Study On Apoptosis Induced By RBTI In Leukemia Cells

Apoptosis is a cell initiative death course, which caused by cells' internaland external environment changes, death signal touching off and theregulation of gene. This course plays an important role for dispelling theaging cell in the organism or having potential cell that unusually grew, andkeeping the organism being in homeostasis. The disorder of internalapoptosis regulation and control mechanism will cause human body manykinds of illnesses. The formation of tumors and the senile dementia belong tothis kind of typical illness. A lot of researchers at present focus on study ofprotease and protease inhibitor in the cell and they think these moleculesmight play a key role in apoptosis. Leukemia is a virulent blood disease,which has serious health implications in humans. Leukemia, as with manyother cancers, tends to spread throughout the body without any symptoms. Atpresent, chemical treatment is the main method for treatment of leukemia,but the side effects are a significant drawback. Although natural productshave long been a fertile source of cures for cancer, there has been a desperateand continuous need for development of new anticancer drugs aimed atkilling cancer cells. It is, therefore, imperative to find a new and effectivemedical treatment that can prevent and cure cancer. It has been reported thatthe Bowman-Birk family of inhibitors, obtained from soybeans and otherlegumes, are potentially nutritionally relevant anti-carcinogens, particularlywith respect to colon cancer. It was proposed that the trypsin inhibitor inbuckwheat seed seems to induce apoptosis of the blood disease cells. But therelated research has not been reported so far. The mechanism of plantprotease inhibitor in inducing tumor cell apoptosis is also not clear.Buckwheat(Fagopyrum esculentum Moench) is planted in many regionscovering Asia, Europe, North America, Austria, South Africa, and other countries in the world, but its birthplace locates in the southwest region ofChina. In many regions it even is the major food. Nutrition abounds inbuckwheat, whose seeds contain some nutritive substances nonexistent orinsufficient in other cereal crops. Buckwheat is known as a valuable sourceof protein, and chemical analyses of hydrolysates indicate that the amino acidcomposition of buckwheat is nutritionally superior to that of cereal grains.Due to its unique chemical and bio-activity components, buckwheat has beenfound to have many uses in food products and medicine. At the present time,research in the area of recombinant protease inhibitors as anti-neoplasticmedical treatments is still not evident. Our Lab obtained a high-purity rBTIby cloning, expression and one-step affinity purification, and the analysisshowed that the recombinant buckwheat protease inhibitors has the samefunction with the native trypsin inhibitors in buckwheat seeds.Here, we investigated the possible effects of a recombinant buckwheattrypsin inhibitor (rBTI) on the induction of apoptosis of the human K562 cellline (chronic myeloid leukemia cells) and HL-60 cell line (acute myeloidleukemia cells), to find out its mechanism of induce apoptosis. First weanalysis the effect of rBTI to K562, HL-60, PBMCs (normal humanperipheral blood mononuclear cells) by MTT (3-[4, 5-dimethylthiazol-2-yl]-2, 5-diphenyltetrazolium bromide) assays, DNAfragmentation and electrophoresis, flow cytometric analysis andmorphological observation of nuclei, the results indicated that rBTI caninduce apoptosis of K562 cells and HL-60 cells. Then using wastern blottinganalysis the realease the cytoChrome C from cytoplasm, using flowcytometric analysis Mitocapture and using Colorimetric assay analysisCaspase-3, find out the possible apoptosis-induction pathways.