Natural Product-Based Glycolysis Inhibitors as a Therapeutic Strategy for Epidermal Growth Factor Receptor-Tyrosine Kinase Inhibitor-Resistant Non-Small Cell Lung Cancer.

Non-small cell lung cancer (NSCLC) is a leading cause of cancer-related deaths worldwide. Targeted therapy against the epidermal growth factor receptor (EGFR) is a promising treatment approach for NSCLC. However, resistance to EGFR tyrosine kinase inhibitors (TKIs) remains a major challenge in its clinical management. EGFR mutation elevates the expression of hypoxia-inducible factor-1 alpha to upregulate the production of glycolytic enzymes, increasing glycolysis and tumor resistance. The inhibition of glycolysis can be a potential strategy for overcoming EGFR-TKI resistance and enhancing the effectiveness of EGFR-TKIs. In this review, we specifically explored the effectiveness of pyruvate dehydrogenase kinase inhibitors and lactate dehydrogenase A inhibitors in combating EGFR-TKI resistance. The aim was to summarize the effects of these natural products in preclinical NSCLC models to provide a comprehensive understanding of the potential therapeutic effects. The study findings suggest that natural products can be promising inhibitors of glycolytic enzymes for the treatment of EGFR-TKI-resistant NSCLC. Further investigations through preclinical and clinical studies are required to validate the efficacy of natural product-based glycolytic inhibitors as innovative therapeutic modalities for NSCLC.

Single-Cell RNA Sequencing Reveals Immuno-Oncology Characteristics of Tumor-Infiltrating T Lymphocytes in Photodynamic Therapy-Treated Colorectal Cancer Mouse Model.

Photodynamic therapy (PDT) has shown promise in reducing metastatic colorectal cancer (CRC); however, the underlying mechanisms remain unclear. Modulating tumor-infiltrating immune cells by PDT may be achieved, which requires the characterization of immune cell populations in the tumor microenvironment by single-cell RNA sequencing (scRNA-seq). Here, we determined the effect of Chlorin e6 (Ce6)-mediated PDT on tumor-infiltrating T cells using scRNA-seq analysis. We used a humanized programmed death-1/programmed death ligand 1 (PD-1/PD-L1) MC38 cell allograft mouse model, considering its potential as an immunogenic cancer model and in combination with PD-1/PD-L1 immune checkpoint blockade. PDT treatment significantly reduced tumor growth in mice containing hPD-1/PD-L1 MC38 tumors. scRNA-seq analysis revealed that the PDT group had increased levels of CD8+ activated T cells and CD8+ cytotoxic T cells, but decreased levels of exhausted CD8+ T cells. PDT treatment also enhanced the infiltration of CD8+ T cells into tumors and increased the production of key effector molecules, including granzyme B and perforin 1. These findings provide insight into immune-therapeutic modulation for CRC patients and highlight the potential of PDT in overcoming immune evasion and enhancing antitumor immunity.

Antitumor Effect of Korean Red Ginseng through Blockade of PD-1/PD-L1 Interaction in a Humanized PD-L1 Knock-In MC38 Cancer Mouse Model.

Blocking immune checkpoints, programmed death-1 (PD-1) and its ligand PD-L1, has proven a promising anticancer strategy for enhancing cytotoxic T cell activity. Although we previously demonstrated that ginsenoside Rg3, Rh2, and compound K block the interaction of PD-1 and PD-L1, the antitumor effect through blockade of this interaction by Korean Red Ginseng alone is unknown. Therefore, we determined the effects of Korean Red Ginseng extract (RGE) on the PD-1/PD-L1 interaction and its antitumor effects using a humanized PD-1/PD-L1-expressing colorectal cancer (CRC) mouse model. RGE significantly blocked the interaction between human PD-1 and PD-L1 in a competitive ELISA. The CD8+ T cell-mediated tumor cell killing effect of RGE was evaluated using murine hPD-L1-expressing MC38 cells and tumor-infiltrating hPD-1-expressing CD8+ T cells isolated from hPD-L1 MC38 tumor-bearing hPD-1 mice. RGE also reduced the survival of hPD-L1 MC38 cells in a cell co-culture system using tumor-infiltrating CD8+ T cells as effector cells combined with hPD-L1 MC38 target cells. RGE or Keytruda (positive control) treatment markedly suppressed the growth of hPD-L1 MC38 allograft tumors, increased CD8+ T cell infiltration into tumors, and enhanced the production of Granzyme B. RGE exhibits anticancer effects through the PD-1/PD-L1 blockade, which warrants its further development as an immunotherapy.

Mulberry Component Kuwanon C Exerts Potent Therapeutic Efficacy In Vitro against COVID-19 by Blocking the SARS-CoV-2 Spike S1 RBD:ACE2 Receptor Interaction.

There has been an immense effort by global pharmaceutical companies to develop anti-COVID-19 drugs, including small molecule-based RNA replication inhibitors via drug repositioning and antibody-based spike protein blockers related to cell entry by SARS-CoV-2. However, several limitations to their clinical use have emerged in addition to a lack of progress in the development of small molecule-based cell entry inhibitors from natural products. In this study, we tested the effectiveness of kuwanon C (KC), which has mainly been researched using in silico docking simulation and can serve as an effective building block for developing anti-COVID-19 drugs, in blocking the spike S1 RBD:ACE2 receptor interaction. KC is a natural product derived from Morus alba L., commonly known as mulberry, which has known antiviral efficacy. Molecular interaction studies using competitive ELISA and the BLItz system revealed that KC targets both the spike S1 RBD and the ACE2 receptor, successfully disrupting their interaction, as supported by the in silico docking simulation. Furthermore, we established a mechanism of action by observing how KC prevents the infection of SARS-CoV-2 spike pseudotyped virus in ACE2/TPRSS2-overexpressing HEK293T cells. Finally, we demonstrated that KC inhibits clinical isolates of SARS-CoV-2 in Vero cells. Future combinations of small molecule-based cell entry inhibitors, such as KC, with the currently prescribed RNA replication inhibitors are anticipated to significantly enhance the efficacy of COVID-19 therapies.

Geraniin Inhibits the Entry of SARS-CoV-2 by Blocking the Interaction between Spike Protein RBD and Human ACE2 Receptor.

The coronavirus disease 2019 (COVID-19) pandemic is caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Despite the development of vaccines, the emergence of SARS-CoV-2 variants and the absence of effective therapeutics demand the continual investigation of COVID-19. Natural products containing active ingredients may be good therapeutic candidates. Here, we investigated the effectiveness of geraniin, the main ingredient in medical plants Elaeocarpus sylvestris var. ellipticus and Nephelium lappaceum, for treating COVID-19. The SARS-CoV-2 spike protein binds to the human angiotensin-converting enzyme 2 (hACE2) receptor to initiate virus entry into cells; viral entry may be an important target of COVID-19 therapeutics. Geraniin was found to effectively block the binding between the SARS-CoV-2 spike protein and hACE2 receptor in competitive enzyme-linked immunosorbent assay, suggesting that geraniin might inhibit the entry of SARS-CoV-2 into human epithelial cells. Geraniin also demonstrated a high affinity to both proteins despite a relatively lower equilibrium dissociation constant (KD) for the spike protein (0.63 µM) than hACE2 receptor (1.12 µM), according to biolayer interferometry-based analysis. In silico analysis indicated geraniin's interaction with the residues functionally important in the binding between the two proteins. Thus, geraniin is a promising therapeutic agent for COVID-19 by blocking SARS-CoV-2's entry into human cells.

Kaempferol and Its Glycoside, Kaempferol 7-O-Rhamnoside, Inhibit PD-1/PD-L1 Interaction In Vitro.

Kaempferol (KO) and kaempferol 7-O-rhamnoside (KR) are natural products from various oriental herbs such as Geranii Herba. Previous studies have reported some biological activities of KO and KR; however, their effects on PD-1/PD-L1 interaction have not been reported yet. To elucidate their inhibitory activities on PD-1/PD-L1 protein-protein interaction (PPI), biochemical assays including competitive ELISA and biolayer interferometry (BLI) systems were performed. Cellular PD-1/PD-L1 blocking activity was measured in a co-culture system with PD-1 Jurkat and PD-L1/aAPC CHO-K1 cells by T-cell receptor (TCR) activation-induced nuclear factor of activated T cells (NFAT)-luciferase reporter assay. The detailed binding mode of action was simulated by an in silico docking study and pharmacophore analysis. Competitive ELISA revealed that KO and its glycoside KR significantly inhibited PD-1/PD-L1 interaction. Cellular PD-1/PD-L1 blocking activity was monitored by KO and KR at non-cytotoxic concentration. Surface plasmon resonance (SPR) and biolayer interferometry (BLI) analysis suggested the binding affinity and direct inhibition of KR against PD-1/PD-L1. An in silico docking simulation determined the detailed mode of binding of KR to PD-1/PD-L1. Collectively, these results suggest that KR could be developed as a potent small molecule inhibitor for PD-1/PD-L1 blockade.

Inhibition of Programmed Death Receptor-1/Programmed Death Ligand-1 Interactions by Ginsenoside Metabolites.

Evidence suggests that programmed death receptor-1/programmed death ligand-1 (PD-1/PD-L1) targeted inhibitors act as an immune checkpoint blockade, indicating that these compounds may be useful in cancer immunotherapy by inhibiting the immune response between T-cells and tumors. Previous studies have shown that ginsenosides can regulate the expression of PD-1 and PD-L1 in target diseases; however, it remains unknown whether ginsenosides act as a blockade of PD-1/PD-L1 interactions. In this study, we used competitive ELISA to investigate 12 ginsenosides for their ability to block PD-1/PD-L1 interactions. In addition, we performed a protein-ligand docking simulation and examined the hydrophobic interactions and hydrogen bonds formed at the interfaces between the ginsenosides and PD-L1/PD-1. Eight out of the 12 ginsenosides studied showed inhibition of PD-1/PD-L1 interactions at 35% at the maximum concentration (1 µM). Among them, Rg3 and Compound K (C-K) demonstrated the highest inhibitory effects. Rg3 and C-K were further identified for their interaction efficacy with PD-1/PD-L1, which supported our results demonstrating the blocking activity of these compounds against PD-1/PD-L1 binding interactions. Collectively, our findings suggest that some ginsenosides, including Rg3 and C-K, inhibit PD-1/PD-L1 binding interactions. Therefore, these compounds may prove useful as part of an overall immuno-oncological strategy.

Immune Checkpoint PD-1/PD-L1 CTLA-4/CD80 are Blocked by Rhus verniciflua Stokes and its Active Compounds.

The bark of Rhus verniciflua Stokes (RVS) has been used to treat cancer in Korean herbal medicine. When we screened for PD-1 and CTLA-4 immune checkpoint inhibitors (PD-1/PD-L1 CTLA-4/CD80) from around 800 herbal extracts using competitive Enzyme-Linked Immunosorbent Assay (ELISA), we found that RVS blocked both the PD-1/PD-L1 and the CTLA-4/CD80 interactions. To identify the active compounds from RVS, we performed bioactivity-guided fractionation, and the ethyl acetate (EtOAc) fraction of RVS proved to be the most effective at blocking the PD-1/PD-L1 and CTLA-4/CD80 interactions. In addition, we isolated and identified 20 major compounds in the EtOAc fraction of RVS and then examined the blocking effects of these 20 compounds on PD-1/PD-L1 and CTLA-4/CD80. Among them, four compounds [eriodictyol (7) > fisetin (9) > quercetin (18) > liquiritigenin (13)] blocked the interaction of PD-1/PD-L1 on competitive ELISA. In addition, four different compounds [protocatechuic acid (2) > caffeic acid (19) > taxifolin (5) > butin (6)] blocked the interaction of CTLA-4/CD80. Our findings suggest that RVS and its components could be used as a potential immune checkpoint inhibitor blockade and could be developed for immuno-oncological therapeutics.

Bee Venom Phospholipase A2, a Novel Foxp3+ Regulatory T Cell Inducer, Protects Dopaminergic Neurons by Modulating Neuroinflammatory Responses in a Mouse Model of Parkinson's Disease.

Foxp3-expressing CD4(+) regulatory T cells (Tregs) are vital for maintaining immune tolerance in animal models of various immune diseases. In the present study, we demonstrated that bee venom phospholipase A2 (bvPLA2) is the major BV compound capable of inducing Treg expansion and promotes the survival of dopaminergic neurons in the 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine mouse model of Parkinson's disease. We associated this neuroprotective effect of bvPLA2 with microglial deactivation and reduction of CD4(+) T cell infiltration. Interestingly, bvPLA2 had no effect on mice depleted of Tregs by injecting anti-CD25 Ab. This finding indicated that Treg-mediated modulation of peripheral immune tolerance is strongly involved in the neuroprotective effects of bvPLA2. Furthermore, our results showed that bvPLA2 directly bound to CD206 on dendritic cells and consequently promoted the secretion of PGE2, which resulted in Treg differentiation via PGE2 (EP2) receptor signaling in Foxp3(-)CD4(+) T cells. These observations suggest that bvPLA2-CD206-PGE2-EP2 signaling promotes immune tolerance through Treg differentiation and contributes to the prevention of various neurodegenerative diseases, including Parkinson's disease.

Neuroprotective effects of bee venom phospholipase A2 in the 3xTg AD mouse model of Alzheimer's disease.

BACKGROUND: Alzheimer's disease (AD) is a severe neuroinflammatory disease. CD4(+)Foxp3(+) regulatory T cells (Tregs) modulate various inflammatory diseases via suppressing Th cell activation. There are increasing evidences that Tregs have beneficial roles in neurodegenerative diseases. Previously, we found the population of Treg cells was significantly increased by bee venom phospholipase A2 (bvPLA2) treatment in vivo and in vitro. METHODS: To examine the effects of bvPLA2 on AD, bvPLA2 was administered to 3xTg-AD mice, mouse model of Alzheimer's disease. The levels of amyloid beta (Aß) deposits in the hippocampus, glucose metabolism in the brain, microglia activation, and CD4(+) T cell infiltration were analyzed to evaluate the neuroprotective effect of bvPLA2. RESULTS: bvPLA2 treatment significantly enhanced the cognitive function of the 3xTg-AD mice and increased glucose metabolism, as assessed with 18F-2 fluoro-2-deoxy-D-glucose ([F-18] FDG) positron emission tomography (PET). The levels of Aß deposits in the hippocampus were dramatically decreased by bvPLA2 treatment. This neuroprotective effect of bvPLA2 was associated with microglial deactivation and reduction in CD4(+) T cell infiltration. Interestingly, the neuroprotective effects of bvPLA2 were abolished in Treg-depleted mice. CONCLUSIONS: The present studies strongly suggest that the increase of Treg population by bvPLA2 treatment might inhibit progression of AD in the 3xTg AD mice.