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Using biocompatible polymer nanofibrous conduits with a controlled drug delivery have attracted much attention for peripheral nerve regeneration. This work was aimed at preparing electrospun poly ( l -lactide-co- D , l -lactide) (PLDLLA) containing multi-walled carbon nanotubes (MWCNTs) and 4-aminopyridine (4-AP)-loaded molecularly imprinted nanoparticles (MIP4-AP) as well as evaluating their performance in in vitro and in vivo assessments. After synthesis of MIP4-AP based on poly (methacrylic acid) with imprinting factor of 1.78, it was incorporated into the PLDLLA/MWCNTs nanofibers to optimize. By adjusting the process variables, the average diameter and electrical conductivity of the nanofibrous sample were 92 nm and 2870 × 10−7 S cm−1, respectively. Afterward, 4-AP release of the optimum sample showed the presence of MIP4-AP leading to initial burst release decrease and plateau level postpone up to 96 h. Moreover, the culture results of PC12 as neuroblastoma cell line on optimal PLDLLA/MWCNTs/MIP4-AP nanofibrous sample revealed the highest cell proliferation without cytotoxicity compared to neat nanofibers. Eventually, the animal model experiment exhibited that the conductive conduit based on the optimum sample was able to repair the rat's sciatic nerve after four weeks in accordance with sciatic function index and histological studies.

Cisplatin treatment confers the relative resistance to MCF-7 cells as compared to other breast cancer cell lines. One principal reason is that chemotherapeutic agents induce autophagy in these cells to inhibit apoptosis. Binding immunoglobulin protein (BiP), a master regulator of unfolded protein response (UPR) and 14-3-3ζ are two critical proteins upregulated in breast cancer rendering resistance to anticancer drugs. They also play pivotal roles in autophagy with crosstalk with the apoptotic pathways of UPR through certain regulators. Thus, BiP and 14-3-3ζ were selected as the candidate targets to enhance cell death and apoptosis. First, cisplatin resistance was induced and determined by MTT assay and qPCR in MCF-7 cells. Then, the apoptosis axis of UPR was activated by knocking down either BiP or 14-3-3ζ and overactivated by co-knockdown of BiP and 14-3-3ζ. Apoptosis assays were performed using flow cytometry, TUNEL assays utilized confocal microscopy followed by western blot analysis and caspase-3 and JNK activities were investigated to assess the outcomes. Finally, an autophagy assay followed by western blotting was performed to study the effects of co-knockdown genes on cell autophagy in the presence and absence of cisplatin. The present data indicated the enhancement of cisplatin sensitivity in MCF-7 cells co-knocked down in BiP and 14-3-3ζ compared with either gene knockdown. Upregulation of JNK and cleaved-PARP1 protein levels as well as caspase-3 and JNK overactivation confirmed the results. A marked attenuation of autophagy and Beclin1 as well as ATG5 downregulation were detected in co-knockdown cells compared to knockdown with either BiP or 14-3-3ζ. Cisplatin sensitization of MCF-7 cells through double-knockdown of BiP and 14-3-3ζ highlights the potential of targeting UPR and autophagy factors to increase the effect of chemotherapy.

In photodynamic therapy (PDT) of cancer both the light and the photosensitizing agent are normally harmless, but in combination they could result in selective tumor killing. Zinc oxide nanoparticles were synthesized and coated with the amino acid cysteine to provide an adequate arm for conjugation with porphyrin photosensitizers (meso-tetra (4-carboxyphenyl) porphyrin [MTCP] and CuMTCP). Porphyrin-conjugated nanoparticles were characterized by TEM, FTIR, and UV-vis, and fluorescence spectrophotometry. The 3-[4, 5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide (MTT) assay was used to measure cell viability in the presence or absence of porphyrin conjugates following UV and X-ray irradiation. The uptake of the porphyrin-conjugated ZnO nanoparticles by cells was detected using fluorescence microscopy. Our results indicated that the survival of T-47D cells was significantly compromised in the presence of ZnO-MTCP-conjugated nanostructures with UV light exposure. Exhibition of cytotoxic activity of ZnO-MTCP for human prostate cancer (Du145) cells occurred at a higher concentration, indicating the more resistant nature of these tumor cells. ZnO-CuMTCP showed milder cytotoxic effects in human breast cancer (T-47D) and no cytotoxic effects in Du145 with UV light exposure, consistent with its lower cytotoxic potency as well as cellular uptake. Surprisingly, none of the ZnO-porphyrin conjugates exhibited cytotoxic effects with X-ray irradiation, whereas ZnO alone exerted cytotoxicity. Thus, ZnO and ZnO-porphyrin nanoparticles with UV or X-ray irradiation may provide a suitable treatment option for various cancers.

Alterations in expression of the DFF40 gene have been reported in some cancers. This study is an in vitro study of the therapeutic effects of gene transfer that lead to elevation in DFF40 expression within T-47D cells in the presence of sulfonamide drugs. In this study, we have constructed a eukaryotic expression vector for DFF40 and transfected it into T-47D cancer cells. We used real time RT-PCR to detect the expression of DFF40 and the MTT assay to determine effects of the sulfonamide drugs acetazolamide, sulfabenzamide, sulfathiazole and sulfacetamide on cell viability in the presence of increased and normal DFF40 levels. Cell cycle distribution was assessed by propidium iodide (PI) staining and the rates of apoptosis by annexin V/PI staining. The DNA laddering analysis was employed to evaluate apoptosis. We observed that overexpression of DFF40 was only effective in decreasing viability in cells incubated with acetazolamide and sulfabenzamide. There was enhanced apoptosis in these groups, particularly with acetazolamide. The cell cycle distribution analysis showed that in the presence of sulfonamide drugs there were no substantial changes in empty-vector or DFF40-transfected cells, except for those cells treated with sulfabenzamide or sulfathiazole. There was no DNA laddering in cells that expressed the empty vector when incubated with sulfonamide drugs. In contrast, we observed DNA laddering in cells that expressed DFF40 in the presence of acetazolamide. Our results have demonstrated that combinatorial use of some sulfonamides such as acetazolamide along with increased expression of DFF40 can potently kill tumor cells via apoptosis and may be beneficial for treatment of some chemoresistant cancers.

RNA interference (RNAi) has become a promising tool for cancer therapy. Small interfering RNAs (siRNAs) can synergistically enhance the cell killing effects of drugs used in cancer treatment. Here we examined the effects of siRNA-mediated DNA fragmentation factor 45 (DFF45) gene silencing on breast cancer cell viability, cell cycle arrest, and apoptosis in the presence and absence of doxorubicin.We designed three siRNAs, which target different regions of the DFF45 mRNA. Gene silencing was confirmed by real time RT-PCR and Western blot analyses. The impact of DFF45 siRNA, doxorubicin, and their combination on the viability, cell cycle and apoptosis of T-47D and MDA-MB-231 breast cancer cells were determined by MTT, PI staining, annexin V binding, caspase-3 activity, DNA laddering, and chromatin condensation assays.Based on flow cytometric analyses, we found that silencing of DFF45 alone had little effect on apoptosis, especially in T-47D cells. However, when used in combination with doxorubicin (0.33 μM) a significant increase (P 0.05) in apoptosis was observed in T-47D and MDA-MB-231 cells, i.e., ~2.5- and 3-fold, respectively. Caspase-3 activity, chromatin condensation, as well as DNA laddering supported increased apoptosis in the combinatorial treatment. Cell cycle arrest in both cell lines occurred at lower levels after siRNA + doxorubicin treatment compared to doxorubicin only.Our data indicate that DFF45 gene silencing, when applied in combination with doxorubicin, may offer a novel therapeutic strategy for the treatment of breast cancer.

The inhibitory effects of acetazolamide on the growth and proliferation of epithelial breast cancer cells (T-47D) were investigated. Analysis of morphological changes indicated little apoptosis in T-47D cells incubated with acetazolamide, according to data from flow cytometry, DNA laddering, and expression of AIF. However, an increase in caspase-3 activity was detected in cells. This was concomitant with an increase in DFF45/DFF40 ratio leading to inhibition of caspase-3 activity, DNA fragmentation and progression of apoptosis. Flow cytometry also confirmed that acetazolamide had no significant effect on cell cycle progression. These results are consistent with lack of change in the expression of cell cycle regulatory proteins p21, p27, cdc2 and cyclinD1. Increased expression of ATG5, p53 and DRAM, along with an increase in BCLN1/Bcl-2 ratio, indicated that acetazolamide inhibited the proliferation of T-47D cells by inducing autophagy. Increased expression of PTEN, along with decreased expression of Akt1, also showed that acetazolamide treatment resulted in death inducing autophagy. Collectively the results indicate that autophagy is an adequate mechanism mediating the anti-cancer effects of acetazolamide in T-47D cells through engagement of p53/DRAM pathway and attenuation of Akt survival signalling.

Nanoparticles of ultra low molecular weight chitosan (ULMWCS)/hyaluronic acid (HA) were prepared by ion gelation. Three independent variables, namely, ratio of concentration of ULMWCS to HA (CS/HA), pH of solution and stirring time were studied to identify their effects on size, polydispersity and zeta potential of prepared nanoparticles using a Box-Behnken design. Results showed that pH and CS/HA have a direct effect on size, while increase of stirring time decreases the size of nanoparticles. Additionally, it was shown that all the independent parameters have direct effects on zeta potential. Also, the minimum polydispersity index was observed at lowest values of CS/HA. The model also predicted that the optimum values are 4.15, 4.14 and 180 (min) for the CS/HA, solution pH and stirring time, respectively. The obtained preparation had a size of 200 nm, polydispersity index of 0.37, and zeta potential of 13.0 mV.

We have evaluated the effects of acetonitrile on the structure and function of bovine carbonic anhydrase II. The potential structural and functional changes in carbonic anhydrase in the presence of different acetonitrile/buffer ratios (0%, 17.5% and 47.5% v/v) were determined using a variety of methods. These included simple spectrophotometric methods to record enzyme velocity, fluorescence measurements and calculation of accessible surface area (ASA) to identify possible alterations in tertiary structure of the protein, CD measurements to search for secondary structure conversions, and thermal scanning to determine structural stability of the protein in different media. The Far-UV CD studies indicated that carbonic anhydrase, for the most part, retains its secondary structure in the presence of acetonitrile. Fluorescence measurements using iodide ion and ANS along with ASA calculations revealed that in the presence of acetonitrile some degree of conformational change occurs in the carbonic anhydrase structure. In addition to the hydrophobic pockets, two additional tryptophanyl residues become exposed to the solvent, thereby increasing the surface hydrophobicity of the protein. These alterations dramatically reduce the catalytic activity, thermal stability, and aggregation velocity of the enzyme. Thus, our results support a molten globule-like structure of carbonic anhydrase in the presence of acetonitrile.

The kinetic parameters of adenosine deaminase such as Km and Ki were determined in the absence and presence of adenine derivatives (R1-R24) in sodium phosphate buffer (50 mM; pH 7.5) solution at 27 degrees C. These kinetic parameters were used for QSAR analysis. As such, we found some theoretical descriptors to which the binding affinity of adenosine deaminase (ADA) towards several adenine nucleosides as inhibitors is correlated. QSAR analysis has revealed that binding affinity of the adenine nucleosides upon interaction with ADA depends on the molecular volume, dipole moment of the molecule, electric charge around the N1 atom, and the highest of positive charge for the related molecules.

The interaction between n-octyl-beta-D-glucopyranoside (octyl glucoside) and bovine liver glutamate dehydrogenase (GDH) was studied using techniques including equilibrium dialysis, UV-spectrophotometry, circular dichroism (CD), fluorescence energy transfer and extrinsic spectrofluorometry in 50 mM sodium phosphate buffer solution (pH 7.6). The equilibrium dialysis experiment showed a higher binding of octyl glucoside to GDH that induces up to 80% enzyme inhibition in 20 mM octyl glucoside solution. The CD study indicated that GDH retains its secondary structure in the presence of octyl glucoside, but loses a degree of its tertiary structure by acquiring a more extended tertiary structure. Measurement of the binding of a hydrophobic fluorescent probe, 1-anilino-naphthalene-8-sulfonate (ANS), to GDH revealed that the binding of ANS to GDH is increased in the presence of octyl glucoside, a finding that may be interpreted in terms of the increment of surface hydrophobic patch(es) of GDH because of its binding to octyl glucoside. Fluorescence energy transfer studies also showed more binding of the reduced coenzyme (NADH) to GDH and the Lineweaver-Burk plots (with respect to NADH) indicate the existence of substrate inhibition in the presence of octyl glucoside. These observations are aimed at explaining the formation of the molten globule-like structure of GDH, which is induced by a non-ionic detergent such as octyl glucoside.

Widespread use of mobile phones has increased the human exposure to electromagnetic fields (EMFs). It is required to investigate the effect of EMFs on the biological systems. In this paper the effect of mobile phone RF (910MHz and 940 MHz) on structure and function of HbA was investigated. Oxygen affinity was measured by sodium dithionite with UV-vis spectrophotometer. Structural changes were studied by circular dichroism and fluorescence spectroscopy. The results indicated that mobile phone EMFs altered oxygen affinity and tertiary structure of HbA. Furthermore, the decrease of oxygen affinity of HbA corresponded to the EMFs intensity and time of exposure.

Kinetic and thermodynamic studies have been made on the effect of acetaminophen on the activity and structure of adenosine deaminase in 50 mM sodium phosphate buffer pH 7.5, at two temperatures of 27 and 37 degrees C using UV spectrophotometry, circular dichroism (CD) and fluorescence spectroscopy. Acetaminophen acts as a competitive inhibitor at 27 degrees C (Ki = 126 microM) and an uncompetitive inhibitor at 37 degrees C (Ki = 214 microM). Circular dichroism studies do not show any considerable effect on the secondary structure of adenosine deaminase by increasing the temperature from 27 to 37 degrees C. However, the secondary structure of the protein becomes more compact at 37 degrees C in the presence of acetaminophen. Fluorescence spectroscopy studies show considerable change in the tertiary structure of the protein by increasing the temperature from 27 to 37 degrees C. Also, the fluorescence spectrum of the protein incubated with different concentrations of acetaminophen show different inhibition behaviors by the effector at the two temperatures.