Soi Moi Chye

Apoptosis Induced by para-Phenylenediamine Involves Formation of ROS and Activation of p38 and JNK in Chang Liver Cells

para‐phenylenediamine (p‐PD) is a suspected carcinogen, but it has been widely used as a component in permanent hair dyes. In this study, the mechanism of p‐PD‐induced cell death in normal Chang liver cells was investigated. The results demonstrated that p‐PD decreased cell viability in a dose‐dependent manner. Cell death via apoptosis was confirmed by enhanced DNA damage and increased cell number in the sub‐G1 phase of the cell cycle, using Hoechst 33258 dye staining and flow cytometry analysis. Apoptosis via reactive oxygen species generation was detected by the dichlorofluorescin diacetate staining method. Mitogen‐activated protein kinase (MAPK) activation was assessed by western blot analysis and revealed that p‐PD activated not only stress‐activated protein kinase (SAPK)/c‐Jun N‐terminal kinases (JNK) and p38 MAPK but also extracellular signal‐regulated kinase (ERK). Cytotoxicity and apoptosis induced by p‐PD were markedly enhanced by ERK activation and selectively inhibited by ERK inhibitor PD98059, thus indicating a negative role of ERK. In contrast, inhibition of p38 MAPK activity with the p38‐specific inhibitor SB203580 moderately inhibited cytotoxicity and apoptosis induction by p‐PD. Similarly, SP600125, an inhibitor of SAPK/JNK, moderately inhibited cytotoxicity and apoptosis induced by p‐PD, thus implying that p38 MAPK and SAPK/JNK had a partial role in p‐PD‐induced apoptosis. Western blot analysis revealed that p‐PD significantly increased phosphorylation of p38 and SAPK/JNK and decreased phosphorylation of ERK. In conclusion, the results demonstrated that SAPK/JNK and p38 cooperatively participate in apoptosis induced by p‐PD and that a decreased ERK signal contributes to growth inhibition or apoptosis.

Anti-adipogenic effects of extracts of Ficus deltoidea var. deltoidea and var. angustifolia on 3T3-L1 adipocytes.

Objective: This study examined the anti-adipogenic effects of extracts of Ficus deltoidea var. deltoidia and var. angustifolia, a natural slimming aid, on 3T3-L1 adipocytes. Methods: Methanol and water extracts of leaves of the F. deltoidea varieties were analyzed to determine their total flavonoid content (TFC) and total phenolic content (TPC), respectively. The study was initiated by determining the maximum non-toxic dose (MNTD) of the methanol and water extracts for 3T3-L1 preadipocytes. Possible anti-adipogenic effects were then examined by treating 2-d post confluent 3T3-L1 preadipocytes with either methanol extract or water extract at MNTD and half MNTD (½MNTD), after which the preadipocytces were induced to form mature adipocytes. Visualisation and quantification of lipid content in mature adipocytes were carried out through oil red O staining and measurement of optical density (OD) at 520 nm, respectively. Results: The TFCs of the methanol extracts were 1.36 and 1.97 g quercetin equivalents (QE)/100 g dry weight (DW), while the TPCs of the water extracts were 5.61 and 2.73 g gallic acid equivalents (GAE)/100 g DW for var. deltoidea and var. angustilofia, respectively. The MNTDs determined for methanol and water extracts were (300.0±28.3) and (225.0±21.2) µg/ml, respectively, for var. deltoidea, while much lower MNTDs [(60.0±2.0) µg/ml for methanol extracts and (8.0±1.0) µg/ml for water extracts] were recorded for var. angustifolia. Studies revealed that the methanol extracts of both varieties and the water extracts of var. angustifolia at either MNTD or ½MNTD significantly inhibited the maturation of preadipocytes. Conclusions: The inhibition of the formation of mature adipocytes indicated that leaf extracts of F. deltoidea could have potential anti-obesity effects.

Tau Proteins and Tauopathies in Alzheimer’s Disease

Alzheimer’s disease (AD) is characterized by progressive memory loss and cognitive function deficits. There are two major pathological hallmarks that contribute to the pathogenesis of AD which are the presence of extracellular amyloid plaques composed of amyloid-β (Aβ) and intracellular neurofibrillary tangles composed of hyperphosphorylated tau. Despite extensive research that has been done on Aβ in the last two decades, therapies targeting Aβ were not very fruitful at treating AD as the efficacy of Aβ therapies observed in animal models is not reflected in human clinical trials. Hence, tau-directed therapies have received tremendous attention as the potential treatments for AD. Tauopathies are closely correlated with dementia and immunotherapy has been effective at reducing tau pathology and improving cognitive deficits in animal models. Thus, in this review article, we discussed the pathological mechanism of tau proteins, the key factors contributing to tauopathies, and therapeutic approaches for tauopathies in AD based on the recent progress in tau-based research.

Ternary and binary copper(II) complexes: synthesis, characterization, ROS-inductive, proteasome inhibitory, and anticancer properties

Abstract Three ternary copper(II) complexes, [Cu(phen)(L-phe)Cl]·2H2O, [Cu(phen)(L-leu)Cl]·4½H2O, and [Cu(phen)(L-tyr)Cl]·3H2O, and four binary copper(II) complexes, [Cu(phen)Cl2], Cu(L-phe)2·½H2O, Cu(L-leu)2·½H2O, and Cu(L-tyr)2·H2O (where phen = 110-phenanthroline, L-phe = L-phenylalanine, L-tyr = L-tyrosine, L-leu = L-leucine and Cl- = chloride), were synthesized and characterized by elemental analysis, spectroscopic techniques (FTIR, UV–visible, fluorescence spectroscopy), magnetic susceptibility, molar conductivity, and lipophilicity measurement. X-ray diffraction determination of a single crystal of [Cu(phen)(L-tyr)Cl] showed two independent molecules in the asymmetric unit, each with the same distorted square pyramidal geometry about copper(II). p-Nitrosodimethylaniline assay revealed that the three ternary complexes were better inducers of reactive oxygen species over time than binary complexes, CuCl2, and free ligands. All the copper(II) complexes in this series inhibited the three proteolytic activities in the order Trypsin-like > Caspase-like > Chymotrypsin-like. In terms of anticancer properties, the copper(II)-phen complexes had GI50 values of less than 4 μM against MCF-7, HepG2, CNE1 and A549 cancer cell lines, more potent than cisplatin.

para-Phenylenediamine induces apoptosis through activation of reactive oxygen species-mediated mitochondrial pathway, and inhibition of the NF-κB, mTOR, and Wnt pathways in human urothelial cells.

para‐Phenylenediamine (PPD) has long been used in two‐thirds of permanent oxidative hair dye formulations. Epidemiological studies and in vivo studies have shown that hair dye is a suspected carcinogen of bladder cancer. However, the toxicity effects of PPD to human bladder remains elusive. In this study, the effects of PPD and its involvement in the apoptosis pathways in human urothelial cells (UROtsa) was investigated. It was demonstrated that PPD decreased cell viability and increased the number of sub‐G1 hypodiploid cells in UROtsa cells. Cell death due to apoptosis was detected using Annexin V binding assay. Further analysis showed PPD generated reactive oxygen species (ROS), induced mitochondrial dysfunction through the loss of mitochondrial membrane potential and increased caspase‐3 level in UROtsa cells. Western blot analysis of PPD‐treated UROtsa cells showed down‐regulation of phosphorylated proteins from NF‐κB, mTOR, and Wnt pathways. In conclusion, PPD induced apoptosis via activation of ROS‐mediated mitochondrial pathway, and possibly through inhibition of NF‐κB, mTOR, and Wnt pathways.

Clinacanthus Nutans Hexane Extracts Induce Apoptosis Through a Caspase-Dependent Pathway in Human Cancer Cell Lines

Background: Clinacanthus nutans (C.nutans) is a plant consumed as a cancer treatment in tropical Asia. Despite the availability of numerous anecdotal reports, evaluation of active anticancer effects has remained elusive. Therefore we here examined antiproliferative, reactive oxygen species (ROS)-inducing and apoptosis mechanisms of whole plant extracts in different cancer cell lines. Methods: Antiproliferative actions of five solvent extracts (hexane, chloroform, ethyl acetate, methanol and water) of C.nutans were tested on non-small cell lung cancer (A549), nasopharygeal cancer (CNE1) and liver cancer (HepG2) cells using MTT assay. The most potent anticancer extract was then assessed by flow cytometry to study cell cycle changes. Intracellular levels of ROS were quantified by DCFH-DA assay. Involvement of the caspase pathway in induction of apoptosis was assessed using caspase assay kits. GC-MS analysis was performed to identify phytoconstituents in the extracts. Results: Hexane and chloroform extracts were antiproliferative against all three cell lines, while the ethyl acetate extract, at 300 µg/mL, was antiproliferative in the CNE1 but not A549 and HepG2 cases. Methanol and water extracts did not inhibit cancer cell proliferation. The most potent anticancer hexane extract was selected for further testing. It induced apoptosis in all three cell lines as shown by an increase in the percentage of cell in sub-G1 phase. Dose-dependent increase in ROS levels in all three cell lines indicated apoptosis to be possibly modulated by oxidative stress. At high concentrations (>100 µg/mL), hexane extracts upregulated caspases 8, 9 and 3/7 across all three cell lines. GC-MS analysis of the hexane extract revealed abundance of 31 compounds. Conclusion: Among the five extracts of C.nutans, that with hexane extract demonstrated the highest antiproliferative activity against all three cancer cell lines tested. Action appeared to be via ion of intracellular ROS, and induction of apoptosis via intrinsic and extrinsic caspase pathways.

Cytotoxic and apoptogenic effects of Strobilanthes crispa Blume extracts on nasopharyngeal cancer cells

The chemotherapeutic agents used to treat nasopharyngeal cancer (NPC) exhibit low efficacy. Strobilanthes crispa Blume is widely used for its anticancer, diuretic and anti‑diabetic properties. The present study aimed to determine the cytotoxic and apoptogenic effects of S. crispa on CNE‑1 NPC cells. A 3‑(4,5‑dimethylthiazol‑2‑yl)‑2,5 diphenyl tetrazolium bromide assay was used to evaluate the cytotoxic effects of S. crispa against CNE‑1 cells. The rate of apoptosis was determined using propidium iodide staining and caspase assays. Ethyl acetate, hexane and chloroform extracts of S. crispa leaves all exhibited cytotoxic effects on CNE‑1 cells, at a half maximal inhibitory concentration (IC50) of 119, 123.5 and 161.7 µg/ml, respectively. In addition, hexane, chloroform and ethyl acetate extracts of S. crispa stems inhibited CNE‑1 cell proliferation, at a IC50 of 49.4, 148.3 and 163.5 µg/ml, respectively. Flow cytometric analysis revealed an increased proportion of cells in the sub G1 phase and a decreased proportion of cells in the G2/M phase, following treatment with the extracts. However, the extracts did not alter the activities of caspase ‑3/7, ‑8 and ‑9. No cytotoxic effect was observed when the cells were treated with the methanol and water extracts of S. crispa stems and leaves. In conclusion, the S. crispa extracts were cytotoxic against CNE‑1 cells and these extracts were able to induce apoptosis, independent of caspase activation.

Enantiomeric pairs of copper(II) polypyridyl-alanine complex salts: anticancer studies

The anticancer properties of two previously characterized pairs of optically pure chiral complex salts [Cu(phen)(ala)(H2O)]X·xH2O (phen = 1.10-phenanthroline; X = NO3−; ala: l-alanine (l-ala) 1 and d-alanine (d-ala) 2; and (X = Cl−; ala: l-ala, 3 and d-ala, 4; x = number of lattice water molecules) are reported herein, together with the crystal structure of the d-enantiomer 4. Unlike cisplatin which is ineffective against MCF-7 cancer cells with the absence of caspase-3 protein expression, these two pairs of complex salts were effective against this cell line and they were able to induce an increase in intracellular ROS, loss in mitochondrial membrane potential, cell cycle arrest mainly at SubG1 phase , caspase-9 activation, and caspase-3/caspase-7-independent apoptosis. Screening of 1 on the NCI-60 panel of human cancer cell lines showed that it was effective against most of the cell lines. MTT-NCI modified assay screening was also done on other cancer cell lines, viz. A549, CNE1, and HepG2, and two normal cell lines, viz. MCF-10A and CHANG. The effects of chirality of these Cu(II) compounds, especially the greater selectivity of d-enantiomers over the l-counterparts, on their anticancer properties are also reported herein.

Single-chain Fv Antibodies for Targeting Neurodegenerative Diseases

BACKGROUND & OBJECTIVE Protein misfolding and aggregation have been considered the common pathological hallmarks for a number of neurodegenerative diseases, including Alzheimers disease (AD), Parkinsons disease (PD) and Huntingtons disease (HD). These abnormal proteins aggregates damage mitochondria and induce oxidative stress, resulting in neuronal cell death. Prolonged neuronal damage activates microglia and astrocytes, development of inflammation reaction and further promotes neurodegeneration. Thus, elimination of abnormal protein aggregates without eliciting any adverse effects are the main treatment strategies. To overcome this, recent studies have deployed single- chain fragment variable antibodies (scFvs) to target the pathological protein aggregates, such as amyloid-beta (Aβ) peptides, α-synuclein (α-syn) and Huntingtin (Htt). To date scFv has been effective at inhibiting abnormal protein aggregates formation in both in vitro and in vivo model system of AD, PD and HD. CONCLUSION Currently active research is still ongoing to improve the scFv gene delivery technology, to further enhance brain penetration, intracellular stability, solubility and efficacy of scFv intrabody.

Niosomes: a review of their structure, properties, methods of preparation, and medical applications

Abstract Target-specific drug-delivery systems for the administration of pharmaceutical compounds enable the localization of drugs to diseased sites. Various types of drug-delivery systems utilize carriers, such as immunoglobulins, serum proteins, synthetic polymers, liposomes, and microspheres. The vesicular system of niosomes, with their bilayer structure assembled by nonionic surfactants, is able to enhance the bioavailability of a drug to a predetermined area for a period. The amphiphilic nature of niosomes promotes their efficiency in encapsulating lipophilic or hydrophilic drugs. Other additives, such as cholesterol, can be used to maintain the rigidity of the niosomes’ structure. This narrative review describes fundamental aspects of niosomes, including their structural components, methods of preparation, limitations, and current applications to various diseases.