Synergistic anticancer effects of co-delivery of linc-RoR siRNA and curcumin using polyamidoamine dendrimers against breast cancer.

Breast cancer resistance to chemotherapy necessitates novel combination therapeutic approaches. Linc-RoR is a long intergenic noncoding RNA that regulates stem cell differentiation and promotes metastasis and invasion in breast cancer. Herein, we report a dual delivery system employing polyamidoamine dendrimers to co-administer the natural compound curcumin and linc-RoR siRNA for breast cancer treatment. Polyamidoamine dendrimers efficiently encapsulated curcumin and formed complexes with linc-RoR siRNA at an optimal N/P ratio. In MCF-7 breast cancer cells, the dendriplexes were effectively internalized and the combination treatment synergistically enhanced cytotoxicity, arresting the cell cycle at the G1 phase and inducing apoptosis. Linc-RoR gene expression was also significantly downregulated. Individual treatments showed lower efficacy, indicating synergism between components. Mechanistic studies are warranted to define the molecular underpinnings of this synergistic interaction. Our findings suggest dual delivery of linc-RoR siRNA and curcumin via dendrimers merits further exploration as a personalized therapeutic approach for overcoming breast cancer resistance.

A simple and efficient method for cytoplasmic production of human enterokinase light chain in E. coli.

Human enterokinase light chain (hEKL) cDNA sequence was designed with the help of codon optimization towards Escherichia coli codon preference and ribosome binding site design and artificially synthesized with a thioredoxin fusion tag at the N-terminal and a five his-tag peptide at the C-terminal. The synthetic hEKL gene was cloned into the pET-15 expression vector and transferred into the three different expression strains of E. coli BL21(DE3), NiCo21, and SHuffle T7 Express. Different growth and induction conditions were studied using a statistical response surface methodology (RSM). Recombinant hEKL protein was expressed at high levels in soluble form with 0.71 mM IPTG after 4 h of induction at 25 °C. Autocatalytic process cleaved TRX tag with enterokinase recognition site by the impure hEKL and yielded the mature enzyme. The target protein was then purified to homogeneity (> 95%) by affinity chromatography. The activity of hEKL was comparable to the commercial enzyme. From 1 L culture, 80 mg pure active hEKL was obtained with the specific activity of 6.25 × 102 U/mg. Three main parameters that help us to produce the enzyme in the folded and active form are the type of strain, SHuffle T7 strain, TRX and histidine fusion tags, and growth conditions including the increase of OD of induction and IPTG concentration and the decrease of induction temperature.

Cationic vesicles for efficient shRNA transfection in the MCF-7 breast cancer cell line.

INTRODUCTION: Novel and safe delivery solutions for RNAi therapeutics are essential to obtain the full potential of cancer gene therapy. METHODS: In this study, cationic vesicular nanocarrier was applied for delivering lnc urothelial carcinoma-associated 1 (lnc UCA1) shRNA expression vector to MCF-7 cells. The physicochemical characteristics, cytotoxicity, and transfection efficiency of cationic vesicles prepared from various molar ratios of amphiphilic surfactant Tween 80 (T), squalene (S), cationic charge lipid didodecyldimethylammonium bromide, and polyethylenimine were investigated. The particle sizes of the vesicles in the nanosize range were determined by dynamic light scattering and transmission electron microscopy. RESULTS: Gel protection assay with agarose gel electrophoresis showed cationic vesicles can protect the shRNA plasmid from DNase 1 enzyme. 3-(4,5-Dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H tetrazolium, inner salt result showed no significant cytotoxicity was caused in MCF-7 cancer cell line by (T:S):polyethylenimine cationic vesicles. 3-(4,5-Dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H tetrazolium, inner salt assay, fluorescence microscope images, and flow cytometry analyses confirmed that (T:S)1,040 µM with 4.3 µg/mL of PEI vesicles provided effective transfection without significant cytotoxicity. Furthermore, we found efficient UCA1 shRNA transfection and significant (P<0.05) cell cycle arrest and apoptosis in MCF-7 cancer cells. CONCLUSION: The novel nonviral vesicular nanocarrier, (T:S)1,040 µM with 4.3 µg/mL of PEI, might be safe and efficient for cancer gene therapy and can be used in further in vitro and in vivo studies.

Wheat stem reserves and salinity tolerance: molecular dissection of fructan biosynthesis and remobilization to grains.

MAIN CONCLUSION: Fructan accumulation and remobilization to grains under salinity can decrease dependency of the wheat tolerant cultivar on current photosynthesis and protect it from severe yield loss under salt stress. Tolerance of plants to abiotic stresses can be enhanced by accumulation of soluble sugars, such as fructan. The current research sheds light on the role of stem fructan remobilization on yield of bread wheat under salt stress conditions. Fructan accumulation and remobilization as well as relative expression of the major genes of fructan metabolism were investigated in the penultimate internodes of 'Bam' as the salt-tolerant and 'Ghods' as the salt-sensitive wheat cultivars under salt-stressed and controlled conditions and their correlations were analyzed. More fructan production and higher efficiency of fructan remobilization was detected in Bam cultivar under salinity. Up-regulation of sucrose: sucrose 1-fructosyltransferase (1-SST) and sucrose: fructan 6-fructosyltransferase (6-SFT) (fructan biosynthesis genes) at anthesis and up-regulation of fructan exohydrolase (1-FEH) and vacuolar invertase (IVR) genes (contributed to fructan metabolism) during grain filling stage and higher expression of sucrose transporter gene (SUT1) in Bam was in accordance with its induced fructan accumulation and remobilization under salt stress. A significant correlation was observed between weight density, WSCs and gene expression changes under salt stress. Based on the these results, increased fructan production and induced stem reserves remobilization under salinity can decrease dependency of the wheat tolerant cultivar on current photosynthesis and protect it from severe yield loss under salt stress conditions.