Polyphenol Mediated Suppression of Hepatocellular Carcinoma (HepG2) Cell Proliferation by Clerodendrum infortunatum L. Root.

OBJECTIVE: Clerodendrum infortunatum L. has long been used in traditional medicine in Sri Lanka for tumours, cancer, and certain skin diseases. The present study aimed to assess the anticancer properties of the aqueous extract of C. infortunatum L. root (AECIR) through the activation of the apoptotic pathway on hepatocellular carcinoma (HepG2) and thus give it a scientific validation. Further, the contribution of polyphenols in antioxidant activity and cell cytotoxicity was investigated. METHODS: Powdered plant material was boiled with water (100°C) to obtained AECIR.  The DPPH assay was used to determine the antioxidant potential. The activity of AECIR on HepG2 and normal rat fibroblast (CC1) cell growth was determined using MTT assay. The morphological changes related to apoptotic pathway was examined by Ethidium Bromide/Acridine Orange (EB/AO), Rhodamine 123 (Rh123) and DNA fragmentation assay. RESULTS: The AECIR demonstrated antioxidant potential with an EC50 of 350.2 ± 1.5 ug/mL for DPPH assay. The HO•, H2O2 and •NO free radical scavenging activity was observed with EC50 of 19.7 ± 2.3, 11.7 ± 0.1 and 273.1 ± 0.9 ug/mL, respectively. The antiproliferative effect of AECIR on HepG2 cells was observed in a time and dose dependent manner with an EC50 of 239.1 ± 1.3 µg/mL while CC1 cells showed a nontoxic effect with an EC50 1062.7 ± 3.4 µg/mL after 24hrs treatment. A significant decrease in antioxidant activity (p<0.001) and 90% HepG2 cell viability was observed with polyphenol removed AECIR compared to the polyphenol present AECIR. The EB/AO uptake, depletion of mitochondrial transmembrane potential, and DNA fragmentation assay results revealed that the apoptosis was induced by AECIR. CONCLUSION: The obtained result of the present study demonstrates that the antioxidant potential and antiproliferative activity of AECIR is attributed to the presence of polyphenols. Furthermore, the findings provide the scientific base for anti-cancer potential of AECIR.

Restoration of mitochondrial structure and function within Helicobacter pylori VacA intoxicated cells.

The Helicobacter pylori vacuolating cytotoxin (VacA) is an intracellular, mitochondrial-targeting exotoxin that rapidly causes mitochondrial dysfunction and fragmentation. Although VacA targeting of mitochondria has been reported to alter overall cellular metabolism, there is little known about the consequences of extended exposure to the toxin. Here, we describe studies to address this gap in knowledge, which have revealed that mitochondrial dysfunction and fragmentation are followed by a time-dependent recovery of mitochondrial structure, mitochondrial transmembrane potential, and cellular ATP levels. Cells exposed to VacA also initially demonstrated a reduction in oxidative phosphorylation, as well as increase in compensatory aerobic glycolysis. These metabolic alterations were reversed in cells with limited toxin exposure, congruent with the recovery of mitochondrial transmembrane potential and the absence of cytochrome c release from the mitochondria. Taken together, these results are consistent with a model that mitochondrial structure and function are restored in VacA-intoxicated cells.

In Vitro Cytotoxic Effect of Aqueous Extracts from Leaves and Rhizomes of the Seagrass Posidonia oceanica (L.) Delile on HepG2 Liver Cancer Cells: Focus on Autophagy and Apoptosis.

Aqueous extracts from Posidonia oceanica's green and brown (beached) leaves and rhizomes were prepared, submitted to phenolic compound and proteomic analysis, and examined for their potential cytotoxic effect on HepG2 liver cancer cells in culture. The chosen endpoints related to survival and death were cell viability and locomotory behavior, cell-cycle analysis, apoptosis and autophagy, mitochondrial membrane polarization, and cell redox state. Here, we show that 24 h exposure to both green-leaf- and rhizome-derived extracts decreased tumor cell number in a dose-response manner, with a mean half maximal inhibitory concentration (IC50) estimated at 83 and 11.5 µg of dry extract/mL, respectively. Exposure to the IC50 of the extracts appeared to inhibit cell motility and long-term cell replicating capacity, with a more pronounced effect exerted by the rhizome-derived preparation. The underlying death-promoting mechanisms identified involved the down-regulation of autophagy, the onset of apoptosis, the decrease in the generation of reactive oxygen species, and the dissipation of mitochondrial transmembrane potential, although, at the molecular level, the two extracts appeared to elicit partially differentiating effects, conceivably due to their diverse composition. In conclusion, P. oceanica extracts merit further investigation to develop novel promising prevention and/or treatment agents, as well as beneficial supplements for the formulation of functional foods and food-packaging material with antioxidant and anticancer properties.

N-Methyl-(2S, 4R)-trans-4-hydroxy-L-proline, the major bioactive compound from Sideroxylon obtusifolium, attenuates pilocarpine-induced injury in cultured astrocytes

Glial cells have been implicated in temporal lobe epilepsy in humans and in its models. Astrocytes are lost in several brain regions after acute seizures induced by pilocarpine and may suffer hyperplasia at subsequent time points. This study investigated the effect of N-methyl-(2S,4R)-trans-4-hydroxy-L-proline (NMP) on astrocytes exposed to cytotoxic concentrations of pilocarpine. Astrocytes were incubated with pilocarpine (half maximal inhibitory concentration (IC50)=31.86 mM) for 24 h. Afterwards, they were treated with NMP at concentrations ranging from 3.12 to 100 μg/mL for 24 h. Cell viability was assessed by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Cytoplasmic reactive oxygen species (ROS) and mitochondrial transmembrane potential (ΔΨm) were analyzed by flow cytometry using 2',7'-dichlorofluorescein diacetate (DCFH-DA) and rhodamine-123 (Rho123), respectively. Expression of glial fibrillary acidic protein (GFAP) and voltage-dependent anion channel-1 (VDAC-1) were measured by western blot. Pilocarpine significantly decreased cell viability and mitochondrial potential and increased ROS concentration significantly by 6.7 times compared to the control. NMP concentrations ≥25 µg/mL protected astrocytes against pilocarpine-induced injury in a concentration-dependent manner. Concomitantly, NMP reduced cytoplasmic ROS accumulation to 27.3, 24.8, and 12.3% in the groups treated with 25, 50, and 100 µg/mL NMP, respectively. NMP also protected mitochondria from pilocarpine-induced depolarization. These effects were associated with improvement of pilocarpine-induced GFAP and VDAC-1 overexpression, which are important biomarkers of astrocyte dysfunction. In conclusion, the improvement of ROS accumulation, VDAC-1 overexpression, and mitochondrial depolarization are possible mechanisms of the NMP protective action on reactive astrocytes.

Monitoring Mitochondrial Function in Mouse Embryonic Stem Cells (mESCs).

Mouse embryonic stem cells (mESCs) can be grown in culture, recapitulating the different states of pluripotency of their in vivo counterparts, with notably different metabolic profiles. mESCs in a naïve pluripotent state present an ambivalent metabolism, using both glycolysis and oxidative phosphorylation as energy sources. Here, we describe a method to evaluate the oxidative function of naïve mESCs using the Seahorse Extracellular Flux Analyzer coupled to flow cytometry analysis of mitochondrial transmembrane potential using the TMRM fluorescence probe, thus assessing both oxygen consumption and mitochondrial membrane potential. This may be a useful protocol for understanding how mitochondrial oxidative function and potential of mESCs change in certain circumstances, and how is it related with various pluripotency/differentiation phenotypes.

Effects of Two-Week Betaine Supplementation on Apoptosis, Oxidative Stress, and Aerobic Capacity after Exhaustive Endurance Exercise.

This study evaluated the effects of 2 weeks of betaine supplementation on apoptosis, oxidative stress, and aerobic capacity after exhaustive endurance exercise (EEE). A double-blind, crossover, and counterbalanced design was adopted, with 10 healthy male participants asked to consume betaine (1.25 g of betaine mixed with 300 mL of sports beverage, twice per day for 2 weeks) or placebo (300 mL of sports beverage). All participants performed a graded exercise test on a treadmill to determine the maximal oxygen consumption (VO2max) before supplementation and then performed the EEE test at an intensity of 80% VO2max after 2 weeks of supplementation. The time to exhaustion, peak oxygen consumption, maximal heart rate, and average heart rate were recorded during the EEE test. Venous blood samples were drawn before, immediately after, and 3 h after the EEE test to assess apoptosis and the mitochondrial transmembrane potential (MTP) decline of lymphocytes as well as the concentrations of thiobarbituric acid reactive substance and protein carbonyl. The results indicated that lymphocyte apoptosis was significantly higher immediately after and 3 h after EEE than before exercise in participants in the placebo trial. However, lymphocyte apoptosis exhibited no significant differences among the three time points in participants in the betaine trial. Moreover, apoptosis in the betaine trial was significantly lower immediately after and 3 h after exercise compared with the placebo trial. No differences were noted for other variables. Thus, 2 weeks of betaine supplementation can effectively attenuate lymphocyte apoptosis, which is elevated by EEE. However, betaine supplementation exhibited no effects on MTP decline, oxidative stress, or aerobic capacity.

Natural products as LSD1 inhibitors for cancer therapy.

Natural products generally fall into the biologically relevant chemical space and always possess novel biological activities, thus making them a rich source of lead compounds for new drug discovery. With the recent technological advances, natural product-based drug discovery is now reaching a new era. Natural products have also shown promise in epigenetic drug discovery, some of them have advanced into clinical trials or are presently being used in clinic. The histone lysine specific demethylase 1 (LSD1), an important class of histone demethylases, has fundamental roles in the development of various pathological conditions. Targeting LSD1 has been recognized as a promising therapeutic option for cancer treatment. Notably, some natural products with different chemotypes including protoberberine alkaloids, flavones, polyphenols, and cyclic peptides have shown effectiveness against LSD1. These natural products provide novel scaffolds for developing new LSD1 inhibitors. In this review, we mainly discuss the identification of natural LSD1 inhibitors, analysis of the co-crystal structures of LSD1/natural product complex, antitumor activity and their modes of action. We also briefly discuss the challenges faced in this field. We believe this review will provide a landscape of natural LSD1 inhibitors.

The inverse relation between mitochondrial transmembrane potential and proteins α-helix in neuronal-like cells under static magnetic field and the role of VDAC.

In this study, a correlation between cell channel α-helices displacement and the mitochondrial transmembrane potential after exposure of 3, 7, 15 and 24 h of neuronal-like cells to a uniform magnetic field at the intensity of 2 mT was shown. Fourier Transform Infrared (FTIR) Spectroscopy and fluorescence techniques were used to analyze the secondary structure of protein content and mitochondrial transmembrane potential, respectively. The main result of this study was represented by a significant inverse relation between the mitochondrial transmembrane potential and the intensity of the Amide I band that can be associated with time exposure. Given that mitochondrial transmembrane potential should be related to the gating state of voltage-dependent anion channel (VDAC) in mitochondrial membrane, this result could have a relevant role in medicine. Indeed, VDAC's irregular behavior can be associated with several varieties of mitochondria-associated pathologies and various forms of cancer and neurodegeneration.

The prospective curative role of lipoxin A4 in induced gastric ulcer in rats: Possible involvement of mitochondrial dynamics signaling pathway.

This study purposed to examine the prospective curative role of lipoxin A4 (LXA4 ) in induced gastric ulcer in rats and explore the possible involvement of mitochondrial dynamics signaling pathway. Forty-eight male Wistar rats were divided into four groups: control, indomethacin (IND), IND + omeprazole (IND + Omez), and IND+ LXA4 groups. At the end of the experiment, the gastric pH, gastric fluid volume, total gastric acidity, ulcer index, and curative index were estimated. The gene expression of mitochondrial related protein 1 and mitofusin 2 were determined. In addition, some mitochondrial parameters include mitochondrial transmembrane potential, complex-I activity and reactive oxygen species were measured. Also, some gastric biochemical parameters, histopathological, and immunohistochemical analyses of the gastric mucosa were determined. We found that IND induced gastric ulcer, as manifested by the biochemical, histopathological, and immunohistochemical analyses. Both Omez and LXA4 treatment for 15 days alleviated the IND-induced gastric ulcer as explored by ameliorating the biochemical, histopathological, and immunohistochemical findings. We concluded that LXA4 mitigated the IND-induced gastric ulcer via improving the mitochondrial dynamic imbalance and mitochondrial dysfunction, in addition to its anti-apoptotic, anti-inflammatory, and antioxidant properties.

Evaluation of apoptotic potential of glyphosate metabolites and impurities in human peripheral blood mononuclear cells (in vitro study).

Glyphosate is used for cereal, vegetable and fruit crops for reducing or inhibiting the growth of weeds as well as a desiccant for various grain crops. That is why, glyphosate has been shown to be accumulated in humans and animals through ingestion of food of both plant and animal origin. The study aimed to assessed the effect of glyphosate, its metabolites: aminomethylphosphonic acid (AMPA), methylphosphonic acid and its impurities: PMIDA, N-methylglyphosate, hydroxymethylphosphonic acid and bis(phosphonomethyl)amine on apoptosis induction in human peripheral blood mononuclear cells (PBMCs). PBMCs were exposed to the compounds studied at the concentrations ranging from 0.01 to 5 mM for 4 h. We have observed an increase in reactive oxygen species (including hydroxyl radical) and cytosolic calcium ions levels as well as reduction of transmembrane mitochondrial potential (ΔΨm) in PBMCs exposed to the compounds examined. All substances studied changed PBMCs membrane permeability, activated caspase-8, -9, -3 and caused chromatin condensation, which showed that they were capable of inducing apoptosis both via extrinsic and particularly intrinsic pathway. Generally the study demonstrated that there were no differences between apoptotic changes induced by glyphosate, its metabolites or impurities, and observed changes were provoked by high concentrations of investigated compounds.

The Cellular Localization of the p42 and p46 Oligoadenylate Synthetase 1 Isoforms and Their Impact on Mitochondrial Respiration.

The importance of the IFN-induced oligoadenylate synthetase (OAS) proteins and the OAS/RNase L pathway in the innate response against viral pathogens is well-established, however the observed differences in anti-viral activity between the human OAS1 p46 and p42 isoforms are not fully understood. The protein expression of these isoforms is determined by the SNP rs10774671, either being an A or a G allele resulting in expression of either the p42 or the p46 isoform. Using fluorescence microscopy and immunoblot analysis of fractionated cell samples, we show here that the CaaX motif is of key importance to the cellular localization. The OAS1 p42 isoform is mainly located in the cytosol, whereas the p46 isoform with a C-terminal CaaX motif is translocated to membranous organelles, like the mitochondria. We furthermore observed differences between p42 and p46 in their effect on mitochondrial physiology using high resolution respirometry and fluorometry. Overexpression of OAS1 p42 and IFN-ß treatment of HeLa cells (AA genotype) resulted in significantly increased respiration, which was not seen with p46 overexpression. The difference in subcellular localization and mitochondrial effect of these two OAS1 isoforms might help to explain the anti-viral mechanisms that differentiate these proteins.

Amyloid-ß-Dependent Inactivation of the Mitochondrial Electron Transport Chain at Low Transmembrane Potential: An Ameliorating Process in Hypoxia-Associated Neurodegenerative Disease?

Cerebral hypoperfusion-induced hypoxia, a condition that impairs oxygen utilization and thus ATP production by mitochondrial oxidative phosphorylation (oxphos), is thought to contribute to neural degeneration in Alzheimer's disease. However, hypoxia upregulates the generation of amyloid-ß (Aß), a group of peptides known to impair/inhibit the electron transport chain (ETC) of reactions that support oxphos in the inner mitochondrial membrane (IMM). This is a hypothesis paper that reconciles the hypoxia-induced upregulation of Aß with Aß's ETC-inhibiting action and, specifically, posits an oxphos-enhancing effect of this inhibition under conditions of newly developing or otherwise mild hypoxia. This effect is typically transient; that is, under conditions of prolonged or severe hypoxia, the oxphos-enhancing activity is overwhelmed by Aß's well-known toxic actions on mitochondria and other cellular components. The hypothesis is motivated by evidence that the IMM transmembrane potential Ψm, an important determinant of ETC activity, exhibits heterogeneity, i.e., a range of values, among a given local population of mitochondria. It specifically proposes that during oxygen limitation, Aß selectively inactivates ETC complexes in mitochondria that exhibit relatively low absolute values of Ψm, thereby suppressing oxygen binding and consumption by complex IV of the ETC in these mitochondria. This effect of Aß on low-Ψm mitochondria is hypothesized to spare hypoxia-limited oxygen for oxphos-enabling utilization by the ETC of the remaining active, higher-Ψm local mitochondria, and thereby to increase overall ATP generated collectively by the local mitochondrial population, i.e., to ameliorate hypoxia-induced oxphos reduction. The protective action of Aß hypothesized here may slow the early development of hypoxia-associated cellular deterioration/loss in Alzheimer's disease and perhaps other neurodegenerative diseases.

Dual-function of Baicalin in nsPEFs-treated Hepatocytes and Hepatocellular Carcinoma cells for Different Death Pathway and Mitochondrial Response.

Nanosecond pulsed electric fields (nsPEFs) is emerged as a potential curative modality to ablate hepatocellular carcinoma (HCC). The application of local ablation is usually limited by insufficiency of liver function. While baicalin, a flavonoid isolated from Scutellaria baicalensis Georgi, has been proven to possess both anti-tumor and protective effects. Our study aimed to estimate different responses of hepatic cancer cells and hepatocytes to the combination of nsPEFs and baicalin. Cell viability, apoptosis and necrosis, mitochondrial transmembrane potential (MTP) and reactive oxygen species (ROS) were examined by CCK-8, FCM, JC-1 and fluorescent probe, respectively. After treatment by nsPEFs, most hepatocytes died by apoptosis, nevertheless, nearly all cancer cells were killed through necrosis. Low concentration of baicalin synergically enhanced nsPEFs-induced suppression and necrosis of HCC cells, nevertheless, the application of baicalin protected normal hepatocytes from the injury caused by nsPEFs, owing to elevating mitochondrial transmembrane potential and reducing ROS generation. Our work provided an advantageous therapy for HCC through the enhanced combination treatment of nsPEFs and baicalin, with which could improve the tumor-ablation effect and alleviate the injury of hepatic tissues simultaneously.

Downregulation of sirtuin 3 by palmitic acid increases the oxidative stress, impairment of mitochondrial function, and apoptosis in liver cells.

Elevated levels of saturated fatty acids show a strong cytotoxic effect in liver cells. Sirtuin 3 (SIRT3), a mitochondrially localized member of NAD+ -dependent deacetylase has been shown to protect hepatocytes against the oxidative stress. The role of SIRT3 on the cytotoxicity caused by fatty acids in liver cells is not fully understood. The aim of this study was to evaluate the expression level of SIRT3, oxidative stress, and mitochondrial impairments in human hepatoma HepG2 cells exposed to palmitic acid (PA). Our results showed that PA treatment caused the deposition of lipid droplets and resulted in an increased expression of tumor necrosis factor-α in a dose-dependent manner. Excessive accumulation of PA induces the reactive oxygen species formation and apoptosis while dissipating the mitochondrial transmembrane potential. The level of SIRT3 expression in both nuclear and mitochondrial fractions in HepG2 cells was decreased with the increase in PA concentrations. However, in the cytosolic fraction, the SIRT3 was undetectable. In conclusion, our results showed that PA caused an increase in inflammation and oxidative stress in HepG2 cells. The exposure of PA also resulted in the decline in transmembrane potential and an increase in apoptosis. The underexpression of nuclear and mitochondrial SIRT3 by PA suggests that the PA target the process that regulates the stress-related gene expression and mitochondrial functions.

Acid-sensing ion channel 1a is involved in ischaemia/reperfusion induced kidney injury by increasing renal epithelia cell apoptosis.

Acidic microenvironment is commonly observed in ischaemic tissue. In the kidney, extracellular pH dropped from 7.4 to 6.5 within 10 minutes initiation of ischaemia. Acid-sensing ion channels (ASICs) can be activated by pH drops from 7.4 to 7.0 or lower and permeates to Ca2+ entrance. Thus, activation of ASIC1a can mediate the intracellular Ca2+ accumulation and play crucial roles in apoptosis of cells. However, the role of ASICs in renal ischaemic injury is unclear. The aim of the present study was to test the hypothesis that ischaemia increases renal epithelia cell apoptosis through ASIC1a-mediated calcium entry. The results show that ASIC1a distributed in the proximal tubule with higher level in the renal tubule ischaemic injury both in vivo and in vitro. In vivo, Injection of ASIC1a inhibitor PcTx-1 previous to ischaemia/reperfusion (I/R) operation attenuated renal ischaemic injury. In vitro, HK-2 cells were pre-treated with PcTx-1 before hypoxia, the intracellular concentration of Ca2+ , mitochondrial transmembrane potential (∆ψm) and apoptosis was measured. Blocking ASIC1a attenuated I/R induced Ca2+ overflow, loss of ∆ψm and apoptosis in HK-2 cells. The results revealed that ASIC1a localized in the proximal tubular and contributed to I/R induced kidney injury. Consequently, targeting the ASIC1a may prove to be a novel strategy for AKI patients.

Cytotoxic Activity of the Histone Deacetylase 3-Selective Inhibitor Pojamide on MDA-MB-231 Triple-Negative Breast Cancer Cells.

We examined the effects of the ferrocene-based histone deacetylase-3 inhibitor Pojamide (N¹-(2-aminophenyl)-N8-ferrocenyloctanediamide) and its two derivatives N¹-(2-aminophenyl)-N6-ferrocenyladipamide and N¹-(2-aminophenyl)-N8-ferroceniumoctanediamide tetrafluoroborate on triple-negative MDA-MB-231 breast cancer cells. Viability/growth assays indicated that only the first two compounds at 70 µM concentration caused an approximate halving of cell number after 24 h of exposure, whereas the tetrafluoroborate derivative exerted no effect on cell survival nor proliferation. Flow cytometric and protein blot analyses were performed on cells exposed to both Pojamide and the ferrocenyladipamide derivative to evaluate cell cycle distribution, apoptosis/autophagy modulation, and mitochondrial metabolic state in order to assess the cellular basis of the cytotoxic effect. The data obtained show that the cytotoxic effect of the two deacetylase inhibitors may be ascribed to the onset of non-apoptotic cell death conceivably linked to a down-regulation of autophagic processes and an impairment of mitochondrial function with an increase in intracellular reactive oxygen species. Our work expands the list of autophagy-regulating drugs and also provides a further example of the role played by the inhibition of autophagy in breast cancer cell death. Moreover, the compounds studied may represent attractive and promising targets for subsequent molecular modeling for anti-neoplastic agents in malignant breast cancer.

Effects of ammonia nitrogen on gill mitochondria in clam Ruditapes philippinarum.

Ammonia nitrogen exposure has been found to significantly increase the early apoptosis rates of gill cells, affect the contents of ATP and disturb expressions of calcium-related genes in clam Ruditapes philippinarum. Mitochondria are the centers for energy production, initiation of apoptosis and calcium signal regulation. It is hypothesized that gill mitochondrion is a target organelle for the ammonia nitrogen. Thus, ATP metabolism together with ATP-consuming functions would be interfered by ammonia exposure. In the present study, mitochondrial transmembrane potential (MTP), ATPase activities, gill functions in clearance and respiration, and histological changes were detected to characterize the effects of ammonia to the gill mitochondria in clam R. philippinarum. Results indicated that ammonia exposure led to significant decreases in MTP, Ca2+-ATPase activity and clearance rates. However, different concentrations of ammonia nitrogen induced different variations on H+, K+-ATPase activity and respiration rates. Histological observation revealed that subacute exposure of ammonia damaged the microstructure of gill tissues. Therefore, ammonia exposure dramatically damaged the normal structure and function of mitochondria, resulting in irreversible damage in energy formation and supply. In addition, it affected Ca2+ and K+ metabolism and inhibited food intake and respiration in clam R. philippinarum.

The therapeutic effects of traditional Chinese medicine Fusu agent in LPS-induced acute lung injury model rats.

PURPOSE: Acute lung injury (ALI) is a common and fatal oxidative stress in the lung, mainly induced by endothelial injury and capillary leakage. In our previous study, "Fusu agent", a traditional Chinese medicine, was found to exert preventive effect on endothelial damage in lipopoly-saccharide (LPS)-induced ALI model rats partially via inhibiting heparanase1 (HPA1) activation and inhibiting the inflammatory factors. However, it is still unknown whether Fusu agent exerts its therapeutic effect in LPS-induced ALI model rats and its potential mechanism. MATERIALS AND METHODS: Rats were injected with LPS (3 mg/kg, intraperitoneally) to induced ALI, and the prepared Fusu agent was given (2, 4 or 6 g/kg) 2 hours after LPS challenge. Twenty-four or 48 hours after Fusu agent administration, the biochemical changes in the plasma and lung tissues and the morphological/histological changes in the lung associated with inflammation and injury were evaluated. Human umbilical vein endothelial cells (HUVECs) were employed to confirm the therapeutic effects of Fusu agent and investigate its mechanisms, that is, affecting ROS accumulation, mitochondrial transmembrane potential (MTP) maintenance and decreasing the expression levels of HPA1. RESULTS: Administration of Fusu agent obviously improved the lung injury and recovered vascular endothelium loss and injury. CD31 signal, which is a specific marker for endothelial vascular lesions, was decreased after Fusu agent treatment in LPS-induced ALI model rats, indicating its therapeutic effect against endothelial surface layer injury. Meanwhile, Fusu agent also decreased HPA1 expression and inflammatory responses. In vitro, Fusu agent-medicated serum decreased injury and cell death induced by LPS in HUVECs by stabilizing MTP and decreasing the leakage of lactate dehydrogenase. Consistently, Fusu agent-medicated serum downregulated HPA1 induced by LPS stimulation. CONCLUSION: These findings suggest that Fusu agent exerts its therapeutic effect in both LPS-induced ALI model rats and HUVECs potentially via suppressing HPA1 expression, and thus exerts prosurvival effect via maintaining MTP and attenuating cell injury.

Cytotoxic and NF-κB and mitochondrial transmembrane potential inhibitory pentacyclic triterpenoids from Syzygium corticosum and their semi-synthetic derivatives.

Syzygium is a large genus of flowering plants, with several species, including the clove tree, used as important resources in the food and pharmaceutical industries. In our continuing search for anticancer agents from higher plants, a chloroform extract of the leaves and twigs of Syzygium corticosum collected in Vietnam was found to be active toward the HT-29 human colon cancer cell line. Separation of this extract guided by HT-29 cells and nuclear factor-kappa B (NF-κB) inhibition yielded 19 known natural products, including seven triterpenoids, three ellagic acid derivatives, two methylated flavonoids, a cyclohexanone, four megastigmanes, a small lactone, and an aromatic aldehyde. The full stereochemistry of (+)-fouquierol (2) was defined for the first time. Biological investigations showed that (+)-ursolic acid (1) is the major cytotoxic component of S. corticosum, which exhibited also potent activities in the NF-κB and mitochondrial transmembrane potential (MTP) inhibition assays conducted, with IC50 values of 31 nM and 3.5 µM, respectively. Several analogues of (+)-ursolic acid (1) were synthesized, and a preliminary structure-activity relationship (SAR) study indicated that the C-3 hydroxy and C-28 carboxylic acid groups and 19,20-dimethyl substitution are all essential in the mediation of the bioactivities observed for this triterpenoid.

Suppression of Nrf2 confers chemosensitizing effect through enhanced oxidant-mediated mitochondrial dysfunction.

AIMS: Transcription factor Nrf2, which regulates the expression of cytoprotective and antioxidant enzymes, contributes to proliferation and resistance to chemotherapy in cancer. The inhibition of Nrf2 can sensitize cholangiocarcinoma (CCA) cells to the cytotoxicity of several chemotherapeutic agents. In this study, we investigated the mechanism of this chemosensitizing effect. MAIN METHODS: KKU-100 cells were used in the study. Nrf2 expression was knocked down by siRNA and expression was validated by reverse transcription and polymerase chain reaction. Cytotoxicity was assessed by sulforhodamine B method. Intracellular reactive oxygen species (ROS) was examined by fluorescent dye, dichlorofluorescin diacetate method and mitochondrial transmembrane potential was assessed by JC1 dye assay. KEY FINDINGS: Cytotoxicity of cisplatin (Cis) in KKU-100 cells was enhanced by knockdown of Nrf2 expression. The enhanced cytotoxic effect was abolished by treatment with N-acetylcysteine, TEMPOL and MnTBAP. Cells with Nrf2 knockdown or Cis treatment increased production of ROS, and ROS was markedly enhanced by a combination of Nrf2 knockdown and Cis. The increased ROS formation was associated with a decrease in mitochondrial transmembrane potential (Δψm), where this decrease was prevented by antioxidant compounds. The loss of Δψm and cell death were prevented by cyclosporine, an inhibitor of mitochondrial permeability transition pore (MPTP). Luteolin inhibited Nrf2 and markedly enhanced cytotoxicity in combination with Cis. SIGNIFICANCE: Inhibition of Nrf2 is a feasible strategy in enhancing antitumor activity of chemotherapeutic agents and improving efficacy of chemotherapy in CCA.