The interrelationship between the accumulation of lipids, protein and the level of acyl carrier protein during the development of Brassica napus L. pollen

Lipid accumulation during pollen and tapetal development was studied using cryostat sections of unfixed anthers from Brassica napus (rapeseed). Diamidino-2-henylindole (DAPI), a DNA fluorochrome, was used to stain the pollen nuclei in order to identify ten stages of pollen development in Brassica. Storage lipids (i.e. triacylglycerides) were stained using the fluorochrome Nile red. Pollen coat lipids are formed in tapetal plastids between the mid-vacuolate and early maturation pollen stages. The pollen coat components, including lipids and a proportion of the proteins, are derived from the remnants of the tapetum, after its rupture, during the second pollen mitosis. Quantitative microfluorometric analyses demonstrated four phases of lipid body accumulation or depletion in the developing pollen cytoplasm. The majority of storage lipids found in the cytoplasm of the mature pollen grain accumulated during the late vacuolate and early maturation stages when the pollen is bicellular. The level of acyl carrier protein, a protein integrally involved in lipid synthesis, was also found to be maximal in the developing pollen during the bicellular pollen stages of development. This coincided with the most active period of lipid accumulation. These data could indicate that the lipids of the pollen are synthesized in situ, by metabolic processes regulated by expression of genes in the haploid genome.

Doxorubicin conjugated to immunomodulatory anticancer lactoferrin displays improved cytotoxicity overcoming prostate cancer chemo resistance and Inhibits tumour development in TRAMP mice

Advanced, metastatic, castration resistant and chemo-resistant prostate cancer has triggered change in the drug development landscape against prostate cancer. Bovine lactoferrin (bLf) is currently attracting attention in clinics for its anti-cancer properties and proven safety profile. bLf internalises into cancer cells via receptor mediated endocytosis, boosts immunity and complements chemotherapy. We employed bLf as an excellent functional carrier protein for delivering doxorubicin (Dox) into DU145 cells, CD44+/EpCAM+ double positive enriched DU145 3D prostaspheres and drug resistant ADR1000-DU145 cells, thus circumventing Dox efflux, to overcome chemo-resistance. Successful bLf-Dox conjugation with iron free or iron saturated bLf forms did not affect the integrity and functionality of bLf and Dox. bLf-Dox internalised into DU145 cells within 6 h, enhanced nuclear Dox retention up to 24 h, and proved significantly effective (p < 0.001) in reducing LC50 value of Dox from 5.3 μM to 1.3 μM (4 fold). Orally fed iron saturated bLf-Dox inhibited tumour development, prolonged survival, reduced Dox induced general toxicity, cardiotoxicity, neurotoxicity in TRAMP mice and upregulated serum levels of anti-cancer molecules TNF-α, IFN-γ, CCL4 and CCL17. The study identifies promising potential of a novel and safer bLf-Dox conjugate containing a conventional cytotoxic drug along with bLf protein to target drug resistance.

Thermodynamics of lipophilic drug binding to intestinal fatty acid binding protein and permeation across membranes

Intestinal fatty acid binding protein (I-FABP) is present at high levels in the absorptive cells of the intestine (enterocytes), where it plays a role in the intracellular solubilization of fatty acids (FA). However, I-FABP has also been shown to bind to a range of non-FA ligands, including some lipophilic drug molecules. Thus, in addition to its central role in FA trafficking, I-FABP potentially serves as an important intracellular carrier of lipophilic drugs. In this study we provide a detailed thermodynamic analysis of the binding and stability properties of I-FABP in complex with a series of fibrate and fenamate drugs to provide an insight into the forces driving drug binding to I-FABP. Drug binding and selectivity for I-FABP are driven by the interplay of protein−ligand interactions and solvent processes. The Gibbs free energies (ΔG°) determined from dissociation constants at 25 °C ranged from −6.2 to −10 kcal/mol. The reaction energetics indicate that drug binding to I-FABP is an enthalpy−entropy driven process. The relationship between I-FABP stability and drug binding affinity was examined by pulse proteolysis. There is a strong coupling between drug binding and I-FABP stability. The effect of an I-FABP protein sink on the kinetics and thermodynamics of tolfenamic acid permeation across an artificial phospholipid membrane were investigated. I-FABP significantly decreased the energy barrier for desorption of tolfenamic acid from the membrane into the acceptor compartment. Taken together, these data suggest that the formation of stable drug−I-FABP complexes is thermodynamically viable under conditions simulating the reactant concentrations likely observed in vivo and maybe a significant biochemical process that serves as a driving force for passive intestinal absorption of lipophilic drugs.