Hesperetin inhibit EMT in TGF-ß treated podocyte by regulation of mTOR pathway.

Renal fibrosis is one of the characteristic features of chronic kidney disease (CKD). Fibrotic change not only impairs the filtration function of the kidney but is also recognized as a marker of end-stage renal disease (ESRD). The epithelial to mesenchymal transition (EMT) is known to play a role in embryonic development and organ formation, but it is getting much attention for its pathological role in the invasion and metastasis of carcinoma. Recently, it has also been reported that EMT plays a role in the formation of fibrosis during chronic inflammation. EMT contribute to the development of the fibrosis in CKD. Moreover, glomerular podocytes and tubular epithelial cells can also undergo mesenchymal transition in CKD. Hesperetin is a flavonoid present in citrus and is well known for its antioxidant and anti-inflammatory properties. In this study, we investigated the effects of hesperetin on the EMT-elicited podocytes. First, we generated an EMT model by treating transforming growth factor (TGF)-ß1, a potent inducer of EMT to the podocytes. TGF-ß1 decreased the expression of epithelial markers such as nephrin, zonula occludens-1 (ZO-1), while it increased the mesenchymal markers, including fibronectin (FN), vimentin, and α-smooth muscle actin (α-SMA) in the podocytes. Hesperetin suppressed EMT-like changes elicited by TGF-ß1. Interestingly, hesperetin did not interfere with the Smad signaling-the classical TGF-ß signaling-pathway, which was confirmed by the experiment with smad 2/3 -/- podocytes. Instead, hesperetin suppressed EMT-like changes by inhibiting the mTOR pathway-one of the alternative TGF-ß signaling pathways. In conclusion, hesperetin has a protective effect on the TGF-ß1 elicited EMT-like changes of podocytes through regulation of mTOR pathway. It could be a good candidate for the suppression of kidney fibrosis in various CKD.

Eclipta prostrata promotes the induction of anagen, sustains the anagen phase through regulation of FGF-7 and FGF-5.

CONTEXT: Eclipta prostrata L. (Asteraceae) (EP) has been widely used for the treatment of skin disease in Asian traditional medicine. OBJECTIVE: This study investigates the potency of EP in promoting hair growth in vivo and in vitro. MATERIALS AND METHODS: C57BL/6N mice were divided into four groups (n = 4) as follows: control (topical treatment of normal saline), topical 3% minoxidil to the dorsal skin of mice for 14 days, and low (1 mg/day) and high (10 mg/day) doses of EP orally administered once a day for 14 days. Dorsal hairs of C57BL/6N mice were depilated to synchronize anagen induction. Hair growth activity was evaluated by gross and microscopic observations. Sections of dorsal skin were stained with haematoxylin and eosin. We also treated the various concentrations of EP (5, 10 and 50 µg/mL) for 24 h on the human dermal papilla cells (HDPs) and examined the effects of EP on the expression of FGF-7 and mTOR signalling. RESULTS: EP enhanced the induction of anagen in the dorsal skin of mice, characterized by the appearance of inner root sheath along with hair shaft, the emergence of hair shaft through the epidermis. EP increased the expression of FGF-7, while decreased the level of FGF-5 in C57/BL6 mice. EP also increased the expression of FGF-7, activated the mTOR signalling in HDPs. DISCUSSION AND CONCLUSIONS: These results suggest that EP has a potency to enhance the growth of hair follicle, promoting hair growth through regulation of FGF-7 and FGF-5.

Hominis Placenta facilitates hair re-growth by upregulating cellular proliferation and expression of fibroblast growth factor-7.

BACKGROUND: Hominis Placenta (HP) known as a restorative medicine in Traditional Chinese Medicine (TCM), has been widely applied in the clinics of Korea and China as an anti-aging agent to enhance the regeneration of tissue. This study was conducted to investigate whether topical treatment of HP promotes hair regrowth in the animal model. METHODS: The dorsal hairs of 8-week-old C57BL/6 mice were depilated to synchronize hair follicles to the anagen phase. HP was applied topically once a day for 15 days. Hair growth was evaluated visually and microscopically. The incorporation of bromodeoxyuridine (BrdU) and expression of proliferating cell nuclear antigen (PCNA), fibroblast growth factor-7 (FGF-7) in dorsal skin tissue was examined by immunohistochemical analysis. Reverse transcription polymerase chain reaction (RT-PCR) was used to measure the mRNA expression of FGF-7. RESULTS: HP exhibited potent hair growth-promoting activity in C57BL/6 mice. Gross examination indicated that HP markedly increased hair regrowth as well as hair density and diameter. Histologic analysis showed that HP treatment enhanced the anagen induction of hair follicles. Immunohistochemical analysis revealed that BrdU incorporation and the expressions of PCNA were increased by treatment of HP. HP treatment significantly increased the expression of FGF-7, which plays pivotal roles to maintain anagen phase both protein and mRNA levels. CONCLUSIONS: Taken together, our results indicate that HP has a potent hair growth-promoting activity; therefore, it may be a good candidate for the treatment of alopecia.

Protective Effects of Alisma orientale Extract against Hepatic Steatosis via Inhibition of Endoplasmic Reticulum Stress.

Endoplasmic reticulum (ER) stress is associated with the pathogenesis of hepatic steatosis. Alisma orientale Juzepzuk is a traditional medicinal herb for diuretics, diabetes, hepatitis, and inflammation. In this study, we investigated the protective effects of methanol extract of the tuber of Alisma orientale (MEAO) against ER stress-induced hepatic steatosis in vitro and in vivo. MEAO inhibited the tunicamycin-induced increase in luciferase activity of ER stress-reporter constructs containing ER stress response element and ATF6 response element. MEAO significantly inhibited tunicamycin-induced ER stress marker expression including GRP78, CHOP, and XBP-1 in tunicamycin-treated Human hepatocellular carcinoma (HepG2) cells and the livers of tunicamycin-injected mice. It also inhibited tunicamycin-induced accumulation of cellular triglyceride. Similar observations were made under physiological ER stress conditions such as in palmitate (PA)-treated HepG2 cells and the livers of high-fat diet (HFD)-induced obese mice. MEAO repressed hepatic lipogenic gene expression in PA-treated HepG2 cells and the livers of HFD obese mice. Furthermore, MEAO repressed very low-density lipoprotein receptor (VLDLR) expression and improved ApoB secretion in the livers of tunicamycin-injected mice or HFD obese mice as well as in tunicamycin or PA-treated HepG2 cells. Alismol, a guaiane-type sesquiterpenes in Alisma orientale, inhibited GRP78 expression in tunicamycin-treated HepG2 cells. In conclusion, MEAO attenuates ER stress and prevents hepatic steatosis pathogenesis via inhibition of expression of the hepatic lipogenic genes and VLDLR, and enhancement of ApoB secretion.

Lipocalin-2 elicited by advanced glycation end-products promotes the migration of vascular smooth muscle cells.

Advanced glycation end-products (AGEs) play key roles in the development of diabetic vascular complications by activating the proliferation and migration of vascular smooth muscle cells. Here, we identified an increase of the migratory properties of human aortic smooth muscle cells (HASMC) through AGE-induced expression of lipocalin-2 (LCN2). Because the AGE-elicited expression of LCN2 was diminished by an antibody against the AGE receptor (RAGE), diphenylene iodonium (DPI), N-acetyl cysteine, LY294002, and SP600125, we suggest that AGEs enhance the expression of LCN2 via a RAGE-NADPH oxidase-reactive oxygen species pathway, leading to the phosphorylation of PI3K-Akt and JNK in HASMCs. In addition, a chromatin immunoprecipitation assay and promoter assay revealed that CCAAT/enhancer binding protein ß is crucial for AGE-induced expression of LCN2. However, any other AGE-related signaling pathway, including ERK1/2, p38, NF-κB, and AP-1, did not affect the AGE- induced expression of LCN2. Knockdown of LCN2 expression by shRNA showed that AGE-elicited LCN2 expression enhanced the invasive and migratory properties of HASMCs, but showed no effect on cell proliferation. Considering the importance of HASMC migration in the development of atherosclerosis, our study provides a novel insight into diabetic vascular complications.

A novel cantharidin analog N-benzylcantharidinamide reduces the expression of MMP-9 and invasive potentials of Hep3B via inhibiting cytosolic translocation of HuR.

Invasion and metastasis are major causes of malignant tumor-associated mortality. The present study aimed to investigate the molecular events underlying inhibitory effect of N-benzylcantharidinamide, a novel synthetic analog of cantharidin, on matrix metalloproteinase-9 (MMP-9)-mediated invasion in highly metastatic hepatocellular carcinoma Hep3B cells. In this investigation, among six analogs of cantharidin, only N-benzylcantharidinamide has the inhibitory action on MMP-9 expression at non-toxic dose. The MMP-9 expression and invasion of Hep3B cells were significantly suppressed by treatment of N-benzylcantharidinamide in a dose-dependent manner. On the other hand, the transcriptional activity of MMP-9 promoter and nuclear levels of NF-κB and AP-1 as the main transcriptional factors inducing MMP-9 expression were not affected by it although the level of MMP-9 mRNA was reduced by treatment of N-benzylcantharidinamide. Interestingly, the stability of MMP-9 mRNA was significantly reduced by N-benzylcantharidinamide-treatment. In addition, the cytosolic translocation of human antigen R (HuR), which results in the increase of MMP-9 mRNA stability through interaction of HuR with 3'-untranslated region of MMP-9 mRNA, was suppressed by treatment of N-benzylcantharidinamide, in a dose-dependent manner. Taken together, it was demonstrated, for the first time, that N-benzylcantharidinamide suppresses MMP-9 expression by reducing HuR-mediated MMP-9 mRNA stability for the inhibition of invasive potential in highly metastatic Hep3B cells.

Regulation of Hippo-YAP signaling axis by Isoalantolactone suppresses tumor progression in cholangiocarcinoma.

Cholangiocarcinoma (CCA) is a devastating malignancy characterized by aggressive tumor growth and limited treatment options. Dysregulation of the Hippo signaling pathway and its downstream effector, Yes-associated protein (YAP), has been implicated in CCA development and progression. In this study, we investigated the effects of Isoalantolactone (IALT) on CCA cells to elucidate its effect on YAP activity and its potential clinical significance. Our findings demonstrate that IALT exerts cytotoxic effects, induces apoptosis, and modulates YAP signaling in SNU478 cells. We further confirmed the involvement of the canonical Hippo pathway by generating LATS1/LATS2 knockout cells, highlighting the dependence of IALT-mediated apoptosis and YAP phosphorylation on the Hippo-LATS signaling axis. In addition, IALT suppressed cell growth and migration, partially dependent on YAP-TEAD activity. These results provide insights into the therapeutic potential of targeting YAP in CCA and provide a rationale for developing of YAP-targeted therapies for this challenging malignancy.

The LKB1-TSSK1B axis controls YAP phosphorylation to regulate the Hippo-YAP pathway.

The Hippo pathway's main effector, Yes-associated protein (YAP), plays a crucial role in tumorigenesis as a transcriptional coactivator. YAP's phosphorylation by core upstream components of the Hippo pathway, such as mammalian Ste20 kinase 1/2 (MST1/2), mitogen-activated protein kinase kinase kinase kinases (MAP4Ks), and their substrate, large tumor suppressor 1/2 (LATS1/2), influences YAP's subcellular localization, stability, and transcriptional activity. However, recent research suggests the existence of alternative pathways that phosphorylate YAP, independent of these core upstream Hippo pathway components, raising questions about additional means to inactivate YAP. In this study, we present evidence demonstrating that TSSK1B, a calcium/calmodulin-dependent protein kinase (CAMK) superfamily member, is a negative regulator of YAP, suppressing cellular proliferation and oncogenic transformation. Mechanistically, TSSK1B inhibits YAP through two distinct pathways. Firstly, the LKB1-TSSK1B axis directly phosphorylates YAP at Ser94, inhibiting the YAP-TEAD complex's formation and suppressing its target genes' expression. Secondly, the TSSK1B-LATS1/2 axis inhibits YAP via phosphorylation at Ser127. Our findings reveal the involvement of TSSK1B-mediated molecular mechanisms in the Hippo-YAP pathway, emphasizing the importance of multilevel regulation in critical cellular decision-making processes.

Marine algal fucoxanthin inhibits the metastatic potential of cancer cells.

Metastasis is major cause of malignant cancer-associated mortality. Fucoxanthin has effect on various pharmacological activities including anti-cancer activity. However, the inhibitory effect of fucoxanthin on cancer metastasis remains unclear. Here, we show that fucoxanthin isolated from brown alga Saccharina japonica has anti-metastatic activity. To check anti-metastatic properties of fucoxanthin, in vitro models including assays for invasion, migration, actin fiber organization and cancer cell-endothelial cell interaction were used. Fucoxanthin inhibited the expression and secretion of MMP-9 which plays a critical role in tumor invasion and migration, and also suppressed invasion of highly metastatic B16-F10 melanoma cells as evidenced by transwell invasion assay. In addition, fucoxanthin diminished the expressions of the cell surface glycoprotein CD44 and CXC chemokine receptor-4 (CXCR4) which play roles in migration, invasion and cancer-endothelial cell adhesion. Fucoxanthin markedly suppressed cell migration in wound healing assay and inhibited actin fiber formation. The adhesion of B16-F10 melanoma cells to the endothelial cells was significantly inhibited by fucoxanthin. Moreover, in experimental lung metastasis in vivo assay, fucoxanthin resulted in significant reduction of tumor nodules. Taken together, we demonstrate, for the first time, that fucoxanthin suppresses metastasis of highly metastatic B16-F10 melanoma cells in vitro and in vivo.

Caffeic acid phenethyl ester inhibits alpha-melanocyte stimulating hormone-induced melanin synthesis through suppressing transactivation activity of microphthalmia-associated transcription factor.

Caffeic acid phenethyl ester (1), a natural compound found in various plants and propolis, is a well-known anti-inflammatory, immunomodulatory, and cytotoxic agent. The present study aimed to investigate the molecular events underlying the antimelanogenic activity of 1 in alpha-melanocyte stimulating hormone (α-MSH)-stimulated B16-F10 melanoma cells. In this investigation, 1 effectively reduced α-MSH-stimulated melanin synthesis by suppressing expression of melanogenic enzymes such as tyrosinase, tyrosinase-related protein-1 (TRP-1), and tyrosinase-related protein-2 (TRP-2), although this compound did not directly inhibit tyrosinase enzyme activity. On the other hand, the expression and nuclear translocation of microphthalmia-associated transcription factor (MITF) as a key transcription factor for tyrosinase expression regulating melanogenesis were not affected by treatment with 1. The upstream signaling pathways including cAMP response element-binding protein (CREB), glycogen synthase kinase-3ß (GSK-3ß), and Akt for activation and expression of MITF were also not influenced by 1. Interestingly, 1 inhibited transcriptional activity of a tyrosinase promoter by suppressing the interaction of MITF protein with an M-box containing a CATGTG motif on the tyrosinase promoter. Given the important role of MITF in melanogenesis, suppression of 1 on the function of MITF to transactivate tyrosinase promoter may present a novel therapeutic approach to treat hyperpigmentation disorders.

Atraumatic Spontaneous Splenic Rupture With Unknown Etiology.

Splenic rupture of all causes is a potentially life-threatening event for patients. The infrequency of atraumatic splenic rupture (ASR) poses a significant diagnostic challenge due to atypical findings. ASR is commonly due to a spleen with an underlying disease process such as malignancy, infection, coagulopathies, or neoplasms. However, ASR without an identifiable cause is rare and poses further complexity. In this case, a 57-year-old woman with a history of hypertension presented to the emergency department complaining of chest pain and was found to have a splenic hematoma. She underwent splenic artery embolization due to her continued hemodynamic instability. The patient was ultimately treated with a splenectomy, as embolization was unsuccessful. Gross pathology revealed no underlying disease processes, nodules, or masses. Splenic hemorrhage due to atraumatic rupture of the spleen is rare and without known pathology. The case illustrates the need for providers to have high clinical suspicion of such a diagnosis to stabilize and surgically manage these patients. Few instances of ASR without an identifiable cause are found in medical literature, and further knowledge of the subject is needed.

Estrogen induced ß-1,4-galactosyltransferase 1 expression regulates proliferation of human breast cancer MCF-7 cells.

Beta 1,4-galactosyltransferase 1 (B4GALT1) synthesizes galactose ß-1,4-N-acetylglucosamine (Galß1-4GlcNAc) groups on N-linked sugar chains of glycoproteins, which play important roles in many biological events, including the proliferation and migration of cancer cells. A previous microarray study reported that this gene is expressed by estrogen treatment in breast cancer. In this study, we examined the regulatory mechanisms and biological functions of estrogen-induced B4GALT1 expression. Our data showed that estrogen-induced expression of B4GALT1 is localized in intracellular compartments and in the plasma membrane. In addition, B4GALT1 has an enzyme activity involved in the production of the Galß1-4GlcNAc structure. The result from a promoter assay and chromatin immunoprecipitation revealed that 3 different estrogen response elements (EREs) in the B4GALT1 promoter are critical for responsiveness to estrogen. In addition, the estrogen antagonists ICI 182,780 and ER-α-ERE binding blocker TPBM inhibit the expression of estrogen-induced B4GALT1. However, the inhibition of signal molecules relating to the extra-nuclear pathway, including the G-protein coupled receptors, Ras, and mitogen-activated protein kinases, had no inhibitory effects on B4GALT1 expression. The knock-down of the B4GALT1 gene and the inhibition of membrane B4GALT1 function resulted in the significant inhibition of estrogen-induced proliferation of MCF-7 cells. Considering these results, we propose that estrogen regulates the expression of B4GALT1 through the direct binding of ER-α to ERE and that the expressed B4GALT1 plays a crucial role in the proliferation of MCF-7 cells through its activity as a membrane receptor.

Time-dependent control of hole-opening degree of porous ZnO hollow microspheres.

Well-designed, monodispersed porous ZnO hollow microspheres with controlled hole-opening were successfully synthesized by a facile two-step solution route at low temperature. The hollow microspheres having average diameter of 3-4 µm showed time-dependent hole-opening, i.e. 4-100% for 15-75 min. The hole-opening percentage increases linearly with time until complete opening. The ZnO hollow microspheres also exhibited a high surface area (34 m(2) g(-1)), a large pore volume (0.19 cm(3) g(-1)) and an average pore diameter of 3.8 nm. A plausible growth mechanism for the formation of ZnO hollow microspheres was also proposed.

Protective Effect of the Fruit Hull of Gleditsia sinensis on LPS-Induced Acute Lung Injury Is Associated with Nrf2 Activation.

The fruit hull of Gleditsia sinensis (FGS) has been prescribed as a traditional eastern Asian medicinal remedy for the treatment of various respiratory diseases, but the efficacy and underlying mechanisms remain poorly characterized. Here, we explored a potential usage of FGS for the treatment of acute lung injury (ALI), a highly fatal inflammatory lung disease that urgently needs effective therapeutics, and investigated a mechanism for the anti-inflammatory activity of FGS. Pretreatment of C57BL/6 mice with FGS significantly attenuated LPS-induced neutrophilic lung inflammation compared to sham-treated, inflamed mice. Reporter assays, semiquantitative RT-PCR, and Western blot analyses show that while not affecting NF-κB, FGS activated Nrf2 and expressed Nrf2-regulated genes including GCLC, NQO-1, and HO-1 in RAW 264.7 cells. Furthermore, pretreatment of mice with FGS enhanced the expression of GCLC and HO-1 but suppressed that of proinflammatory cytokines in including TNF-α and IL-1ß in the inflamed lungs. These results suggest that FGS effectively suppresses neutrophilic lung inflammation, which can be associated with, at least in part, FGS-activating anti-inflammatory factor Nrf2. Our results suggest that FGS can be developed as a therapeutic option for the treatment of ALI.

Suppression of lipopolysaccharide-induced expression of inflammatory indicators in RAW 264.7 macrophage cells by extract prepared from Ginkgo biloba cambial meristematic cells.

CONTEXT: Ginkgo biloba L. (Ginkgoaceae) leaves have been used as an herbal medicine that has a complex range of biological activities. However, when we consider that biological activity of plant extracts is highly variable according to the source, location, and harvest season, technology to obtain the natural products with homogeneity is extremely important. OBJECTIVE: We established the technology to obtain the cambial meristematic cells (CMCs) of Ginkgo biloba, which were expanded in vitro with homogeneity through a suspension culture and then determined the anti-inflammatory activity of fractionated samples prepared from the ethanol extract of CMCs. MATERIALS AND METHODS: We determined the anti-inflammatory activity of samples using lipopolysaccharide (LPS)-stimulated RAW 264.7 macrophage cells. Especially, influence of sample treatment on the expression of various indicators, such as nitric oxide (NO), inducible nitric oxide synthase (iNOS), cyclooxygenase (COX)-2, mitogen-activated protein (MAP) kinases, transcription factor, and cytokines, involved in inflammatory activity was assessed. RESULTS: A fractionated sample demonstrated 53.4% inhibition of LPS-induced NO production from the cells. Additionally, when fractionated samples were treated, iNOS and COX-2 expressions were almost completely suppressed. Fractionated samples also inhibited the phosphorylation of LPS-induced extracellular signal-regulated (ERK) and p38 MAP kinases more than 60%. IκB phosphorylation and subsequent nuclear factor (NF)-κB activation were also suppressed by fractionated samples. The expression of pro-inflammatory cytokines, IL-6 and tumor necrosis factor (TNF)-α, was significantly inhibited by the sample treatment. DISCUSSION AND CONCLUSION: Fractionated samples from the ethanol extract of Ginkgo biloba CMCs could potentially be the source of a powerful anti-inflammatory substance.

Phosphorylation analysis of the Hippo-YAP pathway using Phos-tag.

Phosphorylation is an essential regulatory mechanism in cells that modifies diverse substrates, such as proteins, carbohydrates, lipids, and nucleotides. Protein phosphorylation regulates function, subcellular localization, and protein-protein interactions. Protein kinases and phosphatases catalyze this reversible mechanism, subsequently influencing signal transduction. The dysregulation of protein phosphorylation leads to many diseases, such as cancer, neurodegenerative diseases, and metabolic diseases. Therefore, analyzing the phosphorylation status and identifying protein phosphorylation sites are critical for elucidating the biological functions of specific phosphorylation events. Unraveling the critical phosphorylation events associated with diseases and specific signaling pathways is promising for drug discovery. To date, highly accurate and sensitive approaches have been developed to detect the phosphorylation status of proteins. In this review, we discuss the application of Phos-tag to elucidate the biological functions of Hippo pathway components, with emphasis on the identification and quantitation of protein phosphorylation under physiological and pathological conditions. SIGNIFICANCE: We here provide a comprehensive overview of Phos-tag technique-based strategies to identify phosphorylated proteins at the cellular level in the Hippo-YAP pathway that comprises a major driving force for cellular homeostasis. We clarify the links of applying Phos-tag in elucidating the biological functions of the Hippo pathway components with particular attention to the identification and quantitation of protein phosphorylation under physiological and pathological conditions. We believe that our paper will make a significant contribution to the literature because these detailed phosphorylation modifications and functional diversity of the Hippo pathway components in physiological and pathological processes are only beginning to come to the fore, highlighting the potential for discovering new therapeutic targets. Moreover, this line of research can provide further insight into the inextricable link between phos-tag applications as a molecular tool and cellular signaling modality, offering new directions for an integrated research program toward understanding cellular regulation at the molecular level. Given the broad research and practical applications, we believe that this paper will be of interest to the readership of your journal.

MHY4571, a novel diarylcyclohexanone derivative, exerts anti-cancer activity by regulating the PKA-cAMP-response element-binding protein pathway in squamous cell lung cancer.

BACKGROUND: The protein kinase A (PKA)/cAMP response element-binding protein (CREB) has been suggested to be related to the inhibition of the proliferation of non-small cell lung cancer (NSCLC) cells. This study aimed to investigate the efficacy of a novel diarylcyclohexanone derivative, MHY4571, in regulating the PKA-CREB pathway and to study its anti-tumor role in squamous NSCLC. METHODS: We designed MHY4571 as a novel PKA inhibitor with acceptable in silico ADME properties and tested it in vitro in lung cancer cell lines and in vivo in xenograft and orthotopic mouse models of squamous cell lung carcinoma. RESULTS: MHY4571 inhibited PKA activity (> 70% inhibition) and suppressed the expression of p-PKA and p-CREB dose-dependently. MHY4571 treatment reduced lung cancer cell viability and promoted caspase 3-dependent apoptotic cell death. Orally administered MHY4571 significantly suppressed lung tumor growth in xenograft and orthotopic mouse models. PKA catalytic subunit alpha-silencing by siRNA (siPKA) strongly attenuated CREB phosphorylation; siCREB did not alter PKA protein levels or its phosphorylation, suggesting that PKA is an upstream regulator of CREB activity. MHY4571 acted synergistically with cisplatin (on co-treatment) to induce apoptotic cell death in lung cancer cells. CONCLUSIONS: Our results imply that MHY4571 may be a potential drug candidate for squamous cell lung cancer treatment.

The ethanol extract of Garcinia subelliptica Merr. induces autophagy.

BACKGROUND: Garcinia subelliptica Merr. is a multipurpose coastal tree, the potential medicinal effects of which have been studied, including cancer suppression. Here, we present evidence that the ethanol extract of G. subelliptica Merr. (eGSM) induces autophagy in human lung adenocarcinoma cells. METHODS: Two different human lung adenocarcinoma cell lines, A549 and SNU2292, were treated with varying amounts of eGSM. Cytotoxicity elicited by eGSM was assessed by MTT assay and PARP degradation. Autophagy in A549 and SNU2292 was determined by western blotting for AMPK, mTOR, ULK1, and LC3. Genetic deletion of AMPKα in HEK293 cells was carried out by CRISPR. RESULTS: eGSM elicited cytotoxicity, but not apoptosis, in A549 and SNU2292 cells. eGSM increased LC3-II production in both A549 and, more extensively, SNU2292, suggesting that eGSM induces autophagy. In A549, eGSM activated AMPK, an essential autophagy activator, but not suppressed mTOR, an autophagy blocker, suggesting that eGSM induces autophagy by primarily activating the AMPK pathway in A549. By contrast, eGSM suppressed mTOR activity without activating AMPK in SNU2292, suggesting that eGSM induces autophagy by mainly suppressing mTOR in SNU2292. In HEK293 cells lacking AMPKα expression, eGSM increased LC3-II production, confirming that the autophagy induced by eGSM can occur without the AMPK pathway. CONCLUSION: Our findings suggest that eGSM induces autophagy by activating AMPK or suppressing mTOR pathways, depending on cell types.

Extracts of Perilla frutescens var. Acuta (Odash.) Kudo Leaves Have Antitumor Effects on Breast Cancer Cells by Suppressing YAP Activity.

Yes-associated protein (YAP)/WW domain-containing transcription factor (TAZ) is critical for cell proliferation, survival, and self-renewal. It has been shown to play a crucial oncogenic role in many different types of tumors. In this study, we investigated the antitumor effect of the extracts of Perilla frutescens var. acuta (Odash.) Kudo leaves (PLE) on Hippo-YAP/TAZ signaling. PLE induced the phosphorylation of YAP/TAZ, thereby inhibiting their activity. In addition, the treatment suppresses YAP/TAZ transcriptional activity via the dissociation of the YAP/TAZ-TEAD complex. To elucidate the molecular mechanism of PLE in the regulation of YAP activity, we treated WT and cell lines with gene knockout (KO) for Hippo pathway components with PLE. The inhibitory effects of PLE on YAP-TEAD target genes were significantly attenuated in LATS1/2 KO cells. Moreover, we found the antitumor effect of PLE on MDA-MB-231 and BT549, both of which are triple-negative breast cancer (TNBC) cell lines. PLE reduced the viability of TNBC cells in a dose-dependent manner and induced cell apoptosis. Further, PLE inhibited the migration ability in MDA-MB-231 cells. This ability was weakened in YAP and TEAD-activated clones suggesting that the inhibition of migration by PLE is mainly achieved by regulating YAP activity. Taken together, the results of this study indicate that PLE suppressed cell growth and increased the apoptosis of breast cancer (BC) cells via inactivation of YAP activity in a LATS1/2-dependent manner.

Regulation of TEAD Transcription Factors in Cancer Biology.

Transcriptional enhanced associate domain (TEAD) transcription factors play important roles during development, cell proliferation, regeneration, and tissue homeostasis. TEAD integrates with and coordinates various signal transduction pathways including Hippo, Wnt, transforming growth factor beta (TGFß), and epidermal growth factor receptor (EGFR) pathways. TEAD deregulation affects well-established cancer genes such as KRAS, BRAF, LKB1, NF2, and MYC, and its transcriptional output plays an important role in tumor progression, metastasis, cancer metabolism, immunity, and drug resistance. To date, TEADs have been recognized to be key transcription factors of the Hippo pathway. Therefore, most studies are focused on the Hippo kinases and YAP/TAZ, whereas the Hippo-dependent and Hippo-independent regulators and regulations governing TEAD only emerged recently. Deregulation of the TEAD transcriptional output plays important roles in tumor progression and serves as a prognostic biomarker due to high correlation with clinicopathological parameters in human malignancies. In addition, discovering the molecular mechanisms of TEAD, such as post-translational modifications and nucleocytoplasmic shuttling, represents an important means of modulating TEAD transcriptional activity. Collectively, this review highlights the role of TEAD in multistep-tumorigenesis by interacting with upstream oncogenic signaling pathways and controlling downstream target genes, which provides unprecedented insight and rationale into developing TEAD-targeted anticancer therapeutics.