Knockdown of Bcl-xL Enhances Growth-Inhibiting and Apoptosis-Inducing Effects of Resveratrol and Clofarabine in Malignant Mesothelioma H-2452 Cells

Mcl-1 and Bcl-xL, key anti-apoptotic proteins of the Bcl-2 family, have attracted attention as important molecules in the cell survival and drug resistance. In this study, we investigated whether inhibition of Bcl-xL influences cell growth and apoptosis against simultaneous treatment of resveratrol and clofarabine in the human malignant mesothelioma H-2452 cells. Resveratrol and clofarabine decreased Mcl-1 protein levels but had little effect on Bcl-xL levels. In the presence of two compounds, any detectable change in the Mcl-1 mRNA levels was not observed in RT-PCR analysis, whereas pretreatment with the proteasome inhibitor MG132 led to its accumulation to levels far above basal levels. The knockdown of Bcl-xL inhibited cell proliferation with cell accumulation at G2/M phase and the appearance of sub-G0/G1 peak in DNA flow cytometric assay. The suppression of cell growth was accompanied by an increase in the caspase-3/7 activity with the resultant cleavages of procaspase-3 and its substrate poly (ADP-ribose) polymerase, and increased percentage of apoptotic propensities in annexin V binding assay. Collectively, our data represent that the efficacy of resveratrol and clofarabine for apoptosis induction was substantially enhanced by Bcl-xL-lowering strategy in which the simultaneous targeting of Mcl-1 and Bcl-xL could be a more effective strategy for treating malignant mesothelioma.

Therapeutic effect of clofarabine in children with relapsed or refractory acute lymphoblastic leukemia / 中国当代儿科杂志

<p><b>OBJECTIVE</b>To explore the efficacy and adverse effects of clofarabine for relapsed/refractory acute lymphoblastic leukemia in children.</p><p><b>METHODS</b>Twenty-six pediatric patients with relapsed/refractory acute lymphoblastic leukemia were treated with clofarabine. There were 22 males and 4 females, with a mean age of 9.5 years (ranging from 4 to 17 years). They received clofarabine 52 mg/m2 intravenously over 2 hours daily for 5 days. Thirteen patients received two cycles and one patient received three cycles.</p><p><b>RESULTS</b>In the first cycle of clofarabine, complete remission was obtained in 11 children (42%) and partial remission was obtained in 7 children (27%). Eight children (31%) were considered unresponsive. In the second cycle, 11 (85%) of the 13 children obtained complete remission, 1 (8%) partial remission and 1 (8%) was unresponsive. One child received three cycles and obtained complete remission in each cycle. The common adverse events were myelosuppression, infection, liver dysfunction and gastrointestinal adverse reactions. There were no chemotherapy-related deaths.</p><p><b>CONCLUSIONS</b>Clofarabine is effective in the treatment of children with relapsed/refractory acute lymphoblastic leukemia and its adverse effects can be tolerated. Clofarabine could be a promising new treatment for relapsed/refractory acute lymphoblastic leukemia.</p>

Mechanism for clofarabine inducing autophagic death of acute myelocytic leukemia cell U937 / 中国实验血液学杂志

To explore the mechanism of autophagic death of acute myelocytic leukemia cell U937 induced by clofarabine, the MTT bioassay was used to analyze the growth inhibitory effect and half inhibition concentration on U937 incubated in vitro with different concentrations of clofarabine for 24 and 48 hours, and the flow cytometry was used to detect the autophagy rate of U937. The expression of Beclin 1 in U937 treated by clofarabine for 48h was measured by Western blot. The results indicated that when U937 cells were treated with 0.01 µmol/L and 0.15 µmol/L clofarabine for 48 hours, the proliferation inhibition rate was 46.92% ± 4.24% and 86.10% ± 1.16%, and the half inhibition concentration of clofarabine was 0.022 µmol/L. With 0.01 µmol/L and 0.1 µmol/L clofarabine on U937 for 48 hours, the autophagy rate was 11.0033% ± 1.4387% and 59.4133% ± 3.5409%, and increased in dose-dependent manner (r = 0.99). Meanwhile the Beclin 1 was upregulated along with increase of clofarabine concentration, as compared with control group, the difference was statistically significant (P < 0.05). It is concluded that the different concentrations of clofarabine can significantly inhibit the proliferation of U937 in dose-dependent manner, and the mechanism of autophagic cell death in U937 may be associated with the upregulation of Beclin 1 expression.

Sapacitabine, the prodrug of CNDAC, is a nucleoside analog with a unique action mechanism of inducing DNA strand breaks / 癌症

Sapacitabine is an orally bioavailable prodrug of the nucleoside analog 2'-C-cyano-2'-deoxy-1-β-D-arabino-pentofuranosylcytosine (CNDAC). Both the prodrug and active metabolite are in clinical trials for hematologic malignancies and/or solid tumors. CNDAC has a unique mechanism of action: after incorporation into DNA, it induces single-strand breaks (SSBs) that are converted into double-strand breaks (DSBs) when cells go through a second S phase. In our previous studies, we demonstrated that CNDAC-induced SSBs can be repaired by the transcription-coupled nucleotide excision repair pathway, whereas lethal DSBs are mainly repaired through homologous recombination. In the current work, we used clonogenic assays to compare the DNA damage repair mechanism of CNDAC with two other deoxycytidine analogs: cytarabine, which is used in hematologic malignacies, and gemcitabine, which shows activity in solid tumors. Deficiency in two Rad51 paralogs, Rad51D and XRCC3, greatly sensitized cells to CNDAC, but not to cytarabine or gemcitabine, indicating that homologous recombination is not a major mechanism for repairing damage caused by the latter two analogs. This study further suggests clinical activity and application of sapacitabine that is distinct from that of cytarabine or gemcitabine.

Effect of clofarabine on proliferation and Bcl-2 expression of NB4 cells / 中国实验血液学杂志

The aim of this study was to observe the effect of clofarabine on proliferation of NB4 cells and its possible mechanism. MTT method was used to detect proliferation of NB4 cells treated with clofarabine 0.01 - 0.1 µmol/L for 48 h. The treated with clofarabine 0.01 - 0.1 µmol/L for 24 h, apoptosis rate and Bcl-2 expression of NB4 cells were measured by flow cytometry and Western blot respectively. The results showed that clofarabine inhibited proliferation of NB4 cells in a concentration-depended manner (r = 0.78). After treated with clofarabine for 24 h, apoptosis rate of NB4 cells increased and Bcl-2 expression in NB4 cells decreased obviously (P < 0.05). It is concluded that clofarabine inhibits proliferation of NB4 cells, which may be related with the down-regulation of Bcl-2 and induction of apoptosis.