Suppression Of Aberrant Activation Of NF-κB Pathway In Drug-resistant Leukemia Stem Cells Contributes To Parthenolide-potentiated Reversal Of Drug Resistance In Leukemia

Although many drugs that targeted the specific features of leukemia stem cells (LSCs) have substantial application in the clinical treatment of leukemia, the LSCs relapsed and caused drug-resistant leukemia. Therefore, it is necessary to identify the unique features of LSCs in relapsing and drug-resistant leukemia and also to explore the drugs that directed at these features. Our clinical data have indicated that relapsed patients with acute myeloid leukemia have more abundant proportion of LSCs with enhanced breast cancer resistance protein (BCRP) and P-glycoprotein (P-gp) expression when compared to the untreated patients. The results showed that compared with LSCs derived from sensitive K562 cells, LSCs from drug-resistant K562/ADM cells have much higher chemotherapeutic resistance, and so we termed these cells as “drug-resistant LSCs”. Subsequently, aberrant activation of NF-κB pathway in drug-resistant LSCs was further using gene chip analysis. Also, parthenolide (PTL), which is a specific NF-κB inhibitor, effectively eliminated drug-resistant LSCs and enhanced the sensitivity of K562/ADM cells to doxorubicin-induced apoptosis by down-regulating NF-κB pathway-mediated P-gp expression. These findings make the research area of LSCs more abundant and provide a potential therapeutic strategy for the treatment of refractory and relapsed leukemia.


Cell culture
Doxorubicin-induced and P-glycoprotein-overexpressed multidrug-resistant human leukemia cells (K562/ADM cells) and sensitive cells (K562 cells) were cultured in RPMI1640 (Gibco, Thermo Fisher Scientific, USA) medium supplemented with 10% fetal bovine serum (FBS, GE Healthcare Life Sciences, USA ), 1mM l-glutamine, 50Uml −1 penicillin, and 50Uml −1 streptomycin. The cell cultures were maintained in an incubator at 37°C and 5% CO 2 . To maintain drug resistance, doxorubicin (4.0 mgL −1 ) was supplemented at regular intervals. Cells could be harvested and used for experiments 1 week after the removal of doxorubicin.

Isolation of the LSCs from K562/ADM and K562 cell line by FACS
1×10 8 cells were collected and washed with cold PBS three times. To isolate LSCs from K562/ADM cells and K562 cells, a single-cell suspension of 1×10 6 ml −1 was prepared and stained with anti-CD34-PE-CY5 and Anti-CD38-ECD (Caltag Lab, USA for 15 min in the dark at room temperature. Next, the cells were washed 3 times with cold PBS and were suspended in cold PBS with 2% FBS. The following filtration using 400 mesh strainer, the cells were analyzed and sorted by FCM using the MoFlo system (XDP, Beckman). The cells inside the area of CD34 + and CD38were identified as leukemia stem cells (LSCs).
The enrichment of LSCs was detected by FACS and trypan blue staining.

Gene expression analysis
Human Signal Transduction Pathway Finder TM RT 2 Profiler TM PCR Array (PAHS-014A, Qiagen, USA) was used to screen the differences of 84 key genes representative in 18 different signal transduction pathways between LSC K562 and LSC K562/ADM . Five housekeeping genes (Actb,B2m,Hrpt1,Ldha and Rp1p1) were used for normalization of samples. The data and volcano plot were analyzed and produced using the RT 2 array data analysis web portal (https://dataanalysis2.qiagen.com/pcr).

Proliferation assay
The cells were cultured in the 96-wells plates at a density of 1×10 5 ml -1 and treated with different concentrations of PTL for 24 h-48 h. MTT was added to each well before termination of culture and incubated for 4 h at 37°C in 5% CO 2 . Then, 10%SDS was be added to each well, followed by overnight incubated at 37°C and 5% CO 2 to dissolve the dark blue crystal product. Each sample point was assayed with 4 replica points.
Absorbance at 570 nm (A570) of the solubilized formazan was measured using a Bio-Tek Instruments (KC junior, USA) microplate reader to calculate inhibition rate for cell proliferation and 50% inhibitory concentration (IC 50 ).

Apoptosis assay
The cells were treated with different concentrations of PTL for 24 h. Then 1×10 6 cells were collected and washed with cold PBS. Subsequently, 100μl Annexin V-binding buffer, FITC-conjugated Annexin V and PI were used to stain the cells in each sample for 15 min at room temperature. FACS (Beckman-Coulter Epics XL, USA) was employed to measure the apoptosis rate in the early and terminal phase with 3 replica points.
To assay the apoptosis of LSC in K562/ADM and K562 cell population, the cells were treated with 5μM and 10μM PTL for 24h and marked with PEcy5-CD34, ECD-CD38 and PE-CD123 antibodies at room temperature for 20 min, and then labeled with FITC-Annexin V in 100μl Annexin V-binding buffer for 15min. The percentage of CD34 + CD38 -CD123 + cells was detected by FCM, and then gated with CD34 + CD38 -CD123 + to analyze the apoptotic (Annexin V + ) cells in CD34 + CD38 -CD123 + population. Each sample point was analyzed with 3 replica points.

Wright-Giemsa staining and electron microscope
Wright-Giemsa staining was performed as follows: after designed treatments, cells were reseeded to a coverglass slide and stained with Wright-Giemsa at room temperature for 30min. Changes of morphology were observed using AX80 optical microscope (Olympus, Tokyo, Japan) For electron microscope, cells were fixed, stained, dehydrated, embedded, cut into thin sections with an ultramicrotome and then cell sections were picked up on copper grids. After post-staining, these sections were analyzed with a JEM1230 transmission electron microscope (JEOL, Japan).

Assess the number of LSCs in whole cells population
After designed treatments, cells were labeled with anti-CD38-FITC, anti-CD34-PE-CY5 (Caltag Lab, USA) and anti-CD123-PE. (eBioscience, USA) and incubated at room temperature for 20 min followed by analysis using FACS. The mean percentage of CD34 + CD38 -CD123 + was calculated from 3 independent experiments.

Cell cycle analysis
Cells were collected and washed with cold PBS 3 times. And then, 70% of cold ethanol was added gently to fix cells overnight at -20℃. Before cell-cycle distribution being analyzed by FACS, cells were washed 3 times with cold PBS and stained with PI for 30min at room temperature.

Quantitative real-time PCR
Total RNA was extracted from cells using TRIzol (Invitrogen) and cDNA was synthesized using 1μg RNA with KIT (RR047A, Takara Bio, Otsu, Japan). The mRNA level was

Western blot analysis
After designed treatments, cells were harvested and lysed in Laemmli SDS buffer (62.5 mM Tris-HCL pH 6.8, 25% glycerol, 2% SDS, phosphatase inhibitor and proteinase inhibitor cocktail). The whole lysate were collected and subjected to SDS/PAGE. The proteins were transferred to PVDF membrane. After blocking with 5% non-fat milk in TBST for 1hr, the membrane was probed with primary antibodies and secondary