Acetic acid- and phenyl-p-benzoquinone-induced overt pain-like behavior depends on spinal activation of MAP kinases, PI3K and microglia in mice

The acetic acid and phenyl-p-benzoquinone are easy and fast screening models to access the activity of novel candidates as analgesic drugs and their mechanisms. These models induce a characteristic and quantifiable overt pain-like behavior described as writhing response or abdominal contortions. The knowledge of the mechanisms involved in the chosen model is a crucial step forward demonstrating the mechanisms that the candidate drug would inhibit because the mechanisms triggered in that model will be addressed. Herein, it was investigated the role of spinal mitogen-activated protein (MAP) kinases ERK (extracellular signal-regulated kinase), JNK (Jun N-terminal Kinase) and p38, PI3K (phosphatidylinositol 3-kinase) and microglia in the writhing response induced by acetic acid and phenyl-p-benzoquinone, and flinch induced by formalin in mice. Acetic acid and phenyl-p-benzoquinone induced significant writhing response over 20 min. The nociceptive response in these models were significantly and in a dose-dependent manner reduced by intrathecal pre-treatment with ERK (PD98059), JNK (SB600125), p38 (SB202190) or PI3K (wortmannin) inhibitors. Furthermore, the co-treatment with MAP kinase and PI3K inhibitors, at doses that were ineffective as single treatment, significantly inhibited acetic acid- and phenyl-p-benzoquinone-induced nociception. The treatment with microglia inhibitors minocycline and fluorocitrate also diminished the nociceptive response. Similar results were obtained in the formalin test. Concluding. MAP kinases and PI3K are important spinal signaling kinases in acetic acid and phenyl-p-benzoquinone models of overt pain-like behavior and there is also activation of spinal microglia indicating that it is also important to determine whether drugs tested in these models also modulate such spinal mechanisms. (C) 2012 Elsevier Inc. All rights reserved.

Stimulation of peripheral Kappa opioid receptors inhibits inflammatory hyperalgesia via activation of the PI3K gamma/AKT/nNOS/NO signaling pathway

Background: In addition to their central effects, opioids cause peripheral analgesia. There is evidence showing that peripheral activation of kappa opioid receptors (KORs) inhibits inflammatory pain. Moreover, peripheral mu-opioid receptor (MOR) activation are able to direct block PGE(2)-induced ongoing hyperalgesia However, this effect was not tested for KOR selective activation. In the present study, the effect of the peripheral activation of KORs on PGE(2)-induced ongoing hyperalgesia was investigated. The mechanisms involved were also evaluated. Results: Local (paw) administration of U50488 (a selective KOR agonist) directly blocked, PGE(2)-induced mechanical hyperalgesia in both rats and mice. This effect was reversed by treating animals with L-NMMA or N-propyl-L-arginine (a selective inhibitor of neuronal nitric oxide synthase, nNOS), suggesting involvement of the nNOS/NO pathway. U50488 peripheral effect was also dependent on stimulation of PI3K gamma/AKT because inhibitors of these kinases also reduced peripheral antinociception induced by U50488. Furthermore, U50488 lost its peripheral analgesic effect in PI3K gamma null mice. Observations made in vivo were confirmed after incubation of dorsal root ganglion cultured neurons with U50488 produced an increase in the activation of AKT as evaluated by western blot analyses of its phosphorylated form. Finally, immunofluorescence of DRG neurons revealed that KOR-expressing neurons also express PI3K gamma (congruent to 43%). Conclusions: The present study indicates that activation of peripheral KORs directly blocks inflammatory hyperalgesia through stimulation of the nNOS/NO signaling pathway which is probably stimulated by PI3K gamma/AKT signaling. This study extends a previously study of our group suggesting that PI3K gamma/AKT/nNOS/NO is an important analgesic pathway in primary nociceptive neurons.

High-Fat Diet Obesity Associated With Insulin Resistance Increases Cell Proliferation, Estrogen Receptor, and PI3K Proteins in Rat Ventral Prostate

In this study, we evaluated the effects of obesity and insulin resistance induced by a high-fat diet on prostate morphophysiology, focusing on cell proliferation, expression of androgen (AR) and estrogen receptors (ER) and proteins of the insulin signaling pathway. Adult male Wistar rats were fed a high-fat diet (20% fat) for 15 weeks, whereas control animals received a balanced diet (4% fat). Both groups were then divided and treated for 2 weeks with 1 mg/kg body weight/day of the aromatase inhibitor letrozole or vehicle only. The ventral prostate was analyzed with immunohistochemical, histopathological, stereological, and Western blotting methods. Obese rats showed insulin resistance, hyperinsulinemia, and reduced plasma testosterone levels. The incidence of prostatic intraepithelial neoplasia (PIN) was 2.7 times higher in obese rats and affected 0.4% of the gland compared with 0.1% PIN areas found in control rats. Obesity doubled cell proliferation in both prostate epithelium and stroma. AR content decreased in the prostate of obese rats and estrogen receptor beta (ER beta) increased in this group. Protein levels of insulin receptor substrate 1 and protein kinase B diminished in the obese group, whereas phosphatidylinositol 3-kinase (PI3K) increased significantly. Most structural changes observed in the prostate of obese rats normalized after letrozole treatment, except for increased stromal cell proliferation and ER beta expression, which might be associated with insulin resistance. This experimental model of obesity and insulin resistance induced by a high-fat diet increases cell proliferation in rat prostate. Such alterations are associated with decreased levels of AR and increased ER beta and PI3K proteins. This change can facilitate the establishment of proliferative lesions in rat prostate.

Stimulation of peripheral Kappa opioid receptors inhibits inflammatory hyperalgesia via activation of the PI3Kγ/AKT/nNOS/NO signaling pathway

Abstract Background In addition to their central effects, opioids cause peripheral analgesia. There is evidence showing that peripheral activation of kappa opioid receptors (KORs) inhibits inflammatory pain. Moreover, peripheral μ-opioid receptor (MOR) activation are able to direct block PGE2-induced ongoing hyperalgesia However, this effect was not tested for KOR selective activation. In the present study, the effect of the peripheral activation of KORs on PGE2-induced ongoing hyperalgesia was investigated. The mechanisms involved were also evaluated. Results Local (paw) administration of U50488 (a selective KOR agonist) directly blocked, PGE2-induced mechanical hyperalgesia in both rats and mice. This effect was reversed by treating animals with L-NMMA or N-propyl-L-arginine (a selective inhibitor of neuronal nitric oxide synthase, nNOS), suggesting involvement of the nNOS/NO pathway. U50488 peripheral effect was also dependent on stimulation of PI3Kγ/AKT because inhibitors of these kinases also reduced peripheral antinociception induced by U50488. Furthermore, U50488 lost its peripheral analgesic effect in PI3Kγ null mice. Observations made in vivo were confirmed after incubation of dorsal root ganglion cultured neurons with U50488 produced an increase in the activation of AKT as evaluated by western blot analyses of its phosphorylated form. Finally, immunofluorescence of DRG neurons revealed that KOR-expressing neurons also express PI3Kγ (≅ 43%). Conclusions The present study indicates that activation of peripheral KORs directly blocks inflammatory hyperalgesia through stimulation of the nNOS/NO signaling pathway which is probably stimulated by PI3Kγ/AKT signaling. This study extends a previously study of our group suggesting that PI3Kγ/AKT/nNOS/NO is an important analgesic pathway in primary nociceptive neurons.

adaptive capabilities of the pi3k/akt/mtor pathway in acute myeloid leukemia revealed by the use of selective inhibitors

Because of its aberrant activation, the PI3K/AKT/mTOR signaling pathway represents a pharmacological target in blast cells from patients with acute myelogenous leukemia (AML). Using Reverse Phase Protein Microarrays (RPMA), we have analyzed 20 phosphorylated epitopes of the PI3K/Akt/mTor signal pathway of peripheral blood and bone marrow specimens of 84 patients with newly diagnosed AML. Fresh blast cells were grown for 2 h, 4 h or 20 h untreated or treated with a panel of phase I or phase II Akt allosteric inhibitors, either alone or in combination with the mTOR kinase inhibitor Torin1 or the broad RTK inhibitor Sunitinib. By unsupervised hierarchical clustering a strong phosphorylation/activity of most of the sampled members of the PI3K/Akt/mTOR pathway was observed in 70% of samples from AML patients. Remarkably, however, we observed that inhibition of Akt phosphorylation, as well as of its substrates, was transient, and recovered or even increased far above basal level after 20 h in 60% samples. We demonstrated that inhibition of Akt induces FOXO-dependent insulin receptor expression and IRS-1 activation, attenuating the effect of drug treatment by reactivation of PI3K/Akt. Consistent with this model we found that combined inhibition of Akt and RTKs is much more effective than either alone, revealing the adaptive capabilities of signaling networks in blast cells and highliting the limations of these drugs if used as monotherapy.

Studio della via di segnale PI3K/Akt/mTOR nelle Cellule Dendritiche

Il trapianto allogenico di cellule staminali emopoietiche è spesso l’unica soluzione per la cura di diverse malattie ematologiche. La aGVHD è la complicanza più importante che si può avere a seguito del trapianto allogenico ed è causata dai linfociti T del donatore che riconoscono gli antigeni del ricevente presentati dalle APC. Eliminare o inattivare la APC del ricevente prima del trapianto potrebbe prevenire la aGVHD. Ad oggi non esistono farmaci specifici diretti contro le APC, sono però noti i meccanismi molecolari coinvolti nella sopravvivenza cellulare come la via di segnale di PI3K. In questo lavoro abbiamo testato l’attività di due farmaci, che colpiscono target molecolari della via di PI3K, la rapamicina e la perifosina, sul differenziamento dei monociti a differenti popolazioni di cellule dendritiche (DC), in vitro. La rapamicina riduceva il recupero cellulare delle DC derivate da monociti coltivate in presenza di IL-4 aumentando l’apoptosi, mentre i monociti coltivati in presenza di GM-CSF con o senza IFN-α risultavano resistenti alla rapamicina. Inoltre la rapamicina riduceva l’espressione della molecola costimolatoria CD86 e incrementava l’espressione della molecola CD1a solo nei monociti coltivati con GM-CSF e IL-4. Nelle DC derivate dai monociti in presenza di IL-4 la rapamicina bloccava la produzione di IL-12 e TNF-α e ne alterava la capacità allostimolatoria. La rapamicina non alterava la sopravvivenza e la funzione delle DC circolanti. Il trattamento con perifosina provocava un incremento di apoptosi nei monociti coltivati sia con GM-CSF che con GM-CSF e IL-4. La perifosina bloccava la produzione di TNF-α nelle DC derivate da monociti coltivati nelle diverse condizioni. Questi risultati dimostrano che l’azione della rapamicina è strettamente dipendente dalla presenza dell’IL-4 nel terreno di coltura, in vitro, rispetto alla perifosina e suggeriscono un possibile ruolo della perifosina nella prevenzione della GVHD prima del trapianto allogenico di cellule staminali.

Study of PI3K/Akt signaling pathway as potential molecular target for T-cell acute lymphoblastic leukemia (T-ALL) treatment: pan-inhibition of PI3K catalitic isoforms as better therapeutic approach

Class I phosphatidylinositol 3-kinases (PI3Ks) are heterodimeric lipid kinases consisting of a regulatory subunit and one of four catalytic subunits (p110α, p110β, p110γ or p110δ). p110γ/p110δ PI3Ks are highly enriched in leukocytes. In general, PI3Ks regulate a variety of cellular processes including cell proliferation, survival and metabolism, by generating the second messenger phosphatidylinositol-3,4,5-trisphosphate (PtdIns(3,4,5)P3). Their activity is tightly regulated by the phosphatase and tensin homolog (PTEN) lipid phosphatase. PI3Ks are widely implicated in human cancers, and in particular are upregulated in T-cell acute lymphoblastic leukemia (T-ALL), mainly due to loss of PTEN function. These observations lend compelling weight to the application of PI3K inhibitors in the therapy of T-ALL. At present different compounds which target single or multiple PI3K isoforms have entered clinical trials. In the present research, it has been analyzed the therapeutic potential of the pan-PI3K inhibitor BKM120, an orally bioavailable 2,6-dimorpholino pyrimidine derivative, which has entered clinical trials for solid tumors, on both T-ALL cell lines and patient samples. BKM120 treatment resulted in cell cycle arrest and apoptosis, being cytotoxic to a panel of T-ALL cell lines and patient T-lymphoblasts. Remarkably, BKM120 synergized with chemotherapeutic agents currently used for treating T-ALL patients. BKM120 efficacy was confirmed in in vivo studies to a subcutaneous xenotransplant model of human T-ALL. Because it is still unclear which agents among isoform-specific or pan inhibitors can achieve the greater efficacy, further analyses have been conducted to investigate the effects of PI3K inhibition, in order to elucidate the mechanisms responsible for the proliferative impairment of T-ALL. Overall, these results indicated that BKM120 may be an efficient treatment for T-ALLs that have aberrant up-regulation of the PI3K signaling pathway and strongly support clinical application of pan-class I PI3K rather than single-isoform inhibitors in T-ALL treatment.

A sensitized RNA interference screen identifies a novel role for the PI3K p110γ isoform in medulloblastoma cell proliferation and chemoresistance

Medulloblastoma is the most common malignant brain tumor in children and is associated with a poor outcome. We were interested in gaining further insight into the potential of targeting the human kinome as a novel approach to sensitize medulloblastoma to chemotherapeutic agents. A library of small interfering RNA (siRNA) was used to downregulate the known human protein and lipid kinases in medulloblastoma cell lines. The analysis of cell proliferation, in the presence or absence of a low dose of cisplatin after siRNA transfection, identified new protein and lipid kinases involved in medulloblastoma chemoresistance. PLK1 (polo-like kinase 1) was identified as a kinase involved in proliferation in medulloblastoma cell lines. Moreover, a set of 6 genes comprising ATR, LYK5, MPP2, PIK3CG, PIK4CA, and WNK4 were identified as contributing to both cell proliferation and resistance to cisplatin treatment in medulloblastoma cells. An analysis of the expression of the 6 target genes in primary medulloblastoma tumor samples and cell lines revealed overexpression of LYK5 and PIK3CG. The results of the siRNA screen were validated by target inhibition with specific pharmacological inhibitors. A pharmacological inhibitor of p110γ (encoded by PIK3CG) impaired cell proliferation in medulloblastoma cell lines and sensitized the cells to cisplatin treatment. Together, our data show that the p110γ phosphoinositide 3-kinase isoform is a novel target for combinatorial therapies in medulloblastoma.

The catalytic class I(A) PI3K isoforms play divergent roles in breast cancer cell migration

Transforming growth factor-β (TGFβ) plays an important role in breast cancer metastasis. Here phosphoinositide 3-kinase (PI3K) signalling was found to play an essential role in the enhanced migration capability of fibroblastoid cells (FibRas) derived from normal mammary epithelial cells (EpH4) by transduction of oncogenic Ras (EpRas) and TGFβ1. While expression of the PI3K isoform p110δ was down-regulated in FibRas cells, there was an increase in the expression of p110α and p110β in the fibroblastoid cells. The PI3K isoform p110β was found to specifically contribute to cell migration in FibRas cells, while p110α contributed to the response in EpH4, EpRas and FibRas cells. Akt, a downstream targets of PI3K signalling, had an inhibitory role in the migration of transformed breast cancer cells, while Rac, Cdc42 and the ribosomal protein S6 kinase (S6K) were necessary for the response. Together our data reveal a novel specific function of the PI3K isoform p110β in the migration of cells transformed by oncogenic H-Ras and TGF-β1.

Secretin receptor promotes the proliferation of endocrine tumor cells via the PI3K/AKT pathway

The secretin receptor (SR), a G protein-coupled receptor, mediates the effects of the gastrointestinal hormone secretin on digestion and water homeostasis. Recently, high SR expression has been observed in pancreatic ductal adenocarcinomas, cholangiocellular carcinomas, gastrinomas, and bronchopulmonary carcinoid tumors. Receptor overexpression associates with enhanced secretin-mediated signaling, but whether this molecule plays an independent role in tumorigenesis is currently unknown. We recently discovered that pheochromocytomas developing in rats affected by the MENX (multiple endocrine neoplasia-like) syndrome express at very high-level Sctr, encoding SR. We here report that SR are also highly abundant on the membranes of rat adrenal and extraadrenal pheochromocytoma, starting from early stages of tumor development, and are functional. PC12 cells, the best characterized in vitro pheochromocytoma model, also express Sctr at high level. Thus, we used them as model to study the role of SR in neoplastic transformation. Small interfering RNA-mediated knockdown of Sctr decreases PC12 cells proliferation and increases p27 levels. The proproliferative effect of SR in PC12 cells is mediated, in part, by the phosphatidylinositol 3 kinase (PI3K)/serine-threonine protein kinase (AKT) pathway. Transfection of Sctr in Y1 adrenocortical carcinoma cells, expressing low endogenous levels of Sctr, stimulates cell proliferation also, in part, via the PI3K/AKT signaling cascade. Because of the link between SR and PI3K/AKT signaling, tumor cells expressing high levels of the receptor (MENX-associated primary pheochromocytoma and NCI-H727 human bronchopulmonary carcinoid cells) respond well and in a SR-dependent manner to PI3K inhibitors, such as NVP-BEZ235. The association between SR levels and response to PI3K inhibition might open new avenues for the treatment of tumors overexpressing this receptor.

Novel agents targeting the IGF-1R/PI3K pathway impair cell proliferation and survival in subsets of medulloblastoma and neuroblastoma

The receptor tyrosine kinase (RTK)/phosphoinositide 3-kinase (PI3K) pathway is fundamental for cancer cell proliferation and is known to be frequently altered and activated in neoplasia, including embryonal tumors. Based on the high frequency of alterations, targeting components of the PI3K signaling pathway is considered to be a promising therapeutic approach for cancer treatment. Here, we have investigated the potential of targeting the axis of the insulin-like growth factor-1 receptor (IGF-1R) and PI3K signaling in two common cancers of childhood: neuroblastoma, the most common extracranial tumor in children and medulloblastoma, the most frequent malignant childhood brain tumor. By treating neuroblastoma and medulloblastoma cells with R1507, a specific humanized monoclonal antibody against the IGF-1R, we could observe cell line-specific responses and in some cases a strong decrease in cell proliferation. In contrast, targeting the PI3K p110α with the specific inhibitor PIK75 resulted in broad anti-proliferative effects in a panel of neuro- and medulloblastoma cell lines. Additionally, sensitization to commonly used chemotherapeutic agents occurred in neuroblastoma cells upon treatment with R1507 or PIK75. Furthermore, by studying the expression and phosphorylation state of IGF-1R/PI3K downstream signaling targets we found down-regulated signaling pathway activation. In addition, apoptosis occurred in embryonal tumor cells after treatment with PIK75 or R1507. Together, our studies demonstrate the potential of targeting the IGF-1R/PI3K signaling axis in embryonal tumors. Hopefully, this knowledge will contribute to the development of urgently required new targeted therapies for embryonal tumors.

Targeting survivin via PI3K but not c-akt/PKB by anticancer drugs in immature neutrophils

Myelosuppression is the most common unwanted side effect associated with the administration of anticancer drugs, and infections remain a common cause of death in chemotherapy-treated patients. Several mechanisms of the cytotoxicity of these drugs have been proposed and may synergistically operate in a given cell. Survivin expression has been associated with cancer, but recent reports suggest that this molecule is also expressed in several immature and mature hematopoietic cells. Here, we provide evidence that treatment of immature neutrophils with anticancer drugs reduced endogenous survivin levels causing apoptosis. The anticancer drugs did not directly target survivin, instead they blocked the activity of phosphatidylinositol-3-OH kinase, which regulated survivin expression and apoptosis in these cells. Strikingly, and in contrast to other cells, this pathway did not involve the serine/threonine kinase c-akt/PKB. Moreover, in combination with anticancer drug therapy, rapamycin did not induce increased myelosuppression in an experimental lymphoma mouse model. These data suggest that drugs that block either c-akt/PKB or signaling molecules located distal to c-akt/PKB may preferentially induce apoptosis of cancer cells as they exhibit no cytotoxicity for immature neutrophils.

Biosynthesis and expression of VE-cadherin is regulated by the PI3K/mTOR signaling pathway

Vascular endothelial (VE)-cadherin is an essential protein of adherens junctions of endothelial cells and plays a pivotal role in vascular homeostasis. Mammalian target of rapamycin complex 2 (mTORC2) deficient mice display defects in fetal vascular development. Blocking mTOR or the upstream kinase phosphoinositide 3-kinase (PI3K) led to a dose-dependently decrease of the VE-cadherin mRNA and protein expression. Immunofluorescent staining showed a strongly decreased expression of VE-cadherin at the interface of human umbilical endothelial cells (HUVECs) followed by intercellular gap formation. Herewith, we demonstrated that the expression of VE-cadherin is dependent on mTOR and PI3K signaling.