Synthesis and Cytotoxicity against K562 Cells of 3-O-Angeloyl-20-O-acetyl Ingenol,a Derivative of Ingenol Mebutate

Ingenol mebutate possesses significant cytotoxicity and is clinically used to treat actinic keratosis. However, ingenol mebutate undergoes acyl migration which affects its bioactivity. Compound 3-O-angeloyl-20-O-acetyl ingenol (AAI, also known as 20-O-acetyl-ingenol-3-angelate or PEP008) is a synthetic derivative of ingenol mebutate. In this work, we report the AAI synthesis details and demonstrate AAI has higher cytotoxicity than ingenol mebutate in a chronic myeloid leukemia K562 cell line. Our data indicate that the increased activity of AAI originates from the improved intracellular stability of AAI rather than the increased binding affinity between AAI and the target protein protein kinase Cδ (PKCδ). AAI inhibits cell proliferation, induces G2/M phase arrest, disrupts the mitochondrial membrane potential, and stimulates apoptosis, as well as necrosis in K562 cells. Similar to ingenol mebutate, AAI activates PKCδ and extracellular signal regulated kinase (ERK), and inactivates protein kinase B (AKT). Furthermore, AAI also inhibits JAK/STAT3 pathway. Altogether, our studies show that ingenol derivative AAI is cytotoxic to K562 cells and modulates PKCδ/ERK, JAK/STAT3, and AKT signaling pathways. Our work suggests that AAI may be a new candidate of chemotherapeutic agent.     

JAK Inhibition Prevents DNA Damage and Apoptosis in Testicular Ischemia-Reperfusion Injury via Modulation of the ATM/ATR/Chk Pathway

Testicular ischemia reperfusion injury (tIRI) causes oxidative stress-induced DNA damage leading to germ cell apoptosis (GCA). The aim of the study is to establish a direct link between JAK2 activation and the DNA damage response (DDR) signaling pathways and their role in tIRI-induced GCA using AG490, a JAK2 specific inhibitor. Male Sprague Dawley rats (n = 36) were divided into three groups: sham, unilateral tIRI and tIRI + AG490 (40 mg/kg). During tIRI, augmentation in the phosphorylation levels of the JAK2/STAT1/STAT3 was measured by immunohistochemistry. Observed spermatogenic arrest was explained by the presence of considerable levels of DSB, AP sites and 8OHdG and activation of caspase 9, caspase 3 and PARP, which were measured by colorimetric assays and TUNEL. The ATM/Chk2/H2AX and ATR/Chk1 pathways were also activated as judged by their increased phosphorylation using Western blot. These observations were all prevented by AG490 inhibition of JAK2 activity. Our findings demonstrate that JAK2 regulates tIRI-induced GCA, oxidative DNA damage and activation of the ATM/Chk2/H2AX and ATR/Chk1 DDR pathways, but the cell made the apoptosis decision despite DDR efforts.     

Rapamycin-Induced Apoptosis in HGF-Stimulated Lens Epithelial Cells by AKT/mTOR,ERK and JAK2/STAT3 Pathways

Hepatocyte growth factor (HGF) induced the proliferation of lens epithelial cells (LECs) and may be a major cause of posterior capsule opacification (PCO), which is the most frequent postoperative complication of cataract surgery. To date, several agents that can block LECs proliferation have been studied, but none have been used in clinic. Recently, accumulating evidence has suggested rapamycin, the inhibitor of mTOR (mammalian target of Rapamycin), was associated with the induction of apoptosis in LECs. The purpose of our study was to investigate the potential effects of rapamycin on HGF-induced LECs and the underlying mechanisms by which rapamycin exerted its actions. Using cell proliferation, cell viability and flow cytometric apoptosis assays, we found that rapamycin potently not only suppressed proliferation but also induced the apoptosis of LECs in a dose-dependent manner under HGF administration. Further investigation of the underlying mechanism using siRNA transfection revealed that rapamycin could promote apoptosis of LECs via inhibiting HGF-induced phosphorylation of AKT/mTOR, ERK and JAK2/STAT3 signaling molecules. Moreover, the forced expression of AKT, ERK and STAT3 could induce a significant suppression of apoptosis in these cells after treatment of rapamycin. Together, these findings suggested that rapamycin-induced apoptosis in HGF-stimulated LECs is accompanied by inhibition of AKT/mTOR, ERK and JAK2/STAT3 pathways, which supports its use to inhibit PCO in preclinical studies and provides theoretical foundation for future possible practice.     

ZnO nanoparticle-induced oxidative stress triggers apoptosis by activating JNK signaling pathway in cultured primary astrocytes

It has been documented in in vitro studies that zinc oxide nanoparticles (ZnO NPs) are capable of inducing oxidative stress, which plays a crucial role in ZnO NP-mediated apoptosis. However, the underlying molecular mechanism of apoptosis in neurocytes induced by ZnO NP exposure was not fully elucidated. In this study, we investigated the potential mechanisms of apoptosis provoked by ZnO NPs in cultured primary astrocytes by exploring the molecular signaling pathways triggered after ZnO NP exposure. ZnO NP exposure was found to reduce cell viability in MTT assays, increase lactate dehydrogenase (LDH) release, stimulate intracellular reactive oxygen species (ROS) generation, and elicit caspase-3 activation in a dose- and time-dependent manner. Apoptosis occurred after ZnO NP exposure as evidenced by nuclear condensation and poly(ADP-ribose) polymerase-1 (PARP) cleavage. A decrease in mitochondrial membrane potential (MMP) with a concomitant increase in the expression of Bax/Bcl-2 ratio suggested that the mitochondria also mediated the pathway involved in ZnO NP-induced apoptosis. In addition, exposure of the cultured cells to ZnO NPs led to phosphorylation of c-Jun N-terminal kinase (JNK), extracellular signal-related kinase (ERK), and p38 mitogen-activated protein kinase (p38 MAPK). Moreover, JNK inhibitor (SP600125) significantly reduced ZnO NP-induced cleaved PARP and cleaved caspase-3 expression, but not ERK inhibitor (U0126) or p38 MAPK inhibitor (SB203580), indicating that JNK signaling pathway is involved in ZnO NP-induced apoptosis in primary astrocytes.     

Role of JAK/STAT in Interstitial Lung Diseases; Molecular and Cellular Mechanisms

Interstitial lung diseases (ILDs) comprise different fibrotic lung disorders characterized by cellular proliferation, interstitial inflammation, and fibrosis. The JAK/STAT molecular pathway is activated under the interaction of a broad number of profibrotic/pro-inflammatory cytokines, such as IL-6, IL-11, and IL-13, among others, which are increased in different ILDs. Similarly, several growth factors over-expressed in ILDs, such as platelet-derived growth factor (PDGF), transforming growth factor β1 (TGF-β1), and fibroblast growth factor (FGF) activate JAK/STAT by canonical or non-canonical pathways, which indicates a predominant role of JAK/STAT in ILDs. Between the different JAK/STAT isoforms, it appears that JAK2/STAT3 are predominant, initiating cellular changes observed in ILDs. This review analyzes the expression and distribution of different JAK/STAT isoforms in ILDs lung tissue and different cell types related to ILDs, such as lung fibroblasts and alveolar epithelial type II cells and analyzes JAK/STAT activation. The effect of JAK/STAT phosphorylation on cellular fibrotic processes, such as proliferation, senescence, autophagy, endoplasmic reticulum stress, or epithelial/fibroblast to mesenchymal transition will be described. The small molecules directed to inhibit JAK/STAT activation were assayed in vitro and in in vivo models of pulmonary fibrosis, and different JAK inhibitors are currently approved for myeloproliferative disorders. Recent evidence indicates that JAK inhibitors or monoclonal antibodies directed to block IL-6 are used as compassionate use to attenuate the excessive inflammation and lung fibrosis related to SARS-CoV-2 virus. These altogether indicate that JAK/STAT pathway is an attractive target to be proven in future clinical trials of lung fibrotic disorders.     

Metformin Prevents Renal Fibrosis in Mice with Unilateral Ureteral Obstruction and Inhibits Ang II-Induced ECM Production in Renal Fibroblasts

Renal fibrosis is the final common pathway of chronic kidney disease (CKD), and no effective medication is available clinically for managing its progression. Metformin was initially developed as an anti-diabetic drug and recently gained attention for its potential in the treatment of other diseases. In this study, we investigated its effects on renal fibrosis in a mouse model of unilateral ureteral obstruction (UUO) in vivo and in angiotensin II (Ang II)–treated renal fibroblast NRK-49F cells in vitro. Our data showed that UUO induced renal fibrosis and combined with the activation of ERK signaling, the upregulation of fibronectin, collagen I, and transforming growth factor-β (TGF-β). The administration of metformin inhibited the activation of ERK signaling and attenuated the production of extracellular matrix (ECM) proteins and collagen deposition in the obstructed kidneys. In cultured renal fibroblasts, Ang II increased the expression of fibronectin and collagen I and also activated ERK signaling and TGF-β in a time-dependent manner. Pretreatment of the cells with metformin blocked Ang II–induced ERK signaling activation and ECM overproduction. Our results show that metformin prevents renal fibrosis, possibly through the inhibition of ERK signaling, and may be a novel strategy for the treatment of renal fibrosis.     

What Do We Have to Know about PD-L1 Expression in Prostate Cancer? A Systematic Literature Review. Part 3: PD-L1, Intracellular Signaling Pathways and Tumor Microenvironment

The tumor microenvironment (TME) includes immune (T, B, NK, dendritic), stromal, mesenchymal, endothelial, adipocytic cells, extracellular matrix, and cytokines/chemokines/soluble factors regulating various intracellular signaling pathways (ISP) in tumor cells. TME influences the survival/progression of prostate cancer (PC), enabling tumor cell immune-evasion also through the activation of the PD-1/PD-L1 axis. We have performed a systematic literature review according to the PRISMA guidelines, to investigate how the PD-1/PD-L1 pathway is influenced by TME and ISPs. Tumor immune-escape mechanisms include suppression/exhaustion of tumor infiltrating cytotoxic T lymphocytes, inhibition of tumor suppressive NK cells, increase in immune-suppressive immune cells (regulatory T, M2 macrophagic, myeloid-derived suppressor, dendritic, stromal, and adipocytic cells). IFN-γ (the most investigated factor), TGF-β, TNF-α, IL-6, IL-17, IL-15, IL-27, complement factor C5a, and other soluble molecules secreted by TME components (and sometimes increased in patients’ serum), as well as and hypoxia, influenced the regulation of PD-L1. Experimental studies using human and mouse PC cell lines (derived from either androgen-sensitive or androgen-resistant tumors) revealed that the intracellular ERK/MEK, Akt-mTOR, NF-kB, WNT and JAK/STAT pathways were involved in PD-L1 upregulation in PC. Blocking the PD-1/PD-L1 signaling by using immunotherapy drugs can prevent tumor immune-escape, increasing the anti-tumor activity of immune cells.     

MT1-MMP Cooperates with TGF-β Receptor-Mediated Signaling to Trigger SNAIL and Induce Epithelial-to-Mesenchymal-like Transition in U87 Glioblastoma Cells

Epithelial-to-mesenchymal transition (EMT) recapitulates metastasis and can be induced in vitro through transforming growth factor (TGF)-β signaling. A role for MMP activity in glioblastoma multiforme has been ascribed to EMT, but the molecular crosstalk between TGF-β signaling and membrane type 1 MMP (MT1-MMP) remains poorly understood. Here, the expression of common EMT biomarkers, induced through TGF-β and the MT1-MMP inducer concanavalin A (ConA), was explored using RNA-seq analysis and differential gene arrays in human U87 glioblastoma cells. TGF-β triggered SNAIL and fibronectin expressions in 2D-adherent and 3D-spheroid U87 glioblastoma cell models. Those inductions were antagonized by the TGF-β receptor kinase inhibitor galunisertib, the JAK/STAT inhibitors AG490 and tofacitinib, and by the diet-derived epigallocatechin gallate (EGCG). Transient gene silencing of MT1-MMP prevented the induction of SNAIL by ConA and abrogated TGF-β-induced cell chemotaxis. Moreover, ConA induced STAT3 and Src phosphorylation, suggesting these pathways to be involved in the MT1-MMP-mediated signaling axis that led to SNAIL induction. Our findings highlight a new signaling axis linking MT1-MMP to TGF-β-mediated EMT-like induction in glioblastoma cells, the process of which can be prevented by the diet-derived EGCG.     

AKIP1, a Cardiac Hypertrophy Induced Protein that Stimulates Cardiomyocyte Growth via the Akt Pathway

Cardiac adaptation to unremitting physiological stress typically involves hypertrophic growth of cardiomyocytes, a compensatory response that often fails and causes heart disease. Gene array analysis identified AKIP1 (A Kinase Interacting Protein 1) as a hypertrophic gene and we therefore hypothesized a potential role in the hypertrophic response. We show for the first time that both AKIP1 mRNA and protein levels increased in hypertrophic cardiomyocytes under conditions of sustained cardiac stress, including pressure overload and after myocardial infarction and in vitro in phenylephrine (PE) stimulated neonatal rat ventricular cardiomyocytes (NRVCs). AKIP1 overexpression in NRVCs markedly stimulated hypertrophic growth responses, including significantly increased cell size, augmented cytoskeletal organization and protein synthesis. Although, AKIP1 was not essential for PE induced hypertrophy in NRVCs, it did potentiate neurohormonal induced protein synthesis. AKIP1 did, however, not induce expression of pathological marker genes like ANP and β-MHC. ERK and Akt kinase signaling pathways have been linked to hypertrophy and AKIP1 specifically induced phosphorylation of Akt. This phosphorylation of Akt was essential for activation of ribosomal rpS6 and translation elongation factor eEF2 and this readily explains the increased protein synthesis. Akt inhibition fully blocked AKIP1 induced hypertrophy, showing that this pathway is critically involved. In conclusion, our results show that AKIP1 is induced in hypertrophic hearts and can stimulate adaptive cardiomyocyte growth, which involves Akt signaling.     

Rutaecarpine Increases Nitric Oxide Synthesis via eNOS Phosphorylation by TRPV1-Dependent CaMKII and CaMKKβ/AMPK Signaling Pathway in Human Endothelial Cells

Rutaecarpine (RUT) is a bioactive alkaloid isolated from the fruit of Evodia rutaecarpa that exerts a cellular protective effect. However, its protective effects on endothelial cells and its mechanism of action are still unclear. In this study, we demonstrated the effects of RUT on nitric oxide (NO) synthesis via endothelial nitric oxide synthase (eNOS) phosphorylation in endothelial cells and the underlying molecular mechanisms. RUT treatment promoted NO generation by increasing eNOS phosphorylation. Additionally, RUT induced an increase in intracellular Ca²⁺ concentration and phosphorylation of Ca²⁺/calmodulin-dependent protein kinase kinase β (CaMKKβ), AMP-activated protein kinase (AMPK), and Ca²⁺/calmodulin-dependent kinase II (CaMKII). Inhibition of transient receptor potential vanilloid type 1 (TRPV1) attenuated RUT-induced intracellular Ca²⁺ concentration and phosphorylation of CaMKII, CaMKKβ, AMPK, and eNOS. Treatment with KN-62 (a CaMKII inhibitor), Compound C (an AMPK inhibitor), and STO-609 (a CaMKKβ inhibitor) suppressed RUT-induced eNOS phosphorylation and NO generation. Interestingly, RUT attenuated the expression of ICAM-1 and VCAM-1 induced by TNF-α and inhibited the inflammation-related NF-κB signaling pathway. Taken together, these results suggest that RUT promotes NO synthesis and eNOS phosphorylation via the Ca²⁺/CaMKII and CaM/CaMKKβ/AMPK signaling pathways through TRPV1. These findings provide evidence that RUT prevents endothelial dysfunction and benefit cardiovascular health.     

Heat Killed Lactobacillus reuteri GMNL-263 Reduces Fibrosis Effects on the Liver and Heart in High Fat Diet-Hamsters via TGF-β Suppression

Obesity is one of the major risk factors for nonalcoholic fatty liver disease (NAFLD), and NAFLD is highly associated with an increased risk of cardiovascular disease (CVD). Scholars have suggested that certain probiotics may significantly impact cardiovascular health, particularly certain Lactobacillus species, such as Lactobacillus reuteri GMNL-263 (Lr263) probiotics, which have been shown to reduce obesity and arteriosclerosis in vivo. In the present study, we examined the potential of heat-killed bacteria to attenuate high fat diet (HFD)-induced hepatic and cardiac damages and the possible underlying mechanism of the positive effects of heat-killed Lr263 oral supplements. Heat-killed Lr263 treatments (625 and 3125 mg/kg-hamster/day) were provided as a daily supplement by oral gavage to HFD-fed hamsters for eight weeks. The results show that heat-killed Lr263 treatments reduce fatty liver syndrome. Moreover, heat-killed Lactobacillus reuteri GMNL-263 supplementation in HFD hamsters also reduced fibrosis in the liver and heart by reducing transforming growth factor β (TGF-β) expression levels. In conclusion, heat-killed Lr263 can reduce lipid metabolic stress in HFD hamsters and decrease the risk of fatty liver and cardiovascular disease.     

The Daidzein Metabolite, 6,7,4'-Trihydroxyisoflavone,Is a Novel Inhibitor of PKCα in Suppressing Solar UV-Induced Matrix Metalloproteinase 1

Soy isoflavone is an attractive source of functional cosmetic materials with anti-wrinkle, whitening and skin hydration effects. After consumption, the majority of soy isoflavones are converted to their metabolites in the human gastrointestinal tract. To understand the physiological impact of soy isoflavone on the human body, it is necessary to evaluate and address the biological function of its metabolites. In this study, we investigated the effect of 6,7,4''-trihydroxyisoflavone (6,7,4''-THIF), a major metabolite of daidzein, against solar UV (sUV)-induced matrix metalloproteinases (MMPs) in normal human dermal fibroblasts. MMPs play a critical role in the degradation of collagen in skin, thereby accelerating the aging process of skin. The mitogen-activated protein/extracellular signal-regulated kinase (MEK)/extracellular signal-regulated kinase (ERK), mitogen-activated protein kinase (MKK)3/6/p38 and MKK4/c-Jun N-terminal kinases (JNK) signaling pathways are known to modulate MMP-1 function, and their activation by sUV was significantly reduced by 6,7,4''-THIF pretreatment. Our results also indicated that the enzyme activity of protein kinase C (PKC)α, an upstream regulator of MKKs signaling, is suppressed by 6,7,4''-THIF using the in vitro kinase assay. Furthermore, the direct interaction between 6,7,4''-THIF and endogenous PKCα was confirmed using the pull-down assay. Not only sUV-induced MMP-1 expression, but also sUV-induced signaling pathway activation were decreased in PKCα knockdown cells. Overall, we elucidated the inhibitory effect of 6,7,4''-THIF on sUV-induced MMPs and suggest PKCα as its direct molecular target.     

Cholesteryl ester transfer protein inhibition by PD 140195

The presence of plasma cholesteryl ester transfer protein (CETP) activity may be atherogenic, and, therefore, strategies to inhibit its activity or production may result in a beneficial effect on lipoprotein profiles and the disease process. The current report describes 4-phenyl-5-tridecyl-4H-1,2,4-triazole-3-thiol (PD 140195), a novel CETP inhibitor. The concentration-dependent inhibition of CETP by PD 140195 and the inhibitory monoclonal antibody (Mab) TP2 is demonstrated in a variety ofin vitro assay systems. Molecular models of PD140195 suggest a spatial mimicry of the cholesteryl ester structure. Despite the structural similarity, kinetic studies with a fluorescent cholesteryl ester analog suggest that the inhibition of transfer is not competitive. PD 140195 also selectively inhibited cholesteryl ester but not triglyceride transfer, while the Mab TP2 blocked CETP transfer of both. Studies were carried out to determine whether CETP inhibition observedin vitro could also be demonstratedin vivo. When PD 140195 was intravenously infused to anesthetized rabbits (up to 20 mg/kg), only transient CETP inhibition was observed.In vitro reconstitution studies in the presence of bovine serum albumin resulted in marked reduction of PD 140195 inhibitory activity. Thus, the low activity of PD 140195 in whole plasma probably results from binding to other plasma proteins.     

Biphasic Dose-Dependent G0/G1 and G2/M Cell-Cycle Arrest by Synthetic 2,3-Arylpyridylindole Derivatives in A549 Lung Cancer Cells

A collection of 2,3-arylpyridylindole derivatives were synthesized via the Larock heteroannulation and evaluated for their in vitro cytotoxic activity against A549 human lung cancer cells. Two derivatives expressed good cytotoxicity with IC₅₀ values of 1.18±0.25 μM and 0.87±0.10 μM and inhibited tubulin polymerization in vitro, with molecular docking studies suggesting the binding modes of the compounds in the colchicine binding site. Both derivatives have biphasic cell cycle arrest effects depending on their concentrations. At a lower concentration (0.5 μM), the two compounds induced G0/G1 cell cycle arrest by activating the JNK/p53/p21 pathway. At a higher concentration (2.0 μM), the two derivatives arrested the cell cycle at the G2/M phase via Akt signaling and inhibition of tubulin polymerization. Additional cytotoxic mechanisms of the two compounds involved the decreased expression of Bcl-2 and Mcl-1 antiapoptotic proteins through inhibition of the STAT3 and Akt signaling pathways.