Docetaxel encapsulation in nanoscale assembly micelles of folate-PEG-docetaxel conjugates for targeted fighting against metastatic breast cancer in vitro and in vivo.

Due to the high frequency and mortality of breast cancer, developing efficient targeted drug delivery systems for frightening against this malignancy is among cancer research priorities. The aim of this study was to synthesize a targeted micellar formulation of docetaxel (DTX) using DTX, folic acid (FA) and polyethylene glycol (PEG) conjugates as building blocks. In the current study, two therapeutic polymers consisting of FA-PEG-DTX and PEG-DTX conjugates were synthesized and implemented to form folate-targeted and non-targeted micelles. Dissipative particle dynamics (DPD) method was used to simulate the behavior of the nanoparticle. The anti-cancer drug, DTX was loaded in to the micelles via solvent switching method in order to increase its solubility and stability. The cytotoxicity of the targeted and non-targeted formulations was evaluated against 4T1 and CHO cell lines. In vivo therapeutic efficiency was studied using ectopic tumor model of metastatic breast cancer, 4T1, in Female BALB/c mice. The successful synthesis of therapeutic polymers, FA-PEG-DTX and PEG-DTX were confirmed implementing 1HNMR spectral analysis. The size of DTX-loaded non-targeted and targeted micelles were 176.3 ± 8.3 and 181 ± 10.1 nm with PDI of 0.23 and 0.17, respectively. Loading efficiencies of DTX in non-targeted and targeted micelles were obtained to be 85% and 82%, respectively. In vitro release study at pH = 7.4 and pH = 5.4 showed a controlled and continuous drug release for both formulations, that was faster at pH = 5.4 (100% drug release within 120 h) than at pH = 7.4 (80% drug release within 150 h). The targeted formulation showed a significant higher cytotoxicity against 4T1 breast cancer cells (high expression of folate receptor) within the range of 12.5 to 200 µg/mL in comparison with no-targeted one. However, there was no significant difference between the cytotoxicity of the targeted and non-targeted formulations against CHO cell line as low-expressed cell line. In accordance with in vitro investigation, in vivo studies verified the ideal anti-tumor efficacy of the targeted formulation compared to Taxotere and non-targeted formulation. Based on the obtained data, FA-targeted DTX-loaded nano-micelles significantly increased the therapeutic efficacy of DTX and therefore can be considered as a new potent platform for breast cancer chemotherapy.

Bioinformatics analysis of the genes involved in the extension of prostate cancer to adjacent lymph nodes by supervised and unsupervised machine learning methods: The role of SPAG1 and PLEKHF2.

The present study aimed to identify the genes associated with the involvement of adjunct lymph nodes of patients with prostate cancer (PCa) and to provide valuable information for the identification of potential diagnostic biomarkers and pathological genes in PCa metastasis. The most important candidate genes were identified through several machine learning approaches including K-means clustering, neural network, Naïve Bayesian classifications and PCA with or without downsampling. In total, 21 genes associated with lymph nodes involvement were identified. Among them, nine genes have been identified in metastatic prostate cancer, six have been found in the other metastatic cancers and four in other local cancers. The amplification of the candidate genes was evaluated in the other PCa datasets. Besides, we identified a validated set of genes involved in the PCa metastasis. The amplification of SPAG1 and PLEKHF2 genes were associated with decreased survival in patients with PCa.

Homology Modeling of 5-alpha-Reductase 2 Using Available Experimental Data.

5-Alpha-reductase 2 is an interesting pharmaceutical target for the treatment of several diseases, including prostate cancer, benign prostatic hyperplasia, male pattern baldness, acne, and hirsutism. One of the main approaches in computer aided drug design is structure-based drug discovery. However, the experimental 3D structure of 5-alpha-reductase 2 is not available at present. Therefore, a homology modeling method and molecular dynamics simulation were used to develop a reliable model of 5-alpha-reductase 2 for inhibitor pose prediction and virtual screening. Despite the low sequence identity between the target and template sequences, a useful 3D model of 5-alpha-reductase 2 was generated by the inclusion of experimental data.

Identification of Non-Zinc Binding Inhibitors of MMP-2 Through Virtual Screening and Subsequent Rescoring.

MMP-2 belongs to a large family of proteases called matrix metalloproteinases (MMPs) that degrades type IV collagen, the main structural component of basement membranes and gelatin. The main pathologic role of MMP-2 overexpression is to contribute to the development of cancer through the progression of metastasis and angiogenesis. A structure-based virtual screening was employed to find new inhibitors with possible selectivity for MMP-2. The inhibitory activities of 3 inhibitors (one was not a suitable drug-like hit) among 19 purchased compounds were approved by enzyme inhibition assay. 5 hits were non-zinc-binding inhibitors of MMP-2. The results demonstrated that a computer-aided drug design could be successfully applied for discovering new MMP-2 inhibitors. We found inhibitors with new scaffolds for the inhibition of MMP-2 with some selectivity features that could be used for future lead optimization processes. According to the docked pose and MD simulation, compound 13 was expected to interact with the S1' specificity loop of MMP-2 and had 2 π-π interactions and a stable hydrogen bond with the MMP-2 active site. The key feature of compound 13 could be used to guide the design of new non-zinc-binding inhibitors of MMP-2.

Docking and QSAR Studies of 1,4-Dihydropyridine Derivatives as Anti- Cancer Agent.

BACKGROUND: The multidrug resistance (MDR) of cancer cells has become a great barrier to the success of chemotherapy. OBJECTIVE: In this study, quantitative structure activity relationship (QSAR) modeling was applied to 46 1,4-dihydropyridine structures (DHPs), and some selected compounds were docked. METHODS: QSAR was used to generate models and predict the MDR inhibitory activity for a series of 1,4-dihydropyridines (DHP). The DHPs were built and optimized using the Sybyl program (x1.2 version). Descriptor generation was done by DRAGON package. Docking was carried out using Auto Dock 4.2 software. Multiple linear regression, and partial least square were performed as QSAR modelgeneration methods. External validation, cross-validation (leave one out) and y-randomization were used as validation methods. RESULTS: The constructed model using stepwise-MLR and GA-PLS revealed good statistical parameters. In the final step all compounds were divided into two parts: symmetric (PLS) and asymmetric (MLR) 1,4-dihydropyridines and two other models were built. The square correlation coefficient (R2) and root mean square error (RMSE) for train set for GA-PLS were (R2 = 0.734, RMSE train = 0.26). CONCLUSION: The predictive ability of the models was found to be satisfactory and could be employed for designing new 1,4-dihydropyridines as potent MDR inhibitors in cancer treatment. 1,4- Dihydropyridine ring containing protonable nitrogen as scaffold could be proposed. Sulfur, ester, amide, acyle, ether, fragments are connected to a 1,4-dihydropyridine ring. Phenyl groups (with an electronegative substituent) as a lipophilic part are essential for the inhibitory effect.

Considering Rotatability of Hydroxyl Groups for the Active Site Residues of MMP-13 in Retrospective Virtual Screening Campaigns.

Considering different orientation of hydroxyl and thiol groups of receptor residues such as Thr, Tyr, Ser and Cys is an option available on Glide docking software. This is an attempt that can provide more realistic ligand-receptor interactions. Matrix metalloproteinase 13 (MMP-13) is a suggested target for several diseases including osteoarthritis and cancer. MMP-13 was selected as a receptor with reported flexibility in the active site residues. Four residues in the MMP-13 active site were selected and their hydroxyl groups were made flexible during docking: Tyr(241), Thr(242), Tyr(243) and Thr(244). The ability of retrospective virtual screenings using a rigid receptor for discriminating between actives and decoys were compared to those using receptor with different combination of flexible residues. Statistical analysis of the results and inspecting the binding pose of the ligands suggested that the hydroxyl orientation of Tyr(241), Thr(242), Tyr(243) and Thr(244) (in particular Thr(242) and to a lesser extent Thr(244)) had impacts on the MMP-13 docking results.

A Cross-Docking Study on Matrix Metalloproteinase Family.

BACKGROUND: Matrix metalloproteinases (MMPs) contribute to various physiological and pathophysiological processes. An imbalance in MMP activity causes pathological conditions including inflammatory diseases, cancer, and cardiovascular diseases. Each MMP member has many 3D structures available; therefore, selecting one structure for virtual screening becomes challenging. METHODS: In this study, we used the cross-docking approach to rank the available MMP structures for their probable successful performance in virtual screening. To determine structures that would offer best average RMSD (root mean square deviation), we performed cross-docking studies on 123 holo (protein-ligand) structures of seven MMP enzyme groups (MMP-1, MMP-3, MMP-7, MMP-8, MMP-9, MMP-12, and MMP-13). RESULTS: MMP enzymes with more flexible residues had fewer structures with RMSD < 2.0 A. Further, same resolution and binding affinities, difference in ligand size, and chemotype of the co-crystalized ligand were parameters that could greatly affect the corresponding cross-dock results and the calculated average RMSD for the structures. Four of the six best MMP-12 receptors, which were identified using the average RMSD metric, had the highest EF1% (emrichment factor) in the retrospective enrichment study. CONCLUSION: According to the enrichment results, structures with lower average RMSD have a high probability of being appropriate candidates. These study findings will help in receptor selection for an MMP virtual screening protocol and lead to better enrichment of MMP inhibitors.

Evaluation of the effect of pentoxifylline on erythropoietin-resistant anemia in hemodialysis patients.

Use of recombinant human erythropoietin (rh-Epo) improves hemoglobin (Hgb) in 90-95% of the cases of anemia of chronic kidney disease (CKD). However, it is known that pro-inflammatory cytokines such as interferon-gamma (IFN-γ), tumor necrosis factor-alfa (TNF-α) and interleukin-1 (IL-1) suppress erythropoiesis, resulting in inadequate response to rh-Epo. Pentoxifylline has been shown to have modulatory effects on the immune system. This prospective study to evaluate the effect of pentoxyphylline on erythropoeisis was performed on 15 (eight males, seven females) clinically stable patients who had been on hemodialysis for at least six months with anemia (Hgb of <10.7 g/dL) unresponsive to rh-Epo despite high doses. They were treated with 400 mg pentoxifylline tablets once daily for 12 weeks. Hgb increased after one and two months of drug administration, but significant changes were observed in eight (53%) patients after three months (P <0.05). Our study illustrates a probable new use for an old medicine. Three months treatment with pentoxifylline was seen to increase Hgb significantly in rh-Epo-resistant patients. More prospective studies with a larger sample size are needed to determine the inhibitory role of cytokines on hematopoiesis and exploring new drugs or new drug indications to overcome anemia in advanced renal failure.

A study on druggability of MIA as a promising approach for inhibition of metastasis.

MIA (Melanoma Inhibitory Activity) protein is over expressed in melanoma cells and binds to extracellular matrix proteins as well as to several integrins. These interactions were suggested to promote formation of metastasis. Therefore, abrogation of MIA interaction with other proteins using small molecules might show a diminishing effect on cancer cell invasion. The present study is aimed at the analysis of the integrin-binding site of MIA using molecular docking, followed by a virtual screening for drug-like compounds that show potential as putative inhibitors of MIA-integrin interaction. Results showed that at the proposed binding interface of the MIA-integrin complex, a druggable binding pocket is located. Therefore, the integrin-binding domain of MIA was used as a receptor to screen 2200 drug-like compounds. Next, we analysed the interactions of the identified hit compounds with the MIA binding pocket to find the most important features of the hit compounds.

Evaluation of 11 scoring functions performance on matrix metalloproteinases.

Matrix metalloproteinases (MMPs) have distinctive roles in various physiological and pathological processes such as inflammatory diseases and cancer. This study explored the performance of eleven scoring functions (D-Score, G-Score, ChemScore, F-Score, PMF-Score, PoseScore, RankScore, DSX, and X-Score and scoring functions of AutoDock4.1 and AutoDockVina). Their performance was judged by calculation of their correlations to experimental binding affinities of 3D ligand-enzyme complexes of MMP family. Furthermore, they were evaluated for their ability in reranking virtual screening study results performed on a member of MMP family (MMP-12). Enrichment factor at different levels and receiver operating characteristics (ROC) curves were used to assess their performance. Finally, we have developed a PCA model from the best functions. Of the scoring functions evaluated, F-Score, DSX, and ChemScore were the best overall performers in prediction of MMPs-inhibitors binding affinities while ChemScore, Autodock, and DSX had the best discriminative power in virtual screening against the MMP-12 target. Consensus scorings did not show statistically significant superiority over the other scorings methods in correlation study while PCA model which consists of ChemScore, Autodock, and DSX improved overall enrichment. Outcome of this study could be useful for the setting up of a suitable scoring protocol, resulting in enrichment of MMPs inhibitors.

The effects of pentoxifylline administration on NFΚB P50 transcription factor expression.

BACKGROUND: Pentoxifylline has anti-inflammatory properties and could suppress some inflammatory processes including tumor necrosis factor-alpha (TNF-α) production. We assessed the effects of a two-month administration of pentoxifylline on nuclear factor-kappa B (NFκB) pathways in patients with coronary artery disease (CAD) in which inflammatory pathways, especially NFκB transcription factors, have a critical role. METHODS: A double-blind randomized placebo-controlled study design was used. Forty CAD patients were randomized to either 2 months of pentoxifylline treatment (1200 mg/day) (n = 20) or placebo treatment (n = 20). Blood samples were obtained just before and after two months of treatment. P50 protein concentration in peripheral blood mononuclear cells (PBMCs) was measured by Enzyme Linked ImmunoSorbent Assay (ELISA) method. RESULTS: P50 concentration did not significantly change during two months of pentoxifylline administration. CONCLUSION: Longer pentoxifylline administration is needed to see its favorable effects on NFκB family elements.

Pentoxifylline decreases soluble CD40 ligand concentration and CD40 gene expression in coronary artery disease patients.

CONTEXT AND OBJECTIVE: Increased level of inflammatory mediators plays a central role in the features of coronary artery diseases (CAD). As pentoxifylline could suppress the inflammatory process and has shown some promising beneficial effects in inflammatory diseases, we evaluated the effect of 2 months pentoxifylline administration in patients with CAD. MATERIALS AND METHODS: A randomized placebo-controlled double-blind study design was used. Forty CAD patients (32 males and 8 females) were randomized into either 2 months of pentoxifylline treatment (1200 mg/day) (n = 20) or placebo treatment (n = 20). Blood samples were obtained before and after treatment. Gene expression analysis for mRNA of CD40, p65 and IκBα in peripheral blood mononuclear cells (PBMCs) were performed using real-time reverse-transcription polymerase chain reaction (RT-PCR). Plasma concentration of soluble CD40 (sCD40) ligand as well as protein concentration of p50 were measured by ELISA method. RESULTS: Pentoxifylline decreased CD40 mRNA by 45% (p < 0.05) in PBMCs and sCD40 ligand level in plasma of CAD patients by 34% (p < 0.01). DISCUSSION AND CONCLUSION: Pentoxifylline treatment can suppress the CD40/CD40 ligand system activation in CAD patients. As this system has a role in plaque progression and plaque rupture, pentoxifylline could be a good choice for future studies in preventing cardiovascular events.

Immunohistochemical assessment of MGMT expression and p53 mutation in glioblastoma multiforme.

AIMS AND BACKGROUND: The prognosis of glioblastoma multiforme (GBM) remains poor despite advances in surgery and adjuvant therapies. TP53 and O6-methylguanine-DNA methyltransferase (MGM) are tumor suppressor genes that are implicated in GBM resistance to radiation and chemotherapy. In order to assess the expression of the protein products of these two genes, 50 GBM samples were analyzed in this study. METHODS: Demographic and clinical data along with postsurgery tumor samples from 50 GBM patients were collected from the pathology archive. MGMT and p53 protein expression was evaluated by immunohistochemistry. RESULTS: 52% of cases had mutated p53, predominantly expressed in the nuclei of tumor cells. MGMT immunohistochemistry was negative in 35 (70%) patients and positive in 15 (30%) others. Immunohistochemistry-negative specimens for MGMT expression showed a significantly higher expression of mutant p53 (P = 0.03). CONCLUSION: MGMT expression was significantly lower in cells bearing p53 mutation. This indicates that there is a tendency for p53 activity to decline with MGMT inactivation. However, this study could not deduce which protein was the regulator of the other.

Correlation between cathepsin D serum concentration and carotid intima-media thickness in hemodialysis patients.

AIM: Cardiovascular (CV) disease is the leading cause of death in hemodialysis (HD) patients, and approximately half of mortalities in HD patients are attributed to CV disease. Atherosclerosis is the most frequent cause of CV complications in patients with end-stage renal disease (ESRD). Based on recent studies, cathepsin D has been suggested as a potential marker of atherosclerosis, and we hypothesized that there is an association between serum concentration of cathepsin D and carotid intima-media thickness (CIMT) in hemodialysis patients. METHODS: Thirty-one hemodialysis patients (18 men and 13 women) were enrolled in this study. Serum levels of soluble cathepsin D were measured with an enzyme-linked immunosorbant assay (ELISA) kit. CIMT was determined in each patient. RESULTS: The data showed that there was a positive significant correlation between serum concentration of cathepsin D and CIMT by using Pearson correlation (P value <0.05). CONCLUSION: Based on the results of this study, cathepsin D could be suggested to have a role in the development of atherosclerotic plaques.

Effect of silymarin administration on TNF-α serum concentration in peritoneal dialysis patients.

Chronic inflammation in dialysis patients increases the production of cytokines such as TNF-α, IL-1ß and IFN- IFN-γ and there is evidence of a significant mortality rate in dialysis patients due to inflammation. Overproduction of inflammatory cytokines can induce complications such as atherosclerosis, malnutrition and anaemia, which are mostly resistant to erythropoietin treatment. Cardiovascular disease is the leading cause of death in haemodialysis patients and about half of the mortality is attributable to cardiovascular disease. Silymarin modulates the immune system by inhibition of neutrophil immigration, mast cell immobilization, prostaglandin production and leukotriene synthesis. Furthermore, silymarin suppresses the induction of TNF-α and it was hypothesized that silymarin could decrease the serum concentration of TNF-α in peritoneal dialysis patients, and thus treat anaemia. Fifteen peritoneal dialysis patients were enrolled in this study and serum levels of soluble TNF-α were measured using an enzyme-linked immunosorbant assay (ELISA) kit. Serum TNF-α was found to be decreased in some patients and in the response group, the haemoglobin concentration after 8 weeks of silymarin administration was increased significantly (p < 0.05). Based on the results of this study, it is suggested that silymarin may be useful in the treatment of inflammation for peritoneal dialysis patients.

Association between MGMT promoter hypermethylation and p53 mutation in glioblastoma.

O(6)-methylguanine-DNA methyltransferase (MGMT) is a DNA repair enzyme that removes alkyl groups from the O(6) position of guanine. MGMT is transcriptionally silenced by promoter hypermethylation in several human neoplasia. We used methylation-specific PCR (MSP) to analyze the MGMT promoter methylation status of 50 glioblastoma tumors. Hypermethylation was detected in 24 of 50 (48%) samples. We also analyzed mutant p53 expression by immunohistochemical analysis of glioblastoma tissue samples. A significant association was found between MGMT methylation and p53 mutation status (p< .05). These results suggested that epigenetic inactivation of MGMT plays an important role in the survival of glioblastoma patients and this inactivated gene involved in p53 mutation.

Selective inhibitory effect of adenosine A1 receptor agonists on the proliferation of human tumor cell lines.

BACKGROUND: In this study, the effects of three structural analogues of adenosine upon proliferation of human tumor cells were investigated. Previous research showed a cytotoxic effect of adenosine via A3 receptor and A1 receptor and sometimes this effect was receptor independent. The researches showed a differential cytotoxic effect of adenosine and its A3 agonists on cancerous cells, while other studies demonstrated tumor promoting effect of adenosine and its A1 agonists. The purpose of the present study was the evaluation of the possible selective anti-tumor effect of A1 receptor agonists on cancerous cells. METHODS: The substances of N6-cyclohexyl-adenosine (CHA, A1 agonist), R-isomer of N6 phenylisopropyladenosine (R-PIA, A1 agonist) and N5-eethylcarboxamido-adenosine (NECA, adenosine A1-A2 non-specific agonist) were tested for their anti-proliferative effect using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay method. Hep G2, Hep2, CACO2, ACHN and L929 cell lines were used in this assay. RESULTS: CHA inhibited cell proliferation in three cell lines (in concentration of 5-50 microM) and R-isomer of R-PIA in one cell line (in concentration of 10-50 microM). These effects were inhibited partially by addition of 1,3-Dipropyl-8-cyclopentylxanthine (A1 antagonist). The NECA analogue had no inhibitory effect on the cell proliferations. All of the substances had no cytotoxic effect on L929 cells (mouse connective tissue fibroblast cell line). CONCLUSION: CHA and R-PIA had inhibitory effect on the proliferation of human tumor cell lines partially via A1 receptor, while they didn't show such effect on fibroblast cells. These results suggest that A1 adenosine receptor agonist have a good potential of specific anti-tumor activity.