Computer-Assisted Selective Optimization of Side-Activities—from Cinalukast to a PPARα Modulator

Automated computational analogue design and scoring can speed up hit-to-lead optimization and appears particularly promising in selective optimization of side-activities (SOSA) where possible analogue diversity is confined. Probing this concept, we employed the cysteinyl leukotriene receptor 1 (CysLT₁R) antagonist cinalukast as lead for which we discovered peroxisome proliferator-activated receptor α (PPARα) modulatory activity. We automatically generated a virtual library of close analogues and classified these roughly 8000 compounds for PPARα agonism and CysLT₁R antagonism using automated affinity scoring and machine learning. A computationally preferred analogue for SOSA was synthesized, and in vitro characterization indeed revealed a marked activity shift toward enhanced PPARα activation and diminished CysLT₁R antagonism. Thereby, this prospective application study highlights the potential of automating SOSA.     

Overcoming Obstacles to Targeting Muscarinic Receptor Signaling in Colorectal Cancer

Despite great advances in our understanding of the pathobiology of colorectal cancer and the genetic and environmental factors that mitigate its onset and progression, a paucity of effective treatments persists. The five-year survival for advanced, stage IV disease remains substantially less than 20%. This review examines a relatively untapped reservoir of potential therapies to target muscarinic receptor expression, activation, and signaling in colorectal cancer. Most colorectal cancers overexpress M₃ muscarinic receptors (M₃R), and both in vitro and in vivo studies have shown that activating these receptors stimulates cellular programs that result in colon cancer growth, survival, and spread. In vivo studies using mouse models of intestinal neoplasia have shown that using either genetic or pharmacological approaches to block M₃R expression and activation, respectively, attenuates the development and progression of colon cancer. Moreover, both in vitro and in vivo studies have shown that blocking the activity of matrix metalloproteinases (MMPs) that are induced selectively by M₃R activation, i.e., MMP1 and MMP7, also impedes colon cancer growth and progression. Nonetheless, the widespread expression of muscarinic receptors and MMPs and their importance for many cellular functions raises important concerns about off-target effects and the safety of employing similar strategies in humans. As we highlight in this review, highly selective approaches can overcome these obstacles and permit clinicians to exploit the reliance of colon cancer cells on muscarinic receptors and their downstream signal transduction pathways for therapeutic purposes.     

Thrombospondin-1 in Urological Cancer: Pathological Role,Clinical Significance,and Therapeutic Prospects

Angiogenesis is an important process for tumor growth and progression of various solid tumors including urological cancers. Thrombospondins (TSPs), especially TSP-1, are representative “anti”-angiogenic molecules and many studies have clarified their pathological role and clinical significance in vivo and in vitro. In fact, TSP-1 expression is associated with clinicopathological features and prognosis in many types of cancers. However, TSP-1 is a multi-functional protein and its biological activities vary according to the specific tumor environments. Consequently, there is no general agreement on its cancer-related function in urological cancers, and detailed information regarding regulative mechanisms is essential for a better understanding of its therapeutic effects and prognostic values. Various “suppressor genes” and “oncogenes” are known to be regulators and TSP-1-related factors under physiological and pathological conditions. In addition, various types of fragments derived from TSP-1 exist in a given tissue microenvironment and TSP-1 derived-peptides have specific activities. However, a detailed pathological function in human cancer tissues is not still understood. This review will focus on the pathological roles and clinical significance of TSP-1 in urological cancers, including prostate cancer, renal cell carcinoma, and urothelial cancer. In addition, special attention is paid to TSP-1-derived peptide and TSP-1-based therapy for malignancies.     

S1P Lyase Regulates Intestinal Stem Cell Quiescence via Ki-67 and FOXO3

Background: Reduction of the Sphingosine-1-phosphate (S1P) degrading enzyme S1P lyase 1 (SGPL1) initiates colorectal cancer progression with parallel loss of colon function in mice. We aimed to investigate the effect of SGPL1 knockout on the stem cell niche in these mice. Methods: We performed immunohistochemical and multi-fluorescence imaging on tissue sections of wildtype and SGPL1 knockout colons under disease conditions. Furthermore, we generated SGPL1 knockout DLD-1 cells (SGPL1^−/−M.Ex1) using CRISPR/Cas9 and characterized cell cycle and AKT signaling pathway via Western blot, immunofluorescence, and FACS analysis. Results: SGPL1 knockout mice were absent of anti-Ki-67 staining in the stem cell niche under disease conditions. This was accompanied by an increase of the negative cell cycle regulator FOXO3 and attenuation of CDK2 activity. SGPL1^−/−M.Ex1 cells show a similar FOXO3 increase but no arrest of proliferation, although we found a suppression of the PDK1/AKT signaling pathway, a prolonged G1-phase, and reduced stem cell markers. Conclusions: While already established colon cancer cells find escape mechanisms from cell cycle arrest, in vivo SGPL1 knockout in the colon stem cell niche during progression of colorectal cancer can contribute to cell cycle quiescence. Thus, we propose a new function of the S1P lyase 1 in stemness.     

Sulfonamide Inhibitors of β-Catenin Signaling as Anticancer Agents with Different Output on c-MYC

The Wnt/β-catenin pathway is often found deregulated in cancer. The aberrant accumulation of β-catenin in the cell nucleus results in the development of various malignancies. Specific drugs against this signaling pathway for clinical treatments have not been approved yet. Herein we report inhibitors of β-catenin signaling of potential therapeutic value as anticancer agents. Ethyl 4-((4-(trifluoromethyl)phenyl)sulfonamido)benzoate (compound 14 ) inhibits the effect on Wnt reporter with an IC₅₀ value of 7.0 μM, significantly reduces c-MYC levels, inhibits HCT116 colon cancer cell growth (IC₅₀ 20.2 μM), does not violate Lipinski and Veber rules, and shows predicted Caco-2 and MDCK cell permeability P_app>500 nm s⁻¹. Compound 14 seems to have potential for the development of new anticancer therapies.     

The Potential Role of Spermine and Its Acetylated Derivative in Human Malignancies

Polyamines are essential biomolecules for normal cellular metabolism in humans. The roles of polyamines in cancer development have been widely discussed in recent years. Among all, spermine alongside with its acetylated derivative, N¹, N¹²-Diacetylspermine, demonstrate a relationship with the diagnosis and staging of various cancers, including lung, breast, liver, colorectal and urogenital. Numerous studies have reported the level of spermine in different body fluids and organ tissues in patients with different types of cancers. Currently, the role and the underlying mechanisms of spermine in cancer development and progression are still under investigation. This review summarized the roles of spermine in cancer development and as a diagnostic, prognostic and therapeutic tool in various cancers.     

Salvianolic Acid A,as a Novel ETA Receptor Antagonist,Shows Inhibitory Effects on Tumor in Vitro

Endothelin-1 (ET-1) autocrine and paracrine signaling modulate cell proliferation of tumor cells by activating its receptors, endothelin A receptor (ET_AR) and endothelin B receptor (ET_BR). Dysregulation of ET_AR activation promotes tumor development and progression. The potential of ET_AR antagonists and the dual-ET_AR and ET_BR antagonists as therapeutic approaches are under preclinical and clinical studies. Salvianolic acid A (Sal A) is a hydrophilic polyphenolic derivative isolated from Salvia miltiorrhiza Bunge (Danshen), which has been reported as an anti-cancer and cardio-protective herbal medicine. In this study, we demonstrate that Sal A inhibits ET_AR activation induced by ET-1 in both recombinant and endogenous ET_AR expression cell lines. The IC₅₀ values were determined as 5.7 µM in the HEK293/ET_AR cell line and 3.14 µM in HeLa cells, respectively. Furthermore, our results showed that Sal A suppressed cell proliferation and extended the doubling times of multiple cancer cells, including HeLa, DU145, H1975, and A549 cell lines. In addition, Sal A inhibited proliferation of DU145 cell lines stimulated by exogenous ET-1 treatment. Moreover, the cytotoxicity and cardio-toxicity of Sal A were assessed in human umbilical vein endothelial cells (HUVEC) and Human-induced pluripotent stem cell-derived cardiomyocytes (hiPS-CMs), which proved that Sal A demonstrates no cytotoxicity or cardiotoxicity. Collectively, our findings indicate that Sal A is a novel anti-cancer candidate through targeting ET_AR.     

Molecular Effects of Auto-Antibodies on Angiotensin II Type 1 Receptor Signaling and Cell Proliferation

The angiotensin II (Ang II) type 1 receptor (AT₁R) is involved in the regulation of blood pressure (through vasoconstriction) and water and ion homeostasis (mediated by interaction with the endogenous agonist). AT₁R can also be activated by auto-antibodies (AT₁R-Abs), which are associated with manifold diseases, such as obliterative vasculopathy, preeclampsia and systemic sclerosis. Knowledge of the molecular mechanisms related to AT₁R-Abs binding and associated signaling cascade (dys-)regulation remains fragmentary. The goal of this study was, therefore, to investigate details of the effects of AT₁R-Abs on G-protein signaling and subsequent cell proliferation, as well as the putative contribution of the three extracellular receptor loops (ELs) to Abs-AT₁R signaling. AT₁R-Abs induced nuclear factor of activated T-cells (NFAT) signaling, which reflects G_q/11 and G_i activation. The impact on cell proliferation was tested in different cell systems, as well as activation-triggered receptor internalization. Blockwise alanine substitutions were designed to potentially investigate the role of ELs in AT₁R-Abs-mediated effects. First, we demonstrate that Ang II-mediated internalization of AT₁R is impeded by binding of AT₁R-Abs. Secondly, exclusive AT₁R-Abs-induced G_q/11 activation is most significant for NFAT stimulation and mediates cell proliferation. Interestingly, our studies also reveal that ligand-independent, baseline AT₁R activation of G_i signaling has, in turn, a negative effect on cell proliferation. Indeed, inhibition of G_i basal activity potentiates proliferation triggered by AT₁R-Abs. Finally, although AT₁R containing EL1 and EL3 blockwise alanine mutations were not expressed on the human embryonic kidney293T (HEK293T) cell surface, we at least confirmed that parts of EL2 are involved in interactions between AT₁R and Abs. This current study thus provides extended insights into the molecular action of AT₁R-Abs and associated mechanisms of interrelated pathogenesis.     

Anticancer Potency Studies of Coordination Driven Self‐Assembled Arene–Ru‐Based Metalla‐Bowls

New tetranuclear cationic metalla‐bowls 5 – 7 with the general formula [Ru₄(p‐cymene)₄(N∩N)₂(OO∩OO)₂]⁴⁺ (N∩N=2,6‐bis(N‐(4‐pyridyl carbamoyl)pyridine, OO∩OO=2,5‐dihydroxy‐1,4‐benzoquinonato ( 5 ), OO∩OO=5,8‐dioxydo‐1,4‐naphthaquinonato ( 6 ), OO∩OO=hoxonato ( 7 )) were prepared by the reaction of the respective dinuclear ruthenium complexes 2 – 4 with a bispyridine amide donor ligand 1 in methanol in the presence of AgO₃SCF₃.These new molecular metalla‐bowls were fully characterized by analytical techniques including elemental analysis as well as ¹H and ¹³C NMR and HR‐ESI‐MS spectroscopy. The structure of metalla‐bowl 6 was determined from X‐ray crystal diffraction data. A UV/visible study was also carried out for the entire suite of new complexes. As with recent studies of similar arene–Ru complexes, the inhibition of cell growth by metalla‐bowls was established against SK‐hep‐1 (liver cancer), AGS (gastric cancer), and HCT‐15 (colorectal cancer) human cancer cell lines. Inhibition of cell growth by 6 was found to be considerably stronger against all cancer cell lines than the anticancer drugs, doxorubicin and cisplatin. In particular, in colorectal cancer cells, expression of the cancer suppressor genes APC and p53 was increased following exposure to 6 .     

Impact of Two Neuronal Sigma-1 Receptor Modulators,PRE084 and DMT,on Neurogenesis and Neuroinflammation in an Aβ1–42-Injected,Wild-Type Mouse Model of AD

Alzheimer’s disease (AD) is the most common form of dementia characterized by cognitive dysfunctions. Pharmacological interventions to slow the progression of AD are intensively studied. A potential direction targets neuronal sigma-1 receptors (S1Rs). S1R ligands are recognized as promising therapeutic agents that may alleviate symptom severity of AD, possibly via preventing amyloid-β-(Aβ-) induced neurotoxicity on the endoplasmic reticulum stress-associated pathways. Furthermore, S1Rs may also modulate adult neurogenesis, and the impairment of this process is reported to be associated with AD. We aimed to investigate the effects of two S1R agonists, dimethyltryptamine (DMT) and PRE084, in an Aβ-induced in vivo mouse model characterizing neurogenic and anti-neuroinflammatory symptoms of AD, and the modulatory effects of S1R agonists were analyzed by immunohistochemical methods and western blotting. DMT, binding moderately to S1R but with high affinity to 5-HT receptors, negatively influenced neurogenesis, possibly as a result of activating both receptors differently. In contrast, the highly selective S1R agonist PRE084 stimulated hippocampal cell proliferation and differentiation. Regarding neuroinflammation, DMT and PRE084 significantly reduced Aβ_1–42-induced astrogliosis, but neither had remarkable effects on microglial activation. In summary, the highly selective S1R agonist PRE084 may be a promising therapeutic agent for AD. Further studies are required to clarify the multifaceted neurogenic and anti-neuroinflammatory roles of these agonists.     

Mercury Chloride but Not Lead Acetate Causes Apoptotic Cell Death in Human Lung Fibroblast MRC5 Cells via Regulation of Cell Cycle Progression

Heavy metals are important for various biological systems, but, in excess, they pose a serious risk to human health. Heavy metals are commonly used in consumer and industrial products. Despite the increasing evidence on the adverse effects of heavy metals, the detailed mechanisms underlying their action on lung cancer progression are still poorly understood. In the present study, we investigated whether heavy metals (mercury chloride and lead acetate) affect cell viability, cell cycle, and apoptotic cell death in human lung fibroblast MRC5 cells. The results showed that mercury chloride arrested the sub-G₁ and G₂/M phases by inducing cyclin B1 expression. In addition, the exposure to mercury chloride increased apoptosis through the activation of caspase-3. However, lead had no cytotoxic effects on human lung fibroblast MRC5 cells at low concentration. These findings demonstrated that mercury chloride affects the cytotoxicity of MRC5 cells by increasing cell cycle progression and apoptotic cell death.     

Krill Oil Perturbs Proliferation and Migration of Mouse Colon Cancer Cells in vitro by Impeding Extracellular Signal-Regulated Protein Kinase Signaling Pathway

The prevalence of colorectal cancer (CRC) continues to increase. Treatment of CRC remains a significant clinical challenge, and effective therapies for advanced CRC are desperately needed. Increasing attention and ongoing research efforts have focused on krill oil that may provide health benefits to the human body. Here we report that krill oil exerts in vitro anticancer activity through a direct inhibition on proliferation, colony formation, migration, and invasion of mouse colon cancer cells. Krill oil inhibited the proliferation and colony formation of CT-26 colon cancer cells by causing G0/G1 cell cycle arrest and apoptosis. Cell cycle arrest was attributable to reduction of cyclin D1 levels in krill oil-treated cells. Further studies revealed that krill oil induced mitochondrial-dependent apoptosis of CT-26 cells, including loss of mitochondrial membrane potential, increased cytosolic calcium levels, activation of caspase-3, and downregulation of anti-apoptotic proteins MCL-1 and BCL-XL. Krill oil suppressed migration of CT-26 cells by disrupting the microfilaments and microtubules. Extracellular signal-regulated protein kinase (ERK) plays crucial roles in regulating proliferation and migration of cancer cells. We found that krill oil attenuated the activation of ERK signaling pathway to exert the effects on cell cycle, apoptosis, and migration of colon cancer cells. We speculate that polyunsaturated fatty acids of krill oil may dampen ERK activation by decreasing the phospholipid saturation of cell membrane. Although findings from in vitro studies may not necessarily translate in vivo, our study provides insights into the possibility that krill oil or its components could have therapeutic potential in colon cancer.     

Role of Orai3 in the Pathophysiology of Cancer

The mammalian exclusive Orai3 channel participates in the generation and/or modulation of two independent Ca²⁺ currents, the store-operated current, I_crac, involving functional interactions between the stromal interaction molecules (STIM), STIM1/STIM2, and Orai1/Orai2/Orai3, as well as the store-independent arachidonic acid (AA) (or leukotriene C4)-regulated current I_arc, which involves Orai1, Orai3 and STIM1. Overexpression of functional Orai3 has been described in different neoplastic cells and cancer tissue samples as compared to non-tumor cells or normal adjacent tissue. In these cells, Orai3 exhibits a cell-specific relevance in Ca²⁺ influx. In estrogen receptor-positive breast cancer cells and non-small cell lung cancer (NSCLC) cells store-operated Ca²⁺ entry (SOCE) is strongly dependent on Orai3 expression while in colorectal cancer and pancreatic adenocarcinoma cells Orai3 predominantly modulates SOCE. On the other hand, in prostate cancer cells Orai3 expression has been associated with the formation of Orai1/Orai3 heteromeric channels regulated by AA and reduction in SOCE, thus leading to enhanced proliferation. Orai3 overexpression is associated with supporting several cancer hallmarks, including cell cycle progression, proliferation, migration, and apoptosis resistance. This review summarizes the current knowledge concerning the functional role of Orai3 in the pathogenesis of cancer.     

The Pro-Apoptotic Role of the Regulatory Feedback Loop between miR-124 and PKM1/HNF4α in Colorectal Cancer Cells

Accumulating evidence indicates that miRNA regulatory circuits play important roles in tumorigenesis. We previously reported that miR-124 is correlated with prognosis of colorectal cancer due to PKM-dependent regulation of glycolysis. However, the mechanism by which miR-124 regulates apoptosis in colorectal cancer remains largely elusive. Here, we show that miR-124 induced significant apoptosis in a panel of colorectal cancer cell lines. The mitochondrial apoptosis pathway was activated by miR-124. Furthermore, the pro-apoptotic role of miR-124 was dependent on the status of PKM1/2 level. PKM1 was required for miR-124-induced apoptosis. Via direct protein-protein interaction, PKM1 promoted HNF4α binding to the promoter region of miR-124 and transcribing miR-124. Moreover, HNF4α or PKM1 had a more dramatic effect on colorectal cancer cell apoptosis in the presence of miR-124. However, inhibition of miR-124 blocked cell apoptosis induced by HNF4α or PKM1. These data indicate that miR-124 not only alters the expression of genes involved in glucose metabolism but also stimulates cancer cell apoptosis. In addition, the positive feedback loop between miR-124 and PKM1/HNF4α plays an important role in colorectal cancer cell apoptosis; it suggests that disrupting this regulatory circuit might be a potential therapeutic tool for colorectal cancer treatment.     

Regulation Network of Colorectal-Cancer-Specific Enhancers in the Progression of Colorectal Cancer

Enhancers regulate multiple genes via higher-order chromatin structures, and they further affect cancer progression. Epigenetic changes in cancer cells activate several cancer-specific enhancers that are silenced in normal cells. These cancer-specific enhancers are potential therapeutic targets of cancer. However, the functions and regulation networks of colorectal-cancer-specific enhancers are still unknown. In this study, we profile colorectal-cancer-specific enhancers and reveal their regulation network through the analysis of HiChIP data that were derived from a colorectal cancer cell line and Hi-C and RNA-seq data that were derived from tissue samples by in silico analysis and in vitro experiments. Enhancer–promoter loops in colorectal cancer cells containing colorectal-cancer-specific enhancers are involved in more than 50% of the topological associated domains (TADs) changed in colorectal cancer cells compared to normal colon cells. In addition, colorectal-cancer-specific enhancers interact with 152 genes that are significantly and highly expressed in colorectal cancer cells. These colorectal-cancer-specific enhancer target genes include ITGB4, RECQL4, MSLN, and GDF15. We propose that the regulation network of colorectal-cancer-specific enhancers plays an important role in the progression of colorectal cancer.     

Inositol (1,4,5)-Trisphosphate Receptors in Invasive Breast Cancer: A New Prognostic Tool?

Simple SummaryThe inositol-trisphosphate receptor (IP₃R) is a key player in physiological and pathological intracellular calcium signaling. The objective of the present study was to assess the putative value of the three IP₃R subtypes as prognostic biomarkers in breast cancer. We found that IP₃R3 is the most strongly expressed subtype in breast cancer tissue. Furthermore, IP₃R3 and IP₃R1 are significantly more expressed in invasive breast cancer tissue than in non-tumor tissue. In contrast to IP₃R1 and IP₃R2, the expression of IP₃R3 was positively correlated with prognostic factors including tumor size, regional node invasion, histologic grade, proliferation index, and hormonal status. By analyzing public databases, we found that the expression of all IP₃R subtypes is significantly correlated with the overall survival and disease-free survival of patients with breast cancer. We conclude that relative to the other two IP₃R subtypes, IP₃R3 expression is upregulated in breast cancer and is correlated with prognostic factors. We strongly believe that our results will open up new perspectives with regard to the link between IP₃Rs and breast cancer aggressiveness.AbstractBreast cancer is the leading cause of cancer death among women in worldwide and France. The disease prognosis and treatment differ from one breast cancer subtype to another, and the disease outcome depends on many prognostic factors. Deregulation of ion flux (especially Ca²⁺ flux) is involved in many pathophysiology processes, including carcinogenesis. Inside the cell, the inositol-trisphosphate receptor (IP₃R) is a major player in the regulation of the Ca²⁺ flux from the endoplasmic reticulum to the cytoplasm. The IP₃Rs (and particularly the IP₃R3 subtype) are known to be involved in proliferation, migration, and invasion processes in breast cancer cell lines. The objective of the present study was to evaluate the potential value of IP₃Rs as prognostic biomarkers in breast cancer. We found that expression levels of IP₃R3 and IP₃R1 (but not IP₃R2) were significantly higher in invasive breast cancer of no special type than in non-tumor tissue from the same patient. However, the IP₃R3 subtype was expressed more strongly than the IP₃R1 and IP₃R2 subtypes. Furthermore, the expression of IP₃R3 (but not of IP₃R1 or IP₃R2) was positively correlated with prognostic factors such as tumor size, regional node invasion, histologic grade, proliferation index, and hormone receptor status. In an analysis of public databases, we found that all IP₃Rs types are significantly associated with overall survival and progression-free survival in patients with breast cancer. We conclude that relative to the other two IP₃R subtypes, IP₃R3 expression is upregulated in breast cancer and is correlated with prognostic factors.