KUMAGAI T1,2, KOEFFLER HP2 1 Department of Hematology, Ohme Municipal General Hospital, Tokyo, Japan
2 Division of Hematology/Oncology, Department of Medicine Cedars-Sinai Medical Center, Los Angeles, US
Background:All-trans-retinoic acid (ATRA) is used for the treatment of acute promyelocytic leukemia as a non-chemotherapeutic drug that induces terminal differentiation of leukemia cells. Another seco-steroid, 1,25(OH)2 vitamin D3 [1,25(OH)2D3] and related compounds also have anti-cancer activities against various types of cancer cells by inhibiting proliferation and inducing differentiation of tumor cells in vitro and vivo. Although orally administrated 1,25(OH)2D3had modest usefullness for patients with myelodysplastic syndrome (MDS) in clinical studies, its use was hampered because of hypercalcemia. 19-nor-1,25(OH)2D2 (Paricalcitol) is approved by the FDA for the clinical treatment of secondary hyperparathyroidism in patients with chronic renal failure. Different from other vitamin D analogues, paricalcitol has very little calcemic potential. This prompted us to investigate its anti-proliferative effect against cancer cells.
Methods: We studied anti-proliferative effect of paricalcitol against cancer cell lines in vitro and in vivo. The combinations of the analog with other clinally useful agents were also tested in vitro.
Results: Paricalcitol has antiproliferative effects against human cancer cells including prostate and colon cancer cells, as well as leukemia and multiple myeloma cells by inducing differentiation, cell cycle arrest and apoptosis in vitro and in vivo. Among many combinations with other clinically useful agents tested, paricalcitol in combination with arsenic trioxide has markedly enhanced antiproliferative activity against acute myeloid leukemia cells including acute promyelocytic leukemia cells. In further studies, arsenic trioxide acts as an inhibitor of both 24-hydroxylase which is a negative feedback regulator of vitamin D and the PML-RARalpha, the leukemogenic fusion protein in vitro. This may explain the synergistic effect of the combination against myeloid leukemia cells.
Conclusions: The hypercalcemic side-effect of 1,25(OH)2D3 has been mitigated by less-calcemic vitamin D analogs such as paricalcitol. The analog and its combination with other clinically useful drugs are being investigated with the hope that they may provide a therapeutic approach to cancers with little toxicity.
Purification and properties of a chemotherapeutic enzyme, L-asparaginase, from Pectobacterium carotovorum MTCC 1428. KUMAR S, DASU VV, PAKSHIRAJAN K
Biochemical Engineering Laboratory, Department of Biotechnology
Indian Institute of Technology Guwahati, Guwahati 781309, Assam, INDIA
Background: L-asparaginase is widely used in the chemotherapy. However, the success hitherto has been rather limited and most of the treatments have not been so successful due to various allergic reactions. Some of allergic reactions are mainly due to glutaminase contamination of most L-asparaginase. Hence, the discovery of a glutaminase-free L-asparaginase isolated from an organism that is serologically different from the previously reported ones, but has similar therapeutic effects will be more advantageous. In this communication we report on the purification and properties of glutaminase-free L-asparaginase extracted from P. carotovorum MTCC 1428.
Methods: The production of L-asparaginase from P. carotovorum was studied in the modified M-9 medium at 30°C and 180 rpm for 12 hrs. The harvested cells were ultrasonicated and centrifuged at 20000g for 20 minutes at 4°C to obtain crude extract of L-asparaginase.The purification was carried out by ammonium sulfate fractionation (80% saturation), DEAE cellulose ion exchange chromatography and Sephadex G-100 gel chromatography. The various purification steps were examined using Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) following the method of Laemmli. A Lineweaver-Burk analysis was used to determine kinetic properties of the purified L-asparaginase.
Results: The L-asparaginase was purified to homogeneity from P. carotovorum cells. Different purification steps (including ammonium sulfate fractionation followed by separation on DEAE cellulose chromatography and Sephadex G-100 gel filtration) were applied to the crude culture filtrate to obtain a pure enzyme preparation. The enzyme was purified 70-fold and showed a final specific activity of 1952 IU/mg with a 39% yield. SDS-PAGE gel showed a single protein band after sephadex G 100 gel filtration which revealed the purity of L-asparaginase. Kmand Vmaxvalues of purified L-asparaginase was compareable with the existing one which is used as a drug.
Conclusions: This work gives promising results on the possible production of glutaminase-free L-asparaginase which is particularly important for the development of downstream process for efficient production of L-asparaginase from P. carotovorum in a large scale.
Resistance, Including Carbapenem Resistance, Among Enterobacteriaceae In a University Hospital In Singapore KUMARASINGHE G, JAYASURYA A, JUREEN R, LIN RTP
National University Hospital, Singapore
Background: Our study was designed to detect enzymes such as ESBLs and AmpC in health-care associated strains of Escherichia coli and Klebsiella pneumoniae, using phenotypic methods and to further characterize carbapenem resistant isolates.
Methods: To detect enzymes such as ESBLs and AmpC in Escherichia coli and Klebsiella pneumoniae three-dimensional extract (TDE) method was used. For K. pneumoniae and Enterobacter cloacae with reduced susceptibility to carbapenems a phenotypic disk diffusion bioassay and double disc diffusion methods were used to elucidate the presence of multiple ?-lacatamases such as AmpC, MBLs, and carbapenemases.
Results: When testing 126 isolates that were resistant to third generation cephalosporins (39 E. coli and 87 K. pneumoniae) in 2005, we found AmpC in 28.2 % (11 isolates) of E. coli and in 17.2 % (15 isolates) of K. pneumoniae.
In addition 6 isolates were resistant to carbapenems (5 isolates of K. pneumoniae and 1 isolate of E. cloacae)
A combination of phenotypic tests revealed a likely co-existence of AmpC and ESBL in both E. coli and K. pneumoniae. Resistance to cefepime was observed in 83% of phenotypic AmpC positive isolates (10 out of12 tested isolates).
Multiple ß-lactamases were detected in K. pneumoniae and E. cloacae with reduced susceptibility to carbapenems. The presence of carbapenemases was suspected in isolates with reduced susceptibility to carbapenems, using phenotypical methods. A single isolate of K. pneumoniae was suspected to harbour a carbapenemase and a MBL, using phenotypical methods. K. pneumoniae with phenotypic AmpC and carbapenemases were susceptible to amikacin and trimethoprim sulfamethoxazole.
An increase in resistance to ceftazidime in E. coli isolates was observed in the hospital between 2005 and 2007 while a decrease was observed in K. pneumoniae.
Overall resistance against cefepime was 24.6% in 2007 for E. coli and 39.3% for K. pneumoniae. There were no differences in resistance to ceftriaxone and ceftazidime indicating that AmpC carrying isolates are still rare in the hospital population. However, among isolates from both species there was reduced sensitivity to imipenem.
Conclusions: Results of phenotypic tests for resistance properties may be helpful. Further work is being done to compare the results of phenotypic tests with the outcome of genotypic investigations.
