The Anti-tumoral Effect Of Curcumin Combined With Imatinib On Chronic Myelogenous Leukemia Cell Lines In Vitro

Chronic Myelogenous Leukemia (CML) originates in a pluripotent hematopoetic stem cell of the bone marrow and is characterized by the Philadelphia (Ph) chromosome. With an annual incidence of about ten in 1,000,000,CMLaccounts for most cases of myeloproliferative disease and for 20% of all leukemias, in China, this percentage is about 15-20% and is only inferior to acute myelogenous leukemia (AML) or acute lymphocyte leukemia (ALL). More recently, imatinib mesylate (STI571,Gleevec formerly CGP57148B; Novartis Pharmaceutics, Basel, Switzerland), a potent and highly selective Abl tyrosine kinase inhibitor of the 2-phenyl-amino pyrimidine class, was widely used in clinic for CML patients. Despite the hope that STI571 has generated for many CML patients, development of resistance to this drug is already apparent in some cases, especially if the CML is diagnosed in its later stages. Therefore, novel drugs which can be used alone or in combination with STI571 are highly desirable. To study the anti-tumoral effect of curcumin (Cur) combined with STI571 on chronic myelogenous leukemia cell lines, p210^Bcr-Abl positive of CML cell lines such as K562 cells, STI571 resistant K562/G01 cells and CML CD34⁺ cells were used as the cellular models for drugs screening in vitro.The anti-tumoral effects of cur on K562 cell growth arrestThis part was to study the effects of Cur on K562 cells growth and apoptosis,p210^Bcr-Abl,protein tyrosine phosphorylation and the signaling molecules, furthermore, to investigate the growth inhibition effect of Cur and Bcr-Abl phosphorothioate antisense oligonucleotides (ASODN) to observe the possible mechanisms of Cur on K562 cells. Bcr-Abl positive of K562 cells was used as a cellular model. Methods: 1,Trypan blue exclusive staining was used to observe the cell growth arrest; 2,MTT was used to access the cell growth inhibition; 3,Colony assay was to observe the cell colony forming units(CFUs); 4,AO/EB fluorescent staining was to detect the apoptotic rate; 5,RT-PCR was used to evaluate the gene expression of Bcr-Abl mRNA; 6,Western blot was used to observe the effect of Cur on p210^Bcr-Abl content,protein tyrosine phosphorylation and effects of Cur on K562 cells after Bcr-Abl mRNA was blocked by ASODN.The results showed as follow: 1,Cur inhibited K562 cell growth with time- and dose-dependent manners; 2,Cur inhibited K562 CFUs and showed dose-dependent manner; 3,Cur induced K562 cell apoptosis and showed dose-dependent manner; 4,Comparing to the control, no significant differences of Bcr-Abl mRNA were screened with Cur groups; 5,Cur down-regulated p210^Bcr-Abl and protein tyrosine phosphorylation at a time-and dose-dependent manners; Cur also down-regulated the signaling molecules which are relevant with cell growth and apoptosis such as Pro-caspase-3,p-Erk1/2,Hsp90,C-myc and up-regulated Cytochrome C(Cyt-C); 6,Combination of Cur and ASODN synergistically induced cell apoptosis,inhibition of K562 cells,down-regulation of p210^Bcr-Abl and the signaling molecules which are relevant with cell growth and apoptosis such as NF-КB and Hsp90.The anti-tumoral effects of the combination of Cur and STI571 on K562 cell growth arrestDespite the hope that STI571 has generated for many CML patients, development of resistance to this drug is already apparent in some cases. Therefore, strategies of combination with STI571 are highly desirable. The in vitro anti-tumoral effects of Cur reminded us, whether or not that cur could enhance STI571 in anti-CML response? This study focused on the evaluation of which cur alone or combined with STI571 on K562 cells. Methods: 1,Trypan blue exclusive staining was used to access the cell growth arrest and cytotoxity of drugs was directly observed by microscope; 2,Colony assay was to observe the CFUs; 3,Trypan blue exclusive staining was used to assess the inhibition of K562 cells and the effects of the sequential administrations; 4,Flow cytometry (FCM) was used to detect the penetration on the expression of annexin V/PI; 5,Western blot was used to observe the effects of Cur and STI571 on expression of p210^Bcr-Abl,protein tyrosine phosphorylation and the signaling molecules which are relevant with cell growth and apoptosis;The results showed: 1,Cur synergistically inhibited K562 cell growth, the combined index Q >1.15 for 24 h, while 0.851.15). However, simple addiction or antagonistic effects were observed when Cur was first administered for 24 h while STI571 was administered for another 24 h after Cur(Q<1.15); 4,Combination of Cur and STI571 synergistically induced K562 cell apoptosis; 5,Cur enhanced STI571 to down-regulate p210^Bcr-Abl content,protein tyrosine phosphorylation and the signaling molecules which are relevant with cell growth and apoptosis such as PKC and Hsp90;The anti-tumoral effects of the combination of Cur and STI571 on STI571 resistant K562/G01 cell growth arrestTo further investigate whether or not Cur could be used for STI571 resistant cell line—K562/G01 cells, the original K562 cell line which was cultured in gradually increased concentrations of STI571 over a period of several months to generate their resistance to STI571, was used as the cellular model. Methods: 1,Trypan blue exclusive staining was used to access the cell growth arrest and cytotoxity of drugs was directly observed by microscope; 2,Colony assay was to observe the CFUs; 3,Trypan blue exclusive staining was used to assess the inhibition of K562/G01 cells; 4,FCM was used to detect the penetration on the expression of annexin V/PI; 5,Western blot was used to observe the effects of Cur and STI571 on expression of p210^Bcr-Abl,protein tyrosine phosphorylation and the signaling molecules which are relevant with cell growth and apoptosis;The results showed: 1,Cur alone inhibited K562/G01 cell growth, IC₅₀ of 24 h was (6.5±0.5)μM ; STI571 alone inhibited K562/G01 cells growth, IC₅₀ of 24 h was (10±1.0)μM; Cur synergistically inhibited K562/G01 cells(Q>1.15); the chromatin condensation,nuclear fragmentation were observed after combination under microscope; 2,STI571 (2-16μM) alone gave rise to no effect on colony forming(P>0.05), however, Cur 2.5μM alone exerted significant inhibition on colony forming and enhanced STI571 to inhibit the colony forming at a dose-dependent manner; 3,Combination of Cur and STI571 synergistically induced K562/G01 cell apoptosis; 4,Cur enhanced STI571 to down-regulate p210^Bcr-Abl and the signaling molecules which are relevant with cell growth and apoptosis such as Pro-caspase-3 and NF-КB.The anti-tumoral effects of the combination of Cur and STI571 on CML CD34⁺ cellsCML is a clonal malignancy of the pluripotent hematopoietic stem cell, and CD34⁺ antigen was the glycoprotein that expresses on the membrance of the progenitor cells, it has the strong ability of self-duplication and differentiation, thus could produce the new generation that was completely copies of the original generation. Though CD34⁺ cells are in a very small percentage, they might be the root of occurrence,recurrence and resistance forming. We collected the patient's bone marrow. Written informed consent was obtained from participant before enrollment. CD34⁺ cells were isolated by fluorescence-activated cell sorting (FACS) and Bcr-Abl expression was detected by FCM. Then the CML CD34⁺ cells were used as the cellular model for drugs screening to study cell growth arrest, colony forming, apoptosis and p210^Bcr-Abl expression induced by Cur and STI571. Methods: 1,Trypan blue exclusive staining and MTT were used to observe the cell proliferation; 2,FCM was used to detect the penetration on the expression of annexin V/PI and p210^Bcr-Abl expression was detected by FCM.The results showed: 1,Time- dependent but no significant dose-dependent relationship was observed with STI571 (0.1-0.8μM)to CML CD34⁺ cells for 24-72 h, IC₅₀ for 24 h of STI571 was about 0.8μM which was less sensitive than to K562 cells; 2,Cur had significant dose- and time-dependent manners, IC₅₀ for 24 h of Cur was about 10μM; 3,Both trypan blue exclusive staining and MTT showed, combined index of STI571 ( 0.1-0.8μM) and Cur(5-20μM)to CD34⁺ cells was simple addiction(0.851.15); 4,Increased percentage of apoptotic and nectotic cells was detected for the combination of Cur and STI571 compared to the both treated alone; 5,STI571 exerted no effect on p210^Bcr-Abl expression in CD34⁺ cells (P>0.05 vs control); Cur down-regulated p210^Bcr-Abl expression and this effect was enhanced by combining with STI571.CONCLUSIONS:1,Cur inhibits K562 cells growth and induces cell apoptosis may be correlated with the down-regulation of p210^Bcr-Abl,inhibition of protein tyrosine phosphorylation and the signaling molecules such as p-Erk1/2,c-myc which are relevant with cell growth and apoptosis;2,Cur synergizes STI571 to inhibit K562 cell growth and induce cell apoptosis may be correlated with the down-regulation of p210^Bcr-Abl,inhibition of protein tyrosine phosphorylation and the signaling molecules such as Hsp90,PKC which are relevant with cell growth and apoptosis;3,Cur reverses the resistance of K562/G01 cells to STI571, and synergizes STI571 to inhibit K562/G01 cell growth and induce cell apoptosis;4,Cur inhibits human originated CML CD34⁺ cell growth,induces cell apoptosis, and enhances STI571 to down-regulate the expression of p210^Bcr-Abl , finally inhibit cell growth and induce cell apoptosis.All together, our results indicate that Cur enhances STI571 in vitro to inhibit K562 cell growth,reverse K562/G01 cells to STI571; moreover, Cur synergizies STI571 to inhibit human originated CML CD34⁺ cell growth and induce apoptosis. Cur may be a prosperous agent that can be developed for the treatment of CML.