Apoptosis Of Choriocarcinoma Cell Line JAR Induced By Ubenimex

Ubenimex is a new antineoplastics. Ubenimex, a dipeptide analog, is a low molecular weight agent (N4W308.38) isolated from culture supernatant of Streptomyces olivoreticuli by Umezawa et al in 1976. Chemical tlimula is N- [(2S,3R}-3-amino-2-hydroxy-4-phenylbutyryl]-L-Leucine , and its trade mark is Bestatin. In vitro and in clinical trials, ubenimex in combination with chemotherapy can prolong survival and remission stage in patients suffered from acute nonlymphocytic leukemia in adults. It also obviously affects on other malignants, such as: lung squamous cell carcinomas gastric cancer. esophageal carcinoma~ urinary bladder carcinoma etc. The mechanism of anti-tumor effect may be different from that of usual antineoplastics, with its effective immunomodulating activities, ubenimex may provide a new strategy for treating resistant choriocarcinoma. Initially it is found that ubenimex is a potent inhibitor of aminopeptidase B~ N and Ieucine aminopeptidase. Intensive studies reveals that it has multiple effective immunomodulating activities including T lymphocyte stimulation macrophage activation-, and nature killer cells stimulation etc. Recently both human -4- 2001 ~ non-small-cell lung cancer cell lines and human Ieukemic cell lines induced apoptosis by ubenimex have been reported. However, there have been a few of studies of ubenimex on gestationl trophoblastic tumors, further, no document on ubenimex inducing choriocarcinoma apoptosis. Materials and methods We chose human choriocarcinoma cell line JAR in this study. Treating JAR cells with ubeniniex in five different concentrations that terminal concentration is 25OmglL 500mg/L~ l0O0mg/L~ I500mg/L.. 2000mg/L, and incubated with JAR cells for 24 hours~. 48 hours 72 hours 96 hours. To observe the direct anti-tumor effects of ubenimex on JAR cells, firstly, observing morphological changes of JAR cells with phase-contrast microscope. Secondly, examining the inhibiting action of time-concentration relationship to the cells with MIT method. And thirdly, investigating the function of synthesis and secretion hCG with time-Resolved Fluoimmunoassay (TRFIA). On the other hand, to investigate apoptosis induced by ubenimex in JAR cells, we used transmission and scanning electron microscope to observe the ultrastructral changes. Then flow cytometry (FCM) was performed for measurement of apoptotic peak and phase of cell cycle. TUNEL (terminal- deoxynucleotidyl transferase mediated nick end labeling) which can label DNA fragments was used to detect apoptotic cells in situ. Results I Morphological change Ubenimex treated JAR cells for 24 hours, a few dead cells appeared. Prolonged to 48-72 hours, cell volume decreased, membrane wrinkled, the inter-cellular space broadened. Cells separated each other and -5- 2001 眫4~ suspended in the culture media. The number of dead cells increased according to increasement of time and concentration. Cell shape was round or irregular. 2.MTT metabolism The i...