In the present study, we investigated the in vitro effect of saucernetin-7, which is a dineolignan isolated from Saururus chinensis, on the proliferation, cell cycle-regulation and differentiation of HL-60 human promyelocytic leukemia cells. Saucernetin-7 potently inhibited the proliferation of HL-60 cells in both a dose- and time-dependent manner with an IC50, approximately 5 microM. DNA flow-cytometry indicated that saucernetin-7 markedly induced a G1 phase arrest of HL-60 cells. Among the G1 phase cell cycle-related proteins, the levels of cyclin-dependent kinase (CDK)6 and cyclin D1 were reduced by saucernetin-7, whereas the steady-state levels of CDK2, CDK4, cyclin D2, cyclin D3 and cyclin E were unaffected. The protein and mRNA levels of a CDK inhibitor p21CIP1/WAF1, but not p27KIP1, were markedly increased by saucernetin-7 and p21CIP1/WAF1 induction is likely to occur at the transcriptional level because actinomycin D blocked this induction. In addition, saucernetin-7 markedly enhanced the binding of p21CIP1/WAF1 with CDK2 and CDK6, resulting in the reduced activity of both kinases and the hypophosphorylation of Rb protein. We furthermore suggest that saucernetin-7 is a potent inducer of the differentiation of HL-60 cells, based on observations such as a reduction of the nitroblue tetrazolium level, an increase in the esterase activities and phagocytic activity, morphology changes, and the expression of CD14 and CD66b surface antigens. In conclusion, the onset of saucernetin-7-induced the G0/G1 arrest of HL-60 cells prior to the differentiation is linked to a sharp up-regulation of the p21CIP1/WAF1 level and a decrease in the CDK2 and CDK6 activities. This is the first report demonstrating that saucernetin-7 potently inhibits the proliferation of human promyelocytic HL-60 cells via the G1 phase cell cycle arrest and differentiation induction.

Costunolide has been reported to be a cytotoxic and chemopreventive agent. This work investigated the mechanism of the antiproliferative effect of costunolide and determined that it induced differentiation of the human leukemia cell line HL-60. Costunolide exhibited a potent antiproliferative activity against HL-60 cells. It was also found to be a potent inducer of differentiation in human leukemia derived HL-60 cells through the examination of differentiation markers, as assessed by the reduction of nitroblue-tetrazolium, the increase in esterase activities and phagocytic activity, morphology change and the expression of CD14 and CD66b surface antigens. These results, accompanied by a decline in the expression of c-myc protein, suggest that costunolide induces differentiation of human leukemia cells to granulocytes and monocytes/macrophages lineage.

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The underground or the aerial part of Saururus chinensis (LOUR.) BAILL (Saururaceae) has been used as a folk medicine to treat edema, jaundice, and gonorrhea in Korea.1) Several constituents such as lignans, neolignans, 2) acyclic diterpenes, 3) aristolactams, 4) and particularly manassantin A and B 5) classified as dineolignans have been isolated from the genus Saururus. In addition, the constituent sauchinone and its stereoisomers, a phenylpropanoid (sarisan), lignans (galbacin and saucernetin), 6) diarylbutane lignans, 7) tetrahydrofuran-type sesquilignans, 8) and furanoditerpenes 9) were isolated from S. chinensis. It was reported that sauchinone, a lignan from S. chinensis, attenuated CCl 4 -induced toxicity in primary cultures of rat hepatocytes.10) Further, we previously demonstrated that the methanol extract and its active constituents, saucernetin-7 and saucernetin-8, isolated from S. chinensis inhibited the LPS-induced iNOS and COX-2 expression by blocking NF-kB activation. 11,12) Tumors are diverse and heterogeneous, but all share the ability to proliferate beyond the constraints limiting growth in normal tissue. Deregulated cell proliferation together with suppressed apoptosis and differentiation constitute the minimal commom platform upon which all neoplastic evolution occurs. Based on the understanding of tumor biology in respect to the kinetics of cell populations, two new strategies, induction of differentiation and apoptosis, have recently emerged in the fields of cancer chemoprevention and chemotherapy. Differentiation from malignant or premalignant cells into more mature or normal-like cells as well as apoptosis in multistep carcinogenesis are theoretically amenable to preventive cancer intervention. Thus, compounds capable of inducing differentiation are considered as candidate agents for the prevention and/or treatment of cancer. 13,14) The HL-60 cell line, derived from a patient with acute promyelocytic leukemia, provides a useful model system for studying the cellular and molecular events involved in the differentiation process.15) Certain compounds, known to be efficacious cancer preventative agents, such as interferon, 16) retinoids, 17) 1a,25-(OH) 2 D 3 18) are potent inducers of HL-60 cell differentiation, and appear to be clinically effective against myeloproliferative disorders. Thus, the strategy that manipulates HL-60 cell differentiation has been used as a valid model to discover potential cancer chemopreventive agents in preclinical evaluation. Thus, as a part of our screening program to evaluate the chemopreventive potential effect of natural compounds, we have investigated the effect of saucernetin-8, which was isolated from the underground parts of Saururus chinensis, on HL-60 growth and induction of differentiation. Various biochemical and morphological examinations performed in the present study indicated that saucernetin-8 contains the activity to induce HL-60 cell differentiation. Furthermore, we demonstrated that the expression of p21 CIP1 cyclin dependent kinase inhibito...