Metabolic engineering of Saccharomyces cerevisiae for enhanced taxadiene production.

BACKGROUND: Metabolic engineering enables the sustainable and cost-efficient production of complex chemicals. Efficient production of terpenes in Saccharomyces cerevisiae can be achieved by recruiting an intermediate of the mevalonate pathway. The present study aimed to evaluate the engineering strategies of S. cerevisiae for the production of taxadiene, a precursor of taxol, an antineoplastic drug. RESULT: SCIGS22a, a previously engineered strain with modifications in the mevalonate pathway (MVA), was used as a background strain. This strain was engineered to enable a high flux towards farnesyl diphosphate (FPP) and the availability of NADPH. The strain MVA was generated from SCIGS22a by overexpressing all mevalonate pathway genes. Combining the background strains with 16 different episomal plasmids, which included the combination of 4 genes: tHMGR (3-hydroxy-3-methylglutaryl-CoA reductase), ERG20 (farnesyl pyrophosphate synthase), GGPPS (geranyl diphosphate synthase) and TS (taxadiene synthase) resulted in the highest taxadiene production in S. cerevisiae of 528 mg/L. CONCLUSION: Our study highlights the critical role of pathway balance in metabolic engineering, mainly when dealing with toxic molecules like taxadiene. We achieved significant improvements in taxadiene production by employing a combinatorial approach and focusing on balancing the downstream and upstream pathways. These findings emphasize the importance of minor gene expression modification levels to achieve a well-balanced pathway, ultimately leading to enhanced taxadiene accumulation.

25-Hydroxycholesterol attenuates tumor necrosis factor alpha-induced blood-brain barrier breakdown in vitro.

Intracellular cholesterol metabolism is regulated by the SREBP-2 and LXR signaling pathways. The effects of inflammation on these molecular mechanisms remain poorly studied, especially at the blood-brain barrier (BBB) level. Tumor necrosis factor α (TNFα) is a proinflammatory cytokine associated with BBB dysfunction. Therefore, the aim of our study was to investigate the effects of TNFα on BBB cholesterol metabolism, focusing on its underlying signaling pathways. Using a human in vitro BBB model composed of human brain-like endothelial cells (hBLECs) and brain pericytes (HBPs), we observed that TNFα increases BBB permeability by degrading the tight junction protein CLAUDIN-5 and activating stress signaling pathways in both cell types. TNFα also promotes cholesterol release and decreases cholesterol accumulation and APOE secretion. In hBLECs, the expression of SREBP-2 targets (LDLR and HMGCR) is increased, while ABCA1 expression is decreased. In HBPs, only LDLR and ABCA1 expression is increased. TNFα treatment also induces 25-hydroxycholesterol (25-HC) production, a cholesterol metabolite involved in the immune response and intracellular cholesterol metabolism. 25-HC pretreatment attenuates TNFα-induced BBB leakage and partially alleviates the effects of TNFα on ABCA1, LDLR, and HMGCR expression. Overall, our results suggest that TNFα favors cholesterol efflux via an LXR/ABCA1-independent mechanism at the BBB, while it activates the SREBP-2 pathway. Treatment with 25-HC partially reversed the effect of TNFα on the LXR/SREBP-2 pathways. Our study provides novel perspectives for better understanding cerebrovascular signaling events linked to BBB dysfunction and cholesterol metabolism in neuroinflammatory diseases.

Development of a 2A peptide-based multigene expression system and its application for enhanced production of ganoderic acids in Ganoderma lucidum.

Ganoderma has received much attention for its medicinal value, but the manipulation of multiple genes remains a challenge, hindering the genetic engineering of this species for the development of cell factories. Here, we first showed that the presence of an intron is necessary for the efficient expression of the endogenous cDNA of carboxin-resistant gene (cbx) in G. lucidum. Then, the self-cleaving function of 2 A peptide was investigated in G. lucidum by linking cbx cDNA to the codon-optimized hygromycin B-resistant gene (ophph) using the 2A-peptide sequence. The results showed that cbx cDNA and ophph can be successfully expressed in G. lucidum in a bicistronic manner from a single transcript. Moreover, the expression of both genes was not affected by the order within the 2 A cassette. In addition, simultaneous expression of cbx cDNA, ophph, and codon-optimized yellow fluorescent protein gene (opyfp) was conducted for the first time in G. lucidum using the 2 A peptide-based approach. The developed method was successfully applied to express both cDNA of the 3-hydroxy-3-methylglutaryl coenzyme A reductase (hmgr) and squalene epoxidase gene (se) for enhanced production of ganoderic acids (GAs) in G. lucidum. The engineered strain produced the maximum content of GA-Mk, GA-T, GA-S, and GA-Me were 26.56±3.53,39.58±3.75, 16.54±2.16, and 19.1±1.87 µg/100 mg dry weight, respectively. These values were 3.85-, 4.74-, 3.65-, and 3.23-fold higher than those produced by the control strain. The developed method will be useful for the manipulation of complex metabolic or regulatory pathways involving multiple genes in Ganoderma.

N-SREBP2 Provides a Mechanism for Dynamic Control of Cellular Cholesterol Homeostasis.

Cholesterol is required to maintain the functional integrity of cellular membrane systems and signalling pathways, but its supply must be closely and dynamically regulated because excess cholesterol is toxic. Sterol regulatory element-binding protein 2 (SREBP2) and the ER-resident protein HMG-CoA reductase (HMGCR) are key regulators of cholesterol biosynthesis. Here, we assessed the mechanistic aspects of their regulation in hepatic cells. Unexpectedly, we found that the transcriptionally active fragment of SREBP2 (N-SREBP2) was produced constitutively. Moreover, in the absence of an exogenous cholesterol supply, nuclear N-SREBP2 became resistant to proteasome-mediated degradation. This resistance was paired with increased occupancy at the HMGCR promoter and HMGCR expression. Inhibiting nuclear N-SREBP2 degradation did not increase HMGCR RNA levels; this increase required cholesterol depletion. Our findings, combined with previous physiological and biophysical investigations, suggest a new model of SREBP2-mediated regulation of cholesterol biosynthesis in the organ that handles large and rapid fluctuations in the dietary supply of this key lipid. Specifically, in the nucleus, cholesterol and the ubiquitin-proteasome system provide a short-loop system that modulates the rate of cholesterol biosynthesis via regulation of nuclear N-SREBP2 turnover and HMGCR expression. Our findings have important implications for maintaining cellular cholesterol homeostasis and lowering blood cholesterol via the SREBP2-HMGCR axis.

High expression of RUNX1 in colorectal cancer subtype accelerates malignancy by inhibiting HMGCR.

Colorectal cancer (CRC) presents a complex landscape, characterized by both inter-tumor and intra-tumor heterogeneity. RUNX1, a gene implicated in modulating tumor cell growth, survival, and differentiation, remains incompletely understood regarding its impact on CRC prognosis. In our investigation, we discerned a positive correlation between elevated RUNX1 expression and aggressive phenotypes across various CRC subtypes. Notably, knockdown of RUNX1 demonstrated efficacy in restraining CRC proliferation both in vitro and in vivo, primarily through inducing apoptosis and impeding cell proliferation. Mechanistically, we unveiled a direct regulatory link between RUNX1 and cholesterol synthesis, mediated by its control over HMGCR expression. Knockdown of RUNX1 in CRC cells triggered HMGCR transcriptional activation, culminating in elevated cholesterol levels that subsequently hindered cancer progression. Clinically, heightened RUNX1 expression emerged as a prognostic marker for adverse outcomes in CRC patients. Our findings underscore the pivotal involvement of RUNX1 in CRC advancement and its potential as a therapeutic target. The unique influence of RUNX1 on cholesterol synthesis and HMGCR transcriptional regulation uncovers a novel pathway contributing to CRC progression.

AMPK-mediated regulation of endogenous cholesterol synthesis does not affect atherosclerosis in a murine Pcsk9-AAV model.

BACKGROUND AND AIMS: Dysregulated cholesterol metabolism is a hallmark of atherosclerotic cardiovascular diseases, yet our understanding of how endogenous cholesterol synthesis affects atherosclerosis is not clear. The energy sensor AMP-activated protein kinase (AMPK) phosphorylates and inhibits the rate-limiting enzyme in the mevalonate pathway HMG-CoA reductase (HMGCR). Recent work demonstrated that when AMPK-HMGCR signaling was compromised in an Apoe-/- model of hypercholesterolemia, atherosclerosis was exacerbated due to elevated hematopoietic stem and progenitor cell mobilization and myelopoiesis. We sought to validate the significance of the AMPK-HMGCR signaling axis in atherosclerosis using a non-germline hypercholesterolemia model with functional ApoE. METHODS: Male and female HMGCR S871A knock-in (KI) mice and wild-type (WT) littermate controls were made atherosclerotic by intravenous injection of a gain-of-function Pcsk9D374Y-adeno-associated virus followed by high-fat and high-cholesterol atherogenic western diet feeding for 16 weeks. RESULTS: AMPK activation suppressed endogenous cholesterol synthesis in primary bone marrow-derived macrophages from WT but not HMGCR KI mice, without changing other parameters of cholesterol regulation. Atherosclerotic plaque area was unchanged between WT and HMGCR KI mice, independent of sex. Correspondingly, there were no phenotypic differences observed in hematopoietic progenitors or differentiated immune cells in the bone marrow, blood, or spleen, and no significant changes in systemic markers of inflammation. When lethally irradiated female mice were transplanted with KI bone marrow, there was similar plaque content relative to WT. CONCLUSIONS: Given previous work, our study demonstrates the importance of preclinical atherosclerosis model comparison and brings into question the importance of AMPK-mediated control of cholesterol synthesis in atherosclerosis.

Unravelling effects of phytochemicals from buckwheat on cholesterol metabolism and lipid accumulation in HepG2 cells and its validation through gene expression analysis.

BACKGROUND: The objective of this research was to elucidate the hypocholesterolemic effects of a bioactive compound extracted from buckwheat, and to delineate its influence on the regulatory mechanisms of cholesterol metabolism. The compound under investigation was identified as quercetin. MATERIAL AND RESULTS: In vitro experiments conducted on HepG2 cells treated with quercetin revealed a significant reduction in intracellular cholesterol accumulation. This phenomenon was rigorously quantified by assessing the transcriptional activity of key genes involved in the biosynthesis and metabolism of cholesterol. A statistically significant reduction in the expression of HMG-CoA reductase (HMGCR) was observed, indicating a decrease in endogenous cholesterol synthesis. Conversely, an upregulation in the expression of cholesterol 7 alpha-hydroxylase (CYP7A1) was also observed, suggesting an enhanced catabolism of cholesterol to bile acids. Furthermore, the study explored the combinatory effects of quercetin and simvastatin, a clinically utilized statin, revealing a synergistic action in modulating cholesterol levels at various dosages. CONCLUSIONS: The findings from this research provide a comprehensive insight into the mechanistic pathways through which quercetin, a phytochemical derived from buckwheat, exerts its hypocholesterolemic effects. Additionally, the observed synergistic interaction between quercetin and simvastatin opens up new avenues for the development of combined therapeutic strategies to manage hyperlipidemia.

LY86 facilitates ox-LDL-induced lipid accumulation in macrophages by upregulating SREBP2/HMGCR expression.

LY86, also known as MD1, has been implicated in various pathophysiological processes including inflammation, obesity, insulin resistance, and immunoregulation. However, the role of LY86 in cholesterol metabolism remains incompletely understood. Several studies have reported significant up-regulation of LY86 mRNA in atherosclerosis; nevertheless, the regulatory mechanism by which LY86 is involved in this disease remains unclear. In this study, we aimed to investigate whether LY86 affects ox-LDL-induced lipid accumulation in macrophages. Firstly, we confirmed that LY86 is indeed involved in the process of atherosclerosis and found high expression levels of LY86 in human atherosclerotic plaque tissue. Furthermore, our findings suggest that LY86 may mediate intracellular lipid accumulation induced by ox-LDL through the SREBP2/HMGCR pathway. This mechanism could be associated with increased cholesterol synthesis resulting from enhanced endoplasmic reticulum stress response.

Onion Polyphenols as Multi-Target-Directed Ligands in MASLD: A Preliminary Molecular Docking Study.

A sedentary lifestyle associated with unregulated diets rich in high-calorie foods have contributed to the great prevalence of metabolic dysfunction-associated steatotic liver disease (MASLD) latterly, with up to 60% in the high-risk population and 25% in the general population. The absence of specific pharmacological strategies for this syndrome represents one of the major problems in the management of MASLD patients. Lifestyle interventions and adherence to a healthy diet are the main cornerstones of current therapies. The identification of nutraceuticals useful in the treatment of MASLD appears to be one of the most promising strategies for the development of new effective and safe treatments for this disease. The onion, one of the most widely studied foods in the field of nutraceuticals, serves as an inexhaustible reservoir of potent compounds with various beneficial effects. The following preliminary study analyzes, mediating in silico studies, the iteration of a library of typical onion compounds with 3-hydroxy-3-methylglutaryl-coenzyme A reductase, liver receptors X α and ß, as well as peroxisome proliferator-activated receptors α and γ. In this study, for the first time promising smart molecules from the onion that could have a beneficial action in MASLD patients were identified.

Overcoming statin resistance in prostate cancer cells by targeting the 3-hydroxy-3-methylglutaryl-CoA-reductase.

Prostate cancer is the most prevalent malignancy in men. While diagnostic and therapeutic interventions have substantially improved in recent years, disease relapse, treatment resistance, and metastasis remain significant contributors to prostate cancer-related mortality. Therefore, novel therapeutic approaches are needed. Statins are inhibitors of the 3-hydroxy-3-methylglutaryl-CoA reductase (HMGCR), the rate-limiting enzyme of the mevalonate pathway which plays an essential role in cholesterol homeostasis. Numerous preclinical studies have provided evidence for the pleiotropic antitumor effects of statins. However, results from clinical studies remain controversial and have shown substantial benefits to even no effects on human malignancies including prostate cancer. Potential statin resistance mechanisms of tumor cells may account for such discrepancies. In our study, we treated human prostate cancer cell lines (PC3, C4-2B, DU-145, LNCaP) with simvastatin, atorvastatin, and rosuvastatin. PC3 cells demonstrated high statin sensitivity, resulting in a significant loss of vitality and clonogenic potential (up to - 70%; p < 0.001) along with an activation of caspases (up to 4-fold; p < 0.001). In contrast, C4-2B and DU-145 cells were statin-resistant. Statin treatment induced a restorative feedback in statin-resistant C4-2B and DU-145 cells through upregulation of the HMGCR gene and protein expression (up to 3-folds; p < 0.01) and its transcription factor sterol-regulatory element binding protein 2 (SREBP-2). This feedback was absent in PC3 cells. Blocking the feedback using HMGCR-specific small-interfering (si)RNA, the SREBP-2 activation inhibitor dipyridamole or the HMGCR degrader SR12813 abolished statin resistance in C4-2B and DU-145 and induced significant activation of caspases by statin treatment (up to 10-fold; p < 0.001). Consistently, long-term treatment with sublethal concentrations of simvastatin established a stable statin resistance of a PC3SIM subclone accompanied by a significant upregulation of both baseline as well as post-statin HMGCR protein (gene expression up to 70-fold; p < 0.001). Importantly, the statin-resistant phenotype of PC3SIM cells was reversible by HMGCR-specific siRNA and dipyridamole. Our investigations reveal a key role of a restorative feedback driven by the HMGCR/SREBP-2 axis in statin resistance mechanisms of prostate cancer cells.

Inhibition of mevalonate pathway by macrophage-specific delivery of atorvastatin prevents their pro-inflammatory polarisation.

Adjustment of the cellular metabolism of pro-inflammatory macrophages is essential for their bactericidal function; however, it underlies the development of many human diseases if induced chronically. Therefore, intervention of macrophage metabolic polarisation has been recognised as a potent strategy for their treatment. Although many small-molecule inhibitors affecting macrophage metabolism have been identified, their in vivo administration requires a tool for macrophage-specific delivery to limit their potential side effects. Here, we establish Drosophila melanogaster as a simple experimental model for in vivo testing of macrophage-specific delivery tools. We found that yeast-derived glucan particles (GPs) are suitable for macrophage-specific delivery of small-molecule inhibitors. Systemic administration of GPs loaded with atorvastatin, the inhibitor of hydroxy-methyl-glutaryl-CoA reductase (Hmgcr), leads to intervention of mevalonate pathway specifically in macrophages, without affecting HMGCR activity in other tissues. Using this tool, we demonstrate that mevalonate pathway is essential for macrophage pro-inflammatory polarisation and individual's survival of infection.

Role of hydroxymethylglutharyl-coenzyme A reductase in the induction of stem-like states in breast cancer.

PURPOSE: De novo synthesis of cholesterol and its rate-limiting enzyme, 3-hydroxy-3-methylglutharyl-coenzyme A reductase (HMGCR), is deregulated in tumors and critical for tumor cell survival and proliferation. However, the role of HMGCR in the induction and maintenance of stem-like states in tumors remains unclear. METHODS: A compiled public database from breast cancer (BC) patients was analyzed with the web application SurvExpress. Cell Miner was used for the analysis of HMGCR expression and statin sensitivity of the NCI-60 cell lines panel. A CRISPRon system was used to induce HMGCR overexpression in the luminal BC cell line MCF-7 and a lentiviral pLM-OSKM system for the reprogramming of MCF-7 cells. Comparisons were performed by two-tailed unpaired t-test for two groups and one- or two-way ANOVA. RESULTS: Data from BC patients showed that high expression of several members of the cholesterol synthesis pathway were associated with lower recurrence-free survival, particularly in hormone-receptor-positive BC. In silico and in vitro analysis showed that HMGCR is expressed in several BC cancer cell lines, which exhibit a subtype-dependent response to statins in silico and in vitro. A stem-like phenotype was demonstrated upon HMGCR expression in MCF-7 cells, characterized by expression of the pluripotency markers NANOG, SOX2, increased CD44 +/CD24low/ -, CD133 + populations, and increased mammosphere formation ability. Pluripotent and cancer stem cell lines showed high expression of HMGCR, whereas cell reprogramming of MCF-7 cells did not increase HMGCR expression. CONCLUSION: HMGCR induces a stem-like phenotype in BC cells of epithelial nature, thus affecting tumor initiation, progression and statin sensitivity.

Eugeniin improves cholesterol metabolism in HepG2 cells and Caco-2 cells.

Considering the absence of prior studies on the cholesterol metabolism-improving effects of eugeniin, the present investigation aimed to explore the potential impact of eugeniin on cholesterol metabolism. This study sought to elucidate the molecular mechanisms involved in this process using HepG2 and Caco-2 cells treated with 5 µm eugeniin. The intracellular cholesterol levels in HepG2 and Caco-2 cells were significantly decreased in the 24-h eugeniin-treated group. The protein and messenger ribonucleic acid (mRNA) levels of the low-density lipoprotein receptor (LDLR) were increased, while 3-hydroxy-3-methyl-glutaryl-coenzyme A reductase protein and mRNA levels were decreased in HepG2 cells 6 h of the eugeniin-treated group. Additionally, LDLR protein and mRNA levels were increased in HepG2 cells after 24 h of eugeniin treatment. In Caco-2, the protein and mRNA levels of ATP-binding cassette transporter 1 were increased after 24 h eugeniin treatment. This novel finding indicates that eugeniin improves cholesterol metabolism in human cell cultures.

HMG-CoA reductase degrader, SR-12813, counteracts statin-induced upregulation of HMG-CoA reductase and augments the anticancer effect of atorvastatin.

Statins are cholesterol-lowering drugs that have exhibited potential as cancer therapeutic agents. However, as some cancer cells are resistant to statins, broadening an anticancer spectrum of statins is desirable. The upregulated expression of the statin target enzyme, 3-hydroxy-3-methyl-glutaryl-coenzyme A (HMG-CoA) reductase (HMGCR), in statin-treated cancer cells is a well-known mechanism of statin resistance, which can be counteracted by the downregulation of HMGCR gene expression, or degradation of the HMGCR protein. However, the mechanism by which HMGCR degradation influences the anticancer effects of statins remain unreported. We tested the effect of the HMGCR degrader compound SR-12813 at a concentration that did not affect the growth of eight diverse tumor cell lines. Combined treatment with atorvastatin and a low concentration of SR-12813 led to lowering of increased HMGCR expression, and augmented the cytostatic effect of atorvastatin in both statin-resistant and -sensitive cancer cells compared with that of atorvastatin treatment alone. Dual-targeting of HMGCR using statins and SR-12813 (or similar compounds) could provide an improved anticancer therapeutic approach.

Epigenetic suppression of PGC1α (PPARGC1A) causes collateral sensitivity to HMGCR-inhibitors within BRAF-treatment resistant melanomas.

While targeted treatment against BRAF(V600E) improve survival for melanoma patients, many will see their cancer recur. Here we provide data indicating that epigenetic suppression of PGC1α defines an aggressive subset of chronic BRAF-inhibitor treated melanomas. A metabolism-centered pharmacological screen further identifies statins (HMGCR inhibitors) as a collateral vulnerability within PGC1α-suppressed BRAF-inhibitor resistant melanomas. Lower PGC1α levels mechanistically causes reduced RAB6B and RAB27A expression, whereby their combined re-expression reverses statin vulnerability. BRAF-inhibitor resistant cells with reduced PGC1α have increased integrin-FAK signaling and improved extracellular matrix detached survival cues that helps explain their increased metastatic ability. Statin treatment blocks cell growth by lowering RAB6B and RAB27A prenylation that reduces their membrane association and affects integrin localization and downstream signaling required for growth. These results suggest that chronic adaptation to BRAF-targeted treatments drive novel collateral metabolic vulnerabilities, and that HMGCR inhibitors may offer a strategy to treat melanomas recurring with suppressed PGC1α expression.

The role of HMGCR expression in combination therapy of simvastatin and FAC treated locally advanced breast cancer patients.

OBJECTIVE: Several studies have shown the role of statin added to the patient's chemotherapy regimen and the role of Hydroxymethylglutaryl-CoA Reductase (HMGCR) expression in predicting breast cancer patient outcomes. In our previous study, adding statins improved clinical and pathological responses in LABC patients. Furthermore, we planned to study statin's role as a combination to neoadjuvant chemotherapy (NAC) in treating locally advanced breast cancers on the basis of HMGCR expression. Moreover, we aimed to study the association between the patients' clinicopathological characteristics and HMGCR expression. METHODS: This study is a randomized, double-blinded, placebo-controlled trial in two health centers in Indonesia. Each patient enrolled with written informed consent and then randomized to receive either simvastatin 40 mg/day or a placebo, combined with the fluorouracil, adriamycin, and cyclophosphamide (FAC) NAC. RESULTS: HMGCR was associated with low staging and normal serum cholesterol in the high Ki67 level group (p = 0.042 and p = 0.021, respectively). The pre-and post-chemotherapy tumor sizes are significantly correlated in two groups (HMGCR negative expression, p = 0.000 and HMGCR moderate expression, p = 0.001) with a more considerable average decrease in tumor size compared to HMGCR strong expression group. CONCLUSION: Statin therapy might work better in HMGCR-negative or low-expression tumors, although HGMCR expression is associated with better clinical parameters in our study.

Therapeutic properties and molecular docking study of some phenolic compounds as anti-human lung cancer potential: A biochemical approach.

Chloroxine (5,7-dichloro-8-hydroxyquinoline) is a molecule utilized in some shampoos for the therapy of seborrheic dermatitis of the scalp and dandruff. In this study, we investigated the inhibition effects of 5,7-dichloro-8-hydroxyquinoline and methyl 3,4,5-trihydroxybenzoate compounds on the 3-hydroxy-3-methylglutaryl coenzyme-A (HMG-CoA Reductase) and urease enzymes. We have obtained results for the HMG-CoA Reductase and urease enzymes at the micromolar level. In our study, inhibition result of 5,7-dichloro-8-hydroxyquinoline and Methyl 3,4,5-trihydroxybenzoate on HMG-CoA reductase showed lower values 2.28 ± 0.78 and 33.25 ± 5.04 µg/ml, respectively. Additionally, inhibition result of 5,7-dichloro-8-hydroxyquinoline and methyl 3,4,5-trihydroxybenzoate on urease showed lower values 6.18 ± 1.38 and 8.51 ± 1.35 µg/ml, respectively. Molecular docking calculations were made for their biological activities were compared. In the present work, the structures of the related compounds (1 and 2) were drawn using Gaussian 09 software and done geometry optimization at DFT/B3LYP/6-31G* basis set with aforementioned program. Cytotoxicity potential of these compounds against human lung cancer demonstrated that these compounds had good cytotoxic effects. Both compounds significantly decreased lung cell viability from low doses. In addition, 100 µM dose of all compounds caused significant reductions in lung cell viability. In general, we can say that of the two tested compounds, 5,7-dichloro-8-hydroxyquinoline and methyl 3,4,5-trihydroxybenzoate have cytotoxic effects in all cell types, and this effect is particularly strong in lung cells. Activities were performed at concentrations of 10, 20, 50, 70, and 100 µl and we achieved good results. Lung cell viability (%) value was better at 100 µl concentration and IC50 of them were 54.28 and 48.05 µM.

Pretreatment of hydroethanolic extract of Dillenia indica L. attenuates oleic acid induced NAFLD in HepG2 cells via modulating SIRT-1/p-LKB-1/AMPK, HMGCR & PPAR-α signaling pathways.

ETHNOPHARMACOLOGICAL RELEVANCE: Dillenia indica L. is an edible plant from the Dilleniaceae family present in the forest of India and other Asian countries. Different parts of this plant are being used in the traditional system of medicines for various diseases like diabetes, indigestion, asthma, jaundice, and rheumatic pain by various rural communities. This plant is very common among Khamptis traditional healers, the rural community of the Dhemaji district of Assam, ethnic communities of Dibru-Saikhowa Biosphere Reserve of Northeast, India for various medicinal uses. It is observed as a 'vat' suppressant and 'pitta' boosting medicine in Ayurveda. AIM OF THE STUDY: The aim of this research was to evaluate the effect of hydroethanolic extract of Dillenia indica leaf (DI-HET) against non-alcoholic fatty liver disease (NAFLD) as it is reported effective against jaundice in traditional medicine. We are also planning to see the various molecular mechanisms responsible for its effect if it is efficacious. STUDY DESIGN/METHOD: An in vitro model for NAFLD was employed in this study. For this HepG2 cells were incubated with 100 µM of oleic acid (OA) for 24 h. For evaluation of the effect of DI-HET, the extracts (5 or 10 µg/mL) were pretreated to the OA group. Fenofibrate was the positive control. Various parameters relevant to lipogenesis and ß-oxidation of fatty acids like intracellular lipid accumulation, reactive oxygen species (ROS), mitochondrial stress, and key proteins were studied. RESULTS: DI-HET significantly reduced the intracellular lipid accumulation in OA treated cells. And also substantially decreased the expression of lipogenic proteins and increased ß-oxidation in the OA group. OA induced ROS generation was found to reduce with DI-HET treatment. Western blot analysis showed that the expression of LXR-α, SREBP-1C, SREBP-2, HMGCR, FAS, CD-36, and ACOX-1 were downregulated while that of SIRT-1, p-LKB-, p-AMPK, p-ACC, CPT-1, and PPAR-α upregulated in DI-HET treatment. LCMS/MS analysis showed the presence of polyphenols like naringenin, catechin, epicatechin, shikimic acid, syringic acid, vanillic acid, and kaempferol. CONCLUSION: These results suggest that DI-HET is effective against NAFLD by activation of the SIRT-1/p-LKB-1/AMPK signaling pathway via polyphenols present in the extract.

Identification of HMGCR as the anticancer target of physapubenolide against melanoma cells by in silico target prediction.

Physapubenolide (PB), a withanolide-type compound extracted from the traditional herb Physalis minima L., has been demonstrated to exert remarkable cytotoxicity against cancer cells; however, its molecular mechanisms are still unclear. In this study, we demonstrated that PB inhibited cell proliferation and migration in melanoma cells by inducing cell apoptosis. The anticancer activity of PB was further verified in a melanoma xenograft model. To explore the mechanism underlying the anticancer effects of PB, we carried out an in silico target prediction study, which combined three approaches (chemical similarity searching, quantitative structure-activity relationship (QSAR), and molecular docking) to identify the targets of PB, and found that PB likely targets 3-hydroxy-methylglutaryl CoA reductase (HMGCR), the rate-limiting enzyme of the mevalonate pathway, which promotes cancer cell proliferation, migration, and metastasis. We further demonstrated that PB interacted with HMGCR, decreased its protein expression and inhibited the HMGCR/YAP pathway in melanoma cells. In addition, we found that PB could restore vemurafenib sensitivity in vemurafenib-resistant A-375 cells, which was correlated with the downregulation of HMGCR. In conclusion, we demonstrate that PB elicits anticancer action and enhances sensitivity to vemurafenib by targeting HMGCR.

Flavonoid-statin interactions causing myopathy and the possible significance of OATP transport, CYP450 metabolism and mevalonate synthesis.

3-hydroxy-3-methyl-glutaryl-coenzyme A (HMG-CoA) reductase inhibitors, statins, are a primary treatment for hyperlipidemic cardiovascular diseases which are a leading global cause of death. Statin therapy is life saving and discontinuation due to adverse events such as myotoxicity may lead to unfavourable outcomes. There is no known mechanism for statin-induced myotoxicity although it is theorized that it is due to inhibition of downstream products of the HMG-CoA pathway. It is known that drug-drug interactions with conventional medicines exacerbate the risk of statin-induced myotoxicity, though little attention has been paid to herb-drug interactions with complementary medicines. Flavonoids are a class of phytochemicals which can be purchased as high dose supplements. There is evidence that flavonoids can raise statin plasma levels, increasing the risk of statin-induced myopathy. This could be due to pharmacokinetic interactions involving hepatic cytochrome 450 (CYP450) metabolism and organic anion transporter (OATP) absorption. There is also the potential for flavonoids to directly and indirectly inhibit HMG-CoA reductase which could contraindicate statin-therapy. This review aims to discuss what is currently known about the potential for high dose flavonoids to interact with the hepatic CYP450 metabolism, OATP uptake of statins or their ability to interact with HMG-CoA reductase. Flavonoids of particular interest will be covered and the difficulties of examining herbal products will be discussed throughout.