Alisa Phulukdaree

A549 lung cell line activity of biosynthesized silver nanoparticles using Albizia adianthifolia leaf.

Stable AgNPs were formed in vitro by reacting AgNO3 (aq) solution with the aqueous plant leaf extract. UV-vis revealed the surface plasmon resonance λmax at 448 nm and the absorbance steadily increased in intensity as a function of reaction time. Transmission electron microscope (TEM) and XRD studies were used to characterize the AgNPs; the size was 4-35 nm. Dynamic light scattering (DLS) was used as supporting evidence to determine hydrodynamic size and zeta potential recorded as 80.27 nm and -24.7 mV, respectively. FT-IR spectra suggest that AgNPs are capped with protein molecules and other water soluble phytocompounds such as saponins and glycosides which also behave as stabilizing agents; TEM images indicate a visible layer surrounding the AgNPs. Prominent absorption bands at 3380 and 1642 cm(-1) are assigned to alcohol and carbonyl groups, respectively. (1)H NMR of the neat aqueous plant extract indicates presence of a complex mixture of compounds; however the chemical shift at δ 6.0-8.0 and 1.0-4.0 ppm indicates the presence of few aromatic but abundant aliphatic compounds, respectively. Toxicity of AgNPs on lung cancer cells (A549) and normal healthy peripheral lymphocytes (PLs) at 10 μg/ml and 50 μg/ml was assessed using the MTT, ATP and lactate dehydrogenase assays. Viability data for A549 cells showed a 21% (10 μg/ml) and 73% (50 μg/ml) cell viability after 6h exposure to AgNPs compared to 117% (10 μg/ml) and 109% (50 μg/ml) cell viability of normal peripheral lymphocytes. Lactate dehydrogenase was only significantly altered at 50 μg/ml AgNPs treated cells from 2.43±0.04 units to 0.77±0.04 units.

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

BackgroundSilver 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 (AAAgNP), 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.ResultsSilver 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 AAAgNP 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 AAAgNP 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, AAAgNP 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).ConclusionData suggests that AAAgNP 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.

Mitochondrial and Oxidative Stress Response in HepG2 Cells Following Acute and Prolonged Exposure to Antiretroviral Drugs.

Chronic HIV treatment with antiretroviral drugs has been associated with adverse health outcomes. Mitochondrial toxicity exhibited by nucleoside reverse transcriptase inhibitors (NRTIs) is pinpointed as a molecular mechanism of toxicity. This study evaluated the effect of NRTIs: Zidovudine (AZT, 7.1 μM), Stavudine (d4T, 4 μM) and Tenofovir (TFV, 1.2 μM), on mitochondrial (mt) stress response, mtDNA integrity and oxidative stress response in human hepatoma cells at 24 and 120 h. Markers for mt function, mt biogenesis, oxidative stress parameters, and antioxidant response were evaluated by spectrophotometry, luminometry, flow cytometry, qPCR and western blots. We found that AZT and d4T reduced mtDNA integrity (120 h, AZT: 76.1%; d4T:36.1%, P < 0.05) and remained unchanged with TFV. All three NRTIs, however, reduced ATP levels (AZT: 38%; d4T: 56.4%; TFV: 27.4%, P = 0.01) and mt membrane potential at 120 h (P < 0.005). Oxidative damage and reactive oxygen species (ROS) were increased by TFV and AZT at 24 h, and by d4T at 120 h (P < 0.05). Antioxidant response molecules and mt biogenesis markers were elevated by all NRTIs, with TFV causing the most significant increase (P < 0.05). Data from this study suggest that AZT, d4T and TFV alter mt function. TFV, however, achieves this independently of mtDNA depletion. Furthermore, AZT exerts toxicity soon after exposure as noted from changes at 24 h and d4T exerts greater toxicity over prolonged exposure (120 h). J. Cell. Biochem. 116: 1939–1946, 2015.

Fumonisin B1 modulates expression of human cytochrome P450 1b1 in human hepatoma (Hepg2) cells by repressing Mir-27b

Fumonisin B₁ (FB₁), a common mycotoxin contaminant of maize, is known to inhibit sphingolipid biosynthesis and has been implicated in hepatocellular carcinoma promoting activity in humans and animals. MicroRNAs (miRNA) are small noncoding RNAs that regulate gene expression via translational repression. Human cytochrome P450 (CYP1B1) is highly expressed in oestrogen target tissues and catalyzes the metabolic activation of many procarcinogens. The aim of our study was to investigate the effect of FB₁ on miR-27b suppression and its effect on CYP1B1 modulation in a human hepatoma cell line (HepG2). MiR27b and CYP1B1 expressions were evaluated in HepG2 cells by quantitative PCR. In order to directly assess the effect of miR-27b on CYP1B1 mRNA levels, cells were transfected with the mimic to miR-27b. CYP1B1 protein expression was measured using Western blot. FB₁ significantly down-regulated (11-fold) expression of miR-27b in HepG2 cells; whilst CYP1B1 mRNA and protein expression was significantly upregulated by 1.8-fold and 2.6-fold, respectively. CYP1B1 is post-transcriptionally regulated by miR-27b after HepG2 exposure to FB₁. FB₁-induced modulation of miR-27b in hepatic cells may be an additional mode of hepatic neoplastic transformation.

The effects of vasopressin and oxytocin on methamphetamine-induced place preference behaviour in rats.

Methamphetamine is a highly addictive stimulant drug whose illicit use and resultant addiction has become an alarming global phenomenon. The mesolimbic dopaminergic pathway has been shown to be fundamental to the establishment of addictive behaviour. This pathway, as part of the reward system of the brain, has also been shown to be important in classical conditioning, which is a learnt response. Within the modulation of learning and memory, the neurohypophyseal hormones vasopressin and oxytocin have been reported to play a vital role, with vasopressin exerting a long- term facilitatory effect and oxytocin exerting an inhibitory effect. Therefore we adopted a conditioned place preference model to investigate whether vasopressin V1b receptor antagonist SSR 149415 or oxytocin treatment would cause a decrease in the seeking behaviour in a reinstatement paradigm. Behavioural findings indicated that methamphetamine induced a change in the place preference in the majority of our animals. This change in place preference was not seen when vasopressin was administered during the extinction phase. On the other hand the methamphetamine-induced change in place preference was enhanced during the reinstatement phase in the animals that were treated with oxytocin. Striatal dopamine levels were determined, as methamphetamine is known to increase dopamine transmission in this area. Significant changes in dopamine levels were observed in some of our animals. Rats that received both methamphetamine and oxytocin had significantly higher striatal dopamine than those that received oxytocin alone. Western blot analysis for hippocampal cyclic AMP response element binding protein (CREB) was also conducted as a possible indicator of glutamatergic NMDA receptor activity, a pathway that is important for learning and memory. The Western blot analysis showed no changes in hippocampal pCREB expression. Overall our data led us to conclude that methamphetamine treatment can change place preference behaviour in rats and that this change may be partially restored by vasopressin antagonism, but exaggerated by oxytocin.

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

Gold nanoparticles (AuNPs) 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.

Fumonisin B1 induces global DNA hypomethylation in HepG2 cells - An alternative mechanism of action

Fumonisin B1 (FB1), a common mycotoxin contaminant of maize, is known to inhibit sphingolipid biosynthesis and has been implicated in cancer promoting activity in animals and humans. FB1 disrupts DNA methylation and chromatin modifications in human hepatoma (HepG2) cells. We investigated the effect of FB1 on enzymes, DNA methyltransferases and demethylases, involved in chromatin maintenance and gross changes in structural integrity of DNA in HepG2 cells. We measured: (i) the expression of 84 key genes encoding enzymes known to modify genomic DNA and histones (superarray and qPCR); (ii) protein expression of DNA methyltransferases (DNMT1, DNMT3A and DNMT3B) and the major demethylase (MBD2) (western blotting); (iii) degree of DNA methylation by FACS using anti-5-MeCyt and (iv) DNA migration (single cell gel electrophoresis). FB1 significantly decreased the methyltransferase activities of DNMT1, DNMT3A and DNMT3B, and significantly up regulated the demethylases (MBD2 expression and activity, and KDM5B and KDM5C expression). FACS data showed FB1 significantly increased DNA hypomethylation and resulted in gross changes in structural DNA as evidenced by the Comet assay. We conclude that FB1 induces global DNA hypomethylation and histone demethylation that causes chromatin instability and may lead to liver tumourigenesis.

Biosynthesis of palladium nanoparticles by using Moringa oleifera flower extract and their catalytic and biological properties

The biosynthesis of nanostructured biopalladium nanoparticles (PdNPs) from an aqueous solution of crystalline palladium acetate is reported. For the synthesised PdNPs in solution, an agroforest biomass waste petal of Moringa oleifera derived bis-phthalate was used as natural reducing and biocapping agents. Continuous absorption in the UV region and subsequent brown colour change confirmed the formation of PdNPs. A strong surface plasmon peak for PdNPs occurred at 460nm. PdNPs were characterized by SEM with EDX, FTIR, TEM and DLS. The chemical composition of the aqueous extract was determined by GC-MS coupled with FTIR and 1NMR. The catalytic degradation effect by PdNPs on industrial organic toxic effluents p-nitrophenol (PNP) and methylene blue dye was monitored by UV Spectroscopy. On the other hand PdNPs catalysed the base mediated suzuki coupling reaction for biphenyl synthesis, in water. Moreover, PdNPs were found to be reusable catalysts. Toxicity studies of PdNPs showed that the death of brine shrimp to be <50%. Therefore, PdNPs displayed potential for further anticancer studies via tumour cell lines. The in vitro cytotoxicity evaluation of the extract capped nanoparticles was carried out using human lung carcinoma cells (A549) and peripheral lymphocytes normal cells by MTT cell viability assay. Also, PdNPs showed antibacterial activity against Enterococcus faecalis among the different tested strains, including Bacillus cereus, Staphylococcus aureus, Esherichia coli and Candida albicans, Candida utilis.

GST polymorphisms and early-onset coronary artery disease in young South African Indians

BACKGROUND Glutathione S-transferases (GSTs) detoxify environmental agents which influence the onset and progression of disease. Dysfunctional detoxification enzymes are responsible for prolonged exposure to reactive molecules and can contribute to endothelial damage, an underlying factor in coronary artery disease (CAD). OBJECTIVES We aimed to assess 2 common polymorphic variant isoforms in GSTM1 and GSTP1 of GST in young CAD patients. METHODS All patients (N=102) were South Africans of Indian ancestry, a population associated with high CAD risk. A corresponding age-, sex- and race-matched control group (N=100) was also recruited. Frequency of the GSTM1 +/0 (v. +/0 and 0/0) and GSTP1 A105/G105 (v. wild-type A105/A105) genotypes was assessed by differential polymerase chain reaction (PCR) and PCR restriction fragment length polymorphism (PCR-RFLP), respectively. RESULTS The GSTM1 0/0 and GSTP1 A105/A105 genotypes occurred at higher frequencies in CAD patients compared with the control group (36% v. 18% and 65% v. 48%, respectively). A significant association with CAD was observed in GSTM1 0/0 (OR=2.593; 95% CI 1.353 - 4.971; p=0.0043) and GSTP1 A105/A105 (odds ratio (OR)=0.6011; 95% confidence interval (CI) 0.3803 - 0.9503; p=0.0377). We found a significant association between smoking and CAD; the presence of either of the respective genotypes together with smoking increased the CAD risk (GSTP1 A105 RR=1.382; 95% CI 0.958 - 1.994; p=0.0987 and GSTM1 null RR=1.725; 95% CI 1.044 - 2.851; p=0.0221). CONCLUSION Our findings support the association of genotypes GSTM1 0/0 and GSTP1 A105/A105 and smoking with CAD.

The interleukin-6 -147 g/c polymorphism is associated with increased risk of coronary artery disease in young South African Indian men.

BACKGROUND Interleukin-6 (IL-6) is a proinflammatory cytokine involved in the pathogenesis of chronic inflammatory diseases such as coronary artery disease (CAD). The -174 IL-6 G/C promoter polymorphism influences mRNA levels and protein expression and is implicated in CAD. The Indian population in South Africa, unlike the black community, has a high prevalence of premature CAD. This polymorphism has not been fully explored in this population. The present study assessed the -174 IL-6 G/C polymorphism in young Indian patients with angiographically documented CAD and compared them with age- and gender-matched Indian and black control subjects. METHODS Polymorphic variants were assessed by polymerase chain reaction-restriction fragment length polymorphism, and IL-6 levels were measured using enzyme-linked immunosorbent assay (ELISA). RESULTS The -174 IL-6 C allele was found with a higher frequency (23%) in the total Indian group compared to 2% in the black participants [P<0.0001, odds ratio (OR)=0.05, 95% confidence interval (CI) 0.018-0.14). The difference in frequency was more pronounced when Indian controls were compared to black controls (29% vs. 2%, respectively) (P<0.0001, OR=0.05, 95% CI 0.02-0.17). A significant association between the -174 IL-6 G allele and CAD was found in Indian patients compared to Indian controls (84% in cases vs. 71% in Indian controls; P=0.043, OR=0.47 95% CI 0.23-0.95). Levels of IL-6 in circulation were higher in black controls (6.62±0.63 pg/mL) compared to Indian controls (2.51±0.57 pg/mL) and CAD patients (1.46±0.36 pg/mL) (P<0.0001). Levels of IL-6 were higher in all groups with homozygous -174 IL-6 C alleles, but only significant in the healthy Indian control group (GG 3.73±0.94 pg/mL vs. GC/CC 0.89±0.5 pg/mL, P=0.0001). CONCLUSION The presence of the IL-6 -174 G allele influences levels of IL-6 and increases the risk of CAD in South African Indians.