The mesoionic compound MI-D changes energy metabolism and induces apoptosis in T98G glioma cells.

The mesoionic compound 4-phenyl-5-(4-nitro-cinnamoyl)-1,3,4-thiadiazolium-2-phenylamine chloride (MI-D) impairs mitochondrial oxidative phosphorylation and has a significant antitumour effect against hepatocarcinoma and melanoma. This study evaluated the cytotoxic effect of MI-D on T98G glioblastoma cells and investigated whether the impairment of oxidative phosphorylation promoted by MI-D is relevant to its cytotoxic effect. The effects of MI-D on T98G cells cultured in high glucose Dulbecco's modified Eagle's medium (DMEM) HG (glycolysis-dependent) and galactose plus glutamine-supplemented Dulbecco's modified Eagle's medium (DMEM) GAL (oxidative phosphorylation-dependent) were compared. T98G cells grown in DMEM GAL medium exhibited higher respiration rates and citrate synthase activity and lower lactate levels, confirming the metabolic shift to oxidative phosphorylation in these cells. MI-D significantly decreased the cell viability in a dose-dependent manner in both media; however, T98G cells cultured in DMEM GAL medium were more susceptible. The mesoionic significantly inhibited mitochondrial oxidative phosphorylation of glioma cells in both media. At the same time, lactate levels were not altered, indicating an absence of compensatory glycolysis activation. Additionally, MI-D increased the citrate synthase activity of cells in both media, which in DMEM HG-cultivated cells was followed by citrate accumulation. Apoptosis dependent on caspase-3 mediated the toxicity of MI-D on T98G cells. The higher susceptibility of glioma cells cultured in DMEM GAL medium to MI-D indicates that the impairment of mitochondrial functions is involved in mesoionic cytotoxicity. The results of this study indicate the potential use of MI-D for glioblastoma treatment.

Hypoxia protects against the cell death triggered by oxovanadium-galactomannan complexes in HepG2 cells.

BACKGROUND: Polysaccharides from various sources have been used in traditional medicine for centuries. The beneficial pharmacological effects of plant-derived polysaccharides include anti-tumor activity. METHODS: Here, we evaluated the anti-cancer effect of the MSAGM:VO complex under hypoxic conditions (1% oxygen). MSAGM:VO is a complex of the hydrolysate of galactomannan (MSAGM) from Schizolobium amazonicum with oxovanadium (IV/V). The hepatocellular carcinoma (HCC) cell line HepG2 was selected as HCC are one of the most hypoxic solid tumors. RESULTS: Our results showed that the strong apoptotic activity of MSAGM:VO observed in HepG2 cells under normoxic conditions was completely lost under hypoxic conditions. We found a dynamic balance between the pro- and anti-apoptotic members of the Bcl-2 protein family. The expressions of anti-apoptotic Mcl-1 and Bcl-XL increased in hypoxia, whereas the expression of pro-apoptotic Bax decreased. MSAGM:VO strongly induced autophagy, which was previously characterized as a pro-survival mechanism in hypoxia. These results demonstrate total elimination of the anti-cancer activity of MSAGM:VO with activation of autophagy under conditions of hypoxia. CONCLUSION: Although this study is a proof-of-concept of the impact of hypoxia on the potential of polysaccharides, further study is encouraged. The anti-tumor activity of polysaccharides could be achieved in normoxia or through raising the activity of the immune system. In addition, combination strategies for therapy with anti-autophagic drugs could be proposed.

Unveiling novel targets in melanoma under melanogenesis stimulation and photodynamic therapy by redox proteomics.

Melanogenesis-stimulated B16-F10 cells enter in a quiescent state, present inhibited mitochondrial respiration and increased reactive oxygen species levels. These alterations suggest that these cells may be under redox signaling, allowing tumor survival. The aim of this study was to evaluate redox-modified proteins in B16-F10 cells after melanogenesis stimulation and rose bengal-photodynamic therapy (RB-PDT). A redox proteomics label-free approach based on the biotin switch assay technique with biotin-HPDP and N-ethylmaleimide was used to assess the thiol-oxidized protein profile. Aconitase was oxidized at Cys-448 and Cys-451, citrate synthase was oxidized at Cys-202 and aspartate aminotransferase (Got2) was oxidized at Cys-272 and Cys-274, exclusively after melanogenesis stimulation. After RB-PDT, only guanine nucleotide-binding protein subunit beta-2-like 1 (Gnb2l1) was oxidized (Cys-168). In contrast, melanogenesis stimulation followed by RB-PDT led to the oxidation of different cysteines in Gnb2l1 (Cys-153 and Cys-249). Besides that, glyceraldehyde-3-phosphate dehydrogenase (Gapdh) presented oxidation at Cys-245, peptidyl-prolyl cis-trans isomerase A (Ppia) was oxidized at Cys-161 and 5,6-dihydroxyindole-2-carboxylic acid oxidase (Tyrp1) was oxidized at Cys-65, Cys-30, and Cys-336 after melanogenesis stimulation followed by RB-PDT. The redox alterations observed in murine melanoma cells and identification of possible target proteins are of great importance to further understand tumor resistance mechanisms.

Chemical structure and biological activity of the (1 → 3)-linked ß-D-glucan isolated from marine diatom Conticribra weissflogii.

Several polysaccharides are considered to be "biological response modifiers" (BRM) - these refer to biomolecules that augment immune responses and can be derived from a variety of sources. Microalgae produce a diverse range of polysaccharides and could be an excellent source of BRM. Here, we describe the chemical structure and biological activity of water-soluble polysaccharide isolated from the marine diatom Conticribra weissflogii. Using chemical and NMR spectroscopic methods, the polysaccharide was identified as a (1 â†’ 3)-linked ß-D-glucan with a low proportion of C-6 substitution by single ß-glucose units. The biological activity of this low molecular weight ß-glucan (11.7 kDa) was investigated with respect to glioblastoma cell lines (U87 MG and U251) and macrophages (RAW 264.7). We observed that this ß-D-glucan did not exhibit cytotoxic activity against glioblastoma cells, but did enhance the phagocytic activity of macrophages, suggesting that it possesses immunomodulatory properties.

Effect of sydnone SYD-1 on certain functions of LPS-stimulated macrophages.

In an earlier article, we demonstrated that sydnone SYD-1 (3-[4-chloro-3-nitrophenyl]-1,2,3-oxadiazolium-5-olate) inhibits electron transport in the respiratory chain and uncouples oxidative phosphorylation, and postulated that these effects are probably involved in its antitumor activity. We now report the effect of SYD-1 on certain macrophage functions, considering the important role of these cells in inflammatory response and also the relevant anti-inflammatory activity reported for some sydnones. Incubation of macrophages with SYD-1 (5-100 µM) for 48 h did not affect the cell viability up to a concentration of 50 µM. However, at the highest concentration (100 µM), the compound decreased macrophage viability by ~20%. In assays involving 2 h and 24 h of incubation, SYD-1 (5-100 µM) did not affect the cell viability. The incubation of macrophages with the compound for 2 h promoted a dose-dependent reduction of phagocytic activity of up to ~65% (100 µM). SYD-1 (100 µM) was also able to increase the production of superoxide anion (~50%). In the absence of LPS, SYD-1 decreased NO production dose-dependently by up to ~80% (100 µM). When SYD-1 and LPS were incubated concomitantly, the decrease of NO promoted by SYD was the most pronounced, reaching up to ~98% at the same concentration (50 µM). SYD-1 dose-dependently suppressed IL-6 secretion by LPS-stimulated macrophages, reaching up to ~90% of inhibition at the highest concentration (100 µM). These results indicate that SYD-1 promotes effects similar to those described for anti-inflammatory and immunosuppressive drugs, thus motivating further studies to clarify the mechanisms involved in this activity.

Diagnosis and prediction of COVID-19 severity: can biochemical tests and machine learning be used as prognostic indicators?

OBJECTIVE: This study aimed to implement and evaluate machine learning based-models to predict COVID-19' diagnosis and disease severity. METHODS: COVID-19 test samples (positive or negative results) from patients who attended a single hospital were evaluated. Patients diagnosed with COVID-19 were categorised according to the severity of the disease. Data were submitted to exploratory analysis (principal component analysis, PCA) to detect outlier samples, recognise patterns, and identify important variables. Based on patients' laboratory tests results, machine learning models were implemented to predict disease positivity and severity. Artificial neural networks (ANN), decision trees (DT), partial least squares discriminant analysis (PLS-DA), and K nearest neighbour algorithm (KNN) models were used. The four models were validated based on the accuracy (area under the ROC curve). RESULTS: The first subset of data had 5,643 patient samples (5,086 negatives and 557 positives for COVID-19). The second subset included 557 COVID-19 positive patients. The ANN, DT, PLS-DA, and KNN models allowed the classification of negative and positive samples with >84% accuracy. It was also possible to classify patients with severe and non-severe disease with an accuracy >86%. The following were associated with the prediction of COVID-19 diagnosis and severity: hyperferritinaemia, hypocalcaemia, pulmonary hypoxia, hypoxemia, metabolic and respiratory acidosis, low urinary pH, and high levels of lactate dehydrogenase. CONCLUSION: Our analysis shows that all the models could assist in the diagnosis and prediction of COVID-19 severity.

Cytotoxic effect of xyloglucan and oxovanadium (IV/V) xyloglucan complex in HepG2 cells.

It is well known that the chemical structure of polysaccharides is important to their final biological effect. In this study we investigated the cytotoxic effect of xyloglucan from Copaifera langsdorffii seeds (XGC) and its complex with oxovanadium (XGC:VO) on hepatocellular carcinoma cells (HepG2). After 72 h of incubation, XGC and XGC:VO (200 µg/mL) reduced cell viability in ~20% and ~40%, respectively. At same conditions, only XGC:VO increased in ~20% the LDH enzyme release. In permeabilized cells, incubated with XGC and XGC:VO (200 µg/mL) for 72 h, NADH oxidase activity was reduced by ~45% with XGC and XGC:VO. The succinate oxidase activity was reduced by ~35% with XGC and ~65% with XGC:VO, evidencing that polysaccharide complexation with vanadium could intensify its effects on the respiratory chain. According to this result, the mitochondrial membrane potential was also reduced by ~9% for XGC and ~30% for XGC:VO, when compared to the control group. Interestingly, ATP levels were more elevated for XGC:VO in respect to XGC, probably due the enhance in glycolytic flux evidenced by increased levels of lactate. These results show that the xyloglucan complexation with oxovanadium (IV/V) potentiates the cytotoxic effect of the native polysaccharide, possibly by impairment of oxidative phosphorylation.

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.

Cytotoxicity of xyloglucan from Copaifera langsdorffii and its complex with oxovanadium (IV/V) on B16F10 cells.

The aim of this study was to investigate the effects of xyloglucan extracted from Copaifera langsdorffii seeds (XGC) and its complex with oxovanadium (XGC:VO) in murine melanoma B16F10 cells. The formation of complexes was followed by potentiometric titration and further demonstrated by 51V RMN. The viability and proliferation of B16F10 cells were reduced up 50% by the xyloglucan and its complex, both at 200 µg/mL, from 24 to 72 h. Cytotoxic effects of XGC and XGC:VO do not involve changes in cell cycle progression. Only XGC:VO (200 µg/mL) promoted the cell death evidenced by annexin V stain. XGC increased the respiration and lactate levels in melanoma cells, while XGC:VO reduced about 50% the respiration and levels of pyruvate, without alter the lactate levels, indicating that both xyloglucan preparations interfere with the metabolism of B16F10 cells. No change in activity of the enzyme hexokinase and expression of pyruvate kinase M2 was observed. XGC:VO (200 µg/mL) negatively modulated the expression of the ß subunit of ATP synthase. The results demonstrate that the cytotoxicity of XGC and XGC:VO on murine melanoma B16F10 cells can be related to the impairment of the mitochondrial functions linked to energy provision.

Production of cachexia mediators by Walker 256 cells from ascitic tumors.

In neoplasic cachexia, chemical mediators seem to act as initiators or perpetuators of this process. Walker 256 cells, whose metabolic properties have so far been little studied with respect to cancer cachexia, are used as a model for the study of this syndrome. The main objective of this research was to pinpoint the substances secreted by these cells that may contribute to the progression of the cachectic state. Since inflammatory mediators seem to be involved in the manifestation of this syndrome, the in vitro production of nitric oxide (NO), cytokines (tumor necrosis factor alpha (TNF-alpha) and interleukin-6 (IL-6)), and prostaglandin E2 (PGE2) was evaluated in Walker 256 cells isolated from ascitic tumors. After 4 or 5 h, a significant increase in NO production was observed (2.55 +/- 1.56 and 4.05 +/- 1.99 nmol NO per 10(7) cells, respectively). When isolated from a 6-day-old tumor, a significantly lower production of IL-6 and higher production of TNF-alpha than in cells from a 4-day-old tumor were observed, indicating a relationship between the production of cytokines and the time of tumor development after implantation. Considerable production of PGE(2) by Walker 256 cells isolated from the 6-day-old tumor was also observed. Polyamines were also determined in Walker 256 cells. Levels of putrescine, spermidine, and spermine did not show significant differences in tumors developed during 4 or 6 days. Direct evidence of the release of proinflammatory cytokines and PGE2 by Walker 256 cells suggests that these mediators can drive the cachectic syndrome in the host, the effect being dependent on tumor development time.

Effect of sydnone SYD-1, a mesoionic compound, on energy-linked functions of rat liver mitochondria.

An important antitumour effect of SYD-1 (3-[4-chloro-3-nitrophenyl]-1,2,3-oxadiazolium-5-olate) has been shown. We now report the effects of this mesoionic compound on mitochondrial metabolism. SYD-1 (1.5 micromol mg(-1) protein) dose-dependently inhibited the respiratory rate by 65% and 40% in state 3 using sodium glutamate and succinate, respectively, as substrates. Phosphorylation efficiency was depressed by SYD-1, as evidenced by stimulation of the state 4 respiratory rate, which was more accentuated with glutamate ( approximately 180%) than with succinate ( approximately 40%), with 1.5 micromol mg(-1) protein of SYD-1. As a consequence of the effects on states 3 and 4, the RCC and ADP/O ratios were lowered by SYD-1 using both substrates, although this effect was stronger with glutamate. The formation of membrane electrical potential was inhibited by approximately 50% (1.5 micromol SYD-1mg(-1) protein). SYD-1 interfered with the permeability of the inner mitochondrial membrane, as demonstrated by assays of mitochondrial swelling in the presence of sodium acetate and valinomycin +K(+). SYD-1 (1.5 micromol mg(-1) protein) inhibited glutamate completely and succinate energized-mitochondrial swelling by 80% in preparations containing sodium acetate. The swelling of de-energized mitochondria induced by K(+) and valinomycin was inhibited by 20% at all concentrations of SYD-1. An analysis of the segments of the respiratory chain suggested that the SYD-1 inhibition site goes beyond the complex I and includes complexes III and IV. Glutamate dehydrogenase was inhibited by 20% with SYD-1 (1.5 micromol mg(-1) protein). The hydrolytic activity of complex F(1)F(o) ATPase in intact mitochondria was greatly increased ( approximately 450%) in the presence of SYD-1. Our results show that SYD-1 depresses the efficiency of electron transport and oxidative phosphorylation, suggesting that these effects may be involved in its antitumoural effect.

Degalactosylation of xyloglucans modify their pro-inflammatory properties on murine peritoneal macrophages.

Considering the potential applications of partially degalactosylated xyloglucans as a drug delivery vehicle and reconstruction of tissues, the aim of this study was to investigate whether degalactosylated xyloglucans are immunologically active. The effects of the degalactosylated xyloglucan from seeds of Copaifera langsdorffii (XGCd), Hymenaea courbaril (XGJd), and Tamarindus indica (XGTd) on murine peritoneal macrophages in vitro were evaluated. XGCd, XGJd, and XGTd stimulated NO production in a dose-dependent manner reaching ∼280% for XGTd at 50µg/mL. Regarding cytokines production, XGJd at 50µg/mL increased IL-1ß level by ∼100% and XGCd (10µg/mL) enhanced IL-6 level by 40%. At 10µg/mL, XGTd increased TNF- α and IL-1ß levels by 104 and 2370%, respectively, as compared to the control group. For IL-6, XGTd enhanced this cytokine production by 80% at all concentrations tested. XGTd exhibited the most intensive effects on the production of pro-inflammatory mediators by peritoneal macrophages. All degalactosylated xyloglucans evaluated showed not to be biologically inert. Thus, this finding is relevant for groups that are investigating the use of degalactosylated xyloglucan from T. indica for drug delivery and reconstruction of tissues. The effects observed could contribute to potentiate the immune system against infections or toxicity to tumor cells.

Disrupting glucose-6-phosphate isomerase fully suppresses the "Warburg effect" and activates OXPHOS with minimal impact on tumor growth except in hypoxia.

As Otto Warburg first observed, cancer cells largely favor fermentative glycolysis for growth even under aerobic conditions. This energy paradox also extends to rapidly growing normal cells indicating that glycolysis is optimal for fast growth and biomass production. Here we further explored this concept by genetic ablation of fermentative glycolysis in two fast growing cancer cell lines: human colon adenocarcinoma LS174T and B16 mouse melanoma. We disrupted the upstream glycolytic enzyme, glucose-6-phosphate isomerase (GPI), to allow cells to re-route glucose-6-phosphate flux into the pentose-phosphate branch. Indeed, GPI-KO severely reduced glucose consumption and suppressed lactic acid secretion, which reprogrammed these cells to rely on oxidative phosphorylation and mitochondrial ATP production to maintain viability. In contrast to previous pharmacological inhibition of glycolysis that suppressed tumor growth, GPI-KO surprisingly demonstrated only a moderate impact on normoxic cell growth. However, hypoxic (1% O2) cell growth was severely restricted. Despite in vitro growth restriction under hypoxia, tumor growth rates in vivo were reduced less than 2-fold for both GPI-KO cancer cell lines. Combined our results indicate that exclusive use of oxidative metabolism has the capacity to provide metabolic precursors for biomass synthesis and fast growth. This work and others clearly indicate that metabolic cancer cell plasticity poses a strong limitation to anticancer strategies.

Toxicity of native and oxovanadium (IV/V) galactomannan complexes on HepG2 cells is related to impairment of mitochondrial functions.

Polysaccharides and vanadium compounds have been studied due to their antitumor potential. In this study, the cytotoxic effects of galactomannan preparations on HepG2 cells were investigated. Native galactomannan from S. amazonicum (SAGM) and its modified form (MSAGM) were complexed with oxovanadium resulting in SAGM:VO and MSAGM:VO, respectively. The complexation was confirmed by NMR, FTIR, and AAS. SAGM and MSAGM:VO (250µg/mL) after 72h decreased viability by 51% and 58%, respectively, while the inhibition of the HepG2 cell proliferation was of ∼27% and ∼46%, respectively. SAGM and MSAGM:VO (250µg/mL) significantly inhibited all states of respiration (basal: 85% and 63%; uncoupled: 90% and 70%; and leak: 30% and 58%) after 72h. ROS levels increased by ∼149% after the treatment with MSAGM:VO (250µg/mL) for 72h, while ΔΨm decreased by ∼50%. Our results indicate that galactomannan preparations from S. amazonicum, especially SAGM and the MSAGM:VO complex, could be considered as potential antitumor drugs for further investigations, once they have the ability to make HepG2 cells susceptible to death by affecting vital cellular processes such as respiration and ROS generation.

Leishmanicidal activity of polysaccharides and their oxovanadium(IV/V) complexes.

The parasites of the genus Leishmania cause a range of leishmaniasis diseases, whose treatment is impaired due to intramacrophage parasites living in the mammalian host. Immunostimulation has been considered an important strategy to leishmaniasis treatment. The immunomodulatory effects of the polysaccharides arabinogalactan (ARAGAL), galactomannan (GMPOLY), and xyloglucan (XGJ), as well as their oxovanadium (IV/V) complexes (ARAGAL:VO, GMPOLY:VO, and XGJ:VO) were evaluated on peritoneal macrophages. At 25 µg/mL of GMPOLY:VO and of XGJ:VO, and 10 µg/mL of ARAGAL:VO, nitric oxide (NO) production by the macrophages was not altered compared with the control group. All polymers increased the production of interleukins 1 beta and 6 (IL-1ß and IL-6), but the oxovanadium complexes were more potent activators of these mediators. ARAGAL:VO 10 µg/mL, GMPOLY:VO and XGJ:VO 25 µg/mL led to an increase of 562%, 1054%, and 523% for IL-1ß, respectively. For IL-6 at the same concentration, the levels increased by 539% and 794% for ARAGAL:VO and GMPOLY:VO, respectively. Polysaccharides and their oxovanadium complexes exhibited important leishmanicidal effects on amastigotes of Leishmania (L.) amazonensis. The native and complexed polymers reduced the growth of promastigote-form Leishmania by ∼60%. This effect was reached at concentrations 12 times lower than that observed for Glucantime (300 µg/mL promoted an inhibition of ∼60%). The 50% inhibitory concentration (IC50) values for the complexes were determined. XGJ:VO showed the lowest IC50 value (6.2 µg/mL; 0.07 µg/mL of vanadium), which for ARAGAL:VO was 6.5 µg/mL (0.21 µg/mL of vanadium) and 7.3 µg/mL (0.06 µg/mL of vanadium) for GMPOLY:VO. The upregulation of IL-1ß and IL-6 release and downregulation of NO production by macrophages and the important leishmanicidal effect are essential to stablish their potential use against this pathology.

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.

Acid heteropolysaccharides with potent antileishmanial effects.

The present study investigated in vitro the effects of sulphated heterorhamnan (Go3), iota-/nu-carrageenans (G3d and EHW-I) and arabinogalactan (ARAGAL) polysaccharides on macrophage activation and inhibition of intracellular amastigotes of Leishmania (L.) amazonensis. All the sulphated polysaccharides (Go3, G3d and EHW-I) promoted increased nitric oxide production varying from 71 to 110%. The leishmanicidal activity of all compounds was compared to the inhibition effect of Meglumine Antimoniate at 300µg/mL (∼79%), used as positive control. Inhibition of Leishmania (L.) amazonensis growth was 55% with 5µg/mL of Go3, 50% and 98% to G3d and EHW-I, respectively at 10µg/mL, and 88% with 10µg/mL of ARAGAL. The superoxide anion scavenging activity for the sulphated polysaccharides varied from approximately 30-55% at 10µg/mL. In conclusion, the results of the present study indicate the promising potential of these polysaccharides for the development of new alternative therapeutic agents against leishmaniasis.

Isolation of an arabinogalactan from Endopleura uchi bark decoction and its effect on HeLa cells.

Endopleura uchi is a native plant from the Amazon used in popular medicine to treat myomas. A crude polysaccharide (AGb) obtained from E. uchi bark decoction was purified, yielding a type II arabinogalactan (AG) that was characterized by chemical and spectroscopic methods. AG was evaluated for its cytotoxic effects on HeLa cells. AG (5-500 µg/ml) reduced cell viability at 48 and 72 h (approximately 20%) but not in a dose-dependent manner. Cell proliferation was also reduced by AG, with a 25% inhibition (100 µg/ml) at 72 h. The results suggest that the cytotoxicity exhibited by AG does not involve pathways related to the cell cycle.

Artemisia absinthium and Artemisia vulgaris: a comparative study of infusion polysaccharides.

The aerial parts of Artemisia absinthium and Artemisia vulgaris are used in infusions for the treatment of several diseases. Besides secondary metabolites, carbohydrates are also extracted with hot water and are present in the infusions. The plant carbohydrates exhibit several of therapeutic properties and their biological functions are related to chemical structure. In this study, the polysaccharides from infusions of the aerial parts of A. absinthium and A. vulgaris were isolated and characterized. In the A. absinthium infusion, a type II arabinogalactan was isolated. The polysaccharide had a Gal:Ara ratio of 2.3:1, and most of the galactose was (1 → 3)- and (1 → 6)-linked, as typically found in type II arabinogalactans. In the A. vulgaris infusion, an inulin-type fructan was the main polysaccharide. NMR analysis confirmed the structure of the polymer, which is composed of a chain of fructosyl units ß-(2 ← 1) linked to a starting α-d-glucose unit.

Macrophage activation and leishmanicidal activity by galactomannan and its oxovanadium (IV/V) complex in vitro.

Compounds that activate macrophage antimicrobial activity are potential targets for treatment of leishmaniasis. The present study investigated the in vitro immunomodulatory effects of a galactomannan (GALMAN-A) isolated from seeds of Mimosa scabrella and its oxovanadium (IV/V) complex (GALMAN-A:VO(2+)/VO(3+)) on macrophage activity. GALMAN-A increased nitric oxide levels by ~33% at a concentration of 250µg/ml, while GALMAN-A:VO(2+)/VO(3+) decreased nitric oxide levels by ~33% at a concentration of 50µg/ml. Furthermore, GALMAN-A increased interleukin-1 beta (IL-1ß) and interleukin-6 (IL-6) levels by 5.5 and 2.3 times, respectively, at a concentration of 25µg/ml; at the same concentration, GALMAN-A:VO(2+)/VO(3+) promoted an increase in IL-1ß and IL-6 production by 8 and 5.5 times, respectively. However, neither GALMAN-A nor GALMAN-A:VO(2+)/VO(3+) affected tumor necrosis factor alpha (TNF-α) or interleukin-10 (IL-10) levels. Importantly, both GALMAN-A and GALMAN-A:VO(2+)/VO(3+) exhibited leishmanicidal activity on amastigotes of Leishmania (L.) amazonensis, reaching ~60% activity at concentrations of 100 and 25µg/ml, respectively. These results indicate that GALMAN-A is three times more potent and its oxovanadium complex is twelve times more potent than Glucantime (300µg/ml), which is the drug of choice in leishmaniasis treatment. The IC50 value for GALMAN-A:VO(2+)/VO(3+) was 74.4µg/ml (0.58µg/ml of vanadium). Thus, the significant activation of macrophages and the noted leishmanicidal effect demonstrate the need for further studies to clarify the mechanisms of action of these compounds.