Clinical applications of circulating biomarkers in non-small cell lung cancer.

Despite recent advances in cancer diagnostics and treatment, the mortality associated with lung cancer is still the highest in the world. Late-stage diagnosis, often accompanied by metastasis, is a major contributor to the high mortality rates, emphasizing the urgent need for reliable and readily accessible diagnostic tools that can detect biomarkers unique to lung cancer. Circulating factors, such as circulating tumor DNA and extracellular vesicles, from liquid biopsy have been recognized as diagnostic or prognostic markers in lung cancer. Numerous clinical studies are currently underway to investigate the potential of circulating tumor DNA, circulating tumor RNA, exosomes, and exosomal microRNA within the context of lung cancer. Those clinical studies aim to address the poor diagnostics and limited treatment options for lung cancer, with the ultimate goal of developing clinical markers and personalized therapies. In this review, we discuss the roles of each circulating factor, its current research status, and ongoing clinical studies of circulating factors in non-small cell lung cancer. Additionally, we discuss the circulating factors specifically found in lung cancer stem cells and examine approved diagnostic assays designed to detect circulating biomarkers in lung cancer patients.

The OTX2 Gene Induces Tumor Growth and Triggers Leptomeningeal Metastasis by Regulating the mTORC2 Signaling Pathway in Group 3 Medulloblastomas.

Medulloblastoma (MB) encompasses diverse subgroups, and leptomeningeal disease/metastasis (LMD) plays a substantial role in associated fatalities. Despite extensive exploration of canonical genes in MB, the molecular mechanisms underlying LMD and the involvement of the orthodenticle homeobox 2 (OTX2) gene, a key driver in aggressive MB Group 3, remain insufficiently understood. Recognizing OTX2's pivotal role, we investigated its potential as a catalyst for aggressive cellular behaviors, including migration, invasion, and metastasis. OTX2 overexpression heightened cell growth, motility, and polarization in Group 3 MB cells. Orthotopic implantation of OTX2-overexpressing cells in mice led to reduced median survival, accompanied by the development of spinal cord and brain metastases. Mechanistically, OTX2 acted as a transcriptional activator of the Mechanistic Target of Rapamycin (mTOR) gene's promoter and the mTORC2 signaling pathway, correlating with upregulated downstream genes that orchestrate cell motility and migration. Knockdown of mTOR mRNA mitigated OTX2-mediated enhancements in cell motility and polarization. Analysis of human MB tumor samples (N = 952) revealed a positive correlation between OTX2 and mTOR mRNA expression, emphasizing the clinical significance of OTX2's role in the mTORC2 pathway. Our results reveal that OTX2 governs the mTORC2 signaling pathway, instigating LMD in Group 3 MBs and offering insights into potential therapeutic avenues through mTORC2 inhibition.

Targeting dysregulated lipid metabolism in the tumor microenvironment.

The reprogramming of lipid metabolism and its association with oncogenic signaling pathways within the tumor microenvironment (TME) have emerged as significant hallmarks of cancer. Lipid metabolism is defined as a complex set of molecular processes including lipid uptake, synthesis, transport, and degradation. The dysregulation of lipid metabolism is affected by enzymes and signaling molecules directly or indirectly involved in the lipid metabolic process. Regulation of lipid metabolizing enzymes has been shown to modulate cancer development and to avoid resistance to anticancer drugs in tumors and the TME. Because of this, understanding the metabolic reprogramming associated with oncogenic progression is important to develop strategies for cancer treatment. Recent advances provide insight into fundamental mechanisms and the connections between altered lipid metabolism and tumorigenesis. In this review, we explore alterations to lipid metabolism and the pivotal factors driving lipid metabolic reprogramming, which exacerbate cancer progression. We also shed light on the latest insights and current therapeutic approaches based on small molecular inhibitors and phytochemicals targeting lipid metabolism for cancer treatment. Further investigations are worthwhile to fully understand the underlying mechanisms and the correlation between altered lipid metabolism and carcinogenesis.

Invasive FoxM1 phosphorylated by PLK1 induces the polarization of tumor-associated macrophages to promote immune escape and metastasis, amplified by IFITM1.

BACKGROUND: Understanding the mechanism behind immune cell plasticity in cancer metastasis is crucial for identifying key regulators. Previously we found that mitotic factors regulate epithelial-mesenchymal transition, but how these factors convert to metastatic players in the tumor microenvironment (TME) is not fully understood. METHODS: The clinical importance of mitotic factors was analyzed by heatmap analysis, a KM plot, and immunohistochemistry in lung adenocarcinoma (LUAD) patients. Immunoprecipitation, LC-MS/MS, kinase assay, and site-directed mutagenesis were performed for the interaction and phosphorylation. A tail-vein injection mouse model, Transwell-based 3D culture, microarray analysis, coculture with monocytes, and chromatin immunoprecipitation assays were used to elucidate the function of phosphorylated FoxM1 in metastasis of TME. RESULTS: The phosphorylated FoxM1 at Ser25 by PLK1 acquires the reprogramming ability to stimulate the invasive traits in cancer and influence immune cell plasticity. This invasive form of p-FoxM1 upregulates the expression of IL1A/1B, VEGFA, and IL6 by direct activation, recruiting monocytes and promoting the polarization of M2d-like tumor-associated macrophages (TAMs). Upregulation of PD-L1 in LUAD having phosphomimetic FoxM1 facilitates immune evasion. In invasive LUAD with phosphomimetic FoxM1, IFITM1 is the most highly expressed through the activation of the STING-TBK1-IRF3 signaling, which enhances FoxM1-mediated signaling. Clinically, higher expression of FOXM1, PLK1, and IFITM1 is inversely correlated with the survival rate of advanced LUAD patients, providing a promising therapeutic strategy for the treatment of LUAD. CONCLUSION: FoxM1-based therapy would be a potential therapeutic strategy for LUAD to reduce TAM polarization, immune escape, and metastasis, since FoxM1 functions as a genetic reprogramming factor reinforcing LUAD malignancy in the TME.

PLK1-mediated phosphorylation of ß-catenin enhances its stability and transcriptional activity for extracellular matrix remodeling in metastatic NSCLC.

Rationale: ß-catenin is a component for cell adhesion and a transcriptional coactivator in epithelial-mesenchymal transition (EMT). Previously we found that catalytically active PLK1 drives EMT in non-small cell lung cancer (NSCLC), upregulating extracellular matrix factors including TSG6, laminin γ2, and CD44. To understand the underlying mechanism and clinical significance of PLK1 and ß-catenin in NSCLC, their relationship and function in metastatic regulation were investigated. Methods: The clinical relevance between the survival rate of NSCLC patients and the expression of PLK1 and ß-catenin was analyzed by a KM plot. Immunoprecipitation, kinase assay, LC-MS/MS spectrometry, and site-directed mutagenesis were performed to reveal their interaction and phosphorylation. A lentiviral doxycycline-inducible system, Transwell-based 3D culture, tail-vein injection model, confocal microscopy, and chromatin immunoprecipitation assays were used to elucidate the function of phosphorylated ß-catenin in the EMT of NSCLC. Results: Clinical analysis revealed that the high expression of CTNNB1/PLK1 was inversely correlated with the survival rates of 1,292 NSCLC patients, especially in metastatic NSCLC. In TGF-ß-induced or active PLK1-driven EMT, ß-catenin, PLK1, TSG6, laminin γ2, and CD44 were concurrently upregulated. ß-catenin is a binding partner of PLK1 in TGF-ß-induced EMT and is phosphorylated at S311. Phosphomimetic ß-catenin promotes cell motility, invasiveness of NSCLC cells, and metastasis in a tail-vein injection mouse model. Its upregulated stability by phosphorylation enhances transcriptional activity through nuclear translocation for the expression of laminin γ2, CD44, and c-Jun, therefore enhancing PLK1 expression by AP-1. Conclusions: Our findings provide evidence for the critical role of the PLK1/ß-catenin/AP-1 axis in metastatic NSCLC, implying that ß-catenin and PLK1 may serve as a molecular target and prognostic indicator of the therapeutic response in metastatic NSCLC patients.

Plk2-mediated phosphorylation and translocalization of Nrf2 activates anti-inflammation through p53/Plk2/p21cip1 signaling in acute kidney injury.

The Plk2 is a cellular stress-responsive factor that is induced in response to oxidative stress. However, the roles of Plk2 in acute kidney injury (AKI) have not been clarified. We previously found that Plk2 is an interacting factor of Nrf2 in response to cellular stress, since Plk2 is upregulated in the Nrf2-dependent network. Here, we show that the levels of p53, Plk2, p21cip1, and chromatin-bound Nrf2 were all upregulated in kidney tissues of mice or NRK52E cells treated with either cisplatin or methotrexate. Upregulation of Plk2 by p53 led to an increase of Nrf2 in both soluble and chromatin fractions in cisplatin-treated NRK52E cells. Consistently, depletion of Plk2 suppressed the levels of Nrf2. Of note, Plk2 directly phosphorylated Nrf2 at Ser40, which facilitated its interaction with p21cip1 and translocation into the nuclei for the activation of anti-oxidative and anti-inflammatory factors in response to AKI. Together, these findings suggest that Plk2 may serve as an anti-oxidative and anti-inflammatory regulator through the phosphorylation and activation of Nrf2 to protect kidney cells from kidney toxicants and that Plk2 and Nrf2 therefore work cooperatively for the protection and survival of kidney cells from harmful stresses.

Improved anti-fibrotic effects by combined treatments of simvastatin and NS-398 in experimental liver fibrosis models.

BACKGROUND/AIMS: Efficient anti-fibrotic therapies are required for the treatment of liver cirrhosis. Hydroxymethylglutaryl-coenzyme A reductase inhibitors (statins) and cyclooxygenase-2 (COX-2) inhibitors have been reported to have anti-fibrotic effects. Here, we investigated whether combined treatment with a statin and a COX-2 inhibitor has synergistic anti-fibrotic effects. METHODS: The effects of treatment strategies incorporating both simvastatin and a COX-2 inhibitor, NS-398, were investigated using an immortalized human hepatic stellate cell line (LX-2) and a hepatic fibrosis mouse model developed using thioacetamide (TAA) in drinking water. Cellular proliferation was investigated via 5-bromo-2-deoxyuridine uptake. Pro- and anti-apoptotic factors were investigated through Western blotting and real-time polymerase chain reaction analysis. RESULTS: The evaluation of the anti-proliferative effects on LX-2 cells showed that the observed effects were more pronounced with combination therapy than with single-drug therapy. Moreover, hepatic fibrosis and collagen deposition decreased significantly in TAA-treated mice in response to the combined treatment strategy. The mechanisms underlying the anti-fibrotic effects of the combination therapy were investigated. The effects of the combination therapy were correlated with increased expression levels of extracellular signal-regulated kinase 1/2 signaling molecules, upregulation of the Bax/Bcl-2 signaling pathway, inhibition of the transforming growth factor-ß signaling pathway, and inhibition of tissue inhibitor of matrix metalloproteinases 1 and 2. CONCLUSION: The combination of simvastatin and NS-398 resulted in a synergistic anti-fibrotic effect through multiple pathways. These findings offer a theoretical insight into the possible clinical application of this strategy for the treatment of advanced liver diseases with hepatic fibrosis.

Mitotic protein kinase-driven crosstalk of machineries for mitosis and metastasis.

Accumulating evidence indicates that mitotic protein kinases are involved in metastatic migration as well as tumorigenesis. Protein kinases and cytoskeletal proteins play a role in the efficient release of metastatic cells from a tumor mass in the tumor microenvironment, in addition to playing roles in mitosis. Mitotic protein kinases, including Polo-like kinase 1 (PLK1) and Aurora kinases, have been shown to be involved in metastasis in addition to cell proliferation and tumorigenesis, depending on the phosphorylation status and cellular context. Although the genetic programs underlying mitosis and metastasis are different, the same protein kinases and cytoskeletal proteins can participate in both mitosis and cell migration/invasion, resulting in migratory tumors. Cytoskeletal remodeling supports several cellular events, including cell division, movement, and migration. Thus, understanding the contributions of cytoskeletal proteins to the processes of cell division and metastatic motility is crucial for developing efficient therapeutic tools to treat cancer metastases. Here, we identify mitotic kinases that function in cancer metastasis as well as tumorigenesis. Several mitotic kinases, namely, PLK1, Aurora kinases, Rho-associated protein kinase 1, and integrin-linked kinase, are considered in this review, as an understanding of the shared machineries between mitosis and metastasis could be helpful for developing new strategies to treat cancer.

Dual Targeting of EGFR with PLK1 Exerts Therapeutic Synergism in Taxane-Resistant Lung Adenocarcinoma by Suppressing ABC Transporters.

To overcome the limitations of chemoresistance, combination therapies using druggable targets have been investigated. Our previous studies led us to hypothesize that the downregulation of PLK1 expression or activity can be one strategy to overcome the hurdles of taxane resistance by the downregulation of ABC transporters. To explore this, various versions of PLK1 including a constitutively active version, kinase-dead form, and polo-box domain mutant were expressed in paclitaxel-resistant lung adenocarcinoma (LUADTXR). Targeting PLK1 using shRNA or non-functional mutants downregulated ABCB1, ABCC9, and ABCG2 in LUADTXR cells, which was similar to the downregulation effects from treatment with PLK1 inhibitors. The high expression of EGFR in LUAD led us to administer gefitinib, showing a markedly reduced EGFR level in LUADTXR cells. When gefitinib and PLK1 inhibitors were combined, LUADTXR cells tended to undergo apoptosis more effectively than parental cells, showing a synergistic effect on the downregulation of ABC transporters through c-Myc and AP-1. Clinical data provide evidence for the relevance between survival rates and expressions of PLK1 and EGFR in LUAD patients. Based on these results, we suggest that a combination of gefitinib and PLK1 inhibitors exerts strong synergism in LUADTXR, which helps to overcome the limitations associated with taxanes.

PLK1/vimentin signaling facilitates immune escape by recruiting Smad2/3 to PD-L1 promoter in metastatic lung adenocarcinoma.

The prerequisite function of vimentin for the epithelial-mesenchymal transition (EMT) is not clearly elucidated yet. Here, we show that vimentin phosphorylated by PLK1, triggers TGF-ß-signaling, which consequently leads to metastasis and PD-L1 expression for immune suppression in lung adenocarcinoma. The clinical correlation between expression of both vimentin and PLK1, and overall survival rates of patients was significant in lung adenocarcinoma but not in squamous cell carcinoma. The phosphorylation of vimentin was accompanied by the activation of PLK1 during TGF-ß-induced EMT in lung adenocarcinoma. Among the several phosphorylation sites determined by phospho-proteomic analysis and the site-specific mutagenesis, the phosphorylation at S339 displayed the most effective metastasis and tumourigenesis with the highest expression of PD-L1, compared with that of wild-type and other versions in both 3D cell culture and tail-vein injection metastasis models. Phosphomimetic vimentin at S339 interacted with p-Smad2 for its nuclear localization, leading to the expression of PD-L1. Clinical relevance revealed the inverse correlation between the survival rates of patients and the expressions of VIM, PLK1, and CD274 in primary and metastatic lung adenocarcinoma. Thus, PLK1-mediated phosphorylation of vimentin activates TGF-ß signaling pathway, leading to the metastasis and immune escape through the expression of PD-L1, functioning as a shuttling protein in lung adenocarcinoma.

Combination of Inhibitors of USP7 and PLK1 has a Strong Synergism against Paclitaxel Resistance.

USP7 is a promising target for the development of cancer treatments because of its high expression and the critical functions of its substrates in carcinogenesis of several different carcinomas. Here, we demonstrated the effectiveness of targeting USP7 in advanced malignant cells showing high levels of USP7, especially in taxane-resistant cancer. USP7 knockdown effectively induced cell death in several cancer cells of lung, prostate, and cervix. Depletion of USP7 induced multiple spindle pole formation in mitosis, and, consequently, resulted in mitotic catastrophe. When USP7 was blocked in the paclitaxel-resistant lung cancer NCI-H460TXR cells, which has resistance to mitotic catastrophe, NCI-H460TXR cells underwent apoptosis effectively. Furthermore, combination treatment with the mitotic kinase PLK1 inhibitor volasertib and the USP7 inhibitor P22077 showed a strong synergism through down-regulation of MDR1/ABCB1 in paclitaxel-resistant lung cancer. Therefore, we suggest USP7 is a promising target for cancer therapy, and combination therapy with inhibitors of PLK1 and USP7 may be valuable for treating paclitaxel-resistant cancers, because of their strong synergism.

CRISPR-mediated promoter de/methylation technologies for gene regulation.

DNA methylation on cytosines of CpG dinucleotides is well established as a basis of epigenetic regulation in mammalian cells. Since aberrant regulation of DNA methylation in promoters of tumor suppressor genes or proto-oncogenes may contribute to the initiation and progression of various types of human cancer, sequence-specific methylation and demethylation technologies could have great clinical benefit. The CRISPR-Cas9 protein with a guide RNA can target DNA sequences regardless of the methylation status of the target site, making this system superb for precise methylation editing and gene regulation. Targeted methylation-editing technologies employing the dCas9 fusion proteins have been shown to be highly effective in gene regulation without altering the DNA sequence. In this review, we discuss epigenetic alterations in tumorigenesis as well as various dCas9 fusion technologies and their usages in site-specific methylation editing and gene regulation.

Active PLK1-driven metastasis is amplified by TGF-ß signaling that forms a positive feedback loop in non-small cell lung cancer.

Early findings that PLK1 is highly expressed in cancer have driven an exploration of its functions in metastasis. However, whether PLK1 induces metastasis in vivo and its underlying mechanisms in NSCLC have not yet been determined. Here, we show that the expression of active PLK1 phosphorylated at T210, abundant in TGF-ß-treated lung cells, potently induced metastasis in a tail-vein injection model. Active PLK1 with intact polo-box and ATP-binding domains accelerated cell motility and invasiveness by triggering EMT reprogramming, whereas a phosphomimetic version of p-S137-PLK1 did not, indicating that the phosphorylation status of PLK1 may determine the cell traits. Active PLK1-driven invasiveness upregulated TGF-ß signaling and TSG6 encoded by TNFAIP6. Loss of TNFAIP6 disturbed the metastatic activity induced by active PLK1 or TGF-ß. Clinical relevance shows that PLK1 and TNFAIP6 are strong predictors of poor survival rates in metastatic NSCLC patients. Therefore, we suggest that active PLK1 promotes metastasis by upregulating TGF-ß signaling, which amplifies its metastatic properties by forming a positive feedback loop and that the PLK1/TGF-ß-driven metastasis is effectively blocked by targeting PLK1 and TSG6, providing PLK1 and TSG6 as negative markers for prognostics and therapeutic targets in metastatic NSCLC.

Correction: Active PLK1-driven metastasis is amplified by TGF-ß signaling that forms a positive feedback loop in non-small cell lung cancer.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Roles of the Phosphorylation of Transcriptional Factors in Epithelial-Mesenchymal Transition.

Epithelial-to-mesenchymal transition (EMT) is the first step in the development of the invasive and migratory properties of cancer metastasis. Since the transcriptional reprogramming of a number of genes occurs in EMT, the regulation of EMT transcription factors has been intensively investigated. EMT transcriptional factors are commonly classified by the direct or indirect repression of E-cadherin because one of hallmarks of EMT is the loss of E-cadherin. This facilitates the expression of genes for EMT, tumor invasion, and metastasis. The posttranslational modification of EMT transcriptional factors, such as Snail and Slug, directly regulates their functions, including their stability, nuclear localization, protein-protein interaction, and ubiquitination for the promotion or termination of EMT at the specific points. Here, we discuss how posttranslational modifications regulate gene expression in a dynamic and reversible manner by modifying upstream signaling pathways, focusing in particular on the posttranslational modifications of Snail, Slug, ZEB1, ZEB2, and TWIST1. This review demonstrates that EMT transcription factors regulate metastasis through their posttranslational modifications and that the flexibility and reversibility of EMT can be modified by phosphorylation.

Cotargeting Plk1 and androgen receptor enhances the therapeutic sensitivity of paclitaxel-resistant prostate cancer.

BACKGROUNDS: Despite the clinical success of taxanes, they still have limitations, such as chemoresistance. To overcome the limitations of paclitaxel, genetic alterations and targeting effects of altered genes were observed in paclitaxel-resistant cancer. Because paclitaxel-resistant cancer shows high levels of Plk1, a promising target in chemotherapy, the effectiveness of Plk1 inhibitors in paclitaxel-resistant cancer cells has been investigated. METHODS: Paclitaxel-resistant cancer cells were developed by exposure of stepwise escalating levels of paclitaxel. Genetic alterations were detected by quantitative reverse transcription polymerase chain reaction (qRT-PCR) and immunoblotting. Using a cell viability assay, combined targeting effects for Plk1 and androgen receptor (AR) were determined. Clinical data were analyzed to understand the relationship between Plk1 and AR in prostate cancer patients. RESULTS: Treatment with Plk1 inhibitors markedly reduced the expression of MDR1, MRP1, and Plk1 in the paclitaxel-resistant cancer. Among Plk1 inhibitors, genistein, recently found as a direct Plk1 inhibitor, tended to be more effective in the paclitaxel-resistant prostate cancer than the parental cancer cells, which was related to the suppression of the AR, as well as inhibition of Plk1 activity. A combination of Plk1 inhibitors and AR antagonist bicalutamide exhibited a synergistic effect in LNCaPTXR, as well as LNCaP cells, by inhibiting Plk1 and AR. Analysis of clinical data provides evidence for the relevance between Plk1 and AR in prostate cancer patients, showing that Plk1 and AR are strong predictors of poor survival rates. CONCLUSIONS: We suggest that cotargeting Plk1 and AR would be effective in advanced chemoresistant prostate cancer cells to overcome the limitations associated with paclitaxel.

Molecular Targets of Genistein and Its Related Flavonoids to Exert Anticancer Effects.

Increased health awareness among the public has highlighted the health benefits of dietary supplements including flavonoids. As flavonoids target several critical factors to exert a variety of biological effects, studies to identify their target-specific effects have been conducted. Herein, we discuss the basic structures of flavonoids and their anticancer activities in relation to the specific biological targets acted upon by these flavonoids. Flavonoids target several signaling pathways involved in apoptosis, cell cycle arrest, mitogen-activated protein kinase (MAPK), phosphoinositide 3-kinase (PI3K)/AKT kinase, and metastasis. Polo-like kinase 1 (PLK1) has been recognized as a valuable target in cancer treatment due to the prognostic implication of PLK1 in cancer patients and its clinical relevance between the overexpression of PLK1 and the reduced survival rates of several carcinoma patients. Recent studies suggest that several flavonoids, including genistein directly inhibit PLK1 inhibitory activity. Later, we focus on the anticancer effects of genistein through inhibition of PLK1.

Nrf2-lncRNA controls cell fate by modulating p53-dependent Nrf2 activation as an miRNA sponge for Plk2 and p21cip1.

Long noncoding RNA (lncRNA) capable of controlling antioxidative capacity remains to be investigated. Nuclear factor erythroid-2-related factor 2 (Nrf2) is a central molecule for cellular defense that increases antioxidative capacity. We identified a novel lncRNA named Nrf2-activating lncRNA (Nrf2-lncRNA) transcribed from an upstream region of the microRNA 122 gene (MIR122). Nrf2-lncRNA existed in the cytoplasm, suggestive of its function as a competing endogenous RNA [ceRNA, microRNA (miRNA) sponge]. Nrf2-lncRNA served as a ceRNA for polo-like kinase (Plk) 2 and cyclin-dependent kinase inhibitor 1 (p21cip1) through binding of miRNA 128 and miRNA 224, inducing Plk2/Nrf2/p21cip1 complexation for Nrf2 activation in the cells under p53-activating conditions (i.e., DNA damage and serum deprivation). Nrf2-lncRNA expression was suppressed with the initiation of apoptosis, being a rheostat for cell fate determination. Nrf2-lncRNA levels correlated with the recurrence-free postsurgery survival rate of patients with hepatocellular carcinoma. Collectively, Nrf2-lncRNA promotes Plk2 and p21cip1 translation by competing for specific miRNAs and activating Nrf2 under surviving conditions from oxidative stress, implying that Nrf2-lncRNA serves as a fine-tuning rheostat for cell fate decision.-Joo, M. S., Shin, S.-B., Kim, E. J., Koo, J. H., Yim, H., Kim, S. G. Nrf2-lncRNA controls cell fate by modulating p53-dependent Nrf2 activation as an miRNA sponge for Plk2 and p21cip1.

7-O-Methylwogonin from Scutellaria baicalensis Disturbs Mitotic Progression by Inhibiting Plk1 Activity in Hep3B Cells.

Polo-like kinase 1, a mitotic Ser/Thr kinase, has emerged as a molecular target for the development of anticancer drugs. In this study, we found that polo-like kinase 1 activity was inhibited by 7-O-methylwogonin and related flavones, including baicalein, dihydrobaicalein, and viscidulin II, isolated from Scutellaria baicalensis. Although dihydrobaicalein exhibited the highest polo-like kinase 1 inhibitory activity among the four compounds, it also inhibited other kinases, such as vaccinia-related kinase 2 and polo-like kinase 2. Baicalein and viscidulin II also showed low selectivity to polo-like kinase 1 since they inhibited polo-like kinase 3 and polo-like kinase 2, respectively. However, 7-O-methylwogonin exhibited selective polo-like kinase 1 inhibitory activity, as evidenced from in vitro kinase assays based on fluorescence resonance energy transfer assays and ADP-Glo kinase assays. In addition, examination of mitotic morphology and immunostaining using specific antibodies for the mitotic markers, p-histone H3 and mitotic protein monoclonal 2, in Hep3B cells showed that 7-O-methylwogonin treatment increased mitotic cell populations due to inhibition of mitotic progression as a result of polo-like kinase 1 inhibition. The pattern of 7-O-methylwogonin-induced mitotic arrest was similar to that of BI 2536, a specific polo-like kinase 1 inhibitor. Thus, it was suggested that 7-O-methylwogonin disturbed mitotic progression by inhibiting polo-like kinase 1 activity. These data suggest that 7-O-methylwogonin, a polo-like kinase 1 inhibitor, may be a useful anticancer agent because of its polo-like kinase 1 selectivity and effectiveness.