Muhammad Farid Johan

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

BackgroundTumor-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.MethodsResistant cells harboring the FLT3-ITD were developed by overexposure of MV4-11 to CEP-701, and the effects of 5-Aza treatment were investigated. Apoptosis and cytotoxicity of CEP-701 were determined using Annexin V and MTS assays, respectively. Gene expression was performed by quantitative real-time PCR. STATs activity was examined by western blotting and the methylation profile of SHP-1 was studied using MS-PCR and pyrosequencing analysis. Repeated-measures ANOVA and Kruskal–Wallis tests were used for statistical analysis.ResultsThe cytotoxic dose of CEP-701 on resistant cells was significantly higher in comparison with parental and MV4-11R-cep + 5-Aza cells (p = 0.004). The resistant cells showed a significant higher viability and lower apoptosis compared with other cells (p < 0.001). Expression of SHP-1 was 7-fold higher in MV4-11R-cep + 5-Aza cells compared to parental and resistant cells (p = 0.011). STAT3 was activated in resistant cells. Methylation of SHP-1 was significantly decreased in MV4-11R-cep + 5-Aza cells (p = 0.002).ConclusionsThe restoration of SHP-1 expression induces sensitivity towards CEP-701 and could serve as a target in the treatment of AML. Our findings support the hypothesis that, the tumor-suppressor effect of SHP-1 is lost due to epigenetic silencing and its re-expression might play an important role in re-inducing sensitivity to TKIs. Thus, SHP-1 is a plausible candidate for a role in the development of CEP-701 resistance in FLT3-ITD+ AML patients.

Enhanced induction of cell cycle arrest and apoptosis via the mitochondrial membrane potential disruption in human U87 malignant glioma cells by aloe emodin

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.

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

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.

The first Malay database toward the ethnic-specific target molecular variation

BackgroundThe 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 9th 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).FindingsCurrently, MyHVPDb provides only information about the genetic variations and mutations found in the Malays. In the near future, it will expand for the other Malaysian ethnics as well. The data sets are specified based on diseases or genetic mutation types which have three main subcategories: Single Nucleotide Polymorphism (SNP), Copy Number Variation (CNV) followed by the mutations which code for the common diseases among Malaysians. MyHVPDb has been open to the local researchers, academicians and students through the registration at the portal of MyHVP (http://hvpmalaysia.kk.usm.my/mhgvc/index.php?id=register).ConclusionsThis database would be useful for clinicians and researchers who are interested in doing a study on genomics population and genetic diseases in order to obtain up-to-date and accurate information regarding the population-specific variations and also useful for those in countries with similar ethnic background.

Apoptosis Induction in MV4-11 and K562 Human Leukemic Cells by Pereskia sacharosa (Cactaceae) Leaf Crude Extract

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.

Anti-Proliferative Effects of Dendrophthoe pentandra Methanol Extract on BCR/ABL-Positive and Imatinib-Resistant Leukemia Cell Lines

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.

Restoration of PRG2 Expression by 5-Azacytidine Involves in Sensitivity of PKC-412 (Midostaurin) Resistant FLT3-ITD Positive Acute Myeloid Leukaemia Cells

Background: Tumor-suppressor genes Tumour suppressor genes (TSG) are inactivated by methylation in several cancers including acute myeloid leukemia (AML). Transcriptional silencing of PRG2 gene could be involved in development and progression of cancers. 5-Azacytidine (5-Aza) is a DNA methyltransferase inhibitor that causes DNA de-methylation resulting in re-expression of silenced TSG. Midostaurin (PKC412) is a multitargeted tyrosine kinase inhibitor that potently inhibits FLT3 tyrosine kinase and induces hematological remission in patients with AML. However, majority of AML patients in clinical trials developed resistance to PKC412. Methods: Resistant cells were developed by overexposure of MV4-11 cells to PKC-412 and treated with 5-Aza. Apoptosis and cytotoxicity of PKC-412 were determined using Annexin V and MTS assays, respectively. Gene expression profiling was performed using microarray followed by quantitative real-time PCR. STATs activity was examined using Western bloting and methylation of PRG2 was studied using pyrosequencing analysis. Results: The cytotoxic dose of PKC-412 on resistant cells was significantly higher than parental and MV4-11Rpkc+5-Aza cells (p=0.003). The resistant cells showed significant higher viability and lower apoptotic cells compared to other cells (p<0.001). Expression of PRG2 was more than 300 folds higher in MV4-11R-pkc+5-Aza cells compared to parental and resistant cells (p=0.001). STAT3 was activated in resistant cells. Methylation of PRG2 gene was significantly decreased in MV4-11R-pkc+5-Aza cells. Conclusion: The restoration of PRG2 expression induces sensitivity towards PKC-412 and an increased in cell death. Our findings suggest that PRG2 gene could play a role in the sensitivity to PKC-412 and serve as a target for treatment of AML patients resistant to PKC-412 tyrosine kinase inhibitor.

Re-Expression of Bone Marrow Proteoglycan-2 by 5-Azacytidine is associated with STAT3 Inactivation and Sensitivity Response to Imatinib in Resistant CML Cells

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.

Characterisation and Clinical Significance of FLT3-ITD and non-ITD in Acute Myeloid Leukaemia Patients in Kelantan, Northeast Peninsular Malaysia

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.

Conformational Sensitive Gel Electrophoresis (CSGE) as a method for NPM1 mutational screening in patients with Acute Myeloid Leukaemia

Abstract Introduction Nucleophosmin gene ( NPM1 ) exon 12 is the most common mutated gene found in Acute Myeloid Leukaemia (AML), which is present in 25–35%. NPM1 gene encodes for nucleophosmin (NPM) protein, which contains 294 amino acids. The NPM protein is an abundant nucleolar phosphoprotein that shuttles between the nucleus and cytoplasm. It contributes to promotion of ribosome biogenesis, regulation of centrosomal duplication during cell division, interaction with tumor suppressor gene and control of various nuclear proteins. Objective To screen for NPM1 exon 12 mutationsin AML patients diagnosed at Hospital Universiti Sains Malaysia (HUSM) using Conformational Sensitive Gel Electrophoresis (CSGE). Methods Total genomic DNAs were obtained from bone marrow aspirates or peripheral blood samples taken at diagnosis from 67 AML patients diagnosed at HUSM [French-American-British (FAB) subtypes: M0=1, M1=2, M2=10, M3=22, M4=9,M5=12,M6=2,M7=1, Not Otherwise Spesified (NOS)=8]. Patients were screened for NPM1 exon 12 mutations by CSGE. Cases displaying abnormal CSGE profiles were directly sequenced. Results & Discussion Mutation of NPM1 exon 12 was present in 7/67 AML cases (10%) and all cases were females. They consisted of two M1, two M5b, one M2, one M3 and one NOS FAB subtypes. Most of the mutations identified as insertional (ins) mutation (5/7), 4 patients having ins 962–963 CTGG (2), CATG (1) and CTGT (1). One patient having ins at 969 CATG and followed with stop codon (TGA). One patient having single base deletion (del) at 966 and became TGA (stop codon) while another patient having ins at 956 TGGA and followed with del 958–969. Conclusion Our data showed high frequency of NPM1 in AML-M1 and M5b, which is highly associated in female patients. The CSGE was found to be an inexpensive, simple and reliable test to be used in screening of NPM1 mutations.