Clinical Analysis Of85Cases With Chronic Lymphocytic Leukemia And Effect Of Realgar On MEC-1Cells

Chronic lymphocytic leukemia (CLL) is the most common adult leukemia in Western countries. Although the disease mainly impacts elderly patients, the number of patients less than sixty years old are becoming increasingly more these years. CLL is a highly heterogeneous disease. The immunoglobulin heavy chain variable (IGHV) mutation status, cytogenetic changes, expression of CD38and ZAP-70differ in CLL patients. Furthermore, CLL has a diverse clinical process and distinct prognosis. And the heterogeneity of CLL makes it difficult for the treatment, especially target treatment. Even though survival of CLL has been improved over the past years, no other treatment is effective enough to cure the disease except allogeneic bone marrow transplantation. Especially for IGVH unmated CLL and CLL with disadvantageous cytogenetics, it still remains nearly impossible to prevent the disease from deterioration.Traditional Chinese medicine (TCM) has been used as effective antitumor agents for a long time in ancient China. Yet it is difficult to identify the active ingredient due to its complex composition. Thus the use of TCM was severely restricted in clinical practices after the introduction of western anti-neoplastic methods such as radiotherapy and chemotherapy. Recently the therapeutic efficacy of arsenic trioxide (AS2O3) was proven in the treatment of acute promyelocytic leukemia (APL) and the mechanism was illustrated as well. Consequently, TCM became a hot topic again and attracted much attention in aspects of cancer treatment, especially treatment of malignant hematologic diseases. Besides, many basic researches were conducted and a number of achievements have been obtained so far.Both AS2O3and arsenic sulfide (AS2S2) belong to Chinese arsenic drugs and they have many similarities. For example, AS2S2as well as AS2O3, has a good antitumor effect. It constitutes a formula called Realgar-Indigo naturalis formula (RIF) together with Indigo naturalis, Salvia miltiorrhiza, and Radix psudostellariae. RIF shows a satisfactory efficacy in treating human APL. Besides, AS2S2has been proven to induce apoptosis of chronic myelogenous leukemia (CML) cells through degradation of BCR-ABL. Recent researches showed that AS2O3could induce apoptosis of CLL cells.Therefore the purpose of our study is to analyze the clinical characteristics, laboratory features and main therapeutic methods of the newly diagnosed CLL patients in Shandong Province. In addition, the effect of AS2S2on viability, proliferation and apoptosis of human p53deleted/mutated CLL cell lines MEC-1and the potential mechanism mediating the effect were investigated.Part I Clinical Analysis of85Cases with CLLObjective To analyze the clinical characteristics, laboratory features and main therapeutic methods of the newly diagnosed CLL patients in Shandong Province.Methods A retrospective study of eighty-five newly diagnosed CLL cases was carried out for further analysis of their clinical characteristics, laboratory features and main therapeutic methods. Those patients covered all the CLL patients initially diagnosed and treated in Shandong Provincial Hospital from January2007to December2012. Results Eighty-five CLL patients were diagnosed based on the Chinese guidelines for the diagnosis and management of CLL (2011). Fifty-eight patients (31.8%) were male and twenty-seven cases (68.2%) were female. Male to female ratio was2.15:1and the average age was58±11.6. Patients less than50years old accounted for21.2%of all the cases. Four-fifths of the patients went to see doctors due to obvious symptoms or signs. The main clinical manifestations included lymphadenopathy, fatigue, splenomegaly, abdominal distension and fever. Only20%of the patients were discovered when having health examinations or being examined because of other diseases. According to the Rai system,48cases (56.5%) belonged to the low and intermediate risk group while37cases (43.5%) belonged to the high risk group. Hemoglobin and platelet count were lower in the high risk group than in the low and intermediate risk group. Peripheral blood lymphocyte percentage, bone marrow lymphocyte proportion and serum (32-microglobulin (β2-MG) were higher in the high risk group. The detection rates of CD38, ZAP70, IGVH mutation status and molecular cytogenetics in the CLL patienes were61.2%,35.3%,3.5%and18.8%respectively. About75.3%of the CLL patients needed treatment at diagnosis. Chemotherapy based on use of fludarabine was the main therapeutic method.Conclusions CLL mainly impacts elderly patients. The majority of patients went to see doctors due to obvious symptoms or signs. Routine laboratory examinations (peripheral blood lymphocyte percentage, bone marrow lymphocyte proportion, serum β2-MG, hemoglobin and platelet count) differed between the low and intermediate risk group and the high risk group. The detection of molecular prognostic factors including CD38, ZAP70, IGVH mutation status and molecular cytogenetics needs to be intensified in the future for better evaluation of CLL prognosis. The main treatment of the newly diagnosed CLL patients was based upon use of fludarabine.Part Ⅱ Arsenic Sulfide Induces Apoptosis in Chronic Lymphocytic Leukemia Cell Line MEC-1Objective The aim of our study is to investigate the effect of arsenic sulfide (AS2S2) on the viability, proliferation and apoptosis of human CLL cell line MEC-1. In addition, potential mechanism mediating the effect will be explored in the experiment.Materials and Methods Cultured MEC-1cells were incubated with various concentrations of AS2S2for24h,48h and72h. The WST-8assay was employed to evaluate the effect on the cell viability. The inhibitory effects on cell proliferation were determined by the BrdU cell proliferation ELISA. Apoptotic effect on MEC-1cells was evaluated by Annexin V-FITC/PI dual staining followed by flow cytometry analysis. The quantitative realtime PCR was performed to assess the expression levels of BAX, Bcl-xL, c-Myc and p21mRNA.Results The viability and proliferation were significantly reduced while the apoptotic rates increased in MEC-1cells after they were exposed to AS2S2. Besides, the expression of BAX and c-Myc mRNA was up-regulated and down-regulated respectively in As2S2-treated MEC-1cells.Conclusions AS2S2could inhibit viability and proliferation and induce apoptosis of MEC-1cells in a dose-and time-dependent manner. The effect might depend on the mitochondria apoptosis pathway. Our discovery may be beneficial to find new target therapy of CLL.