Anticancer Activity of Extremely Effective Recombinant L-Asparaginase from Burkholderia pseudomallei.

L-asparaginase (E.C. 3.5.1.1) purified from bacterial cells is widely used in the food industry, as well as in the treatment of childhood acute lymphoblastic leukemia. In the present study, the Burkholderia pseudomallei L-asparaginase gene was cloned into the pGEX-2T DNA plasmid, expressed in E. coli BL21 (DE3) pLysS, and purified to homogeneity using Glutathione Sepharose chromatography with 7.26 purification fold and 16.01% recovery. The purified enzyme exhibited a molecular weight of ~33.6 kDa with SDS-PAGE and showed maximal activity at 50°C and pH 8.0. It retained 95.1, 89.6%, and 70.2% initial activity after 60 min at 30°C, 40°C, and 50°C, respectively. The enzyme reserved its activity at 30°C and 37°C up to 24 h. The enzyme had optimum pH of 8 and reserved 50% activity up to 24 h. The recombinant enzyme showed the highest substrate specificity towards L-asparaginase substrate, while no detectable specificity was observed for L-glutamine, urea, and acrylamide at 10 mM concentration. THP-1, a human leukemia cell line, displayed significant morphological alterations after being treated with recombinant L-asparaginase and the IC50 of the purified enzyme was recorded as 0.8 IU. Furthermore, the purified recombinant L-asparaginase improved cytotoxicity in liver cancer HepG2 and breast cancer MCF-7 cell lines, with IC50 values of 1.53 and 18 IU, respectively.

Loading of some quinoxaline derivatives in poly (l-lactic) acid/Pluronic® F-127 nanofibers enhances their anticancer efficiency and induces a p53 and p21 apoptotic-signaling pathway.

The objective of this study was to evaluate the anticancer activity on cancer cell models of a drug delivery system consisting of poly (l-lactic) acid/Pluronic® F-127 (PLLA/PF127) loaded with the new N-butylpyridoquinoxaline 1,4-dioxide (NBPQD) or 2-amino-3-cyano-6-methylquinoxaline 1,4-dioxide (ACMQD) that was synthesized using an electrospinning process compared to free NBPQD and ACMQD. PLLA/PF127-NBPQD and PLLA/PF127-ACMQD nanofibers were prepared, and their shape, size, Fourier-transform infrared spectroscopy (FTIR), thermogravimetric (TGA) analysis, water contact angel (WCA), drug release, anticancer activity against five human cancer cell lines, and flowcytometeric analyses of cell cycle, p21 and p53 activities were investigated. PLLA/PF127 nanofibers with NBPQD or ACMQD were smooth, and no NBPQD or ACMQD clusters were found on nanofibers surface. FTIR analysis indicated that intermolecular hydrogen bonding between NBPQD or ACMQD and the polymer matrix is present. PLLA/PF127 nanofibers with NBPQD or ACMQD showed quite stable thermal stability with degradation at about 400 °C, and showed high WCA values of 68.72 ± 3.83° and 110.59 ± 0.21°, respectively. They showed higher in vitro anticancer activity towards all investigated cell lines compared to free NBPQD or free ACMQD. The lowest IC50 value for PLLA/PF127-NBPQD was 1.7 µg/ml with colorectal carcinoma (HCT-116) and was 4.5 µg/ml for PLLA/PF127-ACMQD with hepatocellular carcinoma (HepG2). PLLA/PF127 nanofibers with NBPQD or ACMQD increased anticancer efficiency via inducing cancer cell apoptosis through activation of a p53 and p21 apoptotic-signaling pathway.

Pro-inflammatory adipocytokines, oxidative stress, insulin, Zn and Cu: Interrelations with obesity in Egyptian non-diabetic obese children and adolescents.

PURPOSE: To investigate the inter-relationships between adipocytokines, oxidative stress, insulin, Zn and Cu and obesity among Egyptian obese non-diabetic children and adolescents. PATIENTS AND METHODS: 72 obese children and adolescents of both sexes (5-17 years) were recruited for the study. 40 healthy normal non-obese persons of matched ages and sexes were used as control group. Lipid profile, tumor necrosis factor alpha (TNF-α), interleukin-6 (IL-6) and leptin levels were measured. Malondialdehyde (MDA) and reduced glutathione (GSH) concentrations and superoxide dismutase (SOD) activity were estimated. Micronutrients (Zn and Cu) concentrations in addition to insulin and fasting blood sugar (FBS) levels were also evaluated. Estimation of insulin resistance (homeostatic model assessment (HOMA-IR)) was derived from FBS measurements. RESULTS: Significant elevations (P<0.001) in TNF-α, IL-6, leptin, MDA, Cu and FBS levels and significant decreases (P<0.001) in GSH, Zn levels and SOD activity were detected among obese individuals as compared with control group. Insulin and triglyceride levels were significantly increased in obese male children and HDL-cholesterol level was increased significantly in obese adolescent females compared to controls. However, total cholesterol and LDL-cholesterol levels were significantly high in all obese cases as compared with controls. Insulin resistance was detected in 100% of the patients. CONCLUSIONS: We concluded that obesity with pro-inflammatory adipocytokines and hypozincemia together by many mechanisms participate in excessive oxidative stress and are highly associated with inflammation and the development of obesity-related complications. Obesity represents a critical risk factor for development of insulin resistance status.

In vitro upregulation of erythrocytes glucose uptake by Rhaphnus sativa extract in diabetic patients.

In diabetes, both the increase in the oxidative stress and the decrease in the antioxidant defense may elevate the susceptibility of diabetic patients to many pathological complications. So, the aim of the present study was to investigate the effect of superoxide dismutase (SOD) like activity protein, partially purified from radish (Rhaphnus sativa) on uptake of glucose in vitro by erythrocytes of diabetic patients. In hyperglycemic patients, erythrocytes malondialdehyde level was highly significantly increased (P < 0.0001) than that of the control. However, the erythrocytes glutathione content and glutathione reductase activity, were both highly significantly decreased (P < 0.0001) compared to that corresponding control values. The glucose uptake by erythrocytes of diabetic patients was highly significantly decreased (P < 0.0001) with increasing hyperglycemia, while it was highly significantly elevated (p < 0.0001) after addition of the partially purified SOD like activity protein. On the other hand, the malondialdehyde concentration was highly significantly reduced (p < 0.001) on adding the partially purified protein. It thus can be concluded that, an appropriate support for enhancing antioxidant supply, such as SOD like activity protein from natural sources, may help control blood glucose level and may prevent clinical complications of diabetes.