Kamaruddin NN , Mohd Din LH , Jack A , Abdul Manan AF , Mohamad H , Tengku Muhammad TS .

TRGS/1/2015/UMT/01/2/3 Ministry of Higher Education

	Figure 1. Percentage of HepG2-cell growth after treatment with selected A. planci fractions at 5 different concentrations, from 3.13 to 50 µg/mL, for 72 h. The percentage of cell growth was compared with that for the negative control of 1% (v/v) of dimethyl sulfoxide (DMSO), which was used as the carrier. Data obtained are presented as means ± SDs with n = 6. * denotes significantly different compared with DMSO-treated cells (negative control) at p < 0.05. DMSO was used as the carrier to dissolve and dilute the extract. HepG2 cells were treated with vincristine sulfate (VS) as a positive control.
	Figure 2. The effects of selected A. planci fraction on PCSK9 mRNA expression in HepG2 cells. Each value shows mean ± SEM of 3 replicates after the gene expression level of PCSK9 was normalized against that of β-actin, and the value of the untreated control was assigned as 1. The mRNA expression level of PCSK9 for each treatment is relative to the control value. DMSO was used as the carrier to dissolve and dilute the extract. Cells were treated with 1% (v/v) DMSO and berberine sulfate as the negative and positive controls, respectively. ‘*’ denotes statistically significance (p < 0.05) as compared to untreated control.
	Figure 3. The effects of A. planci on the level of LDLR on the HepG2 cell line at 3 different time points: 12, 24, and 36 h, respectively. Cells were either untreated or treated with 3.13, 6.25, or 12.5 µg/mL of the selected fraction for 12, 24, and 36 h. Images were taken using a high-content screening platform (ImageXpress® Micro, Molecular Devices) (A), and analyzed using ImageJ 1.52a; Java 1.8.0_191 (B). Berberine sulfate (BS)-treated cells were used as the positive control. ‘*’ denotes statistically significance (p < 0.05) as compared to untreated control (DMSO).
	Figure 4. The effects of selected A. planci fraction on the level of LDL-C uptake by the HepG2 cell line at 3 different time points: 12, 24, and 36 h, respectively. Cells were either untreated or treated with 3.13, 6.25, or 12.5 µg/mL of the selected fraction for 12, 24, and 36 h. Images were taken using a high-content screening platform (ImageXpress® Micro, Molecular Devices) (A), and analyzed using ImageJ 1.52a; Java 1.8.0_191 (B). Berberine sulfate (BS)-treated cells were used as the positive control. ‘*’ denotes statistically significance (p < 0.05) as compared to untreated control (DMSO).
	Figure 5. Inhibitory effect of A. planci on PCSK9 promoter fragments. Transient transfection was carried out on 7 different PCSK9 promoter constructs (D1–D7) in the HepG2 cell line, and cells were treated with 6.25 µg/mL of the selected fraction. Luciferase assays were then conducted on cell lysates. One-way ANOVA was used for statistical analysis comparing treated transfected HepG2 cells with the untreated control. * denotes statistical significance (p < 0.05) compared with the untreated control.
	Figure 6. Identification of cis-acting elements involved in the downregulatory effects of the selected A. planci fraction on PCSK9 expression. (A) Schematic representation of the predicted potential transcription binding sites on the 5′-end-deletion constructs of human PCSK9 promoter, as well as the mutated PPRE and SRE used in this study. Position −94 is the 3′ end of PCSK9 promoter inserts common to all promoter–reporter constructs. The 5′ ends of the promoter in each construct are marked by the numbers on the left. MatInspector software was used to predict the transcription binding sites on the PCSK9 promoter (PPRE, peroxisome proliferator response element; SRE, sterol regulatory element; Sp1, specificity protein 1; HNF-1, hepatocyte nuclear factor 1; HINFP, histone nuclear factor P). (B) The core nucleotides for the predicted PPRE and SRE on the PCSK9 promoter are presented in capital letters, and the mutated nucleotides within the binding sites are in capital letters, underlined, and highlighted in bold.
	Figure 7. The effects of mutated PPRE and SRE on PCSK9 promoter activity. The activity of the wild-type promoter construct without treatment with the selected A. planci fraction was assigned as the control level (open bar), and the transcriptional activity of promoter constructs in the cells treated with the selected fraction (closed bar) are represented relative to the control value. The data shown are representative of 3 independent experiments, and each experiment was carried out in triplicate (PPRE, peroxisome proliferator response element; SRE, sterol regulatory element; Sp1, specificity protein 1; HNF-1, hepatocyte nuclear factor 1; HINFP, histone nuclear factor P). * Indicates statistical significance (p < 0.05) compared with the untreated control. NS indicates no statistical significance (p > 0.05) between the two compared samples.
	Figure 8. Time-course study on the effects of A. planci on the phosphorylation of MAPK. HepG2 cells were either untreated or treated with 6.25 µg/mL of selected A. planci fraction over a period of 4 h. Isolated cytoplasmic extracts were subjected to Western blot analysis using antibodies against total and phospho-MAPK (A). The levels of protein expression for phospho-MAPK were normalized against those for total MAPK, and the value at each time point represents the fold change of normalized phospho-MAPK protein expression relative to the untreated control, which was assigned a value of 1. * indicates that the value at a particular time point was significantly higher than that of the untreated control (B).
	Figure 9. Time-course study on the effects of A. planci on the phosphorylation of PKCα. HepG2 cells were either untreated or treated with 6.25 µg/mL of selected A. planci fraction over a period of 4 h. Isolated cytoplasmic extracts were subjected to Western blot analysis using antibodies against total and phospho-PKCα (A). The levels of protein expression for phospho-PKCα were normalized against those for total PKCα, and the value at each time point represents the fold change in normalized phospho-PKCα protein expression relative to the untreated control, which is assigned to 1. * Indicates that the value at a particular time point was significantly higher than that of the untreated control (B).
	Figure 10. Effects of pre-treatment with MEK inhibitor (PD98059) (A) and PKCα inhibitor (HA-dihydrochloride) (B) on PCSK9 mRNA level in HepG2 cells treated with A. planci. Assigning the signal for the PCSK9/β-actin ratio in the untreated cells as 1, the expression level of PCSK9 for each dose–response treatment is relative to this control value. Each value shows mean ± SEM of triplicate. * Indicates that the value at a particular time point was significantly higher than that for HepG2 cells treated with the selected A. planci fraction in the absence of inhibitor pre-treatment.

Abidi, Extracellular signal-regulated kinase-dependent stabilization of hepatic low-density lipoprotein receptor mRNA by herbal medicine berberine. 2005, Pubmed

Abidi, Extracellular signal-regulated kinase-dependent stabilization of hepatic low-density lipoprotein receptor mRNA by herbal medicine berberine. 2005, Pubmed
Adorni, Naturally Occurring PCSK9 Inhibitors. 2020, Pubmed
Agabiti Rosei, Management of Hypercholesterolemia, Appropriateness of Therapeutic Approaches and New Drugs in Patients with High Cardiovascular Risk. 2016, Pubmed
Aiman, Statin induced diabetes and its clinical implications. 2014, Pubmed
Asselman, Marine biogenics in sea spray aerosols interact with the mTOR signaling pathway. 2019, Pubmed
Beg, Phosphorylation and modulation of the enzymic activity of native and protease-cleaved purified hepatic 3-hydroxy-3-methylglutaryl-coenzyme A reductase by a calcium/calmodulin-dependent protein kinase. 1987, Pubmed
Bergheanu, Pathophysiology and treatment of atherosclerosis : Current view and future perspective on lipoprotein modification treatment. 2017, Pubmed
Burns, Modulation of PPAR activity via phosphorylation. 2007, Pubmed
Cameron, Berberine decreases PCSK9 expression in HepG2 cells. 2008, Pubmed
Cao, Janus kinase activation by cytokine oncostatin M decreases PCSK9 expression in liver cells. 2011, Pubmed
Careskey, Atorvastatin increases human serum levels of proprotein convertase subtilisin/kexin type 9. 2008, Pubmed
Cartharius, MatInspector and beyond: promoter analysis based on transcription factor binding sites. 2005, Pubmed
Cerda, Effect of statins on lipid metabolism-related microRNA expression in HepG2 cells. 2021, Pubmed
Chambard, ERK implication in cell cycle regulation. 2007, Pubmed
Deichmann, Coenzyme q10 and statin-induced mitochondrial dysfunction. 2010, Pubmed
Dong, Strong induction of PCSK9 gene expression through HNF1alpha and SREBP2: mechanism for the resistance to LDL-cholesterol lowering effect of statins in dyslipidemic hamsters. 2010, Pubmed
Dong, Inhibition of PCSK9 transcription by berberine involves down-regulation of hepatic HNF1α protein expression through the ubiquitin-proteasome degradation pathway. 2015, Pubmed
Drummond, Molecular Control of Atypical Protein Kinase C: Tipping the Balance between Self-Renewal and Differentiation. 2016, Pubmed
Duan, Peroxisome Proliferator-activated receptor γ activation by ligands and dephosphorylation induces proprotein convertase subtilisin kexin type 9 and low density lipoprotein receptor expression. 2012, Pubmed
Farnier, Efficacy and safety of adding alirocumab to rosuvastatin versus adding ezetimibe or doubling the rosuvastatin dose in high cardiovascular-risk patients: The ODYSSEY OPTIONS II randomized trial. 2016, Pubmed
Gao, Pinostrobin Inhibits Proprotein Convertase Subtilisin/Kexin-type 9 (PCSK9) Gene Expression through the Modulation of FoxO3a Protein in HepG2 Cells. 2018, Pubmed
George, Looking into the crystal ball-upcoming drugs for dyslipidemia. 2015, Pubmed
Graversen, Marine n-3 polyunsaturated fatty acids lower plasma proprotein convertase subtilisin kexin type 9 levels in pre- and postmenopausal women: A randomised study. 2016, Pubmed
Hobbs, Is statin-modified reduction in lipids the most important preventive therapy for cardiovascular disease? A pro/con debate. 2016, Pubmed
Horodinschi, Treatment with Statins in Elderly Patients. 2019, Pubmed
Ishii, A ceramide and cerebroside from the starfish asterias amurensis Lütken and their plant-growth promotion activities. 2006, Pubmed , Echinobase
Istvan, Structural mechanism for statin inhibition of 3-hydroxy-3-methylglutaryl coenzyme A reductase. 2002, Pubmed
Jeong, Sterol-dependent regulation of proprotein convertase subtilisin/kexin type 9 expression by sterol-regulatory element binding protein-2. 2008, Pubmed
Kamaruddin, Acanthaster planci Inhibits PCSK9 and Lowers Cholesterol Levels in Rats. 2021, Pubmed , Echinobase
Kourimate, Dual mechanisms for the fibrate-mediated repression of proprotein convertase subtilisin/kexin type 9. 2008, Pubmed
Krum, Statins and chronic heart failure: do we need a large-scale outcome trial? 2002, Pubmed
Lagace, Secreted PCSK9 decreases the number of LDL receptors in hepatocytes and in livers of parabiotic mice. 2006, Pubmed
Lambert, Fasting induces hyperlipidemia in mice overexpressing proprotein convertase subtilisin kexin type 9: lack of modulation of very-low-density lipoprotein hepatic output by the low-density lipoprotein receptor. 2006, Pubmed
Lambert, Plasma PCSK9 concentrations correlate with LDL and total cholesterol in diabetic patients and are decreased by fenofibrate treatment. 2008, Pubmed
Lee, Antioxidative and anticancer activities of various ethanolic extract fractions from crown-of-thorns starfish (Acanthaster planci). 2014, Pubmed , Echinobase
Li, Hepatocyte nuclear factor 1alpha plays a critical role in PCSK9 gene transcription and regulation by the natural hypocholesterolemic compound berberine. 2009, Pubmed
Li, ERK is integral to the IFN-γ-mediated activation of STAT1, the expression of key genes implicated in atherosclerosis, and the uptake of modified lipoproteins by human macrophages. 2010, Pubmed
Liu, Apelin-13 increases expression of ATP-binding cassette transporter A1 via activating protein kinase C α signaling in THP-1 macrophage-derived foam cells. 2013, Pubmed
Ma, Therapeutic targets of hypercholesterolemia: HMGCR and LDLR. 2019, Pubmed
Macchi, Leptin, Resistin, and Proprotein Convertase Subtilisin/Kexin Type 9: The Role of STAT3. 2020, Pubmed
Mohd, Transcriptional regulation of retinol binding protein 4 by Interleukin-6 via peroxisome proliferator-activated receptor α and CCAAT/Enhancer binding proteins. 2020, Pubmed
Newman, Natural Products as Sources of New Drugs from 1981 to 2014. 2016, Pubmed
Nissen, Effect of rosiglitazone on the risk of myocardial infarction and death from cardiovascular causes. 2007, Pubmed
Paton, PCSK9 inhibitors: monoclonal antibodies for the treatment of hypercholesterolemia. 2016, Pubmed
Paunovska, Drug delivery systems for RNA therapeutics. 2022, Pubmed
Rashid, Decreased plasma cholesterol and hypersensitivity to statins in mice lacking Pcsk9. 2005, Pubmed
Rokos, Peroxisome proliferators activate extracellular signal-regulated kinases in immortalized mouse liver cells. 1997, Pubmed
Salna, Pravastatin-Induced Eczematous Eruption Mimicking Psoriasis. 2017, Pubmed
Shapiro, PCSK9: From Basic Science Discoveries to Clinical Trials. 2018, Pubmed
Singer, Hydroxymethylglutaryl-coenzyme A reductase-containing hepatocytes are distributed periportally in normal and mevinolin-treated rat livers. 1984, Pubmed
Sun, Activation of Adiponectin Receptor Regulates Proprotein Convertase Subtilisin/Kexin Type 9 Expression and Inhibits Lesions in ApoE-Deficient Mice. 2017, Pubmed
Tabas, Recent insights into the cellular biology of atherosclerosis. 2015, Pubmed
Toth, Statins: Then and Now. 2019, Pubmed
Verbeek, PCSK9 inhibitors: Novel therapeutic agents for the treatment of hypercholesterolemia. 2015, Pubmed
Wang, A small-molecule inhibitor of PCSK9 transcription ameliorates atherosclerosis through the modulation of FoxO1/3 and HNF1α. 2020, Pubmed
Yan, MG132, a proteasome inhibitor, enhances LDL uptake in HepG2 cells in vitro by regulating LDLR and PCSK9 expression. 2014, Pubmed