Inhibition Of Lipid Metabolism Research Articles

Apigenin inhibits lipid metabolism of hepatocellular carcinoma cells by targeting the histone demethylase KDM1A

BackgroundThe development of cancer is accompanied by metabolic reprogramming, and the liver serves as a central hub for lipid transportation. Apigenin, a plant-derived flavonoid, demonstrates potent anticancer properties across various cancer types and exhibits promising potential as a therapeutic agent for cancer treatment. However, there are limited studies focusing on the downstream targets of apigenin. Moreover, there are few reports on the impact of apigenin in lipid metabolism in liver cancer cells. PurposeThe objective is to elucidate the metabolic mechanism underlying the inhibitory effect of apigenin on liver cancer progression, search for downstream targets and provide reliable data support for the clinical trials of apigenin. MethodsAnticancer effects of apigenin were detected at cellular and molecular levels in vitro, and downstream targets of apigenin, especially metabolic pathway gene, were analyzed by transcriptome. Next, the downstream target of apigenin was verified and the biological function of the downstream target was examined. Finally, the downstream target of apigenin was further verified by restoring target gene expression. ResultsCellular molecular experiments showed that Apigenin inhibited the proliferation, migration, invasion and lipid metabolism of HCC cells. Transcriptome analysis showed apigenin widely regulate histone demethylase, especially, histone H3K4 lysine demethylase 1A (KDM1A). Apigenin treatment inhibits the expression of KDM1A protein and mRNA levels in liver cancer cells, molecular docking predicted the interaction between apigenin and KDM1A. Furthermore, downregulation KDM1A inhibited the proliferation and lipid metabolism of HCC cells, in the same way, overexpressing KDM1A promoted proliferation of HCC cells. Finally, restoring KDM1A expression partially attenuated the effects of apigenin on lipid metabolism in HCC cells. ConclusionIn conclusion, our study provides compelling evidence that apigenin inhibits liver cancer progression and elucidates its mechanism of action in regulating lipid metabolism. Specifically, we found that apigenin suppresses the progression of hepatocellular carcinoma (HCC) cells by downregulating genes involved in lipid metabolism. Additionally, our results indicate that KDM1A acts as a downstream target of apigenin in the inhibition of lipid metabolism in HCC. These findings offer experimental support for the potential use of apigenin as a therapeutic agent for liver cancer, highlighting its relevance in future clinical applications.

Serum LDL Promotes Microglial Activation and Exacerbates Demyelinating Injury in Neuromyelitis Optica Spectrum Disorder

Neuromyelitis optica spectrum disorder (NMOSD) is an autoimmune inflammatory demyelinating disease of the central nervous system (CNS) accompanied by blood-brain barrier (BBB) disruption. Dysfunction in microglial lipid metabolism is believed to be closely associated with the neuropathology of NMOSD. However, there is limited evidence on the functional relevance of circulating lipids in CNS demyelination, cellular metabolism, and microglial function. Here, we found that serum low-density lipoprotein (LDL) was positively correlated with markers of neurological damage in NMOSD patients. In addition, we demonstrated in a mouse model of NMOSD that LDL penetrates the CNS through the leaky BBB, directly activating microglia. This activation leads to excessive phagocytosis of myelin debris, inhibition of lipid metabolism, and increased glycolysis, ultimately exacerbating myelin damage. We also found that therapeutic interventions aimed at reducing circulating LDL effectively reversed the lipid metabolic dysfunction in microglia and mitigated the demyelinating injury in NMOSD. These findings shed light on the molecular and cellular mechanisms underlying the positive correlation between serum LDL and neurological damage, highlighting the potential therapeutic target for lowering circulating lipids to alleviate the acute demyelinating injury in NMOSD.

Aberrant Lipid Metabolic Signatures in Acute Myeloid Leukemia.

Leukemogenesis is a complex process that involves multiple stages of mutation in either hematopoietic stem or progenitor cells, leading to cancer development over time. Acute myeloid leukemia (AML) is an aggressive malignancy that affects myeloid cells. The major disease burden is caused by immature blast cells, which are eliminated using conventional chemotherapies. Unfortunately, relapse is a leading cause of death in AML patients, with 30%-80% experiencing it within 2 years of initial treatment. The dominant cause of relapse in leukemia is the presence of therapy-resistant leukemic stem cells (LSCs). These cells express genes related to stemness that are frequently difficult to eradicate and tend to survive standard treatments. Studies have demonstrated that by targeting the metabolic pathways of LSCs, it is possible to improve outcomes and extend the survival of those afflicted by leukemia. The overwhelming evidence suggests that lipid metabolism is reprogrammed in LSCs, leading to an increase in fatty acid uptake and de novo lipogenesis. Genes regulating this process also play a crucial role in therapy evasion. In this concise review, we summarize the lipid metabolism in normal hematopoietic cells, AML blast cells, and AML LSCs. We also compare the lipid metabolic signatures in de novo versus therapy-resistant AML blast and LSCs. We further discuss the metabolic switches, cellular crosstalk, potential targets, and inhibitors of lipid metabolism that could alleviate treatment resistance and relapse.

O098 Diclofenac alters expression of genes regulating muscle lipid metabolism during resistance exercise training in humans, but does not change IMCL content

Abstract Introduction Diclofenac is an NSAID commonly used off-label to reputedly enhance athletic training adaptation. Diclofenac activates PPAR-γ and upregulates the expression of lipid metabolism genes in vitro. This study investigated the impact of diclofenac administration on IMCL content and muscle genes regulating lipid metabolism during resistance exercise training in healthy volunteers. Methods Following informed consent and screening, 17 healthy, young, exercise-trained males matched for leg strength were allocated to Diclofenac (75mg diclofenac sodium daily, n=9) or Placebo (n=8) groups. Participants performed 5×30 sets of maximal isokinetic (90°/s) knee extensions with the non-dominant leg 3× weekly over 12 weeks. Vastus lateralis biopsies were obtained in the fasted state at baseline and 1, 7, 28, and 84 days of training for histochemical determination of IMCL content and muscle mRNA expression using microfluidic gene cards. IPA identified gene networks altered from baseline. Results There were significant changes in muscle mRNA expression levels associated with the upregulation of muscle lipid metabolism at all time points compared to baseline in Diclofenac. This was not evident in Placebo, where the inhibition of lipid metabolism was predicted by day 84. Mean (±SEM) IMCL content was no different between groups at baseline (7.4±3.8% Placebo vs. 6.9±2.6% Diclofenac), and no time/treatment interactions with resistance exercise training were observed (ηp2=0.07). Conclusion Diclofenac administration had a rapid, robust impact on genes regulating muscle lipid metabolism during 12 weeks of exercise training in humans. However, these responses were not associated with changes in IMCL content and further investigation is warranted.

Abstract 414: Targeting lipid metabolism to improve efficacy of braf-targeted therapy in colorectal cancer

Abstract Background: Aberrant lipid metabolism is associated with poor prognosis in colorectal cancer (CRC). Fatty acid synthase (FASN), a key enzyme of lipogenesis, is overexpressed and a therapeutic target in CRC. Overexpression of CD36, a fatty acid translocase, plays a pro-tumorigenic role in CRC. BRAFV600E is the mutation occurring in about 10-15% of CRC cases. BRAF-targeted therapy is effective, but quickly developed resistance is an issue. Our preliminary data show that development of resistance to BRAF-targeted therapy is associated with an increase in expression of FASN, CD36, accumulation of triglycerides (TGs), and mitochondrial respiration. Therefore, our central hypothesis is that inhibition of lipid metabolism will sensitize CRC cells to BRAF inhibitors and overcome acquired resistance. Methods: We established HT29 cells and primary PT130 and PT2449pt cells resistant to PLX8394, a novel BRAF inhibitor. IC50 curves, PrestoBlue viability, CytoSelect™ 24-Well Cell Invasion, and Triglyceride Assays, Seahorse XF analysis, western blot, and confocal microscopy were used to evaluate differences between parental and resistant cells. RNA-seq and lipid analysis were used to evaluate changes in gene expression and lipids levels. Combination of PLX8394 and TVB3664 (FASN inhibitor) was tested on cell viability in parental and resistant cells. Results: PLX8394 resistant cells have a higher IC50, an increased proliferation, and invasion compared to parental cells. An increase in invasion of resistant cells is associated with a decrease in E-cadherin. RNA sequencing shows a significant increase in FASN expression. Western blot on resistant cells confirms upregulation of FASN, CD36, and other lipogenic markers. Consistently, resistant cells have an increase in levels of triglycerides as compared to parental cells. Seahorse XF Cell Mito Stress Test shows that resistant cells forgo the Warburg effect and instead rely more heavily on oxidative phosphorylation. To further support our hypothesis, FASN shRNA knockdown of FASN in parental HT29 cells is more susceptible to PLX8394 treatment compared to control. We also observed the synergetic effect of PLX8394 and TVB3664 treatment on cell viability in parental cells, but not in resistant cells. Conclusion: Our study demonstrates that resistance to BRAF inhibitors is associated with a significant increase in proliferation, metastasis, and upregulation of lipid metabolism. We show that combination of FASN and BRAF inhibitors has a combinational effect on inhibiting cell viability in parental but not resistant cells, suggesting that the addition of FASN inhibitor to the standard regimen for BRAFV600E mutation positive patients can improve efficacy of these therapies. Additional screening of lipid metabolism-targeted therapies in combination with standard BRAF regimens are needed to develop novel and more efficacious strategies for CRC patients with BRAF mutations. Citation Format: Mariah E. Geisen, Dang he, Chi Wang, Jill M. Kolesar, Yekaterina Zaytseva. Targeting lipid metabolism to improve efficacy of braf-targeted therapy in colorectal cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 414.

Down-regulation of hepatic CLOCK by PPARα is involved in inhibition of NAFLD.

This work aimed to investigate the role of nuclear factor peroxisome proliferator-activated receptor α (PPARα) in modification of circadian clock and their relevance to development of nonalcoholic fatty liver disease (NAFLD). Both male wild-type (WT) and Pparα-null (KO) mice treated with high-fat diet (HFD) were used to explore the effect of PPARα and lipid diet on the circadian rhythm. WT, KO, and PPARα-humanized (hPPARα) mice were treated with PPARα agonist fenofibrate to reveal the hPPARα dependence of circadian locomotor output cycles kaput (CLOCK) down-regulation. The mouse model and hepatocyte experiments were designed to verify the action of PPARα in down-regulating CLOCK and lipid accumulation in vivo and in vitro. Strongest NAFLD developed in mice fed 45%HFD, and it was inhibited in WT mice. The activity rhythm of WT mice was found to be different from that of the KO mice on normal diet and HFD. The core circadian factor CLOCK was down-regulated by HFD in both WT and KO mice in the liver, not in the hypothalamus. More interestingly, hepatic CLOCK was down-regulated by basal PPARα and activated PPARα in dose dependence of fenofibrate. Accordingly, CLOCK down-regulation dependent of PPARα activity was involved in inhibition of lipid metabolism in hepatocytes. Down-regulation of hepatic CLOCK by basal PPARα contributes to tolerance against development of NAFLD. Inhibition of CLOCK by activated PPARα is involved in inhibition of NAFLD by PPARα agonists. KEY MESSAGES: •PPARα inhibited NAFLD development induced by HFD. •PPARα mediated modifications of circadian rhythm and the hepatic circadian factor CLOCK in NAFLD models. •Down-regulation of hepatic CLOCK by basal PPARα contributed to tolerance against development of NAFLD. •Inhibition of CLOCK by activated PPARα was involved in therapeutic actions against fatty liver diseases by PPARα agonists.

Lipophagy As a Mechanism of Resistance in T-ALL Following Energetic Crisis Induced By Oxphos/MCT1 Blockade: Strategies to Eradicate Residual Disease

Acidosis and hypoxia are common hallmarks of cancer associated with poor therapy response and dismal survival. Hypoxia can be due to high demand on oxygen, or due to reduced oxygen supply consequent to pharmacological inhibition of oxidative phosphorylation (OxPhos), causing switch to glycolysis and intracellular acidosis. Both acidosis and hypoxia are known to activate AMPK, stabilize HIF1α, that might orchestrate metabolic switch to alternative sources of energy such as lipophagy, an autophagic degradation of intracellular lipid droplets. We have previously shown that co-inhibition of mitochondrial Complex I and pyruvate-lactate transporter 1 (MCT1) in T-ALL cells is synergistic both in vitro and in vivo (Baran N, et al. ASH 2021). Here we dissect the mechanism of OxPhos/MCT1 blockade (OxPhos/MCT1-i), showing lipophagy as a mechanism of resistance to dual inhibition and provide therapeutic strategy to eradicate the residual disease. In T-ALL cell line models, Complex I/MCT1 blockade led to a significant inhibition of viability and decreased cell proliferation. These effects were accompanied by significant accumulation of reactive oxygen species (ROS) and mitochondrial superoxide, leading to DNA damage and apoptosis (shown by γ-H2AX and cleaved PARP). High level of ROS remained elevated despite co-treatment with ROS-scavengers: ascorbate, α-ketoglutarate, GSH or NAC. To elucidate the mechanisms of cell death, cells were co-treated with inhibitors of autophagy (3-methyladenine, Bafilomycin A, Chloroquine, Wortmannin), apoptosis (QVD-OPH, ZVAD-fmk) and ferroptosis (Ferrostatin1). While all inhibitors improved cell viability, suggesting a mixed contribution of apoptosis, autophagy and ferroptosis, none rescued elevated ROS caused by OxPhos/MCT1-i. Further impairment of mitochondria and Oxphos was evidenced by loss of mitochondrial membrane potential, mitochondria swelling, and reduction of mitochondria number, associated with markers of mitochondria fusion (elevated OPA1, MFN1 and DRP1). Significant reduction of ATP was confirmed by Seahorse and mass spectrometry (MS), indicating TCA cycle blockade and depletion of glutamine, cystathionine and glutathione, likely in response to ROS. ROS and ATP depletion are known to stabilize the hypoxia inducible factor 1 (HIF1α) that controls triglyceride accumulation. Results of GSEA and Western blotting confirmed that dual inhibition induced energetic crisis, activated LKB-AMPK pathway, inhibited mTOR pathway, upregulated fatty acids metabolism, which together with ROS accumulation caused HIF1α stabilization and switch to glycolysis (elevated ECAR by Seahorse and lactate by MS). Glycolysis however, impaired by MCT1 blockade, led to intracellular acidification, reflected by pH Rodo staining. This was associated with significant increase of a key component in lipid turnover acetyl-CoA, and by increased intensity of BODIPY dye, accompanied by increase in level of cellular lysosomes detected by Lysotracker dye, suggesting a possible shift of metabolic dependency toward lipophagy as a remaining source of energy. These results indicate that OxPhos/MCT1-i triggers storage of reduced lipids which subsequently undergo lysosomal degradation during lipophagy, constituting an alternative to OxPhos source of energy. To test if inhibition of lipid metabolism might abrogate the last energetic supply and eradicate residual leukemic cells, T-ALL cells were co-treated with OxPhos/MCT1-i together with Atorvastatin, Etomoxir and Valproate. Concomitant administration of Atorvastatin as well as other inhibitors with both OxPhos/MCT-i significantly reduced cell viability and induced apoptosis, supporting the key role of lipid metabolism in the resistance of T-ALL to dual inhibition. In summary, these results demonstrate that OxPhos/MCT1 inhibition forces switch to lipophagy, sensitizing cells to inhibitors of lipid metabolism, uncovering metabolic checkpoints that can ultimately translate into successful therapies in T-ALL and OxPhos-dependent malignancies. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Phenolic diterpenes from Rosemary supercritical extract inhibit non-small cell lung cancer lipid metabolism and synergise with therapeutic drugs in the clinic.

ClinicalTrials.gov Identifier NCT05080920.

Blue Light Deactivation of Catalase Suppresses Candida Hyphae Development Through Lipogenesis Inhibition.

Hyphae formation is a key step for fungal penetration into epithelial cells and escaping from macrophages or neutrophils. We found that 405 nm light-induced catalase deactivation results in the inhibition of hyphae growth in Candida albicans. The treatment is capable of inhibiting hyphae growth across multiple hyphae-producing Candida species. Metabolic studies on light-treated C. albicans reveal that light treatment results in a strong reduction in both lipid and protein metabolism. A significant decrease in unsaturated and saturated fatty acids was detected through mass spectroscopy, indicating that the suppression of hyphae through light-induced catalase deactivation may occur through inhibition of lipid metabolism. Initial in vivo tests indicate that blue light treatment can suppress the hyphae forming capabilities of C. albicans within murine abrasion infections. Together, these findings open new avenues for the treatment of Candida fungal infections by targeting their dimorphism.

Temporal analysis of melanogenesis identifies fatty acid metabolism as key skin pigment regulator.

Therapeutic methods to modulate skin pigmentation has important implications for skin cancer prevention and for treating cutaneous hyperpigmentary conditions. Towards defining new potential targets, we followed temporal dynamics of melanogenesis using a cell-autonomous pigmentation model. Our study elucidates 3 dominant phases of synchronized metabolic and transcriptional reprogramming. The melanogenic trigger is associated with high MITF levels along with rapid uptake of glucose. The transition to pigmented state is accompanied by increased glucose channelisation to anabolic pathways that support melanosome biogenesis. SREBF1-mediated up-regulation of fatty acid synthesis results in a transient accumulation of lipid droplets and enhancement of fatty acids oxidation through mitochondrial respiration. While this heightened bioenergetic activity is important to sustain melanogenesis, it impairs mitochondria lately, shifting the metabolism towards glycolysis. This recovery phase is accompanied by activation of the NRF2 detoxication pathway. Finally, we show that inhibitors of lipid metabolism can resolve hyperpigmentary conditions in a guinea pig UV-tanning model. Our study reveals rewiring of the metabolic circuit during melanogenesis, and fatty acid metabolism as a potential therapeutic target in a variety of cutaneous diseases manifesting hyperpigmentary phenotype.

Comparative Proteomics Reveals Evidence of Enhanced EPA Trafficking in a Mutant Strain of Nannochloropsis oculata.

The marine microalga Nannochloropsis oculata is a bioproducer of eicosapentaenoic acid (EPA), a fatty acid. EPA is incorporated into monogalactosyldiacylglycerol within N. oculata thylakoid membranes, and there is a biotechnological need to remodel EPA synthesis to maximize production and simplify downstream processing. In this study, random mutagenesis and chemical inhibitor-based selection method were devised to increase EPA production and accessibility for improved extraction. Ethyl methanesulfonate was used as the mutagen with selective pressure achieved by using two enzyme inhibitors of lipid metabolism: cerulenin and galvestine-1. Fatty acid methyl ester analysis of a selected fast-growing mutant strain had a higher percentage of EPA (37.5% of total fatty acids) than the wild-type strain (22.2% total fatty acids), with the highest EPA quantity recorded at 68.5 mg/g dry cell weight, while wild-type cells had 48.6 mg/g dry cell weight. Label-free quantitative proteomics for differential protein expression analysis revealed that the wild-type and mutant strains might have alternative channeling pathways for EPA synthesis. The mutant strain showed potentially improved photosynthetic efficiency, thus synthesizing a higher quantity of membrane lipids and EPA. The EPA synthesis pathways could also have deviated in the mutant, where fatty acid desaturase type 2 (13.7-fold upregulated) and lipid droplet surface protein (LDSP) (34.8-fold upregulated) were expressed significantly higher than in the wild-type strain. This study increases the understanding of EPA trafficking in N. oculata, leading to further strategies that can be implemented to enhance EPA synthesis in marine microalgae.

Quercetin Attenuates Osteoporosis in Orchiectomy Mice by Regulating Glucose and Lipid Metabolism via the GPRC6A/AMPK/mTOR Signaling Pathway.

Quercetin, a flavonoid found in natural medicines, has shown a role in disease prevention and health promotion. Moreover, because of its recently identified contribution in regulating bone homeostasis, quercetin may be considered a promising agent for improving bone health. This study aimed to elucidate the role of quercetin in androgen deprivation therapy-induced osteoporosis in mice. C57BL/6 mice were subjected to orchiectomy, followed by quercetin treatment (75 and 150 mg/kg/d) for 8 weeks. Bone microstructure was then assessed by micro-computed tomography, and a three-point bending test was used to evaluate the biomechanical parameters. Hematoxylin and eosin (H&E) staining was used to examine the shape of the distal femur, gastrocnemius muscle, and liver. The balance motion ability in mice was evaluated by gait analysis, and changes in the gastrocnemius muscle were observed via Oil red O and Masson’s staining. ELISA and biochemical analyses were used to assess markers of the bone, glucose, and lipid metabolism. Western blotting analyses of glucose and lipid metabolism-related protein expression was performed, and expression of the GPCR6A/AMPK/mTOR signaling pathway-related proteins was also assessed. After 8 weeks of quercetin intervention, quercetin-treated mice showed increased bone mass, bone strength, and improved bone microstructure. Additionally, gait analysis, including stride length and frequency, were significantly increased, whereas a reduction of the stride length and gait symmetry was observed. H&E staining of the gastrocnemius muscle showed that the cross-sectional area of the myofibers had increased significantly, suggesting that quercetin improves balance, motion ability, and muscle mass. Bone metabolism improvement was defined by a reduction of serum levels of insulin, triglycerides, total cholesterol, and low-density lipoprotein, whereas levels of insulin-like growth factor-1 and high-density lipoprotein were increased after quercetin treatment. Expression of proteins involved in glucose uptake was increased, whereas that of proteins involved in lipid production was decreased. Moreover, the GPRC6A and the phospho-AMPK/AMPK expression ratio was elevated in the liver and tibia tissues. In contrast, the phospho-mTOR/mTOR ratio was reduced in the quercetin group. Our findings indicate that quercetin can reduce the osteoporosis induced by testosterone deficiency, and its beneficial effects might be associated with the regulation of glucose metabolism and inhibition of lipid metabolism via the GPCR6A/AMPK/mTOR signaling pathway.

Novel Oncogenic Non-Coding RNA：circRIC8B Regulates Lipid Metabolism Via Mir-199b-5p /LPL Axis in Chronic Lymphocytic Leukemia

Objective:During tumor development, energy constraints caused by malnourished microenvironments could exert selective pressure on cancer cells. Tumor cells are driven to metabolic reprogramming to meet the increased demand for energy and metabolites for their rapid proliferation and survival. Chronic lymphocytic leukemia (CLL) is a disease with about 1% of CLL cells proliferating every day which is highly than commonly thought. CLL cells were reported to maintain high levels of proliferation through metabolic changes, but extensive studies did not clearly explain the underlying mechanism of driving genes in CLL metabolism. Circular RNA (circRNA) has recently been shown to play an important role in cell metabolism through lipid accumulation. The purpose of this study is to explore the role of circRNA in lipid metabolism of CLL and provide novel therapeutic targets for CLL.Methods:To analyze circRNAs expression profiles and metabolism map in CLL, peripheral blood mononuclear cells (PBMC) from 53 treatment-naïve CLL patients were collected for transcriptome sequencing. Candidate circRNA circRIC8B in a larger cohort of patients was validated and the clinical characteristics were analyzed. Overexpression and knockdown virus were constructed to infect CLL cells, and untargeted metabolomics was used to find the key lipid metabolic pathway modulating by circRIC8B. The oncogenic functions of circRIC8B were further measured in CLL cell lines (MEC-1 and JVM-3) by performing CCK8 assay, flow cytometry, nile red staining and triglyceride detection. Moreover, we explored the molecular mechanisms of circRIC8B and verified the interactions among circRIC8B, miR-199b-5p and LPL by performing RNA-FISH, RIP, dual-luciferase reporter assay and Western blotting. The killing effects of lipid metabolism inhibitors on CLL cells were detected by CCK8 and flow cytometry.ResultsTranscriptome analysis showed that abnormal lipid metabolism was significantly related to the survival and prognosis of patients with CLL, and circRNAs could be involved in the regulation of lipid metabolism. Kaplan-Meier survival analysis confirmed that patients with higher fatty acid biosynthesis had a significantly lower OS (Figure 1A-B). circRIC8B which is positively correlated with the expression of lipoprotein lipase (LPL) was finally selected for further investigation. qRT-PCR analysis showed that circRIC8B was significantly higher expressed in CLL compared with healthy donors. Moreover, consistent with the sequencing results, circRIC8B was positively correlated with LPL and highly relevant to IGHV region mutation status, which has long been considered as an important prognostic indicator of CLL (Figure 1C). Patients with higher circRIC8B level are associated with worse survival and advanced disease progression (Figure 1D and E). LC-MS/MS results showed that circRIC8B are able to modulated lipid metabolism of CLL cells. Functional analysis demonstrated the promoting role of circRIC8B in cell proliferation. Nile red staining showed lipid accumulation in CLL cells with circRIC8B overexpression increased significantly, while lipid accumulation in circRIC8B knockdown cells decreased significantly, and the quantitative results of triglycerides were similar.Next, we unraveled an original mechanism in CLL that up-regulated circRIC8B was mainly enriched in the cytoplasm, acted as a “sponge” of miR-199b-5p. CCK8 assay, nile red staining showed that the cell viability and lipid accumulating of CLL cell lines were evidently decreased after RNAi of circRIC8B and this result could be reversed by miR-199b-5p inhibitor transfection (Figure 1F-H). In addition, ezetimibe, one of the inhibitors of lipid metabolism was found effectively inhibit the proliferation and promote apoptosis of CLL cells.ConclusionsIn conclusion, as an independent prognostic factor of CLL, circRIC8B was involved in the progress of CLL disease through the miR-199b-5p/LPL axis. In addition, circRIC8B is a key factor in regulating lipid accumulation in CLL, resulting in significant changes in cellular lipid storage, thus supporting the proliferation of CLL cells. Metabolic inhibitor Ezetimibe can effectively block this process and exert anti-tumor functions. This study provides new clues for the role of circRNA in abnormal lipid metabolism of CLL and novel therapeutic strategy for CLL patients. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Diacylglycerol kinase and phospholipase D inhibitors alter the cellular lipidome and endosomal sorting towards the Golgi apparatus

The membrane lipids diacylglycerol (DAG) and phosphatidic acid (PA) are important second messengers that can regulate membrane transport by recruiting proteins to the membrane and by altering biophysical membrane properties. DAG and PA are involved in the transport from the Golgi apparatus to endosomes, and we have here investigated whether changes in these lipids might be important for regulation of transport to the Golgi using the protein toxin ricin. Modulation of DAG and PA levels using DAG kinase (DGK) and phospholipase D (PLD) inhibitors gave a strong increase in retrograde ricin transport, but had little impact on ricin recycling or degradation. Inhibitor treatment strongly affected the endosome morphology, increasing endosomal tubulation and size. Furthermore, ricin was present in these tubular structures together with proteins known to regulate retrograde transport. Using siRNA to knock down different isoforms of PLD and DGK, we found that several isoforms of PLD and DGK are involved in regulating ricin transport to the Golgi. Finally, by performing lipidomic analysis we found that the DGK inhibitor gave a weak, but expected, increase in DAG levels, while the PLD inhibitor gave a strong and unexpected increase in DAG levels, showing that it is important to perform lipidomic analysis when using inhibitors of lipid metabolism.

Crocetin and crocin decreased cholesterol and triglyceride content of both breast cancer tumors and cell lines.

Inhibition of lipid metabolism in breast cancer has been suggested as an effective approach for cancer therapy. Saffron-derived crocetin (Crt) and crocin (Cro) with the known anticancer activity, have shown hypolipidemic effect in diabetes and atherosclerosis. Here, we investigated the effect of Crt/Cro on lipid content in breast cancer. A multi-model approach involving in vivo, in vitro and in silico studies was applied. The 4T1-induced breast cancer in mice was used to investigate the effect of Crt/Cro on cholesterol (Chl) and triglyceride (TG) levels in serum and tumor tissues. The Chl/TG levels were also assessed in the cytosol of MDA-MB-231 and MCF-7 breast cancer cell lines 6, 12 and 24 hr after Crt/Cro treatment. The interaction between Crt/Cro and hydroxymethylglutaryl coenzyme A reductase (HMGCR) was also computed by docking analysis. Crt reduced both serum (p=0.003) and tumor (p=0.011) Chl and TG (p=0.001) levels in mice. Cro reduced TG levels in tumor (p=0.014) and serum (p=0.002) and Chl level in tumor (p=0.013) tissues. Crt reduced both Chl and TG in MDA-MB-231 (p=0.014 and p=0.002, respectively) and MCF-7 (p=0.014 and p=0.002, respectively), after 24 h. Cro reduced both Chl and TG in MDA-MB-231 (p=0.014 and p=0.002, respectively) and MCF-7 (p=0.014 and p=0.002, respectively), after 24 h. Crt binds to the active site of HMGCR with higher affinity (ΔG0=-6.6 kcal/mol) than simvastatin (ΔG0 =-6.0 kcal/mol). Crt and Cro effectively decreased Chl/TG content in the sera of tumor bearing mice, in breast tumors and breast cancer cell lines. Crt showed a higher hypolipidemic potential than Cro. In silico analysis indicated Crt binding in the HMGCR active site.

Abstract 1276: Inhibition of lipid metabolism decreases proliferation and increases radiosensitvity in EGFR-TKI resistant NSCLC cells with bFGF secretion

Abstract Primary and acquired EGFR-TKIs resistance restricts the benefit of EGFR-TKIs to EGFR mutant NSLCLC patients. Recent studies found that metabolic reprogramming might contribute to develop metastasis and drug resistance of tumor cells. Therefore, illustrating the metabolism change is meaningful and targeting the peculiar metabolic pathways might be considered as potential therapeutic strategies for EGFR-TKIs resistant NSCLC. We separated single clones from gefitnib resistant NSCLC cells, by exposing EGFR-mutant EGFR-TKIs sensitive PC-9 cells into increasing concentrations of gefitnib. Among the numerous clones, clone 5 was resistant to gefitinib or AZD9291 and named as PC-9GR-FGF because of high level of bFGF secretion. We found that bFGF promote the transcription of SREBP-1 and its downstream genes, acetyl-CoA carboxylase alpha (ACC), ATP citrate lyase (ACLY) and fatty acid synthase (FASN) in PC-9 cells. Compared to PC-9 cells, higher ACC expression was detected in PC-9GR-FGF cells. Basing on TCGA database (lung Adenocarcinoma, provisional), patients with high ACC expression showed significant shorter PFS (progression-free survival) and OS (overall survival) compared to those with low ACC expression in the EGFR-mutant NSCLC (p<0.05, N=32). Down-regulation of ACC by siRNA or TOFA, a small molecular ACC inhibitor, mederately decreased the proliferation of PC-9 cells whereas strongly decreased proliferation of PC-9GR-FGF cells in vitro and in vivo. Moreover, Reactive oxygen species (ROS) and oxidative stress scavenger, NAC and ferrostatin-1, failed to reverse the effect of ACC inhibition in PC-9 and PC-9GR-FGF cells. Furthermore, after down-regulation or inhibition of ACC, stronger G2/M arrest and more accumulation of DNA damage marker, γH2AX, were found in PC-9GR-FGF cells compared with PC-9 cells. In addition, ACC expression were increased after irradiation of X-ray in PC-9GR-FGF cells, and inhibition of ACC increased accumulation of γH2AX induced by X-rays in PC-9GR-FGF cells. Our research gives a view sight into lipid metabolism changes in EGFR-mutant NSCLC cells before and after acquired EGFR-TKIs resistance, and explores the possibility of combination strategy with radiotherapy and anti-cancer lipid metabolism therapy. Citation Format: Wei Wang, Shunli Peng, Rong Wang, yueyun Ma, Longhui Zhong. Inhibition of lipid metabolism decreases proliferation and increases radiosensitvity in EGFR-TKI resistant NSCLC cells with bFGF secretion [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1276.

Fenofibrate induces human hepatoma Hep3B cells apoptosis and necroptosis through inhibition of thioesterase domain of fatty acid synthase

This study demonstrated that fenofibrate, a lipid-lowering drug, induced a significant time-dependent cytotoxicity of hepatoma Hep3B cells. Hep3B cells are significantly more sensitive to cell killing by fenofibrate than hepatoma HepG2, lung cancer CH27 and oral cancer HSC-3 cells. From the result of docking simulation, fenofibrate can bind excellently to the thioesterase domain of fatty acid synthase (FASN) binding site as orlistat, a FASN inhibitor, acts. The fenofibrate-induced cell cytotoxicity was protected by addition of palmitate, indicating that the cytotoxic effect of fenofibrate is due to starvation of Hep3B cells by inhibiting the formation of end product in the FASN reaction. Inhibition of lipid metabolism-related proteins expression, such as proteins containing thioesterase domain and fatty acid transport proteins, was involved in the fenofibrate-induced Hep3B cell death. Fenofibrate caused S and G2/M cell cycle arrest by inducing cyclin A/Cdk2 and reducing cyclin D1 and E protein levels in Hep3B cells. The anti-tumor roles of fenofibrate on Hep3B cells by inducing apoptosis and necroptosis were dependent on the expression of Bcl-2/caspase family members and RIP1/RIP3 proteins, respectively. These results suggest that fenofibrate has an anti-cancer effect in Hep3B cells and inhibition of lipid metabolism may be involved in fenofibrate-induced Hep3B cells apoptosis and necroptosis.

Catechins Green Tea GMB4 Clone Increases mRNA of ABCA1 through LXR Signaling in Cultured Macrophage Exposed OX-LDL

Inhibition of atherogenesis through inhibition of lipid metabolism has not been explored while other inhibitions through inflammation, endothelial dysfunction and free radicals have done. Inhibition of atherogenesis via inhibition of lipid metabolism can be done through the mechanism of Reverse Cholesterol Transport (RCT). Signaling pathways that play a role in this mechanism is LXR signaling. LXR activation by an LXR agonist led increasing cholesterol efflux. Catechins based on bioinformatics study showed as a potent candidate LXR agonists that can be used as an inhibitor of atherogenesis. This study aims to prove that the administration Catechins green tea Clone GMB4 can prevent atherosclerosis through increasing mechanism cholesterol efflux from macrophage by taking effect of ABCA1, ABCG1, SRB1 gene expression in cultured macrophages were exposed ox-LDL. Long-term goals of the outcome of the research are the use of Catechins Green Tea Clones GMB4 as an inhibitor of atherogenesis so that it can be used as a complementary therapy for the treatment of atherosclerosis and cardiovascular diseases. The research is divided into 5 groups, namely the culture of macrophages without exposed ox-LDL, culture exposed ox-LDL and groups of Catechins dose I, II, III. In vitro study showed that administration of Catechins increases mRNA of ABCA1, whereas mRNA ABCG1 and SRB1 decreased at all three doses given. The result of protein profilling was identified a protein with a molecular weight of 70 kDa by SDS-PGE with silver staining.

Abstract 1674: Novel direct AMPK activator suppresses non-small cell lung cancer through inhibition of lipid metabolism

Abstract Lung cancer is the leading cause of cancer mortality. While non-small cell lung cancer (NSCLC) is the most common histological type of lung cancer and dominates almost 85% of all lung cancer cases. Conventional therapy, such as surgery, chemotherapy, or radiotherapy, scarify normal cells during the curing progress are becoming unfavorable. To avoid harmful side-effect on normal cell, target-based therapy is the mainstream of modern cancer therapy. For example, gefitinib is a clinically-marketed drug which acts as tyrosine kinase inhibitor to inhibit epidermal growth factor receptor (EGFR) activating signaling pathway. For the NSCLC patients who harbor activating substitution from leucine to arginine at amino acid 858 (L858R) point mutation and in-frame exon 19 deletion on EGFR, response well to gefitinib and these two common mutation are used a biomarkers for gefitinib prescription. However, gefitinib-resistance commonly happens due to further substitution mutation from threonine to methionine at amino acid position 790 (T790M) occurred after one year or less gefitinib treatment. Therefore, developing new treatment strategy to tackle gefitinib-resistance is urgently. Abnormal energy metabolism is accepted as new cancer hallmark. Recently, a metabolism rate-limiting enzyme 5'-adenosine menophosphate-activated protein kinase (AMPK) has become a promising anti-cancer target. In this study, we have applied computational docking to perform in silico virtual screening, and we have identified a novel direct AMPK agonist, D561-0775 from a compound library. Using enzyme activation assay, we demonstrated that D561-0775 activates directly on AMPK α subunit. In addition, D561-0775 exhibited significant inhibitory effect on gefitinib-resistant NSCLC cell lines but is less cytotoxicity on normal lung fibroblast cells. D561-0775 also activates AMPK/mTOR signaling pathway, resulting in induction of apoptosis, cell cycle arrest, suppressing glycolysis and cholesterol synthesis in gefitinib-resistant H1975 lung cancer cells. Taken together, D561-0775 has provided clue on a new chemical structure that could be developed as cancer drug for gefitinib-resistant NSCLC patients through inhibition lipid metabolism by directly targeting at AMPK directly. Acknowledgments: This work was supported by Macau Science and Technology Development Fund (Project No: 021/2013/A1, 074/2011/A3 & 082/2013/A3) Citation Format: Xi Chen, Chun Xie, Xing Xing Fan, Ze Bo Jiang, Vincent Wong, Jia Hui Xu, Xiao Jun Yao, Liang Liu, Elaine L. Leung. Novel direct AMPK activator suppresses non-small cell lung cancer through inhibition of lipid metabolism [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1674.

Label-free quantitative proteomic profiling of colon cancer cells identifies acetyl-CoA carboxylase alpha as antitumor target of Citrus limon-derived nanovesicles

We have previously isolated exosome-like nanoparticles from Citrus-limon juice, able to inhibit in vitro and in vivo tumor cell growth. In order to deeply understand the mechanism underlying nanovesicle effects, we performed a proteomic profile of treated colorectal cancer cells. Among the proteins differentially expressed after nanovesicle treatment, we found a significant downregulation of the Acetyl-CoA Carboxylase 1 (ACACA) and we demonstrated that silencing ACACA in cancer cells leads to a reduction of cell growth. Our study proved that the anti-tumor effects of Citrus-limon nanovesicles is partly mediated by lipid metabolism inhibition, in particular via ACACA downregulation. SignificanceThis study represents the attempt to achieve, by a proteomic approach, a better understanding of the role of lemon nanovesicles in affecting colorectal cancer cell growth.