Gypenoside L inhibits hepatocellular carcinoma by targeting the SREBP2-HMGCS1 axis and enhancing immune response.

Hepatocellular carcinoma (HCC) is a malignant tumor that occurs in the liver, with a high degree of malignancy and relatively poor prognosis. Gypenoside L has inhibitory effects on liver cancer cells. However, its mechanism of action is still unclear. This study aims to investigate the inhibitory effects of gypenoside L on HCC in vitro and in vivo, and explore its potential mechanisms. The results showed that gypenoside L reduced the cholesterol and triglyceride content in HepG2 and Huh-7 cells, inhibited cell proliferation, invasion and metastasis, arrested cell cycle at G0/G1 phase, promoted cell apoptosis. Mechanistically, it targeted the transcription factor SREPB2 to inhibit the expression of HMGCS1 protein and inhibited the downstream proteins HMGCR and MVK, thereby regulating the mevalonate (MVA) pathway. Overexpression HMGCS1 led to significant alterations in the cholesterol metabolism pathway of HCC, which mediated HCC cell proliferation and conferred resistance to the therapeutic effect of gypenoside L. In vivo, gypenoside L effectively suppressed HCC growth in tumor-bearing mice by reducing cholesterol production, exhibiting favorable safety profiles and minimal toxic side effects. Gypenoside L modulated cholesterol homeostasis, enhanced expression of inflammatory factors by regulating MHC I pathway-related proteins to augment anticancer immune responses. Clinical samples from HCC patients also exhibited high expression levels of MVA pathway-related genes in tumor tissues. These findings highlight gypenoside L as a promising agent for targeting cholesterol metabolism in HCC while emphasizing the effectiveness of regulating the SREBP2-HMGCS1 axis as a therapeutic strategy.

SREBP2-STARD4 is involved in synthesis of cholesteryl ester stimulated by mono-butyl phthalate in MLTC-1 cells.

Sterol is synthesized from cholesterol which is from the hydrolysis of stored cholesteryl esters. The process of maintaining cholesterol homeostasis is regulated by SREBP2-STARD4. Lots of researches demonstrated that male steroidogenesis could be interfered by di-n-butyl phthalate (DBP) or monobutyl phthalate (MBP). However, mechanisms of MBP exposure in this process have not been uncovered clearly. The objectiveof this study was to explore roles of SREBP2 and STARD4 in cholesteryl estersynthesis stimulated by MBP in mouse Leydig tumor cells (MLTC-1). MLTC-1 exposedto 10-8, 10-7, 10-6, 10-5 M MBP showed that levels of cholestery ester were increased significantly at 10-7 M MBP. Besides, cholesteryl ester synthesis stimulated by MBP was down-regulate when STARD4 or SREBP2 were inhibited. Activity of SREBP2 binding to the promoter of STARD4 was increased after MBP exposure. This study suggests that MBP can increase cholesteryl ester synthesis through SREBP2-STARD4 signal pathway in MLTC-1 cells.

Chitosan oligosaccharides enhance lipid droplets via down-regulation of PCSK9 gene expression in HepG2 cells.

Chitosan oligosaccharides (COS), linear polymers of N-acetyl-D-glucosamine and deacetylated glucosamine, exhibit diverse pharmacological effects such as antimicrobial, antitumor, antioxidant and anti-inflammatory activities. Here, we explored their hypocholesterolemic effects in vivo and the molecular mechanisms of COS in hepatic cells. Our in vivo study of dyslipidemic ApoE-/- male mice showed that COS treatment of 500 mg kg-1 d-1 for 4 weeks clearly reduced the lipid deposits in the aorta and significantly decreased the hepatic proprotein convertase subtilisin/kexin type 9 (PCSK9) protein levels versus HFD groups (p < 0.05). To elucidate the mechanisms behind these effects, the HepG2 cell line was treated with COS. We found that COS (200 µg/ml) increased the amount of cell-surface low-density lipoprotein receptor (LDLR) and enhanced the lipid droplets in HepG2 cells (p < 0.05). The mRNA levels of LDLR and HMG-CoA protein levels were not altered, and the mRNA levels of PCSK9 were down-regulated by COS treatment for 24 h. We also observed that the expression levels of SREBP-2 (125 kD) and HNF-1α were increased in total cell lysates, but nuclear SREBP-2 (nSREBP-2, 68 kD, the active subunit of SREBP-2) levels were decreased and FOXO3a levels increased in nuclear lysates after COS treatment for 24 h. We demonstrated that one of the reasons for regulation of lipid transfer with COS is that FOXO3a levels are up-regulated by COS, leading to a reduction in the PCSK9 promoter binding capacity of HNF-1α and thus suppressing PCSK9 gene expression, up-regulating LDLR levels, and enhancing the lipid droplets in HepG2 cells. In addition, decreased expression of the PCSK9 gene was also contributed to by down-regulation of SREBP-2 by COS. We further confirmed the effect of suppression of PCSK9 expression by COS by utilizing RNA interference to silence HNF-1α and SREBP-2. Finally, to the best of our knowledge, we are the first to demonstrate that PCSK9 expression and LDLR activity are synergistically changed by a combination of HNF-1α and SREBP-2 after COS treatment. Our findings indicate that COS may regulate PCSK9 to modulate hepatic LDLR abundance and activity.

miR-33a Mediates the Anti-Tumor Effect of Lovastatin in Osteosarcoma by Targeting CYR61.

BACKGROUND/AIMS: Preventing cell metastasis is an effective therapeutic strategy to treat osteosarcoma and improve prognosis. Statins have been found to have anticancer effects in addition to their cholesterol-lowering action. As a new target of statins, cysteine-rich 61 (CYR61) was recently identified to promote cell migration and metastasis in osteosarcoma. However, the underlying mechanisms mediating the regulation of CYR61 expression by statins remain unknown. METHODS: Human osteosarcoma cell lines MG63 and SaOS2 were used to clarify the effect of lovastatin on CYR61 expression. Real-time PCR was performed to detect mRNA or microRNA (miRNA) levels and western blot was performed to detect protein levels. Cell invasive ability was determined using Transwell assays. Lentivirus encoding CYR61 cDNA or sterol regulatory element-binding protein 2 (SREBP-2) shRNA was used to upregulate CYR61 expression or downregulate SREBP-2 expression. Binding of the CYR61 3' untranslated region (UTR) and miR-33a was analyzed by luciferase reporter assay. RESULTS: We found that lovastatin treatment decreased CYR61 expression, inhibited cell invasion and altered epithelial-to-mesenchymal-transition (EMT)-related protein expression, while CYR61 overexpression abolished the effect of lovastatin. Moreover, lovastatin increased the expression of SREBP-2 and miR-33a, which were then downregulated by SREBP-2 silencing. Bioinformatics analysis indicated that the CYR61 3'UTR harbored a potential miR-33a binding site and luciferase reporter assay demonstrated that CYR61 was a target of miR-33a in osteosarcoma cells. Furthermore, miR-33a could inhibit cell invasion and alter EMT-related protein expression. SREBP-2 silencing or miR-33a inhibitor upregulated CYR61 expression and reversed the effects of lovastatin on cell invasion and EMT-related proteins. CONCLUSION: Our findings suggest lovastatin suppresses osteosarcoma cell invasion through the SREBP-2/miR-33a/CYR61 pathway.

Novel osmotin inhibits SREBP2 via the AdipoR1/AMPK/SIRT1 pathway to improve Alzheimer's disease neuropathological deficits.

Extensive evidence has indicated that a high rate of cholesterol biogenesis and abnormal neuronal energy metabolism play key roles in Alzheimer's disease (AD) pathogenesis. Here, for we believe the first time, we used osmotin, a plant protein homolog of mammalian adiponectin, to determine its therapeutic efficacy in different AD models. Our results reveal that osmotin treatment modulated adiponectin receptor 1 (AdipoR1), significantly induced AMP-activated protein kinase (AMPK)/Sirtuin 1 (SIRT1) activation and reduced SREBP2 (sterol regulatory element-binding protein 2) expression in both in vitro and in vivo AD models and in Adipo-/- mice. Via the AdipoR1/AMPK/SIRT1/SREBP2 signaling pathway, osmotin significantly diminished amyloidogenic Aß production, abundance and aggregation, accompanied by improved pre- and post-synaptic dysfunction, cognitive impairment, memory deficits and, most importantly, reversed the suppression of long-term potentiation in AD mice. Interestingly, AdipoR1, AMPK and SIRT1 silencing not only abolished osmotin capability but also further enhanced AD pathology by increasing SREBP2, amyloid precursor protein (APP) and ß-secretase (BACE1) expression and the levels of toxic Aß production. However, the opposite was true for SREBP2 when silenced using small interfering RNA in APPswe/ind-transfected SH-SY5Y cells. Similarly, osmotin treatment also enhanced the non-amyloidogenic pathway by activating the α-secretase gene that is, ADAM10, in an AMPK/SIRT1-dependent manner. These results suggest that osmotin or osmotin-based therapeutic agents might be potential candidates for AD treatment.

Sterol regulatory element-binding proteins are transcriptional regulators of the thyroglobulin gene in thyroid cells.

The genes encoding sodium/iodide symporter (NIS) and thyroid peroxidase (TPO), both of which are essential for thyroid hormone (TH) synthesis, were shown to be regulated by sterol regulatory element-binding proteins (SREBP)-1c and -2. In the present study we tested the hypothesis that transcription of a further gene essential for TH synthesis, the thyroglobulin (TG) gene, is under the control of SREBP. To test this hypothesis, we studied the influence of inhibition of SREBP maturation and SREBP knockdown on TG expression in FRTL-5 thyrocytes and explored transcriptional regulation of the TG promoter by reporter gene experiments in FRTL-5 and HepG2 cells, gel shift assays and chromatin immunoprecipitation. Inhibition of SREBP maturation by 25-hydroxycholesterol and siRNA-mediated knockdown of either SREBP-1c or SREBP-2 decreased mRNA and protein levels of TG in FRTL-5 thyrocytes. Reporter gene assays with wild-type and mutated TG promoter reporter truncation constructs revealed that the rat TG promoter is transcriptionally activated by nSREBP-1c and nSREBP-2. DNA-binding assays and chromatin immunoprecipitation assays showed that both nSREBP-1c and nSREBP-2 bind to a SREBP binding motif with characteristics of an E-box SRE at position -63 in the rat TG promoter. In connection with recent findings that NIS and TPO are regulated by SREBP in thyrocytes the present findings support the view that SREBP are regulators of essential steps of TH synthesis in the thyroid gland such as iodide uptake, iodide oxidation and iodination of tyrosyl residues of TG. This moreover suggests that SREBP may be molecular targets for pharmacological modulation of TH synthesis.

Allyl isothiocyanate suppresses the proteolytic activation of sterol regulatory element-binding proteins and de novo fatty acid and cholesterol synthesis.

Sterol regulatory element-binding proteins (SREBPs) are a family of transcription factors that regulate lipid homeostasis by controlling the expression of genes involved in fatty acid and cholesterol synthesis. In this study, we used a stable cell line that expresses a luciferase reporter gene driven by an SRE-containing fatty acid synthase promoter to identify allyl isothiocyanate (AITC), one of the major isothiocyanates in cruciferous vegetables, as a novel SREBP inactivator. We found that AITC downregulated the proteolytic processing of SREBPs and the expression of their target genes in human hepatoma Huh-7 cells. Furthermore, AITC reduced the de novo synthesis of both fatty acids and cholesterol. Our results indicate a novel physiological function of AITC in lipid metabolism regulation.

Synergic hypocholesterolaemic effect of n-3 PUFA and oestrogen by modulation of hepatic cholesterol metabolism in female rats.

n-3 PUFA such as EPA and DHA as well as oestrogen have been reported to decrease blood levels of cholesterol, but their underlying mechanism is unclear. The purpose of this study was to determine the effects of the combination of n-3 PUFA supplementation and oestrogen injection on hepatic cholesterol metabolism. Rats were fed a modified AIN-93G diet with 0, 1 or 2 % n-3 PUFA (EPA+DHA) relative to the total energy intake for 12 weeks. Rats were surgically ovariectomised at week 8, and, after 1-week recovery, rats were injected with 17ß-oestradiol-3-benzoate (E2) or maize oil for the last 3 weeks. Supplementation with n-3 PUFA and E2 injection significantly increased the ratio of the hepatic expression of phosphorylated AMP activated protein kinase (p-AMPK):AMP activated protein kinase (AMPK) and decreased sterol regulatory element-binding protein-2, 3-hydroxy-3-methylglutaryl coenzyme A reductase and proprotein convertase subtilisin/kexin type 9. Supplementation with n-3 PUFA increased hepatic expression of cholesterol 7α-hydroxylase (CYP7A1), sterol 12α-hydroxylase (CYP8B1) and sterol 27-hydroxylase (CYP27A1); however, E2 injection decreased CYP7A1 and CYP8B1 but not CYP27A1. Additionally, E2 injection increased hepatic expression of oestrogen receptor-α and ß. In conclusion, n-3 PUFA supplementation and E2 injection had synergic hypocholesterolaemic effects by down-regulating hepatic cholesterol synthesis (n-3 PUFA and oestrogen) and up-regulating bile acid synthesis (n-3 PUFA) in ovariectomised rats.

Combination of curcumin and piperine prevents formation of gallstones in C57BL6 mice fed on lithogenic diet: whether NPC1L1/SREBP2 participates in this process?

BACKGROUND: A disruption of cholesterol homeostasis characterized by the physical-chemical imbalance of cholesterol solubility in bile often results in formation of cholesterol gallstones. Our earlier studies revealed that curcumin (1000 mg/kg) could prevent formation of gallstones. It has been proved that curcumin is poorly absorbed while piperine is a bioavailability-enhancer. Nevertheless, whether curcumin combined with piperine could enhance the effect of curcumin in preventing gallstones is still awaited. METHOD: C57BL6 mice were fed on a lithogenic diet concomitant with curcumin at 500 or 1000 mg/kg and/or piperine at 20 mg/kg for 4 weeks. The ratio of gallbladder stone formation was recorded and samples of blood, bile, gallbladder, liver and small intestine were also collected. The volume of gallbladder and weight of liver were calculated, and blood and bile samples were analyzed through biochemical methods. Intestinal NPC1L1 and SREBP2 mRNA and protein expression were detected by real-time PCR and Western blot. RESULT: Combining with piperine can significantly enhance the effect of curcumin, thus preventing the development of gallbladder stones, lowering the saturation of blood lipids and cholesterol in bile, as well as decreasing the expression of NPC1L1 and SREBP2 in both mRNA and protein levels. CONCLUSION: Curcumin can prevent the formation of cholesterol gallstones induced by high fat diet in mice and SREBP2 and NPC1L1 may participate in this process. Piperine can increase curcumin's bioavailability, thereby enhancing the effect of curcumin.

Cholesterol accumulation in prostate cancer: a classic observation from a modern perspective.

Prostate cancer (PCa) is the most common cancer in men in developed countries. Epidemiological studies have associated high blood-cholesterol levels with an increased risk of PCa, whilst cholesterol-lowering drugs (statins) reduce the risk of advanced PCa. Furthermore, normal prostate epithelial cells have an abnormally high cholesterol content, with cholesterol levels increasing further during progression to PCa. In this review, we explore why and how this occurs. Concurrent to this observation, intense efforts have been expended in cardiovascular research to better understand the regulators of cholesterol homeostasis. Here, we apply this knowledge to elucidate the molecular mechanisms driving the accumulation of cholesterol in PCa. For instance, recent evidence from our group and others shows that major signalling players in prostate growth and differentiation, such as androgens and Akt, modulate the key transcriptional regulators of cholesterol homeostasis to enhance cholesterol levels. This includes adjusting central carbon metabolism to sustain greater lipid synthesis. Perturbations in cholesterol homeostasis appear to be maintained even when PCa approaches the advanced, 'castration-resistant' state. Overall, this provides a link between cholesterol accumulation and PCa cell growth. Given there is currently no cure for castration-resistant PCa, could cholesterol metabolism be a novel target for PCa therapy? Overall, this review presents a picture that cholesterol metabolism is important for PCa development: growth-promoting factors stimulate cholesterol accumulation, which in turn presents a possible target for chemotherapy. Consequently, we recommend future investigations, both to better elucidate the mechanisms driving this accumulation and applying it in novel chemotherapeutic strategies.

Oxysterols synergize with statins by inhibiting SREBP-2 in ovarian cancer cells.

OBJECTIVE: Determine mechanisms responsible for enhanced statin efficacy in a novel statin combination we name STOX (STatin-OXysterol). METHODS: Ovarian cancer cell lines were treated with combinations of statins and oxysterols. Cell viability was determined by a modified MTT assay. Apoptosis was evaluated by immunoblotting of PARP and DAPI-mediated visualization of apoptotic nuclei. STOX effects on the expression of genes of the mevalonate pathway were assessed by real-time qPCR and immunoblotting. siRNA-mediated gene silencing was used to test the involvement of oxysterol-mediated repression of SREBP-2 in STOX synergy. The impact of statin-mediated inhibition of protein prenylation and on cholesterol homeostasis was evaluated. RESULTS: Oxysterols dramatically enhance cytotoxicity of statins in ovarian cancer cells through increased apoptosis. Decreased expression of SREBP-2 down-regulates the mevalonate pathway and prevents the active statin-induced sterol feedback, enhancing statin toxicity. Comparison of two ovarian cancer cell lines reveals two distinct mechanisms of statin induced toxicity, namely, dependence on protein geranylgeranylation and/or perturbation of cellular cholesterol levels. CONCLUSIONS: We provide evidence of statins' mechanisms of cytotoxicity in different ovarian cancer cells and discovered a new approach to significantly enhance the anti-tumor activity of statins. These observations provide a potential new path to improve statins as a treatment against ovarian cancer with obtainable dosages.

Targeting Cholesterol Biosynthesis with Statins Synergizes with AKT Inhibitors in Triple-Negative Breast Cancer.

Triple-negative breast cancer (TNBC) is responsible for a disproportionate number of breast cancer patient deaths due to extensive molecular heterogeneity, high recurrence rates, and lack of targeted therapies. Dysregulation of the phosphoinositide 3-kinase (PI3K)/AKT pathway occurs in approximately 50% of TNBC patients. Here, we performed a genome-wide CRISPR/Cas9 screen with PI3Kα and AKT inhibitors to find targetable synthetic lethalities in TNBC. Cholesterol homeostasis was identified as a collateral vulnerability with AKT inhibition. Disruption of cholesterol homeostasis with pitavastatin synergized with AKT inhibition to induce TNBC cytotoxicity in vitro in mouse TNBC xenografts and in patient-derived estrogen receptor (ER)-negative breast cancer organoids. Neither ER-positive breast cancer cell lines nor ER-positive organoids were sensitive to combined AKT inhibitor and pitavastatin. Mechanistically, TNBC cells showed impaired sterol regulatory element-binding protein 2 (SREBP-2) activation in response to single-agent or combination treatment with AKT inhibitor and pitavastatin, which was rescued by inhibition of the cholesterol-trafficking protein Niemann-Pick C1 (NPC1). NPC1 loss caused lysosomal cholesterol accumulation, decreased endoplasmic reticulum cholesterol levels, and promoted SREBP-2 activation. Taken together, these data identify a TNBC-specific vulnerability to the combination of AKT inhibitors and pitavastatin mediated by dysregulated cholesterol trafficking. These findings support combining AKT inhibitors with pitavastatin as a therapeutic modality in TNBC. Significance: Two FDA-approved compounds, AKT inhibitors and pitavastatin, synergize to induce cell death in triple-negative breast cancer, motivating evaluation of the efficacy of this combination in clinical trials.

ß-amyloid inhibits protein prenylation and induces cholesterol sequestration by impairing SREBP-2 cleavage.

Accumulation of ß-amyloid (Aß) inside brain neurons is an early and crucial event in Alzheimer's disease (AD). Studies in brains of AD patients and mice models of AD suggested that cholesterol homeostasis is altered in neurons that accumulate Aß. Here we directly investigated the role of intracellular oligomeric Aß(42) (oAß(42)) in neuronal cholesterol homeostasis. We report that oAß(42) induces cholesterol sequestration without increasing cellular cholesterol mass. Several features of AD, such as endosomal abnormalities, brain accumulation of Aß and neurofibrillary tangles, and influence of apolipoprotein E genotype, are also present in Niemann-Pick type C, a disease characterized by impairment of intracellular cholesterol trafficking. These common features and data presented here suggest that a pathological mechanism involving abnormal cholesterol trafficking could take place in AD. Cholesterol sequestration in Aß-treated neurons results from impairment of intracellular cholesterol trafficking secondary to inhibition of protein prenylation. oAß(42) reduces sterol regulatory element-binding protein-2 (SREBP-2) cleavage, causing decrease of protein prenylation. Inhibition of protein prenylation represents a mechanism of oAß(42)-induced neuronal death. Supply of the isoprenoid geranylgeranyl pyrophosphate to oAß(42)-treated neurons recovers normal protein prenylation, reduces cholesterol sequestration, and prevents Aß-induced neurotoxicity. Significant to AD, reduced levels of protein prenylation are present in the cerebral cortex of the TgCRND8 mouse model. In conclusion, we demonstrate a significant inhibitory effect of Aß on protein prenylation and identify SREBP-2 as a target of oAß(42), directly linking Aß to cholesterol homeostasis impairment.

A key regulator of cholesterol homoeostasis, SREBP-2, can be targeted in prostate cancer cells with natural products.

There is growing evidence showing that prostate cancer cells have perturbed cholesterol homoeostasis, accumulating cholesterol to promote cell growth. Consequently, cholesterol-lowering drugs such as statins are being evaluated in prostate cancer treatment. Furthermore, natural products such as betulin (from birch tree bark) and tocotrienol (a minor form of vitamin E) have been shown to lower cholesterol levels. Using these drugs and oxysterols, we have determined which aspects of cholesterol homoeostasis should be targeted in prostate cancer, e.g. cellular cholesterol levels are increased by the transcription factor SREBP-2 (sterol-regulatory-element-binding protein isoform 2), whereas LXR (liver X receptor) promotes cholesterol efflux. Whereas betulin exerted non-specific effects on cell viability, tocotrienols produced a strong direct correlation between SREBP-2 activity and cell viability. Mechanistically, tocotrienols lowered SREBP-2 activity by degrading mature SREBP-2 independently of the proteasome. In contrast, no correlation was seen between LXR activity and cell viability, implying that SREBP-2 is a better target than LXR for prostate cancer treatment. Lastly, androgen-dependent and -independent LNCaP cells were both sensitive to tocotrienols. Overall, this suggests that tocotrienols and other drugs targeting the SREBP-2 pathway are a potential therapeutic option for prostate cancer.

Acetoacetyl-CoA synthetase, a ketone body-utilizing enzyme, is controlled by SREBP-2 and affects serum cholesterol levels.

Ketone bodies have been regarded as an energy source that is mainly produced in the liver, and exported to extrahepatic tissues. However, ketone bodies have also been suggested to be used during the lipogenesis by the ketone body-utilizing enzyme, acetoacetyl-CoA synthetase (AACS). To elucidate the physiological role of AACS in the liver, we investigated the mechanism of transcription of the AACS gene and performed knockdown experiments. We showed that SREBP-2 regulates the expression of AACS and that knockdown of AACS in vivo, by the hydrodynamics method, resulted in the reduction of total blood cholesterol. These results suggest that ketone body metabolism via AACS activity plays an important role in cholesterol homeostasis.

Sterol-regulatory-element-binding protein 2 and nuclear factor Y control human farnesyl diphosphate synthase expression and affect cell proliferation in hepatoblastoma cells.

FDPS (farnesyl diphosphate synthase) catalyses the formation of farnesyl diphosphate, a key intermediate in the synthesis of cholesterol and isoprenylated cellular metabolites. FDPS is also the molecular target of nitrogen-containing bisphosphonates, which are used as bone-antiresorptive drugs in various disorders. In the present study, we characterized the sterol-response element and NF-Y (nuclear factor Y)-binding site in the human FDPS promoter. Using a luciferase assay, electrophoretic mobility-shift assay and chromatin immunoprecipitation assay, we demonstrated that these elements are responsible for the transcription of the FDPS gene, and that its transcriptional activation is mediated by SREBP-2 (sterol-regulatory-element-binding protein 2) and NF-Y. We also investigated whether sterol-mediated FDPS expression is involved in the cell proliferation induced by zoledronic acid, an FDPS inhibitor. We show that the SREBP-2- and NF-Y-mediated regulation of FDPS gene transcription modulates cell proliferation. These results suggest that SREBP-2 and NF-Y are required to trigger cell proliferation through the induction of FDPS expression and that the pharmacological action of zoledronic acid is involved in this pathway.

Design, synthesis, and biological evaluation of novel tetrahydroprotoberberine derivatives to reduce SREBPs expression for the treatment of hyperlipidemia.

Statins play an important role in the treatment of hyperlipidemia, but drug resistance and adverse effects greatly limits their application. To discover new lipid-lowering drugs, three different series of tetrahydroprotoberberine derivatives (THPBs) were designed and synthesized. These compounds were first tested for their effects on viability of HepG2 cells and 21 compounds with the percent of cell viability over 90% were further screened to evaluate their ability to reduce total cholesterol (TC) and triglyceride (TG) levels. Among these derivatives, two compounds displayed significant down-regulation both intracellular of TC and TG content, especially compound 49 exhibited the greatest efficacy. Mechanistically, compound 49 promoted proteasomal degradation of SREBPs. Importantly, compound 49 displayed superior bioavailability (F = 65.1%) and obvious efficacy in the treatment of high fat diet induced obesity in vivo. Therefore, compound 49 is a promising candidate to develop new treatment of hyperlipidemia.

O-GlcNAc inhibits interaction between Sp1 and sterol regulatory element binding protein 2.

O-linked N-acetylglucosamine (O-GlcNAc), a monosaccharide N-acetylglucosamine on the serine and threonine residues of nucleocytoplasmic proteins, is a novel protein modification that is ubiquitous among eukaryotes and implicated in cell regulation. Recent evidence indicates that O-GlcNAc regulates protein-protein interactions. Here we provide evidence that O-GlcNAc interrupts a known interaction between Sp1 and sterol regulatory element binding protein 2 (SREBP2), thereby inhibiting expression of the gene encoding acetyl-CoA synthetase 1, which is involved in lipid synthesis. This study suggests a novel mechanism in which lipid biosynthesis may be regulated by O-GlcNAc.

Pseudoprotodioscin inhibits SREBPs and microRNA 33a/b levels and reduces the gene expression regarding the synthesis of cholesterol and triglycerides.

The extract of Dioscorea zingiberensis C.H. Wright rhizomes is found to be effective in the therapy of cardiovascular disease. Steroidal saponins make substantial contribution. Previous study has proposed that methylprotodioscin (MP) may promote cholesterol efflux by increasing ABCA1 expression. But the other main saponins ingredients are not referred to. The aim of the present work was to reveal the effect and mechanism of protodioscin (PD), MP and pseudoprotodioscin (PPD) on the synthesis-related gene expression of cholesterol and triglycerides. MTT assay apoptosis assay with annexin AV-APC and 7-AAD double staining were performed. MicroRNA assay and qRT-PCR were used to analyze the gene expression which regulates synthesis of cholesterol and triglycerides. Western blot was to demonstrate the levels of target proteins. Cholesterol efflux assay was executed to study the stimulative effect of saponins on cholesterol efflux. In Hep G2 cells, PPD increased ABCA1 protein and mRNA levels, and promoted the effluxion of ApoA-1-mediated cholesterol. The underlying mechanisms involved that PPD inhibited SREBP1c and SREBP2 transcription by decreasing microRNA 33a/b levels. This procedure reciprocally led to the increase of ABCA1 levels. In THP-1 macrophages, PPD showed the similar effect, which reduced HMGCR, FAS and ACC mRNA levels and promoted low density lipoprotein receptor by decreasing the PCSK9 levels. These studies demonstrated that PPD is a potential agent for cholesterol efflux, SREBPs and microRNA 33a/b inhibition, which related to the gene expression for the synthesis of cholesterol and triglycerides.

A lipid-free and insulin-supplemented medium supports De Novo fatty acid synthesis gene activation in melanoma cells.

While investigating the role played by de novo lipid (DNL) biosynthesis in cancer cells, we sought a medium condition that would support cell proliferation without providing any serum lipids. Here we report that a defined serum free cell culture medium condition containing insulin, transferrin and selenium (ITS) supports controlled study of transcriptional regulation of de novo fatty acid (DNFA) production and de novo cholesterol synthesis (DNCS) in melanoma cell lines. This lipid-free ITS medium is able to support continuous proliferation of several melanoma cell lines that utilize DNL to support their lipid requirements. We show that the ITS medium stimulates gene transcription in support of both DNFA and DNCS, specifically mediated by SREBP1/2 in melanoma cells. We further found that the ITS medium promoted SREBP1 nuclear localization and occupancy on DNFA gene promoters. Our data show clear utility of this serum and lipid-free medium for melanoma cancer cell culture and lipid-related areas of investigation.