Bioactive compounds from the medicinal plant, Eurycoma longifolia Jack have been shown to promote anti-proliferative effects on various cancer cell lines. Here we examined the effects of purified eurycomanone, a quassinoid found in Eurycoma longifolia Jack extract, on the expression of selected genes of the A549 lung cancer cells. Eurycomanone inhibited A549 lung cancer cell proliferation in a dose-dependent manner at concentrations ranging from 5 to 20 μg/ml. The concentration that inhibited 50% of cell growth (GI(50)) was 5.1 μg/ml. The anti-proliferative effects were not fully reversible following the removal of eurycomanone, in which 30% of cell inhibition still remained (p<0.0001, T-test). At 8 μg/ml (GI(70)), eurycomanone suppressed anchorage-independent growth of A549 cells by >25% (p<0.05, T-test, n=8) as determined using soft agar colony formation assay. Cisplatin, a chemotherapy drug used for the treatment of non small cell lung cancer on the other hand, inhibited A549 cells proliferation at concentrations ranging from 0.2 μg/ml to 15 μg/ml with a GI(50) of 0.58 μg/ml. The treatment with eurycomanone reduced the abundance expression of the lung cancer markers, heterogeneous nuclear ribonucleoprotein (hnRNP) A2/B1, p53 tumor suppressor protein and other cancer-associated genes including prohibitin (PHB), annexin 1 (ANX1) and endoplasmic reticulum protein 28 (ERp28) but not the house keeping genes. The mRNA expressions of all genes with the exception of PHB were significantly downregulated, 72 h after treatment (p<0.05, T-test, n=9). These findings suggest that eurycomanone at viable therapeutic concentrations of 5-20 μg/ml exhibited significant anti-proliferative and anti-clonogenic cell growth effects on A549 lung cancer cells. The treatment also resulted in suppression of the lung cancer cell tumor markers and several known cancer cell growth-associated genes.
β-Thalassemia/HbE disease has a wide spectrum of clinical phenotypes ranging from asymptomatic to dependent on regular blood transfusions. Ability to predict disease severity is helpful for clinical management and treatment decision making. A thalassemia severity score has been developed from Mediterranean β-thalassemia patients. However, different ethnic groups may have different allele frequency and linkage disequilibrium structures. Here, Thai β0-thalassemia/HbE disease genome-wild association studies (GWAS) data of 487 patients were analyzed by SNP interaction prioritization algorithm, interacting Loci (iLoci), to find predictive SNPs for disease severity. Three SNPs from two SNP interaction pairs associated with disease severity were identifies. The three-SNP disease severity risk score composed of rs766432 in BCL11A, rs9399137 in HBS1L-MYB and rs72872548 in HBE1 showed more than 85% specificity and 75% accuracy. The three-SNP predictive score was then validated in two independent cohorts of Thai and Malaysian β0-thalassemia/HbE patients with comparable specificity and accuracy. The SNP risk score could be used for prediction of clinical severity for Southeast Asia β0-thalassemia/HbE population.
The High-Risk Human Papillomaviruses (HR-HPVs) 16 and 18 are known to cause cervical cancer, which is primarily attributed to E6 and E7 oncoproteins. In addition, recent studies have focused on the vital role of the p130 pocket protein as an oncosuppressor to limit the expression of E2F transcription factors required for cell cycle progression. In view of this, the current study was conducted to investigate the mechanism by which transfection with HPV16/18 E7 leads to the deregulation of the host cell cycle, altering the localisation of p130, and expression of differentiation genes in Human Keratinocytes (HaCaT) cells. Co-immunoprecipitation, Western blot analysis, immunofluorescence microscopy, flow cytometry, quantitative-Polymerase Chain Reaction (qPCR), and the inhibition of p130 by MG132 inhibitor were employed to investigate the loss of p130 and its disruption in HPV 16/18 E7-transfected HaCaT cells. The HPV16- and HPV18-transformed cells, known as CaSki and HeLa, respectively, were also used to complement the ectopic expressions of E7 in HaCaT cells. Normal keratinocytes displayed higher level of p130 expression than HPV-transformed cells. In addition, the immunofluorescence analysis revealed that both HPV 16/18 E7-transfected HaCaT and HPV-transformed cells exhibited higher level of cytoplasmic p130 compared to nuclear p130. A significant increase in the number of S/G2 phase cells in HPV-transformed cells was also recorded since E7 has been shown to stimulate proliferation through the deactivation of Retinoblastoma Protein (pRB)-dependent G1/S checkpoint. Furthermore, the findings recorded the down-regulation of keratinocyte differentiation markers, namely p130, keratin10, and involucrin. The proteasomal degradation of the exported p130 confirmed the cellular localisation pattern of p130, which was commonly observed in cancerous cells. The findings provide strong evidence that the localisation of nuclear p130 nuclear was disrupted by HPV16/18 E7 led to the deregulation of the cell cycle and the impairment of cellular differentiation ultimately lead to cellular transformation.
The presence of multipotent human limbal stromal cells resembling mesenchymal stromal cells (MSC) provides new insights to the characteristic of these cells and its therapeutic potential. However, little is known about the expression of stage-specific embryonic antigen 4 (SSEA-4) and the embryonic stem cell (ESC)-like properties of these cells. We studied the expression of SSEA-4 surface protein and the various ESC and MSC markers in the ex vivo cultured limbal stromal cells. The phenotypes and multipotent differentiation potential of these cells were also evaluated.
It is well-established that HPV E7 proteins, encoded by human papillomavirus (HPV) genes, frequently associated with cervical cancers bind avidly to the retinoblastoma (RB) family of pocket proteins and disrupt their association with members of the E2F transcription factor family. Our previous study showed that the repressive p130-dimerization partner, RB-like, E2F and multi-vulval class (DREAM) complex was disrupted by HPV16 E7 proteins in order to maintain the viral replication in CaSki cells. However, we would like to address whether the activator B-myb-DREAM complex is critical in regulating the replication and mitosis phase since our previous study showed increased B-myb-DREAM expression in HPV-transformed cell lines when compared to control cells.
We previously identified autoantibodies to the endocytic-associated protein Huntingtin-interacting protein 1-related (HIP1R) in diffuse large B-cell lymphoma (DLBCL) patients. HIP1R regulates internalization of cell surface receptors via endocytosis, a process relevant to many therapeutic strategies including CD20 targeting with rituximab. In this study, we characterized HIP1R expression patterns, investigated a mechanism of transcriptional regulation and its clinical relevance in DLBCL patients treated with immunochemotherapy (rituximab, cyclophosphamide, doxorubicin, vincristine and prednisone, R-CHOP). HIP1R was preferentially expressed in germinal center B-cell-like DLBCL (P<0.0001) and inversely correlated with the activated B-cell-like DLBCL (ABC-DLBCL) associated transcription factor, Forkhead box P1 (FOXP1). HIP1R was confirmed as a direct FOXP1 target gene in ABC-DLBCL by FOXP1-targeted silencing and chromatin immunoprecipitation. Lower HIP1R protein expression (≤ 10% tumoral positivity) significantly correlated with inferior overall survival (OS, P=0.0003) and progression-free survival (PFS, P=0.0148) in R-CHOP-treated DLBCL patients (n=157). Reciprocal expression with ≥ 70% FOXP1 positivity defined FOXP1(hi)/HIP1R(lo) patients with particularly poor outcome (OS, P=0.0001; PFS, P=0.0016). In an independent R-CHOP-treated DLBCL (n=233) microarray data set, patients with transcript expression in lower quartile HIP1R and FOXP1(hi)/HIP1R(lo) subgroups exhibited worse OS, P=0.0044 and P=0.0004, respectively. HIP1R repression by FOXP1 is strongly associated with poor outcome, thus further understanding of FOXP1-HIP1R and/or endocytic signaling pathways might give rise to novel therapeutic options for DLBCL.
FOXP2 shares partially overlapping normal tissue expression and functionality with FOXP1; an established diffuse large B-cell lymphoma (DLBCL) oncogene and marker of poor prognosis. FOXP2 is expressed in the plasma cell malignancy multiple myeloma but has not been studied in DLBCL, where a poor prognosis activated B-cell (ABC)-like subtype display partially blocked plasma cell differentiation. FOXP2 protein expression was detected in ABC-DLBCL cell lines, and in primary DLBCL samples tumoral FOXP2 protein expression was detected in both germinal center B-cell-like (GCB) and non-GCB DLBCL. In biopsies from DLBCL patients treated with immunochemotherapy (R-CHOP), ≥ 20% nuclear tumoral FOXP2-positivity (n = 24/158) correlated with significantly inferior overall survival (OS: P = 0.0017) and progression-free survival (PFS: P = 0.0096). This remained significant in multivariate analysis against either the international prognostic index score or the non-GCB DLBCL phenotype (P < 0.05 for both OS and PFS). Expression of BLIMP1, a marker of plasmacytic differentiation that is commonly inactivated in ABC-DLBCL, did not correlate with patient outcome or FOXP2 expression in this series. Increased frequency of FOXP2 expression significantly correlated with FOXP1-positivity (P = 0.0187), and FOXP1 co-immunoprecipitated FOXP2 from ABC-DLBCL cells indicating that these proteins can co-localize in a multi-protein complex. FOXP2-positive DLBCL had reduced expression of HIP1R (P = 0.0348), which is directly repressed by FOXP1, and exhibited distinct patterns of gene expression. Specifically in ABC-DLBCL these were associated with lower expression of immune response and T-cell receptor signaling pathways. Further studies are warranted to investigate the potential functional cooperativity between FOXP1 and FOXP2 in repressing immune responses during the pathogenesis of high-risk DLBCL.
The Runx1 transcription factor cooperates with or antagonizes other transcription factors and plays essential roles in the differentiation and function of T lymphocytes. Previous works showed that Runx1 is expressed in peripheral CD4(+) T cells which level declines after T cell receptor (TCR) activation, and artificial deletion of Runx1 causes autoimmune lung disease in mice. The present study addresses the mechanisms by which Runx1 contributes to the maintenance of peripheral CD4(+) T cell quiescence. Microarray and quantitative RT-PCR analyses were employed to compare the transcriptome of Runx1 -/- CD4(+) T cells to those of unstimulated and TCR-stimulated Runx1 +/- cells. The results identified genes whose expression was modulated similarly by Runx1 deletion and TCR activation. Among them, genes encoding cytokines, chemokines, and Jak/STAT signaling molecules were substantially induced. In Runx1-deleted T cells, simultaneous increases in Il-17A and Rorγc, a known master gene in TH17 differentiation, were observed. In addition, we observed that the loss of Runx1 reduced the transcription of genes encoding quiescence-associated transcription factors, including Foxp1, Foxo1, and Klf2. Interestingly, we identified consensus Runx1 binding sites at the promoter regions of Foxp1, Foxo1, and Klf2 genes, which can be enriched by chromatin immunoprecipitation assay with an anti-Runx1 antibody. Therefore, we suggest that Runx1 may activate, directly or indirectly, the expression of quiescence-associated molecules and thereby contribute to the maintenance of quiescence in CD4(+) T cells.
We investigated the role of protein arginine methylation (PAM) in estrogen receptor (ER)-positive breast cancer cells through pharmacological intervention. Tamoxifen (TAM) or adenosine dialdehyde (ADOX), independently, triggered cell cycle arrest and down-regulated PAM, as reduced protein arginine methyltransferase1 (PRMT1) mRNA and asymmetric dimethylarginine (ADMA) levels. Synergistic effect of these compounds elicited potent anti-cancer effect. However, reduction in ADMA was not proportionate with the compound-induced down-regulation of PRMT1 mRNA. We hypothesized that the disproportionate effect is due to the influence of the compounds on other methyltransferases, which catalyze the arginine dimethylation reaction and the diversity in the degree of drug-protein interaction among these methyltransferases. In silico analyses revealed that independently, ADOX or TAM, binds with phosphatidylethanolamine-methyltransferase (PEMT) or betaine homocysteine-methyl transferase (BHMT); and that the binding affinity of ADOX with PEMT or BHMT is prominent than TAM. These observations suggest that in breast cancer, synergistic effect of ADOX + TAM elicits impressive protective function by regulating PAM; and plausibly, restoration of normal enzyme activities of methyltransferases catalyzing arginine dimethylation could have clinical benefits.
In the present study, two novel series of compounds incorporating naphthyl and pyridyl linker were synthesized and biological assays revealed 5-((6-(2-(5-(2-chlorophenyl)-3-(4-fluorophenyl)-4,5-dihydro-1H-pyrazol-1-yl)-2-oxoethoxy) naphthalene-2-yl)methylene)thiazolidine-2,4-dione (14b) as the most potent dual inhibitors of vascular endothelial growth factors receptor-2 (VEGFR-2) and histone deacetylase 4 (HDAC4). Compounds 13b, 14b, 17f, and 21f were found to stabilize HDAC4; where, pyridyl linker swords were endowed with higher stabilization effects than naphthyl linker. Also, 13b and 14b showed best inhibitory activity on VEGFR-2 as compared to others. Compound 14b was most potent as evident by in-vitro and in-vivo biological assessments. It displayed anti-angiogenic potential by inhibiting endothelial cell proliferation, migration, tube formation and also suppressed new capillary formation in the growing chick chorioallantoic membranes (CAMs). It showed selectivity and potency towards HDAC4 as compared to other HDAC isoforms. Compound 14b (25 mg/kg, i.p.) also indicated exceptional antitumor efficacy on in-vivo animal xenograft model of human colorectal adenocarcinoma (HT-29). The mechanism of action of 14b was also confirmed by western blot.