Punica granatum Seed Essential Oil Suppressed Methadone-Induced Cell Death by Natural Antioxidant Activity.

OBJECTIVES: Methadone is an opioid used in treating chronic and acute pains as well as opioid dependence. It induces death in neural cells. This study investigates Punica granatum oil's effects as a natural antioxidant on methadone-induced cell death. MATERIALS AND METHODS: The cell death index indicating the apoptosis occurrence is calculated using the TUNEL test. Rhodamine123 evaluated mitochondrial membrane permeability. Griess reaction was used to detect nitric oxide production. Furthermore, IL-1ß, IL-6, INFγ, and TNFα inflammatory cytokines were measured using the Rat inflammatory cytokine assay kit, Rat Kit V-Plex, and the caspase-3 activity was calculated through the Caspase-3 Colorimetric Assay Kit. RESULTS: Different treatment processes of Punica granatum oil reduced cell cytotoxicity and cell death index and increased viability and proliferation in methadone-treated PC12 cells. NO production decreased in different treatment processes compared to methadone-induced PC12 cells and decreased IL-1ß, IL-6, INFγ, and TNFα inflammatory cytokines. In these treatment processes, mitochondrial membrane potential increased, and caspase-3 activity decreased compared to methadone-induced PC12 cells. CONCLUSION: Punica granatum essential oil declined methadone-induced cell death in PC12 cells in a dose-dependent manner through suppressing NO production, IL-1ß, IL-6, INF-γ, and TNF-α inflammatory cytokines production, mitochondrial membrane disruption, and caspase-3 activities.

Therapeutic effects of L-arginine, L-carnitine, and mesenchymal stem cell-conditioned medium on endometriosis-induced oocyte poor quality in an experimental mouse model.

AIM: The present study aimed to explore the potential ameliorative effects of L-arginine (LA), L-carnitine (LC), and bone marrow mesenchymal stem cell-conditioned medium (BMSC-CM) on endometriosis (EMS) model in vivo and in vitro. METHODS: The animals were divided into two main groups, normal and EMS-induced mice. Normal and EMS-induced groups were injected with or without LA (250 mg/kg), LC (250 mg/kg), and BMSC-CM (a final volume of 100 µL of CM/mouse). At the end of the study, the level of total antioxidant capacity (TAC), nitric oxide (NO), and total oxidative status (TOS) were measured in plasma. Furthermore, immature oocytes were collected from two groups and cultured in a maturation medium. Subsequently, the rates of in vitro maturation, in vitro fertilization (IVF), and in vitro embryonic development were evaluated. RESULTS: The results revealed that administration of LA, LC, and BMSC-CM ameliorated the oxidative status through maintaining TAC and alleviating TOS and NO levels. More importantly, the maturation and fertilization rates, blastocyst development, and total blastocyst cell numbers significantly increased in LA, LC, and BMSC-CM-administrated groups compared to the control group. In both the normal and EMS groups, the highest IVF, cleavage, and blastocyst percentages were associated with BMSC-CM treatment (p < 0.05). CONCLUSION: Altogether, LA, LC, and BMSC-CM have therapeutic effects on impaired oocyte quality and promote subsequent development in vitro, probably through normalization of nitro-oxidative stress, thus offering potential alternatives to conventional therapies during assisted reproductive technologies for patients with EMS-associated sub/infertility.

In vitro cytotoxicity studies of smart pH-sensitive lamivudine-loaded CaAl-LDH magnetic nanoparticles against Mel-Rm and A-549 cancer cells.

In this study, an effective nano-drug delivery system was prepared by the co-precipitation method via two steps; the preparation of Fe3O4 magnetic nanoparticles and its surface modification with layered double hydroxide (LDH) and loading lamivudine on this nanocarrier (Fe3O4@CaAl-LDH@Lamivudine). The developed nanoparticles (NPs) were characterized by X-ray powder diffraction, scanning electron microscopy, transmission electron microscopy, energy dispersive X-ray analysis, Fourier-transformed infrared spectroscopy, vibrating-sample magnetometry, thermogravimetric analysis, X-ray photoelectron spectroscopy and Brunauer-Emmett-Teller. The prepared system demonstrated an average size of 130 nm. Also, the drug entrapment efficiency was estimated at ∼70%. In vitro, drug release investigations showed a controlled and pH-dependent lamivudine release over 300 min. The in vitro cytotoxic activity of Fe3O4@CaAl-LDH@Lamivudine NPs was explored against Mel-Rm and A-549 cancer cell lines in comparison with lamivudine and nanocarrier using lactate dehydrogenase colorimetric and MTT assay. The results of the MTT assay revealed that the Fe3O4@CaAl-LDH@Lamivudine NPs significantly inhibited the proliferation of Mel-Rm and A-549 cells in a dose-dependent manner. The influences of Fe3O4@CaAl-LDH@Lamivudine on the cancer cell lines by different therapeutic investigation illustrated the remarkable effect in comparison with free drug. Finally, the achieved consequences confirm the anticancer properties of Fe3O4@CaAl-LDH@Lamivudine and indicate that they may be a cost-effective substitute in the treatment of lung and skin cancer.Communicated by Ramaswamy H. Sarma.

Integrative analysis of DNA methylation and gene expression profiles to identify biomarkers of glioblastoma.

Glioblastoma multiforme (GBM) is the most common, most invasive, and malignant type of primary brain tumor with poor prognosis and poor survival rate. Using GSE22891 the expression and methylation status of same GBM patients was evaluated to identify key epigenetic genes in GBM. Using |log2FC| > 1 and FDR 〈 0.05 as the threshold, DEGs including 4910 downregulated and 2478 upregulated were screened and by |log2FC| 〉 0.2 and p-value < 0.05, 3223 DMCs were detected. By merging the results of DEGs and DMCs, 643 genes were selected for network analysis by WGCNA, and based on expression values three modules and by methylation values, one module was selected. Using STRING and Cytoscape databases, PPI network of genes of all modules were constructed separately. According to the PPI network, core genes were picked out. The expression status of core genes was evaluated using GSE77043, GSE42656, GSE30563, GSE22891, GSE15824, and GSE122498, and 50 genes were validated. The methylation status of 50 genes was explored using GSE50923, GSE22891, and GSE36245, and finally, 12 hub genes including ARHGEF7, RAB11FIP4, PPP1R16B, OLFM1, CLDN10, BCAT1, C1QB, C1QC, IFI16, NUP37, PARP9, and PCLAF were selected. Using GEPIA database, the expression and by cBioportal the survival plot and also scatterplot of methylation versus expression of 12 hub genes were extracted based on TCGA. To determine the diagnostic values of the hub genes, the receiver operating characteristic (ROC) curve and the area under the curve (AUC) were extracted based on GSE22891 and GSE122498. Finally, we evaluated the expression level of the genes in tissue of 83 GBM patients and also non-tumoral adjacent (as control) tissues.

A promising dual-drug targeted delivery system in cancer therapy: nanocomplexes of folate-apoferritin-conjugated cationic solid lipid nanoparticles.

Various nano-sized protein and lipid complexes are being investigated as drug delivery systems. The encapsulation of more than one drug in a single nanocomplex carrier could enhance the therapeutic potency and afford synergistic therapeutic effects. In this study, we developed a novel protein-lipid nanocomplex as a controlled drug delivery system for two important cancer drugs, doxorubicin (DOX) and mitoxantrone (MTO). Apoferritin (AFr) functionalized with folic acid (FA) was used to encapsulate DOX to create the targeted protein nanocomplexes (TPNs). The second drug, MTO, was loaded into the cationic solid lipid nanoparticles (cSLN) to form the liposomal drug nanocomplex particles (MTO-cSLNs). Two complexes were then assembled by tight coupling through ionic interactions to obtain the final drug delivery system, the dual-targeted protein-lipid nanocomplexes (DTPLNs). UV-Vis and fluorescence spectroscopy were used for structural characterization of TPNs and DTPLNs. Transmission electron microscopy (TEM) was used for comprehensive analysis of the final DTPLNs. We confirmed that the DTPLNs display desired time-dependent and pH-dependent drug release behaviors. We also demonstrated the improved anti-cancer efficacy of DOX and MTO in their encapsulated DTPLNs as compared to their free forms. Our results provide promising prospects for the application of the DTPLNs as efficient drug delivery systems.

Enhanced Intracellular Delivery of Curcumin by Chitosan-Lipoic Acid as Reduction-Responsive Nanoparticles.

AIMS: Enhancement of anti-tumor activity of the chemotherapeutic agent CUR by redoxsensitive nanoparticle to get a deeper insight into cancer therapy. BACKGROUND: Tumor targetability and stimulus are widely used to study the delivery of drugs for cancer diagnosis and treatment because poor cellular uptake and inadequate intracellular drug release lead to inefficient delivery of anticancer agents to tumor tissue. OBJECTIVE: Studies distinguishing between tumor and normal tissues or redox-sensitive systems using glutathione (GSH) as a significant signal. METHODS: In this study, we designed Chitosan-Lipoic acid Nanoparticles (CS-LANPs) to improve drug delivery for breast cancer treatment by efficient delivery of Curcumin (CUR). The properties of blank CS-LANPs were studied in detail. The size and the Polydispersity Index (PDI) of the CS-LANPs were optimized. RESULTS: The results indicate the mean size and PDI of the blank CS-LANPs were around 249 nm and 0.125, respectively. However, the Drug Loading (DL) and Encapsulation Efficiency (EE) of the CSLANPs were estimated to be about 18.22% and 99.80%, respectively. Compared to non-reductive conditions, the size of reduction-sensitive CS-LANPs increased significantly under reductive conditions. Therefore, the drug release of CS-LANPs in the presence of glutathione was much faster than that of non-GSH conditions .Moreover, the antitumor effect of CS-LANPs on MCF-7 cells was determined in vitro by MTT assay, cell cytotoxicity, Caspase-3 Assay, detection of mitochondrial membrane potential and quantification of apoptosis incidence. CONCLUSION: CS-LANPs showed a remarkably increased accumulation in tumor cells and had a better tumor inhibitory activity in vitro. CS-LANPs could successfully deliver drugs to cancer cells and revealed better efficiency than free CUR.

Folic acid receptor-targeted solid lipid nanoparticles to enhance cytotoxicity of letrozole through induction of caspase-3 dependent-apoptosis for breast cancer treatment.

Chemotherapy using cytotoxic agents, such as letrozole (LTZ), is one of the most effective treatments for hormone-dependent breast cancer. Nevertheless, nonspecific targeting of the drug constructs several remarkable systemic toxicities. In this study, we synthesized solid lipid nanoparticles (SLNs) by solvent emulsification evaporation method as LTZ carriers. Nanoparticles were also modified with a cancer cell-targeting ligand, folic acid (FA), and then characterized. Cell membrane damage and cell viability were investigated by lactate dehydrogenase (LDH) and 3-(4, 5-Dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) assays, respectively. Caspase-3 activity and TUNEL assays were performed to verify induced apoptosis. Scanning electron microscopy (SEM) exhibited uniform and spherical morphology of the SLNs-LTZ and FA-SLNs-LTZ. The X-ray diffraction (XRD) confirmed LTZ was dispersed as amorphous in the SLNs. The cell culture results revealed that FA-SLNs-LTZ was significantly more cytotoxic than SLNs-LTZ and free drug against MCF-7 cancer cells in vitro, with a 50% inhibitory concentration (IC50) value of 81 ± 0.89 nM, but both nanoformulations had negligible cytotoxicity toward MCF-10A normal cells and they showed promising biocompatibility. Taken together, these findings indicated the evidence of apoptosis as a mechanism of cell death. This study suggests the potential of FA-SLNs-LTZ for inducing apoptosis in a target-specific manner with minimal systemic side effects.

Effect of Pentoxifylline on Staurosporine-Induced Neurite Elongation in PC12 Cells.

Objective: Pentoxifylline enhances neurite elongation in PC12 cells. This study investigated the effects of pentoxifylline on staurosporine-induced neurite elongation in PC12 cells. Materials and Methods: There were five treatment groups, including treatment group I (1 nM), treatment group II (10 nM), treatment group III (100 nM), treatment group IV (1uM), and treatment group V (10 mM of pentoxifylline), together with 214 nM staurosporine for a range of time (6, 12 and 24 hours). Cells only treated with staurosporine at a concentration of 214 nM were used as the control group. Cell proliferation, cell death, immunocytochemistry assay, and Total Neurite Length were assessed. Results: The results showed that pentoxifylline increased cell viability (p<0.05) in a dose- and time-dependent manner, and cell death assay showed that cell death decreased in a dose- and time-dependent manner (p<0.05). TNL increased significantly compared with control cells (p<0.05). Immunocytochemistry assay showed that pentoxifylline at low and high concentrations enhanced ß-tubulin III and GFAP protein expression compared with control cells. Conclusion: It can be concluded that pentoxifylline has positive effects on the staurosporine-induced neurite outgrowth process in PC12 cells.

Signal Transduction of Improving Effects of Ibudilast on Methamphetamine Induced Cell Death.

Objective: Interaction of methamphetamine and sigma (σ) receptors lead to up-regulation and activation of these receptors. The σ receptors induced apoptosis in some parts of the brain by increasing calcium, dopamine, ROS, mitochondrial pores and caspase activity. Ibudilast is a phosphodiesterase inhibitor and anti-inflammatory drug, which can decrease the inflammatory cytokines. Also, it has a neuroprotective effect. It seems that ibudilast can reduce the methamphetamine-induced cell death due to inhibition of σ receptors. Materials and Methods: There were seven treatments including; control: culture medium, Treatment 1: 1mM methamphetamine, Treatment 2: 1mM methamphetamine and 1nM ibudilast, Treatment 3: 1mM methamphetamine and 10nM ibudilast, Treatment 4: 1mM methamphetamine and 100nM ibudilast, Treatment 5: 1mM methamphetamine and 1uM ibudilast, Treatment 6: 1mM methamphetamine and 10uM ibudilast, and Treatment 7: 1mM methamphetamine and 100uM ibudilast. Finally, for inhibition of PKA, CREB, IP3 receptor, NMDA receptor, Sigma receptor antagonist, sigma receptor agonist, cells were preincubated with adding H89 dihydrochloride, 666-15, Heparin, Ketamine, BMY 14802, and Pentazocine. MTT and LDH tests were performed for cell viability and cytotoxicity measurement, respectively. In continuing, the caspase activity colorimetric assay kit used for caspase 3 activity diagnosis. Rhodamine-123 performed to detection of mitochondrial membrane potential. TUNEL test used to DNA fragmentation and apoptosis, Fura-2 used to Measurement of (Ca2+) ic and (Ca2+) m, and fluorescence microscope used to Measurement of antioxidant enzyme activities. Results: Ibudilast increased the cell viability and the rhodamine-123 absorbance in methamphetamin-treated PC12 cells. It reduced cell cytotoxicity, caspase 3 activity, ic and m Ca2+ concentration, (OH) generation and DNA fragmentation in all concentrations of 1 nM t0 100 µM (p<0.05) by the optimal concentration of 100 µM, between our tested treatments. Conclusion: Ibudilast as a phosphodiesterase inhibitor can reduce the methamphetamine-induced cell death due to inhibition of σ receptors through cAMP production.

Increasing the anticancer activity of azidothymidine toward the breast cancer via rational design of magnetic drug carrier based on molecular imprinting technology.

Cancer treatment based anticancer drugs face serious obstacles. To prevail these obstacles, an effective targeted drug carrier can be imperative. This study aimed to design rationally an imprinting strategy for the carrying of a model anticancer drug, Azidothymidine via molecular imprinting technology. Considering the identity and affinity of monomers and cross-linkers to AZT, this work succeeded to establish an exclusive procedure to significantly improve the process of imprinting the Azidothymidine. Imprinting process was carried out on the surface of vinyl-modified silica coated Fe3O4 nanoparticles toward the delivery of azidothymidine to targeted tissue by external magnetic field. The resultant carrier was characterized by FT-IR, XRD, VSM, FESEM, EDX, BET, TGA. The AZT loading process on the nanocarrier is followed with Freundlich adsorption isotherm (QMAX:170 mg/g) and pseudo-second order fast adsorption kinetic (5 min). The release process of AZT from nanocarrier was fitted with First-Order and Higuchi dynamic model. Eventually, the involvement of magnetic nanocarrier was investigated on apoptosis in MCF-7 (cancer cell line) and MCF-10 (normal cell line). The cytotoxicity percentage on MCF-7 cells for magnetic nanocarrier was about 49 times greater than the azidothymidine, but did not affect MCF-10 cells. The corresponding results appropriately disclosed that the cytotoxicity of proposed nanocarrier on MCF-7 cells is through the caspase3 activity. The drug loading and release process as well as in-vitro studies of magnetic carrier were compared with bare carrier. This study indicates that the proposed magnetic carrier can be used as a promising drug carrier toward the breast cancer treatment.

Antiproliferative effects of new magnetic pH-responsive drug delivery system composed of Fe3O4, CaAl layered double hydroxide and levodopa on melanoma cancer cells.

In this study, an efficient drug delivery system composed of Fe3O4, CaAl layered double hydroxide (LDH) and l-Dopa has been synthesized through hydrogen bonds between l-Dopa and CaAl-LDH encapsulated Fe3O4 nanoparticles (Fe3O4@CaAl-LDH@l-Dopa). The structural features of Fe3O4@CaAl-LDH@l-Dopa were characterized using XRD, SEM, TEM, EDX, FT-IR, VSM, TGA, XPS, zeta potential analysis and BET. All of the characterization techniques show the uniform high surface area core-shell structure with about 120 nm in average size. Also, the obtained results clearly indicate that this drug delivery system possess high potent for adsorption of l-Dopa (52 wt%) and high drug encapsulation efficiency (71%). The amount of l-Dopa release in low pHs (53.8%) which simulates the environment of cancer cells is greater than higher pHs. The in vitro cytotoxic and anticancer activities of Fe3O4@CaAl-LDH@l-Dopa were investigated against Mel-Rm Cells Melanoma (NCIt: C3224) using LDH colorimetric assay and differential staining cell death assay. The results showed Fe3O4@CaAl-LDH@l-Dopa with a lower concentration of l-Dopa, illustrate a higher cytotoxicity and anticancer activity.

Possible involvement of calcium channels and plasma membrane receptors on Staurosporine-induced neurite outgrowth.

Staurosporine as a protein kinases inhibitor induced cell death or neurite outgrowth in PC12 cells. We investigated the involvement of calcium channel and plasma membrane receptors on staurosporine inducing neurite outgrowth in PC12 cells. PC12 cells were preincubated with NMDA receptor inhibitors (1.8 mM ketamine and 1µM MK801, treatment 1) or L-Type Calcium channels (100 µM nifedipine and 100 µM flavoxate hydrochloride, treatment 2) or calcium-calmoduline kinasses (10 µM trifluoprazine, treatment 3) and nifedipine, MK801, flavoxate hydrochloride and ketamine (treatment4) or without pretreatments (control). Then, the cells were cultured in RPMI culture medium containing 214nM staurosporine for induction of neurite outgrowth. The percentage of Cell cytotoxicity and apoptotic index was assessed. Total neurite length (TNL) and fraction of cell differentiation were assessed. After 24h, the percentage of cell cytotoxicity were increased in treatments 1, 2 and 4 compared with control (p<0.05). After 6h, apoptotic index was similar between all treatments. After 12h, apoptotic index were increased in treatment 4 compared with control (p<0.05). After 24h, apoptotic index were increased in treatments 1, 2 and 4 compared with control (p<0.05). TNL were decreased in treatments 1, 2 and 4 compared with control in different times of assessment (6, 12 and 24 h) (p<0.05). The fraction of cell differentiation were decreased in treatments 1, 2 and 4 compared with control (p<0.05). It can be concluded that the possible involvement of L-type calcium channel and the N-methyl D-aspartate receptor on staurosporine-induced neurite outgrowth process in PC12 cells.

Effects of extracellular calcium concentration on neurite outgrowth in PC12 cells by staurosporine.

Staurosporine as an inhibitor of protein kinases can induce neuronal differentiation in PC12 cells. We investigated the role of extracellular Ca(2+) on staurosporine inducing neurite outgrowth in PC12 cells. The cells were cultured during cell differentiation in the presence of 214 nM staurosporine with 0.0-0.7 Ca(2+)mM as treatment media. We obtained the fraction of neurite-bearing cells, total neurite length and the percentage of cytotoxiciy. The results showed that decrease or increase of extracellular calcium ions resulted in correspondingly significant decrease or increase in total neurite length and cell differentiation in treated cells. With an increase of extracellular calcium concentration from 0.0 to 0.7 mM, the percentage of cytotoxicity of the PC12 cells decreased (p<0.05). Our data suggest that staurosporine uses the extracellular calcium ions to affect on neurite outgrowth.