Cannabinoid and Opioid Receptor Affinity and Modulation of Cancer-Related Signaling Pathways of Machaeriols and Machaeridiols from Machaerium Pers.

Machaeriols and machaeridiols are unique hexahydrodibenzopyran-type aralkyl phytocannabinoids isolated from Machaerium Pers. Earlier studies of machaeriol A (1) and B (2) did not show any affinity for cannabinoid receptor 1 (CB1 or CNR1), although they are structural analogs of psychoactive hexahydrocannabinol. This study comprehensively reports on the affinities of isolated Machaerium Pers. compounds, namely machaeriol A-D (1-4) and machaeridiol A-C (5-7), against cannabinoid (CB1 and CB2) and opioid (κ, δ and µ) receptors. Among the isolated compounds, machaeriol D (4) and machaeridiol A-C (5-7) showed some selective binding affinity for the CB2 receptor, using a radioligand binding assay, with Ki values of >1.3, >1.77, >2.18 and >1.1 µM, respectively. On the other hand, none of the compounds showed any binding to the CB1 receptor. Due to recent reports on the anticancer potential of the endocannabinoid system, compounds 1-7 were tested against a battery of luciferase reporter gene vectors that assess the activity of many cancer-related signaling pathways, including Stat3, Smad2/3, AP-1, NF-κB, E2F, Myc, Ets, Notch, FoxO, Wnt, Hedgehog and pTK in HeLa and T98G glioblastoma cells. Complete dose-response curves have been determined for each compound in both of these cell lines, which revealed that machaeridiol 6 displayed activities (IC50 in µM in HeLa and T98G cells) towards Stat3 (4.7, 1.4), Smad2/3 (1.2, 3.0), AP-1 (5.9, 4.2), NF-κB (0.5, 4.0), E2F (5.7, 0.7), Myc (5.3, 2.0), ETS (inactive, 5.9), Notch (5.3, 4.6), Wnt (4.2, inactive) and Hedgehog (inactive, 5.0). Furthermore, a combination study between machaeriol C (3) and machaeridiol B (6) displayed additive effects for E2F, ETS, Wnt and Hedgehog pathways, where these compounds individually were either minimally active or inactive. None of the compounds inhibited luciferase expression driven by the minimal thymidine kinase promoter (pTK), indicating the lack of general cytotoxicity for luciferase enzyme inhibition at the 50 µM concentration in both of these cell lines. The significance of the inhibition of these signaling pathways via machaeridiol 5-7 and their cross-talk potential has been discussed.

Explicit representation of protein activity states significantly improves causal discovery of protein phosphorylation networks.

BACKGROUND: Protein phosphorylation networks play an important role in cell signaling. In these networks, phosphorylation of a protein kinase usually leads to its activation, which in turn will phosphorylate its downstream target proteins. A phosphorylation network is essentially a causal network, which can be learned by causal inference algorithms. Prior efforts have applied such algorithms to data measuring protein phosphorylation levels, assuming that the phosphorylation levels represent protein activity states. However, the phosphorylation status of a kinase does not always reflect its activity state, because interventions such as inhibitors or mutations can directly affect its activity state without changing its phosphorylation status. Thus, when cellular systems are subjected to extensive perturbations, the statistical relationships between phosphorylation states of proteins may be disrupted, making it difficult to reconstruct the true protein phosphorylation network. Here, we describe a novel framework to address this challenge. RESULTS: We have developed a causal discovery framework that explicitly represents the activity state of each protein kinase as an unmeasured variable and developed a novel algorithm called "InferA" to infer the protein activity states, which allows us to incorporate the protein phosphorylation level, pharmacological interventions and prior knowledge. We applied our framework to simulated datasets and to a real-world dataset. The simulation experiments demonstrated that explicit representation of activity states of protein kinases allows one to effectively represent the impact of interventions and thus enabled our framework to accurately recover the ground-truth causal network. Results from the real-world dataset showed that the explicit representation of protein activity states allowed an effective and data-driven integration of the prior knowledge by InferA, which further leads to the recovery of a phosphorylation network that is more consistent with experiment results. CONCLUSIONS: Explicit representation of the protein activity states by our novel framework significantly enhances causal discovery of protein phosphorylation networks.

Selective targeting of cancer signaling pathways with nanomedicines: challenges and progress.

Cancer is one of the leading causes of death worldwide. Regardless of advances in understanding the molecular mechanics of cancer, its treatment is still lacking and the death rates for many forms of the disease remain the same as six decades ago. Although a variety of therapeutic agents and strategies have been reported, these therapies often failed to provide efficient therapy to patients as a consequence of the inability to deliver right and adequate chemotherapeutic agents to the right place. However, the situation has started to revolutionize substantially with the advent of novel 'targeted' nanocarrier-based cancer therapies. Such therapies hold great potential in cancer management as they are biocompatible, tailored to specific needs, tolerated and deliver enough drugs at the targeted site. Their use also enhances the delivery of chemotherapeutics by improving biodistribution, lowering toxicity, inhibiting degradation and increasing cellular uptake. However, in some instances, nonselective targeting is not enough and the inclusion of a ligand moiety is required to achieve tumor targeting and enhanced drug accumulation at the tumor site. This contemporary review outlines the targeting potential of nanocarriers, highlighting the essentiality of nanoparticles, tumor-associated molecular signaling pathways, and various biological and pathophysiological barriers.

Immuno-MALDI (iMALDI) mass spectrometry for the analysis of proteins in signaling pathways.

INTRODUCTION: Biomarkers are commonly used to stratify cancer patients and guide targeted therapies, but most biomarkers are of a genomic nature. Discrepancies between the genome and proteome and the high rates of drug resistance indicate that proteomic analyses may provide additional critically important information. Here we present immuno-Matrix-Assisted Laser Desorption/Ionization (iMALDI), the combination of immuno-affinity enrichment of peptides followed by direct MALDI-mass spectrometry analysis. iMALDI is a highly sensitive, targeted protein-quantitation technique with the potential to measure clinically relevant signaling-pathway proteins using minimal sample amounts, thus improving upon existing methodologies. Areas covered: We provide a brief overview of the current state of biomarker analysis technologies for modern cancer treatment. We also show the advantages of iMALDI for translating potential new biomarkers into the clinic, factors to consider for iMALDI assay development, and the utility of iMALDI for the quantitation of cell-signaling proteins. Expert commentary: We see targeted mass spectrometry approaches such as iMALDI as an important part of improving patient responses to targeted therapies by providing highly sensitive, accurate, precise, and specific measurements of signaling-pathway proteins, both in tumor cells and in cells from the tumor microenvironment. iMALDI results can be integrated with other -omics data to aid in tumor-targeting therapies and immuno-oncology.

Identification of a Novel GRHL3/HOPX/Wnt/ß-Catenin Proto-oncogenic Axis in Squamous Cell Carcinoma of the Esophagus.

BACKGROUND & AIMS: Esophageal squamous cell carcinoma (ESCC) is an aggressive malignancy with a poor long-term prognosis. The molecular mechanisms underlying the initiation and progression of this tumor are largely unknown. The transcription factor GRHL3 functions as a potent tumor suppressor in SCC of skin, head, and neck. This study aims to determine whether GRHL3 also plays a role in the homeostasis of the esophageal epithelium and in the development of ESCC. METHODS: The effects of Grhl3 deletion on squamous epithelial homeostasis in embryos and adult mice were examined using immunohistochemistry, transmission electron microscopy, and real-time polymerase chain reaction. The conditionally deleted mice were subsequently used to determine susceptibility to ESCC. Whole-transcriptome sequencing (RNA-seq) was performed on ESCC in wild-type and Grhl3 deleted animals. To decipher the signaling pathways, real-time polymerase chain reaction, immunohistochemistry, analysis of chromatin immunoprecipitation sequencing, chromatin immunoprecipitation-polymerase chain reaction, and RNA seq datasets were used. Primary human samples were used to validate the findings in the mouse model. RESULTS: Loss of Grhl3 perturbs the proliferation-differentiation balance in the esophageal epithelium, thereby increasing the susceptibility to esophageal carcinogenesis in adult mice. Grhl3 imparts its tumor suppressor function by regulating the expression of HOPX. We have identified the Wnt/ß-catenin pathway as the downstream effectors of GRHL3 and HOPX through our integrated approach using patient-derived ESCC samples and mouse models. CONCLUSIONS: GRHL3 conveys its tumor suppressor function in ESCC through regulating its target gene HOPX, which limits Wnt/ß-catenin signaling. Targeted therapies to inhibit this pathway could be a potential treatment strategy for ESCC patients with reduced GRHL3 expression.

Moonlight functions of glycolytic enzymes in cancer.

Since an extensive genome research has started, basic principle "one gene-one protein-one function" was significantly revised. Many proteins with more than one function were identified and characterized as "moonlighting" proteins, which activity depend not only on structural peculiarities but also on compartmentation and metabolic environment. It turned out that "housekeeping" glycolytic enzymes show important moonlight functions such as control of development, proliferation, apoptosis, migration, regulation of transcription and cell signaling. Glycolytic enzymes emerged very early in evolution and because of the limited content of genomes, they could be used as ancient regulators for intercellular and intracellular communication. The multifunctionality of the constitutively expressed enzymes began to serve cancer cell survival and growth. In the present review we discuss some moonlight functions of glycolytic enzymes that important for malignant transformation and tumor growth.

Adenosine triphosphate induced cell death: Mechanisms and implications in cancer biology and therapy.

Adenosine triphosphate (ATP) induced cell death (AICD) is a critical cellular process that has garnered substantial scientific interest for its profound relevance to cancer biology and to therapeutic interventions. This comprehensive review unveils the intricate web of AICD mechanisms and their intricate connections with cancer biology. This review offers a comprehensive framework for comprehending the multifaceted role of AICD in the context of cancer. This is achieved by elucidating the dynamic interplay between systemic and cellular ATP homeostasis, deciphering the intricate mechanisms governing AICD, elucidating its intricate involvement in cancer signaling pathways, and scrutinizing validated key genes. Moreover, the exploration of AICD as a potential avenue for cancer treatment underscores its essential role in shaping the future landscape of cancer therapeutics.

CRAC and SK Channels: Their Molecular Mechanisms Associated with Cancer Cell Development.

Cancer represents a major health burden worldwide. Several molecular targets have been discovered alongside treatments with positive clinical outcomes. However, the reoccurrence of cancer due to therapy resistance remains the primary cause of mortality. Endeavors in pinpointing new markers as molecular targets in cancer therapy are highly desired. The significance of the co-regulation of Ca2+-permeating and Ca2+-regulated ion channels in cancer cell development, proliferation, and migration make them promising molecular targets in cancer therapy. In particular, the co-regulation of the Orai1 and SK3 channels has been well-studied in breast and colon cancer cells, where it finally leads to an invasion-metastasis cascade. Nevertheless, many questions remain unanswered, such as which key molecular components determine and regulate their interplay. To provide a solid foundation for a better understanding of this ion channel co-regulation in cancer, we first shed light on the physiological role of Ca2+ and how this ion is linked to carcinogenesis. Then, we highlight the structure/function relationship of Orai1 and SK3, both individually and in concert, their role in the development of different types of cancer, and aspects that are not yet known in this context.

An overview of kinin mediated events in cancer progression and therapeutic applications.

Kinins are bioactive peptides generated in the inflammatory milieu of the tissue microenvironment, which is involved in cancer progression and inflammatory response. Kinins signals through activation of two G-protein coupled receptors; inducible Bradykinin Receptor B1 (B1R) and constitutive receptor B2 (B2R). Activation of kinin receptors and its cross-talk with receptor tyrosine kinases activates multiple signaling pathways, including ERK/MAPK, PI3K, PKC, and p38 pathways regulating cancer hallmarks. Perturbations of the kinin-mediated events are implicated in various aspects of cancer invasion, matrix remodeling, and metastasis. In the tumor microenvironment, kinins initiate fibroblast activation, mesenchymal stem cell interactions, and recruitment of immune cells. Albeit the precise nature of kinin function in the metastasis and tumor microenvironment are not completely clear yet, several kinin receptor antagonists show anti-metastatic potential. Here, we showcase an overview of the complex biology of kinins and their role in cancer pathogenesis and therapeutic aspects.

Epithelial-to-Mesenchymal Transition Signaling Pathways Responsible for Breast Cancer Metastasis.

Breast carcinoma is highly metastatic and invasive. Tumor metastasis is a convoluted and multistep process involving tumor cell disseminating from their primary site and migrating to the secondary organ. Epithelial-mesenchymal transition (EMT) is one of the crucial steps that initiate cell progression, invasion, and metastasis. During EMT, epithelial cells alter their molecular features and acquire a mesenchymal phenotype. The regulation of EMT is centered by several signaling pathways, including primary mediators TGF-ß, Notch, Wnt, TNF-α, Hedgehog, and RTKs. It is also affected by hypoxia and microRNAs (miRNAs). All these pathways are the convergence on the transcriptional factors such as Snail, Slug, Twist, and ZEB1/2. In addition, a line of evidence suggested that EMT and cancer stem like cells (CSCs) are associated. EMT associated cancer stem cells display mesenchymal phenotypes and resist to chemotherapy or targeted therapy. In this review, we highlighted recent discoveries in these signaling pathways and their regulation in breast cancer metastasis and invasion. While the clinical relevance of EMT and breast cancers remains controversial, we speculated a convergent signaling network pivotal to elucidating the transition of epithelial to mesenchymal phenotypes and onset of metastasis of breast cancer cells.

Marine antitumor drugs: status, shortfalls and strategies.

Cancer is considered as one of the deadliest diseases in the medical field. Apart from the preventive therapies, it is important to find a curative measure which holds no loopholes and acts accurately and precisely to curb cancer. Over the past few decades, there have been advances in this field and there are many antitumor compounds available on the market, which are of natural as well as synthetic origin. Marine chemotherapy is well recognized nowadays and profound development has been achieved by researchers to deal with different molecular pathways of tumors. However, the marine environment has been less explored for the production of safe and novel antitumor compounds. The reason is a number of shortfalls in this field. Though ample reviews cover the importance and applications of various anticancerous compounds from marine natural products, in the present review, we have tried to bring the current status of antitumor research based on marine inhibitors of cancer signaling pathways. In addition, focus has been placed on the shortfalls and probable strategies in the arena of marine antitumor drug discovery.

Chemosensitization of Therapy Resistant Tumors: Targeting Multiple Cell Signaling Pathways by Lupeol, A Pentacyclic Triterpene.

BACKGROUND: The resistance of cancer cells to different therapies is one of the major stumbling blocks for successful cancer treatment. Various natural and pharmaceuticals drugs are unable to control drug-resistance cancer cell's growth. Also, chemotherapy and radiotherapy have several side effects and cannot apply to the patient in excess. In this context, chemosensitization to the therapy-resistant cells by non-toxic phytochemicals could be an excellent alternative to combat therapy-resistant cancers. OBJECTIVE: To review the currently available literature on chemosensitization of therapy resistance cancers by Lupeol for clinically approved drugs through targeting different cell signaling pathways. METHODS: We reviewed relevant published articles in PubMed and other search engines from 1999 to 2019 to write this manuscript. The key words used for the search were "Lupeol and Cancer", "Lupeol and Chemosensitization", "Lupeol and Cell Signaling Pathways", "Cancer Stem Cells and Lupeol" etc. The published results on the chemosensitization of Lupeol were compared and discussed. RESULTS: Lupeol chemosensitizes drug-resistant cancer cells for clinically approved drugs. Lupeol alone or in combination with approved drugs inhibits inflammation in different cancer cells through modulation of expression of IL-6, TNF-α, and IFN-γ. Lupeol, through altering the expression levels of BCL-2, BAX, Survivin, FAS, Caspases, and PI3K-AKT-mTOR signaling pathway, significantly induce cell deaths among therapy-resistant cells. Lupeol also modulates the molecules involved in cell cycle regulation such as Cyclins, CDKs, P53, P21, and PCNA in different cancer types. CONCLUSION: Lupeol chemosensitizes the therapy-resistant cancer cells for the treatment of various clinically approved drugs via modulating different signaling pathways responsible for chemoresistance cancer. Thus, Lupeol might be used as an adjuvant molecule along with clinically approved drugs to reduce the toxicity and increase the effectiveness.

Primary cilia and their role in cancer.

Primary cilia are microtubule-based organelles that are expressed on almost all mammalian cells. It has become apparent that these structures are important signaling hubs that serve crucial roles in Wnt, hedgehog, extracellular signal-regulated kinase (ERK)/mitogen-activated protein kinase (MAPK) and Notch signaling pathways. A number of diseases have been found to involve dysfunctional primary cilia; collectively these diseases are called ciliopathies. In recent years, there has been more focus on the association between primary cilia and cancer, including renal, pancreatic and breast cancer. Numerous studies have demonstrated that various types of cancer cells fail to express cilia. Notably, it has also been indicated that a number of renal carcinogens induce a significant loss of cilia in renal epithelial cells. The present review focuses on the existing literature regarding primary cilia and their involvement with cancer signaling pathways, providing a brief overview of the structural features and functions of primary cilia, then discussing the evidence associating primary cilia with cancer, and presenting the available information on the ERK/MAPK, hedgehog and Wnt signaling pathways, and their involvement in primary cilia in association with cancer.

Teneurins: An Integrative Molecular, Functional, and Biomedical Overview of Their Role in Cancer.

Teneurins are large transmembrane proteins originally identified in Drosophila. Their essential role in development of the central nervous system is conserved throughout species, and evidence supports their involvement in organogenesis of additional tissues. Homophilic and heterophilic interactions between Teneurin paralogues mediate cellular adhesion in crucial processes such as neuronal pathfinding and synaptic organization. At the molecular level, Teneurins are proteolytically processed into distinct subdomains that have been implicated in extracellular and intracellular signaling, and in transcriptional regulation. Phylogenetic studies have shown a high degree of intra- and interspecies conservation of Teneurin genes. Accordingly, the occurrence of genetic variants has been associated with functional and phenotypic alterations in experimental systems, and with some inherited or sporadic conditions. Recently, tumor-related variations in Teneurin gene expression have been associated with patient survival in different cancers. Although these findings were incidental and molecular mechanisms were not addressed, they suggested a potential utility of Teneurin transcript levels as biomarkers for disease prognosis. Mutations and chromosomal alterations affecting Teneurin genes have been found occasionally in tumors, but literature remains scarce. The analysis of open-access molecular and clinical datasets derived from large oncologic cohorts provides an invaluable resource for the identification of additional somatic mutations. However, Teneurin variants have not been classified in terms of pathogenic risk and their phenotypic impact remains unknown. On this basis, is it plausible to hypothesize that Teneurins play a role in carcinogenesis? Does current evidence support a tumor suppressive or rather oncogenic function for these proteins? Here, we comprehensively discuss available literature with integration of molecular evidence retrieved from open-access databases. We show that Teneurins undergo somatic changes comparable to those of well-established cancer genes, and discuss their involvement in cancer-related signaling pathways. Current data strongly suggest a functional contribution of Teneurins to human carcinogenesis.

Short peptides interfering with signaling pathways as new therapeutic tools for cancer treatment.

Short peptides have many advantages, such as low molecular weight, selectivity for a specific target, organelles or cells with minimal toxicity. We describe properties of short peptides, which interfere with communication networks in tumor cells and within microenvironment of malignant gliomas, the most common brain tumors. We focus on ligand/receptor axes and intracellular signaling pathways critical for gliomagenesis that could be targeted with interfering peptides. We review structures and efficacy of organelle-specific and cell-penetrating peptides and describe diverse chemical modifications increasing proteolytic stability and protecting synthetic peptides against degradation. We report results of application of short peptides in glioma therapy clinical trials, their rises and falls. The most advanced examples of therapeutics such as short interfering peptides combined with cell-penetrating peptides that show good effectiveness in disease models are presented. It is foreseen that identification of peptides with better clinical properties may improve their success rates in clinical trials.

Establishing a high-throughput and automated cancer cell proliferation panel for oncology lead optimization.

Tumor cell proliferation assays are widely used for oncology drug discovery, including target validation, lead compound identification, and optimization, as well as determination of compound off-target activities. Taking advantage of robotic systems to maintain cell culture and perform cell proliferation assays would greatly increase productivity and efficiency. Here we describe the establishment of automated systems for high-throughput cell proliferation assays in a panel of 13 human tumor cell lines. These cell lines were selected from various types of human tumors containing a broad range of well-characterized mutations in multiple cellular signaling pathways. Standard procedures for cell culture and assay performance were developed and optimized in each cell line. Moreover, in-house developed software (i.e., Toolset, Curvemaster, and Biobars) was applied to analyze the data and generate data reports. Using tool compounds, we have shown that results obtained through this panel exhibit high reproducibility over a long period. Furthermore, we have demonstrated that this panel can be used to identify sensitive and insensitive cell lines for specific cancer targets, to drive cellular structure-activity relationships, and to profile compound off-target activities. All those efforts are important for cancer drug discovery lead optimization.