Integrated Stress Response Potentiates Ponatinib-Induced Cardiotoxicity.

BACKGROUND: Mitochondrial dysfunction is a primary driver of cardiac contractile failure; yet, the cross talk between mitochondrial energetics and signaling regulation remains obscure. Ponatinib, a tyrosine kinase inhibitor used to treat chronic myeloid leukemia, is among the most cardiotoxic tyrosine kinase inhibitors and causes mitochondrial dysfunction. Whether ponatinib-induced mitochondrial dysfunction triggers the integrated stress response (ISR) to induce ponatinib-induced cardiotoxicity remains to be determined. METHODS: Using human induced pluripotent stem cells-derived cardiomyocytes and a recently developed mouse model of ponatinib-induced cardiotoxicity, we performed proteomic analysis, molecular and biochemical assays to investigate the relationship between ponatinib-induced mitochondrial stress and ISR and their role in promoting ponatinib-induced cardiotoxicity. RESULTS: Proteomic analysis revealed that ponatinib activated the ISR in cardiac cells. We identified GCN2 (general control nonderepressible 2) as the eIF2α (eukaryotic translation initiation factor 2α) kinase responsible for relaying mitochondrial stress signals to trigger the primary ISR effector-ATF4 (activating transcription factor 4), upon ponatinib exposure. Mechanistically, ponatinib treatment exerted inhibitory effects on ATP synthase activity and reduced its expression levels resulting in ATP deficits. Perturbed mitochondrial function resulting in ATP deficits then acts as a trigger of GCN2-mediated ISR activation, effects that were negated by nicotinamide mononucleotide, an NAD+ precursor, supplementation. Genetic inhibition of ATP synthase also activated GCN2. Interestingly, we showed that the decreased abundance of ATP also facilitated direct binding of ponatinib to GCN2, unexpectedly causing its activation most likely because of a conformational change in its structure. Importantly, administering an ISR inhibitor protected human induced pluripotent stem cell-derived cardiomyocytes against ponatinib. Ponatinib-treated mice also exhibited reduced cardiac function, effects that were attenuated upon systemic ISRIB administration. Importantly, ISRIB does not affect the antitumor effects of ponatinib in vitro. CONCLUSIONS: Neutralizing ISR hyperactivation could prevent or reverse ponatinib-induced cardiotoxicity. The findings that compromised ATP production potentiates GCN2-mediated ISR activation have broad implications across various cardiac diseases. Our results also highlight an unanticipated role of ponatinib in causing direct activation of a kinase target despite its role as an ATP-competitive kinase inhibitor.

Sortase A-Inhibitory Coumarins from the Folk Medicinal Plant Poncirus trifoliata.

Thirteen coumarins (1-13), including five new compounds (1-5), were isolated from the folk medicinal plant Poncirus trifoliata. Combined spectroscopic analyses revealed that coumarins 1-4 are bis-isoprenylated coumarins with diverse oxidation patterns, while 5 is an enantiomeric di-isoprenylated coumarin. The absolute configurations of the stereogenic centers in the isoprenyl chains were assigned through MTPA and MPA methods, and those of the known compounds triphasiol (6) and ponciol (7) were also assigned using similar methods. These coumarins inhibited significantly Staphylococcus aureus-derived sortase A (SrtA), a transpeptidase responsible for anchoring surface proteins to the peptidoglycan cell wall in Gram-positive bacteria. The present results obtained indicated that the bioactivity and underlying mechanism of action of these coumarins are associated with the inhibition of SrtA-mediated S. aureus adhesion to eukaryotic cell matrix proteins including fibrinogen and fibronectin, thus potentially serving as SrtA inhibitors.

Methods for high-resolution anisotropic finite element modeling of the human head: automatic MR white matter anisotropy-adaptive mesh generation.

This study proposes an advanced finite element (FE) head modeling technique through which high-resolution FE meshes adaptive to the degree of tissue anisotropy can be generated. Our adaptive meshing scheme (called wMesh) uses MRI structural information and fractional anisotropy maps derived from diffusion tensors in the FE mesh generation process, optimally reflecting electrical properties of the human brain. We examined the characteristics of the wMeshes through various qualitative and quantitative comparisons to the conventional FE regular-sized meshes that are non-adaptive to the degree of white matter anisotropy. We investigated numerical differences in the FE forward solutions that include the electrical potential and current density generated by current sources in the brain. The quantitative difference was calculated by two statistical measures of relative difference measure (RDM) and magnification factor (MAG). The results show that the wMeshes are adaptive to the anisotropic density of the WM anisotropy, and they better reflect the density and directionality of tissue conductivity anisotropy. Our comparison results between various anisotropic regular mesh and wMesh models show that there are substantial differences in the EEG forward solutions in the brain (up to RDM=0.48 and MAG=0.63 in the electrical potential, and RDM=0.65 and MAG=0.52 in the current density). Our analysis results indicate that the wMeshes produce different forward solutions that are different from the conventional regular meshes. We present some results that the wMesh head modeling approach enhances the sensitivity and accuracy of the FE solutions at the interfaces or in the regions where the anisotropic conductivities change sharply or their directional changes are complex. The fully automatic wMesh generation technique should be useful for modeling an individual-specific and high-resolution anisotropic FE head model incorporating realistic anisotropic conductivity distributions towards more accurate analysis of bioelectromagnetic problems.

Reduced spatial focality of electrical field in tDCS with ring electrodes due to tissue anisotropy.

For effective stimulation with tDCS, spatial focality of induced electrical field (EF) is one of the important factors to be considered. Recently, there have been some studies to improve the spatial focality via different types of electrodes and their new configurations: some improvements using ring electrodes were reported over the conventional pad electrodes. However, most of these studies assumed isotropic conductivities in the head. In this work, we have investigated the effect of tissue anisotropy on the spatial focality of tDCS with the 4+1 ring electrode configuration via a 3-D high-resolution finite element (FE) head model with anisotropic conductivities in the skull and white matter. By examining the profiles of the induced EF from the head models with isotropic and anisotropic conductivities respectively, we found that the spatial focality of the induced EF significantly drops and get diffused due to tissue anisotropy. Our analysis suggests that it is critical to incorporate tissue anisotropy in the stimulation of the brain via tDCS.

Potentiation of etoposide-induced apoptosis in HeLa cells by co-treatment with KG-135, a quality-controlled standardized ginsenoside formulation.

Our previous studies demonstrated that KG-135, a quality-controlled red ginseng-specific formulation containing approximately equal amounts of three major ginsenosides (Rk1, Rg3 and Rg5), down-regulated G1 cyclin-dependent kinase in HeLa cells. In the present work, we have found that KG-135 potentates cytotoxicity of etoposide by modulating apoptotic signaling. Co-treatment of etoposide and KG-135 markedly elevated the expression and phosphorylation at the serine 15 residue of p53 as well as the cellular levels of Bax and p21(Waf1/Cip1). The increased accumulation and phosphorylation of p53 (Ser15) were attenuated by treatment of cells with wortmannin, a pan-phosphatidylinositol-3 kinase inhibitor. Moreover, co-treatment of etoposide and KG-135 enhanced mitochondrial localization of Bax. Our results indicate that etoposide-induced apoptosis in HeLa cells can be potentiated in the presence of KG-135 through a mechanism that involves the stabilization of p53 and the stimulation of Bax- and p21-mediated apoptotic signaling pathways. These findings suggest that KG-135 represents a useful candidate adjuvant for the treatment of cancers that could potentially minimize the adverse effects of current clinical chemotherapeutics.

Torilin from Torilis japonica inhibits melanin production in alpha-melanocyte stimulating hormone-activated B16 melanoma cells.

Epidermal melanocytes synthesize melanin pigments and transfer them to keratinocytes, which is responsible for skin pigmentation. However, abnormal accumulation of melanin pigments causes hyperpigmentation disorders, which are substantially improved with treatment of tyrosinase inhibitor. In our ongoing study, Torilis japonica DC. (Umbelliferae) was found to inhibit melanin production. A goal of this study is to elucidate the hypopigmenting principle of T. japonica. A sesquiterpene structure of torilin was isolated from the plant extracts via bioassay-guided phytochemical analysis. Torilin dose-dependently inhibited melanin production, with an IC(50) value of 25 microM, in alpha-melanocyte stimulating hormone (alpha-MSH)-activated B16 melanoma cells. Arbutin, a positive control of skin whitener, also inhibited alpha-MSH-induced melanin production with an IC(50) value of 170 microM. As to the mode of action, torilin downregulated alpha-MSH-induced protein levels of tyrosinase without directly inhibiting catalytic activity of the enzyme. Taken together, this study shows that torilin contributes to the hypopigmenting principle of T. japonica, and suggests its pharmacological potential in melanin-associated hyperpigmentation disorders.

Manassantin A and B from Saururus chinensis inhibiting cellular melanin production.

Hyperpigmentation disorders such as freckles and senile lentigines in the skin are associated with abnormal accumulation of melanin pigments. In this study, two lignan constituents were isolated from Saururus chinensis Baill (Saururaceae) as inhibitors of cellular melanin production by bioassay-guided fractionations. The active constituents were manassantin A and B that dose-dependently inhibited melanin production in alpha-melanocyte stimulating hormone (alpha-MSH)-activated melanoma B16 cells with IC(50) values of 13 nm and 8 nm, respectively. Arbutin as a positive control exhibited an IC(50) value of 96 microm on alpha-MSH-induced melanin production. Further, manassantin A inhibited forskolin- or 3-isobutyl-1-methylxanthine (IBMX)-induced melanin production with IC(50) values of 14 nm or 12 nm, respectively. Manassantin A decreased cellular amounts of IBMX-inducible tyrosinase protein but could not affect the catalytic activity of cell-free tyrosinase, a key enzyme in the biosynthetic pathway of melanin pigments. Finally, this study could provide a pharmacological potential of S. chinensis in hyperpigmentation disorders.

Heat-processed neoginseng, KG-135, down-regulates G1 Cyclin-dependent kinase through the proteasome-mediated pathway in HeLa cells.

High temperature heat treatment of ginseng (Panax ginseng, C.A. Meyer) generates KG-135 (heat-processed neoginseng) which contains a mixture of three major ginseng saponins, ginsenosides Rk1, Rg3 and Rg5. Ginsenosides, particularly of the diol-type including Rk1, Rg3 and Rg5, have been shown to induce cell growth arrest in various cell types of human cancer. Herein, we report that KG-135 is able to arrest the cell cycle in human cervix adenocarcinoma HeLa cells. KG-135 arrests cells at the G1 phase of the cell cycle with an IC50 value of 69 microg/ml. The G1 phase arrest is associated with down-regulation of Cyclin D1/Cdk4 and Cyclin B1/Cdc2 activities in cells after treatment with KG-135. Furthermore, down-regulation of G1 Cyclin-dependent kinase activities is kinetically well related to the decreased intracellular protein levels of these kinases. In addition, the decrease in the levels of Cyclin D1/Cdk4 and Cyclin B1, but not of Cdc2, is similarly prevented by co-treatment of cells with MG-132, a potent proteasome inhibitor. Thus, the KG-135-induced arrest of the cell cycle at G1 phase in HeLa cells represents a novel mechanism that involves proteasome-mediated degradation of the Cyclins (Cyclin D1 and B1) and Cdk4 proteins.

Inhibitory effects of kurarinol, kuraridinol, and trifolirhizin from Sophora flavescens on tyrosinase and melanin synthesis.

Previously, it was reported that some prenylated flavonoids contained in the dichloromethane fraction of the ethanolic extract of Sophora flavescens, such as kuraridin, sophoraflavanone G, kurarinone, and kushenol F, are tyrosinase inhibitors; however, based on the level of these inhibitors in the extract, its inhibitory effect on tyrosinase activity was higher than expected. This has led us to further investigate other possible constituents that may contribute to the extract's strong inhibitory activity. The results of this study indicate that kurarinol (1), kuraridinol (2), and trifolirhizin (3), from the ethyl acetate fraction of Sophora extract, can inhibit tyrosinase activity. Compared with kojic acid (16.22+/-1.71 microM), compounds 1-3 possessed potent tyrosinase inhibitory activity with IC(50) values of 8.60+/-0.51, 0.88+/-0.06, and 506.77+/-4.94 microM, respectively. These three compounds were further tested for their inhibitory effects on melanogenesis. In cultured B16 melanoma cells, 1-3 markedly inhibited (>50%) melanin synthesis at 50 microM. This is the first study indicating that 1-3 exert varying degrees of inhibition on tyrosinase-dependent melanin biosynthesis, and therefore, are candidates as skin-whitening agents.