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  1. Enhanced induction of cell cycle arrest and apoptosis via the mitochondrial membrane potential disruption in human U87 malignant glioma cells by aloe emodin
    Ismail S, Haris K, Abdul Ghani AR, Abdullah JM, Johan MF, Mohamed Yusoff AA
    J Asian Nat Prod Res, 2013 Sep;15(9):1003-12.
    PMID: 23869465 DOI: 10.1080/10286020.2013.818982
    Aloe emodin, one of the active compounds found in Aloe vera leaves, plays an important role in the regulation of cell growth and death. It has been reported to promote the anti-cancer effects in various cancer cells by inducing apoptosis. However, the mechanism of inducing apoptosis by this agent is poorly understood in glioma cells. This research is to investigate the apoptosis and cell cycle arrest inducing by aloe emodin on U87 human malignant glioma cells. Aloe emodin showed a time- and dose-dependent inhibition of U87 cells proliferation and decreased the percentage of viable U87 cells via the induction of apoptosis. Characteristic morphological changes, such as the formation of apoptotic bodies, were observed with confocal microscope by Annexin V-FITC/PI staining, supporting our viability study and flow cytometry analysis results. Our data also demonstrated that aloe emodin arrested the cell cycle in the S phase and promoted the loss of mitochondrial membrane potential in U87 cells that indicated the early event of the mitochondria-induced apoptotic pathway.
  2. Evaluation of serological transfusion-transmitted viral diseases and mutliplex nucleic acid testing in malaysian blood donors
    Yaseen SG, Ahmed SA, Johan MF, Kiron R, Daher AM
    Transfus Apher Sci, 2013 Dec;49(3):647-51.
    PMID: 23890575 DOI: 10.1016/j.transci.2013.07.003
    Transmission of infectious diseases is a recognized complication of blood transfusion and blood products. Nucleic acid testing (NAT) may contribute to improved efficiency of blood screening and thereby increase the safety margin for transfused blood.
  3. Silencing of suppressor of cytokine signaling-3 due to methylation results in phosphorylation of STAT3 in imatinib resistant BCR-ABL positive chronic myeloid leukemia cells
    Al-Jamal HA, Jusoh SA, Yong AC, Asan JM, Hassan R, Johan MF
    Asian Pac J Cancer Prev, 2014;15(11):4555-61.
    PMID: 24969884
    BACKGROUND: Silencing due to methylation of suppressor of cytokine signaling-3 (SOCS-3), a negative regulator gene for the JAK/STAT signaling pathway has been reported to play important roles in leukemogenesis. Imatinib mesylate is a tyrosine kinase inhibitor that specifically targets the BCR-ABL protein and induces hematological remission in patients with chronic myeloid leukemia (CML). Unfortunately, the majority of CML patients treated with imatinib develop resistance under prolonged therapy. We here investigated the methylation profile of SOCS-3 gene and its downstream effects in a BCR-ABL positive CML cells resistant to imatinib.

    MATERIALS AND METHODS: BCR-ABL positive CML cells resistant to imatinib (K562-R) were developed by overexposure of K562 cell lines to the drug. Cytotoxicity was determined by MTS assays and IC50 values calculated. Apoptosis assays were performed using annexin V-FITC binding assays and analyzed by flow cytometry. Methylation profiles were investigated using methylation specific PCR and sequencing analysis of SOCS-1 and SOCS-3 genes. Gene expression was assessed by quantitative real-time PCR, and protein expression and phosphorylation of STAT1, 2 and 3 were examined by Western blotting.

    RESULTS: The IC50 for imatinib on K562 was 362 nM compared to 3,952 nM for K562-R (p=0.001). Percentage of apoptotic cells in K562 increased upto 50% by increasing the concentration of imatinib, in contrast to only 20% in K562-R (p<0.001). A change from non-methylation of the SOCS-3 gene in K562 to complete methylation in K562-R was observed. Gene expression revealed down- regulation of both SOCS-1 and SOCS-3 genes in resistant cells. STAT3 was phosphorylated in K562-R but not K562.

    CONCLUSIONS: Development of cells resistant to imatinib is feasible by overexposure of the drug to the cells. Activation of STAT3 protein leads to uncontrolled cell proliferation in imatinib resistant BCR-ABL due to DNA methylation of the SOCS-3 gene. Thus SOCS-3 provides a suitable candidate for mechanisms underlying the development of imatinib resistant in CML patients.

  4. Fulltext Multiplex amplification refractory mutation system (MARMS) for the detection of β-globin gene mutations among the transfusion-dependent β-thalassemia Malay patients in Kelantan, Northeast of Peninsular Malaysia
    Hanafi S, Hassan R, Bahar R, Abdullah WZ, Johan MF, Rashid ND, et al.
    Am J Blood Res, 2014;4(1):33-40.
    PMID: 25232503
    The aim of this study was to adapt MARMS with some modifications to detect beta mutation in our cohort of thalassemia patients. We focused only on transfusion-dependent thalassemia Malay patients, the predominant ethnic group (95%) in the Kelantanese population. Eight mutations were identified in 46 out of 48 (95.83%) beta thalassemia alleles. Most of the patients (54.2%) were compound heterozygous with co-inheritance Cd 26 (G>A). The frequencies of spectrum beta chain mutation among these patients are presented in Table 2. Among the transfusion dependent beta thalassemia Malay patients studied, 26 patients were found to be compound heterozygous and the main alleles were Cd 26 (G>A). Compound heterozygous mutation of Cd 26 (G>A) and IVS 1-5 (G>C) were 12 (46.2%), Cd 26 (G>A) and Cd 41/42 (TTCT) were 9 (34.6%), Cd 26 (G>A) and IVS 1-1 (G>C) were 2 (7.7%) respectively. Meanwhile the minority were made of a single compound heterozygous of Cd 26 (G>A) and Cd 71/72, Cd 26 (>A) and Cd 17 (A>T), Cd 26 (G>A) and -28 (G>A) respectively. Twenty out of forty six patients were shown to have homozygous of IVS 1-5 (G>C) were 2 (10.0%), Cd 26 (G>A) were 15 (75.0%), Cd 19 (A>G) were 1 (5.0%), and IVS 1-1 (G>T) were 2 (10.0%). The beta chain mutations among the Kelantanese Malays followed closely the distribution of beta chain mutations among the Thais and the Malays of the Southern Thailand. The G-C transition at position 5 of the IVS 1-5 mutation was predominant among the Malay patients. In conclusion, this method has successfully identified the mutation spectrum in our cohort of transfusion-dependent beta thalassemia patients, and this method is equally effective in screening for mutation among thalassemia patients.
  5. Apoptosis induction in MV4-11 and K562 human leukemic cells by Pereskia sacharosa (Cactaceae) leaf crude extract
    Asmaa MJ, Al-Jamal HA, Ang CY, Asan JM, Seeni A, Johan MF
    Asian Pac J Cancer Prev, 2014;15(1):475-81.
    PMID: 24528077
    BACKGROUND: Pereskia sacharosa is a genus of cacti widely used in folk medicine for cancer-related treatment. Anti-proliferative effects have been studied in recent years against colon, breast, cervical and lung cancer cell lines, with promising results. We here extended study of anti-proliferative effects to a blood malignancy, leukemia.

    MATERIALS AND METHODS: Two leukemic cell lines, MV4-11 (acute myeloid leukemia) and K562 (chronic myeloid leukemia), were studied. IC50 concentrations were determined and apoptosis and cell cycle regulation were studied by flow cytometric analysis. The expression of apoptosis and cell-cycle related regulatory proteins was assessed by Western blotting.

    RESULTS: P sacharosa inhibited growth of MV4-11 and K562 cells in a dose-dependent manner. The mode of cell death was via induction of intrinsic apoptotic pathways and cell cycle arrest. There was profound up-regulation of cytochrome c, caspases, p21 and p53 expression and repression of Akt and Bcl-2 expression in treated cells.

    CONCLUSIONS: These results suggest that P sacharosa induces leukemic cell death via apoptosis induction and changes in cell cycle checkpoint, thus deserves further study for anti-leukemic potential.

  6. Fulltext Guidelines for nucleic acid detection and analysis in hematological disorders
    Hassan R, Husin A, Sulong S, Yusoff S, Johan MF, Yahaya BH, et al.
    Malays J Pathol, 2015 Aug;37(2):165-73.
    PMID: 26277676 MyJurnal
  7. Fulltext Enhancing SHP-1 expression with 5-azacytidine may inhibit STAT3 activation and confer sensitivity in lestaurtinib (CEP-701)-resistant FLT3-ITD positive acute myeloid leukemia
    Al-Jamal HA, Mat Jusoh SA, Hassan R, Johan MF
    BMC Cancer, 2015;15:869.
    PMID: 26547689 DOI: 10.1186/s12885-015-1695-x
    Tumor-suppressor genes are inactivated by methylation in several cancers including acute myeloid leukemia (AML). Src homology-2 (SH2)-containing protein-tyrosine phosphatase 1 (SHP-1) is a negative regulator of the JAK/STAT pathway. Transcriptional silencing of SHP-1 plays a critical role in the development and progression of cancers through STAT3 activation. 5-Azacytidine (5-Aza) is a DNA methyltransferase inhibitor that causes DNA demethylation resulting in re-expression of silenced SHP-1. Lestaurtinib (CEP-701) is a multi-targeted tyrosine kinase inhibitor that potently inhibits FLT3 tyrosine kinase and induces hematological remission in AML patients harboring the internal tandem duplication of the FLT3 gene (FLT3-ITD). However, the majority of patients in clinical trials developed resistance to CEP-701. Therefore, the aim of this study, was to assess the effect of re-expression of SHP-1 on sensitivity to CEP-701 in resistant AML cells.
  8. Characterisation and Clinical Significance of FLT3-ITD and non-ITD in Acute Myeloid Leukaemia Patients in Kelantan, Northeast Peninsular Malaysia
    Yunus NM, Johan MF, Ali Nagi Al-Jamal H, Husin A, Hussein AR, Hassan R
    Asian Pac J Cancer Prev, 2015;16(12):4869-72.
    PMID: 26163606
    BACKGROUND: Mutations of the FMS-like tyrosine kinase-3 (FLT3) receptor gene may promote proliferation via activation of multiple signaling pathways. FLT3-internal tandem duplication (FLT3-ITD) is the most common gene alteration found in patients diagnosed with acute myeloid leukaemia (AML) and has been associated with poor prognosis.

    MATERIALS AND METHODS: We performed mutational analysis of exons 14-15 and 20 of the FLT3 gene in 54 AML patients using PCR-CSGE (conformational sensitive gel electrophoresis) followed by sequencing analysis to characterise FLT3 mutations in adult patients diagnosed with AML at Hospital USM, Kelantan, Northeast Peninsular Malaysia.

    RESULTS: FLT3 exon 14-15 mutations were identified in 7 of 54 patients (13%) whereas no mutation was found in FLT3 exon 20. Six ITDs and one non-ITD mutation were found in exon 14 of the juxtamembrane (JM) domain of FLT3. FLT3-ITD mutations were associated with a significantly higher blast percentage (p-value=0.008) and white blood cell count (p-value=0.023) but there was no significant difference in median overall survival time for FLT3-ITD+/FLT3-ITD- within 2 years (p-value=0.374).

    CONCLUSIONS: The incidence of FLT3-ITD in AML patients in this particular region of Malaysia is low compared to the Western world and has a significant association with WBC and blast percentage.

  9. Fulltext The first Malay database toward the ethnic-specific target molecular variation
    Halim-Fikri H, Etemad A, Abdul Latif AZ, Merican AF, Baig AA, Annuar AA, et al.
    BMC Res Notes, 2015;8:176.
    PMID: 25925844 DOI: 10.1186/s13104-015-1123-y
    The Malaysian Node of the Human Variome Project (MyHVP) is one of the eighteen official Human Variome Project (HVP) country-specific nodes. Since its inception in 9(th) October 2010, MyHVP has attracted the significant number of Malaysian clinicians and researchers to participate and contribute their data to this project. MyHVP also act as the center of coordination for genotypic and phenotypic variation studies of the Malaysian population. A specialized database was developed to store and manage the data based on genetic variations which also associated with health and disease of Malaysian ethnic groups. This ethnic-specific database is called the Malaysian Node of the Human Variome Project database (MyHVPDb).
  10. Anti-Proliferative Effects of Dendrophthoe pentandra Methanol Extract on BCR/ABL-Positive and Imatinib-Resistant Leukemia Cell Lines
    Zamani A, Mat Jusoh SA, Al-Jamal HA, Sul'ain MD, Johan MF
    Asian Pac J Cancer Prev, 2016 11 01;17(11):4857-4861.
    PMID: 28030911
    Background: Imatinib mesylate, a tyrosine kinase inhibitor specifically targeting the BCR/ABL fusion protein, induces hematological remission in patients with chronic myeloid leukemia (CML). However, the majority of CML patients treated with imatinib develop resistance with prolonged therapy. Dendrophthoe pentandra (L.) Miq. is a Malaysian mistletoe species that has been used as a traditional treatment for several ailments such as smallpox, ulcers, and cancers. Methods: We developed a resistant cell line (designated as K562R) by long-term co-culture of a BCR/ ABL positive CML cell line, K562, with imatinib mesylate. We then investigated the anti-proliferative effects of D. pentandra methanol extract on parental K562 and resistant K562R cells. Trypan blue exclusion assays were performed to determine the IC50 concentration; apoptosis and cell cycle analysis were conducted by flow cytometry. Results: D. pentandra extract had greater anti-proliferative effects towards K562R (IC50= 192 μg/mL) compared to K562 (500 μg/ mL) cells. Upon treatment with D. pentandra extract at the IC50. concentration: K562 but not K562R demonstrated increase in apoptosis and cell cycle arrest in the G2/M phase. Conclusion: D. pentandra methanol extract exerts potent anti-proliferative effect on BCR/ABL positive K562 cells.
  11. Fulltext Genotype-phenotype association of Hbe/β thalassemia disease and the role of genetic modifiers
    HanafI S, Abdullah WZ, Adnan RA, Bahar R, Johan MF, Azman NF, et al.
    Malaysian Journal of Paediatrics and Child Health, 2016;22(1):1-16.
    MyJurnal
    HbE/β-thalassemia is the most common severe form of thalassemia particularly in SEA region including Malaysia and globally, it comprised of a significant severe form of β-thalassemia disorder. It has various clinical manifestations ranging from very mild anemia to severe manifestation similar to beta thalassemia major. Many different syndromes are observed in HbE/β-thalassemia. Several genetic modifiers have been reported to play important role in contributing to phenotypic variability. The true reasons underlying this phenotypic variability remain unknown. The most reliable predictive factor of the disease phenotype is the nature of the beta globin gene mutation itself. However, the degree of severity is also believed to be affected by other genetic modifiers. For instance, high HbF level ameliorates the clinical severity of β thalassemia patients. Therefore, identification of these genetic modifiers is very important. The association of severe clinical manifestation and the specific β-globin gene mutation has been known. But the wide scope and other potential predictors have been only recently appreciated. This review therefore aimed to reveal the potential genetic modifiers of HbE/βthalassemia patients based on the previous reported studies. A better understanding on the mechanisms underlying the variety of phenotypes of this disease may lead to the direction for a better future management plans. This also promotes “personalized medicine” in patient care.
  12. Re-Expression of Bone Marrow Proteoglycan-2 by 5-Azacytidine is associated with STAT3 Inactivation and Sensitivity Response to Imatinib in Resistant CML Cells
    Al-Jamal HAN, Johan MF, Mat Jusoh SA, Ismail I, Wan Taib WR
    Asian Pac J Cancer Prev, 2018 Jun 25;19(6):1585-1590.
    PMID: 29936783
    Background: Epigenetic silencing of tumor suppressor genes (TSG) is involved in development and
    progression of cancers. Re-expression of TSG is inversely proportionate with STAT3 signaling pathways.
    Demethylation of DNA by 5-Azacytidine (5-Aza) results in re-expression of silenced TSG. Forced expression of
    PRG2 by 5-Aza induced apoptosis in cancer cells. Imatinib is a tyrosine kinase inhibitor that potently inhibits BCR/
    ABL tyrosine kinase resulting in hematological remission in CML patients. However, majority of CML patients treated
    with imatinib would develop resistance under prolonged therapy. Methods: CML cells resistant to imatinib were
    treated with 5-Aza and cytotoxicity of imatinib and apoptosis were determined by MTS and annexin-V, respectively.
    Gene expression analysis was detected by real time-PCR, STATs activity examined using Western blot and methylation
    status of PRG2 was determined by pyrosequencing analysis. Result: Expression of PRG2 was significantly higher in
    K562-R+5-Aza cells compared to K562 and K562-R (p=0.001). Methylation of PRG2 gene was significantly decreased
    in K562-R+5-Aza cells compared to other cells (p=0.021). STAT3 was inactivated in K562-R+5-Aza cells which showed
    higher sensitivity to imatinib. Conclusion: PRG2 gene is a TSG and its overexpression might induce sensitivity to
    imatinib. However, further studies are required to evaluate the negative regulations of PRG2 on STAT3 signaling.
  13. Fulltext Apoptotic induction in cCCRF-CEM and HL human leukemic cell lines by 5-azacitidine and trichostatin A
    Aziee, S., Haiyuni, MY, Shafini, MY, Johan, MF, Al-Jamal, HAN, Abdul Wahab, R., et al.
    Journal of Biomedical and Clinical Sciences, 2018;3(1):54-61.
    MyJurnal
    The aims of the study were to investigate the anti-cancer effects of 5-
    Aza and TSA in two leukemic cell lines (CCRF-CEM and HL-60). Inhibition
    concentration of 5-Aza and TSA were measured using trypan blue exclusion
    assay. 5-Aza and TSA at IC50 were treated to both CCRF-CEM and HL-60 cell
    lines for 4-6 days. To confirm the inhibition effects of these agents, Annexin-V
    stained cells were analyzed using flow cytometry to evaluate the apoptotic
    induction. The IC50 values of CCRF-CEM were 2.01±0.1µM and 2.65±0.3µM for
    5-Aza- and TSA-treated, respectively. Whereas, the IC50 values of HL-60 were
    1.98±0.2µM and 2.35±0.2µM for 5-Aza- and TSA-treated, respectively. To
    further substantiate the findings, the time-dependent exposure of both drugs was
    studied. CCRF-CEM cells were reduced to 49.4%±5.0, 49.4%±2.5 and
    41.5%±5.6 by 5-Aza; 56.5%±7.0, 45.3%±4.2 and 40.2%±4.2 by TSA treatment
    at first, third and sixth day. HL-60 cells were reduced to 72.0%±4.5, 51.0%±1.5
    and 40.6%±2.6 by 5-Aza at first, third and sixth day. Meanwhile, HL-60 cells
    reduced to 55.6%±4.5, 45.2%±4.0 and 36.3%±2.9 by TSA at first, second and
    fourth day. Both cell lines were significantly inhibited (p
  14. Anti-Proliferative Effects of Methanol and Water Extracts of Pyrrosia piloselloides on the Hela Human Cervical Carcinoma Cell Line
    Sul ‘ain MD, Zakaria F, Johan MF
    Asian Pac J Cancer Prev, 2019 Jan 25;20(1):185-192.
    PMID: 30678430
    Background: Cervical cancer is one of the most commonly diagnosed neoplasms and a leading cause of cancer
    death among females worldwide. Limitations with conventional medical treatments have driven researchers to
    search for alternative approaches using natural products. This study aimed to detemine potential anti-proliferative
    effects of methanol and water extracts of Pyrrosia piloselloides (P. piloselloides) on the HeLa cell line. Methods:
    3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assays were performed to determine IC50
    concentrations and apoptosis analysis was by flow cytometry. To identify chemical compounds in the extracts, gas
    chromatography-mass spectrometry (GC-MS) was employed. Results: P. piloselloides methanol extracts (PPME) showed
    antiproliferative effects on HeL awith an IC50 of 16.25μg/mL while the P. piloselloides water extract (PPWE) was without
    influence. Neither extract showed any significant effects on apoptosis. GC-MS analysis, revealed 5-hydroxymethylfurfural
    (23.1%), allopurinol (8.66%) and 3, 5-dihydroxy-6-methyl-2,3-dihydropyran-4-one (7.41%) as major components in
    the PPME, while sulfolan-3-ol (10.1%), linoleic acid (9.06%) and β-sitosterol acetate (7.98%) predominated in the
    PPWE case. Conclusion: This first study of P. piloselloides showed PPME to exert potent anti-proliferative effect on
    HeLa cell lines. Further research now needs to be performed to establish the mechanisms of inhibition.
  15. Fulltext Anticancer activity of grassy Hystrix brachyura bezoar and its mechanisms of action: An in vitro and in vivo based study
    Khan AYF, Ahmed QU, Narayanamurthy V, Razali S, Asuhaimi FA, Saleh MSM, et al.
    Biomed Pharmacother, 2019 Jun;114:108841.
    PMID: 30981106 DOI: 10.1016/j.biopha.2019.108841
    Porcupine bezoar (PB) is a calcified undigested material generally found in porcupine's (Hystrix brachyura) gastrointestinal tract. The bezoar is traditionally used in South East Asia and Europe for the treatment of cancer, poisoning, dengue, typhoid, etc. However, limited scientific studies have been performed to verify its anticancer potential to substantiate its traditional claims in the treatment of cancers. Hence, this study was aimed at investigating the in vitro and in vivo anticancer properties of two grassy PB aqueous extract (PB-A and PB-B) using A375 cancer cell line and zebrafish model, respectively. This paper presents the first report on in vitro A375 cell viability assay, apoptosis assay, cell cycle arrest assay, migration assay, invasion assay, qPCR experimental assay and in vivo anti-angiogenesis assay using the grassy PBs. Experimental findings revealed IC50 value are 26.59 ± 1.37 μg/mL and 30.12 ± 3.25 μg/mL for PB-A and PB-B respectively. PBs showed anti-proliferative activity with no significant cytotoxic effect on normal human dermal fibroblast (NHDF). PBs were also found to induce apoptosis via intrinsic pathway and arrest cell cycle at G2/M phase. Additionally, the findings indicated its ability to debilitate migration and invasion of A375 cells. Further evaluation using embryo zebrafish model revealed LC50 = 450.0 ± 2.50 μg/mL and 58.7 ± 5.0 μg/mL for PB-A and PB-B which also exerted anti-angiogenesis effect in zebrafish. Moreover, stearic acid, ursodeoxycholic acid and pregnenolone were identified as possible metabolites that might contribute to the anticancer effect of the both PBs. Overall, this study demonstrated that PB-A and PB-B possess potential in vitro and in vivo anticancer effects which are elicited through selective cytotoxic effect, induction of apoptosis, inhibition of migration and invasion and anti-angiogenesis. This study provides scientific evidence that the porcupine bezoar do possess anti-cancer efficacy and further justifies its traditional utility. However, more experiments with higher vertebrae models are still warranted to validate its traditional claims as an anticancer agent.
  16. Identification and Quantification of Quercetin, A Major Constituent of Artocarpus altilis by Targeting Related Genes of Apoptosis and Cell Cycle: In Vitro Cytotoxic Activity Against Human Lung Carcinoma Cell Lines
    Jalal TK, Khan AYF, Natto HA, Abdull Rasad MSB, Arifin Kaderi M, Mohammad M, et al.
    Nutr Cancer, 2019;71(5):792-805.
    PMID: 30614285 DOI: 10.1080/01635581.2018.1516790
    Nine phenolic compounds were identified and quantified in Artocarpus altilia fruit. One of the main compounds was quercetin, which is the major class of flavonoids has been identified and quantified in pulp part of A. altilis fruit of methanol extract. The aim of this study was to evaluate in vitro cytotoxic assay. Inhibitory concentration 50% concentration was determined using trypan blue exclusion assay. Apoptosis induction and cell cycle regulation were studied by flow cytometric analysis. The expression of apoptosis and cell cycle-related regulatory genes were assessed by RT-qPCR study of the methanol extract of pulp part on human lung carcinoma (A549) cell line. A significant increase of cells at G2/M phases was detected (P 
  17. Transcriptomic Profiles of MV4-11 and Kasumi 1 Acute Myeloid Leukemia Cell Lines Modulated by Epigenetic Modifiers Trichostatin A and 5-Azacytidine
    Asmaa MJS, Al-Jamal HA, Hussein AR, Yahaya BH, Hassan R, Hussain FA, et al.
    Int J Hematol Oncol Stem Cell Res, 2020 Jan 01;14(1):72-92.
    PMID: 32337016
    Background: Acute myeloid leukemia (AML) is the most common form of acute leukemias in adults which is clinically and molecularly heterogeneous. Several risk and genetic factors have been widely investigated to characterize AML. However, the concomitant epigenetic factors in controlling the gene expression lead to AML transformation was not fully understood. This study was aimed to identify epigenetically regulated genes in AML cell lines induced by epigenetic modulating agents, Trichostatin A (TSA) and 5-Azacytidine (5-Aza). Materials and Methods: MV4-11 and Kasumi 1 were treated with TSA and/or 5-Aza at IC50 concentration. Gene expression profiling by microarray was utilized using SurePrint G3 Human Gene Expression v3. Gene ontology and KEGG pathway annotations were analyzed by DAVID bioinformatics software using EASE enrichment score. mRNA expression of the differentially expressed genes were verified by quantitative real time PCR. Results: Gene expression analysis revealed a significant changes in the expression of 24,822, 15,720, 15,654 genes in MV4-11 and 12,598, 8828, 18,026 genes in Kasumi 1, in response to TSA, 5-Aza and combination treatments, respectively, compared to non-treated (p<0.05). 7 genes (SOCS3, TUBA1C, CCNA1, MAP3K6, PTPRC, STAT6 and RUNX1) and 4 genes (ANGPTL4, TUBB2A, ADAM12 and PTPN6) shown to be predominantly expressed in MV4-11 and Kasumi 1, respectively (EASE<0.1). The analysis also revealed phagosome pathway commonly activated in both cell lines. Conclusion: Our data showed a distinct optimal biological characteristic and pathway in different types of leukemic cell lines. These finding may help in the identification of cell-specific epigenetic biomarker in the pathogenesis of AML.
  18. Genetic polymorphisms of HbE/beta thalassemia related to clinical presentation: implications for clinical diversity
    Azman NF, Abdullah WZ, Hanafi S, Diana R, Bahar R, Johan MF, et al.
    Ann Hematol, 2020 Apr;99(4):729-735.
    PMID: 32078010 DOI: 10.1007/s00277-020-03927-5
    HbE/Beta thalassemia (HbE/β-thalassemia) is one of the common genetic disorders in South East Asia. It is heterogeneous in its clinical presentation and molecular defects. There are genetic modifiers which have been reported to influence the disease severity of this disorder. The aim of this study was to determine the genetic polymorphisms which were responsible for the disease clinical diversity. A case-control study was conducted among Malay transfusion-dependent HbE/β-thalassemia patients. Patients who were confirmed HbE/β-thalassemia were recruited and genotyping study was performed on these subjects. Ninety-eight patients were selected and divided into moderate and severe groups based on clinical parameters using Sripichai scoring system (based on hemoglobin level, spleen size, growth development, the age of first transfusion and age of disease presentation). Forty-three (44.9%) and 55 (56.1%) patients were found to have moderate and severe clinical presentation, respectively. Genotyping analysis was performed using Affymetrix 6.0 microarray platform. The SNPs were filtered using PLINK and Manhattan plot by R software. From the GWAS results, 20 most significant SNPs were selected based on disease severity when compared between moderate and severe groups. The significant SNPs found in this study were mostly related to thalassemia complications such as rs7372408, associated with KCNMB2-AS1 and SNPs associated with disease severity. These findings could be used as genetic predictors in managing patients with HbE/β-thalassemia and served as platform for future study.
  19. Fulltext Aberrant Methylation of Tumour Suppressor Gene ADAM12 in Chronic Lympocytic Leukemia Patients: Application of Methylation Specific-PCR Technique
    Mohamad A, Hassan R, Husin A, Johan MF, Sulong S
    Asian Pac J Cancer Prev, 2021 Jan 01;22(1):85-91.
    PMID: 33507683 DOI: 10.31557/APJCP.2021.22.1.85
    OBJECTIVE: Chronic Lymphocytic Leukemia (CLL) is a common leukemia among Caucasians but rare in Asians population. We postulated that aberrant methylation either hypermethylation or partial methylation might be one of the silencing mechanisms that inactivates the tumour suppressor genes in CLL. This study aimed to compare the methylation status of tumour suppressor gene, ADAM12, among CLL patients and normal individuals. We also evaluated the association between methylation of ADAM12 and clinical and demographic characteristics of the participants.

    METHODS: A total of 25 CLL patients and 25 normal individuals were recruited in this study. The methylation status of ADAM12 was determined using Methylation-Specific PCR (MSP); whereas, DNA sequencing method was applied for validation of the MSP results.

    RESULTS: Among CLL patients, 12 (48%) were partially methylated and 13 (52%) were unmethylated. Meanwhile, 5 (20%) and 20 (80.6%) of healthy individuals were partially methylated and unmethylated, respectively. There was a statistically significant association between the status of methylation at ADAM12 and the presence of CLL (p=0.037).

    CONCLUSION: The aberrant methylation of ADAM12 found in this study using MSP assay may provide new exposure to CLL that may improve the gaps involved in genetic epigenetic study in CLL.

  20. Fulltext Thymoquinone Suppresses Cell Proliferation and Enhances Apoptosis of HL60 Leukemia Cells through Re-Expression of JAK/STAT Negative Regulators
    Almajali B, Al-Jamal HAN, Wan Taib WR, Ismail I, Johan MF, Doolaanea AA, et al.
    Asian Pac J Cancer Prev, 2021 Mar 01;22(3):879-885.
    PMID: 33773553 DOI: 10.31557/APJCP.2021.22.3.879
    OBJECTIVE: The natural compound, thymoquinone (TQ) has demonstrated potential anticancer properties in inhibiting cell proliferation and promoting apoptosis in myeloid leukemia cells, breast cancer cells, and others. However, the effect mechanism of TQ on AML cells still not fully understood. In this study, the authors examined the effects of TQ on the expression of JAK/STAT-negative regulator genes SOCS-1, SOCS-3, and SHP-1, and their consequences on cell proliferation and apoptosis in HL60 leukemia cells.

    METHODS: MTT and trypan blue exclusion tests were conducted to determine the 50% inhibitory concentration (IC50) and cell proliferation. FITC Annexin and Guava® reagent were used to study the cell apoptosis and examine the cell cycle phases, respectively. The expression of JAK/STAT-negative regulator genes, SOCS-1, SOCS-3, and SHP-1, was investigated using reverse transcriptase- quantitative PCR (RT-qPCR).

    RESULTS: TQ demonstrated a potential inhibition of HL60 cell proliferation and a significant increase in apoptotic cells in dose and time-dependent manner. TQ significantly induced cycle arrest at G0-G1 phase (P < 0.001) and enhanced the re-expression of JAK/STAT-negative regulator genes.

    CONCLUSION: TQ potentially inhibited HL60 cell proliferation and significantly increased apoptosis with re-expression of JAK/STAT-negative regulator genes suggesting that TQ could be a new therapeutic candidate for leukemia therapy.
    .

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