The expression of Bcl-XL, Bcl-XS and p27Kip1 in topotecan-induced apoptosis in hepatoblastoma HepG2 cell line.
BACKGROUND
To assess the efficacy of topotecan, a topoisomerase I specific inhibitor in S-phase, the reagent-induced apoptosis and cytotoxicity as well as related proteins expression, had been preliminarily investigated in human hepatoblastoma HepG2 cells.


METHODS
Microculture tetrazolium assay (MTT), HE staining, transmission electron microscopy (TEM), flow cytometry (FCM), quantitative immunocytochemistry (QI), gene tranfection and RNAi technology were employed to carry out the exploration.


RESULTS
Topotecan could potently kill HepG2 cells via inducing apoptosis and demonstrated strong cytotoxicity in a time, dose-dependent manner with IC50 of about 95 mu g/L. According to morphologic observation and FCM analyses, it was confirmed that the drug treatment, causing significant S-phase arrest, could trigger a typical interphase apoptosis, the main traits of which were identified as chromatin pycnosis and cytoplasm condensation. It was shown that the expression of Bcl-XL was simultaneously down-regulated with the up-regulation of Bcl-XS in cytoplasm, which was possibly a key downstream event following the topotecan-induced DNA damage in nucleus. The expression level of p27Kip1, a negative regulator in cell cycle at G1/S transient, was also elevated. Transfection of pcDNA 3.1-p27Kip1 into HepG2 cells could abrogate the cytotoxicity in a degree while silence of p27Kip1 with siRNA in drug treatments could significantly increased the chemosensitivity, strongly indicating that the up-regulation of p27Kip1 was not an apoptosis-promoting, but a self-rescue response against drug by moderate G0/G1 arrest.


CONCLUSIONS
Topotecan had potent cytotoxicity against HepG2 cells by triggering an interphase apoptosis possibly mediated by increasing the ratio of Bcl-XS/Bcl-XL. Up-regulation of p27Kip1in TPT treatments could be a protective response for self-rescue and silence of the gene markedly augmented TPT cytotoxicity. Therefore, the experiment in vitro could provide a new idea for the clinical chemotherapy based on the combination of traditional drugs with the specific-siRNA targeted on the protective response gene.
Zhang
Jun
J
Department of Cell Biology and Genetics, Chongqing University of Medical Sciences, Chongqing, People's Republic of China. zhangjun1017@sohu.com
Cheng
Chao
C
He
Chuang-Long
CL
Zhou
Yu-Jian
YJ
Cao
Yang
Y
D000970:Antineoplastic Agents / Q000494:pharmacology*; D017209:Apoptosis / Q000187:drug effects*; D002453:Cell Cycle / Q000187:drug effects; D002460:Cell Line; D049109:Cell Proliferation / Q000187:drug effects; D002470:Cell Survival / Q000187:drug effects; D050760:Cyclin-Dependent Kinase Inhibitor p27; D004264:DNA Topoisomerases, Type I / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005434:Flow Cytometry; D018197:Hepatoblastoma / Q000201:enzymology / Q000378:metabolism* / Q000648:ultrastructure; D006801:Humans; D007150:Immunohistochemistry; D020128:Inhibitory Concentration 50; D047908:Intracellular Signaling Peptides and Proteins / Q000235:genetics / Q000378:metabolism*; D008113:Liver Neoplasms / Q000201:enzymology / Q000378:metabolism* / Q000648:ultrastructure; D046529:Microscopy, Electron, Transmission; D034622:RNA Interference; D015398:Signal Transduction / Q000187:drug effects; D013997:Time Factors; D059004:Topoisomerase I Inhibitors; D019772:Topotecan / Q000494:pharmacology*; D014162:Transfection; D051020:bcl-X Protein / Q000378:metabolism*
D000970:Antineoplastic Agents; C494811:BCL2L1 protein, human; C436291:CDKN1B protein, human; D004791:Enzyme Inhibitors; D047908:Intracellular Signaling Peptides and Proteins; D059004:Topoisomerase I Inhibitors; D051020:bcl-X Protein; D050760:Cyclin-Dependent Kinase Inhibitor p27; D019772:Topotecan; D004264:DNA Topoisomerases, Type I