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Introduction. Current chemotherapy of breast cancer has a wide range of disadvantages, in particular, the development of therapy-related infections and hormonal imbalance. Combination of main cytostatic with glucocorticoids allows to broaden its therapeutic interval and to decrease the total toxicity of the treatment. However, long-term treatment with glucocorticoids leads to the development of severe side effects via activation of multiple molecular mechanisms. Thus, glucocorticoids activate prosurvival mTOR-dependent autophagy. Therefore, the evaluation of PI3K (phosphoinositide 3-kinases) / Akt (protein kinase B) / mTOR (mammalian target of rapamycin) inhibitors as adjuvants for breast cancer therapy is important for optimization of treatment protocol.Aim. Analysis of the effects of PI3K/Akt/mTOR inhibitors, rapamycin, wortmannin and LY-294002 in combination with glucocorticoids in breast cancer cell lines of different subtypes.Materials and methods. We demonstrated the inhibition of PI3K/Akt/mTOR signaling and the autophagy induction after the treatment of breast cancer cells with rapamycin, wortmannin and LY-294002 by Western blotting analysis of Beclin-1, phospho-Beclin-1 (Ser93 and Ser30).Conclusion. PI3K/Akt/mTOR inhibitors in combination with Dexamethasone cooperatively inhibited mTOR signaling and activated autophagy in breast cancer cells in vitro.

Abstract Background Sugammadex is a synthetic γ-cyclodextrin derivative designed to selectively bind to steroidal neuromuscular blocking agents and reverse their effects. Although many cases of sugammadex-induced anaphylaxis have been reported, few studies have investigated the underlying mechanism. Case presentation A 55-year-old Japanese man underwent a laryngectomy under general anesthesia. One month before laryngectomy, he had undergone laryngoscopy under general anesthesia and received sugammadex administration without causing hypersensitivity. He had no history of allergies. The operation was finished without complications. Shortly after sugammadex administration, his blood pressure dropped to approximately 70 mmHg, and his heart rate increased to 110 beats/minute with systemic erythema. Suspecting anaphylaxis, he was treated with the intravenous injection of phenylephrine, d-chlorpheniramine, and hydrocortisone. After these treatments, his cardiovascular condition stabilized. Eight months after the event, skin prick tests and intradermal tests with all agents used during general anesthesia were performed. Intradermal tests showed positive results only for sugammadex. Subsequently, basophil activation tests with CD203c were performed using sugammadex, γ-cyclodextrin, and positive controls (anti-immunoglobulin-E and formyl-methionyl-leucyl-phenylalanine). In addition to both controls, sugammadex, but not γ-cyclodextrin, induced significant upregulation of CD203c expression. We performed additional basophil activation tests with wortmannin, an inhibitor of phosphoinositide 3-kinase, to investigate the mechanism underlying sugammadex-induced basophil activation. The inhibitory effect of wortmannin on basophil activation due to sugammadex was similar to that of anti-immunoglobulin-E, suggesting an immunoglobulin-E-dependent mechanism. Although the patient showed no hypersensitivity after the first exposure of sugammadex, anaphylaxis appeared after the second administration. Because most cases of sugammadex-induced anaphylaxis reportedly appeared after first administration, this seems to be a rare case. Conclusions In the present case, sugammadex-induced anaphylaxis might have occurred through an immunoglobulin-E-dependent mechanism and not involve γ-cyclodextrin as an epitope. Physicians should pay attention to the occurrence of sugammadex-induced anaphylaxis even when the patient has a history of safe administration of sugammadex.

We describe a pre-clinical spinal cord motor neuron injury model that is minimal invasive, reproducible, focal and easily applied to small rodents. Retrograde axonal transport of a pro-apoptotic phosphatidylinosotol 3’-kinase inhibitor, wortmannin, via the sciatic nerve results in loss of ipsilateral lumbar motor neurons proportional to the level of drug administered. Motor neuron loss was detected by choline acetyltransferase (ChAT) immunostaining and with a transgenic thy1-eGFP marker. The short half-life of wortmannin generates minimal wound spread, and wortmannin does not affect axon transport, as determined by co-injection of a pseudorabies virus tracer. Using quantitative transcript analysis, we found that ChAT transcripts significantly decreased at 14 days post-delivery of 1 μg wortmannin, relative to sham controls, and remained low after 90 days. Smaller effects were observed with 200 ng and 100 ng wortmannin. Wortmannin also generated a transient and significant increase in astrocyte Gfap transcripts after 14 days with a return to control levels at 90 days. Treated mice had hind limb spasticity and a forced motor function defect that was quantified using a water exit test. Controls rapidly exit a shallow water tray, and wortmannin treated animals were up to 12-fold slower, a phenotype that persisted for at least 3 months. Thus the focal delivery of wortmannin to motor neurons generates a reproducible and scalable injury that can facilitate quantitative studies on neural regeneration and repair. The efficacy of sciatic nerve suicide transport can also explain neurotoxin-mediated selective loss of motor neurons in diseases such as amyotrophic lateral sclerosis. All procedures were performed at Rutgers under established Institutional Animal Care and Use protocols (eIACUC_TR201800022, approved on March 20, 2018).

Abstract Purpose Contrast-induced acute kidney injury (CI-AKI) resulting from administration of iodinated contrast media (CM) is the third leading cause of hospital-acquired acute kidney injury and is associated with substantial morbidity and mortality. Deteriorated renal microcirculation plays an important role in CI-AKI. Limb ischemic preconditioning (LIPC), where brief and non-injurious ischemia/reperfusion is applied to a limb prior to the administration of the contrast agent, is emerging as a promising strategy for CI-AKI prevention. However, it is not known whether the renal protection of LIPC against CI-AKI is mediated by regulation of renal microcirculation and the molecular mechanisms remain largely unknown. Methods In this study, we examined the renal cortical and medullary blood flow in a stable CI-AKI model using 5/6-nephrectomized (NE) rat. The LIPC and sham procedures were performed prior to the injection of CM. Furthermore, we analyzed renal medulla hypoxia using in vivo labeling of hypoxyprobe. Pharmacological inhibitions and western blotting were used to determine the underlying molecular mechanisms. Results In this study, we found LIPC significantly ameliorated CM-induced reduction of medullary blood flow and attenuated CM-induced hypoxia. PI3K inhibitor (wortmannin) treatment blocked the regulation of medullary blood flow and the attenuation of hypoxia of LIPC. Phosphorylation of Akt/eNOS was significantly decreased via wortmannin treatment compared with LIPC. Nitric oxide synthase-inhibitor [Nω-nitro-l-arginine methyl ester (L-NAME)] treatment abolished the above effects and decreased phosphorylation of eNOS, but not Akt. Conclusions Collectively, the results demonstrate that LIPC ameliorates CM-induced renal vasocontraction and is mediated by activation of PI3K/Akt/eNOS signaling pathway.

Phosphatidylinositol-3 kinase-related kinases (PIKKs) belong to a family of atypical serine/threonine kinases in humans. They actively participate in a diverse set of cellular functions such as meiotic, V(D)J recombination, chromosome maintenance, DNA damage sensing and repair, cell cycle progression and arrest. ATR, ATM, DNA-PKcs, mTOR and hSMG are the members of the PIKK family that play an important role in in cancer cell proliferation, autophagy, and cell survival to radio and chemotherapy. Thereby targeting these PIKK kinases in cancer along with chemo/radiotherapy agents, can help in differential cytotoxicity towards cancer cell over the normal cell. In this review, we compile the various small molecule kinase inhibitors with respect to structural and strategic targeting of PIKK family members. Rapalogs, AZD8055, AZD2014, OSI-027, INK-128, MLN0128, VX970, NVP-BEZ235, Torin2, AZ20, and AZ31 are the diverse scaffolds which have successfully made into the pre-clinical trials either as mono or combinatorial therapy for the treatment of various human cancers. Their synthesis and pre-clinical trial highlight the challenges associated in the development process.

Phosphoinositide 3-kinase (PI3K) takes a key role in a plethora of physiologic processes and controls cell growth, metabolism, immunity, cardiovascular and neurological function, and more. The discovery of wortmannin as the first potent PI3K inhibitor (PI3Ki) in the 1990s provided rapid identification of PI3K-dependent processes, which drove the assembly of the PI3K/protein kinase B (PKB/Akt)/target of rapamycin (mTOR) pathway. Genetic mouse models and first PI3K isoform-specific inhibitors pinpointed putative therapeutic applications. The recognition of PI3K as target for cancer therapy drove subsequently drug development. Here we provide a brief journey through the emerging roles of PI3K to the development of clinical PI3Ki candidates.

A decrease in oxygen concentration is a hallmark of inflammatory reactions resulting from infection or homeostasis disorders. Mast cells interact with extracellular matrix and other cells by adhesion receptors. We investigated the effect of hypoxia on integrin-mediated mast cell adhesion to fibronectin. We found that it was mediated by the α5/β1 receptor and that hypoxia significantly upregulated this process. Hypoxia-mediated increases in mast cell adhesion occurred without increased surface expression of integrins, suggesting regulation by inside-out integrin signaling. Hypoxia also mediated an increase in phosphorylation of Akt, and PI3’kinase inhibitors abolished hypoxia-mediated mast cell adhesion. Hypoxia upregulates the function of integrin receptors by PI3’ kinase-dependent signaling. This process might be important for the location of mast cells at inflammatory sites

Context: Metformin attenuates type-2 diabetes mellitus (T2DM)-induced hepatic dysfunction and altered PI3K/Akt/GLUT-4 signalling in experimental studies. However, its effect on bicuculline-sensitive gamma amino butyric acid (GABA)-A receptor (GABAAR)-mediated calcium-dependent PI3K/Akt/GLUT-4 signalling in liver challenged to T2DM has not been established. Objective: The effectiveness of metformin on bicuculline-sensitive GABAAR-mediated hepatic insulin signalling was carried out in presence or absence of bicuculline (2.0 mg/kg, i.p.) in experimental T2DM rats. Materials and methods: The whole experimental design was divided into three independent sets of experiments. Each set comprised seven groups of six male rats each. T2DM was induced in the animals by administering streptozotocin (45 mg/kg, i.p.) and nicotinamide (110 mg/kg, i.p.) at a time lag of 15 min except control group rats in three experiments. Metformin and/or bicuculline or wortmannin were administered once daily for one week from seventh day of streptozotocin injection in all the experimental sets. Results: Metformin attenuated T2DM-induced hyperglycaemia in glucose (40%) and insulin (50%) tolerance tests in rats. Metformin also attenuated T2DM-induced hyperglycaemia (40%), hyperinsulinaemia (30%), insulin resistance (50%) and β-cell dysfunction (300%) in the animals. Metformin did not attenuate T2DM-induced decrease in rat hepatic intracellular calcium. Further, metformin mitigated T2DM-induced decrease in hepatic phosphorylated Akt and GLUT-4 translocation in the animals. The anti-diabetic activity of metformin was abolished by wortmannin but not with bicuculline co-administration in T2DM animals. Discussion and conclusion: These results suggest that metformin ameliorated T2DM-induced hepatic insulin resistance through bicuculline-sensitive GABAA receptor-independent PI3K/Akt/GLUT-4 signalling pathway in animals.

A rise in antimicrobial resistance demands novel alternatives to antimicrobials for disease control and prevention. As an important component of innate immunity, host defense peptides (HDPs) are capable of killing a broad spectrum of pathogens and modulating a range of host immune responses. Enhancing the synthesis of endogenous HDPs has emerged as a novel host-directed antimicrobial therapeutic strategy. To facilitate the identification of natural products with a strong capacity to induce HDP synthesis, a stable macrophage cell line expressing a luciferase reporter gene driven by a 2-Kb avian β-defensin 9 (AvBD9) gene promoter was constructed through lentiviral transduction and puromycin selection. A high throughput screening assay was subsequently developed using the stable reporter cell line to screen a library of 584 natural products. A total of 21 compounds with a minimum Z-score of 2.0 were identified. Secondary screening in chicken HTC macrophages and jejunal explants further validated most compounds with a potent HDP-inducing activity in a dose-dependent manner. A follow-up oral administration of a lead natural compound, wortmannin, confirmed its capacity to enhance the AvBD9 gene expression in the duodenum of chickens. Besides AvBD9, most other chicken HDP genes were also induced by wortmannin. Additionally, butyrate was also found to synergize with wortmannin and several other newly-identified compounds in AvBD9 induction in HTC cells. Furthermore, wortmannin acted synergistically with butyrate in augmenting the antibacterial activity of chicken monocytes. Therefore, these natural HDP-inducing products may have the potential to be developed individually or in combinations as novel antibiotic alternatives for disease control and prevention in poultry and possibly other animal species including humans.

Alzheimer’s disease (AD) is a progressive neurodegenerative disorder of the brain, characterized by extracellular aggregation of beta-amyloid (Aβ) and hyperphosphorylation of tau causing intraneuronal neurofibrillary tangles (NFTs). There is urgent need to study the interactions between Aβ and tau, especially to solve the question of the pathological cascade. In the present study, we aim to develop a model of organotypic brain slices in which both plaque and tau pathology can be examined. Organotypic brain slices (150 μm thick, coronal, at the hippocampal level) from adult (9 month) wildtype (WT, C57BL/6N) and transgenic AD mice (TG, APP_SweDI) were cultured for 2 weeks. To induce tau hyperphosphorylation 100 nM okadaic acid (OA), 10 μM wortmannin (WM) or both were added to the slices. Hyperphosphorylation of tau was tested at tau-S199, tau-T231 and tau-S396 using Western blot. Our data show that in TG mice with plaques a 50 kDa fragment of tau-S396 was hyperphosphorylated and that OA induced hyperphosphorylation of tau-S199. In WT mice (without plaques) OA caused hyperphosphorylation of a 50 kDa and a 38 kDa tau-T231 form and a 25 kDa sdftau-S396 fragment. The N-methyl-D-aspartate (NMDA) antagonist MK801 (1 μM) did not block these effects. Immunohistochemistry showed diffuse increased tau-S396 and tau-T231-like immunoreactivities at the hippocampal level but no formation of NFTs. Confocal microscopy indicated, that pTau-T231 was preferentially located in cytoplasma surrounding nuclei whereas pTau-S396 was found mainly in nerve fibers and strongly associated with plaques. In conclusion we provide a novel in vitro model to study both plaque and tau hyperphosphorylation but not NFTs, which could be useful to study pathological processes in AD and to screen for drugs.

Introduction: Hyperglycemia is a serious health problem prevailing in diabetes patients. Treatment for hyperglycemia by various oral anti-hyperglycemic drugs have associated with side effects, hence there is growing awareness towards the use of herbal products due to their efficacy,minimal side effects and relatively low costs. This study is designed to evaluate anti-hyperglycemic activity of Caralluma umbellata Haw, which is used as a traditional medicinal plant all over India through in vitro studies. Methods: Methanolic, aqueous and hydro methanolic extracts of Caralluma umbellata were prepared and studied for their anti hyperglycemic activity. The extracts were evaluated for glucose uptake in L6 myotubes in vitro. In addition, the inhibitory activity against alpha amylase and pancreatic lipase was also measured. Results: The methanolic extract (MCU) was found to have significant glucose uptake. Further, MCU was also found to have promising role in inhibiting alpha amylase and pancreatic lipase. Conclusion: The results of present study shows Caralluma umbellata has potential antidiabetic property, thus providing a further scope for study in animal model and understanding the mechanism of action.

Nobiletin is a polymethoxy flavonoid present in fruit tissues of citrus species and has been reported to suppress degranulation of RBL-2H3 cells. However, the detailed mechanism is still unknown. In this study the mechanism of suppressive effect of nobiletin on degranulation of RBL-2H3 cells was examined. Nobiletin suppressed degranulation of both RBL-2H3 cells and bone marrow-derived mast cells induced by antigen stimulation. Nobiletin also suppressed degranulation stimulated by the calcium ionophore A23187 and thapsigargin, a specific inhibitor of the Ca2+-dependent ATPase. Elevation of intracellular free Ca2+ concentration was also suppressed by nobiletin when RBL-2H3 cells were stimulated by either antigen or A23187. Immunoblot analysis revealed that the inhibitory effect results from down-regulated phosphorylation of both phosphatidylinositol 3-kinase (PI3K) and phospholipase Cγ1 in the signaling pathways related to degranulation stimulated by antigen. Furthermore, oral administration of nobiletin suppressed vascular permeability in passive cutaneous anaphylaxis model mice. The tendency in all experiments obtained by treating with nobiletin was highly similar to that obtained by wortmannin, a specific inhibitor of PI3K. These findings suggest that nobiletin suppresses Ca2+ influx and consequently exerts the anti-allergy activity in a similar way to wortmannin.

Wortmannin, a fungal metabolite and an inhibitor of phosphatidylinositol-3 (PI3) and phosphatidylinositol-4 (PI4) kinases, is widely used for the investigation and dissection of vacuolar trafficking routes and for the identification of proteins located at multivesicular bodies (MVBs). In this study we applied wortmannin on internodal cells of the characean green alga, Chara australis. Wortmannin was used at concentrations of 25 and 50 µM which, unlike in other cells, arrested neither constitutive, nor wounding-induced endocytosis via coated vesicles. Wortmannin caused the formation of mixed compartments consisting of MVBs and membranous tubules which were probably derived from the trans-Golgi network (TGN) and within these compartments MVBs fused into larger organelles. Most interestingly, wortmannin also caused pronounced changes in the morphology of the TGNs. After transient hypertrophy, the TGNs lost their coat and formed compact, three-dimensional meshworks of anastomosing tubules containing a central core. These meshworks had a size of up to 4 µm and a striking resemblance to charasomes, which are convoluted plasma membrane domains, and which serve to increase the area available for transporters.Our findings indicate that similar mechanisms are responsible for the formation of charasomes and the wortmannin-induced reorganization of the TGN. We hypothesize that both organelles grow because of a disturbance of clathrin-dependent membrane retrieval due to inhibition of phosphatidylinositol-3 (PI3) and/or phosphatidylinositol-4 (PI4) kinases. This leads to local inhibition of clathrin-mediated endocytosis during charasome formation in untreated cells and to inhibition of vesicle release from the TGN in wortmannin-treated cells, respectively. The morphological resemblance between charasomes and wortmannin-modified TGN compartments suggests that homologous proteins are involved in membrane curvature and organelle architecture.

Epigallocatechin gallate (EGCG), a major polyphenol in green tea, has been studied as an agent against carcinogenesis and Wortmannin is a microbial steroid and it inhibits phosphatidyl inositol 3-kinase (PI3K) pathway. The aim of this study was to investigate the effects of PI3K inhibitor Wortmannin, EGCG and combined treatments on PI3K pathway on human breast cancer cell line MDA-MB-231 using indirect immunohistochemistry method. MDA-MB-231 breast cancer cells were cultured in RPMI-1640 medium containing 10% FBS, 1% l-glutamine and 1% penicillin/streptomycin. Anti-PI3K, anti-AKT, anti-ERK, anti-NFkB, anti-c-jun and anti-EZH2 primary antibodies were used for indirect immunohistochemistry after 24 h administrations of Wortmannin (2.5 µM), EGCG (100 µM) and combination of them. The mean values of the staining intensities (mild, moderate, strong and very strong) and percentage of positively stained cells were calculated using H-Score. The results of this study showed that the combined treatment of Wortmannin and EGCG is more effective on the decreasing of immunoreactivities of PI3K pathway molecules than single administrations. The combined use of these drugs is thought to be advantageous in enhancing the development and efficacy of existing cancer treatments.