Single-cell genomic and transcriptomic landscapes of primary and metastatic colorectal cancer tumors.

BACKGROUND: Colorectal cancer (CRC) ranks as the second-leading cause of cancer-related death worldwide with metastases being the main cause of cancer-related death. Here, we investigated the genomic and transcriptomic alterations in matching adjacent normal tissues, primary tumors, and metastatic tumors of CRC patients. METHODS: We performed whole genome sequencing (WGS), multi-region whole exome sequencing (WES), simultaneous single-cell RNA-Seq, and single-cell targeted cDNA Sanger sequencing on matching adjacent normal tissues, primary tumors, and metastatic tumors from 12 metastatic colorectal cancer patients (n=84 for genomes, n=81 for exomes, n=9120 for single cells). Patient-derived tumor organoids were used to estimate the anti-tumor effects of a PPAR inhibitor, and self-renewal and differentiation ability of stem cell-like tumor cells. RESULTS: We found that the PPAR signaling pathway was prevalently and aberrantly activated in CRC tumors. Blocking of PPAR pathway both suppressed the growth and promoted the apoptosis of CRC organoids in vitro, indicating that aberrant activation of the PPAR signaling pathway plays a critical role in CRC tumorigenesis. Using matched samples from the same patient, distinct origins of the metastasized tumors between lymph node and liver were revealed, which was further verified by both copy number variation and mitochondrial mutation profiles at single-cell resolution. By combining single-cell RNA-Seq and single-cell point mutation identification by targeted cDNA Sanger sequencing, we revealed important phenotypic differences between cancer cells with and without critical point mutations (KRAS and TP53) in the same patient in vivo at single-cell resolution. CONCLUSIONS: Our data provides deep insights into how driver mutations interfere with the transcriptomic state of cancer cells in vivo at a single-cell resolution. Our findings offer novel knowledge on metastatic mechanisms as well as potential markers and therapeutic targets for CRC diagnosis and therapy. The high-precision single-cell RNA-seq dataset of matched adjacent normal tissues, primary tumors, and metastases from CRCs may serve as a rich resource for further studies.

The pleiotropic peroxisome proliferator activated receptors: Regulation and therapeutics.

The Peroxisome proliferator-activated receptors (PPARs) are key regulators of metabolic events in our body. Owing to their implication in maintenance of homeostasis, both PPAR agonists and antagonists assume therapeutic significance. Understanding the molecular mechanisms of each of the PPAR isotypes in the healthy body and during disease is crucial to exploiting their full therapeutic potential. This article is an attempt to present a rational analysis of the multifaceted therapeutic effects and underlying mechanisms of isotype-specific PPAR agonists, dual PPAR agonists, pan PPAR agonists as well as PPAR antagonists. A holistic understanding of the mechanistic dimensions of these key metabolic regulators will guide future efforts to identify novel molecules in the realm of metabolic, inflammatory and immunotherapeutic diseases.

Modulation of PPAR signaling disrupts pancreas development in the zebrafish, Danio rerio.

Peroxisome Proliferator Activated Receptors (PPARs) are transcription factors that regulate processes such as lipid and glucose metabolism. Synthetic PPAR ligands, designed as therapeutics for metabolic disease, provide a tool to assess the relationship between PPAR activity and pancreas development in vivo, an area that remains poorly characterized. Here, we aim to assess the effects of PPAR agonists and antagonists on gene expression, embryonic morphology and pancreas development in transgenic zebrafish embryos. To evaluate developmental perturbations, we assessed gross body and pancreas morphology at 4 days post fertilization (dpf) in response to developmental exposures with PPARα, PPARγ, and PPARß/δ agonists and antagonists at 0, 0.01, 0.1, 1, and 10 µM concentrations. All ligand exposures, with the exception of the PPARα agonist, resulted in significantly altered fish length and yolk sac area. PPARγ agonist and antagonist had higher incidence of darkened yolk sac and craniofacial deformities, whereas PPARα antagonist had higher incidence of pericardial edema and death. Significantly reduced endocrine pancreas area was observed in both PPARγ ligands and PPARα agonist exposed embryos, some of which also exhibited aberrant endocrine pancreas morphology. Both PPARß/δ ligands caused reduced exocrine pancreas length and novel aberrant phenotype, and disrupted gene expression of pancreatic targets pdx1, gcga, and try. Lipid staining was performed at 8 dpf and revealed altered lipid accumulation consistent with isoform function. These data indicate chronic exposure to synthetic ligands may induce morphological and pancreatic defects in zebrafish embryos.

Metabolomic analysis of a selective ABCA1 inducer in obesogenic challenge provides a rationale for therapeutic development.

BACKGROUND: Therapeutic agents with novel mechanisms of action are needed to combat the growing epidemic of type 2 diabetes (T2D) and related metabolic syndromes. Liver X receptor (LXR) agonists possess preclinical efficacy yet produce side effects due to excessive lipogenesis. Anticipating that many beneficial and detrimental effects of LXR agonists are mediated by ABCA1 and SREPB1c expression, respectively, we hypothesized that a phenotypic optimization strategy prioritizing selective ABCA1 induction would identify an efficacious lead compound with an improved side effect profile over existing LXRß agonists. METHODS: We synthesized and characterized a novel small molecule for selective induction of ABCA1 vs. SREBP1c in vitro. This compound was evaluated in both wild-type mice and a high-fat diet (HFD) mouse model of obesity-driven diabetes through functional, biochemical, and metabolomic analysis. FINDINGS: Six weeks of oral administration of our lead compound attenuated weight gain, glucose intolerance, insulin signaling deficits, and adiposity. Global metabolomics revealed suppression of gluconeogenesis, free fatty acids, and pro-inflammatory metabolites. Target identification linked these beneficial effects to selective LXRß agonism and PPAR/RXR antagonism. INTERPRETATION: Our observations in the HFD model, combined with the absence of lipogenesis and neutropenia in WT mice, support this novel approach to therapeutic development for T2D and related conditions.

Relevance of Peroxisome Proliferator Activated Receptors in Multitarget Paradigm Associated with the Endocannabinoid System.

Cannabinoids have shown to exert their therapeutic actions through a variety of targets. These include not only the canonical cannabinoid receptors CB1R and CB2R but also related orphan G protein-coupled receptors (GPCRs), ligand-gated ion channels, transient receptor potential (TRP) channels, metabolic enzymes, and nuclear receptors. In this review, we aim to summarize reported compounds exhibiting their therapeutic effects upon the modulation of CB1R and/or CB2R and the nuclear peroxisome proliferator-activated receptors (PPARs). Concomitant actions at CBRs and PPARα or PPARγ subtypes have shown to mediate antiobesity, analgesic, antitumoral, or neuroprotective properties of a variety of phytogenic, endogenous, and synthetic cannabinoids. The relevance of this multitargeting mechanism of action has been analyzed in the context of diverse pathologies. Synergistic effects triggered by combinatorial treatment with ligands that modulate the aforementioned targets have also been considered. This literature overview provides structural and pharmacological insights for the further development of dual cannabinoids for specific disorders.

Peroxisome proliferator-activated receptors in the pathogenesis and therapies of liver fibrosis.

Liver fibrosis is a dynamic wound-healing process associated with the deposition of extracellular matrix produced by myofibroblasts. HSCs activation, inflammation, oxidative stress, steatosis and aging play critical roles in the progression of liver fibrosis, which is correlated with the regulation of the peroxisome proliferator-activated receptor (PPAR) pathway. As nuclear receptors, PPARs reduce inflammatory response, regulate lipid metabolism, and inhibit fibrogenesis in the liver associated with aging. Thus, PPAR ligands have been investigated as possible therapeutic agents. Mounting evidence indicated that some PPAR agonists could reverse steatohepatitis and liver fibrosis. Consequently, targeting PPARs might be a promising and novel therapeutic option against liver fibrosis. This review summarizes recent studies on the role of PPARs on the pathogenesis and treatment of liver fibrosis.

The Rosmarinus Bioactive Compound Carnosic Acid Is a Novel PPAR Antagonist That Inhibits the Browning of White Adipocytes.

Thermogenic brown and brite adipocytes convert chemical energy from nutrients into heat. Therapeutics that regulate brown adipocyte recruitment and activity represent interesting strategies to control fat mass such as in obesity or cachexia. The peroxisome proliferator-activated receptor (PPAR) family plays key roles in the maintenance of adipose tissue and in the regulation of thermogenic activity. Activation of these receptors induce browning of white adipocyte. The purpose of this work was to characterize the role of carnosic acid (CA), a compound used in traditional medicine, in the control of brown/brite adipocyte formation and function. We used human multipotent adipose-derived stem (hMADS) cells differentiated into white or brite adipocytes. The expression of key marker genes was determined using RT-qPCR and western blotting. We show here that CA inhibits the browning of white adipocytes and favors decreased gene expression of thermogenic markers. CA treatment does not affect ß-adrenergic response. Importantly, the effects of CA are fully reversible. We used transactivation assays to show that CA has a PPARα/γ antagonistic action. Our data pinpoint CA as a drug able to control PPAR activity through an antagonistic effect. These observations shed some light on the development of natural PPAR antagonists and their potential effects on thermogenic response.

PPARs and Angiogenesis-Implications in Pathology.

Peroxisome proliferator-activated receptors (PPARs) belong to the family of ligand-activated nuclear receptors. The PPAR family consists of three subtypes encoded by three separate genes: PPARα (NR1C1), PPARß/δ (NR1C2), and PPARγ (NR1C3). PPARs are critical regulators of metabolism and exhibit tissue and cell type-specific expression patterns and functions. Specific PPAR ligands have been proposed as potential therapies for a variety of diseases such as metabolic syndrome, cancer, neurogenerative disorders, diabetes, cardiovascular diseases, endometriosis, and retinopathies. In this review, we focus on the knowledge of PPAR function in angiogenesis, a complex process that plays important roles in numerous pathological conditions for which therapeutic use of PPAR modulation has been suggested.

Virtual screening of antitumour phytochemical against peroxisome proliferators activated receptor proteins PPARs.

Cancers are caused by the defects in apoptosis process which leads to uncontrolled proliferation, therefore, most attractive drug target discovery strategy is to find ligands which have the ability to activate or regulate the apoptotic machinery. Peroxisome-proliferator-activated receptors (PPARs) are nuclear hormone receptors their over expression is observed in many tumours and contributes to chemotherapy resistance. The goal of this study to scrutinized antitumor phytochemicals from Alysicarpus bupleurifolius, Piper nigrum and Plumeria obtuse and potential energy values render from interactions between active site residues and ligands. The potential phytochemicals with significant binding affinity are ursolic acid, cis-4-decenoic acid and p-coumaric acid respectively most effective compounds in high throughput virtual screening belongs to Plumeria obtuse against PPARs associated with tumour development and progression. This modern drug designing modeling in silico approach, therefore, identifies the potential leads against over expressed tumours.

Inhibitory effect of alpinetin on IL-6 expression by promoting cytosine methylation in CpG islands in the IL-6 promoter region.

BACKGROUND: Alpinetin is a flavonoid which exerts antibacterial and anti-inflammatory functions. In order to prove that the induced methylation is an important mechanism for alpinetin in regulating the expression of inflammatory factor Interleukin-6 (IL-6), we detected the dinucleotide methylation status of CpG islands in the IL-6 promoter region and IL-6 level after treatment of RAW246.7 murine macrophages with alpinetin. METHODS: After RAW246.7 murine macrophages were treated with alpinetin, alpinetin + GW9662 (the peroxisome proliferator-activated receptor (PPAR) antagonist), and alpinetin + DNA methyltransferase 3 alpha (DNMT3A) siRNA for 96 hr, CpG islands were analyzed using time-of-flight mass spectrophotometry (TOF-MS) and bisulfite sequencing polymerase chain reaction (BSP). Dinucleotide methylation status of the CpG islands in the IL-6 promoter region was analyzed by methylation-specific Polymerase Chain Reaction (PCR). IL-6 level was detected using the enzyme-linked immunosorbent assay (ELISA) method. Pearson's correlation analysis was conducted to test for potential correlation between the methylation status of CpG islands in the IL-6 promoter region and IL-6 level in RAW 246.7 cells. RESULTS: Alpinetin promoted dinucleotide methylation status of two CpG islands in the IL-6 promoter region stretching 500-2500 bp upstream of the transcriptional start site (TSS) (p < .05). This promoting effect was more significant for the CpG island stretching 500-1500 bp long. The methylation ratio of dinucleotide at this position was significantly inversely correlated with the level of IL-6 (p < .05). PPAR antagonist GW9662 and interference of DNMT3A could reverse both the alpinetin-induced methylation and inhibitory effects on IL-6 expression. CONCLUSION: Alpinetin could induce dinucleotide methylation status of CpG islands in the IL-6 promoter region by activating methyltransferase, thus inhibiting IL-6 expression in murine macrophages.

Chlorambucil targets BRCA1/2-deficient tumours and counteracts PARP inhibitor resistance.

Due to compromised homologous recombination (HR) repair, BRCA1- and BRCA2-mutated tumours accumulate DNA damage and genomic rearrangements conducive of tumour progression. To identify drugs that target specifically BRCA2-deficient cells, we screened a chemical library containing compounds in clinical use. The top hit was chlorambucil, a bifunctional alkylating agent used for the treatment of chronic lymphocytic leukaemia (CLL). We establish that chlorambucil is specifically toxic to BRCA1/2-deficient cells, including olaparib-resistant and cisplatin-resistant ones, suggesting the potential clinical use of chlorambucil against disease which has become resistant to these drugs. Additionally, chlorambucil eradicates BRCA2-deficient xenografts and inhibits growth of olaparib-resistant patient-derived tumour xenografts (PDTXs). We demonstrate that chlorambucil inflicts replication-associated DNA double-strand breaks (DSBs), similarly to cisplatin, and we identify ATR, FANCD2 and the SNM1A nuclease as determinants of sensitivity to both drugs. Importantly, chlorambucil is substantially less toxic to normal cells and tissues in vitro and in vivo relative to cisplatin. Because chlorambucil and cisplatin are equally effective inhibitors of BRCA2-compromised tumours, our results indicate that chlorambucil has a higher therapeutic index than cisplatin in targeting BRCA-deficient tumours.

Synthesis of novel benzothiazole amides: Evaluation of PPAR activity and anti-proliferative effects in paraganglioma, pancreatic and colorectal cancer cell lines.

The reduced activation of PPARs has a positive impact on cancer cell growth and viability in multiple preclinical tumor models, suggesting a new therapeutic potential for PPAR antagonists. In the present study, the benzothiazole amides 2a-g were synthesized and their activities on PPARs were investigated. Transactivation assay showed a moderate activity of the novel compounds as PPARα antagonists. Notably, in cellular assays they exhibited cytotoxicity in pancreatic, colorectal and paraganglioma cancer cells overexpressing PPARα. In particular, compound 2b showed the most remarkable inhibition of viability (greater than 90%) in two paraganglioma cell lines, with IC50 values in the low micromolar range. In addition, 2b markedly impaired colony formation capacity in the same cells. Taken together, these results show a relevant anti-proliferative potential of compound 2b, which appears particularly effective in paraganglioma, a rare tumor poorly responsive to chemotherapy.

An update about the crucial role of stereochemistry on the effects of Peroxisome Proliferator-Activated Receptor ligands.

Peroxisome Proliferator-Activated Receptors (PPARs) are ligand-activated transcription factors that govern lipid and glucose homeostasis playing a central role in cardiovascular disease, obesity, and diabetes. These receptors show a high degree of stereoselectivity towards several classes of drugs. This review covers the most relevant findings that have been made in the last decade and takes into consideration only those compounds in which stereochemistry led to unexpected results or peculiar interactions with the receptors. These cases are reviewed and discussed with the aim to show how enantiomeric recognition originates at the molecular level. The structural characterization by crystallographic methods and docking experiments of complexes formed by PPARs with their ligands turns out to be an essential tool to explain receptor stereoselectivity.

Significant roles of neuroinflammation in Parkinson's disease: therapeutic targets for PD prevention.

Glial cells outnumber neurons in the brain and play important roles in the neuroinflammation that accompanies brain damage in neurodegenerative diseases. In Parkinson's disease (PD), dopaminergic neuronal loss is accompanied by inflammatory changes in microglia, astrocytes, innate immune cells, and infiltrating peripheral immune cells. Neuroinflammation is probably a fundamental immune response to protect neurons from harm and compensate for neuronal damage, but at the same time, its neurotoxic effects exacerbate neuron damage. Furthermore, neuroinflammatory response is regulated by immune cells, such as microglia, astrocytes, and peripheral immune cells, and by cytokines and chemokines. Accordingly, it is crucial that we understand how such immune cells in the brain regulate neuroinflammatory responses in PD pathology. This review describes the roles played by glia-mediated neuroinflammation in PD, both good and bad, and the therapeutic strategies used to treat PD.

Fenofibrate improves vascular endothelial function and contractility in diabetic mice.

Fenofibrate, a peroxisome proliferator-activated receptors α (PPARα) agonist, reduces vascular complications of diabetic patients but its protective mechanisms are not fully understood. Here we tested the hypothesis that fenofibrate improves vascular endothelial dysfunction by balancing endothelium-dependent relaxation and contractility of the aorta in diabetes mellitus (DM). In streptozotocin-induced diabetic mice, eight weeks of fenofibrate treatment (100 mg/Kg/d) improved endothelium dependent relaxation in the macro- and microvessels, increased nitric oxide (NO) levels, reduced renal damage markers and effects of the vasoconstrictor prostaglandin. Levels of superoxide dismutase and catalase were both reduced and hydrogen peroxide was increased in vehicle-treated DM, but these changes were reversed by fenofibrate treatment. Vasodilation of the aorta after fenofibrate treatment was reversed by PPARα or AMPKα inhibitors. Western blots showed that fenofibrate treatment elevated PPARα expression, induced liver kinase B1 (LKB1) translocation from the nucleus to the cytoplasm and activated AMP-activated protein kinase-α (AMPKα), thus activating endothelial NO synthase (eNOS). Also, fenofibrate treatment decreased NF-κB p65 and cyclooxygenase 2 proteins in aortas. Finally, incubation with indomethacin in vitro improved aortic contractility in diabetic mice. Overall, our results show that fenofibrate treatment in diabetic mice normalizes endothelial function by balancing vascular reactivity via increasing NO production and suppressing the vasoconstrictor prostaglandin, suggesting mechanism of action of fenofibrate in mediating diabetic vascular complications.

The role of AKT and FOXO3 in preventing ovarian toxicity induced by cyclophosphamide.

Cyclophosphamide (CTX) has immunosuppressive effects and has been wildly used as one anti-cancer drug in clinical. Significant toxicity has been noticed particularly in the reproductive system. CTX promotes the maturation of ovarian follicles, decreases follicular reserve, and ultimately lead to ovarian failure or even premature ovarian failure (POF). The placental extract (HPE) has been shown to have some beneficial impact on reproductive system; however, little is known regarding to the effect of HPE on protecting CTX-induced ovarian injury and the mechanism involved. Whether human placental extracts (HPE) has a protective effect on CTX-induced toxicity on ovarian was studied by using a CTX-induced ovarian injury animal model. The effects of HEP on histopathology, the number of atretic follicles, the weight of the ovary, serum hormone levels, and apoptosis in granulosa cells were studied in mice with CTX or control vehicle. Our results have demonstrated that HPE inhibited p-Rictor, reduced the expression of Bad, Bax and PPAR, and activated Akt and Foxo3a (increased their phosphorylation). Mice treated with HPE showed higher ovarian weight, lower number of atretic follicles, higher serum levels of the hormones E2 and progesterone, and lower apoptosis and serum levels of LH and FSH in granulosa cells, than that in the control animal group. Our data show that ovarian injury can be attenuated by HPE. HPE likely protects follicular granulosa cells from undergoing significant apoptosis and reduce atresia follicle formation, therefore, alleviates CTX-induced ovarian injury.

Atorvastatin Inhibits the HIF1α-PPAR Axis, Which Is Essential for Maintaining the Function of Human Induced Pluripotent Stem Cells.

We herein report a novel mechanism of action of statin preparations using a new drug discovery method. Milk fat globule-EGF factor 8 protein (MFG-E8) was identified from the secretory component of mouse embryonic fibroblast (MEF) as a cell adhesion-promoting factor effective for screening active cellular agents of human induced pluripotent stem cells (hiPSCs) in vitro using electrochemical impedance. Our analyses showed that atorvastatin did not cause death in myocardial cells differentiated from hiPSCs but reduced the pluripotent cell survival in vitro when using serum- and albumin-free media, and inhibited the ability to form teratomas in mice. This result could have been already the cytopathic effect of atorvastatin, and complete elimination of hiPSCs was confirmed in the xenotransplantation assay. The administration of atorvastatin to hiPSCs caused the expression of hypoxia inducible factor (HIF)1α mRNA to be unchanged at 6 hr and downregulated at 24 hr. In addition, the inhibition of the survival of hiPSCs was confirmed by HIF1α-peroxisome proliferator-activated receptor (PPAR) axis inhibition. These results suggest that the addition of atorvastatin to hiPSC cultures reduces the survival of pluripotent cells by suppressing the HIF1α-PPAR axis. In summary, the HIF1α-PPAR axis has an important role in maintaining the survival of pluripotent hiPSCs.

The Anticancer Potential of Peroxisome Proliferator-Activated Receptor Antagonists.

The effects on cancer-cell proliferation and differentiation mediated by peroxisome proliferator-activated receptors (PPARs) have been widely studied, and pleiotropic outcomes in different cancer models and under different experimental conditions have been obtained. Interestingly, few studies report and little preclinical evidence supports the potential antitumor activity of PPAR antagonists. This review focuses on recent findings on the antitumor in vitro and in vivo effects observed for compounds able to inhibit the three PPAR subtypes in different tumor models, providing a rationale for the use of PPAR antagonists in the treatment of tumors expressing the corresponding receptors.

A Comparative Study on Selective PPAR Modulators through Quantitative Structure-activity Relationship, Pharmacophore and Docking Analyses.

BACKGROUND: Metabolic syndrome is a matrix of different metabolic disorders which are the leading cause of death in human beings. Peroxysome proliferated activated receptor (PPAR) is a nuclear receptor involved in metabolism of fats and glucose. OBJECTIVE: In order to explore structural requirements for selective PPAR modulators to control lipid and carbohydrate metabolism, the multi-cheminformatics studies have been performed. METHODS: In silico modeling studies have been performed on a diverse set of PPAR modulators through quantitative structure-activity relationship (QSAR), pharmacophore mapping and docking studies. RESULTS: It is observed that the presence of an amide fragment (-CONHRPh) has a detrimental effect while an aliphatic ether linkage has a beneficial effect on PPARα modulation. On the other hand, the presence of an amide fragment has a positive effect on PPARδ modulation, but the aliphatic ether linkage and substituted aromatic ring in the molecular scaffold are very much essential for imparting potent and selective PPARγ modulation. Negative ionizable features (i.e. polar fragments) must be present in PPARδ and α modulators, but a hydrophobic feature is the prime requirement for PPARγ modulation. CONCLUSION: Here, the essential structural features have been explored for selective modulation of each subtype of PPAR in order to design new modulators with improved activity/selectivity.

Peroxisome proliferator-activated receptors as therapeutic targets for heart failure.

Heart failure (HF) is a common clinical syndrome that affects more than 23 million individuals worldwide. Despite the marked advances in its management, the mortality rates in HF patients have remained unacceptably high. Peroxisome proliferator-activated receptors (PPARs) are nuclear transcription regulators, involved in the regulation of fatty acid and glucose metabolism. PPAR agonists are currently used for the treatment of type II diabetes mellitus and hyperlipidemia; however, their role as therapeutic agents for HF remains under investigation. Preclinical studies have shown that pharmacological modulation of PPARs can upregulate the expression of fatty acid oxidation genes in cardiomyocytes. Moreover, PPAR agonists were proven able to improve ventricular contractility and reduce cardiac remodelling in animal models through their anti-inflammatory, anti-oxidant, anti-fibrotic, and anti-apoptotic activities. Whether these effects can be replicated in humans is yet to be proven. This article reviews the interactions of PPARs with the pathophysiological mechanisms of HF and how the pharmacological modulation of these receptors can be of benefit for HF patients.