Targeting Multiple Signaling Pathways Research Articles

Dual Inhibition of SYK and EGFR Overcomes Chemoresistance by Inhibiting CDC6 and Blocking DNA Replication.

Targeting multiple signaling pathways has been proposed as a strategy to overcome resistance to single-pathway inhibition in cancer therapy. A previous study in epithelial ovarian cancers identified hyperactivity of spleen tyrosine kinase (SYK) and epidermal growth factor receptor (EGFR), which mutually phosphorylate and activate each other. Given the potential for pharmacologic inhibition of both kinases with clinically available agents, this study aimed to assess the antitumor efficacy of both pharmacologic and genetic SYK and EGFR co-inhibition using a multifaceted approach to analyze the global phosphoproteome and chemoresistant ovarian cancer cell lines, patient-derived organoids, and xenograft models. Dual inhibition of SYK and EGFR in chemoresistant ovarian cancer cells elicited a highly synergistic antitumor effect. Notably, the combined inhibition strategy activated the DNA damage response, induced G1 cell cycle arrest, and promoted apoptosis. The phosphoproteomic analysis revealed that perturbation of SYK and EGFR signaling induced a significant reduction in both phosphorylated and total protein levels of cell division cycle 6 (CDC6), a crucial initiator of DNA replication. Together, this study offers preclinical evidence supporting dual inhibition of SYK and EGFR as a promising treatment for chemoresistant ovarian cancer that disrupts DNA synthesis by impairing formation of the prereplication complex. These findings warrant further clinical investigation to explore the potential of this combination therapy in overcoming drug resistance and improving patient outcomes.

Curcumin and its Derivatives Targeting Multiple Signaling Pathways to Elicit Anticancer Activity: A Comprehensive Perspective.

The uncontrolled growth and spread of aberrant cells characterize the group of disorders known as cancer. According to GLOBOCAN 2022 analysis of cancer patients in either developed countries or developing countries the main concern cancers are breast cancer, lung cancer, and liver cancer which may rise eventually. Natural substances with dietary origins have gained interest for their low toxicity, anti-inflammatory, and antioxidant effects. The evaluation of dietary natural products as chemopreventive and therapeutic agents, the identification, characterization, and synthesis of their active components, as well as the enhancement of their delivery and bioavailability, have all received significant attention. Thus, the treatment strategy for concerning cancers must be significantly evaluated and may include the use of phytochemicals in daily lifestyle. In the present perspective, we discussed one of the potent phytochemicals, that has been used over the past few decades known as curcumin as a panacea drug of the "Cure-all" therapy concept. In our review firstly we included exhausted data from in vivo and in vitro studies on breast cancer, lung cancer, and liver cancer which act through various cancer-targeting pathways at the molecular level. Now, the second is the active constituent of turmeric known as curcumin and its derivatives are enlisted with their targeted protein in the molecular docking studies, which help the researchers design and synthesize new curcumin derivatives with respective implicated molecular and cellular activity. However, curcumin and its substituted derivatives still need to be investigated with unknown targeting mechanism studies in depth.

RNA Technology to Regenerate and Repair Alveolar Bone Defects

microRNA-200a (miR-200a) targets multiple signaling pathways that are involved in osteogenic differentiation and bone development. However, its therapeutic function in osteogenesis and bone regeneration remains unknown. In this study, we use in vitro and in vivo models to investigate the molecular function of miR-200a overexpression and miR-200a inhibition using a plasmid-based miR inhibitor system (PMIS) on osteogenic differentiation and bone regeneration. Inhibition of miR-200a using PMIS-miR-200a significantly increased osteogenic biomarkers of human embryonic palatal mesenchyme cells and promoted bone regeneration in rat tooth socket defects. In rat maxillary M1 molar extractions, the supporting tooth structures were removed with an implant drill to yield a 3-mm defect in the alveolar bone. A collagen sponge was inserted into the open alveolar defect and PMIS-miR-200a plasmid DNA was added to the sponge and the wound sutured to protect the sponge and close the defect. It was important to remove the existing tooth supporting structure, which can influence alveolar bone regeneration. The alveolar bone was regenerated in 4 wk. The collagen sponge acts to stabilize and deliver the PMIS-miR-200a DNA to cells entering the sponge in the bone defect. We show that mesenchymal stem cells expressing CD90 and Stro-1 enter the sponges, take up the DNA, and express PMIS-miR-200a. PMIS-miR-200a initiates a bone regeneration program in transformed cells in vivo. In vitro inhibition of miR-200a was found to upregulate Wnt and BMP signaling activity as well as Runx2, OCN, Lef-1, Msx2, and Dlx5 associated with osteogenesis. Liver and blood toxicity testing of PMIS-miR-200a–treated rats showed no increase in several biomarkers of liver disease. These results demonstrate the therapeutic function of PMIS-miR-200a for rapid bone regeneration. Furthermore, the studies were designed to demonstrate the ease of use of PMIS-miR-200a in solution and applied using a syringe in the clinic through a simple one-time application.

SGLT2 Inhibitors and Diabetic Kidney Disease: Targeting Multiple and Interrelated Signaling Pathways for Renal Protection.

Almost 20-40% of all patients suffering from diabetes mellitus experience chronic kidney disease, which is related to higher mortality (cardiovascular and all-cause). The implication of several pathophysiological mechanisms (hemodynamic, tubular, metabolic and inflammatory) in the pathogenesis of diabetic kidney disease generates an urgent need to develop multitarget therapeutic strategies to face its development and progression. SGLT2 inhibitors are undoubtedly a practice-changing drug class for individuals who experience type 2 diabetes and diabetic kidney disease. In vitro studies, exploratory research, sub-analyses of large randomized controlled trials, and investigation of several biomarkers have demonstrated that SGLT2 inhibitors achieved multiple beneficial activities, targeting several renal cellular and molecular pathways independent of their antihyperglycemic activity. These mainly include the reduction in intraglomerular pressure through the restoration of TGF, impacts on the renin-angiotensin-aldosterone system, improvement of renal hypoxia, adaptive metabolic alterations in substrate use/energy expenditure, improvement of mitochondrial dysfunction, and reduction of inflammation, oxidative stress and fibrosis. This manuscript thoroughly investigates the possible mechanisms that underlie their salutary renal effects in patients with diabetes, focusing on several pathways involved and the interplay between them. It also explores their upcoming role in ameliorating the evolution of chronic kidney disease in patients with diabetes.

Imipramine Suppresses Tumor Growth and Induces Apoptosis in Oral Squamous Cell Carcinoma: Targeting Multiple Processes and Signaling Pathways.

Oral squamous cell carcinoma (OSCC) has limited treatment options. This study investigated imipramine, a tricyclic antidepressant, as a potential therapy for OSCC using a SAS-bearing xenograft animal model. The SAS-bearing xenograft model evaluated imipramine's impact on tumor growth. The control group received no treatment, while the imipramine-treated group received regular doses. Tumor growth, confirmed by imaging, and histological analysis assessed size and weight. Imipramine's effects on apoptosis, epithelial-to-mesenchymal transition (EMT), and transcription factors (AKT, ERK, STAT3) were analyzed. Imipramine significantly suppressed tumor growth within 6 days of treatment, with sustained activity. Computer tomography (CT) scans and histology confirmed reduced size and weight by imipramine. Imipramine induced apoptosis via caspase-dependent/-independent pathways, inhibited EMT, and down-regulated phosphorylated AKT, ERK, and STAT3. Imipramine shows promise as an effective OSCC therapy, inhibiting tumor growth, inducing apoptosis, and inhibiting EMT. Its impact on transcription factors and modulation of the AKT/ERK/STAT3 pathway suggest a multifaceted approach.

Proteomic Alteration in the Progression of Multiple Myeloma: A Comprehensive Review.

Multiple myeloma (MM) is an incurable hematologic malignancy. Most MM patients are diagnosed at a late stage because the early symptoms of the disease can be uncertain and nonspecific, often resembling other, more common conditions. Additionally, MM patients are commonly associated with rapid relapse and an inevitable refractory phase. MM is characterized by the abnormal proliferation of monoclonal plasma cells in the bone marrow. During the progression of MM, massive genomic alterations occur that target multiple signaling pathways and are accompanied by a multistep process involving differentiation, proliferation, and invasion. Moreover, the transformation of healthy plasma cell biology into genetically heterogeneous MM clones is driven by a variety of post-translational protein modifications (PTMs), which has complicated the discovery of effective treatments. PTMs have been identified as the most promising candidates for biomarker detection, and further research has been recommended to develop promising surrogate markers. Proteomics research has begun in MM, and a comprehensive literature review is available. However, proteomics applications in MM have yet to make significant progress. Exploration of proteomic alterations in MM is worthwhile to improve understanding of the pathophysiology of MM and to search for new treatment targets. Proteomics studies using mass spectrometry (MS) in conjunction with robust bioinformatics tools are an excellent way to learn more about protein changes and modifications during disease progression MM. This article addresses in depth the proteomic changes associated with MM disease transformation.

Conocarpus lancifolius (Combretaceae): Pharmacological Effects, LC-ESI-MS/MS Profiling and In Silico Attributes.

In folklore medicine, Conocarpus lancifolius is used to treat various illnesses. The main objective of this study was a comprehensive investigation of Conocarpus lancifolius leaf aqueous extract (CLAE) for its antioxidant, cardioprotective, anxiolytic, antidepressant and memory-enhancing capabilities by using different in vitro, in vivo and in silico models. The in vitro experimentation revealed that CLAE consumed an ample amount of total phenolics (67.70 ± 0.15 µg GAE/mg) and flavonoids (47.54 ± 0.45 µg QE/mg) with stronger antiradical effects through DPPH (IC50 = 16.66 ± 0.42 µg/mL), TAC (77.33 ± 0.41 µg AAE/mg) and TRP (79.11 ± 0.67 µg GAE/mg) assays. The extract also displayed suitable acetylcholinesterase (AChE) inhibitory (IC50 = 110.13 ± 1.71 µg/mL) activity through a modified Ellman's method. The toxicology examination presented no mortality or any signs of clinical toxicity in both single-dose and repeated-dose tests. In line with the cardioprotective study, the pretreatment of CLAE was found to be effective in relieving the isoproterenol (ISO)-induced myocardial injury in rats by normalizing the heart weight index, serum cardiac biomarkers, lipid profile and various histopathological variations. In the noise-stress-induced model for behavior attributes, the results demonstrated that CLAE has the tendency to increase the time spent in the central zone and elevated open arms in the open field and elevated plus maze tests (examined for anxiety assessment), reduced periods of immobility in the forced swimming test (for depression) and improved recognition and working memory in the novel object recognition and Morris water maze tests, respectively. Moreover, the LC-ESI-MS/MS profiling predicted 53 phytocompounds in CLAE. The drug-likeness and ADMET analysis exhibited that the majority of the identified compounds have reasonable physicochemical and pharmacokinetic profiles. The co-expression of molecular docking and network analysis indicated that top-ranked CLAE phytoconstituents act efficiently against the key proteins and target multiple signaling pathways to exert its cardiovascular-protectant, anxiolytic, antidepressant and memory-enhancing activity. Hence, this artifact illustrates that the observed biological properties of CLAE elucidate its significance as a sustainable source of bioactive phytochemicals, which appears to be advantageous for pursuing further studies for the development of new therapeutic agents of desired interest.

Aloe-emodin targets multiple signaling pathways by blocking ubiquitin-mediated degradation of DUSP1 in nasopharyngeal carcinoma cells.

Aloe-emodin (AE) has been shown to inhibit the proliferation of several cancer cell lines, including human nasopharyngeal carcinoma (NPC) cell lines. In this study, we confirmed that AE inhibited malignant biological behaviors, including cell viability, abnormal proliferation, apoptosis, and migration of NPC cells. Western blotting analysis revealed that AE upregulated the expression of DUSP1, an endogenous inhibitor of multiple cancer-associated signaling pathways, resulting in blockage of the extracellular signal-regulated kinase (ERK)-1/2, protein kinase B (AKT), and p38-mitogen activated protein kinase（p38-MAPK） signaling pathways in NPC cell lines. Moreover, the selective inhibitor of DUSP1, BCI-hydrochloride, partially reversed the AE-induced cytotoxicity and blocked the aforementioned signaling pathways in NPC cells. In addition, the binding between AE and DUSP1 was predicted via molecular docking analysis using AutoDock-Vina software and further verified via a microscale thermophoresis assay. The binding amino acid residues were adjacent to the predicted ubiquitination site (Lys192) of DUSP1. Immunoprecipitation with the ubiquitin antibody, ubiquitinated DUSP1 was shown to be upregulated by AE. Our findings revealed that AE can stabilize DUSP1 by blocking its ubiquitin-proteasome-mediated degradation and proposed an underlying mechanism by which AE-upregulated DUSP1 may potentially target multiple pathways in NPC cells.

Anticancer Potential of Ivermectin: Mechanisms of Action and Therapeutic Implications

Ivermectin is a well-known antiparasitic drug in the macrolide class with a 16-membered ring. It has been used for treating various parasitic diseases, including onchocerciasis, lymphatic filariasis, and strongyloidiasis. The present study aimed to review the mechanisms of action and therapeutic implications of Ivermectin as an anticancer agent. It has been used for over three decades, and its safety has been well-established in humans A growing body of evidence suggests that ivermectin has anticancer properties, making it an attractive candidate for treating various types of cancer. The reason is that ivermectin targets multiple signaling pathways, including the Wnt/β-catenin, PI3K/Akt/mTOR, and STAT3 pathways, to inhibit cancer cell proliferation and induce apoptosis. Inhibition of these pathways by ivermectin leads to suppression of cancer cell growth. Additionally, ivermectin has been shown to induce autophagy, which can lead to programmed cell death in cancer cells. One of the significant advantages of ivermectin as an anticancer drug is its safety profile. Furthermore, it is easily available and affordable, making it a promising alternative to conventional chemotherapy for various types of cancer, including breast, lung, and colon cancer. However, further research is needed to evaluate its clinical effectiveness in humans. Clinical trials are underway to investigate ivermectin's safety and efficacy in cancer treatment. In conclusion, the safety profile and low cost of ivermectin as an anticancer drug have turned it into a feasible alternative to conventional chemotherapy, which needs more investigation.

Licorice (Glycyrrhiza glabra L.)-Derived Phytochemicals Target Multiple Signaling Pathways to Confer Oncopreventive and Oncotherapeutic Effects.

Cancer is a highly lethal disease, and its incidence has rapidly increased worldwide over the past few decades. Although chemotherapeutics and surgery are widely used in clinical settings, they are often insufficient to provide the cure for cancer patients. Hence, more effective treatment options are highly needed. Although licorice has been used as a medicinal herb since ancient times, the knowledge about molecular mechanisms behind its diverse bioactivities is still rather new. In this review article, different anticancer properties (antiproliferative, antiangiogenic, antimetastatic, antioxidant, and anti-inflammatory effects) of various bioactive constituents of licorice (Glycyrrhiza glabra L.) are thoroughly described. Multiple licorice constituents have been shown to bind to and inhibit the activities of various cellular targets, including B-cell lymphoma 2, cyclin-dependent kinase 2, phosphatidylinositol 3-kinase, c-Jun N-terminal kinases, mammalian target of rapamycin, nuclear factor-κB, signal transducer and activator of transcription 3, vascular endothelial growth factor, and matrix metalloproteinase-3, resulting in reduced carcinogenesis in several in vitroand in vivomodels with no evident toxicity. Emerging evidence is bringing forth licorice as an anticancer agent as well as bottlenecks in its potential clinical application. It is expected that overcoming toxicity-related obstacles by using novel nanotechnological methods might importantly facilitate the use of anticancer properties of licorice-derived phytochemicals in the future. Therefore, anticancer studies with licorice components must be continued. Overall, licorice could be a natural alternative to the present medication for eradicating new emergent illnesses while having just minor side effects.

Unraveling the Mechanisms of Clinical Drugs-Induced Neural Tube Defects Based on Network Pharmacology and Molecular Docking Analysis.

Chemotherapeutic agents such as methotrexate (MTX), raltitrexed (RTX), 5-fluorouracil (5-FU), hydroxyurea (HU), and retinoic acid (RA), and valproic acid (VPA), an antiepileptic drug, all can cause malformations in the developing central nervous system (CNS), such as neural tube defects (NTDs). However, the common pathogenic mechanisms remain unclear. This study aimed to explore the mechanisms of NTDs caused by MTX, RTX, 5-FU, HU, RA, and VPA (MRFHRV), based on network pharmacology and molecular biology experiments. The MRFHRV targets were integrated with disease targets, to find the potential molecules related to MRFHRV-induced NTDs. Protein-protein interaction analysis and molecular docking were performed to analyze these common targets. Utilizing the kyoto encyclopedia of genes and genomes (KEGG) signaling pathways, we analyzed and searched the possible causative pathogenic mechanisms by crucial targets and the signaling pathway. Results showed that MRFHRV induced NTDs through several key targets (including TP53, MAPK1, HSP90AA1, ESR1, GRB2, HDAC1, EGFR, PIK3CA, RXRA, and FYN) and multiple signaling pathways such as PI3K/Akt pathway, suggesting that abnormal proliferation and differentiation could be critical pathogenic contributors in NTDs induced by MRFHRV. These results were further validated by CCK8 assay in mouse embryonic stem cells and GFAP staining in embryonic brain tissue. This study indicated that chemotherapeutic and antiepileptic agents induced NTDs might through predicted targets TP53, MAPK1, GRB2, HDAC1, EGFR, PIK3CA, RXRA, and FYN and multiple signaling pathways. More caution was required for the clinical administration for women with childbearing potential and pregnant.

Combined effects on leukemia cell growth by targeting sphingosine kinase 1 and sirtuin 1 signaling.

Targeting multiple signaling pathways is a potential novel therapeutic strategy for the treatment of leukemias. Leukemia cells express high levels of sphingosine kinase 1 (Sphk1) and sirtuin 1 (SIRT1). However, to the best of our knowledge, their interaction and potential synergistic inhibitory effects on the growth and survival of leukemia cells have not been investigated. The present study revealed the role of the Sphk1/S1P/SIRT1 axis in K562, KCL22 and TF1 cells and hypothesized that the inhibition of Sphk1 and SIRT1 had synergistic effects on the growth and survival of leukemia cells. Cell viability was tested using a Cell Counting Kit-8 assay and cell colony forming assay. Cell apoptosis was detected using Annexin V-APC/PI staining. The stages of the cell cycle were measured using PI staining. Protein levels were measured by western blotting. Treatment of leukemia cells with S1P resulted in the upregulation of SIRT1 expression, whereas inhibition of Sphk1 induced SIRT1 downregulation in leukemia cells. Both SKI-II and EX527 actively suppressed growth, blocked cell cycle progression and induced apoptosis of leukemia cells. Furthermore, inhibition of Sphk1 and SIRT1 exhibited suppressive effects on the growth and survival of leukemia cells. Notably, the inhibition of Sphk1 and SIRT1 suppressed cell growth and induced apoptosis of T-315I mutation-harboring cells. Additionally, treatment with SKI-II and EX527 suppressed the ERK and STAT5 pathways in leukemia cells. These data indicated that targeting the Sphk1/S1P/SIRT1 axis may be a novel therapeutic strategy for the treatment of leukemia.

Synergistic Anti-Tumor Activity by Targeting Multiple Signaling Pathways in Ovarian Cancer

Simple SummaryOvarian cancer remains the most lethal gynecological cancer in women. There is a critical need to develop novel strategies that can be used to improve the survival of patients with advanced and recurrent ovarian cancer. Preclinical and early clinical studies with single-targeted agents have shown limited antitumor activity in ovarian cancer. In this study, we found that combined treatment of several FDA-approved targeted drugs—sunitinib, dasatinib, and everolimus—results in simultaneous inhibition of multiple signaling pathways and a better anti-tumor activity than any single treatment. This combination also significantly improves efficacy of paclitaxel in human ovarian cancer. This study may provide a potential combination therapy for the treatment of advanced ovarian cancer.More effective therapy is needed to improve the survival of patients with advanced and recurrent ovarian cancer. Preclinical and early clinical studies with single molecular targeted agents have shown limited antitumor activity in ovarian cancer, likely due to compensation by alternative growth/survival pathways. An emerging strategy in overcoming resistance is to combine inhibitors targeting multiple pathways. In this study, we used a novel strategy of combining several FDA-approved targeted drugs, including sunitinib, dasatinib, and everolimus, in human ovarian cancers. Combination of the tyrosine kinase inhibitor sunitinib with the SRC inhibitor dasatinib showed synergistic anti-tumor activity in human ovarian cancer cells. The increased activity was associated with inhibition of the STAT3, SRC, and MAPK signaling pathways, but not AKT signaling. To inhibit the PI3K/AKT/mTOR pathway, we added the mTOR inhibitor everolimus, which further increased anti-tumor activity in cells. Combined treatment with sunitinib, dasatinib, and everolimus also resulted in greater inhibition of human ovarian tumor growth in mice. Furthermore, the triple combination also synergistically increased the anti-tumor activity of paclitaxel, both in vitro and in vivo. Taken together, our results demonstrate that simultaneous inhibition of several signaling pathways results in better anti-tumor activity compared to inhibiting any of these signaling pathways alone.

Targeting Multiple Signaling Pathways in Cancer: The Rutin Therapeutic Approach.

Multiple dysregulated signaling pathways are implicated in the pathogenesis of cancer. The conventional therapies used in cancer prevention/treatment suffer from low efficacy, considerable toxicity, and high cost. Hence, the discovery and development of novel multi-targeted agents to attenuate the dysregulated signaling in cancer is of great importance. In recent decades, phytochemicals from dietary and medicinal plants have been successfully introduced as alternative anticancer agents due to their ability to modulate numerous oncogenic and oncosuppressive signaling pathways. Rutin (also known as rutoside, quercetin-3-O-rutinoside and sophorin) is an active plant-derived flavonoid that is widely distributed in various vegetables, fruits, and medicinal plants, including asparagus, buckwheat, apricots, apples, cherries, grapes, grapefruit, plums, oranges, and tea. Rutin has been shown to target various inflammatory, apoptotic, autophagic, and angiogenic signaling mediators, including nuclear factor-κB, tumor necrosis factor-α, interleukins, light chain 3/Beclin, B cell lymphoma 2 (Bcl-2), Bcl-2 associated X protein, caspases, and vascular endothelial growth factor. A comprehensive and critical analysis of the anticancer potential of rutin and associated molecular targets amongst various cancer types has not been performed previously. Accordingly, the purpose of this review is to present an up-to-date and critical evaluation of multiple cellular and molecular mechanisms through which the anticancer effects of rutin are known to be exerted. The current challenges and limitations as well as future directions of research are also discussed.

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

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. To review the currently available literature on chemosensitization of therapy resistance cancers by Lupeol for clinically approved drugs through targeting different cell signaling pathways. 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. 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. 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.

Downregulation of STAT3, β-Catenin, and Notch-1 by Single and Combinations of siRNA Treatment Enhance Chemosensitivity of Wild Type and Doxorubicin Resistant MCF7 Breast Cancer Cells to Doxorubicin.

Combinatorial therapeutic strategies using siRNA and small molecules to eradicate tumors are emerging. Targeting multiple signaling pathways decreases the chances of cancer cells switching and adapting new signaling processes that may occur when using a single therapeutic modality. Aberrant functioning of Notch-1, Wnt/β-catenin, and STAT3 proteins and their crosstalk signaling pathways have been found to be involved in tumor survival, drug resistance, and relapse. In the current study, we describe a therapeutic potential of single and combinations of siRNA designed for silencing Notch-1, Wnt/β-catenin, and STAT3 in MCF7_DoxS (wild type) and MCF7_DoxR (doxorubicin resistant) breast cancer cells. The MCF7_DoxR cells were developed through treatment with a gradual increase in doxorubicin concentration, the expression of targeted genes was investigated, and the expression profiling of CD44/CD24 of the MCF7_DoxS and MCF7_DoxR cells were detected by flow cytometry. Both MCF7_DoxS and MCF7_DoxR breast cancer cells were treated with single and combinations of siRNA to investigate synergism and were analyzed for their effect on cell proliferation with and without doxorubicin treatment. The finding of this study showed the overexpression of targeted genes and the enrichment of the CD44−/CD24+ phenotype in MCF7_DoxR cells when compared to MCF7_DoxS cells. In both cell lines, the gene silencing efficacy showed a synergistic effect when combining STAT3/Notch-1 and STAT3/Notch-1/β-catenin siRNA. Interestingly, the chemosensitivity of MCF7_DoxS and MCF7_DoxR cells to doxorubicin was increased when combined with siRNA treatment. Our study shows the possibility of using single and combinations of siRNA to enhance the chemosensitivity of cancer cells to conventional antitumor chemotherapy.

Berberine and Tinospora cordifolia exert a potential anticancer effect on colon cancer cells by acting on specific pathways.

Berberine (BBR) is a natural active principle with potential antitumor activity. The compound targets multiple cell signaling pathways, including proliferation, differentiation, and epithelial–mesenchymal transition. The aim of this study was to elucidate the mechanisms behind the anticancer activity of BBR by comparing the effects of purified BBR with those of the extract of Tinospora cordifolia, a medicinal plant that produces this metabolite. The expression levels of a panel of 44 selected genes in human colon adenocarcinoma (HCA-7) cell line were quantified by real-time polymerase chain reaction (PCR). BBR treatment resulted in a time- and dose-dependent down regulation of 33 genes differently involved in cell cycle, differentiation, and epithelial–mesenchymal transition. The trend was confirmed across the two types of treatment, the two time points, and the different absolute dosage of BBR. These findings suggest that the presence of BBR in T. cordifolia extract significantly contributes to its antiproliferative activity.

Glucocorticoids Inhibit Group 3 Innate Lymphocyte IL-22 Production.

Glucocorticoids (GCs) are commonly prescribed to patients with a variety of inflammatory disorders, including inflammatory bowel disease (IBD). GCs mediate their immunomodulatory effects through many different mechanisms and target multiple signaling pathways. The GC dexamethasone downmodulates innate and adaptive immune cell activation. IBD is the manifestation of a dysregulated immune response involving many different immune cells. Group 3 innate lymphocytes (ILC3s) have critical roles in mucosal inflammation. ILC3s secrete high levels of the cytokine IL-22, promoting epithelial proliferation, antimicrobial peptides, and mucins. In this study, we examined the effects of dexamethasone on IL-22 production by ILC3s. We found that dexamethasone suppressed IL-23-mediated IL-22 production in human and mouse ILC3s. This was mediated in part through dexamethasone modulation of the NF-κB pathway. Inhibition of NF-κB signaling with a small molecule inhibitor also downmodulated IL-23- and IL-1β-mediated IL-22 production in ILC3s. These findings implicate NF-κB as a regulator of IL-22 in ILC3s and likely have repercussions on GC treatment of IBD patients.

Abstract 1963: ARQ 531, a potent reversible BTK inhibitor, exhibits potent antitumor activity in ibrutinib-resistant diffuse large B-cell lymphoma

Abstract Background: B-cell receptor (BCR)-mediated signaling plays an important role in the pathogenesis of a subset of diffuse large B-cell lymphoma (DLBCL). Despite major advances in the treatment, ~40% of the relapsed/refractory DLBCL patients still experience early treatment failure after initial response to chemotherapy. ARQ 531, a reversible inhibitor of BTK and BTK-C481S mutant, also potently suppresses BCR signaling. Here we demonstrate that ARQ 531 targets additional kinases and suppresses multiple oncogenic pathways, this inhibitory potency is coupled to broad anti-tumor activity in DLBCL subtypes including tumors resistant to BCR targeted therapy. Methods: Biochemical inhibition and broad kinase profiling were assessed using recombinant proteins. Binding kinetics and the residence time with BTK and BTK-C481S were measured by Surface Plasmon Resonance (SPR) assay. Binding mode of ARQ 531 with BTK was determined by protein crystallography. Pathway inhibition assessments, in vivo efficacy and in vivo target inhibition were performed in DLBCL tumor models derived from TMD8, SUDHL-4 and DOHH-2 cell lines. Results: ARQ 531 potently inhibited BTK (IC50 = 0.85 nM), the binding potency was accompanied by long residence time (51 min). Crystal structure of BTK/ARQ 531 complex showed that ARQ 531 occupies the ATP-binding pocket. Kinase selectivity profile suggested that ARQ 531 inhibits sub-families of Tec, Src, Trk kinases. Significant anti-proliferative activity (GI50 = < 1µM) was observed in hematological malignant cell lines characterized by addiction to BTK signaling and primarily resistant to ibrutinib. Pathway inhibition analysis suggested that ARQ 531 targets multiple oncogenic signaling pathways in both ABC- and GCB-DLBCL cell lines. Unlike ibrutinib, ARQ 531 suppressed both the upstream activating signals (via inhibition of a select member of Src kinase family) and the downstream signaling pathways (via pAKT and pERK kinases). In GCB-DLBCL cell lines (SUDHL-4 and DOHH-2), ARQ 531 potently suppressed expression of anti-apoptotic c-Myc and BCL6 oncoproteins in a dose dependent fashion, and concomitantly induced apoptotic cleavage of PARP protein. In the ibrutinib-resistant SUDHL-4 mouse xenograft model, ARQ 531 potently suppressed tumor growth (>80% inhibition) compared to the control group when dosed orally at 75 mg/kg. Additionally, preliminary data suggest that ARQ 531 crosses the blood, brain-barrier. Conclusion: ARQ 531 is a potent reversible inhibitor of BTK, its distinct kinase selectivity profile offers significant advantage for simultaneous inhibition of multiple therapeutically relevant targets. ARQ 531 attenuated the expression of key oncogenic drivers via inhibition of downstream BCR activating kinases. These results highlight the therapeutic potential of inhibition of BCR signaling inhibition by ARQ 531 in the treatment of DLBCL. Citation Format: Sudharshan Eathiraj, Yi Yu, Ron Savage, Jennifer A. Woyach, Sean D. Reiff, Amy J. Johnson, Brian Schwartz. ARQ 531, a potent reversible BTK inhibitor, exhibits potent antitumor activity in ibrutinib-resistant diffuse large B-cell lymphoma [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 1963.

Glucocorticoids inhibit group 3 innate lymphocyte IL-22 production

Abstract Glucocorticoids (GCs) are commonly prescribed to patients with a variety of inflammatory disorders, including inflammatory bowel disease (IBD). IBD is the manifestation of a dysregulated mucosal immune response, usually against commensal bacteria, involving many different immune cells. GCs mediate their immunomodulatory effects through many different mechanisms and target multiple signaling pathways. The GC dexamethasone down-modulates both innate and adaptive immune cell activation. Group 3 innate lymphocytes (ILC3s) have critical roles in mucosal inflammation. ILC3s secrete high levels of the cytokine IL-22, promoting epithelial proliferation, antimicrobial peptides and mucins. The effects of dexamethasone on T cell function are well described, however, it is not known if dexamethasone modulates the critical function of ILC3s or if dexamethasone regulates the cytokine IL-22. In this study, we examined the effects of dexamethasone on IL-22 production by ILC3s. We found that dexamethasone suppressed IL-23-mediated IL-22 production in homeostatic and activated human and mouse ILC3s. Dexamethasone did not modulate the IL-23 canonical signaling pathway of JAK2/STAT3, but did suppress phosphorylation of IkBa, an important regulator in NF-kB activation. These findings implicate NF-kB as a regulator of IL-22 production in ILC3s and have likely repercussions on GC treatment of IBD patients.