Synthesis,Cytotoxicity and Antimicrobial Evaluation of New Coumarin‐Tagged β‐Lactam Triazole Hybrid

A series of coumarin‐tagged β‐lactam triazole hybrids ( 10a – 10o ) were synthesized and tested for their cytotoxic activity against MDA‐MB‐231 (triple negative breast cancer), MCF‐7 (estrogen receptor positive breast cancer (ER+)) and A549 (human lung carcinoma) cancer cell lines including one normal cell line, HEK‐293 (human embryonic kidney). Two compounds 10b and 10d exhibited substantial cytotoxic effect against MCF‐7 cancer cell lines with IC₅₀ values of 53.55 and 58.62 μm , respectively. More importantly, compounds 10b and 10d were non‐cytotoxic against HEK‐293 cell lines. Structure–activity relationship (SAR) studies suggested that the nitro and chloro group at the C‐3 position of phenyl ring are favorable for anticancer activity, particularly against MCF‐7 cell lines. Furthermore, antimicrobial evaluation of these compounds revealed modest inhibition of examined pathogenic strains with compounds 10c and 10i being the most promising antimicrobial agents against Pseudomonas aeruginosa and Candida albicans, respectively.     

The Intracellular Domain of Cadherin-11 is not Required for the Induction of Cell Aggregation,Adhesion or Gap-Junction Formation

The cadherin family of cell adhesion molecules demonstrates calcium-dependent hemophilic binding, leading to cellular recognition and adhesion. The adhesion mediated by the classical type 1 cadherins is strengthened through catenin-mediated coupling of the cytoplasmic domain to the cytoskeleton. This cytoskeletal interaction may not be essential for the adhesion promoted by all cadherins, several of which lack cytosolic catenin-binding sequences. Cadherin-11, a classical cadherin, possesses a cytoplasmic domain that interacts with catenins, but may also occur as a variant form expressing a truncated cytoplasmic domain. To study the role of the cytoplasmic sequence in cadherin-11 mediated adhesion we have constructed and expressed a truncated cadherin-11 protein lacking the cytoplasmic domain and unable to bind β-catenin. Expression of the truncated cadherin-11 in MDA-MB-435S human mammary carcinoma cells reduced their motility and promoted calcium-dependent cell aggregation, frequent cell contacts, and functional gap-junctions. We conclude that the intracellular catenin-binding domain of cadherin-11, and by inference cytoskeletal interaction, is not required for the initiation and formation of cell adhesion.     

Docetaxel-loaded nanostructured lipid carriers functionalized with trastuzumab (Herceptin) for HER2-positive breast cancer cells

Abstract

The aim of the present study was to prepare Herceptin targeted nanostructured lipid carriers (NLCs) of docetaxel (DTX). Herceptin was conjugated by chemical and physical methods to NLCs prepared by solvent extraction technique followed by probe sonication. Different types of fatty amines were used in construction of NLCs. The NLCs were characterized for their antibody coupling efficiency, particle size, zeta potential, polydispersity index, drug entrapment efficiency and drug release profiles. The toxicity of NLCs on MDA-MB-468 (HER2 negative receptor) and BT-474 (HER2 positive) breast cancer cell lines was evaluated by MTT assay. Also their cellular uptake was studied by flow-cytometry and fluorescent microscopy. The results showed the NLCs containing stearyl amine had the lowest particle size, the highest zeta potential and antibody coupling efficiency values. Herceptin binding to NLCs led to reduction in zeta potential and drug entrapment efficiency while, particle size increased. The NLCs containing spermine(SP) released DTX slower than other fatty amines. Non-conjugated nanoparticles containing DTX had more toxicity than the free DTX on both cell lines. Herceptin targeted NLCs caused more mortality on BT-474 cells than MDA-MB-468 cells. Flow-cytometry studies revealed enhanced cellular uptake of nanoparticles chemically conjugated by Herceptin on the BT-474 cells. DTX loaded in chemically conjugated NLCs to Herceptin showed more cytotoxic effects than the physically coated nanoparticles. The Herceptin conjugated NLCs seem promising in oriented delivery of DTX to HER2 positive breast cancer cells.     

Role of hypoxia and EGF on expression, activity, localization and phosphorylation of carbonic anhydrase IX in MDA-MB-231 breast cancer cells

Carbonic anhydrase IX (CAIX) is a zinc metalloenzyme that catalyzes the reversible hydration of CO₂. CAIX is overexpressed in many types of cancer, including breast cancer, but is most frequently absent in corresponding normal tissues. CAIX expression is strongly induced by hypoxia and is significantly associated with tumor grade and poor survival. Herein, we show that hypoxia induces a significant increase in CAIX protein in MDA-MB-231 breast cancer cells. Using a unique mass spectrophotometric assay, we demonstrate that CAIX activity in plasma membranes isolated from MDA-MB-231 is correlated with CAIX content. We also show that CAIX exists predominantly as a dimeric, high-mannose N-linked glycoprotein. While there is some evidence that the dimeric form resides specifically in lipid rafts, our data do not support this hypothesis. EGF, alone, did not affect the distribution of CAIX into lipid rafts. However, acute EGF treatment in the context of hypoxia increased the amount of CAIX in lipid rafts by about 5-fold. EGF did not stimulate tyrosine phosphorylation of CAIX, although EGFR and down-stream signaling pathways were activated by EGF. Interestingly, hypoxia activated Akt independent of EGF action. Together, these data demonstrate that the active form of CAIX in the MDA-MB-231 breast cancer cell line is dimeric but that neither lipid raft localization nor phosphorylation are likely required for its dimerization or activity.     

RNA干扰对MDA-MB-231细胞中CaSR基因表达的影响

目的:运用RNA干扰技术,观察siRNA表达载体在乳腺癌细胞MDA-MB-231中对CaSR基因表达的影响。方法:构建靶向CaSR基因的RNA干扰表达载体,用脂质体转染乳腺癌细胞株MDA-MB-231,运用Real-Time荧光定量PCR,Westernblot技术分别从mRNA以及蛋白表达水平检测CaSR基因表达的变化。结果:所构建的质粒载体成功的在MDA-MB-231细胞中抑制了CaSR mRNA及其蛋白的表达。与对照组相比,psiRNA-CaSR载体对CaSR mRNA的抑制率达到65%,对CaSR蛋白抑制率约为70%。结论:实验证明所设计的shRNA片段可以有效地抑制CaSR基因的表达,为下一步研究工作奠定了基础。     

Optimizing coastal and marine spatial planning through the use of high-resolution benthic sensitivity models

Effective management of coastal and marine resources requires knowledge of how community sensitivity varies spatially. With this in mind, we developed a benthic sensitivity index (SI), based on the distribution and abundance of five ecological groups that can be used to assess community tolerance to organic enrichment and other disturbances. The index, projected as a high-resolution map, ranks communities from those dominated by sensitive and ecologically important species (i.e. low SI values) to those composed mainly of tolerant and/or opportunistic species (i.e., high SI values). Applying our model to a multiple-use case study in southeast Brazil, we were able to show considerable variability in the sensitivity of communities across the study area that was relatively stable over time. This allowed us to evaluate the possible direct (i.e., spatially overlapping) and indirect effects (i.e., cumulative changes to the physical environment) of a range of activities on sensitive and ecologically diverse benthic communities. Our approach and the resulting high-resolution maps hold promise for a range of spatial planning applications, including the development of coastal infrastructure, assessments of the representativeness of marine protected areas and other activities such as the selection of appropriate locations for dredge spoil dumping. Overall, we present a novel and transparent way of extrapolating limited survey data to provide spatial and temporal information on the sensitivity of benthic communities in multiple-use coastal and marine areas.     

Facile synthesis of novel substituted aryl-thiazole (SAT) analogs via one-pot multi-component reaction as potent cytotoxic agents against cancer cell lines

In this study, twenty-five (25) substituted aryl thiazoles (SAT) 1–25 were synthesized, and their in vitro cytotoxicity was evaluated against four cancer cell lines, MCF-7 (ER^+ve breast), MDA-MB-231 (ER^−ve breast), HCT116 (colorectal) and HeLa (cervical). The activity was compared with the standard anticancer drug doxorubicin (IC₅₀ = 1.56 ± 0.05 μM). Among them, compounds 1, 4–8, and 19 were found to be toxic to all four cancer cell lines (IC₅₀ values 5.37 ± 0.56–46.72 ± 1.80 μM). Compound 20 was selectively active against MCF7 breast cancer cells with IC₅₀ of 40.21 ± 4.15 μM, whereas compound 19 was active against MCF7 and HeLa cells with IC₅₀ of 46.72 ± 1.8, and 19.86 ± 0.11 μM, respectively. These results suggest that substituted aryl thiazoles 1 and 4 deserve to be further investigated in vivo as anticancer leads.     

Assessment of cytosolic free calcium changes during ceramide-induced cell death in MDA-MB-231 breast cancer cells expressing the calcium sensor GCaMP6m

Alterations in Ca²⁺ signaling can regulate key cancer hallmarks such as proliferation, invasiveness and resistance to cell death. Changes in the regulation of intracellular Ca²⁺ and specific components of Ca²⁺ influx are a feature of several cancers and/or cancer subtypes, including the basal-like breast cancer subtype, which has a poor prognosis. The development of genetically encoded calcium indicators, such as GCaMP6, represents an opportunity to measure changes in intracellular free Ca²⁺ during processes relevant to breast cancer progression that occur over long periods (e.g. hours), such as cell death. This study describes the development of a MDA-MB-231 breast cancer cell line stably expressing GCaMP6m. The cell line retained the key features of this aggressive basal-like breast cancer cell line. Using this model, we defined alterations in relative cytosolic free Ca²⁺ ([Ca²⁺]_CYT) when the cells were treated with C2-ceramide. Cell death was measured simultaneously via assessment of propidium iodide permeability. Treatment with ceramide produced delayed and heterogeneous sustained increases in [Ca²⁺]_CYT. Where cell death occurred, [Ca²⁺]_CYT increases preceded cell death. The sustained increases in [Ca²⁺]_CYT were not related to the rapid morphological changes induced by ceramide. Silencing of the plasma membrane Ca²⁺ ATPase isoform 1 (PMCA1) was associated with an augmentation in ceramide-induced increases in [Ca²⁺]_CYT and also cell death. This work demonstrates the utility of GCaMP6 Ca²⁺ indicators for investigating [Ca²⁺]_CYT changes in breast cancer cells during events relevant to tumor progression, which occur over hours rather than minutes.