proteins, such as HSP70 and HSP27, and also decreased the expression of HSP90/HSP70/BAG3 client proteins, such as BCL2, MCL1, EGFR, MET, and AXL, causing apoptosis of EGFR-TKI-resistant cancer cells. Finally, we demonstrated the efficacy of the HSF1 inhibitor on PC9-ErlR cells expressing mutant EGFR (T790M) in vivo. Collectively, these findings support a targetable HSF1-(HSP90/HSP70/BAG3)-(BCL2/MCL1/EGFR/MET/AXL) pathway to overcome multiple mechanisms of EGFR-TKI resistance.

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.

Emerging evidence has shown that flavonoids extracted from Artemisia have beneficial effects on metabolic disorders. However, whether and how jaceosidin ameliorates insulin resistance and diabetic nephropathy in type 2 diabetes mellitus is largely unknown. For 8 weeks, db/db diabetic mice were fed with or without jaceosidin. Oral jaceosidin supplementation reduced fasting blood glucose levels and insulin resistance through the upregulation of insulin receptor downstream pathways in the liver and skeletal muscles. While jaceosidin did not noticeably alter kidney filtration function, this dietary intervention contributed to attenuating the accumulation of advanced glycation end products in diabetic kidneys. The levels of VEGF-a (vascular endothelial growth factor-a) proteins in the diabetic kidneys were markedly diminished by jaceosidin treatments, which increased the expression and activity of Cu (copper) and Zn-SOD (zinc-superoxide dismutase). Therefore, it is suggested that jaceosidin supplementation elicits antidiabetic effects and treats diabetic nephropathy by augmenting insulin signaling, suppressing fibrosis, and enhancing antioxidant activity.

Inhibition of the signal transducer and activator of transcription 3 (STAT3) signaling pathway is a novel therapeutic strategy to treat human cancers with constitutively active STAT3. During the screening of natural products to find STAT3 inhibitors, we identified 2'-hydroxycinnamaldehyde (HCA) as a STAT3 inhibitor, which was isolated from the stem bark of Cinnamomum cassia. In this study, we found that HCA inhibited constitutive and inducible STAT3 activation in STAT3-activated DU145 prostate cancer cells. HCA selectively inhibited the STAT3 activity by direct binding to STAT3, which was confirmed by biochemical methods, including a pull-down assay with biotin-conjugated HCA, a drug affinity responsive target stability (DARTS) experiment and a cellular thermal shift assay (CETSA). HCA inhibited STAT3 phosphorylation at the tyrosine 705 residue, dimer formation, and nuclear translocation in DU145 cells, which led to a downregulation of STAT3 target genes. The downregulation of cell cycle progression and antiapoptosis-related gene expression by HCA induced the accumulation of cells in the G0/G1 phase of the cell cycle and then induced apoptosis. We also found that reactive oxygen species (ROS) were involved in the HCA-induced inhibition of STAT3 activation and cell proliferation because the suppressed p-STAT3 level was rescued by glutathione or N-acetyl-L-cysteine treatment, which are general ROS inhibitors. These results suggest that HCA could be a potent anticancer agent targeting STAT3-activated tumor cells.

Background/aim Few studies have examined the effect of 2'-hydroxycinnamaldehyde (HCA) on head and neck squamous cell carcinoma (HNSCC) cell invasion. This study examined the role of BMP7 on the anti-migration and anti-invasion activity of HCA using HNSCC cells. Materials and methods Matrigel invasion and wound healing assays were conducted to investigate cell migration or invasion. BMP7 overexpression vector or siRNA mixture was used for transient regulation of gene expression. Results HCA attenuated HNSCC cell migration and spheroids Matrigel invasion without cytotoxicity. mRNA and protein expression of BMP7 increased with HCA treatment. Exogenous BMP7 overexpression without HCA treatment attenuated Matrigel invasion of cells. Furthermore, suppression of BMP7 by siRNA alleviated the inhibitory effect of HCA on the invasion of Matrigel by the cell, indicating that BMP7 is responsible for the anti-migration effect of HCA in HNSCC cells. Conclusion HCA treatment led to a remarkable up-regulation of BMP7, which resulted in the attenuation of HNSCC cell invasion.

It is reported that 2'-hydroxycinnamaldehyde (HCA), isolated from cinnamon, has anti-tumor effects through the modulation of multi-target molecules. In this study, we identified pyruvate kinase M2 (PKM2) as a direct target of HCA by use of biochemical methods including affinity chromatography, drug affinity responsive target stability, and cellular thermal shift assay. PKM2 is up-regulated in multiple cancer types and is considered as a potential target for cancer therapy. HCA binds directly to PKM2 and selectively decreases the phosphorylation of PKM2 at Tyr105, indicating a potential anti-proliferative effect on prostate cancer cells. As a PKM2 activator, HCA increases pyruvate kinase activity by promoting the tetrameric state of PKM2. However, HCA suppresses protein kinase activity of PKM2 by decreasing the phosphorylation at Tyr105. Moreover, this leads to a decrease of PKM2-mediated STAT3 phosphorylation at Tyr705 and a down-regulation of target genes, including MEK5 and cyclin D1. Furthermore, HCA suppresses tumor growth and the release of tumor extracellular vesicles in vivo by inhibiting the phosphorylation of PKM2. Collectively, our results suggest that HCA may be a potential anticancer agent targeting PKM2 in cancer progression.

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.

Ginkgetin is a natural biflavonoid isolated from the leaves of Ginkgo biloba, and is characterized by its anti-inflammatory and anti-viral activities. Although numerous studies state that it has also antitumor activity, the anti-proliferative effect of ginkgetin and the underlying mechanism in breast cancer cells have not yet been investigated. In the present study, ginkgetin inhibited the cell viability of MCF-7 and T-47D cells dose-dependently, and suppressed the expression of the estrogen receptor (ER) at the mRNA and protein levels. Among the targets of the ER, 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase 3 (PFKFB3), cyclin D1 and survivin were also downregulated by ginkgetin treatment. The anti-proliferative effects of ginkgetin were sufficient to suppress the growth by estradiol stimulation. However, ginkgetin did not significantly affect the viability of MDA-MB-231 cells, which are ER-negative cells. Furthermore, the knockdown of the ER and an inhibitor of PFKFB3 significantly sensitized MCF-7 and T-47D cells to ginkgetin. These findings suggest that ginkgetin induces cell death in ER-positive breast cancer cells via the inhibition of ER expression and that it is a promising agent for breast cancer treatment.

New Pt(II) complexes (Pt-1-3) bearing 1,2,5-oxadiazole ligands (1-3) were synthesized, characterized and evaluated for their ability to disrupt STAT3 dimerization. Ligand 3·HCl showed cytotoxic effects on HCT-116 cells (IC50 = 95.2 μM) and a selective ability to interact with STAT3 (IC50 = 8.2 μM) versus STAT1 (IC50 > 30 μM). Its corresponding platinum complex Pt-3 exhibited an increased cytotoxicity (IC50 = 18.4 μM) and a stronger interaction with STAT3 (IC50 = 1.4 μM), leading to inhibition of its signaling pathway. Pt-3 was also evaluated in cell-based assays for its action on p53 expression and on STAT3 phosphorylation. In syngeneic murine Lewis lung carcinoma (LLC) implanted in C57BL/6 mice, Pt-3 showed a higher antitumor activity with fewer side effects than cisplatin.

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.

Adipogenesis involved in hypertrophy and hyperplasia of adipocytes is responsible for expanding the mass of adipose tissues in obese individuals. Peroxisome proliferator-activated receptor γ (PPARγ) and CCAAT/enhancer-binding protein α (C/EBPα) are two principal transcription factors induced by delicate signaling pathways, including signal transducer and activator of transcription 5 (STAT5), in adipogenesis. Here, we demonstrated a novel role of ginkgetin, a biflavone from Ginkgo biloba leaves, as a STAT5 inhibitor that blocks the differentiation of preadipocytes into adipocytes. During the differentiation of 3T3-L1 cells, ginkgetin treatment during the first 2 days markedly inhibited the formation of lipid-bearing adipocytes. PPARγ and C/EBPα expression was decreased in 3T3-L1 cells during adipogenesis following ginkgetin treatment, whereas no change was observed in C/EBPβ or C/EBPδ expression. Inhibition of PPARγ and C/EBPα expression by ginkgetin occurred through the prevention of STAT5 activation during the initiation phase of adipogenesis. In addition, ginkgetin-mediated the inhibition of adipogenesis was recapitulated in the differentiation of primary preadipocytes. Lastly, we confirmed the inhibitory effects of ginkgetin on the hypertrophy of white adipose tissues from high-fat diet-fed mice. These results indicate that ginkgetin is a potential anti-adipogenesis and anti-obesity drug.

We hypothesized that octamer-binding transcription factor 4 (OCT4) inhibition would have therapeutic benefits in testicular germ cell tumors (TGCT). To identify inhibitors of OCT4, a chemical library was screened using a luciferase reporter system under the control of an OCT4 response element. A compound named KRIBB53 was identified based on its blocking of OCT4-dependent luciferase activation. When NCCIT cells were exposed to KRIBB53, the expression levels of OCT4 target genes, such as NANOG and USP44, were inhibited with an IC50 of 13 and 15 μM, respectively. In addition, the levels of OCT4 were decreased by exposing NCCIT cells to KRIBB53, and pretreating the cells with the proteasomal inhibitor MG132 reversed the KRIBB53-induced OCT4 degradation. Biotinyl-KRIBB53 was synthesized and showed comparable activity to KRIBB53 in OCT4 downregulation. Using affinity chromatography assay, KRIBB53 was shown to associate with OCT4 in vitro. Furthermore, the drug affinity responsive target stability (DARTS) assay confirmed unmodified KRIBB53 binding to OCT4. KRIBB53 selectively inhibited proliferation of TGCT cells such as NCCIT and Tera-1 cells but not that of immortalized normal cells. Finally, the administration of KRIBB53 at 30 mg/kg reduced tumor volumes by 77% in the mice xenografted with NCCIT cells relative to their vehicle-treated counterparts. Immunoblotting assays showed that expression of OCT4 was lower in KRIBB53-treated tumor tissues than in control tissues. We provide the first report, to our knowledge, of an OCT4 inhibitor that binds to OCT4 and induces its degradation.

Three new ureido-pyridazinone derivatives, which are structurally related to the known STAT3 inhibitor AVS-0288, were designed by taking into account the structure–activity relationships determined for several ureido-oxadiazole derivatives previously studied by our group. Their synthesis was first attempted through suitable 5-aminopyridazinone intermediates (6a and 6b), which molecular structures were confirmed by means of X-ray diffraction data on 6a. Amine functionalization was unsuccessful, therefore, an alternative method was devised. Dual-luciferase and AlphaScreen-based assays were used to test their activity. The obtained data were rationalized on the basis of a modeling study, which focused our attention on the geometrical preferences of the ureido moiety. Computational results seem to indicate that both the 1,2,5-oxadiazole ring and the extended ZZ arrangement are essential and probably act in a synergistic way to confer significant activity against STAT3.

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.

Signal transducer and activator of transcription 3 (STAT3) is constitutively activated in human cancers. Therefore, STAT3 is a therapeutic target of cancer drug discovery. We previously reported that natural products inhibited constitutively activated STAT3 in human prostate tumor cells. We used a dual-luciferase assay to screen 200 natural products isolated from herbal medicines and we identified ginkgetin obtained from the leaves of Ginkgo biloba L. as a STAT3 inhibitor. Ginkgetin inhibited both inducible and constitutively activated STAT3 and blocked the nuclear translocation of p-STAT3 in DU-145 prostate cancer cells. Furthermore, ginkgetin selectively inhibited the growth of prostate tumor cells stimulated with activated STAT3. Ginkgetin induced STAT3 dephosphorylation at Try705 and inhibited its localization to the nucleus, leading to the inhibition of expression of STAT3 target genes such as cell survival-related genes (cyclin D1 and survivin) and anti-apoptotic proteins (Bcl-2 and Bcl-xL). Therefore, ginkgetin inhibited the growth of STAT3-activated tumor cells. We also found that ginkgetin inhibited tumor growth in xenografted nude mice and downregulated p-STAT3(Tyr705) and survivin in tumor tissues. This is the first report that ginkgetin exerts antitumor activity by inhibiting STAT3. Therefore, ginkgetin is a good STAT3 inhibitor and may be a useful lead molecule for development of a therapeutic STAT3 inhibitor.

Artocarpus altilis (Parkinson) Fosberg has traditionally been used in Indonesia for the treatment of liver cirrhosis, hypertension, and diabetes. In many other countries, it is used for the treatment of malaria, yellow fever, and dengue fever. It has been reported that A. altilis extracts have antiatherosclerotic and cytoprotective effects, but its molecular targets in tumor cells are not yet fully understood. The A. altilis extracts and the partially purified fraction have been shown to inhibit STAT3 activity and the phosphorylation of STAT3 in a dose-dependent manner. To identify the active components, a bioassay-guided isolation of the partially purified fraction resulted in the identification of a geranyl dihydrochalcone, CG901. Its chemical structure was established on the basis of spectroscopic evidence and comparison with published data. The partially purified fraction and the isolated a geranyl dihydrochalcone, CG901, down-regulated the expression of STAT3 target genes, induced apoptosis in DU145 prostate cancer cells via caspase-3 and PARP degradation, and inhibited tumor growth in human prostate tumor (DU145) xenograft initiation model. These results suggest that A. altilis could be a good natural source and that the isolated compound will be a potential lead molecule for developing novel therapeutics against STAT3-related diseases, including cancer and inflammation. Copyright © 2015 John Wiley & Sons, Ltd.

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.

In many pathogenic Gram-negative bacteria, such as Salmonella, Escherichia coli, Yersinia and Chlamydia spp., which cause diseases in humans, the type III secretion system (TTSS) is an important virulence factor that translocates effector proteins into the cytosol of host cells. Thus, the TTSS is a good target for antibacterial agents. Here we used a hemolysis assay to search for TTSS inhibitors and found that a compound from Magnolia obovata called obovatol blocks the TTSS of Salmonella. Obovatol showed potent inhibitory activity (IC50=19.8 μM) against the TTSS-related hemolysis of Salmonella, which was not due to a reduction of bacterial growth. Instead, the compound inhibited bacterial motility, TTSS-related mRNA expression and effector protein secretion. These data demonstrate the inhibitory effect of obovatol on the Salmonella TTSS and suggest that it could be useful for the prevention and supplementary treatment of bacterial infections.

Ginkgetin has been reported to display antitumor activity. However, the relevant pathway integrating cell cycle regulation and signaling pathways involved in growth inhibition in CRC cells remains to be identified. In this study, ginkgetin-treated HCT116 CRC cells exhibited significant dose-dependent growth inhibition with a GI50 value of 4.0 µM for 48-h treatment, together with apoptosis, via G2-phase cell cycle arrest. When HCT116 cells were treated with 10 µM ginkgetin for 48 h, the percentage of cells in G2/M phase increased by 2.2-fold (43.25%) versus the untreated control (19.69%). Ginkgetin regulated the expression of genes that are critically involved in G2 phase arrest cells, such as b‑Myb, CDC2 and cyclin B1. Furthermore, we found that the suppression of b‑Myb expression by ginkgetin was rescued ~5.1-fold by treatment with a miR-34a inhibitor (500 nM) and b‑Myb was downregulated by >80% by 100 nM miR‑34a mimic. These data suggest that the miRNA34a/b‑Myb/cyclin B1 cascade plays a critical role in ginkgetin-induced G2 cell cycle arrest, as well as in the inhibition of HCT116 cell proliferation. Moreover, the administration of ginkgetin (10 mg/kg) reduced tumor volumes by 36.5% and tumor weight by 37.6% in the mice xenografted with HCT116 cells relative to their vehicle-treated counterparts. Therefore, ginkgetin is the first compound shown to regulate b‑Myb by modulating miR-34a, and we suggest the use of ginkgetin as an inducer of G2 arrest for the treatment of CRC.

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.

Since epithelial-mesenchymal transition (EMT) plays a critical role in cancer progression and in maintaining cancer stem cell properties, EMT is emerging as a therapeutic target for inhibiting the metastatic progression of cancer cells. 2'-Hydroxycinnamaldehyde (HCA) and its derivative, 2'-benzoyloxycinnamaldehyde, have recently been suggested as promising therapeutic candidates for cancer treatment. The purpose of this study is to investigate the anti-metastatic effect of HCA on breast cancer and the molecular mechanisms by which HCA regulates the transcriptional program during EMT. HCA induces epithelial reversion at nanomolar concentrations by suppressing Snail via the nuclear translocalization of GSK-3β, which results in the transcriptional upregulation of E-cadherin. HCA also activates the transcription factor KLF17, which suppresses Id-1, indicating that HCA inhibits EMT by multiple transcriptional programs. Further, HCA treatment significantly inhibits lung metastasis in a mouse orthotopic breast cancer model. This study demonstrates the anti-metastatic effect of the non-toxic natural compound HCA through attenuation of EMT in a breast cancer model.

Cinnamaldehyde, an active compound of cinnamon, has been reported to exert various biological functions such as anti-inflammatory and anti-tumor activities. Previously, we showed that 2'-hydroxycinnamaldehyde (HCA) has an inhibitory effect on nitric oxide (NO) production through the inhibition of NF-κB signaling. In an effort to find a more effective anti-atherosclerotic agent, here we evaluated the anti-inflammatory effect of 2'-benzoyloxycinnamaldehyde (BCA) in RAW 264.7 murine macrophage cells. We showed that BCA more effectively inhibited NO production than HCA with less cytotoxicity. We also demonstrated that BCA inhibited the lipopolysaccharide (LPS)-induced expression of iNOS in a concentration-dependent manner. Signal transduction studies showed that BCA significantly inhibited the phosphorylation of SAPK/JNK and AP-1-dependent reporter gene activity. LPS-induced expression levels of JunB, c-Jun and c-Fos were also decreased by BCA treatment. Moreover, the LPS-induced DNA binding activity of AP-1 was markedly inhibited by BCA. The direct injection of BCA into mice inhibited the LPS-induced increase in plasma nitrite levels, confirming the anti-inflammatory effect of BCA in vivo. Overall, these observations suggest that BCA has the potential for use as an anti-atherosclerotic agent.

Jaceosidin is a naturally occurring flavone with pharmacological activity. Jaceosidin, as one of the major constituents of the medicinal herbs of the genus Artemisia, has been shown to exert anticancer, anti-oxidative, anti-inflammatory, and immunosuppressive effects. This study was undertaken to determine the effect of jaceosidin on microglia and neuroinflammation. Microglia are the innate immune cells in the central nervous system, and they play a central role in the initiation and maintenance of neuroinflammation. We report that jaceosidin inhibits inflammatory activation of microglia, reducing nitric oxide (NO) production and proinflammatory cytokine expression. IC50 for NO inhibition was 27 ± 0.4 μM. The flavone also attenuated microglial neurotoxicity in the microglia/neuroblastoma co-culture. Systemic injection of jaceosidin ameliorated neuroinflammation in the mouse model of experimental allergic encephalomyelitis. These results indicate that plant flavone jaceosidin is a microglial inhibitor with anti-neuroinflammation activity.

Phosphatase of regenerating liver (PRL)-3, a member of a subgroup of protein tyrosine phosphatases that can stimulate the degradation of the extracellular matrix, is over-expressed in metastatic colorectal cancer (CRC) relative to primary tumors. To determine whether PRL-3-induced enhancement of migration and invasion is dependent on the expression of matrix metalloproteinases (MMPs), PRL-3 was expressed in DLD-1 human CRC cells. The motility, migration and invasion characteristics of the cells were examined, and metastasis to the lung was confirmed in a nude mouse using PRL-3-overexpressing DLD-1 cells [DLD-1 (PRL-3)]. Migration and invasion of the cells were inhibited by phosphatase and farnesyltransferase inhibitors. Expression of MMPs was enhanced 3- to 10-fold in comparison to control cells, and migration and invasion were partially inhibited by small interfering RNA (siRNA) knockdown of MMP-2, -13 or -14. Importantly, siRNA knockdown of MMP-7 completely inhibited the migration and invasion of DLD-1 (PRL-3) cells, whereas overexpression of MMP-7 increased migration. The expression of MMP-7 was also downregulated by phosphatase and farnesyltransferase inhibitors. It was found that PRL-3 induced MMP-7 through oncogenic pathways including PI3K/AKT and ERK and that there is a relationship between the expression of PRL-3 and MMP-7 in human tumor cell lines. The expression of MMP-13 and -14 was very sensitive to the inhibition of farnesyltransferase; however, the migration and invasion of DLD-1 (PRL-3) cells did not strongly depend on the expression of MMP-13 or -14. These results suggest that the migration and invasion of PRL-3-expressing CRC cells depends primarily on the expression of MMP-7.

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.

Objectives/Hypothesis: As the molecular biology of vestibular schwannoma (VS) is better understood, new means of targeting the pathways involved for intervention in schwannoma cells are being developed. Honokiol, a bioactive constituent of Magnolia officinalis, has attracted attention due to its diverse biological effects. This study was conducted to determine the inhibitory effect of honokiol on schwannoma cell proliferation. Methods: HEI 193 cells were used to investigate the growth-inhibitory effects of honokiol. Cell proliferation was assessed by MTT assays. Apoptosis was measured by flow cytometry analysis and immunofluorescence staining including Hoechst 33342 and TUNEL. Western blot analysis was used to assess the potential inhibition of extracellular signal-regulated kinase (ERK) and AKT signaling by honokiol. Results: Honokiol exhibited significant antiproliferative activity in a dose-dependent manner on HEI 193 cells. Significant apoptosis was detected on schwannoma cells with 7 mg/mL(IC50) honokiol. Western blot analysis showed significant inhibition of ERK phosphorylation. Conclusions: Honokiol, a low molecular weight natural product, inhibits cell proliferation and promotes apoptosis in schwannoma cells by targeting the ERK pathway. Our data suggest that honokiol can be evaluated as a chemotherapeutic agent for VS.

Aim: Thrombosis occurs in the coronary arteries via the activation of platelets, and leads to acute myocardial infarction and sudden death. Obovatol, a major biphenolic component of Magnolia Obovata leaves, displays anti-inflammatory and acyl Co-A cholesterol acyltrasferase inhibitory effects. The purpose of this study was to determine the effects of obovatol on thrombus formation in vivo and platelet activation in vitro and ex vivo.Methods: We investigated the antiplatelet and antithrombotic activities of obovatol in rat carotid arterial thrombosis in vivo along with platelet aggregation in vitro and ex vivo. Its possible cellular mechanism of antiplatelet activity was investigated by testing PLC-γ2 activation, arachidonic acid cascade, calcium mobilization and granule secretion.Results: Oral administration of obovatol prevented carotid thrombosis, but also significantly inhibited collagen-induced platelet aggregation. Obovatol did not change coagulation times, such as activated partial thromboplastin time and prothrombin time, indicating that the antithrombotic effect of obovatol might be due to antiplatelet activity rather than anticoagulation activity. Obovatol inhibited in vitro collagen- and arachidonic acid-induced rabbit platelet aggregation in a concentration-dependent manner (1-10 µM), with IC50 values of 2.4±0.8 and 4.8±0.9 µM, respectively. Obovatol blocked collagen-mediated phospholipase C-γ2 phosphorylation, cytoplasmic calcium mobilization, arachidonic acid liberation and serotonin secretion.Conclusion: Obovatol has a potent antithrombotic effect, which may be due to antiplatelet activity. The antiplatelet activity of obovatol is mediated by inhibition of PLC-γ2 phosphorylation. Thus, obovatol may be a potential candidate to treat cardiovascular disease.

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.