Study On Effects Of Trans-cinnamaldehyde On Myeloid Leukemia Cells In Vitro

PART I Effects of Trans-cinnamaldehyde on the Proliferation and Apoptosis of Acute Myeloid Leukemia CellsObjective To study the effects of trans-cinnamaldehyde (TCA) on the proliferation and apoptosis of acute myeloid leukemia (AML) cells.Methods HL60 AML cells and bone marrow mononuclear cells(BMMNC)from patients with de novo AML and healthy donors were treated by various concentrations of TCA and / orβ-D-Arabinofuranosyl cytosine (AraC). The proliferation of HL60 cells were detected by CCK-8 assay. Flow cytometry was empolyed to detect the cell cycle disribution of HL60 cells. The apoptosis of HL60 cells and BMMNC was measured by Annexin V/PI double labeled, and the apoptosis of BMMNC CD34+ cells were examined by triple labled with CD45/CD34/Annexin V. Western blot assay was employed to quantify the expression of c-myc gene in HL60 cells treated by TCA.The activity of NF-κB was investigated by confocal. Furthermore, the colony formation capability of BMMNC treated by TCA were determined by cytokines-riched methylcellulose culture assay.Results TCA affected the proliferation of HL60 cells in a time- and dose-dependent fashion. Low concentrations of TCA (10μM, 20μM) promoted the growth of HL60 cells at 24 h, different from untreated control significantly (P<0.05). Higher concentrations of TCA inhibited proliferation of HL60 cells. TCA administrated at any concentraion halted proliferation of HL60 cells at 48 h and 72 h. The values of IC₅₀ of TCA on HL60 cells were 78.40μM, 58.13μM and 50.31μM at 24 h, 48 h and 72 h, respectively. High concentrations of TCA (≥80μM) induced more than 10% cells to undergo apoptosis. More apoptosis could be seen in HL60 cells treated by increased concentrations of TCA. Less than 5% HL60 cells underwent apoptosis when treated by TCA with the concentration no more than 60μM for 24 h. The apoptosis of HL60 cells treated by middle concentrations of TCA (40μM, 60μM) increased when the treated time was prolonged. Low concentrations of TCA (10μM, 20μM) could not induce significant apoptosis of HL60 cells at any time. HL60 cells were arrested at G2 /M phase after treated by middle concentraions of TCA for 24 h. Low concentraions of TCA did not affect the cell cycle of HL60 cells at 24 h but accumulated HL60 cell at G2 /M phase at 72 h. c-Myc protein was highly expressed in HL60 cells and TCA inhibited the expression of c-Myc in HL60 cells in a time- and dose- dependent manner. The activity of NF-κB in HL60 cells was inhibited by treatment of 100μM TCA for 4 h. TCA induced apoptosis of AML BMMNC and AML CD34+ cells time- and dose-dependently.The cytotoxicity of TCA on normal BMMNC was slight. TCA inhibited colony formation of AML BMMNC. Furthermore, TCA synergized the cytotoxicity of AraC on HL60 cells, AML BMMNC and AML BMMNC CD34+ cells as well.Conclusion TCA exhibited anti-leukemia charateristic by inhibiting cell cycle progression, inducing apoptosis, deactivating NF-κB and repressing the expression of c-myc gene. TCA leaded AML BMMNC cells and AML CD34+ cells to significantly apoptosis with little cytotoxicity on normal BMMNC. Moreover, TCA synergized the anti-leukemia effect of AraC.PARTâ…¡Effects of Trans-cinnamaldehyde on the Proliferation and Apoptosis of Chronic Myeloid Leukemia CellsObjective To study the effects of trans-cinnamaldehyde (TCA) on the proliferation and apoptosis of chronic myeloid leukemia (CML) cells. Methods K562 CML cells and bone marrow mononuclear cells(MNC)from patients with de novo CML and healthy donors were treated by various concentrations of TCA. The proliferation of K562 cells was detected by CCK-8 assay, Flow cytometry was employed to measure the cell cycle disribution of K562 cells, the apoptosis of K562 cells and primary CML cells, the mitochondrial transmembrane potential (△ψm) and Fas expression of K562 cells. Western blot assay was employed to quantify the expression of c-myc gene and the phosphrylation of Crkl in K562 cells treated by TCA. Real time PCR was employed to quantify the Bcr-Abl mRNA in K562 cells treated by TCA. Furthermore, the colony formation capability of CML BMMNC treated by TCA were determined by cytokines-riched methylcellulose culture assay.Results TCA affected the proliferation of K562 cells in a time- and dose-dependent fashion. Low concentration of TCA (30μM) promoted the growth of K562 cells at 24 h and 48 h, while inhibited proliferation of K562 cells at 72 h, different from untreated control (P<0.01). Higher concentrations of TCA inhibited proliferation of K562 cells. The valueds of IC₅₀ of TCA on K562 cells were 157.42μM at 24 h. Low concentration of TCA (30μM) could not induce apoptosis of K562 cells at any time. Middle concentration of TCA (60μM) induced just a small portion of cells undergoing apoptosis. High concentrations of TCA (≥90μM) leaded more than 10% HL60 cells to apoptosis. More apoptosis could been seen in K562 cells treated by increased concentrations of TCA and prolonged treating time. K562 cells were arrested at G2/M phase significantly by 60μM TCA while slightly by 30μM TCA at 24 h. c-Myc protein was highly expressed and Crkl were highly phosphorylated in K562 cells. TCA inhibited the expression of c-Myc and phosphorylation of Crkl in K562 cells in a time-and dose- dependent manner. TCA caused collapse of mitochondrial transmembrane potential and increased the expression of Fas in K562 cells in a time- and dose- dependent fashion. TCA inhibited the level Bcr-Abl mRNA in a dose dependent manner. TCA induced apoptosis of CML MNC dose dependently. Furthermore, TCA inhibited colony formation of CML MNC in a dose-depedent manner.Conclusion Low concentration of TCA (30μM) exhibited pro-proliferation capability of K562 cells at 24 h and 48 h and anti-leukemia charateristic by inhibiting cell cycle progression. K562 cells treated by middle concentraion of TCA (60μM) were accumulated at G2 /M phase at 24 h and induced to apoptosis at 72 h. High concentraions of TCA (≥90μM ) inhibited proliferation of K562 cells by inducing K562 cells apoptosis in a time- and dose-dependent fashion. Two major death pathways, mitochondrial pathway and Fas pathway, were both involved in the apoptosis of K562 cells induced by TCA. TCA probabily induced apoptosis and proliferation inibition of CML cells by inhibiting the transcription of Bcr-Abl, which resulted in the decreased expression of BCR-ABL protein and diminished the effects of PTK, and subsequently downregulted expression of c-myc. In conclusion, TCA might be a promising drug with strong cytotoxicity on CML cells while little effects on normal BMMNC.PARTâ…¢Trans-cinnamaldehyde induced differentiation of Myeloid Leukemia CellsObjective To study the effects of trans-cinnamaldehyde (TCA) on the differentiation of myeloid leukemia cells.Methods HL60 AML cells and K562 CML cells were treated by TCA and/or all-trans-Retinoic acid(ATRA). The morphology characteristic of cell treated by TCA was investigated by phase contrast microsope and stained by Wright's-Gimsa. Flow cytometry was used to examine the differentiation antigens on TCA treated cells, the cell cycle distribution and apoptosis of TCA treated cells. Western blot assay was employed to quantify the expression of c-myc and p27 in HL60 cells and c-myc expression and phosphorylation level of Crkl in K562 cells. Confocal was employed to detect the expression of p16 and Cdc6 in HL60 cells.Results TCA (≤60μM) induced HL60 to differentiate toward mature granule cells with the upregulation of CD11b on HL60 cells. TCA(≤60μM) induced K562 cells to differentiated toward monocytoid cells with CD14 and CD11b upregulated on K562 cells. Low concentration of TCA (20μM) arrested HL60 cells at G₀/G₁ phase at 72 h without significant apoptosis. Middle concentration of TCA(60μM) accumulated HL60 cells at G2/M at 24 h with the percentage of cells at G₀/G₁ phase decreasing. Middle concentration of TCA (60μM) induced significant apoptosis of HL60 cells at 48 h. Middle concentrations of TCA (60μM) increased the percentage of K562 cells at G2/M phase at 24 h and at 48 h, while decreased the percentage of G₂/M phase at 72 h with significant apoptosis. TCA induced HL60 cells differentiation accompanied by decreased expression of c-Myc protein and increased expression of p27 protein. The expression of c-Myc and phosphorylation level of Crkl in K562 cells decreased when K562 cells were induced to differentiation by TCA. TCA induced upregulated expression and translocation to nuclear of p16 but downregulated expression and translocation to cytoplasm of Cdc6 in differentiated HL60. TCA enhanced the differentiaiton of HL60 cells induction by ATRA.Conclusion Low and middle concentrations of TCA induced myeloid cells to differentiate with different response in differente kind of cells and different concentraion of TCA. The differentiation of HL60 induced by TCA was related to depressed c-Myc protein and upregulated p27 protein. p16 and Cdc6 were involved in the differentiation of HL60 induced by TCA. The diferentiation effects induced by ATRA could be enhanced by TCA in HL60 cells. The differentiation of K562 cells induced by TCA was related to downregulation of c-Myc protein and phosphorylation level of Crkl protein.PARTâ…¥Effects of Trans-cinnamaldehyde on the Adhesion and Migration of Acute Myeloid Leukemia CellsObjective To study the effects of trans-cinnamaldehyde (TCA) on adhesion and migration of acute myeloid leukemia (AML) cells.Methods Expression of CXCR4 on HL60 AML cells treated by TCA was detected by Flow cytometry. The effects of TCA on the migration and invasion of HL60 cells induced by recombinant human stromal cell derived factor-1α(rhSDF-1α) were investigated by transwell assay. HL60 cells were stained by carboxyfluorescein succinimidyl ester (CFSE). Staining of HL60 cells was determined by confocal and Flow cytometry. Confocal was also employed to detected the adhesion of CFSE stained HL60 cells co-cultured with AML bone marrow stromal cell (BMSC) treated by various concentraions of TCA. Secretion of SDF-1αwas quantified by ELISA. The morphologic characteristic of AML BMSC treated by TCA was oberved by phase contrast microscope. Flow cytometry was used to determine the apoptosis of AML BMSC.Results Mean fluorescence intensity(MFI)of CXCR4 on HL60 cells treated by TCA decreased in a time- and dose-dependent manner. The migration and invasion of HL60 cells induced by rhSDF-1αwere inhibited significantly in dose dependently. Bright green fluorescence of CFSE was observed in the cytoplasm and nuclear of CFSE-stained HL60 cells, while no fluorescence could be observed in unstained HL60 cells and AML BMSC in the co-culture system. The adhesion of HL60 cells to the AML BMSC was inhibited by treatment of TCA for 6 h.in a dose-dependent manner. The secretion of SDF-1αby AML BMSC was depressed by TCA treatment for 24 h and 48 h. Furthermore, AML BMSC underwent apoptosis in a time- and dose-dependent manner after treated by TCA as evidenced by cell morphologic change and Flow cytometry.Conclusion In addition to the direct effects on the leukmia cells, TCA impared the protection of AML BMSC and would contribute to the elimination of leukemia cells in bone marrow by inhibiting the chemoattraction and adhesion of leukemia cells to the BMSC via downregulating of CXCR4 expression on AML cells, inhibiting the secretion of SDF-1αby AML BMSC and inducing apoptosis of AML BMSC.PARTâ…¤Regulation of mel18 gene in myeloid leukemia cells by Trans-cinnamaldehydeObjective To investigate the expression and the significance of mel18 gene in acute myeloid leukemia (AML), the efffects of mel18 gene on the proliferation, apoptosis and cell cycle on the leukemia cells as well as the regulation of trans-cinnamaldehyde (TCA) on mel18 gene in AML cells.Methods The expression of mel18 mRNA was detected by reverse transcription polymerase chain reaction (RT-PCR) in leukemia cell lines. Avidin-biotin-peroxidase complex (ABC) immunohistochemistry stain (IHC) was employed to detect Me118 protein in leukemia cell lines, bone marrow mononuclear cells(BMMNC)from patients with de novo AML and healthy donors as well as peripheral blood mononuclear cells (PBMNC) from healthy donors. The change of Mel18 expression was determined by Flow cytometry (FCM) and IHC in HL60 induced to differentiated by phorbol-12-myristate -13- acetaet (PMA). The plasmid pLenti6/V5-mel18 was constructed by gene-recombination technology and confirmed by sequencing. pLenti6/V5-mel18 and pLenti6/V5-LacZ (control) were transfected into the U937 and HL60 cells with Lipofectamine2000? with parent cells as the blank. The change of mel18 mRNA level in different groups was evaluated by RT- PCR while the Mel18 protein was determined by FCM. Proliferation of the cells was analyzed by CCK-8. Flow cytometry was employed to detect the apoptosis and cell cycle distribution. Mel18 expression in HL60 induced to differentiated by TCA was investigated by flow cytometry and immunohistochemistry.Results K562 cells expresses mel18 mRNA strongly, HL60 cells expressed weakly and no mel18 mRNA was detected in U937 cells. Mel18 protein was detected in cytoplasm strong in K562 cells, weak in HL60 cells and normal BMMC. U937 cells doesnot express Mel18 protein. Expression of Mel18 protein couldnot be detected in 58.8 % of AML samples while could be detected in 100% of the normal BMMC samples. The expression of Mel18 increased in HL60 cells induced to differentiate by PMA. The sequence of the recombinant pLenti6/V5-mel18 was proved to be correct according to PCR and the sequence analysis. Expression of mel18 gene increased in U937 cells and HL60 cells after pLenti6/V5-mel18 transfection for 24 h. Transfection of pLenti6/V5-mel18 resulted in halted cell proliferation, increased apoptosis and increased the percentage of cells at G₀/G₁ phase in U937 cells and HL60 cells. The expression of Mel18 increased in HL60 cells and K562 cells undergoing differentiation induced by TCA without changes of location.Conclusion The expression of mel18 gene decreased in AML cells and is correlated to cell differentiation. Overexpression of mel18 gene suppressed the proliferation, arrested cell cycle at G₀/G₁ phase and induced the apoptosis in leukemia cells, which meant mel18 could act as a antitumor gene in AML. TCA increased the expression of mel18 gene by inducing differentiation of leukemia cells.