Discover And Study On Myeloid Leukemia Cell Differentiation Effect And Target Of FTC, A Naturally-occurring Small Molecule Compound

Leukemia is one of the most dangerous threats to human health. Side-effects of chemotherapy and radiotherapy could be severe. Differentiation therapy may turn into another breach to the treatment of leukemia. Existing leukemia cell differentiation inducing agent, most still in the experimental study phase, In addition to ATRA, inducing differentiation of new domestic and foreign agents have not yet entered the clinical treatment of leukemia.FTC, a naturally-occurring small molecule compound, purified from the leaves of Viburnum odoratissimum Ket. ,which had been widely used in traditional medicine. The results showed that FTC induced leukemia cells differentiation with high activity:â‘ Strong differentiation inducing activity: better than ATRA, and similar to PMA, a classic PKC agonist;â‘¡Wide indications: effective in differentiation in APL, a variety of acute and chronic myeloid leukemia and lymphocytic leukemia;â‘¢Broad effective dose range: the effective dose range of FTC was equivalent to ATRA, and higher than PMA, but different with PMA, FTC induced differentiation in a good dose-range manner;â‘£In the dose range of induction of differentiation, FTC can inhibit leukemia cell proliferation and significantly reduce their tumorigenicity;⑤Synergistic with ATRA induced differentiation of leukemia, can be combination therapy.We also found that the specific inhibitors of PKC could completely reversed the differentiation inducing activity of FTC, and can be induced ERK phosphorylation in cells in a time and dose dependent manner , and can be blocked by PKC inhibitor. This showed that FTC induces differentiation of leukemia cells by PKC/ERK signaling pathway.PKC is a multiple gene super family of enzymes that are involved in controlling the function of other proteins through the phosphorylation of hydroxyl groups of serine and threonine amino acid residues on these proteins. PKC enzymes in turn are activated by signals such as increases in the concentration of diacylglycerol or Ca²⁺. Hence PKC enzymes play important roles in several signal transduction cascades. The PKC family consists of at least twelve isozymes. They are divided into three subfamilies, based on their second messenger requirements: conventional (or classical), novel, and atypical. Conventional (c)PKCs contain the isoformsα,βI,βII, andγ. These require Ca²⁺, diacylglycerol (DAG), and phospholipid such as phosphatidylserine for activation. Novel (n)PKCs include theδ,ε,η, andθisoforms, and require DAG, but do not require Ca²⁺ for activation. Thus, conventional and novel PKCs are activated through the same signal transduction pathway as phospholipase C. On the other hand, atypical (a)PKCs (including protein kinase Mζandι/λisoforms) require neither Ca²⁺ nor diacylglycerol for activation. The structure of all PKCs consists of a regulatory domain and a catalytic domain tethered together by a hinge region. The regulatory region consists of C1 and C2 domain. DAG and PMA bind to the C1 domain. We use [3H]PDBu binding assay to determine if FTC could bind to the PKC. The results showed that FTC could block [3H]PDBu binding to PKC in a concentration- dependent manner. Calculating the Ki value of FTC, its affinity to (c)PKCs is higher than (n)PKCs and PKD. Upon activation, protein kinase C enzymes are translocated to the plasma membrane by RACK proteins (membrane-bound receptor for activated protein kinase C proteins). The protein kinase C enzymes are known for their long-term activation: They remain activated after the original activation signal or the Ca²⁺-wave has gone. This is presumably achieved by the production of diacylglycerol from phosphatidylinositol by a phospholipase; fatty acids may also play a role in long-term activation. Like other kinases, PKC catalyzes the transfer of the terminal phosphate from ATP to a protein or peptide substrate. Use ofγ-32P-labeled ATP allows this transfer to monitored by scintillation counting. FTC could active PKC in a concentration-dependent manner. FTC couldn't active PKD.At the cellular level, we observed by confocal microscope that FTC could induce PKC-EGFP translocated to cell membrane within 30 min. Extracted protein from cytoplasm and membrane before and after FTC treatment, we proved by western blot that FTC had effected on PKC membrane translocation.Research has shown that, PKC agonist phorbol ester has the potentially cancer-promoting activity, while antagonists may be tumor suppressor substances. PMA is a typical PKC agonists. Smeared PMA on the skin of mice several times, mouse skin can cause acute inflammation, showed epidermal hyperplasia, redness and edema. Long-term repeated PMA smearing, could induce the occurrence of skin papilloma in mice so as to realize the occurrence and development of chemical carcinogenesis. This showed that the PMA is a tumor-promoting agent.Acute inflammatory reaction is one of the most significant reaction after tumor-promoting agent smeared. Earlier, severer and longer duration of acute inflammation indicated stronger tumor promoting activity. Blocking or inhibiting the tumor promotion process, can achieve the purpose of preventing tumors.We used acute flammatory of mice skin model to determine whether the FTC has the potential carcinogenic activity and whether it has anticancer activity. Acute inflammation in mouse skin mainly results in epidermal hyperplasia, skin redness and edema. The results showed that compared with PMA, FTC has no obvious hyperplasia effect at 200 times doses of PMA. FTC can also antagonize the effect of epidermal hyperplasia induced by PMA, giving the greater dose of the second administration, the smaller dose of the first administration could achieve better antagonistic effect. 12h after applying 5μg PMA, we could see the skin swelling, pale, soft, cut surface jelly-like and edema. The FTC applied showed no edema-like changes in mouse skin. On mouse skin moisture content/dry weight map, we could see that compared with control group, mice skin od PMA group showed obvious edema, but FTC group showed no differences with control group, and within antagonizing epidermal hyperplasia effective dose, FTC could effectively antagonize edema induced by PMA. After applying PMA to the mouse ear 3h, mouse ear showed red, thickened, vascular filling, the helix and the tragus swollen like, while the FTC group and antagonistic group showed only a slight congestion on ear. FTC group and the antagonistic groups achieved marked recovery after 6h. PMA group did not appear recovery after 24h, vascular filling still obvious, no reduction in tragus and helix swelling. This result suggests that, FTC just caused minor, short duration inflammation in mice ear, but PMA induced ear inflammation for a long time. Plus, FTC could antagonize ear inflammation induced by PMA. FTC could significantly inhibited acute inflammation caused by PMA in three aspects of epidermal hyperplasia, skin edema and ear swelling, showing good resistance to mutation effects. It indicated that FTC has no carcinogenic activity and could block the carcinogenic effect of PMA.Prostratin is a PKC agonist, can help AIDS treatment. By detecting the molt-4 cells line CD4/CXCR4 surface antigen, FTC can be proved decrease CD4/CXCR4 positive cells in a time and dose dependented manner, and the effective dose of CXCR4 is lower than the effective dose of CD4.In short, FTC, a naturally-occurring small molecule compound, is a new type of PKC agonist, which induced leukemia cell differentiation through activation of PKC / ERK pathway, which also may be able to assist in AIDS treatment. FTC is also a potential tumor suppressor agent, which has enormous values in new drug development, and provides a novel small molecule probe for the function study of PKC isoforms.