Emerging Immune Checkpoint Molecules on Cancer Cells: CD24 and CD200.

Cancer immunotherapy strategies are based on the utilization of immune checkpoint inhibitors to instigate an antitumor immune response. The efficacy of immune checkpoint blockade, directed at adaptive immune checkpoints, has been demonstrated in select cancer types. However, only a limited subset of patients has exhibited definitive outcomes characterized by a sustained response after discontinuation of therapy. Recent investigations have highlighted the significance of immune checkpoint molecules that are overexpressed in cancer cells and inhibit myeloid lineage immune cells within a tumor microenvironment. These checkpoints are identified as potential targets for anticancer immune responses. Notably, the immune checkpoint molecules CD24 and CD200 have garnered attention owing to their involvement in tumor immune evasion. CD24 and CD200 are overexpressed across diverse cancer types and serve as signaling checkpoints by engaging their respective receptors, Siglec-10 and CD200 receptor, which are expressed on tumor-associated myeloid cells. In this review, we summarized and discussed the latest advancements and insights into CD24 and CD200 as emergent immune checkpoint moieties, further delving into their therapeutic potentials for cancer treatment.

An Improved In Vitro Blood-Brain Barrier Model for the Evaluation of Drug Permeability Using Transwell with Shear Stress.

The development of drugs targeting the central nervous system (CNS) is challenging because of the presence of the Blood-Brain barrier (BBB). Developing physiologically relevant in vitro BBB models for evaluating drug permeability and predicting the activity of drug candidates is crucial. The transwell model is one of the most widely used in vitro BBB models. However, this model has limitations in mimicking in vivo conditions, particularly in the absence of shear stress. This study aimed to overcome the limitations of the transwell model using immortalized human endothelial cells (hCMEC/D3) by developing a novel dish design for an orbital shaker, providing shear stress. During optimization, we assessed cell layer integrity using trans-endothelial electrical resistance measurements and the % diffusion of lucifer yellow. The efflux transporter activity and mRNA expression of junctional proteins (claudin-5, occludin, and VE-cadherin) in the newly optimized model were verified. Additionally, the permeability of 14 compounds was evaluated and compared with published in vivo data. The cell-layer integrity was substantially increased using the newly designed annular shaking-dish model. The results demonstrate that our model provided robust conditions for evaluating the permeability of CNS drug candidates, potentially improving the reliability of in vitro BBB models in drug development.

Wistin Exerts an Anti-Inflammatory Effect via Nuclear Factor-κB and p38 Signaling Pathways in Lipopolysaccharide-Stimulated RAW264.7 Cells.

Inflammation is an immune response to cellular damage caused by various stimuli (internal or external) and is essential to human health. However, excessive inflammatory responses may be detrimental to the host. Considering that the existing drugs for the treatment of inflammatory diseases have various side effects, such as allergic reactions, stomach ulcers, and cardiovascular problems, there is a need for research on new anti-inflammatory agents with low toxicity and fewer side effects. As 4',6-dimethoxyisoflavone-7-O-ß-d-glucopyranoside (wistin) is a phytochemical that belongs to an isoflavonoid family, we investigated whether wistin could potentially serve as a novel anti-inflammatory agent. In this study, we found that wistin significantly reduced the production of nitric oxide and intracellular reactive oxygen species in lipopolysaccharide-stimulated RAW 264.7 cells. Moreover, wistin reduced the mRNA levels of pro-inflammatory enzymes (inducible nitric oxide synthase (iNOS) and cyclooxygenase (COX-2)) and cytokines (interleukin (IL)-1ß and IL-6) and significantly reduced the protein expression of pro-inflammatory enzymes (iNOS and COX-2). Furthermore, wistin reduced the activation of the nuclear factor-κB and p38 signaling pathways. Together, these results suggest that wistin is a prospective candidate for the development of anti-inflammatory drugs.

Physiological Roles of Apoptotic Cell Clearance: Beyond Immune Functions.

The clearance of apoptotic cells is known to be a critical step in maintaining tissue and organism homeostasis. This process is rapidly/promptly mediated by recruited or resident phagocytes. Phagocytes that engulf apoptotic cells have been closely linked to the release of anti-inflammatory cytokines to eliminate inflammatory responses. Defective clearance of apoptotic cells can cause severe inflammation and autoimmune responses due to secondary necrosis of apoptotic cells. Recently accumulated evidence indicates that apoptotic cells and their clearance have important physiological roles in addition to immune-related functions. Herein, we review the current understanding of the mechanisms and fundamental roles of apoptotic cell clearance and the beneficial roles of apoptotic cells in physiological processes such as differentiation and development.

Phytochemicals as Anti-Inflammatory Agents in Animal Models of Prevalent Inflammatory Diseases.

Phytochemicals are known to have anti-inflammatory effects in vitro and in vivo, such as in inflammatory disease model systems. Inflammation is an essential immune response to exogenous stimuli such as infection and injury. Although inflammation is a necessary host-defense mechanism, chronic inflammation is associated with the continuous local or systemic release of inflammatory mediators, non-cytokine mediators, such as ROS and NO, and inflammatory cytokines are strongly implicated in the pathogenesis of various inflammatory disorders. Phytochemicals that exhibit anti-inflammatory mechanisms that reduce sustained inflammation could be therapeutic candidates for various inflammatory diseases. These phytochemicals act by modulating several main inflammatory signaling pathways, including NF-κB, MAPKs, STAT, and Nrf-2 signaling. Here, we discuss the characteristics of phytochemicals that possess anti-inflammatory activities in various chronic inflammatory diseases and review the molecular signaling pathways altered by these anti-inflammatory phytochemicals, with a focus on transcription factor pathways. Furthermore, to evaluate the phytochemicals as drug candidates, we translate the effective doses of phytochemicals in mice or rat disease models into the human-relevant equivalent and compare the human-relevant equivalent doses of several phytochemicals with current anti-inflammatory drugs doses used in different types of chronic inflammatory diseases.

Apoptotic cell clearance in the tumor microenvironment: a potential cancer therapeutic target.

Millions of cells in the human body undergo apoptosis not only under normal physiological conditions but also under pathological conditions such as infection or other diseases related to acute tissue injury. Swift apoptotic cell clearance is essential for tissue homeostasis. Defective clearance of dead cells is linked to pathogenesis of diseases such as inflammatory diseases, atherosclerosis, neurological disease, and cancer. Significance of apoptotic cell clearance has been emerging as an interesting field for disease treatment. Efficient apoptotic cell clearance plays an important role in reducing inflammation through the suppression of inappropriate inflammatory responses under healthy and diseased conditions. However, apoptotic cell clearance related to cancer pathogenesis is more complex in tumor microenvironments. Chronic inflammation resulting from the failure of apoptotic cell clearance can contribute to tumor progression. Conversely, tumor cells can exploit the anti-inflammatory effect of apoptotic cell clearance to generate an immunosuppressive tumor microenvironment. In this review, focus is on the current understanding of apoptotic cell clearance in the tumor microenvironment. Furthermore, we discuss how signaling molecules (PtdSer and PtdSer recognition receptor) mediating apoptotic cell clearance are aberrantly expressed in the tumor microenvironment and their current development state as potential therapeutic targets for clinical cancer therapy.

Understanding the Role of the BAI Subfamily of Adhesion G Protein-Coupled Receptors (GPCRs) in Pathological and Physiological Conditions.

Brain-specific angiogenesis inhibitors (BAIs) 1, 2, and 3 are members of the adhesion G protein-coupled receptors, subfamily B, which share a conserved seven-transmembrane structure and an N-terminal extracellular domain. In cell- and animal-based studies, these receptors have been shown to play diverse roles under physiological and pathological conditions. BAI1 is an engulfment receptor and performs major functions in apoptotic-cell clearance and interacts (as a pattern recognition receptor) with pathogen components. BAI1 and -3 also participate in myoblast fusion. Furthermore, BAI1⁻3 have been linked to tumor progression and neurological diseases. In this review, we summarize the current understanding of the functions of BAI1⁻3 in pathological and physiological conditions and discuss future directions in terms of the importance of BAIs as pharmacological targets in diseases.

Structure-Based Classification and Anti-Cancer Effects of Plant Metabolites.

A variety of malignant cancers affect the global human population. Although a wide variety of approaches to cancer treatment have been studied and used clinically (surgery, radiotherapy, chemotherapy, and immunotherapy), the toxic side effects of cancer therapies have a negative impact on patients and impede progress in conquering cancer. Plant metabolites are emerging as new leads for anti-cancer drug development. This review summarizes these plant metabolites with regard to their structures and the types of cancer against which they show activity, organized by the organ or tissues in which each cancer forms. This information will be helpful for understanding the current state of knowledge of the anti-cancer effects of various plant metabolites against major types of cancer for the further development of novel anti-cancer drugs.

Anti­inflammatory effect of quercetin and galangin in LPS­stimulated RAW264.7 macrophages and DNCB­induced atopic dermatitis animal models.

Flavonols are compounds that have been shown to possess potent anti­inflammatory effects in cellular and animal models of inflammation. In the present study, the anti­inflammatory effects and mechanisms of two natural flavonols, quercetin and galangin, in lipopolysaccharide (LPS)­stimulated RAW264.7 macrophages were investigated. It was identified that quercetin and galangin markedly reduced the production of nitric oxide (NO), inducible NO synthase and interleukin­6, and the nuclear translocation of nuclear factor­κB (NF­κB). In addition, LPS­induced activation of extracellular signal­regulated kinase 1/2 (Erk1/2) and c­Jun N­terminal kinase (JNK) was suppressed by quercetin and galangin. Taken together, these data implied that NF­κB, Erk1/2 and JNK may be potential molecular targets of quercetin and galangin in an LPS­induced inflammatory response. Subsequently, the effects of oral administration of quercetin or galangin, either alone or in combination, in a 2,4­dinitrochlorobenzene­induced atopic dermatitis (AD) mouse model were investigated. As a result, measurements of ear thickness and the levels of serum immunoglobulin E, and histological analysis revealed that the two flavonols led to a decrease in inflammation, whereas, in combination, they were even more effective. These results suggested that quercetin and galangin may be promising therapeutic agents for AD. Additionally, their combination may be a novel therapeutic strategy for the prevention of AD.

Methyl 3-(3-(4-(2,4,4-Trimethylpentan-2-yl)phenoxy)-propanamido)benzoate as a Novel and Dual Malate Dehydrogenase (MDH) 1/2 Inhibitor Targeting Cancer Metabolism.

Previously, we reported a hypoxia-inducible factor (HIF)-1 inhibitor LW6 containing an (aryloxyacetylamino)benzoic acid moiety inhibits malate dehydrogenase 2 (MDH2) using a chemical biology approach. Structure-activity relationship studies on a series of (aryloxyacetylamino)benzoic acids identified selective MDH1, MDH2, and dual inhibitors, which were used to study the relationship between MDH enzyme activity and HIF-1 inhibition. We hypothesized that dual inhibition of MDH1 and MDH2 might be a powerful approach to target cancer metabolism and selected methyl-3-(3-(4-(2,4,4-trimethylpentan-2-yl)phenoxy)propanamido)-benzoate (16c) as the most potent dual inhibitor. Kinetic studies revealed that compound 16c competitively inhibited MDH1 and MDH2. Compound 16c inhibited mitochondrial respiration and hypoxia-induced HIF-1α accumulation. In xenograft assays using HCT116 cells, compound 16c demonstrated significant in vivo antitumor efficacy. This finding provides concrete evidence that inhibition of both MDH1 and MDH2 may provide a valuable platform for developing novel therapeutics that target cancer metabolism and tumor growth.

Ixeris dentata (Thunb. Ex Thunb.) Nakai Extract Inhibits Proliferation and Induces Apoptosis in Breast Cancer Cells through Akt/NF-κB Pathways.

Ixeris dentata (Thunb. Ex Thunb.) Nakai (ID) exhibits various physiological activities, and its related plant derived-products are expected to represent promising cancer therapeutic agents. However, the anticancer effects of ID extract on breast cancer cells classified as estrogen receptor (ER), progesterone receptor (PR), and human epidermal growth factor receptor 2 (HER2) are still unknown. In this study, we investigated the anti-cancer effects and analyzed the molecular mechanism of ID extract in T47D, MCF-7 (ER-, PR-positive, HER2-negative), SK-BR-3(ER-, PR-negative, HER2-positive), and MDA-MB-231 (Triple-negative) through in vitro studies. Additionally, we examined its anti-tumor effects through in vivo studies. Our findings indicated that ID extract-induced apoptosis was mediated via various survival pathways on four breast cancer cells by identifying the factors including Bcl-2 family, phospho-Akt and phospho-nuclear factor-κB (NF-κB). Based on in vitro findings that induced apoptosis via Akt-NF-κB signaling, we investigated the effects of ID extract on mice bearing MDA-MB-231 cells. The results showed that ID extract significantly decreased MDA-MB-231 tumor volume and weight via inducing apoptosis by suppressing phospho-Akt. Overall, these results indicate that ID extract induces apoptosis through the Akt-NFκB signaling pathway in MDA-MB-231 breast cancer cells and tumors, and it may serve as a therapeutic agent for triple-negative human breast cancer.

Anticancer effects of Ixeris dentata (Thunb. ex Thunb.) nakai extract on human melanoma cells A375P and A375SM.

ETHNOPHARMACOLOGICAL RELEVANCE: The plant species Taraxacum coreanum (TC), Youngia sonchifolia (YS), and Ixeris dentata (ID) belong to the family Compositae and are used for medicinal purposes in traditional medicine. However, the anticancer effects of TC, YS, and ID extracts and the underlying molecular mechanisms in melanoma cells have not been elucidated. AIM OF THE STUDY: To investigate the potential anticancer effects of TC, YS, and ID extracts on human melanoma cells and explore the potential pharmacological mechanisms in vitro and in vivo. MATERIALS AND METHODS: In this comparative study, we investigated the effects of TC, YS, and ID extracts on cell proliferation in human melanoma A375P and A375SM cells using MTT[3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] assays. Apoptotic cells were detected by 4',6-diamidino-2-phenylinodole (DAPI) staining. We also investigated whether the growth-inhibitory effects were associated with the induction of apoptosis and whether the mechanisms of cell death were the result of signaling molecules such as p53, Bax, Bcl-2, caspase-9, Poly-ADP ribose polymerase (PARP), and Erk (Extracellular signal-regulated protein kinase) 1/2. The in vivo antitumor effects were evaluated by measuring the tumor volume and weight and performing Terminal deoxynucleotidyl transferase (TdT) dUTP Nick End Labeling (TUNEL) assay and immunohistochemistry (IHC) in tumor xenograft models. RESULTS: TC, YS, and ID extracts effectively inhibited the growth of A375P and A375SM cells. In addition, several apoptotic events were observed following treatment, including DNA fragmentation and chromatin condensation by DAPI staining. The extracts increased p53, Bax, cleaved-caspase-9 and cleaved-PARP expression, whereas the expression of Bcl-2 was decreased in both cell lines. Furthermore, ID extract significantly inhibited the activation of Erk1/2 in both cell lines. Among the three extracts, ID had the strongest apoptotic effects. The administration of ID extract to mice inhibited tumor growth without any toxicity following 4 weeks of treatment. This extract increased the expression of apoptotic cells and p53 protein and decreased phospho-Erk1/2 protein. CONCLUSION: TC, YS, and ID extracts suppress the growth of human melanoma cells through apoptosis. Among these extracts, ID has the strongest anticancer and apoptotic effects. It induces apoptosis through the inhibition of Erk1/2 in A375P and A375SM human melanoma cells and in tumor xenograft models and may be a potential chemotherapeutic agent against melanoma.

Anticancer Effect of Fucoidan on DU-145 Prostate Cancer Cells through Inhibition of PI3K/Akt and MAPK Pathway Expression.

In this study, we showed that PI3K/Akt signaling mediates fucoidan's anticancer effects on prostate cancer cells, including suppression of proliferation. Fucoidan significantly decreased viability of DU-145 cancer cells in a concentration-dependent manner as shown by MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] assay. The drug also significantly increased chromatin condensation, which indicates apoptosis, in a concentration-dependent manner as shown by DAPI (4',6-diamidino-2-phenylindole) staining. Fucoidan increased expression of Bax, cleaved poly-ADP ribose polymerase and cleaved caspase-9, and decreased of the Bcl-2, p-Akt, p-PI3K, p-P38, and p-ERK in a concentration-dependent manner. In vivo, fucoidan (at 5 and 10 mg/kg) significantly decreased tumor volume, and increased apoptosis as assessed by the TUNEL (terminal deoxynucleotidyl transferase dUTP nick end labeling) assay, confirming the tumor inhibitory effect. The drug also increased expression of p-Akt and p-ERK as shown by immunohistochemistry staining. Therefore, fucoidan may be a promising cancer preventive medicine due to its growth inhibitory effects and induction of apoptosis in human prostate cancer cells.

Antitumor and apoptosis-inducing effects of α-mangostin extracted from the pericarp of the mangosteen fruit (Garcinia mangostana L.)in YD-15 tongue mucoepidermoid carcinoma cells.

α-mangostin is a dietary xanthone which has been shown to have antioxidant, anti-allergic, antiviral, antibacterial, anti-inflammatory and anticancer effects in various types of human cancer cells. In the present study, we aimed to elucidate the molecular mechanisms responsible for the apoptosis-inducing effects of α-mangostin on YD-15 tongue mucoepidermoid carcinoma cells. The results from MTT assays revealed that cell proliferation significantly decreased in a dose-dependent manner in the cells treated with α-mangostin. DAPI staining illustrated that chromatin condensation in the cells treated with 15 µM α-mangostin was far greater than that in the untreated cells. Flow cytometric analysis indicated that α-mangostin suppressed YD-15 cell viability by inducing apoptosis and promoting cell cycle arrest in the sub-G1 phase. Western blot analysis of various signaling molecules revealed that α-mangostin targeted the extracellular signal­regulated kinase 1/2 (ERK1/2) and p38 mitogen-activated protein kinase (MAPK) signaling pathways through the inhibition of ERK1/2 and p38 phosphorylation in a dose­dependent manner. α-mangostin also increased the levels of Bax (pro-apoptotic), cleaved caspase-3, cleaved caspase-9 and cleaved-poly(ADP-ribose) polymerase (PARP), whereas the levels of the anti-apoptotic factors, Bcl-2 and c-myc, decreased in a dose-dependent manner. The anticancer effects of α-mangostin were also investigated in a tumor xenograft mouse model. The α-mangostin-treated nude mice bearing YD-15 tumor xenografts exhibited a significantly reduced tumor volume and tumor weight due to the potent promoting effects of α-mangostin on cancer cell apoptosis, as determined by TUNEL assay. Immunohistochemical analysis revealed that the level of cleaved caspase-3 increased, whereas the Ki-67, p-ERK1/2 and p-p38 levels decreased in the α-mangostin­treated mice. Taken together, the findings of our study indicate that α-mangostin induces the apoptosis of YD-15 tongue carcinoma cells through the ERK1/2 and p38 MAPK signaling pathways.