| Interferon- (IFN- ) is one family member of cytokines, regarded as a kind of glycoprotein with many biological properties such as antivirus, antitumor and immunoregulation. It was one of the first choice for the treatment of chronic myeloid leukemia(CML).Clinic evidence showed that it would unearth therapy potential or lessen adverse effects of IFN- a in combination with biological regulators, cytotoxic agents or antivirus agents, but the exact mechanism was not known. Homoharringtonine(HHT)is a plant alkaloid derived from an evergreen tree, has high activity in cure of acute leukemia, which was discovered firstly by our Chinese scientists. In the middle age of twenty century ninetieth, scientists in M.D. Anderson cancer center applied lower dose HHT in the treatment of CML, obtaining positive clinical efficacy. The inhibition of protein synthesis was identified as the most important mechanism of its antileukemia activity. Present study evaluated theinduction of apoptosis determined by HHT alone. In this study, we tried to elucidate the synergistic inhibition of IFN- a and HHT in CML cells and the relative mechanism, to provide a theoretical basis for designing new antitumor strategies.We examined the synergistic effects of IFN- a and HHT on the growth of cell line K562, which is a human chronic myeloid leukemic cell line at blastic phase, by MTT assay. The results showed that the growth of K562 cells could be inhibited when they were treated by IFN- a at1000IU/ml,5000IU/ml,10000IU/ml and 20000IU/ml for 3 days, but correlated analyze indicated that there was no correlation in statistics(r=0.805,p=0.195). The same activity was demonstrated at HHT 5ng/ml,10ng/ml,20ng/ml and 40ng/ml alone, the rate of K562 growth inhibition was 14.48%,23.79%,39.16% and 69.48% respectively in a remarkable dose-dependent manner(r=0.994,p = 0.001), IC50 was 27.35ng/ml. HHT exerted a synergistic effect with IFN- in inhibiting K562 cells growth. The rate of growth inhibition was increase to 19.15%,43.82%,65.27% and 73.77% respectively, with remarkable meaning in statistics(p=0.008 for HHT 5ng/ml, p<0.001 for HHT 10ng/ml,20ng/ml and 40ng/ml), IC50 of HHT decreased to18.72ng/ml. We also found the growth of K562 cells was inhibited by 20ng/ml HHT in combination with 5000IU/ml IFN- in a time-dependent manner(r=0.966,p=0.034).In order to explore the mechanism between the two agents and the induction of apoptosis, we analyzed K562 cells treated with two agents by morphological observation stained with Wright-Giemsa. Characteristic changes for apoptosis emerged in K562 cells after being treated with 5000IU/ml IFN- a in combination with 40ng/ml HHT for 3 days, not with 5000IU/ml IFN- a or 40ng/ml HHT alone. We tried to explore the apoptosis by DNA agarose gel electrophoresis assay further, but DNA ladder was not observed in the DNA agarose gel electrophoresis whether we increased the concentration of HHT and/or treatment time. The outcomes of flow cytometry(FCM) assay according to the positive of PI- Annexin VFIFC showed that necrolic changes of the K562 cells were found with 40ng/ml HHT treatment for 3 days, the rate ofapoptosis/the rate of necrosis was 8.47%/20.12%,and the rate of apoptosis was increased after in combination with 5000IU/ml IFN- a ,the ratio of apoptosis and necrosis was 31.67%/17.04%.The results also showed the apoptotic rate of cells was increased relying on the treatment time. The rates of cellular apoptosis induced by 5000IU/ml IFN- a in combination with 20ng/ml were 9.67% for 24hrs,26.92% for 48hrs and 70.89% for 72hrs.In our study, Krupp modify TRAP assay and RT-PCR were used to assess the telomerase activity and hTERT mRNA expression in K562 cells after agents treatment, to explore their relationship with apoptosis induced by agents. We found that the activity of K562 telomerase was inhibited by 10ng/ml,20ng/ml and 40ng/ml HHT alone or in combination with 5000IU/ml IFN- a respectively within 24 hours , and the suppression was enhanced while applying the two agents simultane... |
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