Design, synthesis and SAR study of Fluorine-containing 3rd-generation taxoids.

It has been shown that the incorporation of fluorine or organofluorine groups into pharmaceutical and agricultural drugs often induces desirable pharmacological properties through unique protein-drug interactions involving fluorine. We have reported separately remarkable effects of the 2,2-difluorovinyl (DFV) group at the C3' position, as well as those of the CF3O and CHF2O groups at the 3-position of the C2-benzoyl moiety of the 2nd- and 3rd-generation taxoids on their potency and pharmacological properties. Thus, it was very natural for us to investigate the combination of these two modifications in the 3rd-generation taxoids and to find out whether these two modifications are cooperative at the binding site in the ß-tubulin or not, as well as to see how these effects are reflected in the biological activities of the new 3rd-generation DFV-taxoids. Accordingly, we designed, synthesized and fully characterized 14 new 3rd-generation DFV-taxoids. These new DFV-taxoids exhibited remarkable cytotoxicity against human breast, lung, colon, pancreatic and prostate cancer cell lines. All of these new DFV-taxoids exhibited subnanomolar IC50 values against drug-sensitive cell lines, A549, HT29, Vcap and PC3, as well as CFPAC-1. All of the novel DFV-taxoids exhibited 2-4 orders of magnitude greater potency against extremely drug-resistant cancer cell lines, LCC6-MDR and DLD-1, as compared to paclitaxel, indicating that these new DFV-taxoids can overcome MDR caused by the overexpression of Pgp and other ABC cassette transporters. Dose-response (kill) curve analysis of the new DFV-taxoids in LCC6-MDR and DLD-1 cell lines revealed highly impressive profiles of several new DFV-taxoids. The cooperative effects of the combination of the 3'-DFV group and 3-CF3O/CHF2O-benzoyl moiety at the C2 position were investigated in detail by molecular docking analysis. We found that both the 3'-DFV moiety and the 3-CF3O/3-CHF2O group of the C2-benzoate moiety are nicely accommodated to the deep hydrophobic pocket of the paclitaxel/taxoid binding site in the ß-tubulin, enabling an enhanced binding mode through unique attractive interactions between fluorine/CF3O/CHF2O and the protein beyond those of paclitaxel and new-generation taxoids without bearing organofluorine groups, which are reflected in the remarkable potency of the new 3rd-generation DFV-taxoids.

Synthesis and applications of S-(trifluoromethyl)-2,8-bis(trifluoromethoxy)dibenzothiophenium triflate (Umemoto reagent IV).

A new, powerful, and easy-to-handle electrophilic trifluoromethylating agent, S-(trifluoromethyl)-2,8-bis(trifluoromethoxy)dibenzothiophenium triflate (Umemoto reagent IV), was developed. Due to the extraordinary electronic effect of trifluoromethoxy group, Umemoto reagent IV was easily synthesized by a one-pot method from readily available 3,3'-bis(trifluoromethoxy)biphenyl. It was shown that Umemoto reagent IV was more powerful than Umemoto reagent II and could trifluoromethylate many kinds of nucleophilic substrates more effectively. In addition, Umemoto reagent IV was successfully utilized for the preparation of trifluoromethyl nonaflate, a useful trifluoromethoxylating agent. The direct conversion of 2,8-bis(trifluoromethoxy)dibenzothiophene to Umemoto reagent IV with triflic anhydride was achieved, albeit in low yield.

Synthesis, Anti-Proliferative Evaluation and Mechanism of 4-Trifluoro Methoxy Proguanil Derivatives with Various Carbon Chain Length.

Among the known biguanide drugs, proguanil has the best antiproliferative activity. In contrast, newly synthesized biguanide derivatives containing fluorine atoms have excellent biological activity, among which trifluoromethoxy compounds show the strongest ability. Preliminary work in our laboratory exhibited that n-heptyl containing proguanil derivatives on one alkyl chain side have better biological activity than those with a shorter carbon chain. However, the relationship between the length of the carbon chain and the activity of the compounds is unknown. In this study, we synthesized 10 new trifluoromethoxy-containing proguanil derivatives with various carbon chain lengths. The phenyl side is fixed as the trifluoromethoxy group with change of carbon chain length in alkyl chain side. It was found that the anti-cancer abilities of 5C-8C with n-pentyl to n-octyl groups was significantly better than that of proguanil in the five human cancer cell lines. The colony formation assay demonstrated that 6C-8C at 0.5 to 1.0 µM significantly inhibited the colony formation of human cancer cell lines, much stronger than that of proguanil. Pharmacologically, 8C activates AMPK, leading to inactivation of the mTOR/p70S6K/4EBP1 pathway. Thus, these novel compounds have a great potential for developing new anti-cancer candidates.

(Trifluoromethoxy)Phenylboronic Acids: Structures, Properties, and Antibacterial Activity.

Three isomers of (trifluoromethoxy)phenylboronic acids were studied in the context of their physicochemical, structural, antimicrobial and spectroscopic properties. They were characterized by 1H, 13C, 11B and 19F NMR spectroscopy. The acidity of all the isomers was evaluated by both spectrophotometric and potentiometric titrations. The introduction of the -OCF3 group influences the acidity, depending, however, on the position of a substituent, with the ortho isomer being the least acidic. Molecular and crystal structures of ortho and para isomers were determined by the single crystal XRD method. Hydrogen bonded dimers are the basic structural motives of the investigated molecules in the solid state. In the case of the ortho isomer, intramolecular hydrogen bond with the -OCF3 group is additionally formed, weaker, however, than that in the analogous -OCH3 derivative, which has been determined by both X-Ray measurements as well as theoretical DFT calculations. Docking studies showed possible interactions of the investigated compounds with LeuRS of Escherichia coli. Finally, the antibacterial potency of studied boronic acids in vitro were evaluated against Escherichia coli and Bacillus cereus.

Phytomodulatory proteins isolated from Calotropis procera latex promote glycemic control by improving hepatic mitochondrial function in HepG2 cells.

The medicinal uses of Calotropis procera are diverse, yet some of them are based on effects that still lack scientific support. Control of diabetes is one of them. Recently, latex proteins from C. procera latex (LP) have been shown to promote in vivo glycemic control by the inhibition of hepatic glucose production via AMP-activated protein kinase (AMPK). Glycemic control has been attributed to an isolated fraction of LP (CpPII), which is composed of cysteine peptidases (95%) and osmotin (5%) isoforms. Those proteins are extensively characterized in terms of chemistry, biochemistry and structural aspects. Furthermore, we evaluated some aspects of the mitochondrial function and cellular mechanisms involved in CpPII activity. The effect of CpPII on glycemic control was evaluated in fasting mice by glycemic curve and glucose and pyruvate tolerance tests. HepG2 cells was treated with CpPII, and cell viability, oxygen consumption, PPAR activity, production of lactate and reactive oxygen species, mitochondrial density and protein and gene expression were analyzed. CpPII reduced fasting glycemia, improved glucose tolerance and inhibited hepatic glucose production in control animals. Additionally, CpPII increased the consumption of ATP-linked oxygen and mitochondrial uncoupling, reduced lactate concentration, increased protein expression of mitochondrial complexes I, III and V, and activity of peroxisome-proliferator-responsive elements (PPRE), reduced the presence of reactive oxygen species (ROS) and increased mitochondrial density in HepG2 cells by activation of AMPK/PPAR. Our findings strongly support the medicinal use of the plant and suggest that CpPII is a potential therapy for prevention and/or treatment of type-2 diabetes. A common epitope sequence shared among the proteases and osmotin is possibly the responsible for the beneficial effects of CpPII.

Exploring the possibility of using fluorine-involved non-conjugated electron-withdrawing groups for thermally activated delayed fluorescence emitters by TD-DFT calculation.

The trifluoromethyl group has been previously explored as a non-conjugated electron-withdrawing group in donor-acceptor thermally activated delayed fluorescence (TADF) emitters. In the present study, we investigate computationally the potential of other fluorine-containing acceptors, trifluoromethoxy (OCF3), trifluoromethylthio (SCF3), and pentafluorosulfanyl (SF5), within two families of donor-acceptor TADF emitters. Time-dependent density functional theory calculations indicate that when only two ortho-disposed carbazole donors are used (Type I molecules), the lowest-lying triplet state possesses locally excited (LE) character while the lowest-lying singlet state possesses charge-transfer character. When five carbazole donors are present in the emitter design (Type II molecules), now both S1 and T1 states possess CT character. For molecules 2CzOCF 3 and 5CzOCF 3 , the singlet energies are predicted to be 3.92 eV and 3.45 eV; however, the singlet-triplet energy gaps, ΔE STs, are predicted to be large at 0.46 eV and 0.37 eV, respectively. The compounds 2CzCF 3 , 2CzSCF 3 , and 2CzSF 5 , from Type I molecules, show significant promise as deep blue TADF emitters, possessing high calculated singlet energies in the gas phase (3.62 eV, 3.66 eV, and 3.51 eV, respectively) and small, ΔE STs, of 0.17 eV, 0.22 eV, and 0.07 eV, respectively. For compounds 5CzSCF 3 and 5CzSF 5 , from Type II molecules, the singlet energies are stabilized to 3.24 eV and 3.00 eV, respectively, while ΔE STs are 0.27 eV and 0.12 eV, respectively, thus both show promise as blue or sky-blue TADF emitters. All these six molecules possess a dense number of intermediate excited states between S1 and T1, thus likely leading to a very efficient reverse intersystem crossing in these compounds.

Design, synthesis and SAR study of 3rd-generation taxoids bearing 3-CH3, 3-CF3O and 3-CHF2O groups at the C2-benzoate position.

It has been shown that inclusion of CF3O and CHF2O groups to drug candidates often improve their pharmacological properties, especially metabolic stability, membrane permeability and PK profile. Moreover, the unique non-spherical structure of the OCHF2 group can provide interesting and beneficial characteristics. Accordingly, new 3rd-generation taxoids, bearing 3-OCF3 or 3-OCF2H (and 3-CH3 for comparison) at the C2 benzoate moiety, were synthesized and their potencies against drug-sensitive and drug-resistant cancer cell lines examined. In this study, our previous SAR studies on 3rd-generation taxoids were expanded to disclose that CH3, CF3O and CHF2O groups are well tolerated at this position and enhance potency, especially against MDR-cancer cell lines so that these taxoids can virtually overcome MDR. These new taxoids exhibit up to 7 times higher cytotoxicity (IC50) than paclitaxel against drug-sensitive cancer cell lines (MCF7 and LCC6-WT) and 2-3 orders of magnitude higher potency than paclitaxel against drug-resistant ovarian, breast and colon cancer cell lines with MDR-phenotype (NCI/ADR, LCC6-MDR and LDL-1), as well as pancreatic cancer cell line, CFPAC-1. Since it has been shown that a bulky group at this position reduces potency, it is noteworthy that rather bulky CF3O and CHF2O groups are well tolerated. Molecular modeling analysis indicated the favorable van der Waals interactions of CF3O and CHF2O groups in the binding site. It is also worthy of note that new taxoids, bearing a CHF2O group at the C2 benzoate position (1-06 series), exhibited the highest potencies against MDR-cancer cell lines and cancer stem cell (CSC)-enriched cancer cell lines. These new 3rd-generation taxoids are promising candidates for highly potent chemotherapeutic agents, as well as payloads for tumor-targeting drug conjugates such as antibody-drug conjugates (ADCs).

Crystalline silica particles cause rapid NLRP3-dependent mitochondrial depolarization and DNA damage in airway epithelial cells.

BACKGROUND: Respirable crystalline silica causes lung carcinomas and many thousand future cancer cases are expected in e.g. Europe. Critical questions are how silica causes genotoxicity in the respiratory epithelium and if new cases can be avoided by lowered permissible exposure levels. In this study we investigate early DNA damaging effects of low doses of silica particles in respiratory epithelial cells in vitro and in vivo in an effort to understand low-dose carcinogenic effects of silica particles. RESULTS: We find DNA damage accumulation already after 5-10 min exposure to low doses (5 µg/cm2) of silica particles (Min-U-Sil 5) in vitro. DNA damage was documented as increased levels of γH2AX, pCHK2, by Comet assay, AIM2 induction, and by increased DNA repair (non-homologous end joining) signaling. The DNA damage response (DDR) was not related to increased ROS levels, but to a NLRP3-dependent mitochondrial depolarization. Particles in contact with the plasma membrane elicited a Ser198 phosphorylation of NLRP3, co-localization of NLRP3 to mitochondria and depolarization. FCCP, a mitochondrial uncoupler, as well as overexpressed NLRP3 mimicked the silica-induced depolarization and the DNA damage response. A single inhalation of 25 µg silica particles gave a similar rapid DDR in mouse lung. Biomarkers (CC10 and GPRC5A) indicated an involvement of respiratory epithelial cells. CONCLUSIONS: Our findings demonstrate a novel mode of action (MOA) for silica-induced DNA damage and mutagenic double strand breaks in airway epithelial cells. This MOA seems independent of particle uptake and of an involvement of macrophages. Our study might help defining models for estimating exposure levels without DNA damaging effects.

High-concentration homocysteine inhibits mitochondrial respiration function and production of reactive oxygen species in neuron cells.

OBJECTIVE: Homocysteine plays critical roles in cellular redox homeostasis, and hyperhomocysteinemia has been associated with multiple diseases, including neurological disorders involving reactive oxygen species-inducing and pro-inflammatory effects of homocysteine that are related to mitochondria. This study investigated the role of homocysteine in regulating mitochondria of neuron cell lines. METHODS: Neuron cells were pre-treated with homocysteine, and then flow cytometry was used to detect reactive oxygen species production and mitochondrial membrane potential, while Seahorse XFp Mito stress assay was used to comprehensively analyze mitochondrial function. RESULTS: The experimental results showed that high-concentration homocysteine diminished carbonyl cyanide-4 (trifluoromethoxy) phenylhydrazone-stimulated oxygen consumption rate and mitochondrial spare respiration capacity in a time- and concentration-dependent manner, and homocysteine also reduced reactive oxygen species in cultured neuron cell lines while no changes in mitochondrial membrane potential were observed. CONCLUSION: These results indicate that homocysteine diminished mitochondrial respiration function in neuron cell lines mediated by its reactive oxygen species-reducing effects, which may underlie the association between hyperhomocysteinemia and human diseases.

Trifluoromethoxypyrazines: Preparation and Properties.

The incorporation of the trifluoromethoxy group into organic molecules has become very popular due to the unique properties of the named substituent that has a "pseudohalogen" character, while the chemical properties of the synthesized compound, especially heterocycles with such a group, are less studied. As trifluoromethoxy-substituted pyrazines are still unknown, we have developed efficient and scalable methods for 2-chloro-5-trifluoromethoxypyrazine synthesis, showing the synthetic utility of this molecule for Buchwald-Hartwig amination and the Kumada-Corriu and Suzuki and Sonogashira coupling reactions. Some comparisons of chlorine atom and trifluoromethoxy group stability in these transformations have been carried out.

Application of ECIS to Assess FCCP-Induced Changes of MSC Micromotion and Wound Healing Migration.

Electric cell-substrate impedance sensing (ECIS) is an emerging technique for sensitively monitoring morphological changes of adherent cells in tissue culture. In this study, human mesenchymal stem cells (hMSCs) were exposed to different concentrations of carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone (FCCP) for 20 h and their subsequent concentration-dependent responses in micromotion and wound healing migration were measured by ECIS. FCCP disrupts ATP synthesis and results in a decrease in cell migration rates. To detect the change of cell micromotion in response to FCCP challenge, time-series resistances of cell-covered electrodes were monitored and the values of variance were calculated to verify the difference. While Seahorse XF-24 extracellular flux analyzer can detect the effect of FCCP at 3 µM concentration, the variance calculation of the time-series resistances measured at 4 kHz can detect the effect of FCCP at concentrations as low as 1 µM. For wound healing migration, the recovery resistance curves were fitted by sigmoid curve and the hill slope showed a concentration-dependent decline from 0.3 µM to 3 µM, indicating a decrease in cell migration rate. Moreover, dose dependent incline of the inflection points from 0.3 µM to 3 µM FCCP implied the increase of the half time for wound recovery migration. Together, our results demonstrate that partial uncoupling of mitochondrial oxidative phosphorylation reduces micromotion and wound healing migration of hMSCs. The ECIS method used in this study offers a simple and sensitive approach to investigate stem cell migration and its regulation by mitochondrial dynamics.

Enantioselective Benzylation and Allylation of α-Trifluoromethoxy Indanones under Phase-Transfer Catalysis.

The organo-catalyzed enantioselective benzylation reaction of α-trifluoromethoxy indanones afforded α-benzyl-α-trifluoromethoxy indanones with a tetrasubstituted stereogenic carbon center in excellent yield with moderate enantioselectivity (up to 57% ee). Cinchona alkaloid-based chiral phase transfer catalysts were found to be effective for this transformation, and both enantiomers of α-benzyl-α-trifluoromethoxy indanones were accessed, depended on the use of cinchonidine and cinchonine-derived catalyst. The method was extended to the enantioselective allylation reaction of α-trifluoromethoxy indanones to give the allylation products in moderate yield with good enantioselectivity (up to 76% ee).

Maternal intake of trans-unsaturated or interesterified fatty acids during pregnancy and lactation modifies mitochondrial bioenergetics in the liver of adult offspring in mice.

The quality of dietary lipids in the maternal diet can programme the offspring to diseases in later life. We investigated whether the maternal intake of palm oil or interesterified fat, substitutes for trans-unsaturated fatty acids (FA), induces metabolic changes in the adult offspring. During pregnancy and lactation, C57BL/6 female mice received normolipidic diets containing partially hydrogenated vegetable fat rich in trans-unsaturated fatty acids (TG), palm oil (PG), interesterified fat (IG) or soyabean oil (CG). After weaning, male offspring from all groups received the control diet until day 110. Plasma glucose and TAG and liver FA profiles were ascertained. Liver mitochondrial function was accessed with high-resolution respirometry by measuring VO2, fluorimetry for detection of hydrogen peroxide (H2O2) production and mitochondrial Ca2+ uptake. The results showed that the IG offspring presented a 20 % increase in plasma glucose and both the IG and TG offspring presented a 2- and 1·9-fold increase in TAG, respectively, when compared with CG offspring. Liver MUFA and PUFA contents decreased in the TG and IG offspring when compared with CG offspring. Liver MUFA content also decreased in the PG offspring. These modifications in FA composition possibly affected liver mitochondrial function, as respiration was impaired in the TG offspring and H2O2 production was higher in the IG offspring. In addition, mitochondrial Ca2+ retention capacity was reduced by approximately 40 and 55 % in the TG and IG offspring, respectively. In conclusion, maternal consumption of trans-unsaturated and interesterified fat affected offspring health by compromising mitochondrial bioenergetics and lipid metabolism in the liver.

Dichlorotrifluoromethoxyacetic Acid: Preparation and Reactivity.

We describe the first gram scale preparation of the reagent dichlorotrifluoromethoxyacetic acid. This stable compound is obtained in five steps starting from the cheap diethylene glycol. The reactivity of the sodium salt of this fluorinated acid was also tested and allowed the preparation of new amides.

Enantioselective Construction of Trifluoromethoxylated Stereogenic Centers by a Nickel-Catalyzed Asymmetric Suzuki-Miyaura Coupling of Secondary Benzyl Bromides.

Trifluoromethoxy-substituted stereogenic centers can be constructed with high enantioselectivity by a nickel-catalyzed Suzuki-Miyaura coupling of readily available α-bromobenzyl trifluoromethyl ethers with a variety of aryl pinacol boronates. The coupling proceeds under mild reaction conditions, and a variety of common functional groups, such as fluoride, chloride, bromide, ester, enolizable ketone, nitro, cyano, amino, and vinyl moieties, were well tolerated. Furthermore, the reaction can be easily scaled up to gram quantities without a decrease in enantioselectivity.

Carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone (FCCP) pre-exposure ensures follicle integrity during in vitro culture of ovarian tissue but not during cryopreservation in the domestic cat model.

PURPOSE: Temporary and reversible downregulation of metabolism may improve the survival of tissues exposed to non-physiological conditions during transport, in vitro culture, and cryopreservation. The objectives of the study were to (1) optimize the concentration and duration of carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone (FCCP-a mitochondrial uncoupling agent) exposures for biopsies of domestic cat ovarian tissue and (2) examine the effects of FCCP pre-exposures on follicle integrity after tissue culture and/or cryopreservation. METHODS: Biopsies of cat ovarian tissue were first treated with various concentrations of FCCP (0, 10, 40, or 200 nM) for 10 or 120 min to determine the most suitable pre-exposure conditions. Based on these results, tissues were pre-exposed to 200 nM FCCP for 120 min for the subsequent studies on culture and cryopreservation. In all experiments and for each treatment group, tissue activity and integrity were measured by mitochondrial membrane potential (relative optical density of rhodamine 123 fluorescence), follicular viability (calcein assay), follicular morphology (histology), granulosa cell proliferation (Ki-67 immunostaining), and follicular density. RESULTS: Ovarian tissues incubated with 200 nM FCCP for 120 min led to the lowest mitochondrial activity (1.17 ± 0.09; P < 0.05) compared to control group (0 nM; 1.30 ± 0.12) while maintaining a constant percentage of viable follicles (75.3 ± 7.8 %) similar to the control group (71.8 ± 11.7 %; P > 0.05). After 2 days of in vitro culture, percentage of viable follicles (78.8 ± 8.9 %) in similar pre-exposure conditions was higher (P < 0.05) than in the absence of FCCP (61.2 ± 12.0 %) with percentages of morphologically normal follicles (57.6 ± 17.3 %) not different from the fresh tissue (70.2 ± 7.1 %; P > 0.05). Interestingly, percentages of cellular proliferation and follicular density were unaltered by the FCCP exposures. Based on the indicators mentioned above, the FCCP-treated tissue fragments did not have a better follicle integrity after freezing and thawing. CONCLUSIONS: Pre-exposure to 200 nM FCCP during 120 min protects and enhances the follicle integrity in cat ovarian tissue during short-term in vitro culture. However, FCCP does not appear to exert a beneficial or detrimental effect during ovarian tissue cryopreservation.

Dodecyl and octyl esters of fluorescein as protonophores and uncouplers of oxidative phosphorylation in mitochondria at submicromolar concentrations.

In our search for fluorescent uncouplers of oxidative phosphorylation, three esters of fluorescein, n-butyl-, n-octyl-, and n-dodecyl-oxycarbonyl-fluorescein (C4-FL, C8-FL, C12-FL) were synthesized and characterized. With increasing liposomal lipid content, the long-chain alkyl derivatives of fluorescein (C8-FL, C12-FL and commercially available C18-FL), but not C4-FL and unsubstituted fluorescein, exhibited an increase in fluorescence polarization reflecting the dye binding to liposomes. C12-FL induced proton permeability in lipid membranes, while C4-FL was inactive. In contrast to C4-FL and C18-FL, C12-FL and C8-FL increased the respiration rate and decreased the membrane potential of isolated rat liver mitochondria with half-maximal effective concentrations of 700nM and 300nM, respectively. The effect of Cn-FL on the respiration correlated with that on proton permeability of the inner mitochondrial membrane, as measured by induction of mitochondria swelling in the potassium acetate medium. Binding of C8-FL to mitochondria depended on their energization, which was apparently associated with pH gradient generation across the inner mitochondrial membrane in the presence of a respiratory substrate. In wild-type yeast cells, C12-FL localized predominantly in plasma membrane, whereas in AD1-8 mutants lacking MDR pumps, it stained cytoplasmic organelles with some preference for mitochondria. Fluorescent uncouplers can be useful as a tool for determining their localization in a cell or distribution between different tissues in a living animal by fluorescent microscopy.

Molecular and bioenergetic differences between cells with African versus European inherited mitochondrial DNA haplogroups: implications for population susceptibility to diseases.

The geographic origins of populations can be identified by their maternally inherited mitochondrial DNA (mtDNA) haplogroups. This study compared human cybrids (cytoplasmic hybrids), which are cell lines with identical nuclei but mitochondria from different individuals with mtDNA from either the H haplogroup or L haplogroup backgrounds. The most common European haplogroup is H while individuals of maternal African origin are of the L haplogroup. Despite lower mtDNA copy numbers, L cybrids had higher expression levels for nine mtDNA-encoded respiratory complex genes, decreased ATP (adenosine triphosphate) turnover rates and lower levels of reactive oxygen species production, parameters which are consistent with more efficient oxidative phosphorylation. Surprisingly, GeneChip arrays showed that the L and H cybrids had major differences in expression of genes of the canonical complement system (5 genes), dermatan/chondroitin sulfate biosynthesis (5 genes) and CCR3 (chemokine, CC motif, receptor 3) signaling (9 genes). Quantitative nuclear gene expression studies confirmed that L cybrids had (a) lower expression levels of complement pathway and innate immunity genes and (b) increased levels of inflammation-related signaling genes, which are critical in human diseases. Our data support the hypothesis that mtDNA haplogroups representing populations from different geographic origins may play a role in differential susceptibilities to diseases.

Availability of the key metabolic substrates dictates the respiratory response of cancer cells to the mitochondrial uncoupling.

Active glycolysis and glutaminolysis provide bioenergetic stability of cancer cells in physiological conditions. Under hypoxia, metabolic and mitochondrial disorders, or pharmacological treatment, a deficit of key metabolic substrates may become life-threatening to cancer cells. We analysed the effects of mitochondrial uncoupling by FCCP on the respiration of cells fed by different combinations of Glc, Gal, Gln and Pyr. In cancer PC12 and HCT116 cells, a large increase in O2 consumption rate (OCR) upon uncoupling was only seen when Gln was combined with either Glc or Pyr. Inhibition of glutaminolysis with BPTES abolished this effect. Despite the key role of Gln, addition of FCCP inhibited respiration and induced apoptosis in cells supplied with Gln alone or Gal/Gln. For all substrate combinations, amplitude of respiratory responses to FCCP did not correlate with Akt, Erk and AMPK phosphorylation, cellular ATP, and resting OCR, mitochondrial Ca(2+) or membrane potential. However, we propose that proton motive force could modulate respiratory response to FCCP by regulating mitochondrial transport of Gln and Pyr, which decreases upon mitochondrial depolarisation. As a result, an increase in respiration upon uncoupling is abolished in cells, deprived of Gln or Pyr (Glc). Unlike PC12 or HCT116 cells, mouse embryonic fibroblasts were capable of generating pronounced response to FCCP when deprived of Gln, thus exhibiting lower dependence on glutaminolysis. Overall, the differential regulation of the respiratory response to FCCP by metabolic environment suggests that mitochondrial uncoupling has a potential for substrate-specific inhibition of cell function, and can be explored for selective cancer treatment.

Progressive stages of mitochondrial destruction caused by cell toxic bile salts.

The cell-toxic bile salt glycochenodeoxycholic acid (GCDCA) and taurochenodeoxycholic acid (TCDCA) are responsible for hepatocyte demise in cholestatic liver diseases, while tauroursodeoxycholic acid (TUDCA) is regarded hepatoprotective. We demonstrate the direct mitochondrio-toxicity of bile salts which deplete the mitochondrial membrane potential and induce the mitochondrial permeability transition (MPT). The bile salt mediated mechanistic mode of destruction significantly differs from that of calcium, the prototype MPT inducer. Cell-toxic bile salts initially bind to the mitochondrial outer membrane. Subsequently, the structure of the inner boundary membrane disintegrates. And it is only thereafter that the MPT is induced. This progressive destruction occurs in a dose- and time-dependent way. We demonstrate that GCDCA and TCDCA, but not TUDCA, preferentially permeabilize liposomes containing the mitochondrial membrane protein ANT, a process resembling the MPT induction in whole mitochondria. This suggests that ANT is one decisive target for toxic bile salts. To our knowledge this is the first report unraveling the consecutive steps leading to mitochondrial destruction by cell-toxic bile salts.