So-Hyeon Park’s research while affiliated with Yonsei University and other places

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Publications (16)


Identification of a novel positive allosteric modulator of PAR1. (A) Dot plot shows the effect of 1813 FDA-approved drugs on enhancing PAR1 activity. (B) Representative traces of intracellular calcium responses to PAR1-AP (30 μM) in the presence or absence of gestodene (10 μM) in HT29 cells. PAR1 was inhibited with vorapaxar (1 μM). (C) Chemical structures of gestodene, levonorgestrel and desogestrel. (D–F) Representative traces of intracellular calcium responses to PAR1-AP (20 μM) in HT29 cells pretreated for 10 min with the indicated concentrations of gestodene, levonorgestrel, and desogestrel. Arrows indicate when PAR1-AP was applied.
Positive allosteric modulation of PAR1 by gestodene. (A) Representative traces of intracellular calcium responses to PAR1-AP (30 μM) in HT29 cells. Cells were treated with the indicated concentrations of gestodene for 10 min prior to PAR1-AP treatment. (B) Summary of dose-response (mean ± S.E., n = 6). (C) Representative traces of intracellular calcium responses to 3 unit/mL thrombin (THB) in HT29 cells. Cells were treated with the indicated concentrations of gestodene for 10 min prior to thrombin treatment. (D) Summary of dose-response (mean ± S.E., n = 3).
Effect of gestodene on the potency of PAR1-AP and the activity of PAR2 and PAR4. (A, B) Representative traces of intracellular calcium responses to PAR1-AP in the presence or absence of gestodene (10 μM) in HT29 cells. Arrows indicate when PAR1-AP was applied. (C) Summary of the dose-response (mean ± S.E., n = 3). **p < 0.01, ***p < 0.001. (D, E) Representative traces of intracellular calcium responses to PAR2-AP and PAR4-AP in HT29 cells. Cells were treated with the indicated concentrations of gestodene for 10 min prior to PAR2-AP (3 μM) or PAR4-AP (30 μM) treatment. PAR2 and PAR4 were inhibited by 30 μM of punicalagin (PCG) and 10 μM of BMS-986120 (BMS), respectively. The dashed line represents full activation of PAR2 and PAR4 by PAR2-AP (10 μM) and PAR4-AP (50 μM), respectively. (F) Representative traces of intracellular calcium responses to thrombin in the presence (solid line) or absence (dashed line) of vorapaxar in HT29 cells. Cells were treated with vorapaxar (1 μM) and gestodene (10 μM) for 10 min prior to THB (10 units/mL) stimulation. PAR1 and PAR4 were inhibited by vorapaxar (1 μM) and BMS (1 μM), respectively. (G) Representative traces of intracellular calcium responses to ADP in MEG-01 cells. Cells were treated with indicated concentrations of gestodene for 10 min prior to ADP stimulation. The dashed line represents intracellular calcium responses induced by 10 μM of ADP. The intracellular calcium elevation induced by 1 μM of ADP was completely inhibited by 100 μM of ticagrelor.
Computational docking simulations of compounds binding to PAR1. (A) Predicted binding models of vorapaxar (blue) and gestodene (green) in complex with PAR1. (B) Close-up view of vorapaxar (blue) interacting with the orthosteric binding site of PAR1, highlighting key residue interactions. (C) Close-up view of gestodene (green) binding to the allosteric site of PAR1, highlighting key residue interactions.
Effect of gestodene on PAR1-AP-induced internalization of PAR1. (A) EGFP-tagged PAR1-expressing HT29 cells were pretreated with gestodene (30 μM) in the presence or absence of vorapaxar (1 μM) for 10 min, prior to stimulation with PAR1-AP (30 μM). Changes in cellular localization of EGFP-tagged PAR1 were observed at the indicated time points. White arrows indicate representative internalized EGFP-PAR1 puncta. Images were captured automatically by Lionheart FX microscope. Scale bar = 10 μm. (B) Summary of the number of puncta per cell. The number of EGFP-PAR1 puncta were quantified using Gen5 software (mean ± S.E., from left to right, n = 73, n = 90, n = 89, n = 71, and n = 79 cells were analyzed). *p < 0.05, ***p < 0.001.

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Gestodene, a novel positive allosteric modulator of PAR1, enhances PAR1-mediated human platelet aggregation
  • Article
  • Full-text available

July 2024

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31 Reads

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1 Citation

So-Hyeon Park

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Il Kwon

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Wan Namkung

Background: Protease-activated receptor 1 (PAR1) is expressed in human platelets and can be activated by low concentrations of thrombin. Vorapaxar, a selective antagonist of PAR1, inhibits thrombin-induced calcium mobilization in human platelet, which is associated with an increased risk of bleeding. Conversely, the administration of a positive allosteric modulator (PAM) of PAR1 may pose a substantial risk of thrombosis due to inducing excessive platelet activation. In this study, we discovered a novel PAM of PAR1 and investigated the effect of enhanced PAR1 activation by PAM of PAR1 on platelet activation. Methods: To find PAMs of PAR1, a cell-based screen was performed in HT29 cells, and finally, gestodene, an oral contraceptive drug (OC), was identified as a novel PAM of PAR1. The mechanism of action of gestodene and its effects on platelet activation were investigated in human megakaryocytic leukemia cell line MEG-01 cells and human platelet. Results: Gestodene enhanced both thrombin- and PAR1-activating peptide (AP)-induced intracellular calcium levels in a dose-dependent manner without altering PAR2 and PAR4 activity. Gestodene significantly increased PAR1-AP-induced internalization of PAR1 and phosphorylation of ERK1/2, and the enhancing effects were significantly blocked by vorapaxar. Furthermore, gestodene potently increased PAR1-AP induced morphological changes in MEG-01 cells. Remarkably, in human blood, gestodene exerted a robust augmentation of PAR1-AP-induced platelet aggregation, and vorapaxar effectively attenuated the gestodene-induced enhancement of platelet aggregation mediated by PAR1. Conclusion: Gestodene is a selective PAM of PAR1 and suggest one possible mechanism for the increased risk of venous thromboembolism associated with OCs containing gestodene.

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Figure 1. Identification of a novel ANO1 inhibitor, hemin. (A) Chemical structure of hemin. (B) The inhibitory effect of hemin was determined via YFP fluorescence quenching assay in PC-3 cells. PC-3 cells expressing mutant YFP (F46L/H148Q/I152L) were treated with the indicated concentrations of hemin for 10 min, and then ANO1 was activated by ATP (100 µM). (C) Dose-response curve; nH = 2.4 (mean ± S.D.; n = 6). (D) The inhibitory effect of hemin on ANO1 channel activity was determined in FRT cells expressing human ANO1. The indicated concentrations of hemin were administered for 10 min prior to ATP (100 µM) treatment. (E) Dose-response curve on ANO1 inhibitory activity; nH = 3.3 (mean ± S.D.; n = 5). (F) Apical membrane currents were measured in FRT cells expressing ANO1. The cells were pre-treated with the indicated concentrations of hemin for 10 min, and the ANO1 chloride currents were activated by ATP (100 µM).
Figure 4. Expression levels of ANO1 protein and effect of hemin on cell viability in PC-3, LNCaP, A549, and HepG2 cells. (A) ANO1 protein expression levels in PC-3, PC-3 ANO1 knockout (KO), LNCaP, A549, and HepG2 cells. (B) Cell viability was assessed in PC-3 and PC-3 ANO1 KO cells. Cells were incubated with hemin at the indicated concentrations for 72 h, and medium was replaced every 24 h (mean ± S.D.; n = 5). * p < 0.05, *** p < 0.001; # p < 0.05 vs. ANO1 KO group control. (C) Cell viability was assessed in PC-3 ANO1 KO cells subjected to either mock transfection or ANO1 transfection. Cells were incubated with 10 µM of hemin for 72 h, and medium was replaced every 24 h (mean ± S.D.; n = 6). ** p < 0.01; # p < 0.05 vs. mock group control. (D-F) LNCaP, A549, and HepG2 cells were treated with hemin at the indicated concentrations for 72 h, and medium was changed every 24 h (mean ± S.D.; n = 5). Cisplatin was treated at 50 µM. *** p < 0.001 vs. control.
Figure 5. Effect of hemin on cell migration in PC-3 and PC-3 ANO1 KO cells. (A) Wound healing assay was performed in PC-3 cells. Cells were treated with the indicated concentrations of hemin, and wound closure was measured every 2 h after wound formation (mean ± S.E., n = 3). (B) Representative images were acquired at 0 and 36 h following administration of hemin at the indicated concentrations. (C) Wound healing assay was performed in PC-3 ANO1 KO cells. Cells were treated with the indicated concentrations of hemin, and wound closure was measured every 2 h after wound formation (mean ± S.E., n = 3). (D) Representative images were acquired at 0, 36 h following administration of hemin at the indicated concentrations. The scale bars represent 300 µm.
Figure 6. Effect of hemin on PARP cleavage and caspase-3 activity in PC-3 cells. (A) PC-3 cells were treated with 1, 3, 10 µM of hemin for 24 h, and then the expression levels of PARP, cleaved-PARP, and β-actin were analyzed via Western blot analysis. (B) Cleaved PARP intensity was normalized to β-actin (mean ± S.E., n = 3). ** p < 0.01 vs. control. (C) Images were taken 24 h after application of 10 µM of hemin in PC-3 cells, and then cells were incubated with 1 µM of caspase-3 substrate (green) and 1 µM of Hoechst 33342 (blue) for 30 min prior to image acquisition. Scale bars represent 200 µm. (D) PC-3 cells were treated with the indicated concentrations of hemin in the presence or absence of 20 µM of Ac-DEVD-CHO for 24 h and then treated with 1 µM of caspase-3 substrate for 30 min to estimate caspase-3 activity. *** p < 0.001.
Figure 7. Effect of hemin on cell cycle in PC-3 cells. (A,B) Cell cycle phases were observed via propidium iodide (PI) staining followed by flow cytometric analysis after cells were treated with 3 µM of hemin for 24 h and 48 h (mean ± S.D.; n = 3). * p < 0.05, ** p < 0.01 vs. control.
Anticancer Effect of Hemin through ANO1 Inhibition in Human Prostate Cancer Cells

May 2024

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20 Reads

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2 Citations

International Journal of Molecular Sciences

Anoctamin1 (ANO1), a calcium-activated chloride channel, is overexpressed in a variety of cancer cells, including prostate cancer, and is involved in cancer cell proliferation, migration, and invasion. Inhibition of ANO1 in these cancer cells exhibits anticancer effects. In this study, we conducted a screening to identify novel ANO1 inhibitors with anticancer effects using PC-3 human prostate carcinoma cells. Screening of 2978 approved and investigational drugs revealed that hemin is a novel ANO1 inhibitor with an IC50 value of 0.45 μM. Notably, hemin had no significant effect on intracellular calcium signaling and cystic fibrosis transmembrane conductance regulator (CFTR), a cyclic AMP (cAMP)-regulated chloride channel, and it showed a weak inhibitory effect on ANO2 at 3 μM, a concentration that completely inhibits ANO1. Interestingly, hemin also significantly decreased ANO1 protein levels and strongly inhibited the cell proliferation and migration of PC-3 cells in an ANO1-dependent manner. Furthermore, it strongly induced caspase-3 activation, PARP degradation, and apoptosis in PC-3 cells. These findings suggest that hemin possesses anticancer properties via ANO1 inhibition and could be considered for development as a novel treatment for prostate cancer.


Anticancer Evaluation of Novel Benzofuran–Indole Hybrids as Epidermal Growth Factor Receptor Inhibitors against Non-Small-Cell Lung Cancer Cells

February 2024

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147 Reads

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1 Citation

Pharmaceuticals

The epidermal growth factor receptor (EGFR), also known as ErbB1 and HER1, belongs to the receptor tyrosine kinase family. EGFR serves as the primary driver in non-small-cell lung cancer (NSCLC) and is a promising therapeutic target for NSCLC. In this study, we synthesized a novel chemical library based on a benzofuran–indole hybrid scaffold and identified 8aa as a potent and selective EGFR inhibitor. Interestingly, 8aa not only showed selective anticancer effects against NSCLC cell lines, PC9, and A549, but it also showed significant inhibitory effects against the double mutant L858R/T790M EGFR, which frequently occurs in NSCLC. In addition, in PC9 and A549 cells, 8aa potently blocked the EGFR signaling pathway, cell viability, and cell migration. These findings suggest that 8aa, a benzofuran–indole hybrid derivative, is a novel EGFR inhibitor that may be a potential candidate for the treatment of NSCLC patients with EGFR mutations.


Gestodene, a Positive Allosteric Modulator of PAR1, Enhances PAR1-Mediated Morphological Changes in MEG-01 Cells

May 2023

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34 Reads

Physiology

Protease-activated receptor 1 (PAR1) is a high-affinity thrombin receptor expressed in human platelets and can be activated by low levels of thrombin. Selective inhibition of PAR1 by vorapaxar significantly inhibits thrombin-induced calcium mobilization in human platelets and increases the risk of bleeding, suggesting that positive allosteric modulator (PAM) of PAR1 may increase the risk of thrombosis. In the present study, we performed a cell-based screening to identify novel PAMs of PAR1 and found gestodene, which acts as PAM of PAR1. Gestodene enhanced both thrombin- and PAR1-AP-induced increases in intracellular calcium levels through PAR1 in a dose-dependent manner, but it did not alter the activity of PAR2 and PAR4. Gestodene increased PAR1-AP-induced internalization of PAR1 receptors and phosphorylation of ERK1/2, and the enhancing effects were significantly blocked by vorapaxar. Furthermore, gestodene potently increased PAR1-AP-induced morphological changes in the human megakaryoblastic leukemia cell line MEG-01 cells, a cellular model of PAR1-mediated morphological changes in platelets. Our results reveal that gestodene is a selective PAM of PAR1 and provide a molecular basis for the risk of venous thromboembolism induced by oral contraceptives including gestodene. This research was supported by a Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education (NRF-2018R1A6A1A03023718). This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.


Figure 1. Inhibition of ANO1 activity by cis-and trans-resveratrol. (A-D) Apical membrane currents were measured in human ANO1-expressing FRT cells. Indicated concentrations of cis-and transresveratrol were treated for 10 min before the application of 100 μM ATP. (B,D) Summary of ANO1 current inhibition (mean ± S.D., n = 4-5). (E,F) The inhibitory effect of cis-and trans-resveratrol on ANO1 activity was measured with a YFP-quenching assay in FRT cells expressing ANO1 and a halide sensor YFP. Indicated concentrations of cis-and trans-resveratrol were added 10 min before the application of iodide solution containing 100 μM ATP. (G) Summary of dose-response (mean ± S.D., n = 4).
Figure 2. Cis-and trans-resveratrol inhibit ANO1 chloride currents. (A) Representative traces of whole-cell recordings of ANO1 in HEK293T cells expressing ANO1. Indicated concentrations of cisresveratrol and 10 μM Ani9 were treated after the activation of ANO1. (B) Whole-cell currents were recorded at a holding potential of −60 mV and pulsed to voltages between ± 100 mV in steps of 20 mV. (C) Summary of current densities measured at +100 mV (mean ± S.D., n = 3-7). (D) Traces of whole-cell recordings of ANO1. Trans-resveratrol and Ani9 were treated as indicated after the activation of ANO1. (E) Whole-cell currents were recorded at a holding potential of -60 mV and pulsed to voltages between ± 100 mV in steps of 20 mV. (F) Summary of current densities measured at +100 mV (mean ± S.D., n = 3-7). ** p < 0.01 vs control.
Figure 3. Effect of cis-and trans-resveratrol on intracellular calcium levels in PC-3 cells. (A,B) Intracellular calcium levels were measured using Fluo-4 NW. Cells were treated with the indicated concentrations of cis-and trans-resveratrol and stimulated with 10 µM ionomycin. (C,D) Cells were pretreated with the indicated concentrations of cis-and trans-resveratrol for 10 min, followed by stimulation with 10 µM ionomycin.
Figure 5. Effect of cis-and trans-resveratrol on cell viability and migration in PC-3 cells. (A,B) Effect of cis-and trans-resveratrol on cell viability. Indicated concentrations of cis-and trans-resveratrol were treated for 72 h and cell viability was estimated with MTS assay (mean ± S.D., n = 5). (C,D) Effect of cis-and trans-resveratrol on cell migration. Cells were treated with indicated concentrations of cisand trans-resveratrol and the wound closure was measured every 2 h for 24 h after wound generation (mean ± S.D., n = 4-5). Scale bars represent 300 µm. * p < 0.05, ** p < 0.01, *** p < 0.001 vs. control.
Inhibition of ANO1 by Cis-and Trans-Resveratrol and Their Anticancer Activity in Human Prostate Cancer PC-3 Cells

January 2023

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119 Reads

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16 Citations

International Journal of Molecular Sciences

Anoctamin1 (ANO1), a calcium-activated chloride channel, is involved in the proliferation, migration, and invasion of various cancer cells including head and neck squamous cell carcinoma, lung cancer, and prostate cancer. Inhibition of ANO1 activity or downregulation of ANO1 expression in these cancer cells is known to exhibit anticancer effects. Resveratrol, a natural polyphenol abundant in wines, grapes, berries, soybeans, and peanuts, shows a wide variety of biological effects including anti-inflammatory, antioxidant, and anticancer activities. In this study, we investigated the effects of two stereoisomers of resveratrol on ANO1 activity and found that cis- and trans-resveratrol inhibited ANO1 activity with different potencies. Cis- and trans-resveratrol inhibited ANO1 channel activity with IC50 values of 10.6 and 102 μM, respectively, and had no significant effect on intracellular calcium signaling at 10 and 100 μM, respectively. In addition, cis-resveratrol downregulated mRNA and protein expression levels of ANO1 more potently than trans-resveratrol in PC-3 prostate cancer cells. Cis- and trans-resveratrol significantly reduced cell proliferation and cell migration in an ANO1-dependent manner, and both resveratrol isomers strongly increased caspase-3 activity, PARP cleavage, and apoptotic sub-G1 phase ratio in PC-3 cells. These results revealed that cis-resveratrol is a potent inhibitor of ANO1 and exhibits ANO1-dependent anticancer activity against human metastatic prostate cancer PC-3 cells.


Synthesis and Biological Evaluation of a Fused Structure of Indolizine and Pyrrolo[1,2-c]pyrimidine: Identification of Its Potent Anticancer Activity against Liver Cancer Cells

November 2022

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111 Reads

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6 Citations

Pharmaceuticals

A highly efficient approach to a new indolizine scaffold fused with pyrrolo[1,2-c]pyrimidine was achieved via one-pot three-component coupling followed by an oxidative cyclization reaction. The simple two-step sequence allowed rapid access to various tetracyclic compounds from commercially available starting materials with the formation of five new bonds. Here, we observed the effects of these compounds on cell viability in HepG2, H1299, HT29, AGS, and A549 cancer cell lines. Interestingly, this fused scaffold had more potent anticancer activity in hepatocellular carcinoma HepG2 and Huh7 cells than other cancer cells. In particular, 5r strongly decreased cell viability in HepG2 and Huh7 cells with an IC50 value of 0.22 ± 0.08 and 0.10 ± 0.11 µM, respectively, but had a very weak inhibitory effect on the cell viability of other cancer cell lines. In addition, 5r significantly inhibited cell migration and induced apoptosis in HepG2 and Huh7 cells via the activation of caspase-3 and cleavage of PARP in a dose-dependent manner. Notably, the co-treatment of 5r with gemcitabine resulted in the significant additional inhibition of cell viability in HepG2 and Huh7 cells. Our results suggest that 5r could be used to develop new chemotype anticancer agents against liver cancers.


Amelioration of SARS-CoV-2 infection by ANO6 phospholipid scramblase inhibition

July 2022

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160 Reads

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21 Citations

Cell Reports

As an enveloped virus, SARS-CoV-2 delivers its viral genome into host cells via fusion of the viral and cell membranes. Here, we show that ANO6/TMEM16F-mediated cell surface exposure of phosphatidylserine is critical for SARS-CoV-2 entry, and that ANO6-selective inhibitors are effective against SARS-CoV-2 infections. Application of the SARS-CoV-2 Spike pseudotyped virus (SARS2-PsV) evokes a cytosolic Ca²⁺ elevation and ANO6-dependent phosphatidylserine externalization in ACE2/TMPRSS2-positive mammalian cells. A high-throughput screening of drug-like chemical libraries identifies three different structural classes of chemicals showing ANO6 inhibitory effects. Among them, A6-001 displays the highest potency and ANO6-selectivity and it inhibits the single-round infection of SARS2-PsV in ACE2/TMPRSS2-positive HEK 293T cells. More importantly, A6-001 strongly inhibits authentic SARS-CoV-2-induced phosphatidylserine scrambling and SARS-CoV-2 viral replications in Vero, Calu-3, and primarily cultured human nasal epithelial cells. These results provide mechanistic insights into the viral entry process and offer a potential target for pharmacological intervention to protect against COVID-19.


Novel positive allosteric modulator of protease‐activated receptor 1 promotes skin wound healing in hairless mice

May 2021

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70 Reads

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8 Citations

Background and Purpose Protease‐activated receptor 1 (PAR1) is a GPCR expressed in several skin cell types, including keratinocyte and dermal fibroblast. PAR1 activation plays a crucial role in the process of skin wound healing such as thrombosis, inflammation, proliferation and tissue repair. In the present study, we identified a novel positive allosteric modulator of PAR1, GB83, and investigated its effect on skin wound healing. Experimental Approach The enhancement of PAR1 activity by GB83 was measured using Fluo‐4 calcium assay. In silico docking analysis of GB83 in PAR1 was performed using dock ligands method (CDOCKER) with CHARMm force field. Effects of GB83 on cell viability and gene expression were observed using MTS assay and quantitative real‐time PCRs, respectively. SKH‐1 hairless mice were used to investigate the wound healing effect of GB83. Key Results We demonstrated that GB83 did not activate PAR1 by itself but strongly enhanced PAR1 activation by thrombin and PAR1‐activating peptide (AP). In silico docking analysis revealed that GB83 can bind to the PAR1 binding site of vorapaxar. GB83 significantly promoted PAR1‐mediated cell viability and migration. In addition, the enhancement of PAR1 activity by GB83 strongly increased gene expression of TGF‐β, fibronectin and type I collagen in vitro and promoted skin wound healing in vivo. Conclusion and Implications Our results revealed that GB83 is the first positive allosteric modulator of PAR1 and it can be a useful pharmacological tool for studying PAR1 and a potential therapeutic agent for skin wound healing.



Figure 3. Identification of novel ANO1 inhibitor, Ani-D2. (A) Inhibitory effect of compounds 1-6 on ANO1 activity was measured in FRT cells expressing human ANO1 and a mutant YFP. The cells were pretreated with compounds 1 (Ani-D1) and 2 (Ani-D2) at 25 μM for 20 min. ANO1 was activated by 100 ATP μM. (B) Representative Western blot analysis of ANO1 in FRT, FRT-ANO1, and PC-3 cells (two independent experiments performed). ANO1 protein expression was measured in FRT, FRT-ANO1, and PC-3 cells. (C) Effect of Ani-D1 and Ani-D2 on the protein expression levels of ANO1 in PC-3 cells. PC-3 cells were treated with 10 μM of Ani-D1 and Ani-D2 for 24 h. (Right) Summary of band intensity. The ANO1 band intensity was normalized to β-actin (mean ± S.E., n = 3). (D) ANO1 mRNA expression levels were determined by real-time PCR in PC-3 cells treated with Ani-D2 for 24 h (mean ± S.E., n = 3). ** p < 0.01.
Figure 4. Characterization of Ani-D2. (A) Effect of Ani-D2 on apical membrane current was observed in FRT cells expressing ANO1. Ani-D2 was applied at the indicated concentrations 20 min prior to ANO1 activation by 100 μM ATP. (B) Summary of dose-responses (mean ± S.E., n = 3-4). (C) Effect of Ani-D2 on intracellular calcium concentration was measured by Fluo-4/NW in FRT cells. The cells were pretreated with 0, 10, 30 μM of Ani-D2 for 20 min prior to the treatment of 100 μM ATP. (D) Effect of Ani-D2 on CFTR chloride channel activity was observed in FRT cells expressing human CFTR. CFTR chloride currents were activated by 10 μM forskolin and inhibited by 10 μM CFTRinh-172.
Figure 7. Effect of Ani-D2 on caspase-3 activity and cleavage of PARP in PC-3 and CAL-27 cells. (A,B) Images were taken after 24 h incubation with Ani-D2. Caspase-3 substrate (green, 2.0 μM) and Hoechst 33342 (blue, 1 μM) were treated for 20 min prior to image acquisition. The white bar represents 200 μm. (C,D) Cells were cultured with Ani-D2 at the indicated concentrations for 24 h, then 2 μM of caspase-3 substrate was treated for 20 min. Caspase-3 activity was inhibited by 10 μM of Ac-DEVD-CHO (mean ± S.E., n = 3-4). (E,F) Cells were cultured with 10 μM of Ani-D2 for 24 h, and expression level of PARP, cleaved PARP, and β-actin were measured by immunoblot analysis (mean ± S.E., n = 3). * p < 0.05, *** p < 0.001.
Cytotoxic effects of compounds 1-6 on human cancer cell line, PC-3.
Novel ANO1 Inhibitor from Mallotus apelta Extract Exerts Anticancer Activity through Downregulation of ANO1

September 2020

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139 Reads

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14 Citations

International Journal of Molecular Sciences

Anoctamin1 (ANO1), a calcium-activated chloride channel, is frequently overexpressed in several cancers, including human prostate cancer and oral squamous cell carcinomas. ANO1 plays a critical role in tumor growth and maintenance of these cancers. In this study, we have isolated two new compounds (1 and 2) and four known compounds (3-6) from Mallotus apelta. These compounds were evaluated for their inhibitory effects on ANO1 channel activity and their cytotoxic effects on PC-3 prostate cancer cells. Interestingly, compounds 1 and 2 significantly reduced both ANO1 channel activity and cell viability. Electrophysiological study revealed that compound 2 (Ani-D2) is a potent and selective ANO1 inhibitor, with an IC50 value of 2.64 μM. Ani-D2 had minimal effect on cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel activity and intracellular calcium signaling. Notably, Ani-D2 significantly reduced ANO1 protein expression levels and cell viability in an ANO1-dependent manner in PC-3 and oral squamous cell carcinoma CAL-27 cells. In addition, Ani-D2 strongly reduced cell migration and induced activation of caspase-3 and cleavage of PARP in PC-3 and CAL-27 cells. This study revealed that a novel ANO1 inhibitor, Ani-D2, has therapeutic potential for the treatment of several cancers that overexpress ANO1, such as prostate cancer and oral squamous cell carcinoma.


Citations (11)


... Hemoglobin (Hb) is the most abundant protein in blood cells and is composed of globin and heme [2]. Globin is an excellent source of various bioactive peptides, and heme is a typical iron-containing porphyrin compound [3], which is highly effective in iron supplementation [4] and has anti-cancer [5], color enhancement [6], and other effects. However, heme is insoluble in water, limiting its wide application in the food industry [7]. ...

Reference:

Enhancement of Antioxidant Activity, Stability, and Structure of Heme-Peptides by L-Lysine
Anticancer Effect of Hemin through ANO1 Inhibition in Human Prostate Cancer Cells

International Journal of Molecular Sciences

... Several studies have reported the anticancer and antiproliferative effects of cisresveratrol across various cancer types (Table 1). For instance, Anoctamin 1 (ANO1), a calcium chloride-activated channel involved in cancer proliferation, migration, and invasion, was downregulated by cis-resveratrol in prostate cancer models [42]. Treatment with 30 µM of cis-resveratrol reduced ANO1 expression and inhibited its activity with an IC50 of 10.6 µM, resulting in a 97% reduction in PC-3 cell proliferation. ...

Inhibition of ANO1 by Cis-and Trans-Resveratrol and Their Anticancer Activity in Human Prostate Cancer PC-3 Cells

International Journal of Molecular Sciences

... Comparing this compound to the three cancer cell types mentioned above, it is noteworthy that HaCaT had little impact on cell viability. The compound In 2022, Kim et al. and the research team successfully synthesized a unique indolizine scaffold merged with a pyrrolo [1,2-c]pyrimidine scaffold using a one-pot oxidative cyclization procedure [33]. The consequence of these compounds on the existence of A549, HT29, HepG2, AGS, and H1299 cancer cell lines were observed by the author. ...

Synthesis and Biological Evaluation of a Fused Structure of Indolizine and Pyrrolo[1,2-c]pyrimidine: Identification of Its Potent Anticancer Activity against Liver Cancer Cells

Pharmaceuticals

... Intriguing open questions include the proteomic composition of the recruited membrane and the full physiological ramifications of these phenomena. TMEM16F (TransMEMbrane protein family 16 member F, encoded by the ANO6 gene) plays a critical role in extracellular vesicle (EV) release 18 , plasma membrane wound repair 19 , developmental and pathological syncytia formation 20,21 , and host cell entry of pathogens including HIV 22 , Ebola virus 23 , and SARS-CoV2 24 . TMEM16F is linked to Scott syndrome, a rare congenital bleeding disorder 14 , where activated platelets from individuals with loss-of-function TMEM16F mutations fail to execute plasma membrane phosphatidylserine scrambling necessary for thrombin production and for the release of EVs that facilitate coagulation 25,26 . ...

Amelioration of SARS-CoV-2 infection by ANO6 phospholipid scramblase inhibition

Cell Reports

... However, it is not clear to what extent this synergy depends on the disruption of MDMX-p53 complexes versus MDM2-p53 complexes at the high dose of nutlin 3a used, and whether downregulation of MDMX by topotecan may also contribute. The small molecule SJ-172550 was identified as an MDMX-p53-selective antagonist 105 that exhibited additive cytotoxicity with nutlin 3a in MDMXamplified retinoblastoma cells. However, subsequent analyses showed that SJ-172550 forms covalent adducts with cysteine residues in the p53-binding domain of both MDM2 and MDMX. ...

Identification and characterization of novel small‐molecule inhibitors of SLC26A9
  • Citing Article
  • May 2021

The FASEB Journal

... These results also suggest that drugs that induce excessive PAR1 activity in platelets have the potential to induce thrombosis. In a previous study, we showed that GB83 is the first small-molecule positive allosteric modulator (PAM) of PAR1 (Seo et al., 2021). However, GB83 is a dual acting modulator of PARs, inducing both PAR1 potentiation and PAR2 activation (Heo et al., 2022b), so it is not appropriate to see a clear effect on thrombus formation by enhancing PAR1 activity. ...

Novel positive allosteric modulator of protease‐activated receptor 1 promotes skin wound healing in hairless mice

... Corydaline inhibits HCC by binding to the druggable pocket of the hEAG1 channel [214,215]. Extracts from Mallotus apelta act as novel ANO1 inhibitors to exhibit anticancer activity [216]. Natural products targeting ion channels hold significant promise for cancer therapy. ...

Novel ANO1 Inhibitor from Mallotus apelta Extract Exerts Anticancer Activity through Downregulation of ANO1

International Journal of Molecular Sciences

... In 2020, Kim et al. synthesized novel quinone-indolizine derivatives from quinones and substituted pyrrole-2carboxaldehyde through domino Michael addition-aldol condensation aromatization [19]. The most promising candidate from the synthesized series isoquinoline containing chlorobenzoyl derivative 40 was evaluated in this study for oral adenosquamous carcinoma cells (CAL-27) and human prostate adenocarcinoma cells (PC-3), with IC 50 (µM) values of 0.028 ± 0.007, 0.024 ± 0.008 respectively. ...

Domino [4+2] Annulation Access to Quinone-Indolizine Hybrids: Anticancer N-Fused Polycycles

The Journal of Organic Chemistry

... Conversely, cathepsin S exacerbated glomerular endothelial injury and albuminuria in a PAR2-dependent manner [91]. Moreover, the novel potent PAR2 antagonist punicalagin significantly ameliorated kidney injury and splenomegaly and reduced the expression of intercellular adhesion molecule 1 (ICAM-1) and vascular adhesion molecule 1 (VCAM-1) in NZB/W F1 lupus mice [92]. These two studies support the injurious role of PAR2 in lupus nephritis. ...

Punicalagin Ameliorates Lupus Nephritis via Inhibition of PAR2

International Journal of Molecular Sciences

... More recent studies have assessed the effects of more specific ANO1 inhibitors. Seo and colleagues identified '3n', a compound derived from a 2,2-dimethyl-2G-chromene motif, that had increased selectivity for inhibiting ANO1, with only weak inhibition of ANO2 [173]. Furthermore, this study showed that 3n induced apoptotic cell death in prostate cancer cells in vitro [173]. ...

Diversity-Oriented Generation and Biological Evaluation of New Chemical Scaffolds Bearing a 2,2-Dimethyl-2H-chromene Unit: Discovery of Novel Potent ANO1 Inhibitors
  • Citing Article
  • June 2020

Bioorganic Chemistry