The comparison of plasma arsenic concentration and urinary arsenic excretion during treatment with Realgar-Indigo naturalis formula and arsenic trioxide in children with acute promyelocytic leukemia.

Realgar-Indigo naturalis formula (RIF) is a traditional Chinese medicine containing As4S4 and effective in treating acute promyelocytic leukemia (APL). The dose of RIF remains to be determined in pediatric patients. Comparison of plasma arsenic concentrations and toxicity between RIF and arsenic trioxide (ATO) treatment in APL may help to establish an appropriate therapeutic dose of RIF for children. From October 2018 to March 2020, 19 pediatric patients with APL treated with SCCLG-APL protocol were included, 9 in RIF group at 135 mg/kg/day orally three times daily, and 10 in ATO group at 0.16 mg/kg/day intravenously over 12 h daily. Peak and trough plasma arsenic concentrations were assayed at D1, 2, 7 and 14 of induction treatment. Urine arsenic excretions were assessed with spot urine samples and the measurements were adjusted using creatinine. Toxicities were compared between two groups. The plasma arsenic concentration reached steady state at D7 either in the RIF or ATO group, and the mean peak and trough concentrations were similar between two groups (P > 0.05), which were 0.54 µmol/L and 0.48 µmol/L in RIF group, and 0.63 µmol/L and 0.51 µmol/L in ATO group, respectively. Urine arsenic excretion rate was positively correlated with the concentration of plasma arsenic. The rates of treatment-related adverse events were similar in two groups. In conclusion, the dose of RIF at 135 mg/kg/day may be an appropriate therapeutic dose in children with APL. Urine arsenic level can be used as an indicator to estimate plasma arsenic concentration. Trial registration www.clinicaltrials.gov NCT02200978.

Effects of methylthiouracil on the proliferation and apoptosis of rat bone marrow stromal cells.

The aim of the present study was to investigate the effects of methylthiouracil (MTU) on the proliferation and apoptosis of rat bone marrow stromal cells (BMSCs). Rat BMSCs were isolated, cultured in vitro and treated with various concentrations of MTU. Cell growth curves were determined using the Cell Counting Kit-8 method and the effect of MTU on BMSCs in a logarithmic growth phase was observed. BMSC apoptosis following MTU treatment was detected by flow cytometry. The experimental results demonstrated that the proliferation-inhibition effect was gradually enhanced with increasing MTU concentrations and the extension of treatment time. Statistically significant differences were observed between the treatment and the control groups (P<0.05). In addition, the BMSC apoptosis rate gradually increased with increasing drug concentrations and treatment time extension; statistically significant differences were observed between the treatment and the control groups (P<0.05). Therefore, the results of the present study demonstrated that MTU inhibited the proliferation of BMSCs and promoted apoptosis, indicating the cytotoxic effects of MTU on BMSCs.

[Inhibition of vegfr-2 gene expression by RNA interference].

This study was aimed to explore a novel potential gene target for therapy of malignant tumor. The recombinant expression plasmids of VEGF/VEGFR-2 were designed, constructed and then transfected into A549 cells by using lipofectamine. The expressions of VEGF/VEGFR-2 mRNA and protein were detected by RT-PCR and Western blot respectively. The cell proliferation was assayed by CCK-8 and the cell apoptosis was detected using Hoechst Staining. The results indicated that as compared with the blank control, pGenesil-1 and scramble groups, the interference effect of pGenesil-1-vegfr-2-shRNA-1 vector group was more obvious. As the expression of endogenous vegfr-2 mRNA decreased, the expression of VEGFR-2 protein decreased correspondingly. The proliferation of A549 cells was inhibited significantly by RNAi at 72 hours (p<0.01). The apoptosis of A549 cells was induced at 48 hours after being transfected with pGenesil-1-vegfr-2-shRNA-1 and the typical apoptosis morphology could be seen by fluorescence microscopy. It is concluded that the expression of vegfr-2 gene is inhibited effectively by vegfr-2 specific shRNA. The proliferation of A549 cells is inhibited significantly and the apoptosis of cells is induced. This result showed that VEGF/VEGFR-2 signaling pathway can be an effective target for the prevention and treatment of malignant tumor.

[Effect of cytochrome C on HL-60 cell apoptosis and its relationship with the relevant genes bcl-2 and bax].

To study the effect of cytochrome C on HL-60 cells in vitro and the mechanism of expression changes of relevant apoptotic genes, the inhibition rate of cytochrome C on HL-60 cells was detected by MTT, the morphology of HL-60 cells was observed by light microscopy and fluorescence microscopy, the changes of apoptosis rate and cell cycle were assayed by flow cytometry (FCM), DNA ladder was investigated on electrophoresis, the expression changes of bax and bcl-2 mRNA were examined by RT-PCR, when HL-60 cells were treated with different concentrations of cytochrome C for 24 hours. The results showed that the inhibition rate increased with increase of the cytochrome C concentration within 0 - 150 mg/L; when treated with 0 - 37.5 mg/L cytochtome C for 24 hours, the percentage of apoptotic HL-60 cells increased with the dose increasing, and the typical apoptotic cells and the apoptotic DNA ladder were observed. At the same time, within this range of concentration, the expression of bcl-2 mRNA decreased gradually and the expression of bax increased gradually. When the cytochrome C concentration was higher than 37.5 mg/L, the percentage of apoptotic HL-60 cells not increased, but decreased, while the cells necrosed. The above metioned results suggested that at certain range of concentration of cytochrome C, apoptosis or necrosis can be induced by cytochrome C, and cell cycle arrests at G(1) phase in HL-60 cells, the percentage of apoptotic cells and the changes of expression of bax and bcl-2 depend on the dose of cytochrome C. The mechanism that cytochtome C induced apoptosis in HL-60 cells may be related to the activation of bax and inhibition of bcl-2.

[Effect of IL-15 on the proliferation, differentiation and anti-apoptosis of CD34+ cells in patients with MDS].

To study the effect of interleukin-15 (IL-15) on the proliferation, differentiation and apoptosis of MDS CD34(+) cells, CD34(+) cells of high enrichment were separated by MACS system, and cultured in liquid media with different concentration of IL-15 in treated group and without IL-15 in the control group. Apoptosis of hematopoietic precursors was assayed by propidium iodine staining and cell by FCM, and the other MDS CD34(+) cells were stained by cytochemical staining after culture. The results showed that after culture with IL-15 the proliferation and differentiation of MDS CD34(+) cells were obviously promoted. It was found the every lineage of mature cells developed, the expressions of cell surface antigens CD71, CD33 and CD19 all increased in the MDS CD34(+) cell treated with IL-15. It is suggested that IL-15 stimulates the proliferation and differentiation of MDS CD34(+) cells, and partly shows anti-apoptosis effects which may be applicable to the therapy MDS.

[Influence of cytochrome C on apoptosis induced by daunorubicine in acute myeloid leukemia (AML) cells].

The purpose was to study the responses of AML cell treated with cytochrome C and to explore the influence of cytochrome C on apoptosis of AML cell induced by daunorudicine (DNR). The differentiation of AML cell was detected by Wright-Giemsa staining and NBT test, the apoptosis of AML cell was assayed by flow cytometry and fluorescence microscopy. The results showed as follows: (1) different concentrations of cytochrome C could induce different effects on AML cells. Concentration of cytochrome C for differentiation was 10 microl/ml, for apoptosis was 20 microl/ml, and for necrosis was 40 microl/ml. (2) the apoptosis of AML cells decreased with the administration of cytochrome C in 10.0 microg/ml before treating AML cells with DNR (P < 0.01), but no change was shown with the administration of cytochrome C in 20.0 microg/ml (P > 0.05). (3) in reverse sequence, administrating of cytochrome C in 10 microl/ml and 20 microl/ml after treating AML cells with DNR, two different concentrations of cytochrome C could increase the apoptosis of AML cells (P < 0.01). It is suggested that cytochrome C may probably affect the apoptosis of AML cells induced by DNR.

[Effect of IL-15 on the proliferation and differentiation of CD34+ cells from MDS patients].

AIM: To explore the effect of IL-15 on proliferation and differentiation of CD34 (+) cells from MDS patients. METHODS: The CD34 (+) cells were separated by magnetic bead-activated cell sorter (MACS) system, and cultured in fluid or methylcellulose semisolid medium. MTT colorimetry was used to examine the inhibitory effect of IL-15 on the proliferation of MDS CD34(+) cells.The numbers of CD34(+)cells and colony formation of CFU-E, BFU-E, CFU-G, CFU-GEMM were counted. The expressions of CD33, CD13, CD71, CD19 and CD3 on cultured cells and the change of cell-cycle were analyzed by FCM. RESULTS: The recovery rate of CD34(+) cells was (75.4+/-5.2) %, the purity of CD34 (+) cells in positive fraction was (90.3+/-6.3) % and the enriched rate was (83.1+/-12.5) % in 11 MDS patients. MTT colorimetry detection showed that IL-15 could promote the proliferation of MDS CD34(+) cells. The optimal time of promotoing CD34(+) cell proliferation by IL-15 was 8 days and optimal dosage of IL-15 was 20 microg/L. After culture for 8 days with 0 microg/L IL-15 (control group) and 20 microg/L IL-15(experimental group), the number of CD34 (+) cells increased by 4.6-fold in control group and 6.3-fold in experimental group (P<0.05, n=5). The colony formation rates of experimental group were significantly higher than those of control group. The expression rates of the CD molecules (except for CD3) on CD34(+) in experimental group were notably higher than those in control group. As compared with control group, much more CD34(+) cells of experimental group were in G(2) and S phase of cell cycle(P<0.05, n=7). CONCLUSION: IL-15 can obviously promote the proliferation and differentiation of CD34(+) cells from MDS patients.