[Effect and Mechanism of Sulforaphane on G2/M Phase Arrest of Acute Myeloid Leukemia KG1a and KG1 Cells].

OBJECTIVE: To investigate the effect of sulforaphane (SFN) on G2/M phase arrest of acute myeloid leukemia cells and its molecular mechanism. METHODS: KG1a and KG1cells were treated by different concentrations of SFN for 48 h. Flow cytometry (FCM) was used to analyze the phase distribution of cell cycle. High-throughput sequencing was used to detect the effect of SFN on the expression of cell cycle related genes in KG1a cells. The mRNA expression of P53, P21, CDC2 and CyclinB1 were detected by qPCR. The protein expression of P53, CDC2, P-CDC2 and CyclinB1 were detected by Western blot. RESULTS: Cells in the G2/M phase were increased from 11.9% to 54.0% in KG1a cells and 18.5% to 83.3% in KG1 cells after treated by SFN (8 µ mol / L) for 48 hours（P<0.001）. KEGG analysis indicated that P53 pathway was enriched in KG1a cells after treated by SFN. The heat-map graph showed that SFN could change the relevant genes of the cell cycle in KG1a cells. After SFN treatment, the mRNA level of P53 and P21 were significantly increased in KG1 and KG1a cells（P<0.05 or P<0.01）. The mRNA level of CDC2 showed a decrease trend with the increasing dose of SFN. At the dosage of 8 µmol /L, the mRNA expression levels of CDC2 was significantly lower than that in control group（P<0.05）. At the same time, the protein level of P53 was significantly increased in KG1 and kG1a cells after treated by SFN（P<0.05）. The protein level of CDC2 showed a decrease trend with the increasing dose of SFN in a dose manner（r=0.9482 and r=0.8977）. The protein levels of CDC2 in SFN 8 and 12 µ mol/L groups were significantly lower than that in control group(P<0.05, P<0.01). The protein levels of P-CDC2 was increased. But the change of mRNA and protein level of CyclinB1 was not significant. CONCLUSION: SFN induces leukemia cells to block in G2/M phase by activating P53 signaling pathway, which can inhibit the expression of CDC2 and the activity of CDC2/cyclinB1.

[Effects of Arsenic Trioxide on K562 Cell Proliferation and Its Mechanisms].

OBJECTIVE: To investigate the effects of arsenic trioxide (As2O3) on K562 cell proliferation by regulating cell cycle protein D1 and cyclin-dependent kinase inhibitor p27kip1. METHODS: MTT was used to detect the effect of As2O3 on K562 cell proliferation, so as to screen out the appropriate drug concentration. Furthermore, the K562 cell apoptosis was observed by microscopy. The expression of CyclinD1 and p27kip1 in K562 cells treated with As2O3 was analyzed by reverse transcription-polymerase chain reaction(RT-PCR), immunohistochemistry and Western blot. RESULTS: As2O3 could inhibit the proliferation of K562 cells in a dose- and time- dependent manner (r= 0.967). And the apoptosis cell number in As2O3 group was significantly higher than that in the control group(P<0.05). As2O3 could markedly inhibit the expression of CyclinD1 in K562 cells(P<0.05), but the expression of P27kip1 was not significantly changed after As2O3 treatment. CONCLUSIONS: As2O3 can induce K562 cell apoptosis and inhibit K562 cell proliferation by regulating the expression of CyclinD1.

[Mannan-Binding Lectin Inhibits Candida Albicans-Induced DC Maturation and Cytokine Secretion].

OBJECTIVE: To investigate the effects of mannan-binding lectin (MBL) on the maturation and cytokine secretion of human dendritic cells (DC) induced by Candida albicans (C. albicans). METHODS: The plastic-adherent mononuclear cells were prepared from the blood of healthy adult volunteers. The human peripheral blood mononuclear cells-derived dendritic cells (MNC-DC) were induced by 5-day-culture in medium supplemented with rhGM-CSF and rhIL-4, and then cultured for 2 days in presence or absence of C. albicans at varying concentration of human MBL ranging from 1 to 20 mg/L. DC's shape and characters were observed under inverted microscopy, the expression of CD83 and CD86 on DC was analyzed by FACS. The levels of TNF-α and IL-6 were detected by ELISA. FACS also was used to investigate the interaction of MBL with immature DC(imDC) and C. albicans. Western blot was used to detect C. albicans-induced IκBα phosphorylation and p65/NF-κB translocation in DC. RESULTS: MBL at higher concentrations (10-20 mg/L) down-regulated the expression of CD83 and CD86 on the monocyte-derived dentritic cells(MoDC) induced by C. albicans, and inhibited the production of TNF-α and IL-6 induced by C. albicans. FACS showed that MBL could not only bind to C. albicans but also bind to imDCs in a Ca2+-dependent manner. Western blot showed that MBL could decrease the phosphorylation of IκBα and the nuclear translocation of p65/ NF-κB. CONCLUSION: MBL may inhibit TNF-α and IL-6 production induced by C. albicans in DC through NF-κB signaling pathways, suggesting that MBL can play some roles in the regulation of C. albicans-induced immune response.

[Mechanism of MBL inhibiting the LPS-induced DC maturation].

The study was aimed to investigate the mechanism of mannan-binding lectin (MBL) on bacterial lipopolysaccharide (LPS)-induced human peripheral blood monocyte-derived dendritic cell (DC) maturation. The monocytes were prepared from the peripheral blood of healthy adult volunteers. The immature dendritic cells (imDC) were induced by 5-day-culture in medium supplemented with rhGM-CSF and rhIL-4. FACS was used to investigate the interaction of MBL with imDC and the impact of MBL on LPS binding to imDC. ELISA and Western blot was used to analyze the interaction of MBL with soluble TLR4 ectodomain protein (sTLR4); Western blot was used to detect LPS-induced NF-κB translocation in imDC. The results showed that MBL could directly bind to imDC in the presence of calcium. sTLR4 protein or LPS could competitively inhibit the binding of MBL to imDC. ELISA and Western blot showed that MBL could evidently bind to sTLR4 protein in a concentration-dependent manner. FACS showed that MBL could competitively inhibit the binding of LPS to imDC by binding to imDC directly. Western blot showed that MBL decreased LPS-induced NF-κB translocation in imDC. It is concluded that MBL may competitively inhibit the binding of LPS to imDC by binding to TLR4 expressed on imDC, resulted in inhibition of LPS-induced DC maturation, suggesting that MBL can regulate DC maturation through ligand-binding. This study provides the good foundation to clarify the mechanism of MBL inhibiting the LPS-induced DC maturation.