Tengku Sifzizul Tengku Muhammad

Cytotoxic, apoptotic and anti-α-glucosidase activities of 3,4-di-O-caffeoyl quinic acid, an antioxidant isolated from the polyphenolic-rich extract of Elephantopus mollis Kunth.

ETHNOPHARMACOLOGICAL RELEVANCE The decoction of the whole plant of Elephantopus mollis Kunth. is traditionally consumed to treat various free radical-mediated diseases including cancer and diabetes. AIM OF THE STUDY This study was initiated to determine whether the most effective antioxidant compound isolated from the whole plant of Elephantopus mollis can also contribute to its claimed traditional values as anticancer and antidiabetes agents. MATERIALS AND METHODS An active antiradical phenolic compound (3,4-di-O-caffeoyl quinic acid) was isolated from the methanol extract (with the highest in polyphenolic content) and their antioxidant activities were compared using four different assays, that are DPPH, FRAP, metal chelating, and β-carotene bleaching tests. The compound was also evaluated for its cytotoxic activity, apoptotic induction and anti-glucosidase efficacies using methylene blue, DeadEnd™ assay and α-glucosidase assays, respectively. RESULTS The compound acted as a greater primary antioxidant than its methanol extract, by having higher ferric reducing activity (EC(50) 2.18±0.05 μg/ml), β-carotene bleaching activity (EC(50) 23.85±0.65 μg/ml) and DPPH scavenging activity (EC(50) 68.91±5.44μg/ml), whereas the methanol extract exhibited higher secondary antioxidant activity as a metal chelator with lower EC(50) value (49.39±3.68 μg/ml) than the compound. Cytotoxicity screening of this compound exhibited a remarkable dose-dependent inhibitory effect on NCI-H23 (human lung adenocarcinoma) cell lines (EC(50) 3.26±0.35 μg/ml) and was found to be apoptotic in nature based on a clear indication of DNA fragmentation. This compound also displayed a concentration-dependent α-glucosidase inhibition with EC(50) 241.80±14.29 μg/ml. CONCLUSIONS The findings indicate the major role of 3,4-di-O-caffeoyl quinic acid to antioxidant capacities of Elephantopus mollis extracts. The compound also exerted apoptosis-mediated cytotoxicity and α-glucosidase inhibitory effects and is thus a promising non toxic agent in treating cancer and type 2 diabetes mellitus.

Cytotoxic Activities of Major Diterpenoid Constituents of Andrographis paniculata. in a Panel of Human Tumor Cell Lines

ABSTRACT The cytotoxic activities of several diterpenoid constituents of the medicinal plant, “hempedu bumi,” or Andrographis paniculata. Nees (Acanthaceae), were evaluated. The seven diterpenoid compounds used were andrographolide, 14-deoxyandrographolide, andrographiside, deoxyandrographiside, 14-deoxy-12-methoxyandrographolide, neoandrographolide, and 14-deoxy-11,12-didehydroandrographolide. The activities of these compounds were evaluated with various human tumor cell lines such as Caov-3 (human ovarian carcinoma cell line), T-47D (human breast carcinoma cell line), Hs-578T (human breast carcinoma cell line), Hep G2 (human hepatocarcinoma cell line), and NCI–H23 (human lung adenocarcinoma cell line). Cell survival was measured using the MTS assay after 72 h of incubation. Andrographolide, neoandrographolide, andrographiside, deoxyandrographiside, and 14-deoxy-12-methoxyandrographolide appeared to be noncytotoxic against all the cell lines. Only 14-deoxyandrographolide and 14-deoxy-11,12-didehydroandrographolide exhibited cytotoxic activities (based on EC50 values), but this was limited to the T-47D cell line (EC50 values of 2.8 µg/ml and 1.5 µg/ml, respectively). As one of the principle diterpenoids of Andrographis paniculata., 14-deoxy-11,12-didehydroandrographolide appeared to be the most potent when compared with the rest of the compounds examined. The effects of 14-deoxy-11,12-didehydroandrographolide on T-47D cells were further confirmed to be nonapoptotic, non-necrotic, but programmed in nature, as demonstrated using a DNA fragmentation detection assay, Trypan blue exclusion assay, and annexin V–propidium iodide staining.

Discovery of new nanomolar peroxisome proliferator-activated receptor γ activators via elaborate ligand-based modeling.

Peroxisome Proliferator-Activated Receptor γ (PPARγ) activators have drawn great recent attention in the clinical management of type 2 diabetes mellitus, prompting several attempts to discover and optimize new PPARγ activators. With this in mind, we explored the pharmacophoric space of PPARγ using seven diverse sets of activators. Subsequently, genetic algorithm and multiple linear regression analysis were employed to select an optimal combination of pharmacophoric models and 2D physicochemical descriptors capable of accessing self-consistent and predictive quantitative structure-activity relationship (QSAR) (r2(71)=0.80, F=270.3, r2LOO=0.73, r2PRESS against 17 external test inhibitors=0.67). Three orthogonal pharmacophores emerged in the QSAR equation and were validated by receiver operating characteristic (ROC) curves analysis. The models were then used to screen the national cancer institute (NCI) list of compounds. The highest-ranking hits were tested in vitro. The most potent hits illustrated EC50 values of 15 and 224 nM.

Identification of Wap65, a human homologue of hemopexin as a copper-inducible gene in swordtail fish, Xiphophorus helleri.

Copper is one of the major heavy metal pollutants found in the aquatic environment. Therefore, it is important for determining the genes that play a key role in copper metabolism in aquatic organisms. This study, thus, aimed to identify a new copper-inducible gene in swordtail fish, Xiphophorus helleri. Using ACP-based RT-PCR coupled with RLM-RACE, we cloned Wap65, a mammalian homologue of hemopexin gene. The gene exhibits high identity at amino acid levels with the Wap65 gene of other fish species (42–68%) and mammalian hemopexin gene (35–37%). In addition, ten cysteine and two histidine residues are conserved in the swordtail fish Wap65 gene. These cysteine residues are vital for structural integrity, and histidine residues provide high binding affinity towards heme. As revealed by RT-PCR, the gene was upregulated in swordtail fish that were exposed to copper in a dose- and time-dependent manner. Therefore, the identification of Wap65, a mammalian homologue of hemopexin, as a new copper-inducible gene will provide greater insight into the role of this gene in copper metabolism.

Cytotoxic Activities of Physalis minima L. Chloroform Extract on Human Lung Adenocarcinoma NCI-H23 Cell Lines by Induction of Apoptosis

Physalis minima L. is reputed for having anticancer property. In this study, the chloroform extract of this plant exhibited remarkable cytotoxic activities on NCI-H23 (human lung adenocarcinoma) cell line at dose- and time-dependent manners (after 24, 48 and 72 h of incubation). Analysis of cell-death mechanism demonstrated that the extract exerted apoptotic programed cell death in NCI-H23 cells with typical DNA fragmentation, which is a biochemical hallmark of apoptosis. Morphological observation using transmission electron microscope (TEM) also displayed apoptotic characteristics in the treated cells, including clumping and margination of chromatins, followed by convolution of the nuclear and budding of the cells to produce membrane-bound apoptotic bodies. Different stages of apoptotic programed cell death as well as phosphatidylserine externalization were confirmed using annexin V and propidium iodide staining. Furthermore, acute exposure to the extract produced a significant regulation of c-myc, caspase-3 and p53 mRNA expression in this cell line. Due to its apoptotic effect on NCI-H23 cells, it is strongly suggested that the extract could be further developed as an anticancer drug.

Immunohistochemical expression of MAP1LC3A and MAP1LC3B protein in breast carcinoma tissues

Autophagy is a protein degradation process within the cell and its deregulation has been linked to various diseases and the formation of cancer. One of the important proteins involved in the autophagy process is microtubule‐associated protein 1 light chain 3 (MAP1LC3). The aims of this study were to determine the MAP1LC3A and MAP1LC3B protein expression in both normal and cancer breast tissues and to determine the relationship between the expression of these proteins and type of tissues. Immunohistochemistry assessments were carried out on tissue microarrays consisting of breast tissues. MAP1LC3A expression was detected in 52/56 of normal breast tissue cores and 65/67 of breast cancer tissue cores. MAP1LC3B expression was detected in 55/56 of normal breast tissue cores and 67/67 of breast cancer tissue cores. MAP1LC3A and MAP1LC3B protein are expressed in the majority of normal and cancer breast tissues. A large number of MAP1LC3A and MAP1LC3B positive breast cancer tissues cores have high proportion of stained cells (81–100%) as compared with normal breast tissues. However, a significantly higher number of breast cancer tissues were found to express the MAP1LC3A protein with strong immunoreactivity as compared with the normal tissues, suggesting that MAP1LC3A may play a role in breast cancer development. J. Clin. Lab. Anal. 23:249–258, 2009.

Swietenia macrophylla extract promotes the ability of Caenorhabditis elegans to survive Pseudomonas aeruginosa infection.

ETHNOPHARMACOLOGICAL RELEVANCE Swietenia macrophylla or commonly known as big leaf mahogany, has been traditionally used as an antibacterial and antifungal agent. AIM OF THE STUDY The unwanted problem of antibiotic resistance in many bacterial species advocates the need for the discovery of the new anti-infective drugs. Here, we investigated the anti-infective properties of Swietenia macrophylla with an assay involving lethal infection of Caenorhabditis elegans with the opportunistic human pathogen Pseudomonas aeruginosa. MATERIALS AND METHODS Using a slow killing assay, Caenorhabditis elegans was challenged with an infective strain of Pseudomonas aeruginosa (PA14). The ability of Swietenia macrophylla seed ethyl acetate extract to promote the survival of infected worms was assessed by comparing the percentage of survival between extract treated and non-treated worm populations. The effect of Swietenia macrophylla towards PA14 growth, Caenorhabditis elegans feeding rate and degree of PA14 colonization in the worm gut was also evaluated. Lastly, using a fluorescent transgenic Caenorhabditis elegans strain and real time PCR, the effect of Swietenia macrophylla on the expression of lys-7, an immune response gene was also investigated. RESULTS Our results demonstrate the ability of Swietenia macrophylla seed ethyl acetate extract in rescuing Caenorhabditis elegans from fatal PA14 infection. Consequently, we showed that the extract promotes the survival without exhibiting any bactericidal effect or perturbation of Caenorhabditis elegans feeding rate. We also showed that Swietenia macrophylla was able to restore the initially repressed lys-7 level in PA14 infected Caenorhabditis elegans. CONCLUSION Swietenia macrophylla extract is able to enhance the ability of Caenorhabditis elegans to survive PA14 infection without directly killing the pathogen. We further showed that the extract boosted the expression of a gene pivotal for innate immunity in Caenorhabditis elegans. Collectively, these findings strongly suggest the presence of compounds within Swietenia macrophylla seed that either reduces Pseudomonas aeruginosa virulence and/or enhance host resistance.

The PPARγ coding region and its role in visceral obesity

Peroxisome proliferator-activated receptor gamma (PPARgamma) is a ligand activated transcription factor, plays many essential roles of biological function in higher organisms. The PPARgamma is mainly expressed in adipose tissue. It regulates the transcriptional activity of genes by binding with other transcription factor. The PPARgamma coding region has been found to be closest to that of monkey in ours and other research groups. Thus, monkey is a more suitable animal model for future PPARgamma studying, although mice and rat are frequently being used. The PPARgamma is involved in regulating alterations of adipose tissue masses result from changes in mature adipocyte size and/or number through a complex interplay process called adipogenesis. However, the role of PPARgamma in negatively regulating the process of adipogenesis remains unclear. This review may help we investigate the differential expression of key transcription factor in adipose tissue in response to visceral obesity-induced diet in vivo. The study may also provide valuable information to define a more appropriate physiological condition in adipogenesis which may help to prevent diseases cause by negative regulation of the transcription factors in adipose tissue.

Cytochrome P450 induction properties of food and herbal-derived compounds using a novel multiplex RT-qPCR in vitro assay, a drug-food interaction prediction tool.

A multiplex RT-qPCR was developed to examine CYP1A2, CYP2D6, and CYP3A4 induction properties of compounds from food and herbal sources. The induction of drug metabolizing enzymes is an important pharmacokinetic interaction with unique features in comparison with inhibition of metabolizing enzymes. Cytochrome induction can lead to serious drug–drug or drug–food interactions, especially if the coadministered drug plasma level is critical as it can reduce therapeutic effects and cause complications. Using this optimized multiplex RT-qPCR, cytochrome induction properties of andrographolide, curcumin, lycopene, bergamottin, and resveratrol were determined. Andrographolide, curcumin, and lycopene produced no significant induction effects on CYP1A2, CYP2D6, and CYP3A4. However, bergamottin appeared to be a significant in vitro CYP1A2 inducer starting from 5 to 50 μmol/L with induction ranging from 60 to 100-fold changes. On the other hand, resveratrol is a weak in vitro CYP1A2 inducer. Examining the cytochrome induction properties of food and herbal compounds help complement CYP inhibition studies and provide labeling and safety caution for such products.

Rapamycin Induces Apoptosis When Autophagy is Inhibited in T-47D Mammary Cells and Both Processes are Regulated by Phlda1

Autophagy is an evolutionarily conserved lysosomal degradation pathway and plays a critical role in the homeostatic process of recycling proteins and organelles. Functional relationships have been described between apoptosis and autophagy. Perturbations in the apoptotic machinery have been reported to induce autophagic cell deaths. Inhibition of autophagy in cancer cells has resulted in cell deaths that manifested hallmarks of apoptosis. However, the molecular relationships and the circumstances of which molecular pathways dictate the choice between apoptosis and autophagy are currently unknown. This study aims to identify specific gene expression of rapamycin-induced autophagy and the effects of rapamycin when the autophagy process is inhibited. In this study, we have demonstrated that rapamycin is capable of inducing autophagy in T-47D breast carcinoma cells. However, when the autophagy process was inhibited by 3-MA, the effects of rapamycin became apoptotic. The Phlda1 gene was found to be up-regulated in both autophagy and apoptosis and silencing this gene was found to reduce both activities, strongly suggests that Phlda1 mediates and positively regulates both autophagy and apoptosis pathways.