Phorbol Ester Research Articles

The massed-spaced learning effect in non-neural human cells

The massed-spaced effect is a hallmark feature of memory formation. We now demonstrate this effect in two separate non-neural, immortalized cell lines stably expressing a short-lived luciferase reporter controlled by a CREB-dependent promoter. We emulate training using repeated pulses of forskolin and/or phorbol ester, and, as a proxy for memory, measure luciferase expression at various points after training. Four spaced pulses of either agonist elicit stronger and more sustained luciferase expression than a single “massed” pulse. Spaced pulses also result in stronger and more sustained activation of molecular factors critical for memory formation, ERK and CREB, and inhibition of ERK or CREB blocks the massed-spaced effect. Our findings show that canonical features of memory do not necessarily depend on neural circuitry, but can be embedded in the dynamics of signaling cascades conserved across different cell types.

Inhibition of the Notch signal transducer CSL by Pkc53E-mediated phosphorylation to fend off parasitic immune challenge in Drosophila.

Notch signalling activity regulates hematopoiesis in Drosophila and vertebrates alike. Parasitoid wasp infestation of Drosophila larvae, however, requires a timely downregulation of Notch activity to allow the formation of encapsulation-active blood cells. Here, we show that the Drosophila CSL transcription factor Suppressor of Hairless [Su(H)] is phosphorylated at Serine 269 in response to parasitoid wasp infestation. As this phosphorylation interferes with the DNA binding of Su(H), it reversibly precludes its activity. Accordingly, phospho-deficient Su(H)S269A mutants are immune-compromised. A screen for kinases involved in Su(H) phosphorylation identified Pkc53E, required for normal hematopoiesis as well as for parasitoid immune response. Genetic and molecular interactions support the specificity of the Su(H)-Pkc53E relationship. Moreover, phorbol ester treatment inhibits Su(H) activity in vivo and in human cell culture. We conclude that Pkc53E targets Su(H) during parasitic wasp infestation, thereby remodelling the blood cell population required for wasp egg encapsulation.

Growth performances of Clarias gariepinus juveniles fed with Jatropha curcas seed meal

Jatropha curcas is an oil producing seed with high nutritional qualities for consumption. However, due to inherent anti-nutritional factors, appropriate processing is necessary to improve its nutrients utilization. This study was aimed at processing J. curcas using solvent-extraction method and evaluating its inclusion effects in the diet for Clarias gariepinus on its growth status. J. curcas meal was extracted using 80 % methanol to remove phorbol esters and other anti-nutrients. Thereafter, five iso-nitrogenous diets (40 % crude protein) were formulated containing J. curcas meal replacements of soybeans meal at 0 % (TRT0), 25 % (TRT25), 50 % (TRT50), 75 % (TRT75) and 100 % (TRT100) and fed to C. gariepinus for 70 days. Results showed that TRT25 fish had the highest mean weight gain and specific growth. The serum alkaline phosphate, alanine aminotransferase, and aspartate aminotransferase levels began to rise significantly (p ≤ 0.05) at 50 % replacement. Likewise, the assessment of gut ecology and morphology indicated that TRT25 had significantly (p ≤ 0.05) the highest gut bacteria colony forming unit (2.60 ×104±0.02cfu/g) and most favorable area of absorption (0.16±0.01 cm2). The histopathological observation of the fish intestine, liver and gills indicated no visible deformity in TRT0 and TRT25 fish. However, various degrees of degenerations were observed in the fish fed with 50 % and higher inclusion of J. curcas. This study showed that methanol-extracted J. curcas meal can be effectively utilized for C. gariepinus at 25 % of replacement.

Topological Heterogeneity of Protein Kinase C Modulators in Human T-Cells Resolved with In-Cell Dynamic Nuclear Polarization NMR Spectroscopy.

Phorbol ester analogs are a promising class of anticancer therapeutics and HIV latency reversing agents that interact with cellular membranes to recruit and activate protein kinase C (PKC) isoforms. However, it is unclear how these esters interact with membranes and how this might correlate with the biological activity of different phorbol ester analogs. Here, we have employed dynamic nuclear polarization (DNP) NMR to characterize phorbol esters in a native cellular context. The enhanced NMR sensitivity afforded by DNP and cryogenic operation reveals topological heterogeneity of 13C-21,22-phorbol-myristate-acetate (PMA) within T cells utilizing 13C-13C correlation and double quantum filtered NMR spectroscopy. We demonstrate the detection of therapeutically relevant amounts of PMA in T cells down to an upper limit of ∼60.0 pmol per million cells and identify PMA to be primarily localized in cellular membranes. Furthermore, we observe distinct 13C-21,22-PMA chemical shifts under DNP conditions in cells compared to model membrane samples and homogenized cell membranes, that cannot be accounted for by differences in conformation. We provide evidence for distinct membrane topologies of 13C-21,22-PMA in cell membranes that are consistent with shallow binding modes. This is the first of its kind in-cell DNP characterization of small molecules dissolved in the membranes of living cells, establishing in-cell DNP-NMR as an important method for the characterization of drug-membrane interactions within the context of the complex heterogeneous environment of intact cellular membranes. This work sets the stage for the identification of the in-cell structural interactions that govern the biological activity of phorbol esters.

Pterocarya hupehensis Skan total flavones ameliorate rheumatoid arthritis in rats by suppressing formation of neutrophil extracellular traps

To investigate the therapeutic mechanism of Pterocarya hupehensis Skan total flavonoids (PHSTF) for rheumatoid arthritis (RA). Twenty-five male SD rats were randomly divided into normal control group, RA model group, PHSTF treatment (45 and 90 mg/kg) groups, and Tripterygium glycosides (TPG) tablet (10 mg/kg) group (n=5). Except for those in the normal control group, all the rats were subjected to collagen-induced arthritis (CIA) modeling using a secondary immunization method, after which PHSTF and TPG were administered via gavage once daily for 4 weeks. After the treatments, serum levels of TNF-α and IL-1β were measured using ELISA, and ankle joint pathologies were assessed with HE staining; the expression of citrullinated histone H3 (Cit-H3), a neutrophil extracellular trap (NET) marker, in the ankle joints was evaluated with immunohistochemistry. In primary cultures of rat peripheral blood neutrophils stimulated with phorbol ester (PMA), the effects of PHSTF (100 and 200 μg/mL) on the expressions of Cit-H3, peptidylarginine deiminase 4 (PADI4), neutrophil elastase (NE), and myeloperoxidase (MPO) were examined with Western blotting; immunofluorescence assay was used to observe Cit-H3 expression and NET formation in the cells. In the CIA rat models, PHSTF significantly alleviated ankle swelling, decreased serum levels of TNF-α and IL-1β, improved histopathological changes in the ankle joints, and reduced Cit-H3 expression in both the serum and ankle joint cartilage. In the isolated rat neutrophils, PHSTF showed no significant effect on cell viability but strongly inhibited PMA-induced NET release. PHSTF can alleviate RA by inhibiting the formation of NETs.

Sex-Specific Skeletal Muscle Gene Expression Responses to Exercise Reveal Novel Direct Mediators of Insulin Sensitivity Change.

Understanding the causal pathways, systems, and mechanisms through which exercise impacts human health is complex. This study explores molecular signaling related to whole-body insulin sensitivity (Si) by examining changes in skeletal muscle gene expression. The analysis considers differences by biological sex, exercise amount, and exercise intensity to identify potential molecular targets for developing pharmacologic agents that replicate the health benefits of exercise. The study involved 53 participants from the STRRIDE I and II trials who completed eight months of aerobic training. Skeletal muscle gene expression was measured using Affymetrix and Illumina technologies, while pre- and post-training Si was assessed via an intravenous glucose tolerance test. A novel gene discovery protocol, integrating three literature-derived and data-driven modeling strategies, was employed to identify causal pathways and direct causal factors based on differentially expressed transcripts associated with exercise intensity and amount. In women, the transcription factor targets identified were primarily influenced by exercise amount and were generally inhibitory. In contrast, in men, these targets were driven by exercise intensity and were generally activating. Transcription factors such as ATF1, CEBPA, BACH2, and STAT1 were commonly activating in both sexes. Specific transcriptional targets related to exercise-induced Si improvements included TACR3 and TMC7 for intensity-driven effects, and GRIN3B and EIF3B for amount-driven effects. Two key signaling pathways mediating aerobic exercise-induced Si improvements were identified: one centered on estrogen signaling and the other on phorbol ester (PKC) signaling, both converging on the epidermal growth factor receptor (EGFR) and other relevant targets. The signaling pathways mediating Si improvements from aerobic exercise differed by sex and were further distinguished by exercise intensity and amount. Transcriptional adaptations in skeletal muscle related to Si improvements appear to be causally linked to estrogen and PKC signaling, with EGFR and other identified targets emerging as potential skeletal muscle-specific drug targets to mimic the beneficial effects of exercise on Si.

Repeated stress to the skin amplifies neutrophil infiltration in a keratin 17- and PKCα-dependent manner.

Neutrophils are the first immune cells to reach inflamed sites and contribute to the pathogenesis of chronic inflammatory skin diseases. Yet, little is known about the pattern of neutrophil infiltration in inflamed skin in vivo and the mechanisms mediating their recruitment. Here, we provide insight into the dynamics of neutrophil infiltration in skin in response to acute or repeated inflammatory stress, highlighting a novel keratinocyte- and keratin 17 (K17)-dependent mechanism that regulates neutrophil recruitment to inflamed skin. We used the phorbol ester TPA and UVB, alone or in combination, to induce sterile inflammation in mouse skin. A single TPA treatment results in a neutrophil influx in the dermis that peaks at 12 h and resolves within 24 h. A subsequent TPA treatment or a UVB challenge, when applied 24 h but not 48 h later, accelerates, amplifies, and prolongs neutrophil infiltration. This transient amplification response (TAR) is mediated by local signals in inflamed skin, can be recapitulated in ex vivo culture, and involves the K17-dependent sustainment of protein kinase Cα (PKCα) activity and release of chemoattractants by stressed keratinocytes. K17 binds RACK1, a scaffold protein essential for PKCα activity. The N-terminal head domain of K17 is crucial for its association with RACK1 and regulation of PKCα activity. Analysis of RNAseq data reveals a signature consistent with TAR and PKCα activation in inflammatory skin diseases. These findings uncover a novel, keratin-dependent mechanism that amplifies neutrophil recruitment in skin under stress, with direct implications for inflammatory skin disorders.

Exploring the Regulation of Cytochrome P450 in SH-SY5Y Cells: Implications for the Onset of Neurodegenerative Diseases.

Human individual differences in brain cytochrome P450 (CYP) metabolism, including induction, inhibition, and genetic variation, may influence brain sensitivity to neurotoxins and thus participate in the onset of neurodegenerative diseases. The aim of this study was to explore the modulation of CYPs in neuronal cells. The experimental approach was focused on differentiating human neuroblastoma SH-SY5Y cells into a phenotype resembling mature dopamine neurons and investigating the effects of specific CYP isoform induction. The results demonstrated that the differentiation protocols using retinoic acid followed by phorbol esters or brain-derived neurotrophic factor successfully generated SH-SY5Y cells with morphological neuronal characteristics and increased neuronal markers (NeuN, synaptophysin, β-tubulin III, and MAO-B). qRT-PCR and Western blot analysis showed that expression of the CYP 1A1, 3A4, 2D6, and 2E1 isoforms was detectable in undifferentiated cells, with subsequent increases in CYP 2E1, 2D6, and 1A1 following differentiation. Further increases in the 1A1, 2D6, and 2E1 isoforms following β-naphthoflavone treatment and 1A1 and 2D6 isoforms following ethanol treatment were evident. These results demonstrate that CYP isoforms can be modulated in SH-SY5Y cells and suggest their potential as an experimental model to investigate the role of CYPs in neuronal processes involved in the development of neurodegenerative diseases.

Tissue-Specific Accumulation Profiles of Phorbol Esters in Response to Abiotic and Biotic Stresses in Jatropha curcas

Jatropha curcas L. (J. curcas), a shrub plant of the Euphorbiaceae family, has received enormous attention as a promising biofuel plant for the production of biodiesel and medical potential in ethnopharmacology. However, the tumor-promoter toxin phorbol esters present in J. curcas raise concerns for health and environmental risk as its large-scale cultivation limits the use of meal obtained after oil extraction for animal feed. Here, we determined the variation of phorbol ester profiles and contents in eight J. curcas tissues by high-performance liquid chromatography (HPLC) and found phorbol esters present in all parts of the plant except the seed shell. We showed tissue-specific patterns of accumulation of phorbol esters and associated terpenoids at the transcriptional level with high transcript levels in reproductive and young tissues. Genes involved in the same module of terpenoids biosynthesis were positively correlated. We further present diverse abiotic and biotic stresses that had different effects on the accumulation of transcripts in terpenoids shared and branched terpenoid pathways in plant seedlings. The fine-tuning of terpenoids biosynthesis may link with ecological functions in plants under extreme environments and defense against pathogens.

Tigliane metabolites in Hyles frass after feeding experiments with TPA and Euphorbia cyparissias

Within the genus Hyles, Euphorbia feeding appears to have evolved twice independently, in H. euphorbiae but also in H. nicaea, a species which had not been studied for its detoxification processes before. Euphorbia is known to contain toxic secondary metabolites, including diterpene esters, preventing most herbivores from feeding on them. We investigated the metabolisation of the standard phorbol ester Phorbol 12-myristate 13-acetate (TPA) and the diterpenes contained in Euphorbia cyparissias by the two species Hyles euphorbiae and Hyles nicaea (subspecies nicaea). For the first time, we report (1) The gut disposition of Hyles n. nicaea for this standard phorbol ester, which is commonly used in cancer and tumour research and (2) The disposition of the food plant tiglinaes of Euphorbia cyparissias after gut passage in both species and (3) Tigliane metabolites in the frass of TPA and of E. cyparissias feeding larvae. For both species around 5–25% of the TPA dose was recovered in the frass of the larvae, along with the metabolites phorbol and phorbol-13-acetate in very small amounts and traces. While the amounts of phorbol found did not differ much between the species frass, phorbol-13-acetate could be found in higher amounts in Hyles n. nicaea frass, indicating a difference in metabolisation. Moreover, enzymatic hydrolysis of TPA to phorbol-13-acetate and phorbol are postulated not to be the main metabolisation pathway, seeing that the amounts found only represent a small fraction of the TPA dose consumed.

Laser-assembled conductive 3D nanozyme film-based nitrocellulose sensor for real-time detection of H2O2 released from cancer cells

In this work, a nanostructured conductive film possessing nanozyme features was straightforwardly produced via laser-assembling and integrated into complete nitrocellulose sensors; the cellulosic substrate allows to host live cells, while the nanostructured film nanozyme activity ensures the enzyme-free real-time detection of hydrogen peroxide (H2O2) released by the sames. In detail, a highly exfoliated reduced graphene oxide 3D film decorated with naked platinum nanocubes was produced using a CO2-laser plotter via the simultaneous reduction and patterning of graphene oxide and platinum cations; the nanostructured film was integrated into a nitrocellulose substrate and the complete sensor was manufactured using an affordable semi-automatic printing approach. The linear range for the direct H2O2 determination was 0.5–80 μM (R2 = 0.9943), with a limit of detection of 0.2 μM. Live cell measurements were achieved by placing the sensor in the culture medium, ensuring their adhesion on the sensors' surface; two cell lines were used as non-tumorigenic (Vero cells) and tumorigenic (SKBR3 cells) models, respectively. Real-time detection of H2O2 released by cells upon stimulation with phorbol ester was carried out; the nitrocellulose sensor returned on-site and real-time quantitative information on the H2O2 released proving useful sensitivity and selectivity, allowing to distinguish tumorigenic cells. The proposed strategy allows low-cost in-series semi-automatic production of paper-based point-of-care devices using simple benchtop instrumentation, paving the way for the easy and affordable monitoring of the cytopathology state of cancer cells.

HIV latency-reversing activity of latex from Euphorbia umbellata (Pax) Bruyns and three diterpenes isolated from this species

Two unusual phorbol esters, namely 20-deoxyphorbol-3,4,12-triacetate-13-phenylacetate (1) and phorbol-3,4,12,13-tetraacetate-20-phenylacetate (2) plus ingol-3,8,12-triacetate-7-phenylacetate (3) were isolated from the latex of Euphorbia umbellata and identified by HRESIMS and 2D NMR. Compound 1 is herein described for the first time. Assignment of the phenylacetyl group at C-7 in compound 3 was suggested by the HMBC and NOESY spectra obtained in pyridine-d5 . In addition to the latex and its distinct terpenoid fractions, the isolated compounds were tested as latent reversal agents against HIV-1-infected J-Lat cells, with reference to phorbol-12-myristate-13-acetate and ingenol-B. Compound 2 reverted 75-80% the viral latency on the GFP-positive cells, resulting EC50 3.70 μg/mL (SI 6.7), while 1 induced 34-40% reactivation at the same concentration range (4-20 µg/mL). The ingol derivative 3 was ineffective. Phorbol esters were confirmed as effective constituents in the latex since the fraction containing them was 2.4-fold more active than the lyophilised latex at the lowest concentration assayed.

Differential regulation of STARD1, STARD4 and STARD6 in the human ovary.

Cells actively engaged in de novo steroidogenesis rely on an expansive intracellular network to efficiently transport cholesterol. The final link in the transport chain is STARD1, which transfers cholesterol to the enzyme complex that initiates steroidogenesis. However, the regulation of ovarian STARD1 is not fully characterized, and even less is known about the upstream cytosolic cholesterol transporters STARD4 and STARD6. Here, we identified both STARD4 and STARD6 mRNAs in the human ovary but only detected STARD4 protein since the primary STARD6 transcript turned out to be a splice variant. Corpora lutea contained the highest levels of STARD4 and STARD1 mRNA and STARD1 protein, while STARD4 protein was uniformly distributed across ovarian tissues. Cyclic AMP analog (8Br-cAMP) and phorbol ester (PMA) individually increased STARD1 and STARD4 mRNA along with STARD1 protein and its phosphoform in cultured primary human luteinized granulosa cells (hGCs). STARD6 transcripts and STARD4 protein were unresponsive to these stimuli. Combining lower doses of PMA and 8Br-cAMP blunted the 8Br-cAMP stimulation of STARD1 protein. Increasing cholesterol levels by blocking its conversion to steroid with aminoglutethimide or by adding LDL reduced the STARD4 mRNA response to stimuli. Sterol depletion reduced the STARD1 mRNA and protein response to PMA. These data support a possible role for STARD4, but not STARD6, in supplying cholesterol for steroidogenesis in the ovary. We demonstrate for the first time how cAMP, PMA and sterol pathways separately and in combination differentially regulate STARD4, STARD6 and STARD1 mRNA levels, as well as STARD1 and STARD4 protein in human primary ovarian cells.

Formaldehyde exacerbates inflammation and biases T helper cell lineage commitment through IFN-γ/STAT1/T-bet pathway in asthma

The correlation between formaldehyde (FA) exposure and prevalence of asthma has been widely reported. However, the underlying mechanism is still not fully understood. FA exposure at 2.0 mg/m3 was found to exacerbate asthma in OVA-induced murine models. IFN-γ, the cytokine produced by T helper 1 (Th1) cells, was significantly induced by FA in serum and bronchoalveolar lavage fluid (BALF) of asthmatic mice, which was different from cytokines secreted by other Th cells. The observation was also confirmed by mRNA levels of Th marker genes in CD4+ T cells isolated from BALF. In addition, increased production of IFN-γ and expression of T-bet in Jurkat T cells primed with phorbol ester and phytohaemagglutinin were also observed with 100 μM FA treatment in vitro. Upregulated STAT1 phosphorylation, T-bet expression and IFN-γ production induced by FA was found to be restrained by STAT1 inhibitor fludarabine, indicating that FA promoted Th1 commitment through the autocrine IFN-γ/STAT1/T-bet pathway in asthma. This work not only revealed that FA could bias Th lineage commitment to exacerbate allergic asthma, but also identified the signaling mechanism of FA-induced Th1 differentiation, which may be utilized as the target for development of interfering strategies against FA-induced immune disorders.

Activation of Peroxisome Proliferator-Activated Receptor-β/δ (PPARβ/δ) in Keratinocytes by Endogenous Fatty Acids.

Nuclear hormone receptors exist in dynamic equilibrium between transcriptionally active and inactive complexes dependent on interactions with ligands, proteins, and chromatin. The present studies examined the hypothesis that endogenous ligands activate peroxisome proliferator-activated receptor-β/δ (PPARβ/δ) in keratinocytes. The phorbol ester treatment or HRAS infection of primary keratinocytes increased fatty acids that were associated with enhanced PPARβ/δ activity. Fatty acids caused PPARβ/δ-dependent increases in chromatin occupancy and the expression of angiopoietin-like protein 4 (Angptl4) mRNA. Analyses demonstrated that stearoyl Co-A desaturase 1 (Scd1) mediates an increase in intracellular monounsaturated fatty acids in keratinocytes that act as PPARβ/δ ligands. The activation of PPARβ/δ with palmitoleic or oleic acid causes arrest at the G2/M phase of the cell cycle of HRAS-expressing keratinocytes that is not found in similarly treated HRAS-expressing Pparb/d-null keratinocytes. HRAS-expressing Scd1-null mouse keratinocytes exhibit enhanced cell proliferation, an effect that is mitigated by treatment with palmitoleic or oleic acid. Consistent with these findings, the ligand activation of PPARβ/δ with GW0742 or oleic acid prevented UVB-induced non-melanoma skin carcinogenesis, an effect that required PPARβ/δ. The results from these studies demonstrate that PPARβ/δ has endogenous roles in keratinocytes and can be activated by lipids found in diet and cellular components.

СРАВНИТЕЛЬНЫЙ АНАЛИЗ ИНГИБИРУЮЩИХ ЭФФЕКТОВ ГИПОМАГНИТНОГО ПОЛЯ И КОМБИНИРОВАННЫХ МАГНИТНЫХ ПОЛЕЙ НА ПРОДУКЦИЮ АКТИВНЫХ ФОРМ КИСЛОРОДА (АФК) НЕЙТРОФИЛАМИ

The article reveals that a decrease in the background production of reactive oxygen species (ROS) in the peritoneal neutrophils of mice after a short-term (40 minutes) stay in hypomagnetic conditions (residual field  10 nT) at physiological temperatures, detected by the method of lucigenin-dependent chemiluminescence, is not accompanied by a violation of chemiluminescent response to respiratory burst activators: formylated peptide N-formyl-Met-Leu-Phe (fMLF) and phorbol ester of phorbol-12-meristat-13-acetate (PMA). These results were obtained by activated chemiluminescence using lucigenin or luminol and various combinations of ROS production activators (PMA and/or fMLF). In contrast, the action of combined parallel constant (induction 60 μT) and alternating (amplitude range 60-180 nT, frequency 49.5 Hz) magnetic fields (CMF) leads to a decrease in the chemiluminescent response to these activators. These data indicate different sources of ROS that respond to certain modes of CMF and hypomagnetic field in neutrophils. The conducted research and the previously obtained results enable to exclude the systems that control the respiratory burst in neutrophils from the main targets and acceptors that respond to short-term deprivation of the magnetic field.

Development and functional testing of a novel in vitro delayed scratch closure assay

As the development of chronic wound therapeutics continues to expand, the demand for advanced assay systems mimicking the inflammatory wound microenvironment in vivo increases. Currently, this is performed in animal models or in in vitro cell-based models such as cell culture scratch assays that more closely resemble acute wounds. Here, we describe for the first time a delayed scratch closure model that mimics some features of a chronic wound in vitro. Chronic wounds such as those suffered by later stage diabetic patients are characterised by degrees of slowness to heal caused by a combination of continued localised physical trauma and pro-inflammatory signalling at the wound. To recreate this in a cell-based assay, a defined physical scratch was created and stimulated by combinations of pro-inflammatory factors, namely interferon, the phorbol ester PMA, and lipopolysaccharide, to delay scratch closure. The concentrations of these factors were characterised for commonly used human keratinocyte (HaCaT) and dermal fibroblast (HDF) cell lines. These models were then tested for scratch closure responsiveness to a proprietary healing secretome derived from human Wharton’s jelly mesenchymal stem cells (MSCs) previously validated and shown to be highly effective on closure of acute wound models both in vitro and in vivo. The chronically open scratches from HaCaT cells showed closure after exposure to the MSC secretome product. We propose this delayed scratch closure model for academic and industrial researchers studying chronic wounds looking for responsiveness to drugs or biological treatments prior to testing on explanted patient material or in vivo.

In vitro toxicity of latex, its terpenoidal fractions and isolated phorbol esters from Euphorbia umbellata (Pax) Bruyns on monocytic and melanoma cells

In Brazil, latex from Euphorbia umbellata (African milk tree) has been increasingly used in folk medicine to treat several types of cancer, including melanoma. The effect of lyophilized latex (LL), its hydroethanolic extract (E80), triterpene (F-TRI)- and diterpene (F-DIT)-enriched fractions, along with six isolated phorbol esters from LL and phorbol 12-myristate 13-acetate (PMA) on J774A.1, THP-1, SK-MEL-28, and B16-F10 cell line viability were evaluated by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) method. The compounds were identified by 2D-NMR and HRESIMS. The effect of the LL, extract and fractions on cell viability was also assessed through a resazurin reduction assay. At 100 μg/ml, LL, and its fractions moderately inhibited J774A.1 (37.5–59.5%) and THP-1 (12.6–43.6%) metabolism. LL (IC50 70 μg/ml) and F-TRI (IC50 68 μg/ml) were barely more effective against B16-F10 cells, and only F-TRI exerted an inhibitory effect on SK-MEL-28 cells (IC50 66–75 μg/ml). The samples did not effectively inhibit THP-1 growth (IC50 69–87 μg/ml, assessed by MTT). B16-F10 was susceptible to PMA (IC50 53 μM) and two 12-phenylacetate esters (IC50 56–60 μM), while SK-MEL-28 growth was inhibited (IC50 58 μM) by one of these kinds of esters with an additional 4β-deoxy structure. Synagrantol A (IC50 39 μM) was as effective as PMA (IC50 47 μM) in inhibiting J774A.1 growth in a dose-dependent manner. Furthermore, an in silico study with target receptors indicated a high interaction of the compounds with the PKC proteins. These results provide useful knowledge on the effect of tigliane-type diterpenes on tumor cell from the perspective of medicinal chemistry.

Keratin 17- and PKCα-dependent amplification of neutrophil influx after repeated stress to skin

Abstract Neutrophils contribute to the pathogenesis of chronic inflammatory skin diseases. Little is known about the source and identity of the signals mediating their recruitment in inflamed skin. We used the phorbol ester TPA and UVB, alone or in combination, to induce sterile inflammation in mouse skin and assess whether keratinocyte-derived signals impact neutrophil recruitment. A single TPA treatment results in a neutrophil influx in the dermis that peaks at 12h and resolves within 24h. A second TPA treatment or a UVB challenge, when applied at 24h but not 48h later, accelerates, amplifies, and prolongs neutrophil infiltration. This transient amplification response (TAR) is mediated by local signals in inflamed skin, can be recapitulated in ex vivo culture, and involves the K17-dependent sustainment of Protein Kinase C α (PKCα) activity and release of neutrophil chemoattractants by stressed keratinocytes. We show that K17 binds RACK1, an essential scaffolding protein for PKCα activity. Analyses of RNAseq data reveal the presence of a transcriptomic signature consistent with TAR and PKCα activation in chronic inflammatory skin diseases including psoriasis, hidradenitis suppurativa, and atopic dermatitis. These findings uncover a novel, transient, and keratin-dependent mechanism that amplifies neutrophil recruitment to the skin under stress, with direct implications for inflammatory skin disorders.

Effect of Kaempferol on Modulation of Vascular Contractility Mainly through PKC and CPI-17 Inactivation.

In this study, we investigated the efficacy of kaempferol (a flavonoid found in plants and plant-derived foods such as kale, beans, tea, spinach and broccoli) on vascular contractibility and aimed to clarify the detailed mechanism underlying the relaxation. Isometric contractions of divested muscles were stored and linked with western blot analysis which was carried out to estimate the phosphorylation of myosin phosphatase targeting subunit 1 (MYPT1) and phosphorylation-dependent inhibitory protein for myosin phosphatase (CPI-17) and to estimate the effect of kaempferol on the RhoA/ROCK/CPI-17 pathway. Kaempferol conspicuously impeded phorbol ester-, fluoride- and a thromboxane mimetic-derived contractions regardless of endothelial nitric oxide synthesis, indicating its direct effect on smooth muscles. It also conspicuously impeded the fluoride-derived elevation in phospho-MYPT1 rather than phospho-CPI-17 levels and phorbol 12,13-dibutyrate-derived increase in phospho-CPI-17 and phospho-ERK1/2 levels, suggesting the depression of PKC and MEK activities and subsequent phosphorylation of CPI-17 and ERK1/2. Taken together, these outcomes suggest that kaempferol-derived relaxation incorporates myosin phosphatase retrieval and calcium desensitization, which appear to be modulated by CPI-17 dephosphorylation mainly through PKC inactivation.