The Molecular Mechanisms Of K562 Cells Apoptosis Induced By Xue Fu Kang And It’s Compositions

Chronic myeloid leukemia (CML) is a hematopoietic disorder characterized by themalignant expansion of bone marrow stem cells. Its cytogenetic hallmark is a reciprocalt(9;22)(q34;q11) chromosomal translocation that creates a derivative 9_q~+ and a small 22_q~-,known as the Philadelphia (Ph) chromosome. The latter harbors the bcr-abl fusion geneencoding a chimeric bcr-abl protein with a deregulated tyrosine kinase activity, theexpression of which has been shown to be necessary and sufficient for the transformedphenotype of CML cells. Otherwise, such as bcl-2 gene family, wt-p53 gene and Fas/FasLare also important genes to influence apoptosis.Xue Fu Kang is a new Chinese medicine, which is composed of Qingdai, Gegen andEzhu. It has been used to treat CML for more than a decade in Jiang Su province hospital ofTCM. It is effective and safety, and also well-tolerated by most patients. Previous study ofour group showed that Xue Fu Kang partially inhibited proliferation as well as inducedapoptosis of K562 cells. Expression of bcr/abl and P210 protein were down-regulated in adose dependent manner. This paper would further investigate molecular mechanisms ofsingle composition of Xue Fu Kang respectively.Objective: To study the effects of single composition of Xue Fu Kang respectively onproliferation and apoptosis of K562 cells, as well as on expression of bcr/abl and bcl-2 gene,the level of P210, bcl-2, wt-p53 and Fas/FasL protein.Methods: K562 cells were handled with different concentrations of Qingdai, Gegen,Ezhu(0 mg/ml、2.5 mg/ml、5 mg/ml、7.5 mg/ml、10 mg/ml、20 mg/ml)and were harvestedin 24 hours. In addition, they were also treated with different time(0、24、48、72、96 h)of 5 mg/ml Xue Fu Kang in K562 cells. The morphological changes were observed throughmicroscope; the changes of cells proliferation was examined by MTT; apoptotic cells weredyed by Hoechest 33258; apoptosis rate was determined with FITC-annexin V/PI doublestaining by flow cytometry; the changes of transcript and protein level of bcr/abl, bcl-2,wt-p53, Fas/FasL were determined by semi-quantitative RT-PCR and Western blot analysisrespectively.Results: 1. Morphological changes showed that with increase of Qingdai, Gegen, Ezhuconcentration, we observed inhibited proliferation and apoptosis characteristics in K562cells. Hoechest exhibited typical morphological changes of apoptotic cells underfluorescent microscope. 2. After treatment with different concentrations Qingdai andGegen, the expression of both bcr/abl mRNA and P210 proteins were decreased in aconcentration-dependent manner in K562 cells, and in a time-dependent decrease with XueFu Kang alone. 3. Both of the transcript and protein level of bcl-2 had no change indifferent treated groups. 4. The wt-p53 protein was up-regulated in Qingdai and Gegengroups, while Fas/FasL in Ezhu group in a concentration-dependent manner.Conclusion: Xue Fu Kang and the single compositions partially inhibitedproliferation and induced apoptosis of K562 cells. Qingdai, Gegen and Ezhu inducedapoptosis in a concentration-dependent manner. The mechanisms of Qingdai and Gegenmight be involved in down-regulation of bcr/abl fusion gene expression and P210 proteinlevel, as well as up-regulation of wt-p53 protein level, while Ezhu up-regulation ofFas/FasL expression. In summary, it suggested that Xue Fu Kang and the singlecompositions induce K562 apoptosis by different signaling pathways.