Effects of 9,10-dihydroxy-4,4-dimethyl-5,8-dihydro-1(4H)-anthracenone derivatives on tumor cell respiration

A series of tricyclic hydroquinones, incorporating a carbonyl group in the ortho position relative to the phenol function, were tested as inhibitors of oxygen uptake against the TA3 mouse carcinoma cell line and its multidrug-resistant variant TA3-MTX-R. The title compound, which proved to be the most active one, also exhibited low micromolar dose-dependent growth inhibition of the human tumor U937 cell line (human monocytic leukemia). A tentative structure-activity relationship is proposed for these substances. A comparison between the cytotoxicities of the title compound and 4,4-dimethyl-5,8-dihydroxynaphthalene-1-one, with their activities as inhibitors of oxygen uptake by the TA3-MTX-R cell line, is presented. Also, the inhibition of oxygen uptake by 6-(4-methylpent-3-enyl)-1,4-naphthoquinone was determined and compared with its reported cytotoxicity toward P-388 (murine lymphocytic leukemia), A-549 (human lung carcinoma), HT-29 (human colon carcinoma), and MEL-28 (human melanoma) cells. The inhibition of oxygen uptake by TA3-MTX-R cells is useful as a quick test for preliminary screening of possible anticancer activity.     

Effects of 4,4-dimethyl-5,8-dihydroxynaphtalene-1-one and 4,4-dimethyl-5,8-dihydroxytetralone derivatives on tumor cell respiration

A set of structurally related compounds incorporating a carbonyl group in the ortho position with regard to a phenol function were tested against the TA3 mouse carcinoma cell line and its multidrug-resistant variant TA3-MTX-R. The series consists of 2'-hydroxyacetophenone, 4'-hydroxyacetophenone 2',5'-dihydroxyacetophenone, 4-acetyl-3,3-dimethyl-5-hydroxy-2-morpholino-2,3-dihydrobenzobfuran, five 4,4-dimethyl-5,8-dioxygenated naphtalene-1-ones and three 4,4-dimethyl-5,8-dioxygenated tetralones. A tentative structure-activity relationship was found for this family of substances, suggesting that a coplanar ortho-carbonyl-1,4-hydroquinone motif is able to cause inhibition of cellular respiration.     

The Degradation of Gallic Acid and its Alkyl Esters by Pullularia pullulans

By elective techniques an isolate of Pullularia pullulans was obtained which possessed the ability to utilize gallic acid (3,4,5-trihydroxybenzoic acid) and its alkyl esters together with other aromatic substrates as the sole carbon source for growth in a mineral salts medium. Quantitative measurements were made of the disappearance of the gallates from culture media and of their metabolism by washed suspensions by the Warburg technique. The pattern of oxygen uptake with various substrates by suspensions adapted to gallic acid was examined in the light of Stanier's simultaneous adaptation postulates in an attempt to identify intermediates of gallic acid metabolism. As a result, β-ketoadipic acid was suspected of being an intermediate. A keto compound was isolated as its 2,4-dinitrophenylhydrazone during gallic acid metabolism but it was not β-ketoadipic acid or levulinic acid.     

The Resistance of Gallic Acid and its Alkyl Esters to Attack by Bacteria able to Degrade Aromatic Ring Structures

Summary: Various bacteria capable of degrading aromatic ring structures were unable to utilize gallic acid, methyl, ethyl or propyl gallates as sole carbon sources for growth when tested in liquid and solid media. A bacterial isolate was obtained which degraded gallic acid but not methyl, ethyl or propyl gallates, although ellagic acid, a major spontaneous degradation product of the gallate esters, was utilized to a limited degree.     

Multiparameter metabolic analysis reveals a close link between attenuated mitochondrial bioenergetic function and enhanced glycolysis dependency in human tumor cells

Increased conversion of glucose to lactic acid associated with decreased mitochondrial respiration is a unique feature of tumors first described by Otto Warburg in the 1920s. Recent evidence suggests that the Warburg effect is caused by oncogenes and is an underlying mechanism of malignant transformation. Using a novel approach to measure cellular metabolic rates in vitro, the bioenergetic basis of this increased glycolysis and reduced mitochondrial respiration was investigated in two human cancer cell lines, H460 and A549. The bioenergetic phenotype was analyzed by measuring cellular respiration, glycolysis rate, and ATP turnover of the cells in response to various pharmacological modulators. H460 and A549 cells displayed a dependency on glycolysis and an ability to significantly upregulate this pathway when their respiration was inhibited. The converse, however, was not true. The cell lines were attenuated in oxidative phosphorylation (OXPHOS) capacity and were unable to sufficiently upregulate mitochondrial OXPHOS when glycolysis was disabled. This observed mitochondrial impairment was intimately linked to the increased dependency on glycolysis. Furthermore, it was demonstrated that H460 cells were more glycolytic, having a greater impairment of mitochondrial respiration, compared with A549 cells. Finally, the upregulation of glycolysis in response to mitochondrial ATP synthesis inhibition was dependent on AMP-activated protein kinase activity. In summary, our results demonstrate a bioenergetic phenotype of these two cancer cell lines characterized by increased rate of glycolysis and a linked attenuation in their OXPHOS capacity. These metabolic alterations provide a mechanistic explanation for the growth advantage and apoptotic resistance of tumor cells. oxygen consumption; oxidative phosphorylation; Warburg effect; real time     

Gallic acid ester derivatives induce apoptosis and cell adhesion inhibition in melanoma cells: The relationship between free radical generation, glutathione depletion and cell death

Malignant melanoma is a lethal disease, and the incidence and mortality associated with it are increasing worldwide. It has a significant tendency to develop both metastasis and resistance to chemotherapy. The tumor cells show abnormal redox regulation, and although the molecular mechanisms involved are not well characterized, they seem to be related to oxidative stress. In a previous study, we showed the antitumoral properties of gallic acid ester derivatives in leukemia cells. Here, we show the effect of octyl, decyl, dodecyl and tetradecyl gallates on B16F10 cells, a melanoma cell line. All compounds induced cytotoxic effects, and the IC₅₀ values obtained were between 7 μM and 17 μM after 48 h of incubation. Cell death occurred through apoptosis, as demonstrated by the genomic DNA fragmentation pattern. The gallates were able to induce significant production of free radicals, deplete both glutathione and ATP, activate NF-κB and promote the inhibition of cell adhesion under the experimental conditions. The glutathione depletion induced by these compounds was related to the inhibition of γ-glutamylcysteine synthase activity. These results suggest that gallates induce tumoral cell death through apoptosis as a consequence of oxidative stress, though they use different mechanisms to do so. These findings are important since melanoma cells are resistant to death because of their high level of antioxidant defense, adhesion capability and propensity to metastasize.     

4-Quinolones cause a selective loss of mitochondrial DNA from mouse L1210 leukemia cells

The 4-quinolone antibiotics nalidixic acid and ciprofloxacin and potent inhibitors of the bacterial type II topoisomerase DNA gyrase. Treatment of mouse L1210 leukemia cells with these drugs resulted in a delayed inhibition of cell proliferation. Prior to inhibition of cell proliferation, there was a time-dependent decrease in the cellular content of mitochondrial DNA (mtDNA). The decrease in mtDNA was associated with a decrease in the rate of mitochondrial respiration and an increase in the concentration of lactate in the growth medium. Inhibition of cell proliferation by 4-quinolones was reversible upon drug washout. However, there was a 2- to 4-day lag before the growth rate returned to normal levels. This was preceeded by an increase in mtDNA content and mitochondrial respiration. These studies suggest that inhibition of mammalian cell proliferation by 4-quinolone drugs is related to the selective depletion of mtDNA. © 1993 Wiley-Liss, Inc.     

Relevance and Therapeutic Possibility of PTEN-Long in Renal Cell Carcinoma

PTEN-Long is a translational variant of PTEN (Phosphatase and Tensin Homolog). Like PTEN, PTEN-Long is able to antagonize the PI3K-Akt pathway and inhibits tumor growth. In this study, we investigated the role PTEN-Long plays in the development and progression of clear cell renal cell carcinoma (ccRCC) and explored the therapeutic possibility using proteinaceous PTEN-Long to treat ccRCC. We found that the protein levels of PTEN-Long were drastically reduced in ccRCC, which was correlated with increased levels of phosphorylated Akt (pAkt). Gain of function experiments showed overexpression of PTEN-Long in the ccRCC cell line 786-0 suppressed PI3K-Akt signaling, inhibited cell proliferation, migration and invasion, and eventually induced cell death. When purified PTEN-Long was added into cultured 786-0 cells, it entered cells, blocked Akt activation, and induced apoptosis involving Caspase 3 cleavage. Furthermore, PTEN-Long inhibited proliferation of 786-0 cells in xenograft mouse model. Our results implicated that understanding the roles of PTEN-Long in renal cell carcinogenesis has therapeutic significance.     

Protective effect of the plasticizer di(2-ethylhexyl) phthalate against damage of the mitochondrial membrane induced by calcium: possible participation of the adenine nucleotide translocator

The effect of di(2-ethylhexyl) phthalate (DEHP) on the response of isolated rat liver mitochondria to Ca2+ was investigated. DEHP was found to inhibit more than 60% of the auto-accelerating release of respiration induced by 100 microM Ca2+, being maximally inhibitory at 40 microM. Prior addition of DEHP also partially inhibited Ca2+-induced swelling of the mitochondrial matrix. However, DEHP did not change the net rate of Ca2+ uptake measured by the steady-state infusion method. DEHP also reduced the rate of adenine nucleotide exchange across the mitochondrial membrane. Another alkyl phthalate and alkyl citrates had similar effects on Ca2+-induced membrane damage, but their potencies depended on the lengths of their alkyl chains. These results suggest that the effects of DEHP and other alkyl esters on mitochondrial functions are mainly based on their actions on membrane lipids surrounding adenine nucleotide translocator (AdNT), resulting in alteration of the interaction between these phospholipids and AdNT, and consequent modification of the state of the protein.     

Effect of retinoic acid on the proliferation and tumorigenicity of mouse mastocytoma cells

Retinoic acid (RA) inhibited the in vitro growth of the mouse mast cell tumor line P₈₁₅ in a dose- and time-dependent manner. The inhibition was accompanied by an increase in the amount of neutral intracellular mucopolysaccharides. Study of cell cycle kinetics showed that exposure to retinoic acid led to a slowing-down of the cell-cycle progression possibly related to a more differentiated cell population disclosed by microscopy with a lower proliferative capacity. In vivo, delays in both tumor appearance and mouse mortality were observed after injecting RA into mice bearing mastocytomas. These results suggest that RA could be of interest in the treatment of human malignant systemic mastocytosis with proliferation of immature mast cells.     

Effects of t-butyl-4-hydroxyanisole and other phenolic antioxidants on tumoral cells and Trypanosoma parasites

The antioxidant food additives 2(3)-tert-butyl-4-hydroxyanisole (BHA), 2,6-di(tert-butyl)-p-cresol (BHT) and the methyl and propyl esters of gallic acid inhibited Trypanosoma cruzi culture growth and oxygen consumption. The I50 values for growth and oxygen uptake with BHA were 0.284 and 0.400 and for BHT 0.083 and 0.235 mM, respectively. Moreover, BHA inhibited the respiration of several tumor cells, as well as of the procyclic and bloodstream trypomastigote forms of T. brucei brucei, with I50 in the range 0.29-0.52 mM. Inhibition of the parasites' oxygen uptake by BHA was not of the pure Michaelis-Menten type, but may be of a mixed form. It is postulated that these compounds are inhibitors because they resemble ubiquinone.     

Use of chemosensitization to overcome fludioxonil resistance in Penicillium expansum

Aim: To overcome fludioxonil resistance of Penicillium expansum, a mycotoxigenic fungal pathogen causing postharvest decay in apple, by using natural phenolic chemosensitizing agents. Methods and Results: Fludioxonil‐resistant mutants of P. expansum were co‐treated with different oxidising and natural phenolic agents. Resistance was overcome by natural phenolic chemosensitizing agents targeting the oxidative stress–response pathway. These agents also augmented effectiveness of the fungicide, kresoxim‐methyl. Results indicated that alkyl gallates target mitochondrial respiration and/or its antioxidation system. Fungal mitochondrial superoxide dismutase (Mn‐SOD) plays a protective role against alkyl gallates. Conclusions: Natural chemosensitizing agents targeting the oxidative stress–response system, such as Mn‐SOD, can synergize commercial fungicides. Significance and Impact of the Study: Redox‐active compounds can serve as potent chemosensitizing agents to overcome resistance and lower effective dosages of fungicides. This can reduce costs with coincidental lowering of environmental and health risks.     

Antifungal activity of alkyl gallates against plant pathogenic fungi

The antifungal activity of alkyl gallates against plant pathogenic fungi was evaluated. All of the fungi tested in this study were susceptible to some alkyl gallates, and the effect of linear alkyl gallates against plant pathogenic fungi was similar to the previously reported effects against Gram-negative and Gram-positive bacteria. We found that branched alkyl gallates showed stronger activity than did linear alkyl gallates with similar log P values. In addition, the antifungal activity of alkyl gallates was correlated with gallate-induced inhibition of the activity of mitochondrial complex II. The antifungal activity of alkyl gallates likely originates, at least in part, from their ability to inhibit the membrane respiratory chain.     

Gallic Acid Protects 6-OHDA Induced Neurotoxicity by Attenuating Oxidative Stress in Human Dopaminergic Cell Line

Gallic acid is one of the most important polyphenolic compounds, which is considered an excellent free radical scavenger. 6-Hydroxydopamine (6-OHDA) is a neurotoxin, which has been implicated in mainly Parkinson’s disease (PD). In this study, we investigated the molecular mechanism of the neuroprotective effects of gallic acid on 6-OHDA induced apoptosis in human dopaminergic cells, SH-SY5Y. Our results showed that 6-OHDA induced cytotoxicity in SH-SY5Y cells was suppressed by pre-treatment with gallic acid. The percentage of live cells (90%) was high in the pre-treatment of gallic acid when compared with 6-OHDA alone treated cell line. Moreover, gallic acid was very effective in attenuating the disruption of mitochondrial membrane potential, elevated levels of intracellular ROS and apoptotic cell death induced by 6-OHDA. Gallic acid also lowered the ratio of the pro-apoptotic Bax protein and the anti-apoptotic Bcl-2 protein in SH-SY5Y cells. 6-OHDA exposure was up-regulated caspase-3 and Keap-1 and, down-regulated Nrf2, BDNF and p-CREB, which were sufficiently reverted by gallic acid pre-treatment. These findings indicate that gallic acid is able to protect the neuronal cells against 6-OHDA induced injury and proved that gallic acid might potentially serve as an agent for prevention of several human neurodegenerative diseases caused by oxidative stress and apoptosis.     

Relationship between the lipophilicity of gallic acid n-alquil esters' derivatives and both myeloperoxidase activity and HOCl scavenging

The gallic acid and several n-alkyl gallates, with the same number of hydroxyl substituents, varying only in the side carbonic chain length, with respective lipophilicity defined through the C log P, were studied. It evidenced the structure-activity relationship of the myeloperoxidase activity inhibition and the hypochlorous acid scavenger property, as well as its low toxicity in rat hepatic tissue. The gallates with C log P below 3.0 (compounds 2-7) were more active against the enzyme activity, what means that the addition of 1-6 carbons (C log P between 0.92 and 2.92) at the side chain increased approximately 50% the gallic acid effect. However, a relationship between the HOCl scavenging capability and the lipophilicity was not observed. With these results it is possible to suggest that the gallates protect the HOCl targets through two mechanisms: inhibiting its production by the enzyme and scavenging the reactive specie.     

Effects of alkyl chain lengths of gallates upon enzymatic wool functionalisation

The covalent grafting of alkyl gallates on wool through a laccase catalysed reaction in 80/20 (v/v, %) aqueous–ethanol mixture provided in a one-step process a multifunctional textile material with antioxidant, antibacterial and water repellent properties. Gallic acid and its alkyl esters ethyl, propyl, octyl and dodecyl gallate have been enzymatically grafted on wool fibres in order to study the effect of alkyl chain length on wool functional modification. The capacity of laccase to oxidise these phenolic compounds in an aqueous–organic medium has been verified by electrochemical techniques. The increase of CH₂, CH₃ groups in the FTIR spectra, together with the XPS analysis of the enzymatically modified fabrics confirmed the covalent grafting of ester gallates on wool. The result obtained in this work for antibacterial, water repellent as well as antioxidant properties show that the length of the alkyl chain of gallates molecule play an important role on wool functionalisation.     

Molecular design of multifunctional antibacterial agents against methicillin resistant Staphylococcus aureus (MRSA)

Antibacterial activity of a series of alkyl gallates (3,4,5-trihydroxybenzoates) against Gram-positive bacteria, especially methicillin resistant Staphylococcus aureus (MRSA) strains was evaluated. Gram-positive bacteria are all susceptible to alkyl gallates. Dodecyl gallate was the most effective against MRSA ATCC 33591 strain with the minimum bactericidal concentration (MBC) of 25 microg/mL (74 microM). The time-kill curve study showed that dodecyl gallate was bactericidal against this MRSA strain at any growth stage. This activity was observed even in the chloramphenicol-treated cells, but the rate of decrease of cell number was slower than that in the exponentially growing cells. The bactericidal activity of medium-chain alkyl gallates was noted in combination with their ability to disrupt the native membrane-associated function nonspecifically as surface-active agents (surfactants) and to inhibit the respiratory electron transport. Subsequently, the same series of alkyl protocatechuates (3,4-dihydroxybenzoates) were studied and the results obtained are similar to those found for alkyl gallates. The length of the alkyl chain is not a major contributor but is related to the activity.