Comparative changes in breast cancer cell proliferation and signalling following somatostatin and cannabidiol treatment.

Breast cancer is the most commonly diagnosed cancer and a leading cause of cancer-related death among women worldwide. Somatostatin (SST) and Cannabinoids have an anti-proliferative and pro-apoptotic effect, but the mechanisms of their actions remain elusive. In the present study, we have evaluated the effects of SST, Cannabidiol (CBD) alone or in combination on receptor expression, cell proliferation and apoptosis and related downstream signalling pathways in MDA-MB-231 and MCF-7 breast cancer cells. The results presented here demonstrate the cell type and agonist-dependent changes in receptor expression at the cell membrane, inhibition of cell proliferation and increased apoptosis following treatment with SST and CBD alone and in combination. In comparison to MDA-MB-231 cells, MCF-7 cells treated with SST alone and in combination with CBD exhibited inhibition of phosphorylated Protein Kinase B (pAKT) and phosphorylated-Phosphoinositide 3-Kinase (pPI3K) expression. Importantly, inhibition of PI3K/AKT activation was accompanied by enhanced PTEN expression in MCF-7 cells. These results highlight the possible interaction between SSTR and CBR subtypes with the implication in the modulation of receptor expression, cell viability and signal transduction pathways in a breast cancer cell type-dependent manner.

Somatostatin and Somatostatin Receptors in Tumour Biology.

Somatostatin (SST), a growth hormone inhibitory peptide, is expressed in endocrine and non-endocrine tissues, immune cells and the central nervous system (CNS). Post-release from secretory or immune cells, the first most appreciated role that SST exhibits is the antiproliferative effect in target tissue that served as a potential therapeutic intervention in various tumours of different origins. The SST-mediated in vivo and/or in vitro antiproliferative effect in the tumour is considered direct via activation of five different somatostatin receptor subtypes (SSTR1-5), which are well expressed in most tumours and often more than one receptor in a single cell. Second, the indirect effect is associated with the regulation of growth factors. SSTR subtypes are crucial in tumour diagnosis and prognosis. In this review, with the recent development of new SST analogues and receptor-specific agonists with emerging functional consequences of signaling pathways are promising therapeutic avenues in tumours of different origins that are discussed.

Somatostatin-Mediated Regulation of Retinoic Acid-Induced Differentiation of SH-SY5Y Cells: Neurotransmitters Phenotype Characterization.

During brain development, neurite formation plays a critical role in neuronal communication and cognitive function. In the present study, we compared developmental changes in the expression of crucial markers that govern the functional activity of neurons, including somatostatin (SST), choline acetyltransferase (ChAT), tyrosine hydroxylase (TH), brain nitric oxide synthase (bNOS), gamma-aminobutyric acid (GABA), glutamic acid decarboxylase (GAD-65) and synaptic vesicle protein synaptophysin (SYP) in non-differentiated and retinoic acid (RA)-induced differentiated SH-SY5Y cells. We further determined the role of SST in regulating subcellular distribution and expression of neurotransmitters. Our results indicate that SST potentiates RA-induced differentiation of SH-SY5Y cells and involves regulating the subcellular distribution and expression of neurotransmitter markers and synaptophysin translocation to neurites in a time-dependent manner, anticipating the therapeutic implication of SST in neurodegeneration.

Cannabinol modulates neuroprotection and intraocular pressure: A potential multi-target therapeutic intervention for glaucoma.

OBJECTIVES: Glaucoma is characterized by progressive damage of the retinal ganglion cells (RGCs), resulting in irreversible vision loss. Cannabinoids (CBs) ameliorate several factors that contribute to the progression of glaucoma, including increased intraocular pressure (IOP), degeneration of RGC and optical nerve (ON) damage. However, a direct correlation of specific CBs with the molecular events pertaining to glaucoma pathology is not well established. Therefore, this study aims to evaluate the role of cannabinol (CBN) on RGC protection, modulation of IOP, and its effects on the level of extracellular matrix (ECM) proteins using both in vitro and in vivo models of glaucoma. METHODS AND RESULTS: When exposed to elevated hydrostatic pressure, CBN, in a dose-dependent manner, protected differentiated mouse 661W retinal ganglion precursor-like cells from pressure-induced toxicity. In human trabecular meshwork cells (hTM), CBN attenuated changes in the ECM proteins, including fibronectin and α-smooth muscle actin (α-SMA), as well as mitogen-activated protein kinases (phospho-ERK1/2) in the presence or absence of transforming growth factor-beta 2 (TGF-ß2) induced stress. Ocular pharmacokinetic parameters were evaluated post-intravitreal (IVT) CBN delivery in vivo. Furthermore, we demonstrated that IVT-administered CBN improved pattern electroretinogram (pERG) amplitudes and reduced IOP in a rat episcleral vein laser photocoagulation model of glaucoma. CONCLUSION: CBN promotes neuroprotection, abrogates changes in ECM protein, and normalizes the IOP levels in the eye. Therefore, our observations in the present study indicate a therapeutic potential for CBN in the treatment of glaucoma.

Somatostatin Ameliorates ß-Amyloid-Induced Cytotoxicity via the Regulation of CRMP2 Phosphorylation and Calcium Homeostasis in SH-SY5Y Cells.

Somatostatin is involved in the regulation of multiple signaling pathways and affords neuroprotection in response to neurotoxins. In the present study, we investigated the role of Somatostatin-14 (SST) in cell viability and the regulation of phosphorylation of Collapsin Response Mediator Protein 2 (CRMP2) (Ser522) via the blockade of Ca2+ accumulation, along with the inhibition of cyclin-dependent kinase 5 (CDK5) and Calpain activation in differentiated SH-SY5Y cells. Cell Viability and Caspase 3/7 assays suggest that the presence of SST ameliorates mitochondrial stability and cell survival pathways while augmenting pro-apoptotic pathways activated by Aß. SST inhibits the phosphorylation of CRMP2 at Ser522 site, which is primarily activated by CDK5. Furthermore, SST effectively regulates Ca2+ influx in the presence of Aß, directly affecting the activity of calpain in differentiated SH-SY5Y cells. We also demonstrated that SSTR2 mediates the protective effects of SST. In conclusion, our results highlight the regulatory role of SST in intracellular Ca2+ homeostasis. The neuroprotective role of SST via axonal regeneration and synaptic integrity is corroborated by regulating changes in CRMP2; however, SST-mediated changes in the blockade of Ca2+ influx, calpain expression, and toxicity did not correlate with CDK5 expression and p35/25 accumulation. To summarize, our findings suggest two independent mechanisms by which SST mediates neuroprotection and confirms the therapeutic implications of SST in AD as well as in other neurodegenerative diseases where the effective regulation of calcium homeostasis is required for a better prognosis.

Role of Somatostatin in the Regulation of Central and Peripheral Factors of Satiety and Obesity.

Obesity is one of the major social and health problems globally and often associated with various other pathological conditions. In addition to unregulated eating behaviour, circulating peptide-mediated hormonal secretion and signaling pathways play a critical role in food intake induced obesity. Amongst the many peptides involved in the regulation of food-seeking behaviour, somatostatin (SST) is the one which plays a determinant role in the complex process of appetite. SST is involved in the regulation of release and secretion of other peptides, neuronal integrity, and hormonal regulation. Based on past and recent studies, SST might serve as a bridge between central and peripheral tissues with a significant impact on obesity-associated with food intake behaviour and energy expenditure. Here, we present a comprehensive review describing the role of SST in the modulation of multiple central and peripheral signaling molecules. In addition, we highlight recent progress and contribution of SST and its receptors in food-seeking behaviour, obesity (orexigenic), and satiety (anorexigenic) associated pathways and mechanism.

Comparative distribution of somatostatin and somatostatin receptors in PTU-induced hypothyroidism.

PURPOSE: Propylthiouracil (PTU)-induced hypothyroidism is a well-established model for assessing hormonal and morphological changes in thyroid as well as other central and peripheral tissues. Somatostatin (SST) is known to regulate hormonal secretion and synthesis in endocrine tissues; however, nothing is currently known about the distribution of SST and its receptor in hypothyroidism. METHOD: In the present study, the comparative immunohistochemical distribution of SST and somatostatin receptors (SSTRs) were analyzed in PTU-induced hypothyroid rats. Rats were treated with PTU for 15 days followed by a co-administration of levothyroxine (LVT) for 15 days. After PTU and LVT treatments (day 30), rats were further administered LVT alone for 15 more days (day 45). The subcellular distribution of SST and SSTR subtypes was determined by peroxidase immunohistochemistry in the thyroid gland collected from control and treated rats. RESULTS: SST and SSTR subtypes were found to be moderately expressed in control thyroid tissues. SST and SSTR subtypes like immunoreactivity increased significantly in follicular and parafollicular epithelial cells in the thyroid of PTU-treated rats. The PTU-induced changes in the expression of SST and SSTR subtypes were suppressed by the administration of the LVT. In addition to thyroid tissues, SST and SSTRs expression was also changed in non-follicular tissues including blood vessels, smooth muscle cells, and connective tissue following treatments. CONCLUSION: The present study revealed a distinct subcellular distribution of SST and SSTR subtypes in the thyroid and provides a new insight for the role of SST and SSTR subtypes in hypothyroidism in addition to its well-established role in negative regulation of hormonal secretion.

Somatostatin and cannabinoid receptors crosstalk in protection of huntingtin knock-in striatal neuronal cells in response to quinolinic acid.

In the present study, we describe the status of somatostatin receptor 2 and 5 (SSTR2 and SSTR5) as well as cannabinoid type 1 receptor (CB1R) in Huntingtin (Htt) knock-in striatal neuronal cells. In mutant Htt (mHtt) knock-in (STHdhQ111/111) and wild type (STHdhQ7/7) striatal neuronal cells, SSTRs and CB1R exhibit prominent cytoplasmic expression and respond to agonist in a receptor specific manner. In response to quinolinic acid (QUIN)-induced toxicity, STHdhQ111/111 cells are more vulnerable and display suppressed cell survival signaling pathways. Receptor-specific agonists protect cells from QUIN-induced toxicity and activate ERK1/2 in both STHdh cells. Co-activation of SSTRs and CB1R resulted in loss of protective effects, delayed ERK1/2 phosphorylation and altered receptor complex composition. These results provide firsthand evidence in support of the protective role of SSTRs in STHdh cells and the possible crosstalk between SSTRs and CB1R in the modulation of excitotoxicity in Huntington's disease.

Somatostatin-Mediated Changes in Microtubule-Associated Proteins and Retinoic Acid-Induced Neurite Outgrowth in SH-SY5Y Cells.

Somatostatin (SST) is a growth hormone inhibitory peptide involved in regulation of several physiological responses of cells including neurotransmission, cell migration, maturation, and neurite formation. In the present study, we examined the role of SST in all-trans retinoic acid (RA)-induced progression of neurite outgrowth in SH-SY5Y cells. We also determined the morphological and developmental changes in prominent intracellular markers of neurite growth including microtubule-associated protein 2 (MAP2), neuron-specific III ß-tubulin (TUJ1), and Tau. Here, we present evidence that SST is a molecular determinant in regulating the transition of SH-SY5Y cells from non-neuronal entity to neuronal phenotype in response to RA. The results from present study reveal that SST changes the distributional pattern of MAP2/Tau and TUJ1, and activates extracellular signal-regulated kinase (ERK1/2) signaling pathway through SST receptors (SSTRs). The expression of MAP2 and Tau remains elevated upon treatment with RA and SST alone or in combination. Importantly, we identified that the cells displaying strong co-expression of SST and TUJ1 are more likely to bear elongated neurite formation than cells devoid of such expression. These findings show that the site-specific expression of MAP2 and TUJ1 is an essential determinant of neurite outgrowth in SH-SY5Y cells in RA-mediated differentiation. Taken together, results presented here further substantiates the role of SST in the promotion of neurite formation and elongation in SH-SY5Y cells in combination with RA. Investigating how SST can improve neurite formation in neurodegenerative disease may help to develop new therapeutic approach in improving cognitive function and memory loss.

Somatostatin Maintains Permeability and Integrity of Blood-Brain Barrier in ß-Amyloid Induced Toxicity.

In Alzheimer's disease (AD), the impaired clearance of ß-amyloid peptide (Aß) due to disrupted tight junction and transporter proteins is the prominent cause of disease progression. Somatostatin (SST) blocks the aggregation of Aß and inflammation whereas reduction of SST levels in the CSF and brain tissue is associated with impaired cognitive function and memory loss. However, the role of SST in preservation of blood-brain barrier (BBB) integrity and functionality in Aß-induced toxicity is not known. In the present study using human CMEC/D3 cells, we demonstrate that SST prevents Aß-induced BBB permeability by regulating LRP1 and RAGE expression and improving the disrupted tight junction proteins. Furthermore, SST abrogates Aß-induced JNK phosphorylation and expression of MMP2. Taken together, results presented here suggest that SST might serve as a therapeutic intervention in AD via targeting multiple pathways responsible for neurotoxicity, impaired BBB function, and disease progression.

Cannabinoid Receptors and the Endocannabinoid System: Signaling and Function in the Central Nervous System.

The biological effects of cannabinoids, the major constituents of the ancient medicinal plant Cannabis sativa (marijuana) are mediated by two members of the G-protein coupled receptor family, cannabinoid receptors 1 (CB1R) and 2. The CB1R is the prominent subtype in the central nervous system (CNS) and has drawn great attention as a potential therapeutic avenue in several pathological conditions, including neuropsychological disorders and neurodegenerative diseases. Furthermore, cannabinoids also modulate signal transduction pathways and exert profound effects at peripheral sites. Although cannabinoids have therapeutic potential, their psychoactive effects have largely limited their use in clinical practice. In this review, we briefly summarized our knowledge of cannabinoids and the endocannabinoid system, focusing on the CB1R and the CNS, with emphasis on recent breakthroughs in the field. We aim to define several potential roles of cannabinoid receptors in the modulation of signaling pathways and in association with several pathophysiological conditions. We believe that the therapeutic significance of cannabinoids is masked by the adverse effects and here alternative strategies are discussed to take therapeutic advantage of cannabinoids.

A stimulus-responsive, in situ-forming, nanoparticle-laden hydrogel for ocular drug delivery.

Most medications targeting optic neuropathies are administered as eye drops. However, their corneal penetration efficiencies are typically < 5%. There is a clear, unmet need for novel transcorneal drug delivery vehicles. To this end, we have developed a stimulus-responsive, in situ-forming, nanoparticle-laden hydrogel for controlled release of poorly bioavailable drugs into the aqueous humor of the eye. The hydrogel is formulated as a composite of hyaluronic acid (HA) and methylcellulose (MC). The amphiphilic nanoparticles are composed of poly(ethylene oxide) (PEO) and poly(lactic acid) (PLA). Experimental design aided the identification of hydrogel composition and nanoparticle content in the formulation, and the formulation reliably switched between thixotropy and temperature-dependent rheopexy when it was tested in a rheometer under conditions that simulate the ocular surface, including blinking. These properties should ensure that the formulation coats the cornea through blinking of the eyelid and facilitate application of the medication as an eye drop immediately prior to the patient's bedtime. We subsequently tested the efficacy of our formulation in whole-eye experiments by loading the nanoparticles with cannabigerolic acid (CBGA). Our formulation exhibits over a 300% increase in transcorneal penetration over control formulations. This work paves the way for the introduction of novel products targeting ocular diseases to the market.

Characterization of somatostatin receptors and associated signaling pathways in pancreas of R6/2 transgenic mice.

The present study describes the status of somatostatin receptors (SSTRs) and their colocalization with insulin (ß), glucagon (α) and somatostatin (δ) producing cells in the pancreatic islets of 11weeks old R6/2 Huntington's Disease transgenic (HD tg) and age-matched wild type (wt) mice. We also determined expression of tyrosine hydroxylase (TH), glutamic acid decarboxylase (GAD) and presynaptic marker synaptophysin (SYP) in addition to signal transduction pathways associated with diabetes. In R6/2 mice, islets are relatively smaller in size, exhibit enhanced expression and nuclear inclusion of mHtt along with the loss of insulin, glucagon and somatostatin expression. In comparison to wt, R6/2 mice display enhanced mRNA for all SSTRs except SSTR2. In the pancreatic lysate, SSTR1, 4 and 5 immunoreactivity decreases whereas SSTR3 immunoreactivity increases with no discernible changes in SSTR2 immunoreactivity. Furthermore, at the cellular level, R6/2 mice exhibit a receptor specific distributional pattern of SSTRs like immunoreactivity and colocalization with ß, α and δ cells. While GAD expression is increased, TH and SYP immunoreactivity was decreased in R6/2 mice, anticipating a cross-talk between the CNS and pancreas in diabetes pathophysiology. We also dissected out the changes in signaling pathway and found decreased activation and expression of PKA, AKT, ERK1/2 and STAT3 in R6/2 mice pancreas. These findings suggest that the impaired organization of SSTRs within islets may lead to perturbed hormonal regulation and signaling. These interconnected complex events might shed new light on the pathogenesis of diabetes in neurodegenerative diseases and the role of SSTRs in potential therapeutic intervention.

Review of recent developments in determining volatile organic compounds in exhaled breath as biomarkers for lung cancer diagnosis.

Lung cancer is the most common cause of cancer deaths, its global incidence is rising, and continuing rises are predicted. The potential to diagnose lung cancers based on the determination of volatile organic compounds (VOCs) in human breath has been attracting increasing attention with the development of new techniques and methodologies. However, despite many reports of VOC profiling in lung cancer patients, little is known about how specific biomarkers relate to the biochemical pathways involved in lung cancer development, and there is still no reliable method for diagnosing lung cancer at the early stages. This review summarizes some of the latest methods used for monitoring biomarkers in lung cancer patients, which could be applicable for clinical diagnosis. Techniques for capturing and pre-concentrating biomarkers, and the technologies used for subsequently determining them, are also discussed.

Regional and subcellular distribution of GABAC ρ3 receptor in brain of R6/2 mouse model of Huntington's disease.

In the present study, we describe the distribution of GABAC ρ3 receptor immunoreactivity in the cortex, striatum and hippocampus of wild type (wt) and 11 weeks old HD transgenic (tg) R6/2 mouse brain. In the brain of wt mice, GABAC ρ3 immunoreactivity is well expressed in neuronal cells, nerve fibers and axonal processes. In comparison to wt, GABAC ρ3 receptor like immunoreactivity decreases significantly in all three brain regions of R6/2 mice. The altered distributional pattern and significant changes in GABAC ρ3 receptor immunoreactivity as seen in the R6/2 mouse brain might be a plausible molecular mechanism for excitotoxicity in HD pathogenesis due to the loss of inhibitory input.

Somatostatin receptor 5 is a prominent regulator of signaling pathways in cells with coexpression of Cannabinoid receptors 1.

Endocannabinoids and somatostatin (SST) play critical roles in several pathophysiological conditions via binding to different receptor subtypes. Cannabinoid receptor 1 (CB1R) and somatostatin receptors (SSTRs) are expressed in several brain regions and share overlapping functions. Whether these two prominent members of G-protein-coupled receptor (GPCR) family interact with each other and constitute a functional receptor complex is not known. In the present study, we investigated the colocalization of CB1R and SSTR5 in rat brain, and studied receptor internalization, interaction and signal transduction pathways in HEK-293 cells cotransfected with human cannabinoid receptor 1 (hCB1R) and hSSTR5. Our results showed that CB1R and SSTR5 colocalized in rat brain cortex, striatum, and hippocampus. CB1R was expressed in SSTR5 immunoprecipitate prepared from the brain tissue lysate, indicating their association in a system where these receptors are endogenously expressed. In cotransfected HEK-293 cells, SSTR5 and CB1R existed in a constitutive heteromeric complex under basal condition, which was disrupted upon agonist treatments. Furthermore, concurrent receptor activation led to preferential formation of SSTR5 homodimer and dissociation of CB1R homodimer. We also discovered that second messenger cyclic adenosine monophosphate and downstream signaling pathways were modulated in a SSTR5-dominant and concentration-dependent manner in the presence of receptor-specific agonist. In conclusion, with predominant role of SSTR5, the functional consequences of crosstalk between SSTR5 and CB1R resulting in the regulation of receptor trafficking and signal transduction pathways open new therapeutic avenue in cancer biology and excitotoxicity.

Cardiac Consequences of Autonomic Dysreflexia in Spinal Cord Injury.

Autonomic dysreflexia (AD), which describes episodic hypertension, is highly prevalent in people with spinal cord injury (SCI). In non-SCI, primary hypertension depresses cardiac contractile reserve via ß-adrenergic mechanisms. In this study, we investigated whether AD contributes to the impairment in cardiac contractile function that accompanies SCI. We induced SCI in rodents and stratified them into sham, SCI, or SCI plus repetitive induction of AD. At 6-week post-SCI, we assessed cardiac function using in vivo (speckle-tracking echocardiography), ex vivo (working heart), and molecular approaches (Western blot). We also provide unique translational insight by comparing the relationship between the number of daily AD events and cardiac function in 14 individuals with cervical SCI. We found SCI and SCI plus repetitive induction of AD exhibited a reduction in left ventricular dimensions at 6-week post-SCI versus preinjury (P<0.049). Compared with sham, SCI exhibited a reduction in peak radial strain along with a down and rightward shift in the Starling curve (P<0.037), both of which were further depressed in SCI plus repetitive induction of AD (P<0.042). In response to ß-adrenergic stimulation, SCI plus repetitive induction of AD exhibited an attenuated increase in contractile indices (P<0.001), despite no differences in ß-receptor expression within the left ventricle. Our clinical data confirm our experimental findings by demonstrating significant associations between the number of daily AD events and markers of systolic and diastolic function along with left ventricular mechanics. Here, we provide the first evidence from a translational perspective that AD exerts insidious effects on cardiac function in rodents and humans with SCI.

Regulation of GABA Equilibrium Potential by mGluRs in Rat Hippocampal CA1 Neurons.

The equilibrium potential for GABA-A receptor mediated currents (EGABA) in neonatal central neurons is set at a relatively depolarized level, which is suggested to be caused by a low expression of K+/Cl- co-transporter (KCC2) but a relatively high expression of Na+-K+-Cl- cotransporter (NKCC1). Theta-burst stimulation (TBS) in stratum radiatum induces a negative shift in EGABA in juvenile hippocampal CA1 pyramidal neurons. In the current study, the effects of TBS on EGABA in neonatal and juvenile hippocampal CA1 neurons and the underlying mechanisms were examined. Metabotropic glutamate receptors (mGluRs) are suggested to modulate KCC2 and NKCC1 levels in cortical neurons. Therefore, the involvement of mGluRs in the regulation of KCC2 or NKCC1 activity, and thus EGABA, following TBS was also investigated. Whole-cell patch recordings were made from Wistar rat hippocampal CA1 pyramidal neurons, in a slice preparation. In neonates, TBS induces a positive shift in EGABA, which was prevented by NKCC1 antisense but not NKCC1 sense mRNA. (RS)-a-Methyl-4-carboxyphenylglycine (MCPG), a group I and II mGluR antagonist, blocked TBS-induced shifts in both juvenile and neonatal hippocampal neurons. While blockade of mGluR1 or mGluR5 alone could interfere with TBS-induced shifts in EGABA in neonates, only a combined blockade could do the same in juveniles. These results indicate that TBS induces a negative shift in EGABA in juvenile hippocampal neurons but a positive shift in neonatal hippocampal neurons via corresponding changes in KCC2 and NKCC1 expressions, respectively. mGluR activation seems to be necessary for both shifts to occur while the specific receptor subtype involved seems to vary.

Human somatostatin receptor-3 distinctively induces apoptosis in MCF-7 and cell cycle arrest in MDA-MB-231 breast cancer cells.

Somatostatin (SST) mediates cytostatic and pro-apoptotic effects through five somatostatin receptors (SSTR1-5). The modest clinical benefits of SST analogs in cancers of different origin such as breast cancer are attributed to diminished SSTRs expression at tumor sites. In the present study, SSTR3 was overexpressed in MCF-7 and MDA-MB-231, and analyzed for downstream signaling molecules associated with cytostatic and cytotoxic effect. Cells overexpressing SSTR3 displayed inhibition of EGF induced proliferation and enhanced antiproliferative effect of SSTR3-specific agonist in comparison to non-transfected cells. SSTR3 overexpression in MCF-7 cells (R3-MCF-7) constitutively enhanced TUNEL staining, PARP-1 and p27(Kip1) expression suggesting apoptosis and cell-cycle arrest. Conversely, R3-MB-231 cells with SSTR3 overexpression exerted cytostatic and were devoid of any cytotoxic effects. The expression of PTP-1C and the status of ERK1/2, p38 and PI3K phosphorylation was modulated in a cell-specific manner. These findings provide new insights in understanding the antiproliferative role of SSTR3 in breast tumor biology.