State Key Laboratory of Medicinal Chemical Biology, College of Pharmacy and Tianjin Key Laboratory of Molecular Drug Research, Nankai University, Haihe Education Park, 38 Tongyan Road, Tianjin 300353, People’s Republic of China

Leukemia stem cells (LSCs), which are challenging to eradicate with standard therapy and are responsible for relapse, are largely to blame for the poor prognosis for patients with acute myeloid leukaemia (AML). New therapeutic approaches that could specifically target LSCs in the treatment of clinical leukaemia and prevent drug resistance are thus urgently required. Only a tiny number of small compounds, nevertheless, have been found to exhibit anti-LSC action.

Acute myeloid leukaemia (AML) patients have dismal prognoses, with overall survival rates for younger patients of 50% and older patients of 1 year or less [1, 2]. Chemotherapy and/or hematopoietic stem cell (HSC) transplantation are frequently used to treat these patients [3]. Effective treatment plans for AML remission and cure are desperately needed. In particular, leukaemia onset, resistance to chemotherapy, prognostic relapse, and worsening are all ascribed to leukaemia stem cells (LSCs), which can develop into either daughter or differentiated cells [2]. LSCs exhibit self-renewal, multipotent differentiation, and relative quiescence characteristics just like HSCs do [4-6].

Due to the LSCs' quiescent state, many clinical chemotherapeutic treatments that target the cell cycle have minimal ability to destroy them. Additionally, the P-glycoprotein is overexpressed on the surface of LSCs [7], which results in drug resistance and the potential for chemotherapeutic medicines to be pumped out of cancer cells. Patients with AML who have LSCs have lower remission rates, shorter disease-free survival times, and shorter overall survival times [8]. It is thought that abnormal regulation of several signal pathways, including the Wnt/-catenin, p53, NF-kB, ROS, and Notch pathways, is essential for the pathogenesis of LSCs. LSCs are eliminated by the restoration of these signal channels [9–13].

In vitro, alantolactone reduced cell growth and triggered apoptosis in a dose-dependent manner. Intriguingly, alantolactone showed negligible cytotoxicity against healthy hematopoietic cells while dramatically inducing apoptosis of CD34 + CD38- cells in primary AML specimens derived from blood samples of AML patients. Alantolactone also prevented the growth of AML colonies in culture. A water-soluble prodrug called DMA-alantolactone was demonstrated to inhibit tumour growth in vivo in a NOD/SCID mice xenograft experiment while causing no discernible harm. These findings showed that alantolactone had a limited impact on healthy hematopoietic cells while preferentially ablating LSCs. To our knowledge, this is the first account of a sesquiterpene lactone of the eudesmane type that exhibits anti-LSCs action.

THP-1, KG1a, HL60, K562, HL60/ADR, and K562/A02 human leukaemia cell lines were grown in 1640 media with 10% foetal bovine serum at 37 °C, 5% CO2 incubator. Ficoll-Paque density gradient separation was used to recover mononuclear cells from the primary human AML samples. The cells were then grown in serum-free IMDM medium for 1 hour before being exposed to various alantolactone doses.

Anti-cancer medication cytotoxicity and cell viability were often assessed using the MTT test. Leukemia cells (1 104 cells/well) were briefly implanted in 96-well plates. The cells were then exposed to alantolactone at several doses, whereas the control group received DMSO treatment. Alantolactone was given a 72-hour treatment before 20 L of MTT solution (5 mg/mL) was added to each well. Each well was then incubated for a further 4 hours at 37 °C with 5% CO2. All of the supernatant was collected after being centrifuged at 1500 rpm for 15 min. DMSO was then applied to each well in order to dissolve the formazan crystal. Using a micro-plate, absorbance was measured at 570 nm.

Apoptosis was measured using flow cytometry, and the manufacturer's instructions were followed when staining KG1a cells for apoptosis using APC-Annexin V and 7-aminoactinomycin (7-AAD) to determine their status. In a six-well plate, 1 105 KG1a cells or 1 106 primary AML mononuclear cells were planted. After a one-hour incubation period, cells were exposed to various concentrations of alantolactone for 24 or 18 hours before being extracted and undergoing three rounds of cold PBS washing. In order to stain the cells, 5 l of APC-Annexin V and 5 l of 7-AAD were added after the cells had been re-suspended in 1 l of binding buffer. After 15 minutes in the dark of incubation, cells were examined using flow cytometry.

By using a density gradient centrifuge, AML mononuclear cells were isolated from AML samples. Magnetic-activated cell sorting CD34 progenitor kit enhanced CD34+ AML cells (Miltenyi Biotech, Auburn, CA, USA). Following enrichment, CD34+ cells were grown in IMDM with 10% foetal calf serum supplement. For rhSCF, rhFlt3, and rhTPO, cytokines were administered at concentrations of 100 ng/mL, 100 ng/mL, and 100 ng/mL, respectively. Then, different alantolactone concentrations were applied to the cells. Cells were collected after 3 days and stained for 30 min. with CD19-PE, CD33-APC, CD3-APC, and CD235a-FITC, respectively. Re-suspended cells underwent flow cytometric examination.

Using flow cytometry, the impact of alantolactone on LSC cells was investigated.From AML patients, the original AML mononuclear cells were derived. Mononuclear cells from primary AML patients were extracted and planted in 24-well plates (1 106 cells/well) with 1 mL of medium. Then, different alantolactone concentrations were applied to the cells. Following an 18-hour treatment, the cells were re-suspended in PBS, stained for 30 minutes with CD34-APC and CD38-PE.cy7, and then re-suspended in 1 binding buffer containing 5 l of Annexin-V-FITC and 5 l of PI.In 1 hour, samples underwent flow cytometric analysis.

IMDM without serum was used to grow mononuclear cells.medium for 18 hours with or without alantolactone or Ara-C. At 200,000 cells per mL, cells were plated.H4434 MethoCult (stem cell). Cells were grown in culture 14 days had passed, and there were 4 colonies formed. tallied in a microscope.

a test for acute toxicity using Kunming mice: DMA-alantolactone was administered orally to five 5-week-old Kunming mice from the Chinese Academy of Sciences in Shanghai, China, at a single dose of 500 mg/kg. The mice's behaviour and appetite were assessed after injection. Every day, the mice's body weight was measured. Cells from the KG1a tumour growth inhibition experiment in naked mice were collected and given one PBS wash. Then, 5-week-old female BALB/c nude mice were injected with KG1a cells (1 107 cells/0.1 mL PBS) (Chinese Academy of Sciences, Shanghai, China). After 20 days, the tumor's volume had grown to 500 mm3, at which point it was removed, leaving a 0.5 mm3 remnant. Mice were then given tiny tumour bits to implant.

Every three days, tumour volumes and weights were assessed using An automatic vernier calliper was used to measure the volumes and weights of the tumours every three days. When the tumor's volume reached 100 mm3, DMA-alantolactone was given orally three times a week at a dose of 100 mg/kg. After 30 days, mice were slaughtered. Calculations were made for tumour growth curves and growth inhibition rates. Each experimental group and control group consisted of five mice. Mice were raised in the Animal Ethics Committee of the Institute of Hematology & Hospital of Blood Diseases, Chinese Academy of Medical Sciences & Peking Union Medical College in accordance with national and international regulations governing animal testing.

Different leukaemia cells' ability to proliferate was decreased by alantolactone in vitro. The cytotoxicity of alantolactone was first investigated in vitro in several leukaemia cell lines, including the human leukaemia cell lines HL60 and K562, their multidrug-resistant counterparts HL60/ADR and K562/A02, and the human AML cell line THP-1. Alantolactone was used to cultivate all leukaemia cells for 72 hours. The HL60 and K562 cells were significantly inhibited by alantolactone, with IC50 values of 3.26 and 2.75 M, respectively. Additionally, activities against the drug-resistant cell lines HL60/ADR (IC50 = 3.28 M) and K562/A02 (IC50 = 2.73 M) were comparable to those against their sensitive cell line counterparts HL60 and K562, while ADR, a clinically used drug, displayed IC50 values for the drug-resistant cell lines that were 205- to 59-fold higher.

Comparing the characteristics of the sensitive cell lines HL60 and K562 to those of HL60/ADR and K562/A02, respectively. With an IC50 value of 2.17 M, alantolactone was effective against the human AML cell line THP-1. In many leukaemia cell lines, alantolactone showed dose-dependent anti-cancer efficacy. According to earlier research, the KG1a cell line is one of the AML progenitor cell types that exhibits significant levels of multidrug resistance and self-renewal potential. Additionally, KG1a cells included a sizable proportion of cells with the immunophenotype CD34+ CD38-, which was thought to be typical of LSCs. As a result, it was determined that KG1a was a cell line that resembled LSCs and was the best cell line for preliminary testing of possible anti-AML stem cell drugs [28–30]. We further investigated alantolactone's effectiveness against KG1a.It is interesting that alantolactone significantly inhibited the growth of KG1a (IC50 = 2.75 M), equivalent to its effects on HL60 and HL60/ADR.

Alantolactone was further investigated to determine its selectivity towards AML cells over normal cells in light of the significant activity against AML cells. Healthy donors were used to obtain normal hematopoietic cells. With an IC50 value of 26.37 M, the results showed that alantolactone had no discernible impact on the viability of normal cells. The selectivity index between KG1a and normal cells was 9.6, indicating that alantolactone might preferentially kill AML cells (IC50 for KG1a = 2.75 M) while being only mildly hazardous to normal hematopoietic cells (IC50 = 26.37 M).Apoptosis and differentiation of stem-like cells were triggered by alantolactone.We looked at the LSC population in the KG1a cell line, which contained 54% CD34+ CD38- cells, according to a report [28].

According to our flow cytometry results, the KG1a cell line had 99.3% CD34+ cells and 37.4% CD34+ CD38- cells, proving that it was an AML stem-like cell line. Alantolactone significantly increased the frequency of lymphocyte cells (CD19), T cells (CD3), and erythroid cells (CD235a) in the differentiation assay while significantly decreasing the frequency of myeloid cells (CD33). Total primary AML cells from AML specimens were cytotoxicly affected by alantolactone in a dose-dependent manner. APC Annexin V and 7-AAD staining were also used to measure apoptosis, together with flow cytometry. In KG1a cells, alantolactone caused apoptosis in a dose-dependent manner.

The apoptotic percentage of KG1a cells was 10.9 0.35% (2.5 M), 20.8 3.12% (5 M), and 36.0 1.56% (10 M) compared to the negative control 8.27 0.92% (DMSO) in KG1a cells after 24 hours of alantolactone culture.

Through the inhibition of NF-kB and its downstream target proteins, alantolactone caused apoptosis. We examined the status of apoptosis by Western blot examination of Bcl-2, Bax, caspase-3, caspase-9, p65, XIAP, FLIP, and PARP in primary CD34+, HL60, and KG1a cells to investigate the mechanism by which alantolactone induces apoptosis. The Bcl-2 family was crucial in controlling apoptosis, and Bcl-2 and Bax protein expression levels were frequently checked for signs of apoptosis. Alantolactone therapy for 24 hours resulted in a significant decrease in the expression of Bcl-2, an apoptosis inhibitor, and an increase in the expression of Bax, an apoptotic protein, as compared to the control group (Fig. 6). In the cell death cascade, caspase-3, caspase-9, and PARP were crucial players. Alantolactone's effects were investigated. With therapy with alantolactone, caspase-3, caspase-9, and PARP cleavages increased. Significantly less p65 was expressed, which was critical for the NF-kB signalling pathway. The levels of XIAP and FLIP, the NF-kB downstream target proteins, decreased concurrently following alantolactone therapy.

One of the most prevalent types of adult malignant myeloid leukaemia is AML. Clinical treatments for younger adults employ a variety of modalities. Nevertheless, improving the survival and cure rates for patients older than 70 years is still difficult [31]. The biggest challenges to treating AML are drug resistance and recurrence. Clinical studies are currently being conducted to find novel anti-AML medications or to target AML treatments [2]. LSCs are a subpopulation of AML cells that have the capacity to self-renew. According to reports, LSCs with the immunophenotype CD34+ CD38- are linked to minimal residual disease (MRD) and are primarily responsible for leukaemia onset, medication resistance, and recurrence.

Theoretically, medication resistance and tumour recurrence could be prevented without harming healthy hematopoietic stem cells by selectively targeting LSCs. Sesquiterpene lactone alantolactone, which was derived from I. helenium L., has been investigated as a possible anticancer agent in numerous cancer lines [34–36]. For the treatment of drug resistance, alantolactone may lower P-glycoprotein expression in the drug-resistant cell line K562/adriamycin [37]. Alantolactone has reportedly been extracted from Chinese plants [38].

This is ourFirst, studies show that alantolactone can target LSCs with specificity. Comparable levels of cytotoxicity were seen across drug-sensitive cell lines K562 and HL60 and drug-resistant cell lines K562/A02 and HL60/ADR with alantolactone. According to reports, LSCs exhibited the traits of multidrug resistance. The cell line KG1a responded favourably to alantolactone treatment despite having a high capacity for drug efflux mediated by P-glycoprotein and a high level of DNR resistance. According to earlier research [28] and the current study, KG1a was a stem-like cell line with a high level of CD34+ and CD34+ CD38.

Treatment with alantolactone at a concentration of 10 M significantly reduced the viability of CD34+ CD38- cells from the majority of specimens. Alantolactone was also more harmful to CD34+ cells CD38– than the total number of primary leukaemia cells. Contrarily, the Ara-C, a commonly used chemotherapeutic medication, showed minimal When evaluated against whole primary leukaemia, apoptosis cells lacking CD34+5 and 10 % concentrations of CD38- cells 10 μM. It's important to remember that CD34+A CD38- cell from the umbilical cord of healthy hematopoietic cells were almost unaffected by alantolactone therapy.at 2.5 and 5 M levels of concentration .

To continue verify the colony-inhibiting effects of alantolactone colony development for the initial AML cells assay was carried out. Alantolactone considerably reduced the In contrast to the control group, DMA-alantolactone significantly reduced tumour size following therapy. In vivo, DMA-alantolactone may inhibit the growth of tumours. Normal hematopoietic stem cells did not express much NF-kB, whereas leukaemia stem cells dramatically overexpressed it [39]. Consequently, NF-kB expression and activity inhibition could be promising therapeutic targets for leukaemia stem cell treatment. Cell apoptosis was significantly influenced by the expression of NF-kB and NF-kB-regulated proteins such p65, Bcl-2, XIAP, and FLIP. Parthenolide and micheliolide were shown to be able to covalently bind to proteins via the Michael acceptor and destroy leukaemia stem cells by blocking the activity of NF-kB [17, 19, 39]. These compounds also have chemically reactive alpha-beta unsaturated lactone moiety.

We hypothesised that substances having There may be some shared protein targets for the alpha-beta unsaturated lactone moiety. Because alantolactone also contains an alpha-beta unsaturated lactone moiety, we hypothesised that it might target AML stem cells by preventing NF-kB from doing its job. The initial research was done to determine the mechanism of alantolactone's anti-AML stem cell action. When compared to the control group, alantolactone therapy resulted in a reduction in the expression of Bcl-2, p65, XIAP, and FLIP and an increase in the expression of Bax. Alantolactone therapy led to an increase in caspase-3, caspase-9, and PARP cleavages. These findings showed that alantolactone primarily suppressed NF-kB and its downstream target proteins, leading to the death of AML progenitor cells.

There may be some shared protein targets for the alpha-beta unsaturated lactone moiety. Because alantolactone also contains an alpha-beta unsaturated lactone moiety, we hypothesised that it might target AML stem cells by preventing NF-kB from doing its job. The initial research was done to determine the mechanism of alantolactone's anti-AML stem cell action. When compared to the control group, alantolactone therapy resulted in a reduction in the expression of Bcl-2, p65, XIAP, and FLIP and an increase in the expression of Bax. Alantolactone therapy led to an increase in caspase-3, caspase-9, and PARP cleavages. These findings showed that alantolactone primarily suppressed NF-kB and its downstream target proteins, leading to the death of AML progenitor cells.

In conclusion, it was first shown that alantolactone, a well-known naturally occurring eudesmane-type sesquiterpene lactone, could be a possible drug that could target LSCs with little harm to normal cells. By inhibiting NF-kB and its downstream target proteins, alantolactone caused apoptosis in AML stem cells. Tumor growth was demonstrated to be inhibited in vivo by DMA-alantolactone, a water-soluble prodrug of alantolactone. Based on these findings, we suggest that alantolactone could be employed as a possible LSC-targeted agent and that eudesmane-type sesquiterpene lactones could serve as a new platform for the development of anti-LSC drugs.

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Yu Zhang . Stem and progenitor cells from acute myeloid leukaemia are preferentially destroyed by alantolactone. World Journal Of Hematology And Oncology 2022.