Background: Acute myeloid leukemia (AML) is a malignant clonal disease of hematopoietic stem cells. The long term survival of AML is not satisfactory, so new treatment should be explored. Here, we show that chidamide(CH), a histone deacetylase inhibitor, combined with decitabine(DE) induces apoptosis of AML cell lines and primary refractory/relapsed AML cells by up-regulating PERP. This may provide a new option for AML treatment.
Methods and results: We first examined the half-inhibitory concentrations (IC50) of chidamide and decitabine against THP-1, MV4-11, HL60 and Kasumi-1 cell lines using MTT (Fig1 A-D). And the drug combination is performed according to the IC50. In the double-drug combination experiment, we used MTT to detect the effect of drugs on the proliferation of the four cell lines (Fig1 E-H), used calcusyn 2.0 software to calculate the synergistic effect (Fig2), flow cytometry to detect apoptosis (Fig3 A-D), and western blot to detect the pro-apoptotic protein (C-CASPASE 3 and C-CASPASE 9) and anti-apoptotic proteins (CASPASE 8, BCL-2 and BCL-XL) (Fig3 E-H). We found that chidamide combined with decitabine synergistically inhibited proliferation of AML cell lines, induced apoptosis, up-regulated pro-apoptotic protein levels and down-regulated anti-apoptotic protein levels.
To investigate this combination therapeutic effect in vivo, we selected 5 refractory/relapsed AML patients, extracted primary AML cells, and used ATP chemiluminescence kit for drug sensitivity test. The results confirmed that four of the five patients with AML showed sensitivity to combinations (Fig4).
To further explore the mechanism of action of CH combination with DE, we performed transcriptome sequencing (Fig5). Analysis of the sequencing results, the gene PERP, which shows the significant difference in the apoptotic pathway, was further examined. The PERP is a new member of the PMP-22/GAS3 family as an apoptosis-associated target of TP53. RT-QPCR and WB verified the role of PERP in apoptosis in DE and CH combination (Fig6). The results showed that the combination could up-regulate the PERP gene than the single drug. When we explored the role of the PERP gene in AML cell lines, we knocked down the PERP gene by lentivirus and detected cell proliferation after infection. Pretreated AML cell lines by lentivirus-infection (Fig7A-F), then we tested for proliferation (Fig7G-I) (Fig8A-C), apoptosis (Fig8D-E), and pro-apoptotic protein expression (Fig8G-I). The results showed that knocking down the PERP gene promoted the proliferation of AML cell lines and attenuated the sensitivity of AML cell lines to chemotherapeutic drugs.
We also compare the mRNA level of PERP between 35 AML patients and 20 normal and found that the PERP mRNA of AML patients was significantly lower than the normal (Fig9).
MV4-11 cells were exposed to CH and DE alone or in combination, and proteomic sequencing was performed to examine the effect of the drug on cellular protein. The result indicates to some extent that CH contributes more to the combined effect. And the drug causes changes in multiple pathways in the cell (Fig10).
Conclusion: Our experiments revealed that CH combined with DE may have therapeutic effects on AML and, to some extent, reveal the mechanism of dual drug combination.
Legends to figures
Fig1. 50% inhibitory concentration (IC50) values of chidamide and decitabine alone treated AML cell lines.
Fig2. Chidamide acts synergistically in AML with DE.
Fig3. Chidamide in combination with decitabine significantly induced apoptosis in AML cell lines.
Fig4. The sensitivity of relapsed or refractory AML primary cells to chidamide and decitabine alone or in combination.
Fig5. Gene expression analysis showed an obvious difference based on treatment.
Fig6. Verify transcriptome sequencing results by real-time QPCR and by western blotting with or without drug treatment.
Fig7. The effect of down-regulation of PERP by Lentivirus-mediated RNAi on AML cells proliferation.
Fig8. PERP knockdown causes AML cells to develop resistance to combination drugs.
Fig9.The level of PERP mRNA in peripheral blood mononuclear cells of AML and normal humans.
Fig10. Proteomics sequencing results show the differentially expressed protein and a cluster analysis of the functions or pathways enriched by differentially expressed proteins in GO and KEGG pathways compared to single agents.
No relevant conflicts of interest to declare.
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