| Homoharringtonine (HHT) is a plant alkaloid derived from the Cephalotaxus forruneii tree. In 70s, it had been confirmed that HHT had favorably therapeutic activity in patients with acute myelogenous leukemia (AML) and chronic myelogenous leukemia (CML). Original studies showed that HHT inhibited protein synthesis in leukemia cells. But further investigations demonstrated that the anti-leukemia activity of HHT was due to induce apoptosis at given concentration, but not at lower-dose level. However, the clinical curative effect of HHT on AML could be achieved at lower dose (0.25-0.5mg/d). In this study we tried to elucidate the anti-tumor mechanisms of lower-dose HHT in order to extend the clinical using of HHT and to some extent, to provide a theoretical basis.We studied the effect of lower-dose (5~10ng/ml) HHT on K562 cell line in vitro, human acute erythroleukemia cell line. Cellular proliferation was detected by tetrazolium salts assay. Our results showed that 5ng/ml and lOng/ml HHT reduced K562 cells growth by 8.77 1.26% and 20.28 1.55% respectively. The longer exposure time was, the stronger inhibitory effect of HHT on cell proliferation was. HHT reduced K562 cells growth by 33.33 3.02% and 58.64 3.88% atconcentration of 5ng/ml and lOng/ml after 12 days in vitro. It indicated that the efficiency of HHT in inhibiting cell growth was in a time dependent manner, and anti-tumor capacity of low dose HHT could be achieved by prolonging exposure time.Additionally, K562 cells were dyed by Annexin VFITC and PI for apoptosis determination by using flow cytometry. The results indicated there was no apoptosis in K562 cells treated with 5ng/ml HHT for 3 to 12 days, and that apoptosis was not detected in K562 cells treated with lOng/ml HHT for 3 and 6 days. Only at 9 and 12 days, 11.8% of the cells became apoptotic. It indicated that the growth inhibition by 5ng/ml HHT was independent of apoptosis. In 6 days, the inhibitory effect of HHT on cell growth also had no relationship with cell apoptosis, but it was possible that there was partial relationship between cell growth inhibiton and cell apoptosis induced by lOng/ml HHT after exposure to longer time.Lately, telomerase has become one of potentially attractive targets for cancer therapy. Human telomerase reverse transcriptase (hTERT) is the key to active telomerase. Several trails have reported that low dose HHT could remarkably inhibit the telomerase activity in two leukemic cell lines in vitro. In order to elucidate the inhibiton of low concentration HHT on the telomerase activity of K562 cells, in this study, Krupp modify TRAP assay and half- quantificational RT-PCR were used to examine the telomerase activity and hTERT mRNA expression respectively. The significant reduction of telomerase activity and hTERT mRNA expression was found after treated with low concentration HHT for 3 to 6 days, and the changes was paralleling with the loss of proliferous capacity. It indicated that the cytotoxicity of lower-dose HHT was achieved by reducing telomerase activity and the expression of hTERT. However, the apoptosis cannot be induced by the inhibition of telomerase activity at given concentration of HHT. Our results suggested that the pathway in apoptosis induced by HHT and in suppression of telomerase activity may be different. The regulatory mechanism of telomerase and hTERT remains unclear. Several trails demonstrated c-myc gene might be responsible for the regulation of hTERT. Reverse transcriptase-polymerase chain reaction analysis showed that HHT could downregulated c-myc gene expression at transcriptional level.In order to explore the relationship between the reduction of hTERT expression induced by HHT and cell mature differentiation, the content of hemoglobin in K562 cells was determined- by the absorption at 570nm wavelength in ultraviolet spectrophotometer. The expression of CDllb and HLA-DR, the membranous marker of mature granulocyte and monocyte, was detected by flow cytometry. Compared to the controls, hemoglobin was increased in the... |
