Cytotoxicity tests of compounds by MTT assay.

<p>Cells were treated with various compounds for 4h, and grown for another 20h. (A)IC50 of HeLa and HepG2 cells (B)HEK-293T cells Data represent the mean± standard deviation (n = 6).</p

A Novel Isoquinoline Derivative Anticancer Agent and Its Targeted Delivery to Tumor Cells Using Transferrin-Conjugated Liposomes

<div><p>We have screened 11 isoquinoline derivatives and α-methylene-γ-butyrolactones using the 3-(4,5-dimethylthi-azol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) cytotoxicity assay in HeLa and HEK-293T cells. Compound 2 was identified as potential anticancer agent. To further improve its therapeutic potential, this agent was incorporated into transferrin (Tf)-conjugated liposomes (LPs) for targeted delivery to tumor cells. We have demonstrated Tf-LP-Compound 2 have superior antitumor activity compared to non-targeted controls and the free drug. These data show Tf-LP-Compound 2 to be a promising agent that warrants further evaluation.</p></div

Fluorescence image of HeLa and HepG2 cells stained with JC-1.

<p>(A)Photograph showing JC-1 red, JC-1 green and merge image. (B)Numerical data were expressed in terms of the ratio of JC-1 aggregates to JC-1 monomers. Data are representative of three independent experiments and expressed as means ±SD, *p, 0.05, **p, 0.01 and ***p, 0.001 as compared with the control.</p

Cellular uptake of LP-Compound 2 and Tf-LP-Compound 2.

<p>Flow cytometry after incubation of LP-Compound 2, Tf-LP-Compound 2 and Tf-LP-Compound 2 with holo Tf. Data represent the mean± standard deviation (n = 3)(**p<0.01 vs LP-Compound 2).</p

FE-SEM images of Tf-LP-Compound 2.

<p>FE-SEM images of Tf-LP-Compound 2.</p

Chemical structure of test compounds.

<p>Chemical structure of test compounds.</p

Size, ζ-potential and Encapsulation efficiency of LPs before and after coupling of Tf at pH 7.4.

<p>Data are shown as means and standard deviation (n = 3).</p

Intracellular localization of LP-Compound 2 and Tf-LP-Compound 2.

<p>Confocal microscopy analysis after 4 h of incubation of LP-Compound 2 and Tf-LP-Compound 2 at 37°C with HeLa cells (A) and HepG2 cells (B). NBD-DOPE fluorescence is shown in green, sulforhodamine B is shown in red, and DAPI nuclear stain is shown in blue.</p

In vitro release of LP-Compound 2, Tf-LP-Compound 2 and free Compound 2.

<p>Drug release during 24 h incubation in PBS (0.5%v/v Tween 80) at 37 degree C, mean SD values (n = 3) are presented.</p

Table_1_Comparative transcriptomic and lipidomic analyses indicate that cold stress enhanced the production of the long C18–C22 polyunsaturated fatty acids in Aurantiochytrium sp..docx

Aurantiochytrium sp. belonging to Thraustochytrids are known for their capacity to produce long-chain polyunsaturated fatty acids (PUFAs). However, effects of cold stress accompanied with staged-temperature control on the fatty acid metabolism in Aurantiochytrium sp. were rarely studied. In this study, cold stress (15°C, 5°C) was applied for Aurantiochytrium sp., with the physiological responses (morphology, growth, fatty acid profiling) and gene expression related FA synthesis, lipid metabolism, and regulatory processes was observed. Results showed that there is a significant change for the lipid types under 5°C (251 species) and 15°C (97 species) treatment. The 5°C treatment was benefit for the C18–C22 PUFAs with the yield of docosahexaenoic acid (DHA) increased to 1.25 times. After incubation at 15°C, the accumulation of eicosadienoic acid (EA) (20:2) was increased to 2.00-fold. Based on transcriptomic and qPCR analysis, an increase in genes involved in fatty acid synthase (FAS) and polyketide synthase (PKS) pathways was observed under low-temperature treatment. With upregulation of 3-ketoacyl-CoA synthase (2.44-fold), ketoreductase (2.50-fold), and dTDP-glucose 4,6-Dehydratase (rfbB) (2.31-fold) involved in PKS pathway, the accumulation of DHA was enhanced under 5°C. While, FAS and fatty elongase 3 (ELO) involved in the FAS pathway were upregulated (1.55-fold and 2.45-fold, respectively) to accumulate PUFAs at 15°C. Additionally, glycerol-3-phosphate acyltransferase (GPAT), lysophospholipid acyltransferase (LPAT), phosphatidic acid phosphatase (PAP), phosphatidylserine synthase (PSS), and phosphatidylserine decarboxylase (PSD) involved in glycerophospholipid biosynthesis were upregulated at 5°C increasing the accumulation of phosphatidic acid (PA), phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylglycerol (PG), and phosphatidylinositol (PI). However, glycolysis and the TCA cycle were inhibited under 5°C. This study provides a contribution to the application of two-staged temperature control in the Aurantiochytrium sp. fermentation for producing cold stress-enhancing PUFAs, in order to better understand the function of the key genes for future genetic engineering.</p