The toxicity of 1,3,4-thiadiazolium mesoionic derivatives on hepatocarcinoma cells (HepG2) is associated with mitochondrial dysfunction.

Mesoionic compounds, 4-phenyl-5-(4-X-cinnamoyl)-1,3,4-thiadiazolium-2-phenylamine chloride derivatives (MI-J: X = OH; MI-D: X = NO2), possess significant antitumor and cytotoxic effects on several cancer cells. In this work, we evaluated the cytotoxicity of MI-J and MI-D on human hepatocellular carcinoma (HepG2 cells) grown in either high glucose (HG) or galactose medium (GAL) to clarify whether the effects of mesoionics on mitochondrial bioenergetics are associated with their cytotoxicity in these cells. MI-J and MI-D (5-50 µM) decreased the viability of HepG2 cells in a dose- and time-dependent manner, as assessed by MTT, LDH release and dye with crystal violet assays. Both compounds at lower (5 µM) and intermediate (25 µM) concentrations were more toxic to cells grown in GAL medium. MI-J inhibited the basal state of respiration in HepG2 cells cultured in HG and GAL media; however, in GAL medium, this effect occurred at the lowest concentration (5 µM). A leak-state stimulus was observed only after incubation with MI-J (5 µM) for GAL medium. MI-D stimulated and inhibited the leak state in cells grown in HG medium at concentrations of 5 µM and 25 µM, respectively. In cells cultured in GAL medium, respiration was strongly inhibited by MI-D at the highest concentration (25 µM). In contrast, at 5 µM, the mesoionic inhibited the basal and uncoupled states at 30% and 50%, respectively. The inhibition of the basal state by MI-J and MI-D was consistent with the increase in lactate levels in both media, which was higher for the GAL medium. Both mesoionics slightly decreased pyruvate levels only in cells cultured in GAL medium. Additionally, MI-J (25 µM) reduced the ATP amount in cells cultured in both media, while MI-D (25 µM) promoted a reduction only in cells grown in GAL medium. Our results show that MI-J and MI-D depress mitochondrial respiration and consequently change metabolism and reduce ATP levels, effects associated with their toxicity in hepatocarcinoma cells.

Antineoplastic activity of a novel ruthenium complex against human hepatocellular carcinoma (HepG2) and human cervical adenocarcinoma (HeLa) cells.

Novel metal complexes have received much attention recently because of their potential anticancer activity. Notably, ruthenium-based complexes have emerged as good alternatives to the currently used platinum-based drugs for cancer therapy, with less toxicity and fewer side effects. The beneficial properties of Ru, which make it a highly promising therapeutic agent, include its variable oxidative states, low toxicity, and high selectivity for cancer cells. The present study evaluated the cytotoxic effects of a ruthenium complex, namely cis-[Ru(1,10-phenanthroline)2(imidazole)2]2+ (RuC), on human hepatocellular carcinoma (HepG2) and human cervical adenocarcinoma (HeLa) cells and analyzed metabolic parameters. RuC reduced HepG2 and HeLa cell viability at all tested concentrations (10, 50, and 100 nmol/L) at 48 h of incubation, based on the MTT, Crystal violet, and neutral red assays. The proliferation capacity of HepG2 cells did not recover, whereas HeLa cell proliferation partially recovered after RuC treatment. RuC also inhibited all states of cell respiration and increased the levels of the metabolites pyruvate and lactate in both cell lines. The cytotoxicity of RuC was higher than cisplatin (positive control) in both lineages. These results indicate that RuC affects metabolic functions that are related to the energy provision and viability of HepG2 and HeLa cells and is a promising candidate for further investigations that utilize models of human cervical adenocarcinoma and mainly hepatocellular carcinoma.

Effects of a new antiprotozoal drug, N,N'-diphenyl-4-methoxy-benzamidine, on energy-linked functions of rat liver mitochondria.

Amidines are chemically characterized by the presence of two nitrogen atoms that bind to the same carbon atom in its structure. Several biological activities have been ascribed to these compounds. Pentamidine, an aromatic diamidine, is effective in the treatment against Pneumocystis carinii and leishmaniasis, but it can also have severe side effects. New amidine derivatives have been synthesized, among them N,N'-diphenyl-4-methoxy-benzamidine (methoxyamidine), which is effective against Leishmania amazonensis (LD50 = 20 µM) and Trypanosoma cruzi (LD50 = 59 nM). In the present study, methoxyamidine toxicity was evaluated in isolated rat liver mitochondria at the same range of concentrations that exert antiprotozoal activity. In these organelles, actively oxidizing glutamate + malate inhibited state 3 respiration (25 nmol mg-1 of protein) by ∼15%. The sites of inhibition in the respiratory chain were complex I and the segment between ubiquinone and complex III. Methoxyamidine also stimulated state 4 respiration by ∼32% and ∼43% at 50 and 65 nmol mg-1 of protein, respectively. Its uncoupling effect was confirmed by a dose-dependent increase in oxygen consumption in state 4 respiration that was induced by oligomycin, reaching up to ∼69% (65 nmol mg-1 of protein) and an increase in ATPase activity in intact mitochondria by ∼27% and ∼83% at 50 and 65 nmol mg-1 protein, respectively. Swelling that was supported by the oxidation of glutamate + malate in the presence of sodium acetate was reduced by methoxyamidine by ∼16% and 32% at 50 and 65 nmol mg-1 protein, respectively. Mitochondrial swelling in the absence of substrate and in the presence of K+ and valinomycin was inhibited by ∼20% at the same concentrations, suggesting that methoxyamidine affects mitochondrial membrane permeability and fluidity. Our data show that methoxyamidine has slight effects on the energy-linked functions of isolated mitochondria at concentrations that correspond to the LD50 against Leishmania amazonensis and Trypanosoma cruzi. These findings may prompt further studies that evaluate methoxyamidine toxicity in vivo.

Cytotoxic effect of a mannogalactoglucan extracted from Agaricus bisporus on HepG2 cells.

A mannogalactoglucan (RK2-Ab; Mw 1.8×104gmol-1) composed by Man (27.3%), Gal (24.4%) and Glc (48.3%) was extracted and characterized from Agaricus bisporus, and its biological activity was evaluated on human hepatocarcinoma cells (HepG2). The partially-O-methylated alditol acetates together with the NMR data suggest the main chain to be composed of α-d-Galp (32.8%) and ß-d-Glcp (37.0%) units (1→6)-linked, with ß-d-Manp (14.6%), as non-reducing end units, substituting the side chains at O-2 (α-d-Galp units; 3.3%) and O-2 and O-4 (ß-d-Glcp units; 3.6%). (1→2)-linked ß-d-Glcp (2.7%) and ß-d-Manp (6.0%) can also be observed. RK2-Ab reduced cellular viability of HepG2 cells, by both, the MTT and lactate dehydrogenase release assays, promoted the increase of cytochrome c release and decrease of ATP content. Suggesting that the mannogalactoglucan from A. bisporus may have antitumor activity by inducing apoptosis by the mitochondrial death pathway, and could be used in cancer therapy.

Impairment of oxidative phosphorylation increases the toxicity of SYD-1 on hepatocarcinoma cells (HepG2).

Toxicity of the SYD-1 mesoionic compound (3-[4-chloro-3-nitrophenyl]-1,2,3-oxadiazolium-5-olate) was evaluated on human liver cancer cells (HepG2) grown in either high glucose (HG) or galactose (GAL) medium, and also on suspended cells kept in HG medium. SYD-1 was able to decrease the viability of cultured HepG2 cells in a dose-dependent manner, as assessed by MTT, LDH release and dye with crystal violet assays, but no effect was observed on suspended cells after 1-40 min of treatment. Respiration analysis was performed after 2 min (suspended cells) or 24 h (cultured cells) of treatment: no change was observed in suspended cells, whereas SYD-1 inhibited as well basal, leak and uncoupled states of the respiration in cultured cells with HG medium. These inhibitions were consistent with the decrease in pyruvate level and increase in lactate level. Even more extended results were obtained with HepG2 cells grown in GAL medium where, additionally, the ATP amount was reduced. Furthermore, SYD-1 appears not to be transported by the main ABC multidrug transporters. These results show that SYD-1 is able to change the metabolism of HepG2 cells, and suggest that its cytotoxicity is related to impairment of mitochondrial metabolism. Therefore, we may propose that SYD-1 is a potential candidate for hepatocarcinoma treatment.

New, highly potent and non-toxic, chromone inhibitors of the human breast cancer resistance protein ABCG2.

Breast cancer resistance protein (BCRP/ABCG2) is one of the major transporters involved in the efflux of anticancer compounds, contributing to multidrug resistance (MDR). Inhibition of ABCG2-mediated transport is then considered a promising strategy for overcoming MDR in tumors. We recently identified a chromone derivative, namely MBL-II-141 as a selective ABCG2 inhibitor, with relevant in vivo activity. Here, we report the pharmacomodulation of MBL-II-141, with the aim of identifying key pharmacophoric elements to design more potent selective and non-toxic inhibitors. Through rational structural modifications of MBL-II-141, using simple and affordable chemistry, we obtained highly active and easily-made inhibitors of ABCG2. Among the investigated compounds, derivative 4a, was found to be 3-fold more potent than MBL-II-141. It was similarly efficient as the reference inhibitor Ko143 but with the advantage of a lower intrinsic cytotoxicity, and therefore constitutes the best ABCG2 inhibitor ever reported displaying a very high therapeutic ratio.

Selective Cytotoxicity of 1,3,4-Thiadiazolium Mesoionic Derivatives on Hepatocarcinoma Cells (HepG2).

In this work, we evaluated the cytotoxicity of mesoionic 4-phenyl-5-(2-Y, 4-X or 4-X-cinnamoyl)-1,3,4-thiadiazolium-2-phenylamine chloride derivatives (MI-J: X=OH, Y=H; MI-D: X=NO2, Y=H; MI-4F: X=F, Y=H; MI-2,4diF: X=Y=F) on human hepatocellular carcinoma (HepG2), and non-tumor cells (rat hepatocytes) for comparison. MI-J, M-4F and MI-2,4diF reduced HepG2 viability by ~ 50% at 25 µM after 24-h treatment, whereas MI-D required a 50 µM concentration, as shown by 3-(4,5-dimethythiazol-2-yl)-2,5-diphenyltetrazolium bromide assays. The cytotoxicity was confirmed with lactate dehydrogenase assay, of which activity was increased by 55, 24 and 16% for MI-J, MI-4F and MI-2,4diF respectively (at 25 µM after 24 h). To identify the death pathway related to cytotoxicity, the HepG2 cells treated by mesoionic compounds were labeled with both annexin V and PI, and analyzed by flow cytometry. All compounds increased the number of doubly-stained cells at 25 µM after 24 h: by 76% for MI-J, 25% for MI-4F and MI-2,4diF, and 11% for MI-D. It was also verified that increased DNA fragmentation occurred upon MI-J, MI-4F and MI-2,4diF treatments (by 12%, 9% and 8%, respectively, at 25 µM after 24 h). These compounds were only weakly, or not at all, transported by the main multidrug transporters, P-glycoprotein, ABCG2 and MRP1, and were able to slightly inhibit their drug-transport activity. It may be concluded that 1,3,4-thiadiazolium compounds, especially the hydroxy derivative MI-J, constitute promising candidates for future investigations on in-vivo treatment of hepatocellular carcinoma.

Sydnone SYD-1 affects the metabolic functions of isolated rat hepatocytes.

Previously, we demonstrated that sydnone SYD-1 (3-[4-chloro-3-nitrophenyl]-1,2,3-oxadiazolium-5-olate) impairs the mitochondrial functions linked to energy provision and suggested that this effect could be associated with its antitumor activity. Herein, we evaluated the effects of SYD-1 (25 and 50 µM) on rat hepatocytes to determine its cytotoxicity on non-tumor cells. SYD-1 (25 and 50 µM) did not affect the viability of hepatocytes in suspension after 1-40 min of incubation. However, the viability of the cultured hepatocytes was decreased by ∼66% as a consequence of treatment with SYD-1 (50 µM) for 18 h. Under the same conditions, SYD-1 promoted an increase in the release of LDH by ∼19%. The morphological changes in the cultured cells treated with SYD-1 (50 µM) were suggestive of cell distress, which was demonstrated by the presence of rounded hepatocytes, cell fragments and monolayer impairment. Furthermore, fluorescence microscopy showed an increase in the annexin label after treatment with SYD-1 (50 µM), suggesting that apoptosis had been induced in these cells. SYD-1 did not affect the states of respiration in the suspended hepatocytes, but the pyruvate levels were decreased by ∼36%, whereas the lactate levels were increased by ∼22% (for the 50 µM treatment). The basal and uncoupled states of respiration of the cultured hepatocytes were inhibited by ∼79% and ∼51%, respectively, by SYD-1 (50 µM). In these cells, SYD-1 (50 µM) increased the pyruvate and lactate levels by ∼84% and ∼16%, respectively. These results show that SYD-1 affects important metabolic functions related to energy provision in hepatocytes and that this effect was more pronounced on cells in culture than those in suspension.

The antioxidant effect of the mesoionic compound SYD-1 in mitochondria.

The sydnone SYD-1 (3-[4-chloro-3-nitrophenyl]-1,2,3-oxadiazolium-5-olate] possesses important antitumor activity against Sarcoma 180 and Ehrlich tumors. We previously showed that SYD-1 depresses mitochondrial phosphorylation efficiency, which could be involved in its antitumoral activity. Considering the important role of mitochondria in the generation of reactive oxygen species (ROS) and the involvement of ROS in cell death mechanisms, we evaluated the effects of SYD-1 on oxidative stress parameters in rat liver mitochondria. SYD-1 (0.5 and 0.75µmolmg(-1) protein) inhibited the lipoperoxidation induced by Fe(3+)/ADP-oxoglutarate by approximately 75% and promoted total inhibition at the highest concentration tested (1.0µmolmg(-1) protein). However, SYD-1 did not affect lipoperoxidation started by peroxyl radicals generated by α-α'-azodiisobutyramidine dihydrochloride. The mesoionic compound (0.25-1.0µmolmg(-1) protein) demonstrated an ability to scavenge superoxide radicals, decreasing their levels by 9-19%. The activities of catalase and superoxide dismutase did not change in the presence of SYD-1 (0.25-1.0µmolmg(-1) protein). SYD-1 inhibited mitochondrial swelling dependent on the formation/opening of the permeability transition pore induced by Ca(2+)/phosphate by approximately 30% (1.0µmolmg(-1) protein). When Ca(2+)/H2O2 were used as inducers, SYD-1 inhibited swelling only by approximately 12% at the same concentration. NADPH oxidation was also inhibited by SYD-1 (1.0µmolmg(-1) of protein) by approximately 48%. These results show that SYD-1 is able to prevent oxidative stress in isolated mitochondria and suggest that the antitumoral activity of SYD-1 is not mediated by the increasing generation of ROS.

Cytotoxic effect of Agaricus bisporus and Lactarius rufus ß-D-glucans on HepG2 cells.

The cytotoxic activity of ß-D-glucans isolated from Agaricus bisporus and Lactarius rufus fruiting bodies was evaluated on human hepatocellular carcinoma cells (HepG2). NMR and methylation analysis suggest that these ß-d-glucans were composed of a linear (1→6)-linked and a branched (1→3), (1→6)-linked backbone, respectively. They both decreased cell viability at concentrations of up to 100 µg mL(-1), as shown by MTT assay. The amount of LDH released and the analysis of cell morphology corroborated these values and also showed that the ß-D-glucan of L. rufus was more cytotoxic to HepG2 cells than that of A. bisporus. The treatment of HepG2 cells with L. rufus and A. bisporus ß-D-glucans at a dose of 200 µg mL(-1) for 24h promoted an increase of cytochrome c release and a decrease of ATP content, suggesting that these polysaccharides could promote cell death by apoptosis. Both ß-D-glucans were tested against murine primary hepatocytes at a dose of 200 µg mL(-1). The results suggest that the L. rufus ß-d-glucan was as cytotoxic for hepatocytes as for HepG2 cells, whereas the A. bisporus ß-D-glucan, under the same conditions, was cytotoxic only for HepG2 cells, suggesting cell selectivity. These results open new possibilities for use of mushroom ß-D-glucans in cancer therapy.

Interaction of 1,3,4-thiadiazolium mesoionic derivatives with mitochondrial membrane and scavenging activity: Involvement of their effects on mitochondrial energy-linked functions.

The aim of this work was to assess the significance of the interaction of the 1,3,4-thiadiazolium derivatives MI-J, MI-4F and MI-2,4diF with mitochondrial membrane and their effects on energy-linked functions. Mitochondrial swelling in the absence of substrate was inhibited by all derivatives; however, the fluorine derivatives were most effective. MI-4F decreased swelling by ~32% even at the lowest concentration (65 nmol mg(-1) protein), reaching ~67% at the concentration of 130 nmol mg(-1) protein. Swelling of mitochondria in the presence of oxidizable substrates was also strongly decreased by all derivatives. This effect was more pronounced when using glutamate plus malate, and also fluorine derivatives, which promoted complete inhibition at all concentrations (6.5-130 nmol mg(-1) protein). Swelling occurred when succinate was the substrate in the presence of MI-J (6.5-65 nmol mg(-1) protein); however, the shrinkage rate was strongly decreased. MI-4F and MI-2,4diF also inhibited swelling, with total inhibition occurring at a concentration of 65 nmol mg(-1) protein. Lipid peroxidation induced by Fe(3+)-ADP/2-oxoglutarate in isolated mitochondria was inhibited time- and dose-dependently by the derivatives, reaching complete inhibition at the highest concentration (80 nmol mg(-1) protein). However, when lipid peroxidation was initiated by peroxyl radicals generated from AAPH, the inhibition was less intense, reaching ~50%, ~40% and ~58% with MI-J, MI-4F and MI-2,4diF (80 nmol mg(-1) protein), respectively. The mesoionic compounds also showed superoxide radical scavenging ability of ~22%, ~32% and ~40% (80 nmol mg(-1) protein), respectively. Fluorescence polarization experiments showed that the derivatives are able to enter the bilayer, decreasing its fluidity in the hydrophobic DMPC membrane region and ordering the fluid phase. Our results suggest that MI-J, MI-4F and MI-2,4diF interact significantly, albeit in different modes, with mitochondrial membrane, and that fluorine derivatives seem to alter the membrane's properties more markedly.

Comparative study of the effects of 1,3,4-thiadiazolium mesoionic derivatives on energy-linked functions of rat liver mitochondria.

The main goal of this work was to investigate the relationship between the effects of three new 1,3,4-thiadiazolium mesoionic derivatives on mitochondrial bioenergetics and their previously described chemical structure and antimelanoma activity. The 4-phenyl-5-(2'-Y, 4'-X or 4'-X-cinnamoyl)-1,3,4-thiadiazolium-2-phenylamine chlorides differed from each other only in the cinnamoyl ring substituent: MI-J, X=OH; MI-F, X=F; MI-2,4diF X=Y=F. The state 3 respiratory rate was strongly decreased by all derivatives, reaching total inhibition of MI-4F and MI-2,4diF (130nmolmg(-1) protein), when glutamate plus malate were used as substrate. State 3 inhibition was less accentuated with succinate as substrate. Analyses of segments of the respiratory chain indicated complexes I and IV as sites inhibited by the derivatives. State 4 respiration was strongly stimulated by the three derivatives, and was characterized as an uncoupling effect, which was more intense for MI-4F. This stimulus was so pronounced that the values of RCC and ADP/O ratio were only calculated for the lowest concentration (6.5nmolmg(-1) protein). In intact mitochondria, the ATPase activity was increased dramatically by approximately 120%, approximately 207% and approximately 261% for MI-J, MI-4F and MI-2,4diF (32.5nmolmg(-1) protein), respectively. In conclusion, the presence of fluorine substituent in the cinnamoyl ring intensifies the effect of mesoionic compounds on mitochondrial functions and, in this context, hydrophobicity is more important than the electronic effect, which was correlated to antimelanoma activity described previously for these compounds.

Celecoxib prevents tumor growth in an animal model by a COX-2 independent mechanism.

PURPOSE: Nonsteroidal antiinflammatory drugs (NSAIDs) have been shown to reduce cell growth in several tumors. Among these possible antineoplastic drugs are cyclooxygenase- 2 (COX-2)-selective drugs, such as celecoxib, in which antitumoral mechanisms were evaluated in rats bearing Walker-256 (W256) tumor. METHODS: W256 carcinosarcoma cells were inoculated subcutaneously (10(7) cells/rat) in rats submitted to treatment with celecoxib (25 mg kg(-1)) or vehicle for 14 days. Tumor growth, body-weight gain, and survival data were evaluated. The mechanisms, such as COX-2 expression and activity, oxidative stress, by means of enzymes and lipoperoxidation levels, and apoptosis mediators were also investigated. RESULTS: A reduction in tumor growth and an increased weight gain were observed. Celecoxib provided a higher incidence of survival compared with the control group. Cellular effects are probably COX-2 independent, because neither enzyme expression nor its activity, measured by tumoral PGE(2), showed significant difference between groups. It is probable that this antitumor action is dependent on an apoptotic way, which has been evaluated by the expression of the antiapoptotic protein Bcl-xL, in addition to the cellular changes observed by electronic microscopy. Celecoxib has also a possible involvement with redox homeostasis, because its administration caused significant changes in the activity of oxidative enzymes, such as catalase and superoxide dismutase. CONCLUSION: These results confirm the antitumor effects of celecoxib in W256 cancer model, contributing to elucidating its antitumoral mechanism and corroborating scientific literature about its effect on other types of cancer.