Golgi complex is brefeldin A resistant in multidrug resistant cells.

The multidrug resistance (MDR) is one of the main reasons for chemotherapeutic failures in cancer patients. The overexpression of mdr1 gene product, P-glycoprotein (Pgp), leads to the appearance of resistant tumor cells. In the previous paper (Erokhina, 1997) we have demonstrated that the first stages of Pgp-mediated MDR are accompanied by the reorganization of cytoskeleton elements and the vacuolar system. These data were true for two independently isolated sublines of Syrian hamster embryo fibroblasts transformed by Raus sarcoma virus. In this study, we continued the investigation of the properties of the vacuolar system in Pgp-expressing cells. Brefeldin A (BFA), which is not a Pgp substrate, affects different elements of the vacuolar system and blocks vesicular transport. Our data demonstrate that BFA has different effects on parental and resistant cells. In parental cells, the Golgi apparatus and vesicular transport are sensitive to BFA, while in resistant sublines, BFA affects the vesicular transport but not the Golgi apparatus structure. We discuss the existence of similar and different BFA targets in parental and resistant cells and their role in the evolution of multidrug resistance mechanisms.     

Mitochondrial Aconitase is a Transglutaminase 2 Substrate: Transglutamination is a Probable Mechanism Contributing to High-Molecular-Weight Aggregates of Aconitase and Loss of Aconitase Activity in Huntington Disease Brain

Transglutaminase activity was found to be present in highly purified non-synaptosomal rat brain mitochondria. A 78-kDa protein in these organelles was shown to be a transglutaminase 2 substrate, and incubation of a non-synaptosomal mitochondrial lysate with transglutaminase 2 yielded high-M_r proteins. The 78-kDa protein was identified as mitochondrial aconitase by MALDI-TOF analysis. Aconitase activity was decreased in a dose-dependent manner when non-synaptosomal rat brain mitochondria were incubated with transglutaminase 2. Transglutaminase activity is increased about 2-fold in the mitochondrial fraction of HD caudate. Moreover, Western blotting of the mitochondrial fraction revealed that most of the mitochondrial aconitase in HD caudate is present as high-M_r aggregates. Aconitase activity was previously shown to be decreased in Huntington disease (HD) caudate (a region severely damaged by the disease). The present findings suggest that an increase of transglutaminase activity in HD caudate may contribute to mitochondrial dysfunction by incorporating aconitase into inactive polymers.     

Cellular mechanisms of multidrug resistance of tumor cells

Multidrug resistance (MDR) is the protection of a tumor cell population against numerous drugs differing in chemical structure and mechanisms of influence on the cells. MDR is one of the major causes of failures of chemotherapy of human malignancies. Recent studies show that the molecular mechanisms of MDR are numerous. Cellular drug resistance is mediated by different mechanisms operating at different steps of the cytotoxic action of the drug from a decrease of drug accumulation in the cell to the abrogation of apoptosis induced by the chemical substance. Often several different mechanisms are switched on in the cells, but usually one major mechanism is operating. The most investigated mechanisms with known clinical significance are: a) activation of transmembrane proteins effluxing different chemical substances from the cells (P-glycoprotein is the most known efflux pump); b) activation of the enzymes of the glutathione detoxification system; c) alterations of the genes and the proteins involved into the control of apoptosis (especially p53 and Bcl-2).     

Influence of human tumor suppressor PTEN on sensitivity of malignant cells to anticancer drugs

The influence of the human tumor suppressor PTEN on sensitivity of tumor cells to cytostatic drugs was studied. Rat ras-transformed (N-ras ^Asp12 ) fibroblasts were stably transfected with a full-size PTEN gene. Transfected clone was characterized by an enhanced expression of PTEN and a more normal phenotype in comparison with the parental cells. The effect of transient transfection with PTEN on the sensitivity of several malignant cell lines to the cytostatic drugs colchicine and adriablastine was studied. These drugs differ from each other in action mechanisms and intracellular targets. The tumor cell lines tested in this study included parental cell lines and stable sublines possessing drug resistance due to overexpression of P-glycoprotein. In all cell lines, introduction of exogenous PTEN caused a decrease in proliferation rates. This indicated that transgene was active. The chemosensitivity of some drug-resistant sublines was changed after PTEN transfection, but the drug sensitivity of parental cell lines remained unaffected. The effect of PTEN overexpression on chemosensitivity of malignant cells to cytostatic drugs was found to depend both on their mechanisms of action and on the origin of transfected cells. Our data suggest that PTEN is involved into the molecular mechanisms of drug resistance in cells studied.     

Non-canonical functions of the cellular transporter P-glycoprotein

P-glycoprotein/ABCB1 (Pgp) is a well known protein of cell defense system. It is localized in cell membrane and pumps different drugs out of various cells using ATP energy. Its overexpression is associated with the development of multidrug resistance (MDR) in cancer cells. The data showing that Pgp also has other functions appeared recently, and this review surveys these data. In particular, (1) Pgp can protect cells from apoptosis; it suppresses the expression of endogenous protein TRAIL and decreases the activity of caspases 8 and 3; (2) Pgp is able to act as an outwardly directed flippase; (3) Pgp participates in a proper development of the innate immune response to intracellular pathogens and in the development of inflammation; (4) functionally active Pgp can be transferred from drug-resistant to drug-sensitive cells by microvesicles (MV). This is a new way of the Pgp-mediated MDR emergence in populations of tumor cells. Thus, Pgp functions as a regulator of some cellular processes. Molecular mechanisms of the Pgp influence on tumor cell viability are related not only with the drug efflux but also with some other functions.     

Induction of P-glycoprotein functional activity and multidrug resistance by a soluble factor(s) produced by some mammalian cells.

In the previous study we have found that Djungarian hamster fibroblasts with high levels of multidrug resistance (MDR) (colchicine-resistance index RI of 1000 to 42000) produce soluble factor(s) communicating MDR to the drug-sensitive cells of the same species by elevating the functional activity of P-glycoprotein (Pgp). Here we have shown that these cells can influence human tumor cells in the same fashion. Rat hepatoma McA RH7777 cells and their colchicine-resistant derivatives are shown to produce a factor with similar effects (induction of MDR and Pgp functional activity in the drug-sensitive cells). These effects seem to depend on the drug resistance level of the donor cells. Our results show that induction of the Pgp-mediated MDR is not species-specific and the tumor cells with intrinsic MDR (arising from the tissue with a high level of Pgp expression) can produce a factor(s) communicating this type of drug resistance to the sensitive cells.     

Participation of mTOR in the regulation of multidrug resistance of tumor cells

Molecular mechanisms of the influence of PI3K/Akt/PTEN/mTOR-signaling pathway on survival of tumor cells treated with cytotoxic drugs was studied using rapamycin (Rapa), mTOR specific inhibitor, and 9 human tumor cell lines of different origin and with different Akt kinase activity. Three of these cell lines were selected for drug resistance due to P-glycoprotein (Pgp or ABCB1) overexpression. Rapa inhibited phosphorylation of mTOR downstream effectors. Rapa sensitivity of the cells was Akt-dependent but did not correlate with ABCB1 overexpression. Suppression of mTOR function increased drug resistance in 8 out of 9 cell lines studied. The influence of Rapa on the ABC-transporter gene expression was examined. It was shown that in half of the cell lines studied Rapa exerted differential effects on the amount of ABC-transporter proteins: in some cases the protein amount decreased and in others, increased. The amount of mRNA remained unchanged. These data suggest that mTOR can regulate ABC transporters at the level of translation.     

Dietary macronutrients modulate the fatty acyl composition of rat liver mitochondrial cardiolipins

The interaction of dietary fats and carbohydrates on liver mitochondria were examined in male FBNF1 rats fed 20 different low-fat isocaloric diets. Animal growth rates and mitochondrial respiratory parameters were essentially unaffected, but mass spectrometry-based mitochondrial lipidomics profiling revealed increased levels of cardiolipins (CLs), a family of phospholipids essential for mitochondrial structure and function, in rats fed saturated or trans fat-based diets with a high glycemic index. These mitochondria showed elevated monolysocardiolipins (a CL precursor/product of CL degradation), elevated ratio of trans-phosphocholine (PC) (18:1/18:1) to cis-PC (18:1/18:1) (a marker of thiyl radical stress), and decreased ubiquinone Q₉; the latter two of which imply a low-grade mitochondrial redox abnormality. Extended analysis demonstrated: i) dietary fats and, to a lesser extent, carbohydrates induce changes in the relative abundance of specific CL species; ii) fatty acid (FA) incorporation into mature CLs undergoes both positive (>400-fold) and negative (2.5-fold) regulation; and iii) dietary lipid abundance and incorporation of FAs into both the CL pool and specific mature tetra-acyl CLs are inversely related, suggesting previously unobserved compensatory regulation. This study reveals previously unobserved complexity/regulation of the central lipid in mitochondrial metabolism.     

Qualitative characterization of the rat liver mitochondrial lipidome using LC–MS profiling and high energy collisional dissociation (HCD) all ion fragmentation

Lipids play multiple roles essential for proper mitochondrial function, from their involvement in membrane structure and fluidity, cellular energy storage, and signaling. Lipids are also major targets for reactive species, and their peroxidation byproducts themselves mediate further damage. Thousands of lipid species, from multiple classes and categories, are involved in these processes, suggesting lipid quantitative and structural analysis can help provide a better understanding of mitochondrial physiological status. Due to the diversity of lipids that contribute to and reflect mitochondrial function, analytical methods should ideally cover a wide range of lipid classes, and yield both quantitative and structural information. We developed a high resolution LC–MS method that is able to monitor the major lipid classes found in biospecimens (i.e. biofluids, cells and tissues) with relative quantitation in an efficient, sensitive, and robust manner while also characterizing individual lipid side-chains, by all ion high energy collisional dissociation fragmentation and chromatographic alignment. This method was used to profile the liver mitochondrial lipids from 192 rats undergoing a dietary macronutrient study in which changes in mitochondria function are related to changes in the major fat and glycemic index component of each diet. A total of 381 unique lipids, spanning 5 of the major LIPID MAPS defined categories, including fatty acyls, glycerophospholipids, glycerolipids, sphingolipids and prenols, were identified in mitochondria using the non-targeted LC–MS analysis in both positive and negative mode. The intention of this report is to show the breadth of this non-targeted LC–MS profiling method with regards to its ability to profile, identify and characterize the mitochondrial lipidome and the details of this will be discussed.