| A novel SAHA-bendamustine hybrid induces apoptosis of leukemia cellsObjective:Development of novel drugs with powerful biological activity and low toxicity has been a hot area for the research of cancer therapy. Hybrid anticancer drugs are of great therapeutic interests as they can potentially overcome the deficiencies of conventional chemotherapy drugs and improve the efficacy. We reported a novel hybrid NL-101 which comprises the chemoactive groups of histone deacetylase inhibitor SAHA and alkylating agent bendamustine. In this study, we tested the in vitro and in vivo anticancer effect of NL-101 on acute myeloid leukemia cells, and investigated its possible related mechanisms.Methods:Cell inhibition rate of leukemia cells treated with NL-101, SAHA and bendamustine was determined by MTT assay. The inhibitory concentration of the drug uesd to cause 50% inhibition of cell growth (IC50) was calculated by SPSS statistical software. The combination index (CI) of SAHA and bendamustine was calculated by CompuSyn software. The cell cycle distribution and apoptosis rate were detected by flow cytometry. Western blot analysis was used to analyze the level of acetylated H3 as well as cell signaling proteins related apoptosis and DNA damage, including y-H2AX, PARP, caspase-3, Bax, Bcl-2 and Bcl-xL. Bone marrow samples of AML patients and healthy donors were isolated by Ficoll gradient solution and the morphology was observed with Wright staining. For in vivo experiment, transplantable mice leukemia model co-expressing AML1-ETO and HyC-KITD816v were generated by retroviral infection. Leukemia mice received NL-101, bendamustine and SAHA treatment. The body weight and the infiltration of GFP+cells were monitored, and the survival time of each mouse was recorded.Results:In vitro study:(1) The synergistical effect of SAHA and bendamustine was proved in leukemia cells. (2) NL-101 exhibited efficient anti-proliferative activity on myeloid leukemia cells especially Kasumi-1 and NB4 cells with resistance on non-malignant cells. The potency of NL-101 showed far superior to that of bendamustine, while similar with SAHA. (3) NL-101 caused S phase arrest and significant apoptosis of Kasumi-1 and NB4 cells. (4) The activation of caspase-3 dependent apoptosis pathway and members of Bcl-2 family proteins contributed to its cytotoxic effect. In addition, NL-101 presented both the properties of HDAC inhibition and DNA damage, as assessed by the acetylation of histone H3 and DNA double-strand breaks marker y-H2AX. (5) NL-101 induced apoptosis and DNA damage in primary cells from acute myeloid leukemia (AML) patients.In vivo study:(1) Recipient mice co-expressing AML1-ETO and HyC-KITD816V developed aggressive acute leukemia with extensive infiltration of immature blast cells (c-Kit+, Gr1-, CD11b-) in hematopoietic organs. (2) NL-101 treatment could reduce the infiltration of leukemia cells and significantly prolong the survival time of leukemia mice showing the enhanced efficacy than bendamustine and SAHA. (3) Recipient mice could tolerate the NL-101 treatment without significant weight loss.Conclusions:The novel SAHA-bendamustine compound NL-101 exhibited potent anti-proliferative and pro-apoptotic activities towards AML cells both in vitro and in vivo. It possessed the dual mechanisms of HDAC inhibition and DNA damage.
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