Displaying publications 21 - 40 of 226 in total

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  1. Antibodies against prM protein distinguish between previous infection with dengue and Japanese encephalitis viruses
    Cardosa MJ, Wang SM, Sum MS, Tio PH
    BMC Microbiol, 2002 May 5;2:9.
    PMID: 12019028
    In Southeast Asia, dengue viruses often co-circulate with other flaviviruses such as Japanese encephalitis virus, and due to the presence of shared antigenic epitopes it is often difficult to use serological methods to distinguish between previous infections by these flaviviruses.
    Matched MeSH terms: Blotting, Western
  2. Fulltext Helicobacter pylori recombinant UreG protein: cloning, expression, and assessment of its seroreactivity
    Khalilpour A, Osman S, Yunus MH, Santhanam A, Vellasamy N, Noordin R
    BMC Res Notes, 2014;7:809.
    PMID: 25406411 DOI: 10.1186/1756-0500-7-809
    Helicobacter pylori is a human pathogen and during the process of infection, antigens from the bacterium elicit strong host humoral immune responses. In our previous report, native H. pylori UreG protein showed good reactivity with sera from H. pylori patients. This study was aimed at producing the recombinant form of the protein (rUreG) and determining its seroreactivities.
    Matched MeSH terms: Blotting, Western
  3. Fulltext Recombinant dense granular protein (GRA5) for detection of human toxoplasmosis by Western blot
    Ching XT, Lau YL, Fong MY, Nissapatorn V, Andiappan H
    Biomed Res Int, 2014;2014:690529.
    PMID: 24987700 DOI: 10.1155/2014/690529
    Toxoplasma gondii infects all warm-blooded animals, including humans, causing serious public health problems and great economic loss for the food industry. Commonly used serological tests require costly and hazardous preparation of whole Toxoplasma lysate antigens from tachyzoites. Here, we have evaluated an alternative method for antigen production, which involved a prokaryotic expression system. Specifically, we expressed T. gondii dense granular protein-5 (GRA5) in Escherichia coli and isolated it by affinity purification. The serodiagnostic potential of the purified recombinant GRA5 (rGRA5) was tested through Western blot analysis against 212 human patient serum samples. We found that rGRA5 protein was 100% specific for analysis of toxoplasmosis-negative human sera. Also, rGRA5 was able to detect acute and chronic T. gondii infections (sensitivities of 46.8% and 61.2%, resp.).
    Matched MeSH terms: Blotting, Western/methods*
  4. Fulltext Induction of apoptosis of 2,4',6-trihydroxybenzophenone in HT-29 colon carcinoma cell line
    Lay MM, Karsani SA, Malek SN
    Biomed Res Int, 2014;2014:468157.
    PMID: 24579081 DOI: 10.1155/2014/468157
    2,4',6-Trihydroxy-4-methoxybenzophenone was isolated from the ethyl acetate fraction of Phaleria macrocarpa (Scheff.) Boerl. fruits. It was found to inhibit cell proliferation in HT-29 human colon carcinoma cell line but caused little damage to WRL-68 normal human liver and MRC-5 normal human fibroblast lung cell lines. The compound was found to sharply affect the viability of HT-29 cells in a dose- and time-dependent manner. HT-29 cells treated with the compound showed morphological changes under microscopic examination such as cell shrinkage, membrane blebbing, DNA fragmentation, and the occurrence of apoptotic nuclei. The percentage of early apoptotic, late apoptotic, and dead or necrotic cells was determined by flow cytometry using annexin V-FTIC/PI staining. In addition, flow cytometry showed that, when the HT-29 cells were treated with 115 µM of the compound, it resulted in G0/G1 phase arrest in a time-dependent manner. Western blot revealed an upregulation of PUMA, Bak, Bcl-2, and Mcl-1 proteins suggesting that the compound induced apoptosis in HT-29 cells by regulating these proteins.
    Matched MeSH terms: Blotting, Western
  5. Fulltext Dengue patients exhibit higher levels of PrM and E antibodies than their asymptomatic counterparts
    Yeo AS, Rathakrishnan A, Wang SM, Ponnampalavanar S, Manikam R, Sathar J, et al.
    Biomed Res Int, 2015;2015:420867.
    PMID: 25815314 DOI: 10.1155/2015/420867
    Dengue virus infection is a common tropical disease which often occurs without being detected. These asymptomatic cases provide information in relation to the manifestation of immunological aspects. In this study, we developed an ELISA method to compare neutralizing effects of dengue prM and E antibodies between dengue patients and their asymptomatic household members. Recombinant D2 premembrane (prM) was constructed, cloned, and tested for antigenicity. The recombinant protein was purified and tested with controls by using an indirect ELISA method. Positive dengue serum samples with their asymptomatic pair were then carried out onto the developed ELISA. In addition, commercially available recombinant envelope (E) protein was used to develop an ELISA which was tested with the same set of serum samples in the prM ELISA. Asymptomatic individuals showed preexisting heterotypic neutralizing antibodies. The recombinant prM was antigenically reactive in the developed ELISA. Dengue patients had higher prM and E antibodies compared to their household members. Our study highlights the neutralizing antibodies levels with respect to dengue prM and E between dengue patients and asymptomatic individuals.
    Matched MeSH terms: Blotting, Western
  6. Galactose-binding lectin from the seeds of champedak (Artocarpus integer): sequences of its subunits and interactions with human serum O-glycosylated glycoproteins
    Abdul Rahman M, Anuar Karsani S, Othman I, Shafinaz Abdul Rahman P, Haji Hashim O
    Biochem Biophys Res Commun, 2002 Jul 26;295(4):1007-13.
    PMID: 12127996
    Our group has previously reported the isolation, partial characterisation, and application of a Galbeta1-3GalNAc- and IgA1-reactive lectin from the seeds of champedak (Artocarpus integer). In the present study, we have subjected the purified lectin to reverse-phase high performance liquid chromatography and sequenced its subunits. Determination of the N-terminal sequence of the first 47 residues of the large subunit demonstrated at least 95% homology to the N-terminal sequence of the alpha chains of a few other galactose-binding Artocarpus lectins. The two smaller subunits of the lectin, each comprised of 21 amino acid residues, demonstrated minor sequence variability. Their sequences were generally comparable to the beta chains of the other galactose-binding Artocarpus lectins. When used to probe human serum glycopeptides that were separated by two-dimensional gel electrophoresis, the lectin demonstrated strong apparent interactions with glycopeptides of IgA1, hemopexin, alpha2-HS glycoprotein, alpha1-antichymotrypsin, and a few unknown glycoproteins. Immobilisation of the lectin to Sepharose generated an affinity column that may be used to isolate the O-glycosylated serum glycoproteins.
    Matched MeSH terms: Blotting, Western
  7. Demonstration of antigenic similarities and variations in excretory/secretory antigens of Toxoplasma gondii
    Rahmah N, Anuar AK
    Biochem Biophys Res Commun, 1992 Aug 31;187(1):294-8.
    PMID: 1520310
    C57BL/6 mice were orally infected with different doses of cysts of ME49 strain of Toxoplasma gondii to produce groups of acutely and chronically infected mice. Sera were obtained at different periods post-infection. SDS-PAGE was ran with excretory/secretory antigens of ME49 and RH strains of T. gondii, followed by Western blot analyses using the above sera and anti- IgA, IgM, IgG as conjugates. The SDS-PAGE profiles of the two antigens were similar. However the antigenic bands showed variations in all blots, most evidently in IgA blots of chronic sera. IgG blots showed greatest similarities in reactive bands. In IgM blots, more common bands were shown in chronic sera than in acute sera. Variations and similarities in prominence of some bands and time of their appearance were also noted, especially in IgM and IgG blots of chronic sera. Thus antigenic variations and similarities are present in excretory/secretory products of different strains of T. gondii.
    Matched MeSH terms: Blotting, Western
  8. Demonstration of an antigenic protein specific for Salmonella typhi
    Ismail A, Hai OK, Kader ZA
    Biochem Biophys Res Commun, 1991 Nov 27;181(1):301-5.
    PMID: 1958200
    Current studies were undertaken to determine the presence of a specific antigenic protein on the outer membrane of Salmonella typhi. Immunoblot analysis using sera from patients with fevers revealed that the 50 kD band was specifically recognized only by typhoid sera. The 50 kD band located on the outer membrane is protein by nature and is not a Vi (capsular), dH (flagellar), or O9 (somatic) antigen of S. typhi. These results indicate the usefulness of the specific antigen in the development of a serodiagnostic test for typhoid fever since antibodies of both the IgM and IgG class responses were obtained.
    Matched MeSH terms: Blotting, Western
  9. Intracellular production of IFN-alpha 2b in Lactococcus lactis
    Bayat O, Baradaran A, Ariff A, Mohamad R, Rahim RA
    Biotechnol Lett, 2014 Mar;36(3):581-5.
    PMID: 24185903 DOI: 10.1007/s10529-013-1390-4
    Human interferon alpha (IFN-α) was expressed in two strains of Lactococcus lactis by aid of two promoters (P32 and Pnis) giving rise to two recombinant strains: MG:IFN and NZ:IFN, respectively. The expression of IFN was confirmed by ELISA and western blotting. Highest production was achieved using glucose for growth of both recombinant strains with nisin, used for induction of the recombinant strain with Pnis promoter, at 30 ng/ml. The optimum time for MG:IFN was 9 h and for NZ:IFN was 4.5 h. The highest productions by MG:IFN and NZ:IFN were 1.9 and 2.4 μg IFN/l, respectively. Both of the expressed IFNs showed bioactivities of 1.9 × 10(6) IU/mg that were acceptable for further clinical studies.
    Matched MeSH terms: Blotting, Western
  10. Cloning and in silico characterization of two signal peptides from Pediococcus pentosaceus and their function for the secretion of heterologous protein in Lactococcus lactis
    Baradaran A, Sieo CC, Foo HL, Illias RM, Yusoff K, Rahim RA
    Biotechnol Lett, 2013 Feb;35(2):233-8.
    PMID: 23076361 DOI: 10.1007/s10529-012-1059-4
    Fifty signal peptides of Pediococcus pentosaceus were characterized by in silico analysis and, based on the physicochemical analysis, (two potential signal peptides Spk1 and Spk3 were identified). The coding sequences of SP were amplified and fused to the gene coding for green fluorescent protein (GFP) and cloned into Lactococcus lactis pNZ8048 and pMG36e vectors, respectively. Western blot analysis indicated that the GFP proteins were secreted using both heterologous SPs. ELISA showed that the secretion efficiency of GFP using Spk1 (0.64 μg/ml) was similar to using Usp45 (0.62 μg/ml) and Spk3 (0.58 μg/ml).
    Matched MeSH terms: Blotting, Western
  11. Characterization of secreted recombinant Toxoplasma gondii surface antigen 2 (SAG2) heterologously expressed by the yeast Pichia pastoris
    Fong MY, Lau YL, Zulqarnain M
    Biotechnol Lett, 2008 Apr;30(4):611-8.
    PMID: 18043869
    The surface antigen 2 (SAG2) gene of the protozoan parasite, Toxoplasma gondii, was cloned and extracellularly expressed in the yeast Pichia pastoris. The effectiveness of the secreted recombinant SAG2 (rSAG2-S) as a serodiagnosis reagent was assessed by western blots and ELISA. In the western blot assay, rSAG2-S reacted with all Toxoplasma-antibody positive human serum samples but not with Toxoplasma-negative samples. In the ELISA, rSAG2-S yielded sensitivity rates ranging from 80% (IgG negative, IgM positive) to 100% (IgG positive, IgM negative). In vivo experiments showed that serum from mice immunized with rSAG2-S reacted specifically with the native SAG2 of T. gondii. These mice were protected when challenged with live cells of T. gondii.
    Matched MeSH terms: Blotting, Western
  12. Expression and purification of the matrix protein of Nipah virus in baculovirus insect cell system
    Masoomi Dezfooli S, Tan WS, Tey BT, Ooi CW, Hussain SA
    Biotechnol Prog, 2016 Jan-Feb;32(1):171-7.
    PMID: 26519022 DOI: 10.1002/btpr.2192
    Nipah virus (NiV) causes fatal respiratory illness and encephalitis in humans and animals. The matrix (M) protein of NiV plays an important role in the viral assembly and budding process. Thus, an access to the NiV M protein is vital to the design of viral antigens as diagnostic reagents. In this study, recombinant DNA technology was successfully adopted in the cloning and expression of NiV M protein. A recombinant expression cassette (baculovirus expression vector) was used to encode an N-terminally His-tagged NiV M protein in insect cells. A time-course study demonstrated that the highest yield of recombinant M protein (400-500 μg) was expressed from 107 infected cells 3 days after infection. A single-step purification method based on metal ion affinity chromatography was established to purify the NiV M protein, which successfully yielded a purity level of 95.67% and a purification factor of 3.39. The Western blotting and enzyme-linked immunosorbent assay (ELISA) showed that the purified recombinant M protein (48 kDa) was antigenic and reacted strongly with the serum of a NiV infected pig.
    Matched MeSH terms: Blotting, Western
  13. Anti-tumour promoter activity in Malaysian ginger rhizobia used in traditional medicine
    Vimala S, Norhanom AW, Yadav M
    Br. J. Cancer, 1999 Apr;80(1-2):110-6.
    PMID: 10389986
    Zingiberaceae rhizomes commonly used in the Malaysian traditional medicine were screened for anti-tumour promoter activity using the short-term assay of inhibition of 12-O-tetradecanoyl phorbol-13-acetate (TPA)-induced Epstein-Barr virus early antigen (EBV-EA) in Raji cells. The inhibition of TPA-induced EBV-EA was detected using the indirect immunofluorescence assay (IFA) and Western blot technique. The indirect IFA detected the expression/inhibition of EBV-EA-D (diffused EA antigen), whereas the Western blot technique detected the expression/inhibition of both EBV-EA-D and EA-R (restricted EA antigen). Seven rhizomes were found to possess inhibitory activity towards EBV activation, induced by TPA; they are: Curcuma domestica, C. xanthorrhiza, Kaempferia galanga, Zingiber cassumunar, Z. officinale, Z. officinale (red variety), and Z. zerumbet. A cytotoxicity assay was carried out to determine the toxicity of the Zingiberaceae rhizome extracts. The rhizome extracts that exhibited EBV activation inhibitory activity had no cytotoxicity effect in Raji cells. Therefore, the present study shows that several Zingiberaceae species used in Malaysian traditional medicine contain naturally occurring non-toxic compounds that inhibit the EBV activation, which, if further investigated, could contribute in the development of cancer prevention methods at the tumour-promoting stage.
    Matched MeSH terms: Blotting, Western
  14. Rapamycin synergizes cisplatin sensitivity in basal-like breast cancer cells through up-regulation of p73
    Wong SW, Tiong KH, Kong WY, Yue YC, Chua CH, Lim JY, et al.
    Breast Cancer Res Treat, 2011 Jul;128(2):301-13.
    PMID: 20686837 DOI: 10.1007/s10549-010-1055-0
    Recent gene expression profiling studies have identified five breast cancer subtypes, of which the basal-like subtype is the most aggressive. Basal-like breast cancer poses serious clinical challenges as there are currently no targeted therapies available to treat it. Although there is increasing evidence that these tumors possess specific sensitivity to cisplatin, its success is often compromised due to its dose-limiting nephrotoxicity and the development of drug resistance. To overcome this limitation, our goal was to maximize the benefits associated with cisplatin therapy through drug combination strategies. Using a validated kinase inhibitor library, we showed that inhibition of the mTOR, TGFβRI, NFκB, PI3K/AKT, and MAPK pathways sensitized basal-like MDA-MB-468 cells to cisplatin treatment. Further analysis demonstrated that the combination of the mTOR inhibitor rapamycin and cisplatin generated significant drug synergism in basal-like MDA-MB-468, MDA-MB-231, and HCC1937 cells but not in luminal-like T47D or MCF-7 cells. We further showed that the synergistic effect of rapamycin plus cisplatin on basal-like breast cancer cells was mediated through the induction of p73. Depletion of endogenous p73 in basal-like cells abolished these synergistic effects. In conclusion, combination therapy with mTOR inhibitors and cisplatin may be a useful therapeutic strategy in the treatment of basal-like breast cancers.
    Matched MeSH terms: Blotting, Western
  15. Fulltext Benzylidene derivatives of andrographolide inhibit growth of breast and colon cancer cells in vitro by inducing G(1) arrest and apoptosis
    Jada SR, Matthews C, Saad MS, Hamzah AS, Lajis NH, Stevens MF, et al.
    Br J Pharmacol, 2008 Nov;155(5):641-54.
    PMID: 18806812 DOI: 10.1038/bjp.2008.368
    BACKGROUND AND PURPOSE: Andrographolide, the major phytoconstituent of Andrographis paniculata, was previously shown by us to have activity against breast cancer. This led to synthesis of new andrographolide analogues to find compounds with better activity than the parent compound. Selected benzylidene derivatives were investigated for their mechanisms of action by studying their effects on the cell cycle progression and cell death.
    EXPERIMENTAL APPROACH: Microculture tetrazolium, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) and sulphorhodamine B (SRB) assays were utilized in assessing the in vitro growth inhibition and cytotoxicity of compounds. Flow cytometry was used to analyse the cell cycle distribution of control and treated cells. CDK1 and CDK4 levels were determined by western blotting. Apoptotic cell death was assessed by fluorescence microscopy and flow cytometry.
    KEY RESULTS: Compounds, in nanomolar to micromolar concentrations, exhibited growth inhibition and cytotoxicity in MCF-7 (breast) and HCT-116 (colon) cancer cells. In the NCI screen, 3,19-(2-bromobenzylidene) andrographolide (SRJ09) and 3,19-(3-chloro-4-fluorobenzylidene) andrographolide (SRJ23) showed greater cytotoxic potency and selectivity than andrographolide. SRJ09 and SRJ23 induced G(1) arrest and apoptosis in MCF-7 and HCT-116 cells, respectively. SRJ09 downregulated CDK4 but not CDK1 level in MCF-7 cells. Apoptosis induced by SRJ09 and SRJ23 in HCT-116 cells was confirmed by annexin V-FITC/PI flow cytometry analysis.
    CONCLUSION AND IMPLICATIONS: The new benzylidene derivatives of andrographolide are potential anticancer agents. SRJ09 emerged as the lead compound in this study, exhibiting anticancer activity by downregulating CDK4 to promote a G(1) phase cell cycle arrest, coupled with induction of apoptosis.
    Matched MeSH terms: Blotting, Western
  16. Fulltext miR-145 supports cancer cell survival and shows association with DDR genes, methylation pattern, and epithelial to mesenchymal transition
    Manvati S, Mangalhara KC, Kalaiarasan P, Chopra R, Agarwal G, Kumar R, et al.
    Cancer Cell Int, 2019;19:230.
    PMID: 31516387 DOI: 10.1186/s12935-019-0933-8
    Background: Despite several reports describing the dual role of miR-145 as an oncogene and a tumor suppressor in cancer, not much has been resolved and understood.

    Method: In this study, the potential targets of miR-145 were identified bio-informatically using different target prediction tools. The identified target genes were validated in vitro by dual luciferase assay. Wound healing and soft agar colony assay assessed cell proliferation and migration. miR-145 expression level was measured quantitatively by RT-PCR at different stages of breast tumor. Western blot was used to verify the role of miR-145 in EMT transition using key marker proteins.

    Result: Wound healing and soft agar colony assays, using miR-145 over-expressing stably transfected MCF7 cells, unraveled its role as a pro-proliferation candidate in cancerous cells. The association between miR-145 over-expression and differential methylation patterns in representative target genes (DR5, BCL2, TP53, RNF8, TIP60, CHK2, and DCR2) supported the inference drawn. These in vitro observations were validated in a representative set of nodal positive tumors of stage 3 and 4 depicting higher miR-145 expression as compared to early stages. Further, the role of miR-145 in epithelial-mesenchymal (EMT) transition found support through the observation of two key markers, Vimentin and ALDL, where a positive correlation with Vimentin protein and a negative correlation with ALDL mRNA expression were observed.

    Conclusion: Our results demonstrate miR-145 as a pro-cancerous candidate, evident from the phenotypes of aggressive cellular proliferation, epithelial to mesenchymal transition, hypermethylation of CpG sites in DDR and apoptotic genes and upregulation of miR-145 in later stages of tumor tissues.

    Matched MeSH terms: Blotting, Western
  17. Activation of phosphatidylinositol 3-kinase/Akt signaling by EGF downregulates membranous E-cadherin and β-catenin and enhances invasion in nasopharyngeal carcinoma cells
    Yip WK, Seow HF
    Cancer Lett, 2012 May 28;318(2):162-72.
    PMID: 22182447 DOI: 10.1016/j.canlet.2011.12.018
    Dysregulation of E-cadherin and β-catenin function in cell-cell adhesion is common in nasopharyngeal carcinoma (NPC) and correlates with metastatic disease. In this study, we examined the role of EGF-activated phosphatidylinositol 3-kinase (PI3K)-Akt signaling in E-cadherin and β-catenin regulation. We found that reduced membranous E-cadherin and β-catenin expression was positively correlated with Akt phosphorylation in NPC tissues. EGF treatment disrupted cell-cell adhesion and resulted in mesenchymal morphological features in NPC cell lines (TW01, TW04, and TW06). Western blot analysis showed that the E-cadherin protein level was partially reduced in TW04 cells only and the β-catenin levels were not considerably affected upon EGF treatment. In contrast, quantitative real-time RT-PCR showed that the E-cadherin, but not β-catenin, mRNA levels were markedly reduced by EGF in all cell lines. Immunofluorescent staining revealed that E-cadherin and β-catenin appeared to be markedly reduced on the cell surface and more localized in the cytoplasm. Inhibition of PI3K by LY294002 did not abolish the EGF-induced downregulation of E-cadherin protein or mRNA in TW04 cells but moderately increased the β-catenin protein level in TW01 cells and mRNA level in TW06 cells. However, LY294002 substantially restored or increased cell surface E-cadherin and β-catenin in all EGF-treated cell lines, in concordance with the inhibition of cell morphological changes. Moreover, LY294002 significantly blocked EGF-driven cell invasion, correlating with the elevation of membranous E-cadherin and β-catenin levels. In conclusion, EGF-induced epithelial-to-mesenchymal transition may not be only dependent on downregulation of E-cadherin protein/mRNA but also on mislocalization of E-cadherin and β-catenin. The mechanisms involved may be related, at least in part, to the PI3K-Akt pathway.
    Matched MeSH terms: Blotting, Western
  18. Fulltext N-Ethyl-n-Nitrosourea Induced Leukaemia in a Mouse Model through Upregulation of Vascular Endothelial Growth Factor and Evading Apoptosis
    Aliyu A, Shaari MR, Ahmad Sayuti NS, Reduan MFH, Sithambaram S, Noordin MM, et al.
    Cancers (Basel), 2020 Mar 13;12(3).
    PMID: 32183192 DOI: 10.3390/cancers12030678
    Chemical carcinogens are commonly used to investigate the biology and prognoses of various cancers. This study investigated the mechanism of leukaemogenic effects of n-ethyl-n-nitrosourea (ENU) in a mouse model. A total of 14 3-week-old male Institute of Cancer Research (ICR)-mice were used for the study. The mice were divided into groups A and B with seven mice each. Group A served as the control while group B received intraperitoneal (IP) injections of 80 mg/kg ENU twice with a one-week interval and were monitored monthly for 3 months for the development of leukaemia via blood smear examination. The mice were sacrificed humanely using a CO2 chamber. Blood, spleen, lymph nodes, liver, kidney and lung samples were collected for blood smear examination and histopathological evaluation. The expression of angiogenic protein (VEGF), and pro and anti-apoptotic proteins (BCL2 and BAX), was detected and quantified using Western blot technique. Leukaemia was confirmed by the presence of numerous blast cells in the peripheral blood smear in group B. Similarly, the VEGF and BCL2 proteins were significantly (p < 0.05) upregulated in group B compared to A. It is concluded that IP administration of 80 mg/kg ENU induced leukaemia in ICR-mice 12 weeks post administration through upregulation of angiogenic and anti-apoptotic proteins: VEGF and BCL2.
    Matched MeSH terms: Blotting, Western
  19. Thymoquinone induces apoptosis and increase ROS in ovarian cancer cell line
    Taha MM, Sheikh BY, Salim LZ, Mohan S, Khan A, Kamalidehghan B, et al.
    Cell Mol Biol (Noisy-le-grand), 2016 May 30;62(6):97-101.
    PMID: 27262811
    Nigella sativa is also known for its properties as a traditional herbal healing for many ailments. In this study, the anticancer properties of thyomquinone (TQ), the active ingredient of N. sativa, were studied using ovarian cancer cell line (Caov-3 cells). The anti-proliferative activity of TQ was determined using MTT and the apoptosis was investigated using Flowcytometry and Annexin-V Assays. Multiparameteric cytotoxicity bioassays were used to quantify the changes in cell permeability and mitochondrial membrane potential. Reactive oxygen species (ROS) and apoptosis-involved cell markers were examined to verify cell death mechanism. The MTT-assay showed that TQ induces anti-proliferative activity on Caov-3 with an IC50 of 6.0±0.03 μg/mL, without any cytotoxic activity towards WRL-68 normal hepatocytes. A significant induction of early phase of apoptosis was shown by annexin-V analysis. Treatment of Caov-3 cells with TQ induces decreases in plasma membrane permeability and mitochondrial membrane potential. Visible decrease in the nuclear area was also observed. A significant decrease is observed in Bcl-2 while Bax is down-regulated. TQ-triggered ROS-mediated has found to be associated with Hsp70 dysregulation, an indicator of oxidative injury. We found that TQ induced anti-cancer effect involves intrinsic pathway of apoptosis and cellular oxidative stress. Our results considered collectively indicated that thyomquinone may be a potential agent for ovarian cancer drug development.
    Matched MeSH terms: Blotting, Western
  20. Helicobacter pylori outer inflammatory protein A (OipA) suppresses apoptosis of AGS gastric cells in vitro
    Al-Maleki AR, Loke MF, Lui SY, Ramli NSK, Khosravi Y, Ng CG, et al.
    Cell. Microbiol., 2017 12;19(12).
    PMID: 28776327 DOI: 10.1111/cmi.12771
    Outer inflammatory protein A (OipA) is an important virulence factor associated with gastric cancer and ulcer development; however, the results have not been well established and turned out to be controversial. This study aims to elucidate the role of OipA in Helicobacter pylori infection using clinical strains harbouring oipA "on" and "off" motifs. Proteomics analysis was performed on AGS cell pre-infection and postinfection with H. pylori oipA "on" and "off" strains, using liquid chromatography/mass spectrometry. AGS apoptosis and cell cycle assays were performed. Moreover, expression of vacuolating cytotoxin A (VacA) was screened using Western blotting. AGS proteins that have been suggested previously to play a role or associated with gastric disease were down-regulated postinfection with oipA "off" strains comparing to oipA "on" strains. Furthermore, oipA "off" and ΔoipA cause higher level of AGS cells apoptosis and G0/G1 cell-cycle arrest than oipA "on" strains. Interestingly, deletion of oipA increased bacterial VacA production. The capability of H. pylori to induce apoptosis and suppress expression of proteins having roles in human disease in the absence of oipA suggests that strains not expressing OipA may be less virulent or may even be protective against carcinogenesis compared those expressing OipA. This potentially explains the higher incidence of gastric cancer in East Asia where oipA "on" strains predominates.
    Matched MeSH terms: Blotting, Western
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