Said I. Ismail

Thymoquinone in liposomes: a study of loading efficiency and biological activity towards breast cancer

Thymoquinone (2-isopropyl-5-methyl-1,4-benzoquinone) is a herbal-derived drug with potential chemopreventive and chemotherapeutic activity. However, thymoquinone suffers from high hydrophobicity causing poor solubility which limits its bioavailability and high lipophilicity causing poor formulation characteristics. Liposomes are versatile drug carriers that can be used to solve problems of drug solubility, instability, and bio-distribution. In this study, we were able to prepare thymoquinone-loaded liposomes (TQ-LP) and thymoquinone loaded in liposomes modified with Triton X-100 (XLP) with diameters of about 100 nm, and entrapment efficiency of more than 90% for TQ-LP and of 49.6% for XLP. The TQ-LP liposomes were effective in suppressing the proliferation of breast cancer cell lines MCF-7 and T47D, and at the same time exerting very low toxicity on normal periodontal ligament fibroblast. Altogether, this report describes the first successful encapsulation of thymoquinone into liposome; which maintains stability, improves bioavailability and maintains its anticancer activity.

Hotspot mutations of PIK3CA and AKT1 genes are absent in multiple myeloma

The phosphatidylinositol-3-kinases PI3K/AKT pathway regulates many growth and survival mechanisms in the cell, and it has been implicated in development and progression of many human cancers, including multiple myeloma. Recently, many reports have revealed that the PIK3CA gene which encodes the p110 catalytic subunit of PI3K kinase is mutated in many human malignancies. To investigate the oncogenic role of PI3K/AKT pathway in multiple myeloma we sequenced three hot exons: exons 9 and 20 of PIK3CA gene and exon 3 of AKT1 gene in 27 multiple myeloma patients. Our results indicate the absence of the four hot spot mutations E542K, E545K, H1047R and E17K in all studied cases. These findings suggest that PI3K/AKT mutations may not play a major role in multiple myeloma.

UDP-glucuronosyltransferase 1A4 (UGT1A4) polymorphisms in a Jordanian population

Glucuronidation is one of the most important phase II metabolic pathways. It is catalyzed by a family of UDP-glucuronosyltransferase enzymes (UGTs). One of the subfamilies is UGT1A. Allele frequencies in UGT1A4 differ among ethnic groups. The aim of this study was to determine the allelic frequency of two most common defective alleles: UGT1A4*2 and UGT1A4*3 in a Jordanian population. A total of 216 healthy Jordanian Volunteers (165 males and 51 females) were included in this study. Genotyping for UGT1A4*1, UGT1A4*2 and UGT1A4*3 was done using a well established polymerase chain reaction–restriction fragment length polymorphism test. Among 216 random individuals studied for UGT1A4*2 mutation there were 26 individuals who were heterozygous, giving a prevalence of 12% and an allele frequency of 6.5%. Only one individual was homozygous for UGT1A4*2. The UGT1A4*3 mutation was detected as heterozygous in 9 of 216 individuals indicating a prevalence of 4.2% and allele frequency of 3.5%. Three individuals were homozygous for the UGT1A4*3 indicating a prevalence of 1.4%. The prevalence of UGT1A4*2 is similar to the Caucasians but different from other populations whilst the UGT1A4*3 prevalence in the Jordanian population is distinct from other populations. Our results provide useful information for the Jordanian population and for future genotyping of Arab populations in general.

Methylenetetrahydrofolate reductase genotype association with the risk of follicular lymphoma

The metabolism of folate is essential in DNA synthesis, and polymorphisms of genes involved in such metabolism have been implicated in many types of cancer. Among these, the methylene tetrahydrofolate reductase gene (MTHFR) encodes an enzyme that converts folate to a methyl donor used for DNA methylation. We studied the association between the different genotypes of the two most common MTHFR polymorphisms, C677T and A1298C, and the risk of follicular lymphoma (FL). For this purpose, 55 previously diagnosed FL patients and 170 normal control subjects were examined using polymerase chain reaction followed by restriction fragment length polymorphism. The frequency of the A1298C CC homozygous mutant genotype was significantly higher in patients with FL than in control subjects (OR = 3.51, 95% CI = 1.39-8.86, P = 0.008). No such association was found for the heterozygous A1298C AC genotype (OR = 1.08, 95% CI = 0.55-2.12, P = 0.83). On the other hand, no significant association was found for either the C677T CT heterozygous genotype (OR = 0.79, 95% CI = 0.42-1.51, P = 0.49) or the C677T TT homozygous mutant genotype (OR = 0.55, 95% CI = 0.12-2.65, P = 0.46). The present findings add to the very few reports suggesting a link between the A1298C CC homozygous MTHFR genotype and a higher risk of developing FL, and the first such in a Jordanian population.

The transforming mutation E17K/AKT1 is not a major event in B-cell-derived lymphoid leukaemias

Despite the major role of the AKT/PKB family of proteins in the regulation of many growth and survival mechanisms in the cell, and the increasing evidence suggesting that AKT disruption could play a key role in many human malignancies, no major mutations of AKT genes had been reported, until very recently when Carpten et al reported a novel transforming mutation (E17K) in the pleckstrin homology domain of the AKT1 gene in solid tumours. Several laboratories are now screening for this mutation in different malignancies, and, recently, the mutation was described by Malanga et al in 1.9% of lung cancer patients. Considering the importance of the PI3K/AKT pathway in mediating survival and antiapoptotic signals in the B-cell types of chronic lymphocytic leukaemia (CLL) and acute lymphoblastic leukaemia (ALL), we sequenced the AKT1 exon 3 for the above mentioned mutation in 87 specimens, representing 45 CLLs, 38 ALLs and 4 prolymphocytic leukaemia (PLL) cases, which are all of B-cell origin. Our results show that the mutation E17K/AKT1 was not detected in the pleckstrin homology domain of AKT1 of the investigated cases. We conclude that this mutation is not a major event in B-cell-derived lymphoid leukaemias.

Incidence of bcr‑abl fusion transcripts in healthy individuals.

Bcr‑abl fusion transcripts, resulting from translocation t(9;22), are hallmarks of Philadelphia chromosome positive (Ph+) leukemias. This translocation is detected in >90% of patients with chronic myelogenous leukemia and ~20% of acute lymphoblastic leukemia patients, which predominantly express the p210 and p190 proteins, respectively. Although the occurrence of t(9;22) in healthy individuals has been previously demonstrated, the number of studies is limited and the results are inconsistent. The present study screened for the presence of bcr‑abl transcripts in the blood of a group of healthy individuals using a sensitive‑nested reverse transcription polymerase chain reaction (RT‑PCR) assay. Samples were collected from 189 healthy volunteers (145 adults and 44 children). RNA was reverse transcribed and amplified by two rounds of PCR, amplifying the two common variants of bcr‑abl transcripts, p190 and p210. While the bcr‑abl p190 transcript was not detected, the p210 transcript was detected in ~10% of samples. Notably, the incidence of p210 translocation was higher in males (12.2%) compared with females (7.7%) and males were 2.4 times more likely to have the translocation. A significant incidence was also observed in adults compared with children, where adults were 6 times more likely to have the translocation. The presence of bcr‑abl transcripts in the blood of a significant proportion of healthy individuals should be considered in long‑term investigations to establish its exact association with the risk of developing leukemia. Furthermore, the current assays should be revised to consider the proportion of normal samples carrying the p210 transcripts when making a differential diagnosis.

Synthesis and biological activity assays of some new N1-(flavon-7-yl)amidrazone derivatives and related congeners.

A series of new N1-(flavon-7-yl)amidrazones incorporating N-piperazines and related congeners were synthesized by reacting the hydrazonoyl chloride derived from 7-aminoflavone and 7-amino-2-methylchromen-4-one with the appropriate piperazine. The chemical structures of the newly prepared compounds were confirmed by elemental analyses, (1)H NMR, (13)C NMR, and ESI-HRMS spectral data. The antitumor activity of these compounds was evaluated on breast cancer (MCF-7 and T47D) and Leukemic (K562) cell lines by a cell viability assay utilizing the tetrazolium dye 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT). Although with varying degrees, a significant growth inhibitory and cytotoxic effect was observed on all three cancer cell lines. Among the compounds tested compounds, 5a, 15a, and 18b, were the most active against T47D cell line with IC(50) values of 1.42, 1.92, and 2.92 μM, respectively. By using other cancer cell lines and with further characterization of their biological mechanism of action, these compounds could prove to be useful candidates as anticancer drugs.

Identification of atypical PML-RARA breakpoint in a patient with acute promyelocytic leukemia.

Acute promyelocytic leukemia (APL) of the M3 subtype is characterized by translocation t(15;17) that generates the PML-RARA fusion gene. Depending on the breakpoint position in the PML gene, 3 main fusion transcripts usually result. These breakpoints are bcr1 and bcr3 in introns 6 and 3, respectively, and bcr2 in exon 6. This report describes a rare atypical bcr2 breakpoint in a patient with morphological, cytogenetic and molecular features of APL. The presence of t(15;17) was first revealed by fluorescent in situ hybridization. Molecular analysis by reverse transcription polymerase chain reaction using primers for different PML-RARA junctions showed bands with different sizes compared with those generated from the three classical breakpoints, namely bcr1, bcr2 and bcr3. However, sequence analysis confirmed the presence of a bcr2 transcript with an atypical breakpoint within exon 6. The patient responded well to treatment and is now in complete remission. However, suggesting a favorable prognosis associated with such a rare transcript is difficult as more similar cases are needed to confirm such a conclusion. This article also stresses the importance of sequencing unusual polymerase chain reaction products to confirm their nature.

Allele and Genotype Frequencies of the Polymorphic Methylenetetrahydrofolate Reductase and Colorectal Cancer among Jordanian Population

BACKGROUND Methylenetetrahydrofolate reductase (MTHFR) is involved in DNA synthesis and repair. We here aimed to investigate two common polymorphisms, C677T and A1298C, with genotype and haplotype frequencies in colorectal cancer (CRC) cases among Jordanian. MATERIALS AND METHODS 131 CRC cases were studied for MTHFR C677T and A1298C polymorphisms, compared to 117 controls taken from the general population, employing the PCR-RFLP technique. RESULTS We found the frequency of the three different genotypes of MTHFR C677T among Jordanians to be CC: 61.7%, CT: 35.2%, and TT 3.1% among CRC cases and 50.9%, 38.8% and 10.3% among controls. Carriers of the TT genotype were less likely to have CRC (OR=0.25; 95%CI: 0.076-0.811; p=0.021) as compared to those with the CC genotype. Genotype analysis of MTHFR A12987C revealed AA: 38.9%, AC: 45%, and CC 16% among CRC cases and 37.4%, 50.4% and 12.2% among controls. There was no significant association between genetic polymorphism at this site and CRC. Haplotype analysis of MTHFR polymorphism at the two loci showed differential distribution of the TA haplotype (677T-1298A) between cases and controls. The TA haplotype was associated with a decreased risk for colorectal cancer (OR=0.6; 95% CI: 0.4-0.9, p=0.03). CONCLUSIONS The genetic polymorphism of MTHFR at 677 and the TA haplotype may modulate the risk for CRC development among the Jordanian population. Our findings may reflect an importance of genes involved in folate metabolism in cancer risk.

Thymoquinone Efficiently Inhibits the Survival of EBV-Infected B Cells and Alters EBV Gene Expression

Epstein–Barr virus (EBV) is a human virus with oncogenic potentials that is implicated in various human diseases and malignancies. In this study, the modulator activity of the potent herbal extract drug thymoquinone on EBV was assessed in vitro. Thymoquinone was tested for cytotoxicity on human cells of lymphoblastoid cells, Raji Burkitt’s lymphoma, DG-75 Burkitt’s lymphoma, peripheral blood mononuclear cells, and periodontal ligament fibroblast. Apoptosis induction was analyzed via TUNEL assay and activity studies of caspase-3. The effect of thymoquinone on EBV gene expression was determined using real-time polymerase chain reaction. We report here, for the first time, a promising selective inhibitory affect of thymoquinone on EBV-infected B cell lines in vitro, compared with lower activity on EBV negative B cell line and very low toxicity on human peripheral blood mononuclear cells and periodontal ligament fibroblasts. Moreover, the drug was found to efficiently suppress the RNA expression of EBNA2, LMP1, and EBNA1 genes. Specifically, EBNA2 expression levels were the most affected indicating that this gene might have a major contribution to thymoquinone potency against EBV infected cells. Overall, our results suggest that thymoquinone has the potential to suppress the growth of EBV-infected B cells efficiently.