Clinical study of compound Zhebei particles target of anti AML in LSC
|Beijing University of Traditional Chinese Medicine
|Traditional Chinese Medicine
|Leukemia stem cells Molecular biomarkers CompoundZhebei Granule Acute myeloid leukemia
This article contains two parts. From literature review, leukemia stem cell (LSC) which has self renewal capacity and infinite proliferation potential is the root of acute leukemia. LSC is in stationary stage, reluctant to most chemotherapy drugs, and easily causes multidrug resistance in leukemia cells. LSC concentrates in bone marrow niche where chemotherapy drugs can’t reach and owns natural resistance characteristics, so LSC can avoid drug’s attack to be a risk factor of relapsed acute leukemia. When most acute leukemia patients are in clinical complete remission stage, minimal residual diseases including LSC could repeatedly activate into cell cycles, realize self renewal and infinitely proliferated, then acute leukemia patients will become relapsed. In clinical observation, some refractory acute leukemia patients turn out to serious myelosuppression after chemotherapy, but they will relapse quickly with mass leukemia cells possessing over expression of drug-resistant related proteins in peripheral blood or bone marrow after drugs withdrawl. This indicates that LSC has biological characteristics of high proferation potential and multidrug resistance. Targeting on leukemia stem cell may be one of best therapies to overcome leukemia cells’multidrug resistance. In previous studies, Compound Zhebei Granule plus chemotherapy can improve clinical efficacy of refractory acute leukemia, which curative mechanisms rely on inducing cell apoptosis, down regulating over expression resistant proteins on membrane surface and adjusting abnormal changes of enzymes about multidrug resistance. These studies focus on leukemia cells and lack further researching on its function of leukemia stem cell.Objective: this thesis is to investigate the efficacy of Compound Zhebei Granule plus chemotherapy on acute myeloid LSC’s surface markers, and to detect the expression of PI3K/Akt/mTOR and NF-κB signal pathway biomarkers (PTEN, PI3K, mTOR, NF-κB) and bone marrow peripheral microenviroment’ scell surface marker (CD44) related with bone marrow cells in patients with refractory acute myeloid leukemia.Methods:From December,2011to August,2013, all cases who were candidates for refractory acute myeloid leukemia criteria were included from department of oncology and hematology in Dongzhimen hospital affiliated Beijing University of Traditional Chinese Medicine, department of hematology in Renmin hospital affiliated Beijing University and department of hematology in Langfang hospital of Chinese medicine. The common chemotherapy has HAE, MA, CAG, AA, EA, FLAG, et al. Compound Zhebei Granules are composed of fritillaria12grams,ligust icum9grams and tetrandr ine9grams, and is made into granule by Beijing tcmages pharmaceutical company limited. Compound Zhebei Granules were taken before chemotherapy in three days, once one bag, and twice a day, lasting fourteen days, twenty-one days a treatment cycle. Two milliliters bone marrow were analyzed by flow cytometry to evaluate the quantity of CD34+CD123+、CD33+CD123+leukemia stem cell. Thirty patients had undergone Compound Zhebei Granules plus chemotherapy and were abstracted bone marrow twice, the other twenty three patients were abstracted bone marrow once. Twenty patients in department of oncology and hematology in Dongzhimen hospital affiliated Beijing University of Traditional Chinese Medicine have abstracted bone marrow biopsy, then to do immunohis tochemistry to detect PTEN, PI3K, mTOR, NF-κB and CD44in all the samples. Results:①The proportions of CD34+CD123+, CD33+CD123+cells in refractory AML respectively are11.78%,12.86%, the correlation coefficient between them is0.953, both of them have significant correlation (p<0.001).②Eighty-three cases of refractory acute leukemia had been tested the proportions of CD34+CD123+, CD33+CD123+cel Is. According to diagnostic criteria and therapeutic standards, the proportions of LSC in refractory AML patients who were under remission stations are0.66%,0.92%, which have a significant decrease when compared with the proportions of leukemia stem cell in refractory AML patients who were under relapsed stations.③Thirty refractory AML patients had gone through Compound Zhebei Granules plus chemotherapy. Compared with the prior chemotherapy group (17.05%,17.71%), the proportions of LSC cells in post-treatment group (5.49%,5.94%) have a significant decrease (p<0.05). To further investigation, the proportions of leukemia stem cell in twenty-five patients who were under relapsed stations also had a significant decrease when compared with the prior chemotherapy group. Ten patients in twenty-five who were refractory AML patients under relapsed stations had accomplished completely release with the effects of Compound Zhebei Granules plus chemotherapy, and the remission rate is40%. When compared with the unreleased group, the proportions of CD34+CD123+, CD33+CD123+cel Is have a statistical significant reduction (p<0.01)in clinical remission.④Twenty refractory AML pat ients who have biopsy bone marrow were divided into nine patients who were in remission stations and eleven patients who were unreleased stations. The positive expression rates of PTEN, mTOR, NF- kB, CD44and PI3K in nine remission patients were33.3%,33.3%,77.8%,22.2%and0%, meanwhile in eleven relapsed patients, the positive expression rates were63.6%,18.2%,90.9%,63.6%and0%. There were statistical differences in the percentage and mean optical density for PTEN, CD44between the two groups (p<0.05). Also the average optical density of PTEN, mTOR, NF-κB, CD44and PI3K in nine remission patients were91.16,393.58,642.66,441.78,217.28, meanwhile in eleven relapsed patients, average optical density of PTEN, mTOR, NF-κB, CD44and PI3K were292.51,223.00,775.35,840.06,316.21. There were statistical differences in the percentage and mean optical density for PTEN, CD44between the two groups (p<0.05). Conclusions:①The proportions of CD34+CD123+, CD33+CD123+cells have significant correlation and could be defined as LSC②The proportion of LSC could be utilized to judge acute leukemia patients station of sustained remission.③Compound Zhebei Granule can reduce the ratio of CD34+CD123+,CD33+CD123+LSC to improve the efficacy of refractory and relapse acute leukemia when combining chemotherapy, and Compound Zhebei Granule plus chemotherapy has a brilliant application prospect in the efficacy of refractory and relapse acute leukemia.④High expreesion of CD44could be an important index for diagnosis and prognosis in acute myeloid leukemia. PI3K/Akt/mTOR and NF-κB signal pathway in leukemia cells need further investigation.