Phorbol Ester Research Articles

Characterization of the Activity of Croton tiglium Oil in Hetter's Very Heavy Phenol-Croton Oil Chemical Peels.

Croton oil (CO) is used by dermatologists and plastic surgeons in deep chemical peels. It is mixed with phenol, water, and a soap in Baker-Gordon's or Hetter's formulas. There is controversy as to whether CO or phenol is the active agent in the dermal effect of deep chemical peels. To better clarify the role of CO in deep peels, by identification of active compounds in commercially available CO in the United States and biological effects in vivo. Liquid chromatography-tandem mass spectrometry on CO and a domestic pig model experiment using 3 different formulas: G1: 5% Septisol (SEP), G2: 1.6% croton oil in 35% phenol with 5% SEP, and G3: 35% phenol with 5% SEP. Liquid chromatography-tandem mass spectrometry indicated the presence of phorbol esters. G1 was null overall. Extent of the coagulative necrosis: G2 > G3. Vascular ectasia: G2 > G3. Inflammation pattern: intense neutrophilic inflammatory band in G2 versus mild, sparse, perivascular mononuclear cell infiltrate in G3. Neocollagenesis: pronounced in G2, negligible in G3. Coagulative necrosis of the epidermis, superficial fibroblasts, and vasculature can be attributed to the action of phenol. Phorbol esters on CO could be responsible for the dense deep acute inflammation and the distinctive neocollagenesis.

Lophira alata Suppresses Phorbol Ester-Mediated Increase in Cell Growth via Inhibition of Protein Kinase C-α/Akt in Glioblastoma Cells.

Medicinal plants serve as sources of compounds used to treat other types of cancers. The root of the plant Lophira alata (Ochnaceae) has been used as a component of traditional herbal decoctions administered to cancer patients in southwestern Nigeria. However, the mechanism of the cytotoxic effects of Lophira alata alone or in the presence of phorbol ester has not been investigated in brain tumor cells. This study aimed to examine the cytotoxic potential of the methanolic fraction of Lophira alata root on malignant glioma invasive cellular growth and survival. The methanolic fraction of Lophira alata (LAM) was subjected to high-performance liquid chromatography to determine the fingerprints of the active molecules. The antiproliferative effects of Lophira alata were assessed using the MTT and LDH assays. Protein immunoblots were carried out to test the effects of Lophira alata, alone or in the presence of phorbol ester, on survival signaling pathways, such as Akt, mTOR, and apoptotic markers such as PARP and caspases. The methanolic fraction of Lophira alata (LAM) induced a concentration-dependent and time-dependent decrease in glioma cell proliferation. In addition, LAM attenuated phorbol ester-mediated signaling of downstream targets such as Akt/mTOR. Gene silencing using siRNA targeting PKC-alpha attenuated LAM-mediated downregulation of Akt. In addition, LAM induced both PARP and caspase cleavages. The HPLC fingerprint of the fraction indicates the presence of flavonoids. LAM decreases cell proliferation and induces apoptosis in glioma cell lines and thus could serve as a therapeutic molecule in the management of gliomas.

Abstract PS17-46: Pkc agonism restricts immune suppression and promotes antigen cross-presentation in triple negative breast cancer

Abstract Immunotherapy has revolutionized cancer treatment showing unprecedented long-term antitumor responses. However, most patients do not respond to immunotherapies due at least partly to immune suppression. Immunotherapy non-responders have high levels of circulating myeloid-derived suppressor cells (MDSCs)- an immunosuppressive innate cell population that suppresses both innate and adaptive immunity. Triple negative breast cancer (TNBC) is the most aggressive subtype of breast cancers with poor responses to conventional therapies. TNBC patients harbor higher levels of MDSC populations compared to non-TNBC breast cancer patients. Consequently, TNBC and other solid tumor patients who have high levels of circulating MDSCs respond poorly to immunotherapy. On the other hand, cross-presenting dendritic cells (DCs) are essential to generate an antitumor immune response. Breast cancer patients who harbor higher numbers of these DCs have a better prognosis than patients with lower DC numbers. Several strategies aiming at achieving an effective combination with immunotherapy are under active investigation. The central dogma of these strategies consists of inducing T cells into “immunologically cold tumors” which are defined by having low neoantigen burden and a paucity of T cells and DCs. Hence, strategies that enhance cross-presenting DCs and T cell antitumor potential while altering MDSC’s suppressive function are likely to be effectively combined with immunotherapy for a maximum therapeutic benefit. Protein Kinase C (PKC) is a family of enzymes that play a critical role in cell signaling controlling the balance between survival and cell death. With the discovery in 1980s that PKC is a receptor for the tumor-promoting phorbol esters, the dogma that PKC is an oncoprotein was fueled. This led to more than three decades of failed clinical trials trying to inhibit PKC in cancer. Recent evidence suggests that PKC isozymes are generally inactivated in cancer and that most mutations affecting PKC isozymes are in fact loss of function mutations. This suggests that PKC is a tumor suppressor rather than an oncoprotein and that strategies in cancer treatment should focus on restoring PKC, rather than inhibiting it. To date, the role of PKC isozymes in antitumor immunity is unknown. Herein, our novel data suggest that PKC agonism using established agonists reduced MDSC generation from bone marrow (BM) progenitors specifically via activation of the PKC delta (PKCδ) isoform. PKC agonism induced MDSC differentiation to cross-presenting CD103+ DCs both ex-vivo and in vivo. Additionally, PKC agonist-treated purified MDSCs lost their suppressive capacity on CD8+ T cells in both in vitro and in vivo suppression assays. In contrast, PKC agonism significantly increased the generation of cross-presenting DCs (cDC1) from BM progenitors. Treatment of TNBC-bearing C57BL/6J mice with PKC agonist PEP005 markedly reduced tumor burden by decreasing the frequencies of M-MDSCs in tumor, spleen, and bone marrow while increasing cDC1 frequencies in tumor and spleen. These findings propose PKC as a common pathway in myeloid cells to tip the balance from immune suppression to effective antitumor immunity. Citation Format: Mehdi Chaib, Jeremiah Holt, Laura Sipe, Ajeeth Pingili, Deidre Daria, Liza Makowski. Pkc agonism restricts immune suppression and promotes antigen cross-presentation in triple negative breast cancer [abstract]. In: Proceedings of the 2020 San Antonio Breast Cancer Virtual Symposium; 2020 Dec 8-11; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2021;81(4 Suppl):Abstract nr PS17-46.

Deciphering the LRRK code: LRRK1 and LRRK2 phosphorylate distinct Rab proteins and are regulated by diverse mechanisms.

Autosomal dominant mutations in LRRK2 that enhance kinase activity cause Parkinson's disease. LRRK2 phosphorylates a subset of Rab GTPases including Rab8A and Rab10 within its effector binding motif. Here, we explore whether LRRK1, a less studied homolog of LRRK2 that regulates growth factor receptor trafficking and osteoclast biology might also phosphorylate Rab proteins. Using mass spectrometry, we found that in LRRK1 knock-out cells, phosphorylation of Rab7A at Ser72 was most impacted. This residue lies at the equivalent site targeted by LRRK2 on Rab8A and Rab10. Accordingly, recombinant LRRK1 efficiently phosphorylated Rab7A at Ser72, but not Rab8A or Rab10. Employing a novel phospho-specific antibody, we found that phorbol ester stimulation of mouse embryonic fibroblasts markedly enhanced phosphorylation of Rab7A at Ser72 via LRRK1. We identify two LRRK1 mutations (K746G and I1412T), equivalent to the LRRK2 R1441G and I2020T Parkinson's mutations, that enhance LRRK1 mediated phosphorylation of Rab7A. We demonstrate that two regulators of LRRK2 namely Rab29 and VPS35[D620N], do not influence LRRK1. Widely used LRRK2 inhibitors do not inhibit LRRK1, but we identify a promiscuous inhibitor termed GZD-824 that inhibits both LRRK1 and LRRK2. The PPM1H Rab phosphatase when overexpressed dephosphorylates Rab7A. Finally, the interaction of Rab7A with its effector RILP is not affected by LRRK1 phosphorylation and we observe that maximal stimulation of the TBK1 or PINK1 pathway does not elevate Rab7A phosphorylation. Altogether, these findings reinforce the idea that the LRRK enzymes have evolved as major regulators of Rab biology with distinct substrate specificity.

Monitoring casbene synthase in Jatropha curcas tissues using targeted proteomics

BackgroundCasbene synthase (CS) is responsible for the first committed step in the biosynthesis of phorbol esters (PE) in the Euphorbiaceae. PE are abundant in the seeds of the biofuel crop Jatropha curcas and its toxicity precludes the use of the protein-rich cake obtained after oil extraction as an animal feed and the toxicity of the fumes derived from burning PE containing biofuel is also a matter of concern. This toxicity is a major hindrance to exploit the potential of this crop as a source of raw material to produce biodiesel. For this reason, the current research on J. curcas is mainly focused on the understanding of the biosynthesis and site of synthesis of PE, as an avenue for the development of genotypes unable to synthesize PE in its seeds.ResultsHere, we present targeted proteomics assays (SRM and PRM) to detect and quantify CS in leaves, endosperm, and roots of two J. curcas genotypes with contrasting levels of PE. These assays were based on the use of reference isotopic labeled synthetic peptides (ILSP) predicted from 12 gene models of CS from the J. curcas genome.ConclusionOur targeted proteomics methods were able to detect and quantify, for the first time, CS gene products and demonstrate the distribution of CS isoforms only in roots from J. curcas genotypes with a high and low concentration of PE. These methods can be expanded to monitor CS, at the protein level, in different tissues and genotypes of J. curcas.

Ubash3b promotes TPA-mediated suppression of leukemogenesis through accelerated downregulation of PKCδ protein

Acquired drug-resistance, often involving downregulation or mutations in the target protein, is a major caveat in precision medicine. Understanding mechanisms of resistance to therapeutic drugs may unravel strategies to overcome or prevent them. We previously identified phorbol ester (PE) compounds such as TPA that induce Protein Kinase δ (PKCδ), thereby suppressing leukemogenesis. Here we identified erythroleukemia cell lines that resist PEs and showed that reduced PKCδ protein expression underlies drug resistance. Reduced level of PKCδ in resistant cell lines was due to its phosphorylation followed by protein degradation. Indeed, proteasome inhibition prevented PE-induced loss of PKCδ. Accordingly, a combination of TPA and the proteasome inhibitor ALLN significantly suppressed leukemia in a mouse model of leukemia. PKCδ downregulation by TPA was independent of the downstream MAPK/ERK/P38/JNK pathway. Instead, expression of ubiquitin-associated and SH3 domain-containing protein b (Ubash3b) was induced by TPA, which leads to PKCδ protein dephosphorylation and degradation. This specific degradation was blocked by RNAi-mediated depletion of Ubash3b. In drug-sensitive leukemic cells, TPA did not induce Ubash3b, and consequently, PKCδ levels remained high. A PE-resistant cell line derived from PE-treated sensitive cells exhibited very low PKCδ expression. In these drug resistance cells, a Ubash3b independent mechanism led to PKCδ degradation. Thus, PE compounds in combination with proteasome or specific inhibitors for Ubash3b, or other factors can overcome resistance to TPA, leading to durable suppression of leukemic growth. These results identify Ubash3b as a potential target for drug development.

PHLPPing the balance: restoration of protein kinase C in cancer.

Protein kinase signalling, which transduces external messages to mediate cellular growth and metabolism, is frequently deregulated in human disease, and specifically in cancer. As such, there are 77 kinase inhibitors currently approved for the treatment of human disease by the FDA. Due to their historical association as the receptors for the tumour-promoting phorbol esters, PKC isozymes were initially targeted as oncogenes in cancer. However, a meta-analysis of clinical trials with PKC inhibitors in combination with chemotherapy revealed that these treatments were not advantageous, and instead resulted in poorer outcomes and greater adverse effects. More recent studies suggest that instead of inhibiting PKC, therapies should aim to restore PKC function in cancer: cancer-associated PKC mutations are generally loss-of-function and high PKC protein is protective in many cancers, including most notably KRAS-driven cancers. These recent findings have reframed PKC as having a tumour suppressive function. This review focusses on a potential new mechanism of restoring PKC function in cancer - through targeting of its negative regulator, the Ser/Thr protein phosphatase PHLPP. This phosphatase regulates PKC steady-state levels by regulating the phosphorylation of a key site, the hydrophobic motif, whose phosphorylation is necessary for the stability of the enzyme. We also consider whether the phosphorylation of the potent oncogene KRAS provides a mechanism by which high PKC expression may be protective in KRAS-driven human cancers.

Removing phorbol esters from the biomass to add extra value to the byproduct from deoiling seeds of Jatropha curcas in the biodiesel industry

Either pressing or solvent extraction of Jatropha curcas seed oil results in great amounts of cake as a byproduct. The direct use of these fresh biomasses threats the health of mammals as they contain phorbol esters (PEs), a highly toxic class of substance. Five different treatments were bench-assayed to degrade PEs: (i) ammonium hydroxide, (ii) urea, (iii) heat, (iv) ultraviolet radiation, and (v) gamma radiation. All used methods were evaluated for their efficiency on removing PEs from the biomass resulting from deoiling seeds of J. curcas. The treatments were variably effective in reducing PEs contents to nontoxic levels. Aqueous ammonium hydroxide solution (3% w/w) at 70 °C was found to reduce the contents of PEs down to 0.084 mg g−1 (cake) and 0.083 mg g−1 (bran). The treatment with an aqueous solution NH4OH 7% w/w with heating at 90 °C led to the most effective reduction, rendering PEs contents as low as 0.063 mg g−1 (cake) and 0.066 mg g−1 (bran). These are below the critical toxicity threshold, namely 0.1 mg g−1, which is found in seeds of nontoxic J. curcas varieties. The corresponding results from cytotoxicity tests and assessments of nutritional characteristics confirmed that these treated samples have become safe enough, making this affordable technology potentially scalable to be used in the feeding of livestock at the industrial level.

Design, synthesis and biological evaluation of novel benzoxaborole derivatives as potent PDE4 inhibitors for topical treatment of atopic dermatitis.

In this work, a series of structurally novel benzoxaborole derivatives were designed, synthesized and biologically evaluated as PDE4 inhibitors for battling atopic dermatitis (AD). Among them, the majority exhibited superior PDE4B inhibitory activities to that of the lead compound Crisaborole, an approved PDE4 inhibitor. In particular, 72, the most potent PDE4B inhibitor throughout this series, displayed 136-fold improved enzymatic activity (IC50=0.42nM) as compared to Crisaborole (IC50=57.20nM), along with favorable isoform specificity. In the phorbol ester (PMA)-induced mouse ear oedema model, 72 exerted remarkably greater efficacy than Crisaborole at the same dosage (P<0.05). Moreover, the ointment of 72 exerted dramatically enhanced therapeutic potency than the ointment of Crisaborole (P<0.05) in the calcipotriol-induced mouse AD model. In addition to the potent invitro and invivo activity, 72 displayed favorable safety in the repeated oral dose toxicity study and did not exhibit phototoxicity. With the above attractive biological performance, 72 is worthy of further functional investigation as a novel anti-AD therapeutic agent.

Protein kinase C evokes quantal catecholamine release from PC12 cells via activation of L-type Ca2+ channels.

Application of the phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA) to PC12 cells under resting conditions evoked quantal catecholamine secretion, as detected amperometrically. This effect was not mimicked by 4alpha-phorbol-12,13-didecanoate, another phorbol ester, which is inactive with respect to protein kinase C activation, and was prevented by the protein kinase C inhibitor bisindolylmaleimide. TPA also caused a rise of [Ca(2+)](i) in Fura-2-loaded PC12 cells, and again this was not mimicked by 4alpha-phorbol-12,13-didecanoate and could be blocked by bisindolylmaleimide. TPA-evoked secretion was entirely dependent on extracellular Ca(2+) and was fully abolished by nifedipine, as were TPA-induced rises of [Ca(2+)](i). Resting membrane potential, monitored using perforated patch recordings, was unaffected by TPA. However, a small (6-8 mV) hyperpolarizing shift in the voltage dependence of Ca(2+) channel currents (determined using whole-cell patch clamp recordings) was induced by TPA, and this could be fully prevented by nifedipine. In contrast to results with depolarizing stimuli, which evoke exocytosis because of Ca(2+) influx through N-type channels in these cells, the present results indicate that protein kinase C activation leads directly to quantal catecholamine secretion in the absence of depolarizing stimuli via a selective shift in the activation of L-type Ca(2+) channels.

Efficient Vortex-assisted Extraction of Phorbol Esters from Jatropha Leaves and Correlation Between Leaves and Seeds in Phorbol EstersContent

ABSTRACT This work aimed to optimise and validate the vortex-assisted extraction with high-performance liquid chromatography-ultraviolet detection (HPLC-UV) method to quantify phorbol esters (PEs) in Jatropha leaves. Additionally, to evaluate the correlation between PEs content in leaves and seeds. The results of PEs content were expressed as equivalent of the major Jatropha phorbol ester, 12-deoxy-16-hydroxyphorbol-4'-[12',14'-butadienyl]-6'-[16',18',20'-nonatrienyl]-bicyclo[3.1.0]-hexane-(13-O)-2'-[carboxylate]-(16-O)-3'-]8'-butenoic-10']ate (DHPB) . One-variable-at-time strategy and 25-1V factorial fractional design were employed to determine the effects of solvent type, volume, stirring frequency, extraction time and cycles on yield. The quantification was performed using HPLC-UV. The optimum conditions were three extractions with 2% tetrahydrofuran:dichloromethane (1:1) in methanol (THF:DCM/MeOH). The sample:solvent ratio was 1 mg:25 μL, and the vortex stirring was 2200 rpm for 5 minutes. The method showed good linearity (R2 0.999), recoveries (97 to 105%), selectivity and repeatability (RSD 1.93-7.95%). In addition, good limits of detection and quantification of 2.19 and 6.65 ng μL"1, respectively, were noted. DHPB content in leaves and seeds ranged from 0.01 to 0.38 mg g"1 and 0.41 to 1.82 mg g"1, respectively. There is a statistically significant and positive linear relationship between leaves and seeds for DHPB content. These results may have practical application in analysing and predicting the amount of PEs in Jatropha leaves for environmental safety studies and the selection of better varieties for genetic improvement. Keywords: correlation, Jatropha leaves; Jatropha seeds; phorbol esters; vortex-assisted.

Degradation of Phorbol Esters on the Jatropha curcas Linn. Seed by Biological Detoxification

The application of fermentation is one of methods to increase food quality biologically. Availability of Jatropha curcas residual from oil factory be focused as a soybean meal or fish meal replacement. On the other hand, J. curcas residuals possess a toxic compound as well. This study aimed to examine the effect of Aspergillus niger on the nutrition and harmful content of J. curcas as a potential ingredient of feed. In brief, J. curcas residual was fermented with a detoxification method at 3 d, 5 d, and 7 d. Crude protein, fat, and crude fiber content were assessed to discover the biological responses of J. curcas post–fermentation while phorbol ester was evaluated to toxic content post–detoxification. The results showed that crude protein and fat content were highest on 7 d post–fermentation but it was no significant difference (p &gt; 0.05). While crude fiber content showed significant difference which the 3 d fermentation had the highest content of fiber. For phorbol ester content, 3 d fermentation showed a better result than the control group (p &lt; 0.05). The present findings suggest that A. niger is recommendable as starter to reduce fiber and toxic content of J. curcas residual at 3 d fermentation.

Comparison of orosomucoid-1 immunoexpression and angiogenesis between oral squamous cell carcinoma cases with different histological grades.

Background:Oral squamous cell carcinoma (OSCC) is the most common malignancy in this region, and thus, further elucidation of its tumoral mechanisms is important. One of the main roles of the acute-phase protein orosomucoid-1 (ORM1) is the promotion of angiogenesis, which is key for tumor nutrition and growth.Aim:Our aim was to evaluate the immunohistochemical expression of ORM1 and the angiogenic activity indicated by microvascular density (MVD) in OSCC samples according to histological grade.Materials and Methods:Formalin-fixed, paraffin-embedded sections from 45 OSCC cases were submitted to immunohistochemistry: 25 were well-differentiated OSCC, 18 were moderately differentiated OSCC and 2 were poorly differentiated OSCC. ORM1 staining was evaluated by a semiquantitative method, and CD34-positive blood vessels were quantified to calculate the MVD. The results were statically analyzed.Results:All cases exhibited immunoexpression of ORM1 and CD34. However, no significant differences were found between the expression of both markers among the histological grades. In addition, the presence of ORM1 in inflammatory cells and in the extracellular matrix was detected in most cases.Conclusion:These results suggest that the induction of angiogenesis is not the main role of ORM1 in OSCC and may be associated with the regulation of the immune/inflammatory response or the transport of protumoral molecules, such as sialyl-Lewis X or phorbol esters, which requires confirmation in future studies.

The oncogenic role of protein kinase D3 in cancer.

Protein kinase D3 (PRKD3), a serine/threonine kinase, belongs to protein kinase D family, which contains three members: PRKD1, PRKD2, and PRKD3. PRKD3 is activated by many stimuli including phorbol esters, and G-protein-coupled receptor agonists. PRKD3 promotes cancer cell proliferation, growth, migration, and invasion in various tumor types including colorectal, gastric, hepatic, prostate, and breast cancer. Accumulating data supports that PRKD3 is a promising therapeutic target for treatment of cancer. This review discusses the functions and mechanisms of PRKD3 in promoting tumorigenesis and tumor progression of various tumor types as well as the latest developments of small-molecule inhibitors selection for PRKD/PRKD3.

Transcriptional activation of urokinase by the Krüppel-like factor Zf9/COPEB activates latent TGF-β1 in vascular endothelial cells

Understanding the regulation of genes controlling fibrinolysis and matrix homeostasis is essential for elucidating the basis of tissue repair. A recently described novel Krüppel-like factor, Zf9, is up-regulated in acute liver injury in activated hepatic stellate cells. Because Zf9 can be induced widely, its activity was examined in vascular endothelium, a key cell in vascular injury. Zf9 is induced as an immediate-early response gene in bovine aortic endothelial cells (BAECs) following treatment with serum or phorbol ester. Zf9 transcriptionally activates urokinase plasminogen activator (uPA). Recombinant Zf9-GST binds to wild-type but not mutated 'GC-box' motifs within the human uPA promoter (-63 to -32), with greatest affinity to the middle of 3 contiguous GC boxes. Transient transfection of Zf9 drives transactivation of a full-length uPA promoter- and GC box-construct, but not a uPA promoter-construct devoid of GC boxes. Transactivation of uPA by Zf9 is also supported in Drosophila S2 cells. Most importantly, transiently transfected Zf9 up-regulates endogenous uPA messenger RNA and activity in BAECs, resulting in increased bioactive transforming growth factor-beta (TGF-beta) via enhancement of proteolytic activation of the latent molecule. Furthermore, concomitant expression of Zf9 and uPA proteins was observed in arterial endothelial cells after balloon injury in rats, suggesting a potential role of Zf9 in uPA expression not only in vitro but also in vivo. These findings suggest a role of Zf9 in the injury response by enhancing uPA synthesis and subsequent activation of latent TGF-beta. (Blood. 2000;95:1309-1316)

Regulation of monocyte migration by amphoterin (HMGB1)

AbstractAmphoterin (HMGB1) is a 30-kD heparin-binding protein involved in process extension and migration of cells by a mechanism involving the receptor for advanced glycation end products (RAGE). High levels of amphoterin are released to serum during septic shock. We have studied the expression of amphoterin in monocytes and the role of amphoterin and RAGE in monocyte transendothelial migration. Un-activated monocytes in suspension did not reveal amphoterin on their surface, but adherent monocytes exported amphoterin to the cell surface. Immunohistochemical staining of arterial thrombi in vivo revealed amphoterin in mononuclear cells and in surrounding extracellular matrix. Amphoterin was secreted from phorbol ester and interferon-γ (IFN-γ)-activated macrophages, and the secretion was inhibited by blocking the adenosine 5′-triphosphate (ATP)-binding cassette transporter-1, a member of the multidrug resistance protein family. Amphoterin was specifically adhesive for monocytes in peripheral blood leukocyte adhesion assay. Adhesion caused an extensive spreading of cells, which was inhibited by the dominant-negative RAGE receptor (soluble ectodomain of RAGE), and adhesion up-regulated chromogranin expression in monocytes, also suggesting a RAGE-dependent interaction. Monocyte transendothelial migration was efficiently inhibited by anti-amphoterin and anti-RAGE antibodies and by the soluble RAGE. We suggest that amphoterin is an autocrine/paracrine regulator of monocyte invasion through the endothelium. (Blood. 2004;104:1174-1182)

Mutations in 3β-hydroxysteroid-δ8, δ7-isomerase paradoxically benefit epidermal permeability barrier homeostasis in mice.

Inherited or acquired blockade of distal steps in the cholesterol synthetic pathway results in ichthyosis, due to reduced cholesterol production and/or the accumulation of toxic metabolic precursors, while inhibition of epidermal cholesterol synthesis compromises epidermal permeability barrier homeostasis. We showed here that 3β-hydroxysteroid-δ8, δ7-isomerase-deficient mice (TD), an analog for CHILD syndrome in humans, exhibited not only lower basal transepidermal water loss rates, but also accelerated permeability barrier recovery despite the lower expression levels of mRNA for epidermal differentiation marker-related proteins and lipid synthetic enzymes. Moreover, TD mice displayed low skin surface pH, paralleled by increased expression levels of mRNA for sodium/hydrogen exchanger 1 (NHE1) and increased antimicrobial peptide expression, compared with wild-type (WT) mice, which may compensate for the decreased differentiation and lipid synthesis. Additionally, in comparison with WT controls, TD mice showed a significant reduction in ear thickness following challenges with either phorbol ester or oxazolone. However, TD mice exhibited growth retardation. Together, these results demonstrate that 3β-hydroxysteroid-δ8, δ7-isomerase deficiency does not compromise epidermal permeability barrier in mice, suggesting that alterations in epidermal function depend on which step of the cholesterol synthetic pathway is interrupted. But whether these findings in mice could be mirrored in humans remains to be determined.

Chemical analysis of callus extracts from toxic and non-toxic varieties of Jatropha curcas L.

Jatropha curcas L. belongs to Euphorbiaceae family, and it synthesizes flavonoid and diterpene compounds that have showed antioxidant, anti-inflammatory, anticancer, antiviral, antimicrobial, antifungal and insecticide activity. Seeds of this plant accumulate phorbol esters, which are tigliane type diterpenes, reported as toxic and, depending on its concentration, toxic and non-toxic varieties has been identified. The aim of this work was to characterize the chemical profile of the extracts from seeds, leaves and callus of both varieties (toxic and non-toxic) of Jatropha curcas, to verify the presence of important compounds in dedifferentiated cells and consider the possibility of using these cultures for the massive production of metabolites. Callus induction was obtained using NAA (1.5 mg L−1) and BAP (1.5 mg L−1) after 21 d for both varieties. Thin layer chromatography analysis showed differences in compounds accumulation in callus from non-toxic variety throughout the time of culture, diterpenes showed an increase along the time, in contrast with flavonoids which decreased. Based on the results obtained through microQTOF-QII spectrometer it is suggested a higher accumulation of phorbol esters, derived from 12-deoxy-16-hydroxy-phorbol (m/z 365 [M+H]+), in callus of 38 d than those of 14 d culture, from both varieties. Unlike flavonoids accumulation, the MS chromatograms analysis allowed to suggest lower accumulation of flavonoids as the culture time progresses, in callus from both varieties. The presence of six glycosylated flavonoids is also suggested in leaf and callus extracts derived from both varieties (toxic and non-toxic), including: apigenin 6-C-α-L-arabinopyranosyl-8-C-β-D-xylopyranoside (m/z 535 [M+H]+), apigenin 4′-O-rhamnoside (m/z 417 [M+H]+), vitexin (m/z 433 [M+H]+), vitexin 4′-O-glucoside-2″-O-rhamnoside (m/z 741 [M+H]+), vicenin-2 (m/z 595 [M+H]+), and vicenin-2,6″-O-glucoside (m/z 757 [M+H]+).

Anaerobic Digestion of aqueous Jatropha seed oil extraction residues and phorbol ester degradation

Aqueous Jatropha Oil Extraction Residue (AJOR) is a byproduct from a novel technique for Jatropha seed oil extraction. Its potential for biogas production, its effects on process stability and the reduction of toxic phorbol esters were investigated. AJOR had a total solid and volatile solid content of 24.3% and 92% and contained 35.5% of crude protein and 17.5% of crude fat, based on total solids. The specific methane yield was measured with the Hohenheim Biogas Yield Test at 406 LCH4 kg−1VS. In continuous digestion, 405 LCH4 kg−1VS was reached, but was accompanied by an excessive volatile organic acids to total inorganic carbon ratio at an organic loading rate of 2.64 kgVS m−3 d−1. After a reduction of the organic loading rate to 1.7 kgVS m−3 d−1, the specific methane yield amounted to 440 LCH4 kg−1VS and the fermentation process became stabilized. Phorbol esters per FM decreased by 97–98%.

ADAM17-Deficient Pluripotent Stem Cell-Derived Natural Killer Cells Possess Improved Antibody-Dependent Cellular Cytotoxicity and Antitumor Activity

Antibody-dependent cellular cytotoxicity (ADCC) is a key pathway that mediates natural killer (NK) cell cytotoxicity against antibody-opsonized target cells. This process helps mediate the therapeutic efficacy of anti-tumor antibodies. On NK cells, ADCC occurs via engagement of antibody-coated target cells with activating receptor FcγRIIIa, or CD16a, leading to proinflammatory cytokine upregulation, degranulation, and target cell death. Upon cellular activation, the CD16a ectodomain is cleaved from the NK cell surface by A Disintegrin and Metalloprotease-17 (ADAM17). Cleavage of the ectodomain prevents further antibody binding and signaling through CD16a, which dampens NK cell activity. Blocking activation-induced ADAM17-mediated CD16a cleavage has been previously demonstrated to augment ADCC activity and provides a novel strategy to improve efficacy of therapeutic antibodies in combination with adoptive transfer of engineered NK cells. To further define the ability of ADAM17 to regulate NK cell activity, we have generated and characterized ADAM17-deficient (ADAM17-KO) NK cells derived from CRISPR/Cas9-modified human induced pluripotent stem cells (iPSCs). ADAM17-KO iPSCs successfully differentiate into hematopoietic progenitor cells, then to NK cells that uniformly express typical NK cell surface markers including CD56, CD94, NKG2D, NKp44, and NKp46. ADAM17-KO iPSC-NKs are functional and kill K562 erythroleukemia cells comparable to wildtype iPSC-derived NK cells (WT iPSC-NK cells) and healthy donor-derived peripheral blood NK cells (PB-NK cells) in vitro. Surprisingly, upon differentiation, ADAM17-KO iPSC-NK cells express ~20% lower CD16a surface expression compared to WT iPSC-NK cells, but stably retain CD16a expression after enrichment for CD16a+ cells and over 6 weeks of expansion in culture. WT iPSC-NKs and PB-NKs rapidly lose CD16a surface expression upon stimulation with phorbol esters, while ADAM17 KO iPSC-NK cells maintain over 90% CD16a expression after this stimulation. Additionally, a significantly higher proportion of ADAM17-KO iPSCs express TNF-α (71%) and CD62L (L-Selectin) (36%) - two other known ADAM17 substrates, on the cell surface after stimulation with phorbol esters for 4 hours compared to WT iPSC-NK (7% TNF-α+, 2% L-Selectin+) and PB-NK (2% TNF-α+, 1% L-Selectin+). CD16a+ ADAM17-KO iPSC-NK cells mediate increased CD107a (45%) and IFNγ (39%) expression when co-incubated with RAJI B-lymphoma cells in the presence of the anti-CD20 antibody rituximab, compared to CD16a+ WT iPSC-NK (32% CD107a+, 11% IFNγ) and PB-NK (37% CD107a+, 7% IFNγ) cells. Similarly, CD16a+ ADAM17-KO iPSC-NK cells upregulate increased CD107a (29%) and IFNγ (42%) expression when co-incubated with CAL27 squamous cell carcinoma cells in the presence of the anti-EGFR antibody cetuximab, compared to CD16a+ WT iPSC-NK (12% CD107a+, 8% IFNγ) and PB-NK (14% CD107a+, 6% IFNγ). Long-term (24 hour) cytotoxicity assay against RAJI cells in the presence of rituximab demonstrates higher cytotoxicity in CD16a+ ADAM17-KO iPSC-NK cells compared to CD16a+ WT iPSC-NK and CD16a+ PB-NK cells over time (see associated figure). In vivo studies to determine the therapeutic efficacy of ADAM17-KO iPSC-NK cells compared to WT iPSC-NK and PB-NK cells are ongoing. Together, these studies demonstrate ADAM17-KO iPSC-NK cells derived from a renewable source of gene-edited iPSCs possess enhanced ADCC potential, and provide a promising candidate to be used for standardized, off-the-shelf NK cell-based therapies in conjunction with therapeutic antibodies. Figure Disclosures Blum: Fate Therapeutics: Current Employment. Kaufman:Fate Therapeutics: Consultancy.