The Study Of HDN-1, A ETP Compound Derived From Antarctic Fungal, On Anti-leukemia Effects And Its Mechanism

Acute myelogenous leukemia (AML) is a malignant clonal hematological diseases, mainly characterized by cell excessive proliferation and differentiation retardation. According to the change of specific genes and different stages of differentiation in cell, AML can be divided into eight subtypes (MO to M7), where the acute myeloid leukemia part mature and acute promyelocytic leukemia (APL) belong to M2 and M3 type.Currently, it is a hot point how to improve the treatment of leukemia patients in complete remission rate and disease-free survival. Although there has made great progress of the molecule-targeted treatment of AML, the clinical drug resistance appeared gradually with unclear mechanisms. Although all-trans retinoic acid (ATRA) is encouraging in APL therapy by induction of the differentiation of APL cells, many complications occured associated with the treatment. Therefore, the development of new drug for acute myeloid leukemia remains to be a challenge.HDN-1 which belongs to the epipolythiodioxopiperazine is derived from the antarctic soil fungi Oidiodendron truncatum GW3-13 with a novel molecular structure and has independent intellectual property rights. Our early studies showed that HDN-1 bound to the C-terminal of Hsp90 and subsequently degraded Hsp90 client proteins. On this basis, in the present paper, HDN-1 is evaluated proliferation inhibition in various tumor cells and is exposed to be most cytotoxic in acute myeloid part mature leukemia cells HL-60. Furthermore, HDN-1 is found to induce differentiation of HL-60 in vitro, then, anti-leukemia effect was further assessed in HL-60 cells xenograft mice. Finally, mechanisms about the growth inhibition and differentiation of HL-60 cells induced by HDN-1 are investigated in this paper.Part 1. HDN-1 induced apoptosis in HL-60 cells1. MTT assay is applied to measure the proliferation inhibition of HDN-1 on variety of tumor cells.The results show that the HDN-1 can inhibit cell growth in liver cancer, lung cancer, breast cancer etc. The IC50 is in 0.03 μmol·L-1～3 μmol·L-1 level. The results of Mouse immune cell proliferation inhibition experiment showed that there is no inhibition of mice immune cells after the treatment of HDN-1. All above illustrate that HDN-1 has a far-ranging anti-tumor activity in vitro and is most cytotoxic in HL-60 cells. HDN-1 is selective in tumor cells without affecting immune cell activity and the immune function.2. Hoechst 33342 staining shows that HDN-1 can induce the nucleus pycnosis, fragmentation and apoptotic bodies formation of HL-60 cells. The DNA fragments in the size of 180-200bp produced by HDN-1 can be observed by Genomic DNA agarose gel electrophoresis. Western Blotting is applied to detect the apoptotic proteins expression in HL-60 cells. After roled by different concentrations of HDN-1 for 24 hours, compared with vehicle group, Caspase-9, Caspase-3, Caspase-8 and PARP cleaved with varying degrees and activation of caspase is dramatically inhibited by caspase specific inhibitor. Pro-apoptotic protein Bax is up-regulated significantly accompanied by the decrease of anti-apoptotic protein Bcl-2 and Mcl-1 and the expression of γ-H2AX increased. All these results indicate that HDN-1 induces apoptosis of HL-60 leukemia cells depends both on the death receptor pathway and the mitochondrial pathway.Part 2. HDN-1 promoted terminal differentiation of HL-60 cells1. Under the Giemsa staining, we find that HDN-1 can change the HL-60 nuclear morphology, reduce nuclear-cytoplasmic ratio and generate kidney-shaped and segmented granulocytes gradually. Moreover, glycogen in HL-60 cells are detected by periodic acid Schiff reaction(PAS). The result shows that HDN-1 rise the PAS positive rate in both time-and dose-dependent manners. All above suggest that HDN-1 promoted terminal differentiation of HL-60 cells.2. Flow Cytometry is applied to investigate expression of cell-surface antigens. HDN-1 promote HL-60 cells’differentiation as reflected by increasing expression of the granulocyte differentiation marker CD11b and myeloid leukemia marker CD13. There are no changes of mature monocytic marker CD 14. Overall, this series of results show that HDN-1 could induce granulocyte differentiation of HL-60 cells.3. We compare the HDN-1 with all-trans retinoic acid (ATRA) in promoting differentiation of HL-60 cells by PAS staining by Giemsa staining. Statistical results point out that HDN-1 take effect in a shorter time and lower concentrations. In other words, HDN-1 tend to gain the advantage over ATRA in HL-60 cells differentiation.4. Combined effect of HDN-1 and ATRA in cell proliferation inhibition of HL-60 cells. As a result, the combination index (CI) is calculated and this interactions became synergistic (most data with C1 values<1) in HL-60 cells. Moreover, using the dose-response curves, a normalized isobologram was constructed, most all the experimental data points below and to left the diagonal line, which means the two compounds are synergism in treatment of HL-60 cellsPart 3. The anti-leukemia activity of HDN-1 in vivoIn this part, we successfully build the NOD-SCID mouse acute myelogenous leukemia model, and design experimental proposal through the preliminary experiment. There are six groups:a blank group, the vehicle group, the HDN-1 groups (1mg/kg,0.5mg/kg), intravenous injection, daily administration for consecutive 12 days. Experimental results show that the HDN-1 high and low doses inhibition rates are 18% and 46%. HDN-1 can significantly inhibit the growth of HL-60 xenograft tumors, have a good anti-tumor activity and less impact on the body weight of mice.Part 5. Research of the mechanisms on HDN-1 inducting differentiation and apoptosis of HL-60 cell.1. Co-chaperones play important roles in heat shock protein 90 as a molecular chaperone which is essential in many cellular processes. Among the co-chaperones, Hsp70 is an important one which can bind to C-terminalof Hsp90, form chaperone complex and fold or maturate of many client proteins. Our research in this part by using Co-Immunoprecipitation indicate that HDN-1 can inhibit the combination of Hsp90 and Hsp70. In other words HDN-1 bind to C-terminal of Hsp90 in order to influence the effect of Molecular chaperone Hsp90.2. There exsist many client proteins in leukemia cells. To further investigate anti-leukemia mechanism of HDN-1, Western Blotting is applied to detect the effect of HDN-1 on Hsp90 clients proteins with respect to cell apoptosis and differentiation in leukemia cells. The result suggest that HDN-1 induce the degration of Akt, p-Akt, Erk, p-Erk, CyclinDl, Bcr-Abl, Stat3, p-Stat3 and inhibited their activities in order to influence molecules downstream. It maybe the mechanisms on HDN-1 inducting differentiation and apoptosis of HL-60 cell.In conclusion, HDN-1 has a broad-spectrum anti-tumor activity and is most cytotoxic in acute myeloid part mature leukemia HL-60 cells. HDN-1 and ATRA become synergism to make higher growth inhibition of HL-60 cells. HDN-1 can induce apoptosis and promote granulocyte differentiation of HL-60 cells, which is the fist time to find that the ETPs could induce leukemia cells differentiation. Compared with ATRA, HDN-1 take effect in a shorter time and lower concentrations. HDN-1 have a significant anti-xenograft tumor activity in vivo and the mechanisms may related to binding with C-terminal of Hsp90 so that inhibit the effect of molecular chaperone. Our study provide an effective basis for further exploration of HDN-1 anti-leukemia mechanisms and lay the foundation for the development of medicines of ETPs.