Muhamad Noor Alfarizal Kamarudin

Swietenia macrophylla King induces mitochondrial-mediated apoptosis through p53 upregulation in HCT116 colorectal carcinoma cells

ETHNOPHARMACOLOGICAL RELEVANCE Swietenia macrophylla King is a traditional herb used to treat various diseases including hypertension, diabetes and cancer. Previous study demonstrated its anti-tumor effect but the potential mechanisms have not been clearly defined. The current study was to further investigate the underlying mechanism of ethyl acetate fraction of Swietenia macrophylla (SMEAF)-induced anti-proliferative effect and apoptosis in HCT116 colorectal carcinoma cell. MATERIALS AND METHODS Cell viability was evaluated in HCT116 cells by trypan blue exclusion assay. Apoptotic cell death was detected by Hoechst 33342/propidium iodide (PI) staining and intracellular reactive oxygen species (ROS) was analyzed by flow cytometry. The apoptotic gene and protein expression were determined by Real-time quantitative PCR (q-PCR) and immunofluorescence staining using flow cytometry, respectively. RESULTS SMEAF significantly inhibited HCT116 cell viability and induced apoptosis in a dose-dependent manner. SMEAF-induced apoptosis was triggered by the activation of p53 and intracellular reactive oxygen species (ROS) production. Moreover, the significant increase in p53 was accompanied by a decrease murine double minute 2 (MDM2) expression. SMEAF significantly increased the expression of the Bax protein resulting in a markedly elevated Bax/Bcl-2 ratio which may have triggered the mitochondrial apoptotic pathway, resulting in caspase-3/7 and caspase-9 activation. CONCLUSION These results suggested that SMEAF exerts its antitumor activity in HCT116 cells by activating proapoptotic signaling pathway through intracellular ROS formation triggering the mitochondrial-mediated pathway via p53 activation.

Phytochemistry and Biology of Loranthus parasiticus Merr, a Commonly Used Herbal Medicine

Loranthus parasiticus Merr (L. parasiticus) is a member of Loranthaceae family and is an important medicinal plant with a long history of Chinese traditional use. L. parasiticus, also known as Sang Ji Sheng (in Chinese), benalu teh (in Malay) and baso-kisei (in Japanese), is a semiparasitic plant, which is mostly distributed in the southern and southwestern regions of China. This review aims to provide a comprehensive overview of the ethnomedicinal use, phytochemistry and pharmacological activity of L. parasiticus and to highlight the needs for further investigation and greater global development of the plants medicinal properties. To date, pharmacological studies have demonstrated significant biological activities, which support the traditional use of the plant as a neuroprotective, tranquilizing, anticancer, immunomodulatory, antiviral, diuretic and hypotensive agent. In addition, studies have identified antioxidative, antimutagenic, antiviral, antihepatotoxic and antinephrotoxic activity. The key bioactive constituents in L. parasiticus include coriaria lactone comprised of sesquiterpene lactones: coriamyrtin, tutin, corianin, and coriatin. In addition, two proanthocyanidins, namely, AC trimer and (+)-catechin, have been recently discovered as novel to L. parasiticus. L. parasiticus usefulness as a medicinal plant with current widespread traditional use warrants further research, clinical trials and product development to fully exploit its medicinal value.

(+)-Catechin Attenuates NF-κB Activation Through Regulation of Akt, MAPK, and AMPK Signaling Pathways in LPS-Induced BV-2 Microglial Cells

(+)-Catechin is a flavanol that possesses various health and medicinal values, which include neuroprotection, anti-oxidation, antitumor and antihepatitis activities. This study investigated the modulatory effects of (+)-catechin on the lipopolysaccharides (LPS)-stimulated BV-2 cells. (+)-catechin attenuated LPS-induced inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) and inhibited microglial NO and ROS production. Additionally, (+)-catechin suppressed the production of tumor necrosis factor-α (TNF-α) and interleukin (IL)-6, while augmenting IL-4. (+)-catechin attenuated LPS-induced nuclear factor-κB (NF-κB) p65 nuclear translocation via the inhibition of IκB-α phosphorylation. Moreover, (+)-catechin blocked the activation of Akt and its inhibition was shown to play a crucial role in LPS-induced inflammation in BV-2 microglial cells. (+)-catechin also attenuated the LPS-induced phosphorylation of extracellular signal-regulated kinase (ERK1/2), and p-38 mitogen activated protein kinases (p38 MAPK) and specific inhibitors of ERK1/2 (UO126) and p38 MAPK (SB202190) subsequently down-regulated the expression of the proinflammatory mediators iNOS and COX-2. Further mechanistic study revealed that (+)-catechin acted through the amelioration of the LPS-induced suppression of adenosine monophosphate-activated protein kinase (AMPK) activity. Taken together, our data indicate that (+)-catechin exhibits anti-inflammatory effects in BV-2 cells by suppressing the production of proinflammatory mediators and mitigation of NF-κB through Akt, ERK, p38 MAPK, and AMPK pathways.

Aqueous Fraction of Nephelium ramboutan-ake Rind Induces Mitochondrial-Mediated Apoptosis in HT-29 Human Colorectal Adenocarcinoma Cells

The aim of this study was to investigate the cytotoxic and apoptotic effects of Nephelium ramboutan-ake (pulasan) rind in selected human cancer cell lines. The crude ethanol extract and fractions (ethyl acetate and aqueous) of N. ramboutan-ake inhibited the growth of HT-29, HCT-116, MDA-MB-231, Ca Ski cells according to MTT assays. The N. ramboutan-ake aqueous fraction (NRAF) was found to exert the greatest cytotoxic effect against HT-29 in a dose-dependent manner. Evidence of apoptotic cell death was revealed by features such as chromatin condensation, nuclear fragmentation and apoptotic body formation. The result from a TUNEL assay strongly suggested that NRAF brings about DNA fragmentation in HT-29 cells. Phosphatidylserine (PS) externalization on the outer leaflet of plasma membranes was detected with annexin V-FITC/PI binding, confirming the early stage of apoptosis. The mitochondrial permeability transition is an important step in the induction of cellular apoptosis, and the results clearly suggested that NRAF led to collapse of mitochondrial transmembrane potential in HT-29 cells. This attenuation of mitochondrial membrane potential (Δψm) was accompanied by increased production of ROS and depletion of GSH, an increase of Bax protein expression, and induced-activation of caspase-3/7 and caspase-9. These combined results suggest that NRAF induces mitochondrial-mediated apoptosis.

(R)-(+)-α-Lipoic acid protected NG108-15 cells against H2O2-induced cell death through PI3K-Akt/GSK-3β pathway and suppression of NF-κβ-cytokines

Alpha-lipoic acid, a potent antioxidant with multifarious pharmacological benefits has been reported to be neuroprotective in several neuronal models and used to treat neurological disorders such as Alzheimer’s disease. Nonetheless, conclusive mechanisms of alpha-lipoic acid for its protective effects particularly in NG108-15 cells have never been investigated. In this study, the intricate neuroprotective molecular mechanisms by (R)-(+)-alpha-lipoic acid (R-LA) against H2O2-induced cell death in an in vitro model of neurodegeneration were elucidated. Pretreatment with R-LA (2 hours) significantly increased NG108-15 cell viability as compared to H2O2-treated cells and mitigated the induction of apoptosis as evidenced by Hoechst 33342/propidium iodide staining. R-LA (12.5–50 μM) aggrandized the reduced glutathione over glutathione disulfide ratio followed by a reduction in the intracellular reactive oxygen species level and an increase in mitochondrial membrane potential following H2O2 exposure. Moreover, pretreatment with R-LA stimulated the activation of PI3K-Akt through mTORC1 and mTORC2 components (mTOR, rictor and raptor) and production of antiinflammatory cytokine, IL-10 which led to the inactivation of glycogen synthase kinase-3β (GSK-3β) and reduction of both Bax/Bcl2 and Bax/Bcl-xL ratios, accompanied by inhibition of the cleaved caspase-3. Additionally, this observation was preceded by the suppression of NF-κβ p65 translocation and production of proinflammatory cytokines (IL-6 and TNF-α). The current findings accentuate new mechanistic insight of R-LA against apoptogenic and brain inflammatory factors in a neuronal model. These results further advocate the therapeutic potential of R-LA for the treatment of neurodegenerative diseases.

Anti-neuroinflammatory Activity of Elephantopus scaber L. via Activation of Nrf2/HO-1 Signaling and Inhibition of p38 MAPK Pathway in LPS-Induced Microglia BV-2 Cells

Elephantopus scaber L. (family: Asteraceae) has been traditionally utilized as a folkloric medicine and scientifically shown to exhibit anti-inflammatory activities in various in vivo inflammatory models. Given the lack of study on the effect of E. scaber in neuroinflammation, this study aimed to investigate the anti-neuroinflammatory effect and the underlying mechanisms of ethyl acetate fraction from the leaves of E. scaber (ESEAF) on the release of pro-inflammatory mediators in lipopolysaccharide (LPS)-induced microglia cells (BV-2). Present findings showed that ESEAF markedly attenuated the translocation of NF-κB to nucleus concomitantly with the significant mitigation on the LPS-induced production of NO, iNOS, COX-2, PGE2, IL-1β, and TNF-α. These inflammatory responses were reduced via the inhibition of p38. Besides, ESEAF was shown to possess antioxidant activities evident by the DPPH and SOD scavenging activities. The intracellular catalase enzyme activity was enhanced by ESEAF in the LPS-stimulated BV-2 cells. Furthermore, the formation of ROS induced by LPS in BV-2 cells was reduced upon the exposure to ESEAF. Intriguingly, the reduction of ROS was found in concerted with the activation of Nrf2 and HO-1. It is conceivable that the activation promotes the scavenging power of antioxidant enzymes as well as to ameliorate the inflammatory response in LPS-stimulated BV-2 cells. Finally, the safety profile analysis through oral administration of ESEAF at 2000 mg/kg did not result in any mortalities, adverse effects nor histopathologic abnormalities of organs in mice. Taken altogether, the cumulative findings suggested that ESEAF holds the potential to develop as nutraceutical for the intervention of neuroinflammatory disorders.

Mitigation of H2O2-Induced Mitochondrial-Mediated Apoptosis in NG108-15 Cells by Novel Mesuagenin C from Mesua kunstleri (King) Kosterm

This study was aimed to isolate and evaluate neuroprotective compounds from the hexane extract of the bark of Mesua kunstleri (Clusiaceae) on H2O2-induced apoptosis in NG108-15 cells. Five 4-phenylcoumarins were isolated by using various chromatographic techniques via neuroprotective activity-guided fractionation and isolation from the active hexane extract. The chemical structures of the isolated compounds were confirmed by NMR spectroscopic data interpretation and comparison with literature values. Cell viability data demonstrated that mesuagenin C 3 significantly increased cell viability. Hoechst 33342/PI staining illustrated mesuagenin C 3 was able to abate the nuclear shrinkage, chromatin condensation and formation of apoptotic bodies. Pretreatment with mesuagenin C 3 reduced total annexin V positive cells and increased the level of intracellular glutathione (GSH). Mesuagenin C 3 attenuated membrane potential (Δψm), reduced Bax/Bcl-2 ratio and inactivated of caspase-3/7 and -9. These results indicated that mesuagenin C 3 could protect NG108-15 cells against H2O2-induced apoptosis by increasing intracellular GSH level, aggrandizing Δψm, and modulating apoptotic signalling pathway through Bcl-2 family and caspase-3/7 and -9. These findings confirmed the involvement of intrinsic apoptotic pathway in H2O2-induced apoptosis and suggested that mesuagenin C 3 may have potential therapeutic properties for neurodegenerative diseases.

Antiproliferative and apoptosis inducing effects of citral via p53 and ROS-induced mitochondrial-mediated apoptosis in human colorectal HCT116 and HT29 cell lines

Despite various anticancer reports, antiproliferative and apoptosis inducing activity of citral in HCT116 and HT29 cells have never been reported. This study aimed to evaluate the cytotoxic and apoptosis inducing effects of citral in colorectal cancer cell lines. The citral-treated cells were subjected to MTT assay followed by flow cytometric Annexin V-FITC/PI, mitochondrial membrane potential and intracellular reactive oxygen species (ROS) determination. The apoptotic proteins expression was investigated by Western blot analysis. Citral inhibited the growth of HCT116 and HT29 cells by dose- and time-dependent manner without inducing cytotoxicity in CCD841-CoN normal colon cells. Flow cytometric analysis showed that citral (50-200μM; 24-48h) induced the externalization of phoshpotidylserine and reduced the mitochondrial membrane potential in HCT116 and HT29 cells. Citral elevated intracellular ROS level while attenuating GSH levels in HCT116 and HT29 cells which were reversed with N-acetycysteine (2mM) pre-treatment indicating that citral induced mitochondrial-mediated apoptosis via augmentation of intracellular ROS. Citral induced the phosphorylation of p53 protein and the expression of Bax while decreasing Bc-2 and Bcl-xL expression which promoted the cleavage of caspase-3. Collectively, our data suggest that citral induced p53 and ROS-mediated mitochondrial-mediated apoptosis in human colorectal cancer HCT116 and HT29 cells.

α-Lipoic acid protects against LPS-induced BV-2 activation and MPTP-induced toxicity in SH-SY5Y neuronal cells

WCN 2013 No: 2364 Topic: 5 — Dementia α-Lipoic acid protects against LPS-induced BV-2 activation and MPTP-induced toxicity in SH-SY5Y neuronal cells H. Abdul Kadir, M.N.A. Kamarudin, N.A. Mohd Raflee, T. Shabab. Institute of Biological Sciences, Faculty of Science, University of Malaya, Kuala Lumpur, Malaysia Background and objectives: α-Lipoic acid (LA), a natural dithiol compound is a powerful antioxidant that has been used to treat various neural disorders. Microglial activation has been implicated in chronic neuroinflammation leading to neurodegenerative diseases such as Alzheimers and Parkinsons diseases. For the first time, the anti-inflammatory effects of LA and its possible mechanisms in LPSstimulated inflammation in microglial BV2 cells and MPTP-induced toxicity in SH-SY5Y were investigated. Material and methods: Cell viability was measured by using MTT assay. Flow cytometry chip beads array (CBA), Western blot and ICC were used to analyse NO, ROS, PGE2, TNF-α, IL-1β, IL-6, IFN-γ, iNOS, COX-2, CCL21 expression and the involvement of signalling pathways such as MAPK cascades, pMTOR-PI3K-Akt and NF-κβ. The protective effects of LA on BV-2 and SH-SY5Y co-cultured model induced with LPS and MPTP were further evaluated. Results: LA significantly attenuated LPS-induced iNOS, NO, ROS, PGE2, TNF-α, IL-1β, IL-6, IFN-γ and COX-2 expression as shown in CBA and Western blot. LA also suppressed the expression of CCL21, a pro-inflammatory chemokine in both LPS-treated BV-2 and MPTPtreated SH-SY5Y. Moreover, LA inhibited Iκβα degradation and thus, prevented p65 NF-κβ translocation in BV-2 and SH-SY5Y cells. LA increased cell viability and rescued co-cultured SH-SY5Y cells from MPTP-induced toxicity and apoptosis. Conclusion: The anti-inflammatory properties of LA prevented excessive microglia (BV-2) activation and thus, protected SH-SY5Y cells from LPS and MPTP induced toxicity by downregulating proinflammatory proteins through PI3K-Akt pathway. This suggests a therapeutic potential of LA for the treatment of neurodegenerative diseases. doi:10.1016/j.jns.2013.07.1249 Abstract — WCN 2013 No: 1156 Topic: 5 — Dementia Patient and caregiver adherence and persistence to the rivastigmine patch in a non-interventional clinical study M. Riepe, J. Weinman, R. Brady, C. Strohmaier, B. Mueller. University of Ulm, Ulm, Germany; Institute of Psychiatry, Kings College London, UK; Royal Free London Foundation Hospital Trust, London, UK; Novartis Pharma AG, Basel, Switzerland; Novartis Pharma GmbH, Nuernberg, Germany Background: Rivastigmine transdermal patch provides smooth drug delivery and therefore increased tolerance at similar efficacy compared to the oral formulation. An earlier registration study for the patch (IDEAL) with 1059 caregivers demonstrated that more than 70% caregivers prefer rivastigmine transdermal patch to capsules. Objective: To identify patient and caregiver-related factors associated with adherence, persistence and caregiver satisfactionwith rivastigmine patch. Methods: In this non-interventional trial, adherence and persistence were assessed after three and six months of patch treatment. Relevant factors from literature for persistence and adherence to medication were validated prior to study enrolment. Results: 127 caregivers answered the questionnaires, 3 months (visit 1) and 110 caregivers after 6 months (visit 2) after first prescription of rivastigmine patch. Mean MMSE score of the patients was 20.8 after 3 months of treatment. Physician ratings for CGI-I scores for efficacy suggested patients as ‘improved’ (66.2%). Caregivers were adherent to patch use at both visit 1 and visit 2 respectively, (‘never’ omitted 74.8%; 78.2% or ‘never’ paused for a while, 73.2%; 74.5%) and agreed with ease of its application. Efficacy was rated from caregivers for visit 1 and visit 2 respectively, as ‘satisfied’ for memory (62.2%; 56.4%), activities (69.3%; 66.4%) and behavior and emotions (65.4%; 64.5%). Valuations of tolerability were deemed as ‘good’ (90.5% and 90%) at visit 1 and visit 2 respectively. Conclusion: Caregivers achieved reasonably high adherence rates to rivastigmine patch. This may be linked to its good efficacy outcomes and favourable tolerability profile. Reference [1] Winblad B, et al. Int J Geriatr Psychiatry 2007;22:485–91. doi:10.1016/j.jns.2013.07.1250 Abstract — WCN 2013 No: 1169 Topic: 5 — Dementia Ease of use of rivastigmine patch can help manage medication for caregivers and patients with Alzheimers disease WCN 2013 No: 1169 Topic: 5 — Dementia Ease of use of rivastigmine patch can help manage medication for caregivers and patients with Alzheimers disease M. Riepe, J. Weinman, R. Brady, C. Strohmaier, B. Mueller. University of Ulm, Ulm, Germany; Institute of Psychiatry, Kings College London, UK; Royal Free London Foundation Hospital Trust, London, UK; Novartis Pharma AG, Basel, Switzerland; Novartis Pharma GmbH, Nuernberg, Germany Background: Rivastigmine transdermal patch provides smooth drug delivery and better satisfaction to caregivers and patients compared to oral formulation. An earlier study demonstrated that more than 70% caregivers prefer the rivastigmine transdermal patch to the capsule. Objective: To identify patient and caregiver-related factors associated with adherence, persistence and caregiver satisfaction to rivastigmine patch. Methods: In this non-interventional, clinical and psychosocial study, caregiver or patient-related factors impacting on rivastigmine patch Abstracts / Journal of the Neurological Sciences 333 (2013) e292–e357 e338

Mesuagenin C mitigated LPS-induced neuroinflammation in BV-2 and NG108-15 cells through pMTOR-PI3K-Akt and ccl21 downregulation

WCN 2013 No: 2753 Topic: 5 — Dementia А critical role for molecular chaperones in Alzheimers disease Z. Milicevic, B. Spremo-Potparevic, L. Zivkovic, V. Bajic. Department of Endocrinology and Molecular Biology, Vinca Institute for Nuclear Sciences, Belgrade, Serbia; Department of Biology, School of Pharmacy, University of Belgrade, Belgrade, Serbia; Department of Biomedicines, Institute for Pharmacology Research and Development,