Screening Of Active Ingredients From Chinese Herbs Against Acute Lymphoblastic Leukemia And Exploring Their Mechanisms

Acute lymphoblastic leukemia (ALL) is a form of hematological malignancy resulting from progressive accumulation of lymphoid precursors whose differentiation into functional mature cells is arrested. It is true that acute leukemia, especially children acute lymphoblastic leukemia might be treated successfully with drugs; however the development of drug resistance and side effects severely influence the therapeutic effect. Therefore it is essential to develop new alternative agents with minimal side-effects and less resistance for ALL prevention and therapy. Based on the literature review and analysis,30 Chinese herbs were selected for further study in this thesis. The aim of the present research is to seek the active ingredients of anti-ALL from Chinese herbs for potential new candidate of anti-leukemia drugs with few side effects and explore their underlying mechanisms.Screening of the active ingredients from Chinese herb:Samples were isolated from Chinese herbs by chemical methods. The proliferative inhibition of samples against human ALL cell line Reh, human APL cell line HL-60 and human lymphoma cell line Raji were evaluated by MTT assay.66 samples from 3 herds such as Uvaria grandiflora Roxb, Duchesnea indica, Cyrtomium fortunei, Paris polyphylla were tested, the results demonstrated that ethanol extracts from indigo naturalis, Cortex Moutan, Pyrola, and both of aqueous extracts and methanol extracts from Duchesnea indica specifically inhibited the growth of HL-60; ethanol extract of Rhizoma Paris had significant inhibition against Raji. While protopanoxadiol(PPD) and zeylenone showed remarkable inhibitory effects in all test cell lines and had stronger effects against Reh, the IC50 values at 48h were 11.5 and 0.52μg/mL, respectively. In addition 3-Methyl-butyrylphloroglucinol from Cyrtomium fortunei, ursolic acid from Fructus Gardeniae, protopanaxatriol from American ginseng had the significant inhibition agains Reh as well.Study on the anti-ALL effect of active ingredients in vitro:The anti-cancer effects of PPD against a variety of cancer cells and normal cell-peripheral blood mononuclear cells (PBMC) were comparable by MTT assay. The results showed that PPD had the strongest effects to ALL cells Reh and RS4;11 among tested cell lines, the IC50 values of PPD on Reh and RS4;11 were 24.15 and 27.19 u M respectively, with little toxicity to PBMC(IC50:115.76 u M). The anti-ALL actions of PPD were determined with morphologic observation, DNA content, apoptotic assay and expression of cell surface markers by flow cytometry etc. The results showed that PPD inhibited Reh and RS4; 11 in a time and dose dependent manners. PPD(24-36μM) also blocked cell cycle progression from G0/G1 phase with down-regulation of cyclin D1, CDK4, up-regulation of P21. PPD induced cell differentiation as well. However, cell apoptosis was not affected. To evaluate the anti-ALL effect of zeylenone, MTT assay, AO/EB staining, flow cytometry and Weston-blot assay were used. The results showed that zeylenone had the potent efficiency against 12 cell lines and PBMC Of the test cell lines, Reh and RS4; 11 had the most sensitivity to zeylenone and PBMC had the less toxicity. Zeylenone decreased survival, inhibited proliferation, induced apoptosis, and led to G1 cell cycle arrest in dose-time dependent manners in both cell lines. It also activated apoptotic proteins (caspase-3,-8,-9), increased the expression of apoptotic protein Bax and decreased the expression of anti-apototic protein Bcl-2. Cell cycle arrest was associated with down-regulation of cyclin D1 and up-regulation of P21.Experiments on the anti-leukemia effect of PPD in vivo:24 hours after DBA/2 mice were implanted (i.p) murine lymphocytic leukemia L1210 cells, PPD (10,20, 40mg/kg) were given intrastracally for ten days, cisplatin (0.1mg/kg) was injected by tail vain as positive group on dl, d5 and d10 respectively. The experimental results showed that PPD could improve the general conditions of mice, inhibit the growth of leukemia xenograft tumors. PPD also prolonged the life time of mice with L1210 cells, decreased the number of WBC of peripheral blood and the infiltration of leukemia cells in marrow. Moreover, PPD reduced the infiltration of inflammatory cells in liver and kidney and improved the immune functions of mice as well. The effect of PPD on L1210 cell in vitro also was envaluated. The results showed that PPD inhibited the growth of L1210 in a dose and time dependent manners and arrested the cell cycle at G0/G1 phase, which were in accord with the results of Reh and RS4;11.In conclusion, the screening expereiments of 66 samples from 30 Chinese herbs demonstrated that PPD and zeylenone might have the strongest effects to ALL by the sensitivity assay of in cell modes. The anti-ALL effects of PPD were confirmed by in vitro and in invo. The investigation of the mechanisms of anti-cancer effects showed that PPD inhibited the proliferation, arrested the cell cycle and induced the differentiation of leukemia cells. Moreover the anti-cancer effects of zeylenone were associated with inducing apoptosis and arresting the cell cycle. Additionally, it is addressing that the differentiated effect of PPD and apoptotic action of zeylenone against Reh and RS4;11 cells, which might provide new viewpoint for the anti-cancer drug development. Those results laid foundation for PPD and zeylenone to be research as a potential therapeutic agent after further investigation.