Displaying publications 1 - 20 of 83 in total

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  1. Novel PEX1 mutations in fibroblasts from children with Zellweger spectrum disorders exhibit temperature sensitive characteristics
    Liang JS, Hung KL, Lin LJ, Ong WP, Keng WT, Lu JF
    Epilepsy Behav, 2023 Aug;145:109266.
    PMID: 37385119 DOI: 10.1016/j.yebeh.2023.109266
    Zellweger spectrum disorders (ZSD) are rare autosomal recessive disorders caused by defects in peroxisome biogenesis factor (PEX; peroxin) genes leading to impaired transport of peroxisomal proteins with peroxisomal targeting signals (PTS). Four patients, including a pair of homozygotic twins, diagnosed as ZSD by genetic study with different clinical presentations and outcomes as well as various novel mutations are described here. A total of 3 novel mutations, including a nonsense, a frameshift, and a splicing mutation, in PEX1 from ZSD patients were identified and unequivocally confirmed that the p.Ile989Thr mutant PEX1 exhibited temperature-sensitive characteristics and is associated with milder ZSD. The nature of the p.Ile989Thr mutant exhibited different characteristics from that of the other previously identified temperature-sensitive p.Gly843Asp PEX1 mutant. Transcriptome profiles under nonpermissive vs. permissive conditions were explored to facilitate the understanding of p.Ile989Thr mutant PEX1. Further investigation of molecular mechanisms may help to clarify potential genetic causes that could modify the clinical presentation of ZSD.
    Matched MeSH terms: Fibroblasts/metabolism
  2. Fulltext Hydroxylated polymethoxyflavones reduce the activity of pancreatic lipase, inhibit adipogenesis and enhance lipolysis in 3T3-L1 mouse embryonic fibroblast cells
    Ahmad B, Friar EP, Vohra MS, Khan N, Serpell CJ, Garrett MD, et al.
    Chem Biol Interact, 2023 Jul 01;379:110503.
    PMID: 37084996 DOI: 10.1016/j.cbi.2023.110503
    Hydroxylated polymethoxyflavones (HPMFs) have been shown to possess various anti-disease effects, including against obesity. This study investigates the anti-obesity effects of HPMFs in further detail, aiming to gain understanding of their mechanism of action in this context. The current study demonstrates that two HPMFs; 3'-hydroxy-5,7,4',5'-tetramethoxyflavone (3'OH-TetMF) and 4'-hydroxy-5,7,3',5'-tetramethoxyflavone (4'OH-TetMF) possess anti-obesity effects. They both significantly reduced pancreatic lipase activity in a competitive manner as demonstrated by molecular docking and kinetic studies. In cell studies, it was revealed that both of the HPMFs suppress differentiation of 3T3-L1 mouse embryonic fibroblast cells during the early stages of adipogenesis. They also reduced expression of key adipogenic and lipogenic marker genes, namely peroxisome proliferator-activated receptor-gamma (PPARγ), CCAAT/enhancer-binding protein α and β (C/EBP α and β), adipocyte binding protein 2 (aP2), fatty acid synthase (FASN), and sterol regulatory element-binding protein 1 (SREBF 1). They also enhanced the expression of cell cycle genes, i.e., cyclin D1 (CCND1) and C-Myc, and reduced cyclin A2 expression. When further investigated, it was also observed that these HPMFs accelerate lipid breakdown (lipolysis) and enhance lipolytic genes expression. Moreover, they also reduced the secretion of proteins (adipokines), including pro-inflammatory cytokines, from mature adipocytes. Taken together, this study concludes that these HPMFs have anti-obesity effects, which are worthy of further investigation.
    Matched MeSH terms: Fibroblasts/metabolism
  3. Fulltext Doxycycline Interferes with Zika Virus Serine Protease and Inhibits Virus Replication in Human Skin Fibroblasts
    Chong Teoh T, J Al-Harbi S, Abdulrahman AY, Rothan HA
    Molecules, 2021 Jul 16;26(14).
    PMID: 34299596 DOI: 10.3390/molecules26144321
    Zika virus (ZIKV) represents a re-emerging threat to global health due to its association with congenital birth defects. ZIKV NS2B-NS3 protease is crucial for virus replication by cleaving viral polyprotein at various junctions to release viral proteins and cause cytotoxic effects in ZIKV-infected cells. This study characterized the inhibitory effects of doxycycline against ZIKV NS2B-NS3 protease and viral replication in human skin cells. The in silico data showed that doxycycline binds to the active site of ZIKV protease at a low docking energy (-7.8 Kcal/mol) via four hydrogen bonds with the protease residues TYR1130, SER1135, GLY1151, and ASP83. Doxycycline efficiently inhibited viral NS2B-NS3 protease at average human temperature (37 °C) and human temperature with a high fever during virus infection (40 °C). Interestingly, doxycycline showed a higher inhibitory effect at 40 °C (IC50 = 5.3 µM) compared to 37 °C (9.9 µM). The virus replication was considerably reduced by increasing the concentration of doxycycline. An approximately 50% reduction in virus replication was observed at 20 µM of doxycycline. Treatment with 20 µM of doxycycline reduced the cytopathic effects (CPE), and the 40 µM of doxycycline almost eliminated the CPE of human skin cells. This study showed that doxycycline binds to the ZIKV protease and inhibits its catalytic activity at a low micro-molecular concentration range. Treatment of human skin fibroblast with doxycycline eliminated ZIKV infection and protected the cells against the cytopathic effects of the infection.
    Matched MeSH terms: Fibroblasts/metabolism*
  4. Fulltext The Role of Calcium in Wound Healing
    Subramaniam T, Fauzi MB, Lokanathan Y, Law JX
    Int J Mol Sci, 2021 Jun 17;22(12).
    PMID: 34204292 DOI: 10.3390/ijms22126486
    Skin injury is quite common, and the wound healing is a complex process involving many types of cells, the extracellular matrix, and soluble mediators. Cell differentiation, migration, and proliferation are essential in restoring the integrity of the injured tissue. Despite the advances in science and technology, we have yet to find the ideal dressing that can support the healing of cutaneous wounds effectively, particularly for difficult-to-heal chronic wounds such as diabetic foot ulcers, bed sores, and venous ulcers. Hence, there is a need to identify and incorporate new ideas and methods to design a more effective dressing that not only can expedite wound healing but also can reduce scarring. Calcium has been identified to influence the wound healing process. This review explores the functions and roles of calcium in skin regeneration and reconstruction during would healing. Furthermore, this review also investigates the possibility of incorporating calcium into scaffolds and examines how it modulates cutaneous wound healing. In summary, the preliminary findings are promising. However, some challenges remain to be addressed before calcium can be used for cutaneous wound healing in clinical settings.
    Matched MeSH terms: Fibroblasts/metabolism
  5. Fulltext (E)-N-(2-(3, 5-Dimethoxystyryl) phenyl) furan-2-carboxamide (BK3C231) induces cytoprotection in CCD18-Co human colon fibroblast cells through Nrf2/ARE pathway activation
    Tan HH, Thomas NF, Inayat-Hussain SH, Chan KM
    Sci Rep, 2021 02 26;11(1):4773.
    PMID: 33637843 DOI: 10.1038/s41598-021-83163-7
    Cytoprotection involving the nuclear factor erythroid 2-related factor 2 (Nrf2)/antioxidant response element (ARE) signaling pathway is an important preventive strategy for normal cells against carcinogenesis. In our previous study, the chemopreventive potential of (E)-N-(2-(3, 5-Dimethoxystyryl) phenyl) furan-2-carboxamide (BK3C231) has been elucidated through its cytoprotective effects against DNA and mitochondrial damages in the human colon fibroblast CCD-18Co cell model. Therefore this study aimed to investigate the molecular mechanisms underlying BK3C231-induced cytoprotection and the involvement of the Nrf2/ARE pathway. The cells were pretreated with BK3C231 before exposure to carcinogen 4-nitroquinoline N-oxide (4NQO). BK3C231 increased the protein expression and activity of cytoprotective enzymes namely NAD(P)H:quinone oxidoreductase 1 (NQO1), glutathione S-transferase (GST) and heme oxygenase-1 (HO-1), as well as restoring the expression of glutamate-cysteine ligase catalytic subunit (GCLC) back to the basal level. Furthermore, dissociation of Nrf2 from its inhibitory protein, Keap1, and ARE promoter activity were upregulated in cells pretreated with BK3C231. Taken together, our findings suggest that BK3C231 exerts cytoprotection by activating the Nrf2 signaling pathway which leads to ARE-mediated upregulation of cytoprotective proteins. This study provides new mechanistic insights into BK3C231 chemopreventive activities and highlights the importance of stilbene derivatives upon development as a potential chemopreventive agent.
    Matched MeSH terms: Fibroblasts/metabolism
  6. In Vitro Methods to Evaluate the Effects of Mesenchymal Stem Cells on TGF-β1-Induced Pulmonary Fibrosis
    Lan YW, Chen CM, Chong KY
    Methods Mol Biol, 2021;2269:83-92.
    PMID: 33687673 DOI: 10.1007/978-1-0716-1225-5_6
    A co-culture model of mesenchymal stem cells (MSCs) and fibroblasts is an efficient and rapid method to evaluate the anti-fibrotic effects of MSCs-based cell therapy. Transforming growth factor (TGF)-β1 plays a key role in promotion of fibroblast activation and differentiation which can induce collagen deposition, increase ECM production in lung tissue, eventually resulted in pulmonary fibrosis. Here, we use this co-culture system and examine the ECM production in activated fibroblasts by western blot and quantitative real-time analysis to understand the therapeutic effects of MSCs.
    Matched MeSH terms: Fibroblasts/metabolism*
  7. Cancer-associated fibroblasts of colorectal cancer and their markers: updates, challenges and translational outlook
    Musa M, Ali A
    Future Oncol, 2020 Oct;16(29):2329-2344.
    PMID: 32687721 DOI: 10.2217/fon-2020-0384
    Accumulation of cancer-associated fibroblasts (CAFs) in the tumor microenvironment is associated with poor prognosis and recurrence of colorectal cancer (CRC). Despite their prominent roles in colorectal carcinogenesis, there is a lack of robust and specific markers to classify the heterogeneous and highly complex CAF populations. This has resulted in confusing and misleading definitions of CAFs in cancer niche. Advancements in molecular biology approaches have open doors to reliable CAF marker detection methods in various solid tumors. These discoveries would contribute to more efficient screening, monitoring and targeted therapy of CRC thus potentially will reduce cancer morbidity and mortality rates. This review highlights current scenarios, dilemma, translational potentials of CAF biomarker and future therapeutic applications involving CAF marker identification in CRC.
    Matched MeSH terms: Cancer-Associated Fibroblasts/metabolism*
  8. Fulltext Concentration Dependent Effect of Human Dermal Fibroblast Conditioned Medium (DFCM) from Three Various Origins on Keratinocytes Wound Healing
    Maarof M, Chowdhury SR, Saim A, Bt Hj Idrus R, Lokanathan Y
    Int J Mol Sci, 2020 Apr 22;21(8).
    PMID: 32331278 DOI: 10.3390/ijms21082929
    Fibroblasts secrete many essential factors that can be collected from fibroblast culture medium, which is termed dermal fibroblast conditioned medium (DFCM). Fibroblasts isolated from human skin samples were cultured in vitro using the serum-free keratinocyte-specific medium (Epilife (KM1), or define keratinocytes serum-free medium, DKSFM (KM2) and serum-free fibroblast-specific medium (FM) to collect DFCM-KM1, DFCM-KM2, and DFCM-FM, respectively). We characterised and evaluated the effects of 100-1600 µg/mL DFCM on keratinocytes based on attachment, proliferation, migration and gene expression. Supplementation with 200-400 µg/mL keratinocyte-specific DFCM-KM1 and DFCM-KM2 enhanced the attachment, proliferation and migration of sub-confluent keratinocytes, whereas 200-1600 µg/mL DFCM-FM significantly increased the healing rate in the wound healing assay, and 400-800 µg/mL DFCM-FM was suitable to enhance keratinocyte attachment and proliferation. A real-time (RT2) profiler polymerase chain reaction (PCR) array showed that 42 genes in the DFCM groups had similar fold regulation compared to the control group and most of the genes were directly involved in wound healing. In conclusion, in vitro keratinocyte re-epithelialisation is supported by the fibroblast-secreted proteins in 200-400 µg/mL DFCM-KM1 and DFCM-KM2, and 400-800 µg/mL DFCM-FM, which could be useful for treating skin injuries.
    Matched MeSH terms: Fibroblasts/metabolism*
  9. Single-cell analysis on stromal fibroblasts in the microenvironment of solid tumours
    Musa M
    Adv Med Sci, 2020 Mar;65(1):163-169.
    PMID: 31972467 DOI: 10.1016/j.advms.2019.12.001
    Besides malignant cells, the tumour microenvironment consists of various stromal cells such as cancer-associated fibroblasts (CAFs) and myofibroblasts. Accumulation of heterogeneous populations of stromal cells in solid tumours is associated with lower survival rates and cancer recurrence in patients. Certain limitations presented by conventional experimental designs and techniques in cancer research have led to poor understanding of the fundamental basis of cancer niche. Recent developments in single-cell techniques allow more in-depth studies of the tumour microenvironment. Analyses at the single-cell level enables the detection of rare cell types, characterization of intra-tumour cellular heterogeneity and analysis of the lineage output of malignant cells. This subsequently, provides valuable insights on better diagnostic methods and treatment avenues for cancer. This review explores the recent advancements and applications of single-cell technologies in cancer research pertaining to the study of stromal fibroblasts in the microenvironment of solid tumours.
    Matched MeSH terms: Cancer-Associated Fibroblasts/metabolism
  10. Fulltext Bioassay-Guided Different Extraction Techniques of Carica papaya (Linn.) Leaves on In Vitro Wound-Healing Activities
    Soib HH, Ismail HF, Husin F, Abu Bakar MH, Yaakob H, Sarmidi MR
    Molecules, 2020 Jan 24;25(3).
    PMID: 31991676 DOI: 10.3390/molecules25030517
    Herbal plants are traditionally utilized to treat various illnesses. They contain phytochemicals that can be extracted using conventional methods such as maceration, soxhlet, and boiling, as well as non-conventional methods including ultrasonic, microwave, and others. Carica papaya leaves have been used for the treatment of dengue, fungal, and bacterial infections as well as an ingredient in anti-aging products. Phytochemicals analysis detected the presence of kaempferol, myricetin, carpaine, pseudocarpaine, dehydrocarpaine I and II, ferulic acid, caffeic acid, chlorogenic acid, β-carotene, lycopene, and anthraquinones glycoside. Conventional preparation by boiling and simple maceration is practical, simple, and safe; however, only polar phytochemicals are extracted. The present study aims to investigate the effects of three different non-conventional extraction techniques (ultrasonic-assisted extraction, reflux, and agitation) on C. papaya phytochemical constituents, the antioxidant capacity, and wound-healing activities. Among the three techniques, the reflux technique produced the highest extraction yield (17.86%) with the presence of saponins, flavonoids, coumarins, alkaloids, and phenolic metabolites. The reflux technique also produced the highest 2, 2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging with an IC50 value of 0.236 mg/mL followed by ultrasonic-assisted extraction (UAE) (IC50: 0.377 mg/mL) and agitation (IC50: 0.404 mg/mL). At tested concentrations (3.125 µg/mL to 500 µg/mL), all extracts do not exhibit a cytotoxicity effect on the human skin fibroblast, HSF1184. Interestingly, reflux and UAE were active fibroblast proliferators that support 85% (12.5 µg/mL) and 41% (6.25 µg/mL) better cell growth, respectively. Additionally, during the early 24 h of the scratch assay, the migration rate at 12.5 µg/mL was faster for all extracts with 51.8% (reflux), 49.3% (agitation), and 42.5% (UAE) as compared to control (21.87%). At 48 h, proliferated cells covered 78.7% of the scratch area for reflux extract, 63.1% for UAE, 61% for agitation, and 42.6% for control. Additionally, the collagen synthesis was enhanced for 31.6% and 65% after 24 and 48 h of treatment for reflux. An HPLC-MS/MS-QTOF (quadruple time-of-flight) analysis of reflux identified nine phytochemicals, including carpaine, kaempferol 3-(2G-glucosylrutinoside), kaempferol 3-(2″-rhamnosylgalactoside), 7-rhamnoside, kaempferol 3-rhamnosyl-(1->2)-galactoside-7-rhamnoside, luteolin 7-galactosyl-(1->6)-galactoside, orientin 7-O-rhamnoside, 11-hydroperoxy-12,13-epoxy-9-octadecenoic acid, palmitic amide, and 2-hexaprenyl-6-methoxyphenol. The results suggested that reflux was the best technique as compared to ultrasonic and agitation.
    Matched MeSH terms: Fibroblasts/metabolism*
  11. Fulltext In vivo acute toxicity and anti-gastric evaluation of a novel dichloro Schiff base: Bax and HSP70 alteration
    Saremi K, Rad SK, Khalilzadeh M, Hussaini J, Majid NA
    Acta Biochim Biophys Sin (Shanghai), 2020 Jan 02;52(1):26-37.
    PMID: 31889181 DOI: 10.1093/abbs/gmz140
    Chlorine is shown to possess anti-gastric ulcer activity, since it can inactivate Helicobacter pylori, which is regarded as one of the most common risk factors for causing gastric problems. In the current study, the gastroprotective property of a novel dichloro-substituted Schiff base complex, 2, 2'- [-1, 2-cyclohexanediylbis(nitriloethylidyne)] bis(4-chlorophenol) (CNCP), against alcohol-induced gastric lesion in SD rats was assessed. SD rats were divided into four groups, i.e. normal, ulcer control, testing, and reference groups. Ulcer area, gastric wall mucus, and also gastric acidity of the animal stomachs were measured. In addition, antioxidant activity of CNCP was evaluated and its safe dose was identified. Immunohistochemistry staining was also carried to evaluate two important proteins, i.e. Bcl2-associated X protein (Bax) and heat shock protein 70 (HSP70). Moreover, the activities of super oxide dismutase and catalase, as well as the levels of prostaglandin E2 (PGE2) and malondialdehyde (MDA) were also measured. Antioxidant activity of CNCP was approved via the aforementioned experiments. Histological evaluations showed that the compound possesses stomach epithelial defense activity. Additionally, periodic acid-Schiff staining exhibited over-expression of HSP70 and down-expression of Bax protein in the CNCP-treated rats. Moreover, CNCP caused deceased MDA level and elevated PGE2 level, and at the same time increased the activities of the two enzymes.
    Matched MeSH terms: Fibroblasts/metabolism
  12. Chlorella vulgaris modulates the expression of senescence-associated genes in replicative senescence of human diploid fibroblasts
    Jaafar F, Durani LW, Makpol S
    Mol Biol Rep, 2020 Jan;47(1):369-379.
    PMID: 31642042 DOI: 10.1007/s11033-019-05140-8
    Human diploid fibroblasts (HDFs) cultured in vitro have limited capacity to proliferate after population doubling is repeated several times, and they enter into a state known as replicative senescence or cellular senescence. This study aimed to investigate the effect of Chlorella vulgaris on the replicative senescence of HDFs by determining the expression of senescence-associated genes. Young and senescent HDFs were divided into untreated control and C. vulgaris-treated groups. A senescence-associated gene transcription analysis was carried out with qRT-PCR. Treatment of young HDFs with C. vulgaris reduced the expression of SOD1, CAT and CCS (p 
    Matched MeSH terms: Fibroblasts/metabolism
  13. Fulltext RNA-Seq analysis revealed genes associated with UV-induced cell necrosis through MAPK/TNF-α pathways in human dermal fibroblast cells as an inducer of premature photoaging
    Alafiatayo AA, Lai KS, Ahmad S, Mahmood M, Shaharuddin NA
    Genomics, 2020 01;112(1):484-493.
    PMID: 30946891 DOI: 10.1016/j.ygeno.2019.03.011
    Exposing the skin to solar UV radiation induces cascades of signaling pathways and biological alterations such as redox imbalance, suppression of antioxidant genes and programmed cell death. Therefore, the aim of this study was to use RNA-Seq to unravel the effects of UV radiation on Normal Human Adult Fibroblast cells (NHDF). Cells were exposed to UV (20 mJ/cm2 for 3 mins) and incubated for 24 h. Total mRNA from the cells generated libraries of 72,080,648 and 40,750,939 raw reads from UV-treated and control cells respectively. Of the differentially expressed genes (DEGs) produced 2,007 were up-regulated and 2,791 were down-regulated (fold change ≥2, p 
    Matched MeSH terms: Fibroblasts/metabolism
  14. Linkage analysis and whole exome sequencing reveals AHNAK2 as a novel genetic cause for autosomal recessive CMT in a Malaysian family
    Tey S, Shahrizaila N, Drew AP, Samulong S, Goh KJ, Battaloglu E, et al.
    Neurogenetics, 2019 08;20(3):117-127.
    PMID: 31011849 DOI: 10.1007/s10048-019-00576-3
    Charcot-Marie-Tooth (CMT) disease is a form of inherited peripheral neuropathy that affects motor and sensory neurons. To identify the causative gene in a consanguineous family with autosomal recessive CMT (AR-CMT), we employed a combination of linkage analysis and whole exome sequencing. After excluding known AR-CMT genes, genome-wide linkage analysis mapped the disease locus to a 7.48-Mb interval on chromosome 14q32.11-q32.33, flanked by the markers rs2124843 and rs4983409. Whole exome sequencing identified two non-synonymous variants (p.T40P and p.H915Y) in the AHNAK2 gene that segregated with the disease in the family. Pathogenic predictions indicated that p.T40P is the likely causative allele. Analysis of AHNAK2 expression in the AR-CMT patient fibroblasts showed significantly reduced mRNA and protein levels. AHNAK2 binds directly to periaxin which is encoded by the PRX gene, and PRX mutations are associated with another form of AR-CMT (CMT4F). The altered expression of mutant AHNAK2 may disrupt the AHNAK2-PRX interaction in which one of its known functions is to regulate myelination.
    Matched MeSH terms: Fibroblasts/metabolism
  15. Fabrication and evaluation of bacterial nanocellulose/poly(acrylic acid)/graphene oxide composite hydrogel: Characterizations and biocompatibility studies for wound dressing
    Chen XY, Low HR, Loi XY, Merel L, Mohd Cairul Iqbal MA
    J Biomed Mater Res B Appl Biomater, 2019 08;107(6):2140-2151.
    PMID: 30758129 DOI: 10.1002/jbm.b.34309
    Graphene oxide (GO) is a potential material for wound dressing due to its excellent biocompatibility and mechanical properties. This study evaluated the effects of GO concentration on the synthesis of bacterial nanocellulose (BNC)-grafted poly(acrylic acid) (AA)-graphene oxide (BNC/P(AA)/GO) composite hydrogel and its potential as wound dressing. Hydrogels were successfully synthesized via electron-beam irradiation. The hydrogels were characterized by their mechanical properties, bioadhesiveness, water vapor transmission rates (WVTRs), water retention abilities, water absorptivity, and biocompatibility. Fourier transform infrared analysis showed the successful incorporation of GO into hydrogel. Thickness, gel fraction determination and morphological study revealed that increased GO concentration in hydrogels leads to reduced crosslink density and larger pore size, resulting in increased WVTR. Thus, highest swelling ratio was found in hydrogel with higher amount of GO (0.09 wt %). The mechanical properties of the composite hydrogel were maintained, while its hardness and bioadhesion were reduced with higher GO concentration in the hydrogel, affirming the durable and easy removable properties of a wound dressing. Human dermal fibroblast cell attachment and proliferation studies showed that biocompatibility of hydrogel was improved with the inclusion of GO in the hydrogel. Therefore, BNC/P(AA)/GO composite hydrogel has a potential application as perdurable wound dressing. © 2019 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater 107B: 2140-2151, 2019.
    Matched MeSH terms: Fibroblasts/metabolism
  16. Fulltext Important Metabolites in Maintaining Folate Cycle, Homocysteine, and Polyamine Metabolism Associated with Ranibizumab Treatment in Cultured Human Tenon's Fibroblasts
    Munirah Md Noh S, Hamimah Sheikh Abdul Kadir S, Vasudevan S
    Biomolecules, 2019 06 22;9(6).
    PMID: 31234474 DOI: 10.3390/biom9060243
    The anti-fibrotic properties of ranibizumab have been well documented. As an antagonist to vascular endothelial growth factor (VEGF), ranibizumab works by binding and neutralizing all active VEGF-A, thus limiting progressive cell growth and proliferation. Ranibizumab application in ocular diseases has shown remarkable desired effects; however, to date, its antifibrotic mechanism is not well understood. In this study, we identified metabolic changes in ranibizumab-treated human Tenon's fibroblasts (HTFs). Cultured HTFs were treated for 48 h with 0.5 mg/mL of ranibizumab and 0.5 mg/mL control IgG antibody which serves as a negative control. Samples from each group were injected into Agilent 6520 Q-TOF liquid chromatography/mass spectrometer (LC/MS) system to establish the metabolite expression in both ranibizumab treated cells and control group. Data obtained was analyzed using Agilent Mass Hunter Qualitative Analysis software to identify the most regulated metabolite following ranibizumab treatment. At p-value < 0.01 with the cut off value of two-fold change, 31 identified metabolites were found to be significantly upregulated in ranibizumab-treated group, with six of the mostly upregulated having insignificant role in fibroblast cell cycle and wound healing regulations. Meanwhile, 121 identified metabolites that were downregulated, and seven of the mostly downregulated are significantly involved in cell cycle and proliferation. Our findings suggest that ranibizumab abrogates the tissue scarring and wound healing process by regulating the expression of metabolites associated with fibrotic activity. In particular, we found that vitamin Bs are important in maintaining normal folate cycle, nucleotide synthesis, and homocysteine and spermidine metabolism. This study provides an insight into ranibizumab's mechanism of action in HTFs from the perspective of metabolomics.
    Matched MeSH terms: Fibroblasts/metabolism*
  17. Fulltext Development of a luciferase/luciferin cell proliferation (XenoLuc) assay for real-time measurements of Gfp-Luc2-modified cells in a co-culture system
    Teow SY, Liew K, Che Mat MF, Marzuki M, Abdul Aziz N, Chu TL, et al.
    BMC Biotechnol, 2019 06 14;19(1):34.
    PMID: 31200673 DOI: 10.1186/s12896-019-0528-4
    BACKGROUND: In vitro modelling of cancer cells is becoming more complex due to prevailing evidence of intimate interactions between cancer cells and their surrounding stroma. A co-culture system which consists of more than one cell type is physiologically more relevant and thus, could serve as a useful model for various biological studies. An assay that specifically detects the phenotypic changes of cancer cells in a multi-cellular system is lacking for nasopharyngeal carcinoma (NPC).

    RESULTS: Here, we describe a luciferase/luciferin (XenoLuc) assay that could specifically measure changes in the proliferation of cancer cells in the co-culture system using two modified NPC patient-derived tumour xenograft (PDTXs) cells: Xeno284-gfp-luc2 and XenoB110-gfp-luc2. Through this assay, we are able to show that the growth of NPC xenograft cells in both two-dimensional (2D) and three-dimensional (3D) models was enhanced when co-cultured with normal human dermal fibroblasts (NHDFs). In addition, potential applications of this assay in in vitro drug or inhibitor screening experiments are also illustrated.

    CONCLUSIONS: XenoLuc assay is specific, sensitive, rapid and cost-effective for measuring the growth of luciferase-expressing cells in a co- or multiple-culture system. This assay may also be adapted for tumour microenvironment studies as well as drug screening experiments in more complex 3D co-culture systems.

    Matched MeSH terms: Fibroblasts/metabolism*
  18. Establishment of epithelial and fibroblast cell cultures and cell lines from primary renal cancer nephrectomies
    Yap NY, Ong TA, Morais C, Pailoor J, Gobe GC, Rajandram R
    Cell Biol Int, 2019 Jun;43(6):715-725.
    PMID: 31062478 DOI: 10.1002/cbin.11150
    Renal cell carcinoma (RCC) is one of the most lethal urogenital cancers and effective treatment of metastatic RCC remains an elusive target. Cell lines enable the in vitro investigation of molecular and genetic changes leading to renal carcinogenesis and are important for evaluating cellular drug response or toxicity. This study details a fast and easy protocol of establishing epithelial and fibroblast cell cultures or cell lines concurrently from renal cancer nephrectomy tissue. The protocol involves mechanical disaggregation, collagenase digestion and cell sieving for establishing epithelial cells while fibroblast cells were grown from explants. This protocol has been modified from previous published reports with additional antibiotics and washing steps added to eliminate microbial contamination from the surgical source. Cell characterisation was carried out using immunofluorescence and quantitative polymerase chain reaction. Eleven stable epithelial renal tumour cell lines of various subtypes, including rare subtypes, were established with a spontaneous immortalisation rate of 21.6% using this protocol. Eight fibroblast cell cultures grew successfully but did not achieve spontaneous immortalisation. Cells of epithelial origin expressed higher expressions of epithelial markers such as pan-cytokeratin, cytokeratin 8 and E-cadherin whereas fibroblast cells expressed high α-smooth muscle actin. Further mutational analysis is needed to evaluate the genetic or molecular characteristics of the cell lines.
    Matched MeSH terms: Fibroblasts/metabolism
  19. Fulltext Role of Hyaluronan in Human Adipogenesis: Evidence from in-Vitro and in-Vivo Studies
    Wilson N, Steadman R, Muller I, Draman M, Rees DA, Taylor P, et al.
    Int J Mol Sci, 2019 May 31;20(11).
    PMID: 31151314 DOI: 10.3390/ijms20112675
    Hyaluronan (HA), an extra-cellular matrix glycosaminoglycan, may play a role in mesenchymal stem cell differentiation to fat but results using murine models and cell lines are conflicting. Our previous data, illustrating decreased HA production during human adipogenesis, suggested an inhibitory role. We have investigated the role of HA in adipogenesis and fat accumulation using human primary subcutaneous preadipocyte/fibroblasts (PFs, n = 12) and subjects of varying body mass index (BMI). The impact of HA on peroxisome proliferator-activated receptor gamma (PPARγ) expression was analysed following siRNA knockdown or HA synthase (HAS)1 and HAS2 overexpression. PFs were cultured in complete or adipogenic medium (ADM) with/without 4-methylumbelliferone (4-MU = HA synthesis inhibitor). Adipogenesis was evaluated using oil red O (ORO), counting adipogenic foci, and measurement of a terminal differentiation marker. Modulating HA production by HAS2 knockdown or overexpression increased (16%, p < 0.04) or decreased (30%, p = 0.01) PPARγ transcripts respectively. The inhibition of HA by 4-MU significantly enhanced ADM-induced adipogenesis with 1.52 ± 0.18- (ORO), 4.09 ± 0.63- (foci) and 2.6 ± 0.21-(marker)-fold increases compared with the controls, also increased PPARγ protein expression (40%, (p < 0.04)). In human subjects, circulating HA correlated negatively with BMI and triglycerides (r = -0.396 (p = 0.002), r = -0.269 (p = 0.038), respectively), confirming an inhibitory role of HA in human adipogenesis. Thus, enhancing HA action may provide a therapeutic target in obesity.
    Matched MeSH terms: Fibroblasts/metabolism
  20. Fulltext Therapeutic challenges and current immunomodulatory strategies in targeting the immunosuppressive pancreatic tumor microenvironment
    Looi CK, Chung FF, Leong CO, Wong SF, Rosli R, Mai CW
    J Exp Clin Cancer Res, 2019 Apr 15;38(1):162.
    PMID: 30987642 DOI: 10.1186/s13046-019-1153-8
    BACKGROUND: Pancreatic cancer is one of the most lethal type of cancers, with an overall five-year survival rate of less than 5%. It is usually diagnosed at an advanced stage with limited therapeutic options. To date, no effective treatment options have demonstrated long-term benefits in advanced pancreatic cancer patients. Compared with other cancers, pancreatic cancer exhibits remarkable resistance to conventional therapy and possesses a highly immunosuppressive tumor microenvironment (TME).

    MAIN BODY: In this review, we summarized the evidence and unique properties of TME in pancreatic cancer that may contribute to its resistance towards immunotherapies as well as strategies to overcome those barriers. We reviewed the current strategies and future perspectives of combination therapies that (1) promote T cell priming through tumor associated antigen presentation; (2) inhibit tumor immunosuppressive environment; and (3) break-down the desmoplastic barrier which improves tumor infiltrating lymphocytes entry into the TME.

    CONCLUSIONS: It is imperative for clinicians and scientists to understand tumor immunology, identify novel biomarkers, and optimize the position of immunotherapy in therapeutic sequence, in order to improve pancreatic cancer clinical trial outcomes. Our collaborative efforts in targeting pancreatic TME will be the mainstay of achieving better clinical prognosis among pancreatic cancer patients. Ultimately, pancreatic cancer will be a treatable medical condition instead of a death sentence for a patient.

    Matched MeSH terms: Cancer-Associated Fibroblasts/metabolism
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