MicroRNA-Mediated Drug Repurposing Unveiled Potential Candidate Drugs for Prolactinoma Treatment.

INTRODUCTION: Prolactinomas, also called lactotroph adenomas, are the most encountered type of hormone-secreting pituitary neuroendocrine tumors in the clinic. The preferred first-line therapy is a medical treatment with dopamine agonists (DAs), mainly cabergoline, to reduce serum prolactin levels, tumor volume, and mass effect. However, in some cases, patients have displayed DA resistance with aggressive tumor behavior or are faced with recurrence after drug withdrawal. Also, currently used therapeutics have notorious side effects and impair the life quality of the patients. METHODS: Since the amalgamation of clinical and laboratory data besides tumor histopathogenesis and transcriptional regulatory features of the tumor emerges to exhibit essential roles in the behavior and progression of prolactinomas; in this work, we integrated mRNA- and microRNA (miRNA)-level transcriptome data that exploit disease-specific signatures in addition to biological and pharmacological data to elucidate a rational prioritization of pathways and drugs in prolactinoma. RESULTS: We identified 8 drug candidates through drug repurposing based on mRNA-miRNA-level data integration and evaluated their potential through in vitro assays in the MMQ cell line. Seven repurposed drugs including 5-fluorocytosine, nortriptyline, neratinib, puromycin, taxifolin, vorinostat, and zileuton were proposed as potential drug candidates for the treatment of prolactinoma. We further hypothesized possible mechanisms of drug action on MMQ cell viability through analyzing the PI3K/Akt signaling pathway and cell cycle arrest via flow cytometry and Western blotting. DISCUSSION: We presented the transcriptomic landscape of prolactinoma through miRNA and mRNA-level data integration and proposed repurposed drug candidates based on this integration. We validated our findings through testing cell viability, cell cycle phases, and PI3K/Akt protein expressions. Effects of the drugs on cell cycle phases and inhibition of the PI3K/Akt pathway by all drugs gave us promising output for further studies using these drugs in the treatment of prolactinoma. This is the first study that reports miRNA-mediated repurposed drugs for prolactinoma treatment via in vitro experiments.

The Repertoire of Glycan Alterations and Glycoproteins in Human Cancers.

Cancer as the leading cause of death worldwide has many issues that still need to be addressed. Since the alterations on the glycan compositions or/and structures (i.e., glycosylation, sialylation, and fucosylation) are common features of tumorigenesis, glycomics becomes an emerging field examining the structure and function of glycans. In the past, cancer studies heavily relied on genomics and transcriptomics with relatively little exploration of the glycan alterations and glycoprotein biomarkers among individuals and populations. Since glycosylation of proteins increases their structural complexity by several orders of magnitude, glycome studies resulted in highly dynamic biomarkers that can be evaluated for cancer diagnosis, prognosis, and therapy. Glycome not only integrates our genetic background with past and present environmental factors but also offers a promise of more efficient patient stratification compared with genetic variations. Therefore, studying glycans holds great potential for better diagnostic markers as well as developing more efficient treatment strategies in human cancers. While recent developments in glycomics and associated technologies now offer new possibilities to achieve a high-throughput profiling of glycan diversity, we aim to give an overview of the current status of glycan research and the potential applications of the glycans in the scope of the personalized medicine strategies for cancer.

The potential use of natural products to negate hepatic, renal and neuronal toxicity induced by cancer therapeutics.

Different types of chemotherapeutics are used for cancer treatment. These drugs act on several signal pathways, lead to programmed cell death, and damage cancer cells. Although many specific mechanisms of action have been suggested for chemotherapeutics, there are still gaps in understanding their effects. They may affect different components of the cell, particularly proteins with specific functions, such as enzymes. Recently, targeted and immuno therapies were introduced for treatment of different cancers. However, many cancer patients still depend on traditional and well-known drugs. Doxorubicin and platinum-based drugs are among the most frequently used chemotherapeutics. They are highly cytotoxic for cancer cells, but they also act on healthy cells. Hence, it is crucial to understand the mechanisms involved in order to decrease their side effects. Natural products, many of which are also available over-the-counter, may be considered to decrease various cancer drug-induced side effects. This review focuses on the use of these compounds to overcome side effects of chemotherapeutics, primarily doxorubicin and cisplatin, in the liver, kidney, and neuronal systems.

Indocyanine green based fluorescent polymeric nanoprobes for in vitro imaging.

Indocyanine green (ICG) provides an advantage in the imaging of deep tumors as it can reach deeper location without being absorbed in the upper layers of biological tissues in the wavelengths, which named "therapeutic window" in the tissue engineering. Unfortunately, rapid elimination and short-term stability in aqueous media limited its use as a fluorescence probe for the early detection of cancerous tissue. In this study, stabilization of ICG was performed by encapsulating ICG molecules into the biodegradable polymer composited with poly(l-lactic acid) and poly(ε-caprolactone) via a simple one-step multiaxial electrospinning method. Different types of coaxial and triaxial structure groups were performed and compared with single polymer only groups. Confocal microscopy was used to image the encapsulated ICG (1 mg/mL) within electrospun nanofibers and in vitro ICG uptake by MIA PaCa-2 pancreatic cancer cells. Stability of encapsulated ICG is demonstrated by the in vitro sustainable release profile in PBS (pH = 4 and 7) up to 21 days. These results suggest the potential of the ability of internalization and accommodation of ICG into the pancreatic cell cytoplasm from in vitro implanted ICG-encapsulated multiaxial nanofiber mats. ICG-encapsulated multilayer nanofibers may be promising for the local sustained delivery system to eliminate loss of dosage caused by direct injection of ICG-loaded nanoparticles in systemic administration.

Intraluminal fluid infusion in a rat jejunum ischemia/reperfusion model is associated with improved tissue perfusion and less mucosal damage.

OBJECTIVE: This study used an experimental model mimicking early postoperative enteral feeding after the transfer of free jejunal flap and tested the hypothesis that jejunal infusion with dextrose or saline is associated with improved tissue perfusion and/or less mucosal damage after ischemia/reperfusion (IR) injury. METHODS: Thirty-five male Sprague Dawley rats were randomly divided into five groups: sham group (no IR and no intraluminal infusion); IR control group (IR but not intraluminal infusion); IR plus intraluminal 0.9% NaCl infusion or 5% dextrose or 10% dextrose infusion groups. A jejunal segment of each rat was isolated. The animals had jejunal ischemia for 40 min, reperfusion, and intestinal infusion on the basis of their allocation. Jejunal tissue perfusion was measured with laser Doppler flowmetry at one hour and two hours after reperfusion, after which the animals were sacrificed and tissue samples were obtained for the scoring of histological damage at superficial and cryptic epithelium, villus structure, and inflammatory cell infiltration and tissue nitric oxide (NO), interleukin (IL)-1, IL-6, and matrix metalloproteinase-1 (MMP) level measurements. RESULTS: At 1 h of reperfusion, IR plus 5% dextrose and 10% dextrose groups both had significantly higher perfusion rates than the IR control group (384.8 ± 26.7 and 462.4 ± 44.7 versus 270.3 ± 34.2 PU, respectively, p < 0.05 for both). These differences were maintained at 2 h of reperfusion (p < 0.05 for both). Saline infusion, however, resulted in improved tissue perfusion only at the early phase of reperfusion. Intraluminal infusion with dextrose solution, either 5% or 10%, was associated with higher tissue NO, IL-1, and IL-6 levels than that in the sham group (p < 0.05 for all). In addition, intraluminal infusion of any fluid resulted in less severe histological damage (8.1 ± 0.9 versus 5.8 ± 1.0, 5.4 ± 0.9, and 5.2 ± 1.9, for IR plus saline, 5% dextrose and 10% dextrose groups, respectively, p < 0.05 for all). CONCLUSIONS: Intraluminal infusion of fluids, particularly dextrose solutions, may be protective against IR injury as demonstrated by improved tissue perfusion and less histological damage. In addition, increases in tissue NO, IL-1, and IL-6 levels in association with dextrose infusion may be explained by the activation of pro-inflammatory and anti-inflammatory protective pathways. These support early enteral feeding after free jejunum flap transfers; however, further studies are warranted.

Proteomic approach for understanding milder neurotoxicity of Carfilzomib against Bortezomib.

The proteasomal system is responsible for the turnover of damaged proteins. Because of its important functions in oncogenesis, inhibiting the proteasomal system is a promising therapeutic approach for cancer treatment. Bortezomib (BTZ) is the first proteasome inhibitor approved by FDA for clinical applications. However neuropathic side effects are dose limiting for BTZ as many other chemotherapeutic agents. Therefore second-generation proteasome inhibitors have been developed including carfilzomib (CFZ). Aim of the present work was investigating the mechanisms of peripheral neuropathy triggered by the proteasome inhibitor BTZ and comparing the pathways affected by BTZ and CFZ, respectively. Neural stem cells, isolated from the cortex of E14 mouse embryos, were treated with BTZ and CFZ and mass spectrometry was used to compare the global protein pool of treated cells. BTZ was shown to cause more severe cytoskeletal damage, which is crucial in neural cell integrity. Excessive protein carbonylation and actin filament destabilization were also detected following BTZ treatment that was lower following CFZ treatment. Our data on cytoskeletal proteins, chaperone system, and protein oxidation may explain the milder neurotoxic effects of CFZ in clinical applications.

Proteasome Inhibitors in Cancer Therapy and their Relation to Redox Regulation.

Redox homeostasis is important for the maintenance of cell survival. Under physiological conditions, redox system works in a balance and involves activation of many signaling molecules. Regulation of redox balance via signaling molecules is achieved by different pathways and proteasomal system is a key pathway in this process. Importance of proteasomal system on signaling pathways has been investigated for many years. In this direction, many proteasome targeting molecules have been developed. Some of them are already in the clinic for cancer treatment and some are still under investigation to highlight underlying mechanisms. Although there are many studies done, molecular mechanisms of proteasome inhibitors and related signaling pathways need more detailed explanations. This review aims to discuss redox status and proteasomal system related signaling pathways. In addition, cancer therapies targeting proteasomal system and their effects on redox-related pathways have been summarized.

Repair of critical size defects using bioactive glass seeded with adipose-derived mesenchymal stem cells.

Bioactive glass has been demonstrated as a biocompatible bone substitute. However bone healing process can be prolonged due to late resorption of the material. Adipose derived stem cells (ASC) have osteogenic differentiation potential and hence can be a cell source for bone regeneration. The aim of this study was to test whether combination of bioactive glass with ASCs would enhance bone regeneration. Following creation of critical sized defects on the calvaria of 32 Wistar rats, the animals were randomly divided into four groups: Group C (control): Defects were left untreated; Group G: Defects were covered with autologous bone graft; Group BG: Defects were filled with bioactive glass; Group BG/ASC: Defects were filled with bioactive glass seeded with ASCs. The defect size was significantly greater in Group C compared to all other groups. Bone density was significantly lower in Group C compared to Group G and Group BG/ASC. Bone regeneration score of Group C was significantly lower than other groups. Group BG/ASC demonstrated lamellar bone and havers canal formation. The results of this study demonstrated that bioactive glass implanted with ASC is a biocompatible construct stimulating radiologically and histologically evident bone regeneration similar to autologous bone grafting. © 2016 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 105B: 1002-1008, 2017.

Proteasome inhibitors in cancer therapy: Treatment regimen and peripheral neuropathy as a side effect.

Proteasomal system plays an important role in protein turnover, which is essential for homeostasis of cells. Besides degradation of oxidized proteins, it is involved in the regulation of many different signaling pathways. These pathways include mainly cell differentiation, proliferation, apoptosis, transcriptional activation and angiogenesis. Thus, proteasomal system is a crucial target for treatment of several diseases including neurodegenerative diseases, cystic fibrosis, atherosclerosis, autoimmune diseases, diabetes and cancer. Over the last fifteen years, proteasome inhibitors have been tested to highlight their mechanisms of action and used in the clinic to treat different types of cancer. Proteasome inhibitors are mainly used in combinational therapy along with classical chemo-radiotherapy. Several studies have proved their significant effects but serious side effects such as peripheral neuropathy, limits their use in required effective doses. Recent studies focus on peripheral neuropathy as the primary side effect of proteasome inhibitors. Therefore, it is important to delineate the underlying mechanisms of peripheral neuropathy and develop new inhibitors according to obtained data. This review will detail the role of proteasome inhibition in cancer therapy and development of peripheral neuropathy as a side effect. Additionally, new approaches to prevent treatment-limiting side effects will be discussed in order to help researchers in developing effective strategies to overcome side effects of proteasome inhibitors.

Quercetin-Induced Cell Death in Human Papillary Thyroid Cancer (B-CPAP) Cells.

In this study, we have investigated the antiproliferative effect of quercetin on human papillary thyroid cancer cells and determined the apoptotic mechanisms underlying its actions. We have used different concentrations of quercetin to induce apoptosis and measured cell viability. Apoptosis and cell cycle analysis was determined by flow cytometry using Annexin V and propidium iodide. Finally, we have measured changes in caspase-3 and cleaved poly(ADP-ribose) polymerase (PARP) protein expression levels as hallmarks of apoptosis and Hsp90 protein expression level as a marker of proteasome activity in treated and control cells. Quercetin treatment of human papillary thyroid cancer cells resulted in decreased cell proliferation and increased rate of apoptosis by caspase activation. Furthermore, it was demonstrated that quercetin induces cancer cell apoptosis by downregulating the levels of Hsp90. In conclusion, we have shown that quercetin induces downregulation of Hsp90 expression that may be involved in the decrease of chymotrypsin-like proteasome activity which, in order, induces inhibition of growth and causes cell death in thyroid cancer cells. Thus, quercetin appears to be a promising candidate drug for Hsp90 downregulation and apoptosis of thyroid cancer cells.

Nrf2 silencing to inhibit proteolytic defense induced by hyperthermia in HT22 cells.

Nrf2 pathway has been known to be protective against cancer progression however recent studies have revealed that the antioxidant activity of Nrf2 contributes to chemotherapy resistance. For many years, hyperthermia has been used as an additional therapy to increase the efficiency of chemotherapy and radiotherapy. Besides the positive effects of hyperthermia during treatment procedure, thermotolerance has been found to develop against heat treatment. Although the involved molecular mechanisms have not been fully clarified, heat shock proteins (HSP) and proteasome activity are known to be involved in the acquisition of thermotolerance. The aim of this study was to investigate the potential beneficial effects of combining hyperthermia with Nrf2 silencing to inhibit molecular mechanisms leading to induction of defense mechanisms in transcription level. Following heat treatment of HT22 cells, HSP70 and the proteasome levels and as well as proteasome activity were found to be elevated in the nucleus. Our results demonstrated that Nrf2 silencing reduced defense mechanisms against heat treatment both in antioxidant and proteolytic manner and Nrf2 may be a potential target for therapeutic approach in order to improve the beneficial effects of hyperthermia in cancer therapy.

Potential role of proteasome on c-jun related signaling in hypercholesterolemia induced atherosclerosis.

Atherosclerosis and its complications are major causes of death all over the world. One of the major risks of atherosclerosis is hypercholesterolemia. During atherosclerosis, oxidized low density lipoprotein (oxLDL) regulates CD36-mediated activation of c-jun amino terminal kinase-1 (JNK1) and modulates matrix metalloproteinase (MMP) induction which stimulates inflammation with an invasion of monocytes. Additionally, inhibition of proteasome leads to an accumulation of c-jun and phosphorylated c-jun and activation of activator protein-1 (AP-1) related increase of MMP expression. We have previously reported a significant increase in cluster of differentiation 36 (CD36) mRNA levels in hypercholesterolemic rabbits and shown that vitamin E treatment prevented the cholesterol induced increase in CD36 mRNA expression. In the present study, our aim is to identify the signaling molecules/transcription factors involved in the progression of atherosclerosis following CD36 activation in an in vivo model of hypercholesterolemic (induced by 2% cholesterol containing diet) rabbits. In this direction, proteasomal activities by fluorometry and c-jun, phospo c-jun, JNK1, MMP-9 expressions by quantitative RT-PCR and immunoblotting were tested in aortic tissues. The effects of vitamin E on these changes were also investigated in this model. As a result, c-jun was phosphorylated following decreased proteasomal degradation in hypercholesterolemic group. MMP-9 expression was also increased in cholesterol group rabbits contributing to the development of atherosclerosis. In addition, vitamin E showed its effect by decreasing MMP-9 levels and phosphorylation of c-jun.

Effects of vitamin E on peroxisome proliferator-activated receptor γ and nuclear factor-erythroid 2-related factor 2 in hypercholesterolemia-induced atherosclerosis.

Atherosclerosis and associated cardiovascular complications such as stroke and myocardial infarction are major causes of morbidity and mortality. We have previously reported a significant increase in mRNA levels of the scavenger receptor CD36 in aortae of cholesterol-fed rabbits and shown that vitamin E treatment attenuated increased CD36 mRNA expression. In the present study, we further investigated the redox signaling pathways associated with protection against atherogenesis induced by high dietary cholesterol and correlated these with CD36 expression and the effects of vitamin E supplementation in a rabbit model. Male albino rabbits were assigned to either a control group fed with a low vitamin E diet alone or a test group fed with a low vitamin E diet containing 2% cholesterol in the absence or presence of daily intramuscular injections of vitamin E (50mg/kg). To elucidate the mechanisms by which vitamin E supplementation alters the effects of hypercholesterolemia in rabbit aortae, we measured peroxisome proliferator-activated receptor γ (PPARγ), ATP-binding cassette transporter A1 (ABCA1), and matrix metalloproteinase-1 (MMP-1) mRNA levels by quantitative RT-PCR and the expression of MMP-1, nuclear factor-erythroid 2-related factor 2 (Nrf2), and glutathione S-transferase α (GSTα) protein by immunoblotting. The increased MMP-1 and decreased GSTα expression observed suggests that a cholesterol-rich diet contributes to the development of atherosclerosis, whereas vitamin E supplementation affords protection by decreasing MMP-1 and increasing PPARγ, GSTα, and ABCA1 levels in aortae of rabbits fed a cholesterol-rich diet. Notably, protein expression of Nrf2, the antioxidant transcription factor, was increased in both the cholesterol-fed and the vitamin E-supplemented groups. Although Nrf2 activation can promote CD36-mediated cholesterol uptake by macrophages, the increased induction of Nrf2-mediated antioxidant genes is likely to contribute to decreased lesion progression. Thus, our study demonstrates that Nrf2 can mediate both pro- and antiatherosclerotic effects.

The effect of resveratrol on signal transduction pathways and the role of pro-apoptotic Bax protein on apoptosis in HCT- 116 colon carcinoma cell lines.

Colon carcinoma is the third among the cancer related deaths. The role of pro-apoptotic Bax protein on the resveratrol related apoptosis, mitochondrial membrane potential and signal pathways has not been identified in colon carcinoma cells. In this direction, HCT-116 bax positive and HCT-116 negative cell lines were utilized to detect the apoptotic effect and I?B, MEK1 and STAT 3 signal transduction pathways of resveratrol. The impact on the cell viability and IC50 value of resveratrol has been determined via WST-1 viability assay. The ratio of apoptosis has been evaluated via flow cytometry following Annexin V/Propidium iodide (PI) double staining. Changes in the mitochondrial membrane potential have been analyzed by flow cytometry and JC-1 fluorometric staining. IkB, MEK1 and STAT3 molecules were measured by Enspire device. Data showed the IC50 value for resveratrol as 50M. According to the flow cytometry, apoptosis ratio has been determined as 29.65% in the experimental group of bax positive cells, as 13.98% in the experimental group of bax negative cells. Changes in the membrane potential has been established as 8.62% in the experimental group of bax positive cells, as 97.98% in the experimental group of bax negative cells. When the obtained data from Enspire device was reviewed; bax positive cells I?B phosphorylations were found as as 5.22 for experimental groups; MEK1 phosphorylations were found as and as 1.15 for experimental groups; STAT 3 phosphorylations were found as and as 2.52 for experimental groups. In HCT-116 bax negative cells, I?B phosphorylation were and 2.71 in experimental groups; MEK1 phosphorylation were 1.18 in experimental groups; STAT 3 phosphorylation were 1.54 in experimental groups. Our data show that Bax protein plays role in the apoptotic effect of resveratrol by altering mitochondrial membrane potential and mitochondrial membrane permeability, signal transduction and the absence of Bax increase the sensitivity of HCT-116 colon carcinoma cells to apoptosis.

Heat shock proteins and proteasomal degradation in normal and tumor cells.

Proteasomal degradation of oxidized proteins is a crucial mechanism to prevent the accumulation of cellular damage. The removal of the damage is generally a required process for healthy organisms to keep the integrity while in cancer cells the situation may be different. In normal conditions, cancer cells have higher proteasome activity compared to normal cells. During cancer treatment, cellular damage by chemotherapy is an expected process to be able to kill the tumor cells. And the accumulation of this damage accompanied by the decrease in protein repair and removal systems may increase the efficacy of the cancer therapy. Heat shock proteins (Hsp) as molecular chaperones are involved in the folding, activation and assembly of a variety of proteins. Among these Hsp40, Hsp70 and Hsp90 are believed to act as a chaperone system to regulate the proteasomal degradation. In this study, we tested the role of heat stress response on the proteasomal degradation of oxidized proteins. We used two different cell lines to observe the difference in normal and tumor cells. First the effect of heat stress (42°C, 1h) were tested in terms of protein oxidation tested by protein carbonyl formation and proteasomal degradation. The results were extremely different in normal fibroblast cells and hippocampal tumor cells. In the same direction, the expressions of Hsp40, Hsp70 and Hsp90 were affected in a different manner in two cell lines, will be discussed in detail. Supported by TUBITAK COST-CM1001-110S281.

The role of heat stress on the age related protein carbonylation.

Since the proteins are involved in many physiological processes in the organisms, modifications of proteins have important outcomes. Protein modifications are classified in several ways and oxidative stress related ones take a wide place. Aging is characterized by the accumulation of oxidized proteins and decreased degradation of these proteins. On the other hand protein turnover is an important regulatory mechanism for the control of protein homeostasis. Heat shock proteins are a highly conserved family of proteins in the various cells and organisms whose expressions are highly inducible during stress conditions. These proteins participate in protein assembly, trafficking, degradation and therefore play important role in protein turnover. Although the entire functions of each heat shock protein are still not completely investigated, these proteins have been implicated in the processes of protection and repair of stress-induced protein damage. This study has focused on the heat stress related carbonylated proteins, as a marker of oxidative protein modification, in young and senescent fibroblasts. The results are discussed with reference to potential involvement of induced heat shock proteins. This article is part of a Special Issue entitled: Protein Modifications. BIOLOGICAL SIGNIFICANCE: Age-related protein modifications, especially protein carbonylation take a wide place in the literature. In this direction, to highlight the role of heat shock proteins in the oxidative modifications may bring a new aspect to the literature. On the other hand, identified carbonylated proteins in this study confirm the importance of folding process in the mitochondria which will be further analyzed in detail.