Effects of triptolide on RIZ1 expression, proliferation, and apoptosis in multiple myeloma U266 cells.
OBJECTIVE
To investigate the effects of triptolide on proliferation and apoptosis as well as on the expression of RIZ1 in the human multiple myeloma cell line U266 in vitro.


METHODS
The effect of triptolide on the growth of U266 cells was studied by MTT assay. Apoptosis was detected by Hoechst 33258 staining and Annexin V/PI double-labeled flow cytometry, and caspase-3 mRNA was measured by RT-PCR. Western blotting, flow cytometry and RT-PCR were used to assess the expression of RIZ1, and the location and expression of H3K9me1 were detected by confocal microscopy and Western blotting.


RESULTS
Triptolide significantly inhibited the proliferation of U266 cells in a time- and concentration-dependent manner (the IC(50) value for a 24-h exposure was 157.19+/-0.38 nmol/L). Triptolide induced typical apoptotic morphological changes. Triptolide 40, 80, and 160 nmol/L treatment induced significant caspase-3-dependent apoptosis compared with control group (10.5%+/-1.23%, 37.9%+/-2.45%, and 40.5%+/-2.30% vs 3.8%+/-1.98%, P<0.05). Compared with peripheral blood monocular cells (PBMC) from healthy donors, the protein expression of RIZ1 in U266 cells was relatively low, but the mRNA and protein expression of RIZ1 were strikingly increased by triptolide in a concentration-dependent manner. Triptolide increased the protein expression of RIZ1 and RIZ1 methylates histone H3 lysine 9 in U266 cells.


CONCLUSIONS
Triptolide increased the protein expression of RIZ1, inhibited the proliferation, and induced caspase-dependent apoptosis in U266 cells.
Department of Hematology, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, China.
Fei
F
Zhao
Yan
Y
Chen
Ling-lan
LL
Zeng
Rui
R
Li
Rong
R
Zeng
Lu
L
Wen
Yuan
Y
Liu
Chun
C
Zhang
D018906:Antineoplastic Agents, Alkylating / Q000494:pharmacology*; D017209:Apoptosis / Q000187:drug effects*; D006690:Bisbenzimidazole / Q000032:analysis; D015153:Blotting, Western; D045744:Cell Line, Tumor; D049109:Cell Proliferation / Q000187:drug effects*; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D004224:Diterpenes / Q000494:pharmacology*; D004365:Drugs, Chinese Herbal / Q000494:pharmacology; D004852:Epoxy Compounds / Q000494:pharmacology; D005434:Flow Cytometry; D005456:Fluorescent Dyes / Q000032:analysis; D011495:Histone-Lysine N-Methyltransferase; D006657:Histones / Q000378:metabolism; D006801:Humans; D051034:Inhibitor of Apoptosis Proteins / Q000235:genetics; D008745:Methylation; D009101:Multiple Myeloma / Q000188:drug therapy / Q000235:genetics* / Q000473:pathology; D009687:Nuclear Proteins / Q000235:genetics / Q000378:metabolism*; D010616:Phenanthrenes / Q000494:pharmacology*; D014157:Transcription Factors / Q000235:genetics / Q000378:metabolism*
D018906:Antineoplastic Agents, Alkylating; D004268:DNA-Binding Proteins; D004224:Diterpenes; D004365:Drugs, Chinese Herbal; D004852:Epoxy Compounds; D005456:Fluorescent Dyes; D006657:Histones; D051034:Inhibitor of Apoptosis Proteins; D009687:Nuclear Proteins; D010616:Phenanthrenes; D014157:Transcription Factors; C001899:triptolide; D011495:Histone-Lysine N-Methyltransferase; C093563:PRDM2 protein, human; D006690:Bisbenzimidazole