Browsing by Author "Bandara, M.M.K."

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Camptothecin enhances c-Myc-mediated endoplasmic reticulum stress, leading to cytoprotective autophagy
(Faculty of Science, University of Kelaniya, Sri Lanka, 2016) Dilshara, M.G.; Bandara, M.M.K.; Kumara, M.H.S.R.; Molagoda, I.M.N.; Jayasooriya, R.G.P.T.; Kim, G.Y.
Camptothecin (CPT) is known to selectively inhibit the nuclear enzyme DNA topoisomerase I which catalyzes the relaxation of negatively supercoiled DNA and thus leads to cell cycle arrest and apoptosis through DNA damages. However, whether CPT induces endoplasmic reticulum (ER) stress and autophagy has not been clearly understood. The LNCaP cells were cultured at 37°C in a 5% CO2-humidified incubator and maintained in RPMI 1640 culture medium containing 10% FBS and antibiotics mixtures. Total RNA was isolated from LNCaP cells using Easy-Blue total RNA extraction kit (iNtRON Biotechnology, Sungnam, Republic of Korea.) according to the manufacturer′s instruction. Total cell extracts were separated on polyacrylamide gels and standard procedures were used to transfer them to the nitrocellulose membranes. Cells were seeded on a 24-well plate at a density of 1 × 105 cells/ml and transfected c-Myc-, JNK-, and eIF2α-specific silencing RNA (siRNA, Santa Cruz Biotechnology) for 24 h. Present study first reported that CPT enhanced DNA-binding activity of c-Myc in LNCaP cells according to electronic mobility shift assay and transient knockdown of c-Myc completely abrogated reactive oxygen species (ROS) generation which was resulting in accumulation of ER stress-regulating proteins such as PERK, eIF2α, ATF4, and CHOP. These observations suggests that CPT-induced c-Myc triggered ER stress along with the PERK-eIF2α-ATF4-CHOP pathway by increasing ROS generation. Moreover, CPT promoted formation of autophagy accompanied by increasing autophagic proteins such as beclin-1 and Atg7. Transfection of eIF2α-targeted siRNA attenuated CPTinduced beclin-1 and Atg7 expression. Treatment with autophagy inhibitors such as 3-methyladenine and bafilomycin A1 downregulated relative cell viability in response to CPT, which indicate that CPT induces ER stress-mediated cytoprotective autophagy. Additionally, CPT significantly induced AMPK phosphorylation as a result of intracellular Ca2+ release. Moreover, CPT phosphorylated JNK and activated DNA-binding activity of AP-1, and knockdown of JNK abolished the expression level of beclin-1 and Atg7, implying that the JNK-AP-1 pathway is a potent mediator on CPT-induced autophagy. Our findings indicate that CPT promotes ROS-mediated ER stress through the PERK-eIF2α-ATF-4-CHOP pathway, which enhances cytoprotective autophagy, resulting from the Ca2+-AMPK pathway and the JNK-AP-1 pathway.
Isolation and identification of naphthalene degrading bacteria from a polluted environment
(Faculty of Science, University of Kelaniya, Sri Lanka, 2016) Bandara, M.M.K.; Bamunuarachchige, T.C.
Hydrocarbon pollution is one of the major environmental problems and this has led to studies on use of hydrocarbon degrading bacteria for bioremediation. Isolation of hydrocarbon degrading bacteria from a polluted environment is plausible, because the environmental conditions can have a selection pressure on the type of bacteria that can reside in a particular contaminated environment. Naphthalene is considered to be a potent pollutant which is an intermediate product of hydrocarbon combustion. Toxicity, low volatility and recalcitrance to degradation and high affinity for sediments make naphthalene bio-degradation an important intervention. This study reports the isolation of indigenous naphthalene degrading bacteria from hydrocarbon contaminated soil near a filling station. Aqueous soil extracts were prepared using three soil samples collected near a filling station in Anuradapura. To prepare the Naphthalene supplemented Bushnell Hass (BH) agar plates, a portion (0.3 ml) of naphthalene solution (2% w/v) in methanol was added to the bottom agar surface and swirled to obtain an even distribution. Then 0.1 ml soil extract was mixed with 0.9 ml of molten BH agar medium and poured on bottom agar. The plates were then swirled to spread molten agar medium over bottom agar with a PAH layer. All plates were incubated at room temperature for 3-6 weeks and the colony count of naphthalene degrading bacteria was recorded daily. Isolated bacterial colonies were identified according to the Bergey’s manual of determinative bacteriology. Appearance of bacterial colonies was observed after fifteenth day, showing a slow degradation and number of colonies gradually increased with the time (Figure 1). Gradual increase of number of colonies shows the ability of bacteria to acquire the naphthalene degrading capability with time. This can be resulted due to the decrease in naphthalene concentration below the toxicity level or ability to acquire naphthalene degrading capability with the time of incubation. Isolated bacterial strains were identified as Staphylococcus aureus, Staphylococcus sp. and Pseudomonas sp. and they have the potential to be used in bioremediation of environments polluted with naphthalene.
Silibinin sensitizes TRAIL-mediated apoptosis by upregulating DR5 through reactive oxygen species mediated endoplasmic reticulum stress-Ca2+-CaMKII-Sp1 pathway
(Faculty of Science, University of Kelaniya, Sri Lanka, 2016) Dilshara, M.G.; Bandara, M.M.K.; Jayawardana, B.C.; Vidanarachchi, J.K.; Jayasooriya, R.G.P.T.; Kim, G.Y.
The cytotoxic effect of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) makes TRAIL a promising candidate for the treatment of various cancers. However, some cancers are resistant to the effects of TRAIL. Therefore, in this study, we addressed how silibinin enhances TRAIL-mediated apoptosis in various cancer cells. A549, U937 and HCT116 cells were plated at a density of 1 × 105 cells/well for overnight and treated with 75 ng/ml TRAIL either in the absence or presence of silibinin for 24 h. MTT assay was done to check the cell viability. Apoptotic cells were determined by the annexin-V+ staining (R&D Systems). Whole-cell lysates were prepared by PRO-PREP protein extraction solution (iNtRON Biotechnology, Sungnam, Republic of Korea). Cytoplasmic and nuclear protein extracts were prepared using NE-PER nuclear and cytosolic extraction reagents (Pierce, Rockford, IL). Combined treatment with silibinin and TRAIL (silibinin/TRAIL) induced apoptosis accompanied by the activation of caspase-3, caspase-8, caspase-9, and Bax and cytosolic accumulation of cytochrome c Anti-apoptotic proteins such as Bcl-2, IAP-1, and IAP-2 were inhibited as well. Silibinin triggered TRAIL-induced apoptosis in A549 cells through upregulation of death receptor 5 (DR5). Pretreatment with DR5/Fc chimeric protein and DR5-targeted small interfering RNA (siRNA) significantly blocked silibinin/TRAIL-mediated apoptosis in A549 cells. Furthermore, silibinin increased the production of reactive oxygen species (ROS), which led to the induction of TRAIL-mediated apoptosis through DR5 upregulation. 5mM concentration of N-acetyl-L-cysteine (NAC) and glutathione (GSH) reversed the apoptosis-inducing effects of TRAIL which are known antioxidants. Silibinin further induced endoplasmic reticulum (ER) stress as was indicated by the increase in ER marker proteins such as PERK, eIF2α, and ATF-4, which stimulate the expression of CCAAT/enhancer binding protein homologous protein (CHOP). CHOP-targeted siRNA eliminated the induction of DR5 and resulted in a significant decrease in silibinin/TRAIL-mediated apoptosis. We also found that silibinin/TRAIL-induced apoptosis was accompanied with intracellular influx of Ca2+, which was stimulated by ER stress and the Ca2+ chelator, ethylene glycol tetraacetic acid (EGTA). Ca2+/calmodulin-dependent protein kinase (CaMKII) inhibitor, K252a, blocked silibinin/TRAIL-induced DR5 expression along with TRAIL-mediated apoptosis. Accordingly, it was demonstrated that ROS/ER stressmediated CaMKII regulated Sp1, which is an important transcription factor for DR5 expression. Our results revealed that silibinin enhanced TRAIL-induced apoptosis by upregulating DR5 expression through the ROS-ER stress-CaMKII-Sp1 axis.