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  1. Fulltext Bcl-xL silencing induces alterations in hsa-miR-608 expression and subsequent cell death in A549 and SK-LU1 human lung adenocarcinoma cells
    Othman N, In LL, Harikrishna JA, Hasima N
    PLoS One, 2013;8(12):e81735.
    PMID: 24339958 DOI: 10.1371/journal.pone.0081735
    Bcl-xL is an anti-apoptotic protein that is frequently found to be overexpressed in non-small cell lung cancer leading to an inhibition of apoptosis and poor prognosis. Recently, the role of miRNAs in regulating apoptosis and cell survival during tumorigenesis has become evident, with cancer cells showing perturbed expression of various miRNAs. In this study, we utilized miRNA microarrays to determine if miRNA dysregulation in bcl-xL silenced lung adenocarcinoma cells could be involved in regulating cell death. Short interfering RNA-based transfection of A549 and SK-LU1 lung adenocarcinoma cells was successful in inducing a reduction in bcl-xL expression levels, resulting in a decrease in cell viability. A total of 10 miRNAs were found to be significantly differentially expressed when compared between siRNA-transfected and non-transfected cells including hsa-miR-181a, hsa-miR-769-5p, hsa-miR-361-5p, hsa-miR-1304 and hsa-miR-608. When overexpression studies on hsa-miR-608 was performed via transfection of miRNA mimics, cell death was found to be induced in A549 and SK-LU1 cells in comparison to untreated cells. This effect was reversed when knockdown studies involving anti-sense inhibitors were introduced. Combination of siRNA based silencing of bcl-xL (siBcl-xL) followed by anti-sense inhibitor transfection led to a decrease in the apoptotic population of A549 and SK-LU1 cells in comparison to cells only treated with siBcl-xL, illustrating the connection between bcl-xL, hsa-miR-608 and cell death. Gene target prediction analysis implicated the PI3K/AKT, WNT, TGF-β, and ERK signaling pathways as targets of bcl-xL induced miRNA alterations. We have demonstrated that bcl-xL silencing in A549 and SK-LU1 cells leads to the occurrence of cell death through the dysregulation of specific miRNAs. This study also provides a platform for anti-sense gene therapy whereby miRNA expression can be exploited to increase the apoptotic properties in lung adenocarcinoma cells.
    Matched MeSH terms: Cell Survival/genetics
  2. Dynamics of PEGylated-dextran-spermine nanoparticles for gene delivery to leukemic cells
    Amini R, Jalilian FA, Abdullah S, Veerakumarasivam A, Hosseinkhani H, Abdulamir AS, et al.
    Appl Biochem Biotechnol, 2013 Jun;170(4):841-53.
    PMID: 23615733 DOI: 10.1007/s12010-013-0224-0
    Leukemic cells are hard-to-transfect cell lines. Many transfection reagents which can provide high gene transfer efficiency in common adherent cell lines are not effective to transfect established blood cell lines or primary leukemic cells. This study aims to examine a new class of cationic polymer non-viral vector, PEGylated-dextran-spermine (PEG-D-SPM), to determine its ability to transfect the leukemic cells. Here, the optimal conditions of the complex preparation (PEG-D-SPM/plasmid DNA (pDNA)) were examined. Different weight-mixing (w/w) ratios of PEG-D-SPM/pDNA complex were prepared to obtain an ideal mixing ratio to protect encapsulated pDNA from DNase degradation and to determine the optimal transfection efficiency of the complex. Strong complexation between polymer and pDNA in agarose gel electrophoresis and protection of pDNA from DNase were detected at ratios from 25 to 15. Highest gene expression was detected at w/w ratio of 18 in HL60 and K562 cells. However, gene expression from both leukemic cell lines was lower than the control MCF-7 cells. The cytotoxicity of PEG-D-SPM/pDNA complex at the most optimal mixing ratios was tested in HL60 and K562 cells using MTS assay and the results showed that the PEG-D-SPM/pDNA complex had no cytotoxic effect on these cell lines. Spherical shape and nano-nature of PEG-D-SPM/pDNA complex at ratio 18 was observed using transmission electron microscopy. As PEG-D-SPM showed modest transfection efficiency in the leukemic cell lines, we conclude that further work is needed to improve the delivery efficiency of the PEG-D-SPM.
    Matched MeSH terms: Cell Survival/genetics
  3. Fulltext Mutant p53-R273H mediates cancer cell survival and anoikis resistance through AKT-dependent suppression of BCL2-modifying factor (BMF)
    Tan BS, Tiong KH, Choo HL, Chung FF, Hii LW, Tan SH, et al.
    Cell Death Dis, 2015;6:e1826.
    PMID: 26181206 DOI: 10.1038/cddis.2015.191
    p53 is the most frequently mutated tumor-suppressor gene in human cancers. Unlike other tumor-suppressor genes, p53 mutations mainly occur as missense mutations within the DNA-binding domain, leading to the expression of full-length mutant p53 protein. Mutant p53 proteins not only lose their tumor-suppressor function, but may also gain new oncogenic functions and promote tumorigenesis. Here, we showed that silencing of endogenous p53-R273H contact mutant, but not p53-R175H conformational mutant, reduced AKT phosphorylation, induced BCL2-modifying factor (BMF) expression, sensitized BIM dissociation from BCL-XL and induced mitochondria-dependent apoptosis in cancer cells. Importantly, cancer cells harboring endogenous p53-R273H mutant were also found to be inherently resistant to anoikis and lack BMF induction following culture in suspension. Underlying these activities is the ability of p53-R273H mutant to suppress BMF expression that is dependent on constitutively active PI3K/AKT signaling. Collectively, these findings suggest that p53-R273H can specifically drive AKT signaling and suppress BMF expression, resulting in enhanced cell survivability and anoikis resistance. These findings open the possibility that blocking of PI3K/AKT will have therapeutic benefit in mutant p53-R273H expressing cancers.
    Matched MeSH terms: Cell Survival/genetics
  4. Fulltext Knockdown of Tousled‑like kinase 1 inhibits survival of glioblastoma multiforme cells
    Ibrahim K, Abdul Murad NA, Harun R, Jamal R
    Int J Mol Med, 2020 Aug;46(2):685-699.
    PMID: 32468002 DOI: 10.3892/ijmm.2020.4619
    Glioblastoma multiforme (GBM) is an aggressive type of brain tumour that commonly exhibits resistance to treatment. The tumour is highly heterogenous and complex kinomic alterations have been reported leading to dysregulation of signalling pathways. The present study aimed to investigate the novel kinome pathways and to identify potential therapeutic targets in GBM. Meta‑analysis using Oncomine identified 113 upregulated kinases in GBM. RNAi screening was performed on identified kinases using ON‑TARGETplus siRNA library on LN18 and U87MG. Tousled‑like kinase 1 (TLK1), which is a serine/threonine kinase was identified as a potential hit. In vitro functional validation was performed as the role of TLK1 in GBM is unknown. TLK1 knockdown in GBM cells significantly decreased cell viability, clonogenicity, proliferation and induced apoptosis. TLK1 knockdown also chemosensitised the GBM cells to the sublethal dose of temozolomide. The downstream pathways of TLK1 were examined using microarray analysis, which identified the involvement of DNA replication, cell cycle and focal adhesion signalling pathways. In vivo validation of the subcutaneous xenografts of stably transfected sh‑TLK1 U87MG cells demonstrated significantly decreased tumour growth in female BALB/c nude mice. Together, these results suggested that TLK1 may serve a role in GBM survival and may serve as a potential target for glioma.
    Matched MeSH terms: Cell Survival/genetics
  5. Silencing of PROS1 induces apoptosis and inhibits migration and invasion of glioblastoma multiforme cells
    Che Mat MF, Abdul Murad NA, Ibrahim K, Mohd Mokhtar N, Wan Ngah WZ, Harun R, et al.
    Int J Oncol, 2016 Dec;49(6):2359-2366.
    PMID: 27840905 DOI: 10.3892/ijo.2016.3755
    Glioblastoma multiforme (GBM) is an aggressive brain tumor and most patients have poor prognosis. Despite many advances in research, there has been no significant improvement in the patient survival rate. New molecular therapies are being studied and RNA interference (RNAi) therapy is one of the promising approaches to improve prognosis and increase survival in patients with GBM. We performed a meta‑analysis of five different microarray datasets and identified 460 significantly upregulated genes in GBM. Loss‑of‑function screening of these upregulated genes using LN18 cells was performed to identify the significant target genes for glioma. Further investigations were performed using siRNA in LN18 cells and various functional assays were carried out on the selected candidate gene to understand further its role in GBM. We identified PROS1 as a candidate gene for GBM from the meta‑analysis and RNAi screening. Knockdown of PROS1 in LN18 cells significantly induced apoptosis compared to siPROS1‑untreated cells (p<0.05). Migration in cells treated with siPROS1 was reduced significantly (p<0.05) and this was confirmed with wound-healing assay. PROS1 knockdown showed substantial reduction in cell invasion up to 82% (p<0.01). In addition, inhibition of PROS1 leads to decrease in cellular proliferation by 18%. Knockdown of PROS1 in LN18 cells caused activation of both of the extrinsic and intrinsic apoptotic pathways. It caused major upregulation of FasL which is important for death receptor signaling activation and also downregulation of GAS6 and other members of TAM family of receptors. PROS1 may play an important role in the development of GBM through cellular proliferation, migration and invasion as well as apoptosis. Targeting PROS1 in GBM could be a novel therapeutic strategy in GBM treatment.
    Matched MeSH terms: Cell Survival/genetics
  6. Fulltext Co-visualization of DNA damage and ion traversals in live mammalian cells using a fluorescent nuclear track detector
    Kodaira S, Konishi T, Kobayashi A, Maeda T, Ahmad TA, Yang G, et al.
    J Radiat Res, 2015 Mar;56(2):360-5.
    PMID: 25324538 DOI: 10.1093/jrr/rru091
    The geometric locations of ion traversals in mammalian cells constitute important information in the study of heavy ion-induced biological effect. Single ion traversal through a cellular nucleus produces complex and massive DNA damage at a nanometer level, leading to cell inactivation, mutations and transformation. We present a novel approach that uses a fluorescent nuclear track detector (FNTD) for the simultaneous detection of the geometrical images of ion traversals and DNA damage in single cells using confocal microscopy. HT1080 or HT1080-53BP1-GFP cells were cultured on the surface of a FNTD and exposed to 5.1-MeV/n neon ions. The positions of the ion traversals were obtained as fluorescent images of a FNTD. Localized DNA damage in cells was identified as fluorescent spots of γ-H2AX or 53BP1-GFP. These track images and images of damaged DNA were obtained in a short time using a confocal laser scanning microscope. The geometrical distribution of DNA damage indicated by fluorescent γ-H2AX spots in fixed cells or fluorescent 53BP1-GFP spots in living cells was found to correlate well with the distribution of the ion traversals. This method will be useful for evaluating the number of ion hits on individual cells, not only for micro-beam but also for random-beam experiments.
    Matched MeSH terms: Cell Survival/genetics
  7. Transcript, methylation and molecular docking analyses of the effects of HDAC inhibitors, SAHA and Dacinostat, on SMN2 expression in fibroblasts of SMA patients
    Mohseni J, Al-Najjar BO, Wahab HA, Zabidi-Hussin ZA, Sasongko TH
    J Hum Genet, 2016 Sep;61(9):823-30.
    PMID: 27251006 DOI: 10.1038/jhg.2016.61
    Several histone deacetylase inhibitors (HDACis) are known to increase Survival Motor Neuron 2 (SMN2) expression for the therapy of spinal muscular atrophy (SMA). We aimed to compare the effects of suberoylanilide hydroxamic acid (SAHA) and Dacinostat, a novel HDACi, on SMN2 expression and to elucidate their acetylation effects on the methylation of the SMN2. Cell-based assays using type I and type II SMA fibroblasts examined changes in transcript expressions, methylation levels and protein expressions. In silico methods analyzed the intermolecular interactions between each compound and HDAC2/HDAC7. SMN2 mRNA transcript levels and SMN protein levels showed notable increases in both cell types, except for Dacinostat exposure on type II cells. However, combined compound exposures showed less pronounced increase in SMN2 transcript and SMN protein level. Acetylation effects of SAHA and Dacinostat promoted demethylation of the SMN2 promoter. The in silico analyses revealed identical binding sites for both compounds in HDACs, which could explain the limited effects of the combined exposure. With the exception on the effect of Dacinostat in Type II cells, we have shown that SAHA and Dacinostat increased SMN2 transcript and protein levels and promoted demethylation of the SMN2 gene.
    Matched MeSH terms: Cell Survival/genetics
  8. Fulltext Molecular analysis of ciprofloxacin resistance mechanisms in Malaysian ESBL-producing Klebsiella pneumoniae isolates and development of mismatch amplification mutation assays (MAMA) for rapid detection of gyrA and parC mutations
    Al-Marzooq F, Mohd Yusof MY, Tay ST
    Biomed Res Int, 2014;2014:601630.
    PMID: 24860827 DOI: 10.1155/2014/601630
    Ninety-three Malaysian extended-spectrum β-lactamase (ESBL)-producing Klebsiella pneumoniae isolates were investigated for ciprofloxacin resistance. Two mismatch amplification mutation (MAMA) assays were developed and used to facilitate rapid detection of gyrA and parC mutations. The isolates were also screened for plasmid-mediated quinolone resistance (PMQR) genes including aac(6')-Ib-cr, qepA, and qnr. Ciprofloxacin resistance (MICs 4- ≥ 32  μ g/mL) was noted in 34 (37%) isolates, of which 33 isolates had multiple mutations either in gyrA alone (n = 1) or in both gyrA and parC regions (n = 32). aac(6')-Ib-cr was the most common PMQR gene detected in this study (n = 61), followed by qnrB and qnrS (n = 55 and 1, resp.). Low-level ciprofloxacin resistance (MICs 1-2  μ g/mL) was noted in 40 (43%) isolates carrying qnrB accompanied by either aac(6')-Ib-cr (n = 34) or a single gyrA 83 mutation (n = 6). Ciprofloxacin resistance was significantly associated with the presence of multiple mutations in gyrA and parC regions. While the isolates harbouring gyrA and/or parC alteration were distributed into 11 PFGE clusters, no specific clusters were associated with isolates carrying PMQR genes. The high prevalence of ciprofloxacin resistance amongst the Malaysian ESBL-producing K. pneumoniae isolates suggests the need for more effective infection control measures to limit the spread of these resistant organisms in the hospital.
    Matched MeSH terms: Cell Survival/genetics
  9. Fulltext Targeting the B-cell lymphoma 2 anti-apoptotic proteins for cervical cancer treatment
    Abdul Rahman SF, Xiang Lian BS, Mohana-Kumaran N
    Future Oncol, 2020 Oct;16(28):2235-2249.
    PMID: 32715755 DOI: 10.2217/fon-2020-0389
    The B-cell lymphoma 2 (BCL-2) anti-apoptotic proteins have become attractive therapeutic targets especially with the development of BH3-mimetics which selectively target these proteins. However, it is important to note that expression levels of the anti-apoptotic proteins and their relevance in inhibiting apoptosis varies between different cell lineages. This addiction to certain anti-apoptotic proteins for survival, can be determined with various techniques and targeted effectively with selective BH3-mimetics. Studies have highlighted that anti-apoptotic proteins BCL-XL and MCL-1 are crucial for cervical cancer cell survival. Co-targeting BCL-XL and MCL-1 with selective BH3-mimetics yielded promising results in cervical cancer cell lines. In this review, we focus on the expression levels of the anti-apoptotic proteins in cervical cancer tissues and how to possibly target them with BH3-mimetics.
    Matched MeSH terms: Cell Survival/genetics
  10. Improvement of isolated caprine islet survival and functionality in vitro by enhancing of PDX1 gene expression
    Hani H, Allaudin ZN, Mohd-Lila MA, Sarsaifi K, Rasouli M, Tam YJ, et al.
    Xenotransplantation, 2017 05;24(3).
    PMID: 28397308 DOI: 10.1111/xen.12302
    BACKGROUND: Dead islets replaced with viable islets are a promising offer to restore normal insulin production to a person with diabetes. The main reason for establishing a new islet source for transplantation is the insufficiency of human donor pancreas while using xenogeneic islets perhaps assists this problem. The expression of PDX1 is essential for the pancreas expansion. In mature β-cells, PDX1 has several critical roles such as glucose sensing, insulin synthesis, and insulin secretion. In this study, we aimed to evaluate the expression of pancreatic duodenal homeobox-1 (PDX1) in treated caprine islets in culture and to assess the protective effects of antioxidant factors on the PDX1 gene in cultured caprine islets.

    MATERIALS AND METHODS: Purified islets were treated with serum-free, serum, IBMX, tocopherol, or IBMX and tocopherol media. Quantitative polymerase chain reaction and Western blotting were carried out to compare the expression levels of PDX1 in treated purified islets cultured with different media.

    RESULTS: Islets treated with IBMX/tocopherol exhibited the highest fold change in the relative expression of PDX1 on day 5 post-treatment (relative expression: 6.80±2.08), whereas serum-treated islets showed the lowest fold changes in PDX1 expression on day 5 post-treatment (0.67±0.36), as compared with the expression on day 1 post-treatment. Insulin production and viability tests of purified islets showed superiority of islet at supplemented serum-free media with IBMX/tocopherol compared to other cultures (53.875%±1.59%).

    CONCLUSIONS: Our results indicated that supplemented serum-free medium with tocopherol and IBMX enhances viability and PDX1 gene expression compared to serum-added and serum-free media.

    Matched MeSH terms: Cell Survival/genetics
  11. Fulltext Mitochondrial DNA alterations may influence the cisplatin responsiveness of oral squamous cell carcinoma
    Aminuddin A, Ng PY, Leong CO, Chua EW
    Sci Rep, 2020 May 12;10(1):7885.
    PMID: 32398775 DOI: 10.1038/s41598-020-64664-3
    Cisplatin is the first-line chemotherapeutic agent for the treatment of oral squamous cell carcinoma (OSCC). However, the intrinsic or acquired resistance against cisplatin remains a major obstacle to treatment efficacy in OSCC. Recently, mitochondrial DNA (mtDNA) alterations have been reported in a variety of cancers. However, the role of mtDNA alterations in OSCC has not been comprehensively studied. In this study, we evaluated the correlation between mtDNA alterations (mtDNA content, point mutations, large-scale deletions, and methylation status) and cisplatin sensitivity using two OSCC cell lines, namely SAS and H103, and stem cell-like tumour spheres derived from SAS. By microarray analysis, we found that the tumour spheres profited from aberrant lipid and glucose metabolism and became resistant to cisplatin. By qPCR analysis, we found that the cells with less mtDNA were less responsive to cisplatin (H103 and the tumour spheres). Based on the findings, we theorised that the metabolic changes in the tumour spheres probably resulted in mtDNA depletion, as the cells suppressed mitochondrial respiration and switched to an alternative mode of energy production, i.e. glycolysis. Then, to ascertain the origin of the variation in mtDNA content, we used MinION, a nanopore sequencer, to sequence the mitochondrial genomes of H103, SAS, and the tumour spheres. We found that the lower cisplatin sensitivity of H103 could have been caused by a constellation of genetic and epigenetic changes in its mitochondrial genome. Future work may look into how changes in mtDNA translate into an impact on cell function and therefore cisplatin response.
    Matched MeSH terms: Cell Survival/genetics
  12. Fulltext Artonin E Induces Apoptosis via Mitochondrial Dysregulation in SKOV-3 Ovarian Cancer Cells
    Rahman MA, Ramli F, Karimian H, Dehghan F, Nordin N, Ali HM, et al.
    PLoS One, 2016;11(3):e0151466.
    PMID: 27019365 DOI: 10.1371/journal.pone.0151466
    Artonin E is a prenylated flavonoid isolated from the stem bark of Artocarpus elasticus Reinw.(Moraceae). This study aimed to investigate the apoptotic mechanisms induced by artonin E in a metastatic human ovarian cancer cell line SKOV-3 in vitro. MTT assay, clonogenic assay, acridine orange and propidium iodide double staining, cell cycle and annexin V analyses were performed to explore the mode of artonin E-induced cell death at different time points. DNA laddering, activation of caspases-3, -8, and -9, multi-parametric cytotoxicity-3 analysis by high-content screening, measurement of reactive oxygen species generation, and Western blot were employed to study the pathways involved in the apoptosis. MTT results showed that artonin E inhibited the growth of SKOV-3 cells, with IC50 values of 6.5±0.5 μg/mL after 72 h treatment, and showed less toxicity toward a normal human ovarian cell line T1074, with IC50 value of 32.5±0.5 μg/mL. Results showed that artonin E induced apoptosis and cell cycle arrest at the S phase. This compound also promoted the activation of caspases-3, -8, and -9. Further investigation into the depletion of mitochondrial membrane potential and release of cytochrome c revealed that artonin E treatment induced apoptosis via regulation of the expression of pro-survival and pro-apoptotic Bcl-2 family members. The expression levels of survivin and HSP70 proteins were also down regulated in SKOV-3 cells treated with artonin E. We propose that artonin E induced an antiproliferative effect that led to S phase cell cycle arrest and apoptosis through dysregulation of mitochondrial pathways, particularly the pro- and anti-apoptosis signaling pathways.
    Matched MeSH terms: Cell Survival/genetics
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