Induction of apoptosis by novel inosine monophosphate dehydrogenase, 3-hydrogenkwadaphnin

Abstract

Today’s medical word is highly dependent on severel natural products (e.g. taxol) in fighting different kinds of cancer and in constantly working to found new compounds. In this respect, 3-hydrogenkwadaphnin is a new diterpene ester isolated from Dendrostellera lessertii (Thymelaceae). It has been previously shown that this new compound has high anti-tumor activity and the capability of arresting the growing leukemic cells at G1/S phase of their progression cycle, which finally leads to cell death. Further studies revealed 3-hydrogenkwadaphnin decreased both DNA synthesis and inosine monophosphate dehydrogenase (IMPDH) activity in K562 and HL-60 cells. Here we decided to interrogate the mechanism (s) and mode of cytotoxic effects of this compound. Inosine monophosphate dehydrogenase is the key regulatory and the rate-limiting enzyme of the de novo biosynthetic pathway of purines. Apparently, the activity of this enzyme significantly increases in tumor cells in proportion to the proliferation. We hypothesized that inhibition of IMPDH and consequently depletion of guanine nucleotide pool is responsible of cytotoxic effect with characteristic of apoptosis. HL-60 and K562 leukemia cells were treated with increasing doses of 3-hydrogenkwadaphnin. Thereafter, the mode of its cytotoxic action was determined by Acridine orange/Ethedium bromide double staining, DNA fragmentation, sub-G1 flow cytometry and electron microscopic. Involvement of guanine nucleotide pool was tested by co-provision of guanosine and deoxyguanosine that replead GTP and dGTP pool size, respectively. 3-hydrogenkwadaphnin at low doses (10 nM) enhanced fragment of the apoptotic HL-60 cells within 72 hours of treatment. Higher doses (25 nM) led to rapid induction of apoptosis, which could be detected as early as 24 h. The majority (85%) of the treated HL-60 cells died by apoptosis, 72 hours after exposure to 3-hydrogenkwadaphnin. Althought K562 cells are generally more resistant to a variety of apoptotic stimuli, to our surprising 3-hydrogenkwadaphnin was equally effective in this cell line as compared to HL-60. 3-hydrogenkwadaphnin-induced apoptosis was almost totally prevented by supplementation with guanosine, but not with adenosine or deoxyguanosine, indicating the specific effect of 3-hydrogenkwadaphnin in reducing the pool size of GTP. The results indicate that 3-hydrogenkwadaphnin decreases GTP pool size and depletion of this pool, and not dGTP, is probably the cause of cell death through apoptosis. Induction of apoptosis is a major goal of chemotherapy. GTP depletion induced apoptosis is selective for tumor cells; thereby 3-hydrogenkwadaphnin could be presented as a powerful drug for treatment of cancer, especially leukemia.