Centella asiatica Modulates Nrf-2 Antioxidant Mechanisms and Enhances Reactive Oxygen Species-Mediated Apoptotic Cell Death in Leukemic (THP-1) Cells.

Centella asiatica is commonly used in traditional medicine owing to its many therapeutic properties including but not limited to antioxidant and antitumor potential. This study examined the antioxidant and antiproliferative effects of its crude (C) and fractionated (C3) ethanolic leaf extracts in THP-1 cells. In THP-1 cells, C and C3 cytotoxicity was evaluated (WST-1 viability assay; 24 h; [0.2-3 mg/mL]) and half maximal inhibitory concentration was obtained. Malondialdehyde (MDA; spectrophotometry), mitochondrial depolarization (Δψm), intracellular reactive oxygen species (IROS; flow cytometry), glutathione (GSH), oxidized GSH (GSSG) concentrations, adenosine triphosphate (ATP) levels, caspase activities (luminometry) and DNA fragmentation (single cell gel electrophoresis assay) were evaluated. Protein expression and gene expression was quantified by Western blotting and quantitative polymerase chain reaction, respectively. THP-1 cell viability was dose-dependently reduced by C and C3. MDA, IROS, GSH, and Δψm were increased and ATP was decreased by C and C3 (P < .01). Antioxidant gene expression, Nrf-2 protein expression, and GSSG levels (P < .01) were increased by C, but were decreased by C3. C and C3 elevated caspase activity and DNA damage (P < .0001), whereas they decreased glutathione peroxidase and Bcl-2 protein expressions (P < .003). c-PARP protein expression and c-myc gene expression was decreased by C, whereas they were increased by C3 (P < .002). C3 reduced OGG-1 gene expression (P < .0003). Antioxidant responses were increased by C, whereas they were decreased by C3. Both C and C3 exerted antiproliferative effects in THP-1 cells by enhancing apoptosis. Of note, C3 more effectively induced apoptosis.

Moringa oleifera Aqueous Leaf Extract Induces Cell-Cycle Arrest and Apoptosis in Human Liver Hepatocellular Carcinoma Cells.

Aim: Hepatocellular carcinoma is one of the leading global epidemics. A medicinal tree, Moringa oleifera (MO), has been part of traditional treatments including cancer therapies. We investigated the apoptosis inducing effects of MO crude aqueous leaf extract (MOE) in human liver hepatocellular carcinoma (HepG2) cells. Methods: HepG2, PBMCs and Hek293 cell viability was evaluated using MTT assay. Oxidative stress and DNA damage was determined using TBARS and comet assays, respectively. Apoptosis was assessed by caspase-9, -3/7 activities and ATP levels (luminometry). Cell cycle, γH2AX, and cleaved PARP-1 were determined (flow cytometry). Protein expression of c-myc, Bax, p-Bcl2, Smac/DIABLO, Hsp70, SRp30a and cleaved PARP-1 was assessed using western blotting. Results: MOE displayed minimal toxicity in PBMCs and Hek293 cells for 24 h. HepG2 cells were exposed to MOE (24 h) and an IC50 (4.479 mg/mL) was determined. MOE significantly increased lipid peroxidation, DNA damage and γH2AX levels. A significant decrease in G1, S and G2-M phase was seen. Significant increase in SRp30a protein expression activated caspase-9. Caspase-9 and -3/7 was significantly increased with significant decrease in ATP levels. Apoptosis was confirmed with significant decrease in c-myc, p-Bcl2 and Hsp70 protein expression and a significant increase in Bax, Smac/DIABLO and PARP-1 cleavage. Conclusion: MOE induces cell-cycle arrest and apoptosis in cancerous HepG2 cells.

Withania somnifera modulates cancer cachexia associated inflammatory cytokines and cell death in leukaemic THP-1 cells and peripheral blood mononuclear cells (PBMC's).

BACKGROUND: Cancer and inflammation are associated with cachexia. Withania somnifera (W. somnifera) possesses antioxidant and anti-inflammatory potential. We investigated the potential of an aqueous extract of the root of W. somnifera (WRE) to modulate cytokines, antioxidants and apoptosis in leukaemic THP-1 cells and peripheral blood mononuclear cells (PBMC's). METHODS: Cytotoxcity of WRE was determined at 24 and 72 h (h). Oxidant scavenging activity of WRE was evaluated (2, 2-diphenyl-1 picrylhydrazyl assay). Glutathione (GSH) levels, caspase (- 8, - 9, - 3/7) activities and adenosine triphosphate (ATP) levels (Luminometry) were thereafter assayed. Tumour necrosis factor-α (TNF-α), interleukin (IL)-6, IL-1ß and IL-10 levels were also assessed using enzyme-linked immunosorbant assay. RESULTS: At 24 h, WRE (0.2-0.4 mg/ml) decreased PBMC viability between 20 and 25%, whereas it increased THP-1 viability between 15 and 23% (p < 0.001). At 72 h, WRE increased PBMC viability by 27-39% (0.05, 0.4 mg/ml WRE) whereas decreased THP-1 viability between 9 and 16% (0.05-0.4 mg/ml WRE) (p < 0.001). Oxidant scavenging activity was increased by WRE (0.05-0.4 mg/ml, p < 0.0001). PBMC TNF-α and IL-10 levels were decreased by 0.2-0.4 mg/ml WRE, whereas IL-1ß levels were increased by 0.05-0.4 mg/ml WRE (p < 0.0001). In THP-1 cells, WRE (0.05-0.4 mg/ml) decreased TNF-α, IL-1ß and IL-6 levels (p < 0.0001). At 24 h, GSH levels were decreased in PBMC's, whilst increased in THP-1 cells by 0.2-0.4 mg/ml WRE (p < 0.0001). At 72 h, WRE (0.1-0.4 mg/ml) decreased GSH levels in both cell lines (p < 0.0001). At 24 h, WRE (0.2-0.4 mg/ml) increased PBMC caspase (-8, -3/7) activities whereas WRE (0.05, 0.1, 0.4 mg/ml) increased THP-1 caspase (-9, -3/7) activities (p < 0.0001). At 72 h, PBMC caspase (-8, -9, -3/7) activities were increased at 0.05-0.1 mg/ml WRE (p < 0.0001). In THP-1 cells, caspase (-8, -9, -3/7) activities and ATP levels were increased by 0.1-0.2 mg/ml WRE, whereas decreased by 0.05 and 0.4 mg/ml WRE (72 h, p < 0.0001). CONCLUSION: In PBMC's and THP-1 cells, WRE proved to effectively modulate antioxidant activity, inflammatory cytokines and cell death. In THP-1 cells, WRE decreased pro-inflammatory cytokine levels, which may alleviate cancer cachexia and excessive leukaemic cell growth.

Centella asiatica Fraction-3 Suppresses the Nuclear Factor Erythroid 2-Related Factor 2 Anti-Oxidant Pathway and Enhances Reactive Oxygen Species-Mediated Cell Death in Cancerous Lung A549 Cells.

Centella asiatica is a tropical medicinal plant that is commonly used in traditional medicine. Medicinal properties of C. asiatica include anti-oxidant, anti-inflammatory, and anti-cancer activity. We investigated the anti-oxidant and anti-proliferative/cytotoxic effects of a semi-purified fraction of C. asiatica ethanolic leaf extract (C3) in cancerous lung A549 cells. C3 was obtained by silica column fractionation and identified by using thin-layer chromatography and gas chromatography mass spectrometry. Cytotoxicity of C3 in A549 cells was evaluated (cell viability assay-WST-1; 24 h; [0.2-3 mg/mL]) to determine an inhibitory concentration (IC50). Intracellular reactive oxygen species (IROS), mitochondrial membrane potential (flow cytometry), malondialdehyde (MDA), lactate dehydrogenase (LDH) (spectrophotometry), glutathione (GSH), oxidised glutathione (GSSG), adenosine triphosphate levels, caspase activity (luminometry), and DNA damage (comet assay) were evaluated. Protein expression (Nrf-2, p53, Bax, Bcl-2, and HSP-70) and gene expression (Nrf-2, GPx, SOD, CAT, c-myc, and OGG-1) were quantified by western blotting and quantitative polymerase chain reaction (qPCR), respectively. C3 dose dependently decreased A549 cell viability. The IC50 of C3 increased MDA, IROS, mitochondrial depolarization, LDH, caspase (-8, -9, -3/7) activity, DNA damage, GSH levels, Nrf-2 protein expression, HSP-70 protein expression, and OGG-1 gene expression (P < .05). GSSG levels, anti-oxidant (Nrf-2, GPx, SOD) gene expression, p53, Bax, and Bcl-2 protein expression were decreased by C3 (P < .02). C3 diminished the anti-oxidant gene expression and induced anti-proliferative/cytotoxic effects in A549 cells.

Centella asiatica modulates cancer cachexia associated inflammatory cytokines and cell death in leukaemic THP-1 cells and peripheral blood mononuclear cells (PBMC's).

BACKGROUND: Cancer cachexia is associated with increased pro-inflammatory cytokine levels. Centella asiatica (C. asiatica) possesses antioxidant, anti-inflammatory and anti-tumour potential. We investigated the modulation of antioxidants, cytokines and cell death by C. asiatica ethanolic leaf extract (CLE) in leukaemic THP-1 cells and normal peripheral blood mononuclear cells (PBMC's). METHODS: Cytotoxcity of CLE was determined at 24 and 72 h (h). Oxidant scavenging activity of CLE was evaluated using the 2, 2-diphenyl-1 picrylhydrazyl (DPPH) assay. Glutathione (GSH) levels, caspase (-8, -9, -3/7) activities and adenosine triphosphate (ATP) levels (Luminometry) were then assayed. The levels of tumour necrosis factor-α (TNF-α), interleukin (IL)-6, IL-1ß and IL-10 were also assessed using enzyme-linked immunosorbant assay. RESULTS: CLE decreased PBMC viability between 33.25-74.55% (24 h: 0.2-0.8 mg/ml CLE and 72 h: 0.4-0.8 mg/ml CLE) and THP-1 viability by 28.404% (72 h: 0.8 mg/ml CLE) (p < 0.0001). Oxidant scavenging activity was increased by CLE (0.05-0.8 mg/ml) (p < 0.0001). PBMC TNF-α and IL-10 levels were decreased by CLE (0.05-0.8 mg/ml) (p < 0.0001). However, PBMC IL-6 and IL-1ß concentrations were increased at 0.05-0.2 mg/ml CLE but decreased at 0.4 mg/ml CLE (p < 0.0001). In THP-1 cells, CLE (0.2-0.8 mg/ml) decreased IL-1ß and IL-6 whereas increased IL-10 levels (p < 0.0001). In both cell lines, CLE (0.05-0.2 mg/ml, 24 and 72 h) increased GSH concentrations (p < 0.0001). At 24 h, caspase (-9, -3/7) activities was increased by CLE (0.05-0.8 mg/ml) in PBMC's whereas decreased by CLE (0.2-0.4 mg/ml) in THP-1 cells (p < 0.0001). At 72 h, CLE (0.05-0.8 mg/ml) decreased caspase (-9, -3/7) activities and ATP levels in both cell lines (p < 0.0001). CONCLUSION: In PBMC's and THP-1 cells, CLE proved to effectively modulate antioxidant activity, inflammatory cytokines and cell death. In THP-1 cells, CLE decreased pro-inflammatory cytokine levels whereas it increased anti-inflammatory cytokine levels which may alleviate cancer cachexia.

The Antiproliferative Effect of Moringa oleifera Crude Aqueous Leaf Extract on Human Esophageal Cancer Cells.

Esophageal cancer (EC) is commonly diagnosed in South Africa (SA), with high incidences occurring in SA's black population. Moringa oleifera (MO), a multipurpose tree, is used traditionally for its nutritional and medicinal properties. It has been used for the treatment of a variety of ailments, including cancer. We investigated the antiproliferative effect of MO crude aqueous leaf extract (MOE) on a cancerous esophageal cell line (SNO). SNO cells were exposed to a range of MOE dilutions to evaluate cytotoxicity (MTT assay). Oxidative stress was determined using the TBARS assay. The comet assay was used to assess DNA damage. We then determined cell death mechanisms by measuring phosphatidylserine (PS) externalization (flow cytometry), caspase-3/7 and caspase-9 activities, and adenosine triphosphate (ATP) levels (luminometry). Protein expression of Smac/DIABLO and PARP-1 was determined by western blotting. SNO cells were treated with a range of MOE dilutions to obtain an IC50 value of 389.2 µg/mL MOE (24 h), which was used in all subsequent assays. MOE significantly increased lipid peroxidation (P < .05) and DNA fragmentation (P < .0001) in SNO cells. The induction of apoptosis was confirmed by the increase in PS externalization (P < .0001), caspase-9 (P < .05) and caspase-3/7 (P = .22) activities, and decreased ATP levels (P < .0001). MOE significantly increased both the expression of Smac/DIABLO protein and cleavage of PARP-1, resulting in an increase in the 24-kDa fragment (P < .001). MOE possesses antiproliferative effects on SNO EC cells by increasing lipid peroxidation, DNA fragmentation, and induction of apoptosis.

Moringa oleifera Gold Nanoparticles Modulate Oncogenes, Tumor Suppressor Genes, and Caspase-9 Splice Variants in A549 Cells.

Gold nanoparticles (AuNP's) facilitate cancer cell recognition and can be manufactured by green synthesis using nutrient rich medicinal plants such as Moringa oleifera (MO). Targeting dysregulated oncogenes and tumor suppressor genes is crucial for cancer therapeutics. We investigated the antiproliferative effects of AuNP synthesized from MO aqueous leaf extracts (MLAuNP ) in A549 lung and SNO oesophageal cancer cells. A one-pot green synthesis technique was used to synthesise MLAuNP . A549, SNO cancer cells and normal peripheral blood mononuclear cells (PBMCs) were exposed to MLAuNP and CAuNP to evaluate cytotoxicity (MTT assay); apoptosis was measured by phosphatidylserine (PS) externalization, mitochondrial depolarization (ΔΨm) (flow cytometry), caspase-3/7, -9 activity, and ATP levels (luminometry). The mRNA expression of c-myc, p53, Skp2, Fbw7α, and caspase-9 splice variants was determined using qPCR, while relative protein expression of c-myc, p53, SRp30a, Bax, Bcl-2, Smac/DIABLO, Hsp70, and PARP-1 were determined by Western blotting. MLAuNP and CAuNP were not cytotoxic to PBMCs, whilst its pro-apoptotic properties were confirmed in A549 and SNO cells. MLAuNP significantly increased caspase activity in SNO cells while MLAuNP significantly increased PS externalization, ΔΨm, caspase-9, caspase-3/7 activities, and decreased ATP levels in A549 cells. Also, p53 mRNA and protein levels, SRp30a (P = 0.428), Bax, Smac/DIABLO and PARP-1 24 kDa fragment levels were significantly increased. Conversely, MLAuNP significantly decreased Bcl-2, Hsp70, Skp2, Fbw7α, c-myc mRNA, and protein levels and activated alternate splicing with caspase-9a splice variant being significantly increased. MLAuNP possesses antiproliferative properties and induced apoptosis in A549 cells by activating alternate splicing of caspase-9. J. Cell. Biochem. 117: 2302-2314, 2016. © 2016 Wiley Periodicals, Inc.

Trigonella foenum-graecum Seed Extract, 4-Hydroxyisoleucine, and Metformin Stimulate Proximal Insulin Signaling and Increase Expression of Glycogenic Enzymes and GLUT2 in HepG2 Cells.

BACKGROUND: Fenugreek (Trigonella foenum-graecum) is globally recognized for its medicinal properties and hypoglycemic effects. The seed extract as well as its active compound, 4-hydroxyisoleucine (4-OH-Ile), have been shown to reduce hyperglycemic insulin resistance. The mechanism by which this occurs has not been investigated in human liver cells (HepG2) in comparison to the antihyperglycemic drug, metformin. METHODS: We investigated the effects of an aqueous fenugreek seed extract (FSE), 4-OH-Ile, and metformin in HepG2 cells relative to insulin as a positive control. Cells were treated with FSE and 4-OH-Ile at 100 ng/mL under normoglycemic (5 mM glucose) and hyperglycemic (30 mM glucose) conditions for 72 hr. Tyrosine phosphorylation of insulin receptor-ß (IR-ß), protein kinase B (Akt), glycogen synthase kinase-3α/ß (GSK-3α/ß), and glucose transporter 2 (GLUT2) was determined by western blotting. Gene expression of sterol regulatory element-binding protein 1c (SREBP1c), GLUT2, glycogen synthase (GS), and glucokinase (GK) was evaluated by quantitative polymerase chain reaction, and supernatant glucose levels were measured using the Piccolo biochemistry analyzer. RESULTS: Under normo- and hyperglycemic conditions, FSE, 4-OH-Ile, insulin (100 ng/mL), and metformin (2 mM) caused a significant increase in phosphorylation of IR-ß, Akt, GSK-3α/ß, and GLUT2. Glucose uptake, however, was most significantly increased in FSE-treated cells during both conditions. FSE induced the most significant changes in downstream insulin signaling, GS, GK, SREBP1c, and GLUT2 expression compared to 4-OH-Ile, metformin, and insulin. In addition, FSE significantly increased glucose uptake. CONCLUSIONS: Collectively, these findings provide a mechanism by which FSE exerts antihyperglycemic effects similar to metformin and insulin that occurs via enhanced insulin signaling, gene expression, and increasing glucose uptake.

The antiproliferative effect of Moringa oleifera crude aqueous leaf extract on cancerous human alveolar epithelial cells.

BACKGROUND: The incidence of lung cancer is expected to increase due to increases in exposure to airborne pollutants and cigarette smoke. Moringa oleifera (MO), a medicinal plant found mainly in Asia and South Africa is used in the traditional treatment of various ailments including cancer. This study investigated the antiproliferative effect of MO leaf extract (MOE) in cancerous A549 lung cells. METHODS: A crude aqueous leaf extract was prepared and the cells were treated with 166.7 µg/ml MOE (IC50) for 24 h and assayed for oxidative stress (TBARS and Glutathione assays), DNA fragmentation (comet assay) and caspase (3/7 and 9) activity. In addition, the expression of Nrf2, p53, Smac/DIABLO and PARP-1 was determined by Western blotting. The mRNA expression of Nrf2 and p53 was assessed using qPCR. RESULTS: A significant increase in reactive oxygen species with a concomitant decrease in intracellular glutathione levels (p < 0.001) in MOE treated A549 cells was observed. MOE showed a significant reduction in Nrf2 protein expression (1.89-fold, p < 0.05) and mRNA expression (1.44-fold). A higher level of DNA fragmentation (p < 0.0001) was seen in the MOE treated cells. MOE's pro-apoptotic action was confirmed by the significant increase in p53 protein expression (1.02-fold, p < 0.05), p53 mRNA expression (1.59-fold), caspase-9 (1.28-fold, p < 0.05), caspase-3/7 (1.52-fold) activities and an enhanced expression of Smac/DIABLO. MOE also caused the cleavage and activation of PARP-1 into 89 KDa and 24 KDa fragments (p < 0.0001). CONCLUSION: MOE exerts antiproliferative effects in A549 lung cells by increasing oxidative stress, DNA fragmentation and inducing apoptosis.

Silver nanoparticles of Albizia adianthifolia: the induction of apoptosis in human lung carcinoma cell line.

BACKGROUND: Silver nanoparticles (AgNP), the most popular nano-compounds, possess unique properties. Albizia adianthifolia (AA) is a plant of the Fabaceae family that is rich in saponins. The biological properties of a novel AgNP, synthesized from an aqueous leaf extract of AA (AA(AgNP)), were investigated on A549 lung cells. Cell viability was determined by the MTT assay. Cellular oxidative status (lipid peroxidation and glutathione (GSH) levels), ATP concentration, caspase-3/-7, -8 and -9 activities were determined. Apoptosis, mitochondrial (mt) membrane depolarization (flow cytometry) and DNA fragmentation (comet assay) were assessed. The expression of CD95 receptors, p53, bax, PARP-1 and smac/DIABLO was evaluated by flow cytometry and/or western blotting. RESULTS: Silver nanoparticles of AA caused a dose-dependent decrease in cell viability with a significant increase in lipid peroxidation (5-fold vs. control; p = 0.0098) and decreased intracellular GSH (p = 0.1184). A significant 2.5-fold decrease in cellular ATP was observed upon AA(AgNP) exposure (p = 0.0040) with a highly significant elevation in mt depolarization (3.3-fold vs. control; p < 0.0001). Apoptosis was also significantly higher (1.5-fold) in AA(AgNP) treated cells (p < 0.0001) with a significant decline in expression of CD95 receptors (p = 0.0416). Silver nanoparticles of AA caused a significant 2.5-fold reduction in caspase-8 activity (p = 0.0024) with contrasting increases in caspase-3/-7 (1.7-fold vs. control; p = 0.0180) and -9 activity (1.4-fold vs. control; p = 0.0117). Western blots showed increased expression of smac/DIABLO (4.1-fold) in treated cells (p = 0.0033). Furthermore, AA(AgNP) significantly increased the expression of p53, bax and PARP-1 (1.2-fold; p = 0.0498, 1.6-fold; p = 0.0083 and 1.1-fold; p = 0.0359 respectively). CONCLUSION: Data suggests that AA(AgNP) induces cell death in the A549 lung cells via the mt mediated intrinsic apoptotic program. Further investigation is required to potentiate the use of this novel compound in cancer therapy trials.

The cytotoxic effects of Scilla nervosa (Burch.) Jessop (Hyacinthaceae) aqueous extract on cultured HepG2 cells.

ETHNOPHARMACOLOGICAL RELEVANCE: Bulbs of Scilla nervosa, a medicinal plant indigenous to Southern Africa, are traditionally used in aqueous decoctions to treat a diverse range of illnesses. The bulbs contain homoisoflavanones and stilbenoids. Little information is known about the plant's toxicity on the liver, a major detoxifying organ. This study investigated the effects of an aqueous extract of the bulbs in cultured HepG2 liver cells, a model system for investigating the toxicity of xenobiotics. MATERIALS AND METHODS: The concentration that reduced cell viability to 50% (IC(50)) after 24h treatment was derived. Potential mechanisms of toxicity using the IC(50) were investigated as changes in metabolic activity, apoptosis, oxidative damage and DNA fragmentation. In addition, cytochrome P450 3A4 (CYP3A4) activity, which is implicated in drug metabolism and interactions, was also assayed. RESULTS: Cell viability decreased in a concentration-dependent manner and the IC(50) was determined as 0.03 mg/mL. Treating the cells at the IC(50) for 24h resulted in increased intracellular ATP levels, no significant change in phosphatidylserine externalisation, increased caspase-8 activity, decreased caspase-9 activity, no significant change in mitochondrial membrane potential, increased lipid peroxidation, evidence for genotoxicity as demonstrated by DNA fragmentation, and slightly induced CYP3A4 activity. CONCLUSION: Results suggest that liver cells are sensitive to an aqueous extract of the bulbs and there is an increased potential to induce apoptosis, oxidative stress and genotoxicity in vitro.